L-gulonolactone oxidase in the bodies of fish. VC plays a significant role in maintaining the physiological functions and in improving the growth performance, immunity, and survival of fish. In this study, zebrafish (Danio rerio) were treated with 8.2, 509.6, and 1007.5 mg/kg VC diets for 2 weeks, and the muscle samples were collected for gene expression analysis and biochemical index analysis. The results indicated that 509.6 and 1007.5 mg/kg VC diets inhibited glycogen synthase kinase-3β (GSK-3β) expression and induced the expression of β-catenin in the muscle of zebrafish. The mRNA expression of CCAAT/enhancer-binding protein α (C/EBPα) and fatty acid synthase (FAS), FAS activity, and the content of glycerol and triglyceride (TG) were decreased in the muscle by 509.6 and 1007.5 mg/kg VC diets. In addition, GSK-3β RNA interference was observed in zebrafish fed with 8.2 and 1007.5 mg/kg VC diets. It was found that GSK-3β RNA interference induced the mRNA expression of β-catenin but decreased the mRNA expression of C/EBPα and FAS, FAS activity, as well as the content of glycerol and TG in the muscle of zebrafish. In ZF4 cells, the mRNA expression of GSK-3β, C/EBPα, and FAS was decreased, but β-catenin expression was increased by 0.1 and 0.5 mmol/L VC treatments in vitro. The glycerol and TG content, and FAS activity in ZF4 cells were decreased by 0.1 and 0.5 mmol/L VC treatments. Moreover, the result of western blot indicated that the protein expression level of GSK-3β was significantly decreased and that of β-catenin was significantly increased in ZF4 cells treated with 0.1 and 0.5 mmol/L VC. The results from in vivo and in vitro studies corroborated that VC exerted the lipid-lowering effect through GSK-3β/β-catenin signaling in zebrafish.Vitamin C (VC) is an essential nutrient for most fish species because of the absence of  L-gulonolactone oxidase in the fish body (Oncorhynchus mykiss)  (Oreochromis niloticus ×O. aureus) (Morone chrysops ×M. saxatilis) (Pseudosciaena crocea), the VC requirement was 28.2 mg/kg diet based on the survival, whereas it was 87.0 mg/kg diet based on the VC content in liver  based on growth was 102.6∼109.5 mg/kg (Anguilla japonica) (Vitamin (VC) is an essential nutrient for most fish species because of the absence of ish body . In the  mykiss) , channelnctatus) , hybrid  aureus) , and hybxatilis) . For larin liver . Dietary.5 mg/kg , and it aponica) .As an essential nutrient for fish species, VC plays a significant role in maintaining the normal physiological functions . It has In the canonical Wnt signaling pathway, without Wnt ligands, β-catenin is continuously ubiquitinylated for degradation through glycogen synthase kinase-3β (GSK-3β). Upon Wnt ligand binding to receptors at membrane, a cascade of events impedes GSK-3β activity . The ensCyprinus carpio)  (Danio rerio) by an in vivo and in vitro study.In fish species, muscle is one of the main sites for lipid storage . Adipocy carpio) . In our maximus) . HoweverTable 1. Then the different contents of L-Ascorbate-2-phosphate  were added to the basal diets. The content of VC in the diet was measured with high efficiency liquid chromatography, and 8.2, 509.6, and 1007.5 mg/kg VC was detected in three diets, respectively.The basal diet was formulated according to Zebrafish (AB strain) were maintained at a light:dark photoperiod (12-h:12-h) at 28°C in the dechlorinated water in the flow-through tanks. The zebrafish were, on average, ∼3.4 cm in length and 6 months of age. Twelve glass tanks were randomly distributed with 72 male zebrafish. Animals were fed twice daily with a commercial diet  and were allowed to get acclimated for 15 days. Subsequently, the diet containing each level of VC was randomly assigned to four tanks. Fish were fed to apparent satiation twice daily (08:00 and 18:00).g for 15 min at 4°C, and the supernatants were collected for biochemical analysis. All animal experiments were approved by Shandong University of China’s Institutional Animal Care Committee in accordance with the Guidelines for Proper Conduct of Animal Experiments (Science Council of China).Two weeks later, fish were anesthetized with 0.1 g/L MS222, and four independent white muscle samples were collected from four tanks, of which each sample was pooled from the dorsum of three fish. Four muscle samples were frozen in liquid nitrogen and stored at -80°C for molecular biology analysis. In addition, the muscle from the other three fish were sampled and pooled for the biochemical analysis. Four samples were homogenized in cold saline with a glass homogenizer and centrifuged at 4000 ×in vitro transcription were performed in water at 37°C in a solution containing T7 RNA polymerase (Takara), 100 mM DTT, RNase Inhibitor, 5 × transcription buffer, 2.5 mM rNTP, and the template. A UV spectrophotometer (Nano View) was used to detect the concentration of dsRNA. Then 72 male zebrafish were randomly distributed into 12 glass tanks, which included four groups and each group included three tanks: group 1 (DEPC water + 8.2 mg/kg VC), group 2 (RNAi + 8.2 mg/kg VC), group 3 (DEPC water + 1007.5 mg/kg VC), and group 4 (RNAi + 1007.5 mg/kg VC). For the groups of GSK-3β RNA interference (group 2 and group 4), each fish received intraperitoneal injection of 25 μL dsRNA (8ng/μL). For the control groups (group 1 and group 3), each fish was injected with 25 μL DEPC water. The diet containing 8.2 mg/kg VC was fed to the fish in group 1 and group 2, and the diet containing 1007.5 mg/kg VC was fed to fish in group 3 and group 4, respectively. Seven days later, fish were sampled for VC treatment in vivo.Firstly, the open reading frame (ORF) of GSK-3β gene (NM_131381.1) was cloned with the primers . According to the method of 2). ZF4 cells were cultured in 0, 0.1, and 0.5 mmol/L VC diluted in the basal culture medium for 48 h. Then cells were collected for gene expression analysis and biochemical analysis. The experiment was repeated three times.ZF4 cells of zebrafish were purchased from China Zebrafish Resource Center (CZRC) , and maintained in DMEM:F12 (Gibco) supplemented with 10% fetal bovine serum at 28°C . The primer sequences for GSK-3β, β-catenin, FAS, CCAAT/enhancer-binding protein α (C/EBPα), and reference gene (β-actin)  and SYBR® Premix Ex TaqTM II  were used to carry out real-time PCR. The real-time PCR program was set as follows: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min. The amplification efficiency was detected, and the 2T-ΔΔC method was employed to analyze the differences of relative gene expression in each sample by using β-actin as the internal reference gene  and transcribed to cDNA by PrimeScriptβ-actin)  were lisnce gene .ZF4 cells were cultured in basal culture medium containing 0, 0.1, and 0.5 mmol/L VC for 48 h. After the cells were fixed with paraformaldehyde and permeabilized with Triton X-100, cells were incubated with antibodies, anti-GSK-3β , and anti-β-catenin , respectively. Then cells were stained with the fluorescein isothiocyanate (FITC) dye secondary antibody . The negative controls with only secondary antibody were also performed. Finally, the samples were incubated with 5 μM DRAQ5™  nuclear stain, and a laser scanning confocal microscope  was used to detect the immunofluorescence.ZF4 cells were cultured in basal culture medium containing 0, 0.1, and 0.5 mmol/L VC for 48 h. The ZF4 cell samples were prepared using the method described by post hoc test was carried out to calculate the interaction of the two factors, “RNAi” and “VC content”, on the levels of gene expression and biochemical index. P < 0.05 was considered statistically significant.Data were expressed as mean values ± standard error of mean (s.e.m). The statistical analyses were conducted using SPSS 16.0 . The homogeneity of variances among groups and normality were tested, and the differences among samples were compared using one-way analysis of variance (ANOVA) followed by Tukey’s test. In addition, for GSK-3β RNA interference experiment, two-way ANOVA followed by the appropriate P < 0.05) . However, the mRNA expression of β-catenin was significantly induced by 1007.5 mg/kg VC treatment (P < 0.05) . The mRNA expression of β-catenin in 509.6 mg/kg VC group was higher than the control but no significant difference was observed . In addition, the mRNA expression of C/EBPα and FAS in the muscle of zebrafish was significantly decreased by 509.6 and 1007.5 mg/kg VC treatments (P < 0.05) . No significant difference in the mRNA expression of C/EBPα and FAS was observed between the diets of 509.6 and 1007.5 mg/kg VC .Compared with the control group, the mRNA expression of GSK-3β in the muscle of zebrafish was significantly inhibited by 509.6 and 1007.5 mg/kg VC treatments for 2 weeks (P < 0.05) . However, there was no significant difference between 509.6 and 1007.5 mg/kg VC treatments . The activity of FAS in the muscle was significantly decreased by 509.6 and 1007.5 mg/kg VC treatments (P < 0.05), and there was significant difference between these two groups (P < 0.05) .Compared with the control, the content of glycerol and TG in the muscle was significantly decreased by 509.6 and 1007.5 mg/kg VC treatments (P < 0.05) . However, the mRNA expression of β-catenin was significantly increased by GSK-3β interference in the zebrafish fed with 8.2 and 1007.5 mg/kg VC diets (P < 0.05) . Furthermore, the mRNA expression of C/EBPα and FAS was significantly decreased by GSK-3β interference fed with 1007.5 mg/kg VC diet (P < 0.05) . Two-way ANOVA revealed that the factor RNAi had significant effect on the expression levels of GSK-3β, β-catenin, C/EBPα, and FAS (P < 0.05) (Supplementary Table 1). However, no significant difference was found on the factor VC content and the interaction factor (RNAi and VC content) (Supplementary Table 1).Compared with the control, the mRNA expression of GSK-3β was significantly decreased by GSK-3β RNA interference in the muscle of zebrafish fed with 8.2 and 1007.5 mg/kg VC diets a week later (P < 0.05) , and GSK-3β interference significantly affected the activity of FAS in the muscle of zebrafish fed with 8.2 and 1007.5 mg/kg VC diets (P < 0.05) . Two-way ANOVA analysis indicated that the factor RNAi had significant effect on FAS activity and the content of glycerol and TG (P < 0.05), but no significant difference was found on the interaction factor (RNAi and VC content) (Supplementary Table 2).The content of glycerol and TG was significantly decreased by GSK-3β RNA interference in the muscle of zebrafish fed with 8.2 and 1007.5 mg/kg VC diets (P < 0.05), whereas β-catenin expression was significantly induced by 0.1 and 0.5 mmol/L VC treatments (P < 0.05) . However, the mRNA expression of C/EBPα and FAS in the ZF4 cells of zebrafish was significantly decreased by 0.1 and 0.5 mmol/L VC treatments (P < 0.05) .Compared with the control group, the mRNA expression of GSK-3β was significantly decreased (P < 0.05) . The content of TG and FAS activity in ZF4 cells was significantly decreased by 0.1 and 0.5 mmol/L VC treatments (P < 0.05) .Compared with the control group, the content of glycerol in ZF4 cells was significantly decreased by 0.5 mmol/L VC treatment . It was found that the immunofluorescence of β-catenin was increased, whereas the immunofluorescence of GSK-3β was decreased with increasing VC concentrations . The result of western blot indicated that the protein expression level of total GSK-3β was significantly decreased (P < 0.05) and that of total β-catenin was significantly increased in ZF4 cells treated with 0.1 and 0.5 mmol/L VC (P < 0.05) . Moreover, the phosphorylation level of GSK-3β was significantly increased (P < 0.05) and that of β-catenin was significantly decreased in ZF4 cells treated with 0.1 and 0.5 mmol/L VC (P < 0.05) .The immunofluorescent assay was carried out to detect the effect of VC on the protein expression of GSK-3β and β-catenin in ZF4 cells .The treatment of VC induced the expression of β-catenin by inhibiting GSK-3β expression. GSK-3β/β-catenin signaling exerted the lipid-lowering effect by controlling FAS activity and lipid profile in zebrafish (DL contributed to the conception and design of the work. HY, DL, XZ, and QP contributed to data acquisition, analysis, and interpretation. DL, XZ, and QP drafted the work and revised it critically.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.


To summarize the four recent sodium-glucose cotransporter 2 inhibitor (SGLT2i) trials: Dapagliflozin Effect on CardiovascuLAR Events (DECLARE-TIMI 58), CANagliflozin CardioVascular Assessment Study (CANVAS) Program, Empagliflozin Cardiovascular Outcome Event Trial in Type 2 Diabetes Mellitus Patients–Removing Excess Glucose (EMPA–REG OUTCOME), Canagliflozin and Renal Events in Diabetes with Established Nephropathy Clinical Evaluation (CREDENCE), and explore the potential determinants for their cardiovascular, renal, and safety outcomes.2 for DECLARE-TIMI 58, CANVAS, EMPA–REG OUTCOME, and CREDENCE, respectively). Additionally, CREDENCE had considerably higher rates of albuminuria  were 927, 12.3, and 13.1 mg/g for CREDENCE, CANVAS, and DECLARE-TIMI 58, respectively; EMPA–REG OUTCOME had 59.4% UACR < 30, 28.6% UACR > 30–300, 11.0% UACR > 300 mg/g).The composite renal outcome event rates per 1000 patient-years for drug and placebo, as well as the corresponding relative risk reductions, were 3.7, 7.0, 47%; 5.5, 9.0, 40%; 6.3, 11.5, 46%; 43.2, 61.2, 30% for DECLARE-TIMI 58, CANVAS, EMPA–REG OUTCOME, and CREDENCE, respectively . The major adverse cardiovascular (CV) event rates per 1000 patient-years for drug and placebo, as well as the corresponding relative risk reductions, were 22.6, 24.2, 7%; 26.9, 31.5, 14%; 37.4, 43.9, 14%; 38.7, 48.7, 20% for DECLARE-TIMI 58, CANVAS, EMPA–REG OUTCOME, and CREDENCE, respectively. DECLARE-TIMI 58 had the fewest cardiorenal events and CREDENCE the most. These differences were presumably due to varying inclusion criteria resulting in DECLARE-TIMI 58 having the best baseline renal filtration function and CREDENCE the worst (mean estimated glomerular filtration rate 85.2, 76.5, 74, 56.2 mL/min/1.73 mDapagliflozin, empagliflozin, and canagliflozin have internally and externally consistent and biologically plausible class effects on cardiorenal outcomes. Baseline renal filtration function and degree of albuminuria are the most significant indicators of risk for both CV and renal events. Thus, these two factors also anticipate the greatest clinical benefit for SGLT2i. Type 2 diabetes mellitus (T2DM) is significantly associated with cardiovascular disease (CVD) and is a risk factor for heart failure (HF); diabetic patients are hospitalized for HF approximately four times more frequently than nondiabetic patients –5. T2DM The 2008 United States Food and Drug Administration (FDA) antidiabetic drug guidance required cardiovascular outcome trials (CVOTs) for novel antihyperglycemic medications to demonstrate that new drugs would not increase the risk for MI, stroke, or CV death . The FDAThree SGLT2i  have been studied in CVOTs; canagliflozin has also been studied in an additional randomized clinical trial involving patients with diabetic kidney disease –23. ThisWe reviewed the relevant trials’ original methodology and results papers. The methodological details and outcomes of the trials will be reviewed in the Results section below. As some p-values were not provided in all trials, we calculated them from the HR and 95% CI . We calc2 [calculated using the Modification of Diet in Renal Disease (MDRD) equation]; empagliflozin is indicated for T2DM patients with eGFR ≥ 45 mL/min/1.73 m2  EMPA–REG OUTCOME patients had established CVD, most commonly stable coronary artery disease Fig. . The mea/1.73 m2 . There w2, initiation of renal-replacement therapy (RRT), or renal death] occurred in 1.7% of empagliflozin patients compared to 3.1% of placebo patients   occurred in 10.5% of empagliflozin patients compared to 12.1% of placebo patients  ; canagliflozin is indicated for T2DM patients with eGFR ≥ 45 mL/min/1.73 m2 and contraindicated for patients with eGFR < 30 mL/min/1.73 m2  rate per 1000 patient-years was 5.5 in empagliflozin patients compared to 9.0 in placebo patients   rate per 1000 patient-years was 26.9 for canagliflozin patients compared to 31.5 for placebo patients   and contraindicated for patients with eGFR < 30 mL/min/1.73 m2  year follow-up period . Males ≥/1.73 m2 , 34. Inv2, 1265 (7.4%) patients had eGFR < 60, and 15,895 (92.6%) had eGFR > 60 mL/min/1.73 m2. The median UACR was 13.1  mg/g; 11,652 (67.9%) patients had UACR < 30 mg/g, 4023 (23.4%) had UACR > 30–300 mg/g, and 1169 (6.8%) had UACR > 300 mg/g. Although ACEi/ARB use was not required, they were used in 13,950 (81.3%) patients.In DECLARE-TIMI 58, 6974 (40.6%) patients had established CVD, most commonly stable coronary disease Fig. . The mea2, ESRD , or renal/CV death] occurred in 1.5% of dapagliflozin patients compared to 2.8% of placebo patients   occurred in 8.8% of dapagliflozin patients compared to 9.4% of placebo patients   and investigators planned to include ~ 60% of patients with eGFR between 30 and 60 mL/min/1.73 m2. Additionally, patients were required to have albuminuria, defined as UACR > 300–5000 mg/g. Patients were not required to have prior CVD. Notably, the trial was stopped early as it met the pre-specified efficacy criteria for premature cessation.The Canagliflozin and Renal Events in Diabetes with Established Nephropathy Clinical Evaluation randomized double-blind controlled trial (CREDENCE) assigned 4401 patients with T2DM and CKD to 100 mg of canagliflozin or placebo daily over a 2.62 year median follow-up period . Study p2, 2631 (59.8%) patients had eGFR < 60 mL/min/1.73 m2, and 1769 (40.2%) had eGFR > 60 mL/min/1.73 m2. In striking contrast to the three CVOT trials, the median UACR was 927  mg/g; 31 (0.7%) patients had UACR < 30 mg/g, 496 (11.3%) had UACR > 30–300 mg/g, 3371 (76.6%) had UACR > 300–3000 mg/g, and 503 (11.4%) had UACR > 3000 mg/g. Stable ACEi/ARB use was required for ≥ 4 weeks prior to randomization and RAASi were used in 4395 (99.9%) patients.In CREDENCE, 2220 (50.4%) patients had established CVD and ~ 16% had a baseline history of HF , or renal/CV death] occurred in 11.1% of canagliflozin patients compared to 15.4% of placebo patients  , ESRD  compared to DECLARE-TIMI 58, CANVAS, and EMPA–REG OUTCOME   in a previous review . We arguNote that eGFR is more likely to identify CKD in older patients whereas UACR/albuminuria is more likely to identify it in younger patients . AdditioBaseline renal filtration function appears to play a major role in predicting cardiorenal outcomes, perhaps more so than prior CVD. Even though CREDENCE was not planned as a CVOT and thus only 50.4% of its population had prior CVD , CREDENCE still had, for example, a two-fold increase in MACE compared to DECLARE-TIMI 58. This is supported by findings that SGLT2i decreased CV risk depending on baseline renal filtration function but not prior CVD status—lower function was associated with greater reductions in HHF .We considered variance in trial methodologies as determinants for differences in results. Although the four trials had comparable CV event definitions due to FDA regulatory guidance, their composite renal outcome definitions varied according to sponsor choice Table . For exaInterestingly, the CREDENCE and EMPA–REG OUTCOME placebo groups had similar MACE incidence rates , despite different baseline UACR and eGFR. We expect this can be attributed to the balance of baseline CVD vs. renal risk: CREDENCE had significantly higher renal risk but only 50.4% prior CVD whereas EMPA–REG had nearly 100% prior CVD.The composite renal outcome RRR is another intriguing result when comparing the four trials. In a reversal of the trend seen with the other outcomes, CREDENCE had the smallest RRR and DECLARE-TIMI 58 the largest (Fig. We considered other sources of confounding for differences among the trials. All four trials had substantial RAASi use: approximately 80% in the three CVOTs and 99.9% in CREDENCE. Thus, we do not believe differential rates of RAASi can explain the contrasts between the trials. Notably, the high rates of RAASi use indicate that the patients were well-treated at baseline. This should ease skepticism about the real-world therapeutic opportunity for SGLT2i, as any benefits due to the SGLT2i can be viewed as being additional to those from RAASi therapy.We balanced our views on efficacy with the safety data. The four trials demonstrated several general safety trends Tables  and 3. SSGLT2i have demonstrated a host of positive effects of interest for future research. In animal models of T2DM female mice, empagliflozin ameliorated kidney injury by promoting glycosuria, and possibly by reducing systemic and renal artery stiffness; canagliflozin attenuated the progression of atherosclerosis, reducing hyperlipidemia, hyperglycemia, and inflammation by lowering the expression of some inflammatory molecules , 49. Of Giugliano et al.  studied Dapagliflozin, empagliflozin, and canagliflozin have internally and externally consistent class effects on cardiorenal outcomes and similar safety profiles. Baseline renal filtration function and degree of albuminuria are the most significant indicators of risk for both CV and renal events. Thus, these two factors also anticipate the greatest clinical benefit for SGLT2i.


Babesia and Theileria has become increasingly urgent due to parasite resistance to current drugs. Ivermectin (IVM) was the world’s first endectocide, capable of killing a wide variety of parasites and vectors, both inside and outside the body. It is currently authorized to treat onchocerciasis, lymphatic filariasis, strongyloidiasis, and scabies. The current study documented the efficacy of IVM on the growth of Babesia and Theileria in vitro and in vivo.Treatment is the principle way to control and eliminate piroplasmosis. The search for new chemotherapy against Babesia species, including B. bovis, B. bigemina, B. divergens, B. caballi, and Theileria equi, the combination with diminazene aceturate (DA), clofazimine (CF), and atovaquone (AQ) on in vitro cultures, and on the multiplication of a B. microti-infected mouse model. The cytotoxicity of compounds was tested on Madin–Darby bovine kidney (MDBK), mouse embryonic fibroblast (NIH/3 T3), and human foreskin fibroblast (HFF) cell lines.The fluorescence-based assay was used for evaluating the inhibitory effect of IVM on four 50) values determined for IVM against B. bovis, B. bigemina, B. divergens, B. caballi, and T. equi were 53.3 ± 4.8, 98.6 ± 5.7, 30.1 ± 2.2, 43.7 ± 3.7, and 90.1 ± 8.1 μM, respectively. Toxicity assays on MDBK, NIH/3 T3, and HFF cell lines showed that IVM affected the viability of cells with a half-maximal effective concentration (EC50) of 138.9 ± 4.9, 283.8 ± 3.6, and 287.5 ± 7.6 μM, respectively. In the in vivo experiment, IVM, when administered intraperitoneally at 4 mg/kg, significantly (p < 0.05) inhibited the growth of B. microti in mice by 63%. Furthermore, combination therapies of IVM–DA, IVM–AQ, and IVM–CF at a half dose reduced the peak parasitemia of B. microti by 83.7%, 76.5%, and 74.4%, respectively. Moreover, this study confirmed the absence of B. microti DNA in groups treated with combination chemotherapy of IVM + DA and IVM + AQ 49 days after infection.The half-maximal inhibitory concentration  contains supplementary material, which is available to authorized users. Babesia, a genus of Apicomplexa /10  was thawed and injected into two mice intraperitoneally. The parasitemia was monitored every day by microscopy, and when the parasitemia was over 30%, the mice were sacrificed, and blood was collected by cardiac puncture. After that, phosphate-buffered saline was used to dilute the blood to obtain an inoculum containing 1 × 107/mL of B. microti iRBCs. Forty-five female 8-week-old BALB/c mice were caged in nine groups. The mice in group 1 were left uninfected to act as the negative control, while groups 2–9 were injected intraperitoneally (i.p.) with 0.5 mL of inoculum (1 × 107B. microti iRBCs). When the average parasitemia in all mice reached 1%, drug treatment was initiated for 5 days. The mice in group 2 were administered 0.2 mL of DDW intraperitoneally to act as the positive control, while groups 3 and 4 were i.p. injected with 0.2 mL of 25 mg/kg DA and 4 mg/kg of IVM single treatment, respectively. Groups 5 and 6 received 0.2 mL 20 mg/kg AQ and 20 mg/kg CF single treatment orally, respectively. Groups 7–9 were treated with combinations of IVM + DA, IVM + AQ, and IVM + CF, respectively. The parasitemia and hematocrit were monitored every 2 and 4 days for 45 days by microscopy and a hematology analyzer , respectively. On day 49, all mice were anesthetized, and the blood was collected by cardiac puncture for PCR detection of parasites. The experiment was conducted two times. The significant differences between groups were determined by independent Student’s t test and one-way ANOVA Tukey’s test using GraphPad Prism version 5.0 for Windows . A p value of < 0.05 was considered statistically significant.The in vivo inhibitory effects of IVM were evaluated against escribed . BrieflyB. microti small-subunit rRNA (ss-rRNA) gene was carried out in accordance with the previously described protocol [A nested PCR (nPCR) targeting the protocol , 35 afte50 values of IVM, DA, AQ, and CF were determined using the nonlinear regression curve fit in GraphPad Prism . Differences in parasitemia, hematology profiles, and body weight were analyzed using an independent Student’s t test and one-way ANOVA Tukey’s test using GraphPad Prism version 5.0 for Windows . A p value < 0.05 was considered statistically significant.The ICAll experiments were approved by the Animal Welfare Committee and performed in accordance with standards for the care and management of experimental animals as stipulated by Obihiro University of Agriculture and Veterinary Medicine .Additional file 1:Table S1. Concentrations of ivermectin combined with diminazene aceturate and atovaquone against Babesia and Theileria parasites in vitro (DOCX 21 kb)Additional file 2:Table S2. Calculation of weighted average of combination Index values (DOCX 17 kb)Additional file 3:Table S3. The IC50 and selectivity index of DA, AQ, and CF (DOCX 19 kb)


E)-6,6′-bis are described. The fully conjugated bis-phenanthroline molecule is expected to offer exciting new physical and chemical properties, and should form the basis of novel metal coordination complexes as a consequence of the dual N,N′-1,10-phenanthroline chelating moieties situated on the opposite ends of the molecule.The preparation and structural characterization of the diazo-diphenanthroline compound, ( E and Z forms and so could have applications in optical memory and logic devices or as molecular machines. Herein we report the formation and crystal structure of a highly unusual novel diazo-diphenanthroline compound, C24H14N6O2·2CHCl3.Phenanthroline ligands are important metal-binding molecules which have been extensively researched for applications in both material science and medicinal chemistry. Azobenzene and its derivatives have received significant attention because of their ability to be reversibly switched between the  Compound 1 crystallizes with two trichloromethane solvate molecules (1·2CHCl3). The fully conjugated bis-phenanthroline molecule 1 is expected to offer exciting new physical and chemical properties as a stand-alone organic molecule, and it will also birth a plethora of interesting metal coordination complexes as a consequence of the dual N,N′-1,10-phenanthroline chelating moieties situated on the opposite ends of the molecule.Herein, we detail the preparation and structural characterization of the purple diazo-diphenanthroline compound,  Å].Compound ne Fig. 1. The molThe bond lengths indicate some delocalization through the central part of the molecule. The C6—O1and C5—N2 bonds are short  and the N=N bond, at 1.316 (4) Å, is significantly longer than in most free diazo molecules [mean of 1.24 (4) Å for 2730 CSD entries].x, 1 − y, 1 − z) and the axis of the stack runs parallel to the a axis. The shortest ring centroid–centroid distance is 3.5154 (15) Å between the C6 rings; however, there is a more direct overlap between the diazo group and an imine group in the next layer   Fig. 3. The moset al., 2014et al., 2008et al., 19941·2CHCl3.There are three published examples of molecules containing the 2,2′-dihydroxyazobenzene core . UV/vis spectra of 1·2CHCl3 recorded in ethanol, methanol, CHCl3 and CH2Cl2 are given in Fig. 41 exhibits significant solvatochromism showing a broad band in CHCl3 and CH2Cl2 with λmax at 543 nm. This band undergoes a bathochromic shift and separates into two bands in the alcohols with λmax values at 643 nm and 600 nm in ethanol and at 636 nm and 591 nm in methanol. No accurate measurement of the extinction coefficient could be made as 1 was not fully soluble in the solvents and precipitation from the solvent occurred upon standing. The solubility of 1 was so low that only a very poorly resolved 1H NMR of the compound was obtained in d4-methanol showing a series of peaks in the region expected for the phenanthroline H-atom signals and no definitive assignments of the peaks could be made. Interestingly, 1 was more soluble in strongly acidic solutions due to the protonation of one or more of the N atoms. Compound 1 dissolved in CF3COOD to form a bright-red solution. Six signals are observed in the 1H NMR spectrum in the region associated with the phenanthroline peaks. This finding is consistent with a compound which has a centre of symmetry, as found for the crystal structure, and suggests that at room temperature 1 remains in the E form in this solvent.Compound 1 from the reaction mixture is unclear; however, the formation of isonicotinic acid N′-(pyridine-4-carbonyl)-hydrazide (2) from the mixture is an indication that radical chemistry is occurring. Isoniazid is well known to react to form radical species and these radicals are important in its role as an anti-tuberculosis drug  dihydrochloride but these were unsuccessful. However, although the reaction to form 1 was low yielding, attempts to make 1 by reaction of 6-amino-1,10-phenanthrolin-5-ol and 6-nitroso-1,10-phenananthrolin-5-ol using known conditions to form diazo compounds . p-Tolouenesulfonic acid  was added and the solution refluxed for 6 h. The resulting suspension was filtered whilst hot and the filtrate allowed to stand in the dark overnight. Precipitated yellow (Z)-N′- ylidene)isonicotinohydrazide  was filtered off and the bright-orange filtrate was concentrated to ca 10 cm3 using rotary evaporation and then allowed to stand in the dark. Over a period of four weeks, the bright-orange filtrate changed to a dark-green suspension. This mixture was heated to reflux and filtered whilst hot to give a green filtrate (see below) and a dark-purple powder. The powder was dissolved in CHCl3 and allowed to crystallize over several days to produce dark-purple crystals of (E)-6,6′-bis trichloromethane disolvate (1·2CHCl3) . Upon leaving the above green filtrate to evaporate further white isonicotinic acid N′-(pyridine-4-carbonyl)-hydrazide (compound 2) precipitated. The supernatent was decanted off and the solid dissolved in hot acetone. This colourless solution was evaporated to dryness on a rotary evaporator to give 2 .Phendione  was added to solution of isoniazid  in EtOH  (1.2CHCl3): 3065, 2108, 1583, 1568, 1500, 1472, 1415, 1338, 1284, 1227, 1162, 1027, 795, 739, 717. 1H NMR (protonated-1) : δ 9.60 , PhenH 9.56 , 9.47 , 9.37 , 8.57 , 8.38 , 4.12 . Elemental analysis calculated for 1·2CHCl3 : C 47.52, H 2.45, N 12.79%; found: C 47.73, H 2.52, N 12.77%.Compound 2 has been previously reported  3435, 3210, 3045, 1682, 1642, 1546, 1489, 1406, 1299, 838, 751. 1H NMR : δ 8.80 , 7.42 . 13C NMR : δ 165.6 (C=O), 150.0 (PyrC), 140.5 (PyrC), 121.8 (PyrC). MS: Calculated m/z for C12H10N4O2: (M + H)+ 243.0877; found: (M + H)+ 243.0882; difference (ppm): 2.15.Compound Uiso(H) = 1.5Ueq(C) for methyl H atoms or 1.2Ueq(C) otherwise. The H atom (H1A) bonded to oxygen was located in a difference-Fourier map and its coordinates were refined with Uiso(H) = 1.5Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698901900954X/jj2213sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S205698901900954X/jj2213Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698901900954X/jj2213Isup3.cmlSupporting information file. DOI: 1937969CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Andrias davidianus) iridovirosis, leading to severe infectious disease and huge economic losses. However, the infection mechanism by GSIV is far from clear. In this study, a Chinese giant salamander muscle (GSM) cell line is used to investigate the mechanism of cell death during GSIV infection. Microscopy observation and DNA ladder analysis revealed that DNA fragmentation happens during GSIV infection. Flow cytometry analysis showed that apoptotic cells in GSIV-infected cells were significantly higher than that in control cells. Caspase 8, 9, and 3 were activated in GSIV-infected cells compared with the uninfected cells. Consistently, mitochondria membrane potential (MMP) was significantly reduced, and cytochrome c was released into cytosol during GSIV infection. p53 expression increased at an early stage of GSIV infection and then slightly decreased late in infection. Furthermore, mRNA expression levels of pro-apoptotic genes participating in the extrinsic and intrinsic pathway were significantly up-regulated during GSIV infection, while those of anti-apoptotic genes were restrained in early infection and then rose in late infection. These results collectively indicate that GSIV induces GSM apoptotic cell death involving mitochondrial damage, caspases activation, p53 expression, and pro-apoptotic molecules up-regulation.Chinese giant salamander iridovirus (GSIV) is the causative pathogen of Chinese giant salamander ( Apoptosis, pyroptosis, and necroptosis are the three major ways of programmed cell death following virus infection, and apoptosis is the most extensively studied programmed cell death during the process ,2,3. HosRanavirus in the family Iridoviridae that displays icosahedral symmetry with a 120–200 nm diameter [Iridovirus belongs to the genus diameter . It has diameter ,17,18. Idiameter ,22,23. Adiameter . The lardiameter .Andrias davidianus), one of the largest amphibian species in the world, is of great evolutionary importance and classified as a critically endangered species by the International Union for Conservation of Nature and Natural Resources [Chinese giant salamander  are investigated. Our results indicate that GSIV infection evokes typical apoptosis by both extrinsic and intrinsic pathways, with mitochondrial changes, caspases activation, p53 expression, and pro-apoptotic factors up-regulation. Our findings may contribute to elucidate the mechanism of GSIV infection.Microscopy observation showed that GSIV induced host cell death gradually within 48 hours . CompareDNA fragmentation was examined in GSIV-infected cells. A DNA ladder assay revealed that DNA fragmentation begins at 6 h p.i. which was similar to staurosporine treated cells, and fragmentation intensity increased along with the infection time A. Furthep < 0.01) higher than that in control cells  were observed in GSM cells with GSIV infection A, which ol cells B.p < 0.05) increased (about 2.2-fold) at 12 h p.i. compared to that in control cells, and reached a peak level of 3.1-fold at 24 h p.i. Activity of caspase 8 didn’t increase till 48 h p.i., which rose up to about 7.7-fold in comparison to control cells  as early as 6 h p.i., continuously rose up at 12 h (2.6-fold) and 24 h p.i. (4.2-fold), and peaked at 48 h p.i. (5.9-fold) in comparison to that in control group  increased to 18.3% ± 3.2% and further increased up to 21.5% ± 2.9% and 34.4% ± 2.3% at 24 h and 48 h p.i., respectively, in compared to that in control group  and R2 . GSIV infected GSM cells exhibited weakened red fluorescent signals A compareol group B.Loss of MMP results in membrane permeabilization and cytochrome c release. To investigate whether cytochrome c release occurred in GSM cells during GSIV infection, cytosolic proteins of GSIV infected cells and control cells at 24 h p.i. were extracted to detect the presence of cytochrome c by Western blot. The results exhibited that cytochrome c was found in cytosolic proteins of GSIV infected cells . In contTo clarify whether p53 participated in this process, p53 expression was detected by Western blot. The results showed that expression of p53 protein kept a low level at the beginning, then increased at 12 h post GSIV infection, and slightly decreased at 24 h and 48 h p.i. .p < 0.01) at 12 h p.i. and peaked at 24 h p.i. (p < 0.05) at 12 h, went back to the original level 24 h p.i. and then increased significantly (p < 0.01) at 48 h p.i. (p < 0.05) from 12 h to 24 h p.i. and then increased from 24 h to 48 h p.i.  . Ligand–ironment ,34. Pro-ironment . In currironment . We founIt has been reported that p53 participates in the defense against viral infection by activating cell-cycle arrest or apoptosis via the transcription of target genes . p53-depIn conclusion, we demonstrate the elementary mechanism of cell death by GSIV infection in the present study. It is apparent that a GSIV infection induces typical apoptosis in GSM cells. Moreover, signals in both the extrinsic and intrinsic pathways are involved in GSIV-induced apoptosis. In addition, p53 also participates in this process. These results provide new insights into the regulatory mechanism of GSIV infection and contribute to find the potential strategy for preventing iridovirus infection.The giant salamander muscle (GSM) cell line was generously presented by Prof. Qiya Zhang, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan and grown in medium 199  supplemented with 10% fetal bovine serum at 20 °C. The GSIV was originally isolated and identified from diseased Chinese giant salamanders by our laboratory .® Supermix, 0.2 μL of each primer (10 M) and 7.6 μL H2O. The ddPCR cycle profile included 1 cycle at 95 °C for 5 min, then 40 cycle at 95 °C for 30 s, 55 °C for 1 min, then 4 °C for 5 min, 90 °C for 5 min.At 0, 6, 12, 24, and 48 h post GSIV infection (MOI = 0.5), GSM cells were harvested and washed with PBS. Virus DNA were isolated with Viral DNA Kit  according to the manufactures’s protocols. ddPCR was performed in an QX200TM Droplet Digital PCRTM system  using QX200 ddPCR EvaGreen Dye Kit  with primers MCP-F (5’-GCGGTTCTCACACGCAGTC-3’) /MCP-R (5’-ACGGGAGTGACGCAGGTGT-3’). The ddPCR mixture consisted of 2 μL of the diluted cDNA sample, 10 μL 2 × QX200 ddPCR EvaGreenGSM cells were grown on 20-mm circular coverslips and cultured in 12-wells for 24 h. Then GSIV was inoculated onto GSM cells at a multiplicity of infection (MOI) of 0.5 for 24 h, and GSM cells without GSIV infection were set as control and with staurosporine-treatment (30 min) were set as positive control. The cells were fixed with 4% paraformaldehyde for 20 min and washed three times with PBS. The cells were stained with DAPI (10 µg/mL)  for 10 min at room temperature in the dark. After washing as above, the cells were observed with a fluorescent microscope .g for 5 min. Then the supernatant was mixed with equivalent 100% (v/v) ethanol and transferred to an separation column. After washed with 70% ethanol and dried, DNA was dissolved in double-distilled water and then analyzed by 2% agarose gel electrophoresis.The confluent monolayer of GSM cells were infected with GSIV at a MOI of 0.5. At 6, 12, 24, and 48 h post-infection, the infected GSM cells and the control were harvested and the total genomic DNA was extracted as reported previously [An in situ cell death detection kit  was used to further analyze apoptosis based on labeling of DNA strand breaks. After grown on 20-mm circular coverslips for 24 h, GSM cells were infected with or without GSIV at a MOI of 0.5 for another 24 h. The cells were fixed with 4% PFA for 1 h at room temperature and permeabilized with 0.1% Triton X-100 for 2 min on ice. After rinsed twice with PBS, the cells were incubated with TUNEL reaction mixture in above detection kit for 1 h at 37 °C in the dark and then rinsed three times with PBS. The cells were observed with a fluorescent microscope .At 6, 12, 24, and 48 h post GSIV infection (MOI = 0.5), the infected GSM cells and the control were harvested and washed with PBS. Cells were then suspended with 210 µL staining solution containing annexin V-FITC and PI  and incubated at room temperature for 20 min in the dark. After washing, the fluorescence signals were determined by a FACScan flow cytometry . FlowJo 7.6.1 was used for flow cytometric analysis.To explore the function of mitochondrion during GSIV infection, JC-1 was used to evaluate the alteration of mitochondrial membrane potential according to the manufacturer’s protocols. GSM cells were cultured in 24-wells plate for 24 h, and then infected with GSIV at a MOI of 0.5. At 12, 24, and 48 h post-infection, GSM cells were harvested, washed and resuspended in fresh culture medium. The cells were incubated with JC-1 dye solution  for 20 min. After washed three times, the cells were resuspended in JC-1 buffer and quantified by flow cytometry . FlowJo 7.6.1 was used for flow cytometric analysis.The activities of caspase 3, 8, and 9 were detected using a fluorometric protease assay kit  following the manufacturer’s instruction. Briefly, GSM cells were cultured in 24-wells plate for 24 h, and then infected with GSIV at a MOI of 0.5. Then infected and control GSM cells were harvested at 0, 6, 12, 24, and 48 h post-infection and incubated with FITC-DEVD-FMK (for caspase 3 detection), FITC-IETD-FMK (for caspase 8 detection), or FITC-LEHD-FMK (for caspase 9 detection) at 20 °C for 1 h, separately. After washed three times with wash buffer, samples were analyzed by flow cytometry . FlowJo 7.6.1 was used for flow cytometric analysis.TM XRS+ imaging system . β-actin was treated as a reference.Samples of cytosolic proteins of GSM cells with or without GSIV infection (MOI of 0.5) were prepared using a cell mitochondria isolation kit  following the manufacturer’s instruction. Proteins were separated by 12% SDS-PAGE and then electro-transferred to 0.22 µm PVDF membranes with a semi-dry blotter . The membranes were blocked subsequently in TBST  containing 5% skim milk at room temperature for 2 h. The membranes were then respectively incubated with an anti-cytochrome c rabbit polyclonal antibody  at a dilution of 1:500, and anti-β-actin antibody  at a dilution of 1:1000 at 4 °C overnight. After three times of washing with TBST, the membranes were respectively incubated with HRP-labeled goat-anti-rabbit IgG  and goat-anti-mouse IgG  for 1 h. After rinsed with TBST for three times, the membranes were incubated in Western lighting-ECL substrate system (Perkin Elmer) before exposure to a ChemiDocProtein samples of GSM cells with GSIV infection (MOI = 0.5) for different time points were prepared as above. After separated by SDS-PAGE, transferred to PVDF membranes and blocked, the membranes were then respectively incubated with an anti-p53 mouse antibody  at a dilution of 1:1000, and anti-β-actin antibody  at a dilution of 1:1000 at 4 °C overnight. After washed and incubated with HRP-labeled goat-anti-mouse IgG , the membranes were detected as above. β-actin was treated as a reference.−ΔΔCT) with β-actin as an internal reference. The primers designed for qRT-PCR were as follows: AdFas-F: 5′-AAAGGCAGTCTGAATGTCCA-3′, AdFas-R: 5′-TGGCAAATGGTATTCGCTTC-3′; AdTNFR1-F: 5′-GAATGCTCCAATGGGAAACC-3′, AdTNFR1-R: 5′-GCATCTCCCTCCAATAGACC-3′; AdBid-F: 5′-CATGAGCTTGTAACTGAGCTG-3′, AdBid-R: 5′-CAATCTCAGCCAGCTTTTGT-3′; AdBax-F: 5′-CCCCCAAGGAGGTGTTCTTC-3′, AdBax-R: 5′-AGTAAAAGAGCGCCACCACA-3′; AdBcl-W-F: 5′-GGACTTTGTGTGGTACAAGC-3′, AdBcl-W-R: 5′-TCCTTATTCATGCTCTCGCC-3′; AdBcl-xL-F: 5′-AGTTTGAACTGAGGTATCGC-3′, AdBcl-xL-R: 5′-CAATGTTCCCTACGAGTCCC-3′; Adβ-actin-F: 5′-TGAACCCAAAAGCCAACCGAGAAAAGAT-3′, Adβ-actin-R: 5′-TACGACCAGAGGCATACAGGGACAGGAC-3′.qRT-PCR was performed as reported previously . Brieflyt test, and statistical significance was defined as p < 0.05 and extremely significant as p < 0.01.The results were expressed as the mean ± standard deviation (SD) and statistical analyses were carried out with SPSS 17.0 software . Comparisons between groups were analyzed with the two-sample Student’s


Height remained stable during early childhood (median height − 0.6 (IQR − 1.4 to 0.1) SDS for girls and − 0.4 (IQR − 1.6 to 0.2) SDS for boys at 6 years of age). Patients were of normal height adjusted weight at 6 years with a median age and sex-adjusted body mass index (ISO-BMI) of 20.9 (IQR 19.3 to 25.0) for girls and 22.1 (IQR 20.7 to 25.6) for boys. Higher (≥ 50 mg/kg) cumulative post-portoenterostomy prednisolone dosage resulted in 0.18 SDS lower height per treatment week  compared to lower dosage (< 50 mg/kg).We addressed growth of biliary atresia (BA) patients living with native livers between ages 0–6 and effects of post-surgical corticosteroid treatment on growth. Growth charts of 28 BA patients born in Finland between 1987 and 2017 were retrospectively evaluated. Dosage and length of corticosteroid treatment and hydrocortisone substitution were reviewed. At birth, BA patients were shorter (median height − 0.6 (interquartile range (IQR) − 1.3 to − 0.1) SDS, Conclusion: BA patients grow normally during early childhood. As high postoperative corticosteroid dosage has a short-term negative effect on height, very high dosages should be avoided. The incidence of BA is varied between 1:8000 and 1:10000 reported in Asia to 1:17000–1:20000 in Northern Europe [Biliary atresia (BA) is an idiopathic fibro-obstructive cholangiopathy manifesting in infancy . The incn Europe –28. Untrn Europe , 41. Livn Europe .Following PE, commonly used adjunct postoperative management includes ursodeoxycholic acid, prophylactic antibiotic treatment, and nutritional management , 47, 49.We included term BA patients born in Finland between 1987 and 2017 who had normalized their bilirubin < 20 μmol/l after PE, were at least 4 months old, and were living with their native livers as of 31st of December 2017. Data on gestational age, birth height, and weight, associated congenital malformations, BA-type , age at Height and weight at birth as well as at 3, 6, 9, 12, 15, 18, 21, and 24 months and at yearly intervals between 2 and 20 years of age were retrospectively collected from medical records. Height and weight at PE and at 1, 2, 3, 4, 5, 6, 9, 12, 15, 18, 21, and 24 months after PE were also retrieved from medical records. For the first year, measurements performed within a ± 4-week period of the exact time point were accepted but for growth data after portoenterostomy, for the first 12 months, the nearest available measurement was accepted if it was within a ± 2-week period of the exact time point. After 12 months, measurements within a ± 6-week period of the except time point were accepted.2), normal weight (17–25 kg/m2), overweight (> 25 kg/ m2), and obesity (> 30 kg/m2) [Height was analyzed as standard deviation scores (SDS) from the mean of age- and sex-adjusted national values . Normal 0 kg/m2) . While c0 kg/m2) .Cumulative dosage of glucocorticoid treatment after PE, the length and dosage of subsequent hydrocortisone substitution regimen, and measured serum cortisol values after corticosteroid treatment were reviewed from patient records. Corticosteroid dosage was converted to equivalents of prednisolone, and total cumulative dosage of prednisolone (mg) per weight (kg) was calculated. In converting, 5 mg of prednisolone was considered to be equivalent to 5 mg of prednisone, 20 mg of hydrocortisone, 670 μg of dexamethasone, and 4 mg of methylprednisolone . A high P < 0.05.Values are expressed as medians with interquartile range (IQR) unless otherwise stated. Wilcoxon signed-rank test was used to compare matched groups. Growth of patients was modeled using linear mixed models . Models During the study period, 96 patients were diagnosed with BA in Finland. Of these, 30 patients had died, 28 were alive with a liver transplant (median age at transplantation 1.45 (0.84 to 2.51) years), 3 were born preterm and 1 had a birthweight < 2500 g. Thus, 34 patients fulfilled the initial inclusion criteria. Six patients were excluded as follows: two because of missing height measurements at birth and four because both parents were non-European. As shown in Table n = 28, P < 0.001) than the general population. Median height SDS of patients with mid-parental target height (MPH) data (n = 20) was − 0.2 (− 1.2 to 0.2), which was 0.1 SDS below their median MPH of − 0.1 (− 0.4 to 0.4) SDS .At birth, BA patients were shorter (median height − 0.6 (− 1.3 to − 0.1) SDS for all patients, n = 13); their median SDS changed from − 0.6 (− 1.5 to − 0.1) at birth to − 1.8 (− 2.5 to − 0.8) at 3 months while girls’ (n = 15) median SDS changed from − 0.7 (− 0.7 to 0.4) SDS to − 1.4 (− 1.8 to − 1.1) SDS .Growth rate appeared to slow down during the first 3 months, as median height for all patients at 3 months was − 1.70 (− 2.3 to − 1.1) SDS. Change in growth rate was more drastic for boys  SDS while boys’ median height changed from − 0.9 (− 1.3 to 0.0) SDS to − 0.4 (− 1.6 to 0.2) SDS.At birth, patients were of normal weight with median 0.0 (− 5.0 to 5.5) DW% for all, 2.0 (− 5.0 to 6.0) DW% for girls and 0.0 (− 6.0 to 4.5) DW% for boys. During the first 2 years, both genders remained of normal weight with median of 2.5 (− 1.0 to 10.5) DW% for all, − 1.0 (− 3.5 to 4.0) DW% for girls and 8.0 (2.5 to 17.5) DW% for boys at 2 years of age  ISO-BMI at 6 years. Boys’ weight decreased slightly with − 1.06  ISO-BMI yearly with median ISO-BMI at 22.1 (20.7 to 25.6) at 6 years , i.e., on the upper margin of normal range. There was a distinct difference between genders; girls were of normal weight with median of 21.1 (19.7 to 23.3) ISO-BMI while boys were overweight with a median of 25.9 (24.1 to 32.3) ISO-BMI  days. Serum cortisol values were suppressed after glucocorticoid treatment in 12 out of 19 patients (63%) with measured values.Complete data for postsurgical corticosteroid treatment was available for 24 out of 28 patients, including 3 patients not treated with corticosteroids. Corticosteroid treatment started at median 5 (range 4–17) days after PE at median age of 70 (range 18–167) days. The median length of corticosteroid-treatment was 18.5 (range 0–49) days. Sixteen patients received hydrocortisone substitution. Total median cumulative corticosteroid dosage was 277.6 (186.6 to 374.2) mg and 63.6 (47.7 to 74.6) mg/kg of prednisolone excluding hydrocortisone substitution. Median hydrocortisone dosage during the first 30 days of substitution was 8.2 (range 5.0–21.2) mg/mβ − 1.51, SE 0.19, P < 0.001) SDS shorter than the general population. A growth model for cumulative prednisolone dosage showed 0.16  SDS decrease in height for each 100 mg of prednisolone. In a mixed model, there was 0.18  SDS decrease in height for each 100 mg of prednisolone while hydrocortisone substitution caused faint 0.07  SDS increase in height per treatment week. A weight-adjusted dosage model showed negative effect on height with 0.25  SDS decrease for each 100 mg of prednisolone per treatment week. In a mixed model, weight-adjusted dosage of prednisolone demonstrated 0.27  SDS decrease for each 100 mg of prednisolone per treatment week while hydrocortisone substitution caused 0.07  SDS increase in height per week. A higher total corticosteroid dosage (> 50 mg/kg) resulted in 0.18  SDS lower height per treatment week compared to lower dosage. The effect remained in a model including higher corticosteroid dosage  and hydrocortisone substitution . Suppression of cortisol production caused 1.37  SDS decrease per week in height compared to unsuppressed patients (Table .At PE (median age 65 IQR 26 to 90) days), patients were 1.51  DW% at time of portoenterostomy. A growth model for total cumulative prednisolone showed no effect on weight, nor did a growth model combining cumulative prednisolone and hydrocortisone substitution (Table ). A growth model with weight-adjusted corticosteroid dosage showed no effect of prednisolone on weight nor did a model combining weight-adjusted dosage and hydrocortisone substitution  DW% increase in weight per treatment week compared to lower dosage, the effect remained  in a mixed model including hydrocortisone substitution . Suppression of cortisol production had no effect  of children had lower weight-for-height percentile than BMI-for-age percentile at the same age .Patients in our study are shorter than general population and their mid-parental target height at birth. BA associated with cytomegalovirus infection has been described to have worse clinical outcome with increased mortality , althougz-scores but normal weight-for-height z-scores for 32 BA patients in early childhood. However, as 21 out of 32 patients in their cohort had signs of cirrhosis although patients were clinically stable, it is likely that our study group consists of healthier patients. Karrer et al. [z-scores at follow-up in 28 BA patients living with native livers, these patients were also considerable older adolescents compared to our cohort (median age 13.4 (range 10.2–22.2) years vs 8.1 (range 0.3–19.7) years in our cohort). Ng et al. [z-scores for 219 BA patients (median age 9.7 (range 5.1–17.9)) living with native livers at least 5 years after portoenterostomy. Similarly to our findings, Arvay et al. [z-scores inversely correlated with age [Published data assessing growth of nonjaundiced children with BA living with native livers in early childhood are sparse. Sokol et al.  found der et al.  reportedr et al. . Hadzić r et al.  found nog et al.  reportedz-scores for length from portoenterostomy (69 patients) until 24 months of age (35 patients) and for weight from portoenterostomy (70 patients) until 18 months of age (42 patients) for BA patients treated with post-surgical corticosteroids (cumulative prednisolone dosage 116 mg/kg). They also found significant difference between length z-scores at 1, 2, and 3 months after portoenterostomy between patient groups treated with corticosteroids or placebo (70 patients in each group) [Postnatal corticosteroids have been used for prevention and treatment of chronic lung disease in preterm infants since 1980s . In preth group) , 21, inas of age  patientsh group)  possiblyOur study has some limitations. Because data were collected retrospectively, we were unable to capture complete growth data for all patients at all time points. Secondly, patients’ nutritional status before PE and energy and protein intake during follow-up could not be assessed reliably.In conclusion, we found that BA patients surviving with their native livers are shorter than their background population at birth. Their height gain stays stable during early childhood. High post-PE corticosteroid treatment dosage has a negative impact on height gain and an observable positive impact on weight gain compared to lower dosage, but these effects are of short duration. However, as there are conflicting results regarding the beneficial effects of postsurgical corticosteroid treatment for BA patients, it is wise to minimize the possible risks of treatment and to avoid very high corticosteroid dosages.


Adaptive deep brain stimulation (DBS) devices aim to personalize stimulation delivery by following the current state of symptom‐specific neural signals during different activities of daily living . This approach is not yet suitable for clinical practice, and groundwork is needed. The first essential steps for establishing adaptive DBS comprise the capacity for measurements in chronically implanted patients (to avoid the “stunning effect”)ClinicalTrials.gov: NCT03422757). The recordings were performed in a 55‐year‐old woman suffering from akinetic‐rigid PD for 8 years and admitted to the hospital for battery replacement after 4 years of subthalamic DBS . After 30‐minute recordings (baseline) in stim‐off/meds‐off condition , we set the new AlphaDBS device (Newronika Srl)r = 0.62, P < 0.0001) during the daytime, which diminished during night sleep  in freely moving, chronically stimulated patients. They further corroborate the hypothesis that oscillations in the β‐frequency range might be used as a levodopa‐related biomarker for adaptive DBS paradigms, as they are present during active stimulation and years after surgery. We also provide for the first time preliminary evidence that interhemispheric subthalamic coupling changes between wakefulness and sleep can be monitored and possibly serve as an additional behavior‐specific biomarker. These findings pave the way for testing different adaptive stimulation paradigms for STN‐DBS and prompt a more accurate definition of symptom‐related and behavior‐specific biomarkers in PD.1) Research project: A. Conception, B. Organization, C. Execution;2) Statistical Analysis: A. Design, B. Execution, C. Review and Critique;3) Manuscript: A. Writing of the first draft, B. Review and Critique.M.A.: 1B, 1C, 2A, 2B, 3AC.P.: 1B, 1C, 2CB.M.: 1BM.T.: 1CC.P.: 1BA.C.: 1C, 2A, 2C, 3BN.G.P.: 3BR.C.: 1BM.P.: 1CS.M.: 1A, 1B, 3BA.P.: 1AP.R.: 1BS.B.: 1BD.S.: 1CJ.V.: 1A, 3BG.P.: 1B, 3BI.U.I.: 1A, 1B, 2C, 3A, 3B


Methanobacterium formicicum and Methanothermobacter wolfeii, were tested for their ability to survive long-term (~4 year) exposure to freeze/thaw cycles varying in both temperature and duration, with implications both for climate change on Earth and possible life on Mars.Although the martian environment is currently cold and dry, geomorphological features on the surface of the planet indicate relatively recent (<4 My) freeze/thaw episodes. Additionally, the recent detections of near-subsurface ice as well as hydrated salts within recurring slope lineae suggest potentially habitable micro-environments within the martian subsurface. On Earth, microbial communities are often active at sub-freezing temperatures within permafrost, especially within the active layer, which experiences large ranges in temperature. With warming global temperatures, the effect of thawing permafrost communities on the release of greenhouse gases such as carbon dioxide and methane becomes increasingly important. Studies examining the community structure and activity of microbial permafrost communities on Earth can also be related to martian permafrost environments, should life have developed on the planet. Here, two non-psychrophilic methanogens,  On Earth, permafrost is defined by temperature and refers to any rocks, soil, or sediments that remain at or below 0 °C for at least two years in a row ,2. Perma4) from substrates such as carbon dioxide (CO2) and hydrogen (H2), among others. Methanogens are often significant members of microbial permafrost communities ). Many studies also indicate a shift in the dominant methanogenic orders between frozen and thawed samples, and although methanogenic communities typically vary by environment [Methanobacterium formicicum is evident in the dominance of a Methanobacterium species across three separate ancient permafrost samples tested , providg et al. ). The exExperiments utilizing freeze/thaw cycles have come under scrutiny for not accurately representing temperature changes seen in nature . HoweverM. formicicum and Methanothermobacter wolfeii. These experiments incorporated the use of varying amounts of liquid medium and sand in order to provide additional stress(es) to the microorganisms and to incorporate environmental factors, in an attempt to mimic a possible subsurface environment.The experiments conducted here aimed to investigate the effect of long-term (~4 year) freeze/thaw cycles varying in temperature and duration on the methane production of two non-psychrophilic methanogen species within the order Methanobacteriales, M. formicicum (OCM 55), MS medium supplemented with sodium formate (designated MSF medium) [M. wolfeii (OCM 36), MM medium [4Cl, 1.0 g MgCl2·6H2O, 0.4 g CaCl2·2H2O, 0.4 g K2HPO4·3H2O, 1.0 mg resazurin, 5.0 mg Na2-EDTA·2H2O, 1.5 mg CoCl2·6H2O, 1.0 mg MnCl2©4H2O, 1.0 mg FeSO4·7H2O, 1.0 mg ZnCl2, 0.4 mg AlCl3·6H2O, 0.3 mg Na2WO4·H2O, 0.2 mg CuCl2·2H2O, 0.2 mg NiSO4·6H2O, 0.1 mg H2SeO3, 0.1 mg H3BO3, 0.1 mg NaMoO4·2H2O, 2.0 g yeast extract, 2.0 g trypticase peptone, 0.5 g sodium 2-mercaptoethanesulfonate and 0.25 g sodium formate. MM is a minimal medium that contains the same components as MSF medium except yeast extract, trypticase peptone, sodium 2-mercaptoethanesulfonate and sodium formate. All of the media were sparged with 100% CO2 gas prior to distribution into test tubes.Methanogens were initially obtained from the Oregon Collection of Methanogens (OCM) . Two methanogen species were cultured in their respective anaerobic growth medium:  medium)  and M. wM medium . MSF med2:H2 Coy Anaerobic Chamber  following the methods of Kendrick and Kral [2S) were added to each tube to remove residual oxygen [M. formicicum; MM medium: M. wolfeii). The tubes were then pressurized with 2 bar H2 and placed at the organisms’ respective incubation temperatures . Deviations from this standard protocol for each experiment are given in For each experiment, growth media were prepared under anaerobic conditions in a 90:10 COand Kral . In geneand Kral . The medl oxygen . Each tuMedia were prepared as described  were prepared as described (see M. formicicum (n = 4). The MM test tubes were inoculated with 0.5 mL of MM medium containing M. wolfeii (n = 4). One test tube for each medium type was not inoculated. After inoculation, each tube was pressurized with 2 bar H2 gas. The tubes were next subjected to varying freeze/thaw cycles at temperatures of 55 °C, 37 °C, 22 °C, 4 °C, −15 °C, and −80 °C  was inoculated with 0.5 mL from the corresponding tube in Transfer Set 2 . The MM test tubes were inoculated with 0.5 mL of MM medium containing M. wolfeii (n = 3). After inoculation, each tube was pressurized with 2 bar H2 gas. The tubes were next subjected to varying freeze/thaw cycles at temperatures of 55 °C, 37 °C, 22 °C, 4 °C, −15 °C, and −80 °C  were prepared, as noted above  and M. wolfeii (n = 5) were initially grown in their respective anaerobic growth media  and were inoculated with 0.5 mL culture. Tubes were pressurized with 2 bar H2 and incubated at the organisms’ growth temperature  for 17 days. The cultures were then exposed to varying freeze/thaw cycles for 126 days . Cultures of 126 days . On Day 126 days . The tubM. formicicum or M. wolfeii) after 140 days’ incubation at 4 °C (data not shown). M. formicicum was able to produce methane at 22 °C, however, M. wolfeii was not capable of any methane production after 140 days at 22 °C (Methane production did not occur for either methanogen species (at 22 °C . M. wolfeii and M. formicicum consisted of n = 4 replicates (one tube in each set of 5 was not inoculated). Both transfer sets for M. wolfeii consisted of n = 5 tubes. The transfer sets for M. formicicum both consisted of n = 4 tubes.The original sets for both M. formicicum all displayed ~3% methane production after 5 days (data not shown). After an additional 9 days at 37 °C, methane abundance was much more varied between replicates  after 14 days’ incubation at 55 °C  for the four tubes within the Original Set after the initial incubation period of 18 days at 4 °C followed by 7 days at 37 °C , while the other three produced 6–11% methane, resulting in a large error bar for this set as shown in For M. formicicum and M. wolfeii were not identical  after 14 days’ incubation at 55 °C. The methane abundance within these two cultures increased with another 14 days’ incubation at 55 °C , with a third culture producing ~7% methane  may be possible during brief exposures to 22 °C (for M. formicicum) or higher. The optimum temperature for growth for M. formicicum is 37 °C, but certain strains are capable of growth down to 20–25 °C [M. wolfeii did not display growth below 37 °C with optimal growth occurring between 55 °C and 65 °C [M. wolfeii displayed methane (>1%) production after only 7 days’ incubation at room temperature, despite the lack of methane production over 140 days at room temperature in Expt. 1  was not expected during the brief exposures to 4 °C during the subsequent freeze/thaw cycling experiments. Growth was possible at room temperature (22 °C) for rmicicum , but not20–25 °C . When innd 65 °C . Interes Expt. 1 . This phn et al. ). Methanogenium frigidum, which grows maximally at 15 °C with a doubling time of 2.9 days [Methanolobus psychrophilus, with an optimum temperature of 18 °C [Methanococcoides burtonii, isolated from Ace Lake in Antarctica, are capable of growth at low temperatures  only after growth is initiated at higher temperature  [M. frigidum at 4 °C using H2/CO2 with and without acetate were unsuccessful in this lab.Psychrotolerant methanogens exist in pure culture that can actively grow at temperatures down to 1 °C ,77,78,792.9 days  and Methof 18 °C . At low , 20 °C) . AttemptM. formicicum, and a thermophile, M. wolfeii to a variety of extreme temperature changes over time . This is surprising given the extent of freeze/thaw cycling and the classification of the organism as a thermophile. Replicates of M. formicicum subjected to the same conditions failed to produce appreciable methane (>1%) after 28 days’ incubation at 37 °C. In Experiment 3, one replicate of M. wolfeii produced 0.70% methane, but all the remaining replicates, as well as the five replicates for M. formicicum, failed to produce any methane after incubation at the organisms’ optimal growth temperatures for 28 days. Methane production for cultures of M. wolfeii in Experiment 4 was more varied with one replicate measuring 12.8% methane, another replicate measuring 31.0% methane, and three replicates measuring 0% methane after 14 days’ incubation at 55 °C. Cultures of M. formicicum in Expt. 4 failed to produce any methane following incubation for 28 days. There are a few possible explanations for this: 1. There were no surviving cells within those cultures (or an insufficient number of cells within cultures before freezing); 2. The rate of freezing was lethal to the microorganisms; or 3. The cells are subject to a significant lag phase and methane production may be delayed. In a study using M. barkeri, Gunnigle et al. [2/CO2 as substrates produced the lowest optical density and methane measurements, compared to methanol as a substrate. However, the strain of M. barkeri used was previously adapted for use of methanol as a substrate and that may account for the poor performance of the organism under H2/CO2 conditions [Methanobacterium. These two species also only grew via hydrogenotrophic methanogenesis (utilizing H2 as an energy source and CO2 as a carbon source). The two species used in the experiments described here are also solely hydrogenotrophic methanogens. Certain permafrost environments are dominated by hydrogenotrophic methanogenesis [These experiments also included long-term survival, with tubes exposed to freeze/thaw cycles, then stored at −80 °C for over three years . In Expee et al.  noticed nditions . Additionditions . Howevernditions . Interesogenesis ,52,53,92ogenesis . Howeverogenesis  interpreM. barkeri (a mesophile), M. frigidium  and Methanobacterium spec. MC-20 (isolated from non-permafrost sediments). After exposure to diurnal freeze/thaw cycles between −75 °C and 20 °C for 22 days, the three permafrost strains had the highest survival , whereas the survival rate for the reference organisms was exceptionally low . Additionally, methane production following exposure was significantly decreased for the three reference strains, whereas the permafrost strains had similar methane production before and after exposure [To our knowledge, the only other freeze/thaw study using pure cultures of methanogens was conducted by Morozova et al.  and focuexposure . Howeverexposure , may not+, thereby increasing the pH of the acidic environment; and 3. Indirectly increasing methane emissions by increased water-filled pore space within the soil [Sand was utilized in Experiments 2 and 3 to mimic a near-subsurface environment, as may be applicable on Mars. In certain environments, sand also makes up a significant fraction of the permafrost, compared to silt or clay . Additiothe soil . Analysithe soil . Thus, tUnlike Earth, Mars is considered to be a planet completely covered in permafrost ,96. HoweM. wolfeii cells under extreme cold temperatures, as compared to the other methanogen tested here, remain unknown. Enhanced survival may be attributable to the presence of DEAD-box RNA helicases, which are believed to function as cold stress proteins within other methanogenic species including the psychrotolerant M. burtonii [M. psychrophilus [Methanococcus jannaschii [Thermococcus kodakaraensis, which typically grows at lower temperatures (optimum growth temperature range for genus Thermococcus: 75–95 °C) than the hyperthermophiles within the closely-related genus Pyrococcus (optimum growth temperature range: 95–103 °C), which lack any orthologs [M. wolfeii. Ding et al. [Methanothermobacter thermoautotrophicus ∆H during growth at temperatures below optimal  and after cold shock at 4 °C. Ultimately, additional experiments are required to assess the true nature of cold-tolerance in M. wolfeii.The reasons for the increased survival of burtonii , the psyrophilus , and thennaschii . Shimadannaschii  suggest rthologs ,105. Howg et al.  have alsmcrA gene, as has been performed in certain environmental studies. The experiments described here subjected a mesophilic and a thermophilic methanogen to long-term exposure (~4 year) to extreme temperature changes varying in duration. The data demonstrate that extreme temperature change (between −80 °C and 37/55 °C) over time is not necessarily lethal to non-psychrophilic methanogens, with resumed metabolism (methane production) possible once warmer temperatures are achieved. These results complement environmental studies, which show that methanogens remain significant community members within both current and ancient permafrost samples, and that methanogenic permafrost communities also feature active, non-psychrophilic species. Should life have arisen on Mars, permafrost may constitute a potentially habitable environment. Further insight into survival and growth in response to extreme temperature changes could be achieved through additional growth proxies such as direct cell counts or transcriptomics analyzing the expression of the


We found that IL-10 significantly potentiated basal glutamatergic excitatory synaptic transmission within 15 min after application. Obtained results revealed a presynaptic nature of the effect, as IL-10 in a dose-dependent manner significantly increased the frequency but not the amplitude of mEPSC. Further, we tested the effect of IL-10 on mEPSC in a model of homeostatic synaptic plasticity (HSP) induced by treatment of primary hippocampal culture with 1 µM of tetrodotoxin (TTX) for a 24 h. It was found that 15 min application of IL-10 at established HSP resulted in enhanced mEPSC frequency, thus partially compensating for a decrease in the mEPSC frequency associated with TTX-induced HSP. Next, we studied if IL-10 can influence induction of HSP. We found that co-incubation of IL-10 with 1 µM of TTX for 24 h induced synaptic scaling, significantly increasing the amplitude of mEPSC and Ca2+ responses to application of the AMPA agonist, 5-Fluorowillardiine, thus facilitating a compensatory postsynaptic mechanism at HSP condition. Our results indicate that IL-10 potentiates synaptic activity in a dose- and time-dependent manner exerting both presynaptic (short-term exposure) and postsynaptic (long-term exposure) action. Obtained results demonstrate involvement of IL-10 in the regulation of basal glutamatergic synaptic transmission and plasticity at normal conditions.Anti-inflammatory cytokines are known to exert neuroprotective action ameliorating aberrant neuronal network activity associated with inflammatory responses. Yet, it is still not fully understood if anti-inflammatory cytokines play a significant role in the regulation of synaptic activity under normal conditions. Thus, the aim of our study was to investigate the effect of Interleukin-10 (IL-10) on neuronal synaptic transmission and plasticity. For this we tested the effect of IL-10 on miniature excitatory postsynaptic currents (mEPSC) and intracellular Ca It has n = 7 vs. 3.11 ± 0.71 Hz before application in control, n = 7; p < 0.05 with paired t-test, p < 0.01 with Kolmogorov–Smirnov test, n = 7 vs. −16.92 ± 2.54 pA before application in control, n = 7; p > 0.05 with paired t-test, p > 0.05 with Kolmogorov–Smirnov test, To study the effect of IL-10 on glutamatergic excitatory synaptic transmission we tested how 15 min application of this anti-inflammatory cytokine affects frequency and amplitude of miniature excitatory postsynaptic currents (mEPSC) in neurons from primary hippocampal neuro-glial culture A. It wasn = 8 vs. 0.33 ± 0.09 Hz before application in 24 h TTX-treated group, n = 8; p < 0.05 with paired t-test, p < 0.01 with Kolmogorov–Smirnov test, n = 7 vs. 20.59 ± 2.99 pA before application in 24 h TTX-treated group, n = 7; p > 0.05 with paired t-test, p > 0.05 with Kolmogorov–Smirnov test, Further we studied the effect of IL-10 on homeostatic plasticity by measuring frequency and amplitude of mEPSC from neurons that were incubated with 1 µM of tetrodotoxin (TTX) for 24 h prior to the experiment. It was found that, similarly to its effect on basal synaptic transmission, IL-10 added for 15 min was able to increase frequency of mEPSC with no effect on its amplitude A. Thus, n = 12 vs. 0.49 ± 0.16 Hz in 24 h TTX-treated group, n = 10; p < 0.01 with one-way ANOVA followed by a Bonferroni posthoc test, n = 12 vs. 0.71 ± 0.33 Hz in 24 h TTX + 10 ng/mL Il-10-treated group, n = 10; p < 0.01 with one-way ANOVA followed by a Bonferroni posthoc test, n = 10 vs. 0.71 ± 0.33 Hz in 24 h TTX + 10 ng/mL IL-10-treated group, n = 10; p > 0.05 with one-way ANOVA followed by a Bonferroni posthoc test, n = 12 vs. −20.61 ± 2.19 pA in 24 h TTX-treated group, n = 10; p > 0.05 with one-way ANOVA followed by a Bonferroni posthoc test, n = 12 vs. -32.17 ± 5.6 pA in 24 h TTX + 10 ng/mL IL-10-treated group, n = 10; p < 0.01 with one-way ANOVA followed by a Bonferroni posthoc test, n = 10 vs. -32.17 ± 5.6 pA in 24 h TTX + 10 ng/mL IL-10-treated group, n = 10; p < 0.05 with one-way ANOVA followed by a Bonferroni posthoc test, Next we studied whether IL-10 is capable of exerting any effect on the formation of homeostatic plasticity. For this we compared frequency and amplitude of mEPSC from neurons incubated for 24 h with 1 µM of TTX alone to those of neurons that in addition to 1 µM of TTX, were co-incubated with 10 ng/mL of IL-10 for the same period of time A. A 24 h2+ signals ([Ca2+]i) in Fura-2 loaded neurons  in a dose-dependent manner. Reduction in both mIPSC frequency and amplitude was evident within several minutes after IL-10 administration . In the Our results are in line with previously published observations showing the importance of cytokines in the regulation of basal excitatory glutamatergic synaptic transmission. It was found that pro-inflammatory cytokines such as TNF-α and IL-18 effectively enhance basal synaptic activity increasing both mEPSC frequency and amplitude ,18. On ti signals in response to the application of 5-Fluorowillardiine, a selective agonist of AMPA receptors. We found that 24 h co-incubation of IL-10 with TTX was able to effectively modulate a dose-dependent effect of 5-Fluorowillardiine on neuronal [Ca2+]i,, shifting the dose-dependent curve leftward and leading to a reduction in EC50 values and an increase in the Hill coefficient, thus resulting in neuronal sensitization to 5-Fluorowillardiine. Previously we have found that ~50 min incubation with IL-10 ameliorated hypoxia-induced downregulation of hippocampal AMPA GluR1 subunit and did not have any affect on GluR2 subunit expression level i in neurons was assessed by measuring fluorescence intensity of ratiometric Ca2+ sensitive probe using Fura-2 and analyzed, as previously described [4, 1.25 KH2PO4, 1.4 CaCl2, 10 glucose and 10 HEPES, pH 7.4. Registration of fluorescent signals was made with a fluorescent system using an inverted fluorescence microscope Axio Observer Z1 equipped with a high-speed mono-chrome CCD camera Hamamatsu ORCA-Flash 2.8. Lambda DG-4 Plus illuminator  was used as a source of excitation for fluorescence. To excite and record the Fura-2 fluorescence, we used a 21HE filter set  with the excitation filters BP340/30 and BP387/15, FT409 separator and BP510/90 emission filter.The level of [Caescribed ,35. Briet-test or analysis of variance followed by Bonferroni correction for multiple comparisons or Kolmogorov–Smirnov test. P ≤ 0.05 was considered significantly different.All values are given as mean ± SEM. All data were statistically analyzed with GraphPad Prism 7 . Comparison of experimental groups was done with paired


Gracilaria coronopifolia synbiotic and verified the efficacy by small intestinal cells (Caco-2). We also developed a functional material that promotes intestinal health and prevents intestinal inflammation. G. coronopifolia was used as a red seaweed prebiotic, and Bifidobacterium bifidums, B. longum subsp. infantis, B. longum subsp. longum, Lactobacillus acidophilus, and L. delbrueckii subsp. bulgaricus were mixed for the seaweed’s synbiotics. G. coronopifolia synbiotics were nontoxic to Caco-2 cells, and the survival rate was 101% to 117% for a multiplicative effect on cell survival. After cells were induced by H2O2, the levels of reactive oxygen species (ROS) increased to 151.5%, but after G. coronopifolia synbiotic treatment, decreased to a range between 101.8% and 109.6%. After cells were induced by tumor necrosis factor α, the ROS levels increased to 124.5%, but decreased to 57.7% with G. coronopifolia symbiotic treatment. G. coronopifolia synbiotics could effectively inhibit the production of ROS intestinal cells under oxidative stress (induced by H2O2 and tumor necrosis factor α (TNF-α)), which can reduce the damage of cells under oxidative stress. Functioning of intestinal cells could be improved by inhibiting the production of inflammatory factor substances (interleukin 8) with G. coronopifolia symbiotic treatment. Also, gastrointestinal diseases may be retarded by a synbiotic developed from G. coronopifolia to promote intestinal health and prevent intestinal inflammation.The market contains only limited health care products that combine prebiotics and probiotics. In this study, we developed a seaweed-based  Gracilaria coronopifolia (G. coronopifolia), belonging to the family Gracilariaceae, is one of the most common types of eatable endemic Hawaiian marine red seaweed . In. In27]. 5 cells/mL according to the value. Cells were cultured at 100 μL/well in 96-well plates. After incubation at 37 °C with 5% CO2 for 24 h, the original culture solution was eliminated, and 100 μL of different concentrations (0.1–1000 μg/mL) of sample solutions was added, then continued to incubate at 37 °C with 5% CO2 for 24 h. Next, 100 μL MTT  was added to each well, and the reaction was performed at 37 °C for 4 h, and finally, 100 μL DMSO was added. The cell membrane was lysed and uniformly mixed to ensure complete dissolution of the blue-violet crystal. After standing for 40 min, the absorbance wavelength 570 nm was read by an ELISA reader. The sample was replaced with the culture medium as the control group and set to 100%, and the other blank group was the culture medium containing no cells and samples [After cells were collected by centrifugation at 1500 rpm for 5 min, cell density was calculated by using a cell counter, and cells were adjusted to 2 × 10 samples . We calctotal is the measured resistance value, Rblank is the blank film resistance value, and A is the membrane area.TEER provides ion flow resistance information across a single monolayer cell that correlates with the integrity of tight junctions between cells . Caco-2 2O2) at a final concentration of 400 μM, and 5 × 105 cells/mL were added to 12-well plates (containing 2% serum medium) and incubated in a constant temperature incubator at 37 °C, with 5% CO2 for 12 h. At 30 min before the end of the reaction, 20 μM of the fluorescent dye 2’,7’-dichlorofluorescin diacetate (DCFH-DA) was added into the culture medium. After the nonpolar substance DCFH-DA infiltrates into the cells, it can be transformed by intracellular esterification to the non-fluorescent polar substance DCFH; DCFH is oxidized by H2O2 in the cell after it enters the cell and becomes a fluorescent substance, 2’,7’-dichlorodihydrofluorescein (DCF), for indirect quantification of intracellular ROS production. After the cells and DCFH-DA were reacted at 37 °C for 30 min, they were centrifuged for 10 min, 10,000 rpm and the supernatant was removed, then cells were further washed twice with PBS, suspended in 0.5 mL PBS, and placed in a 96-well black plate with a fluorescence analyzer  for detecting fluorescence response  [The experimental sample, hydrogen peroxide  of samples was injected into the inner chamber, and 2 mL of RPMI medium was added to the outer chamber, and further incubated in a 37 °C, 5% CO2 cell incubator for 3 h. Next, 100 ng/mL of TNF-α was added into the outer chamber for 24 h. The suspension was collected and stored at −20 °C. The cytokine human IL-8 was detected by using a human assay kit (431505).The cultured Caco-2 cell concentration was adjusted to 3 × 10A 96-well test plate was prepared with a monoclonal antibody, and 100 μL capture antibody diluted with PBS was added to each well, placed overnight at a temperature of 4 °C. The solution in each well was aspirated, then rinsed with wash buffer. An amount of 200 μL blocking buffer was added into each well and reacted at room temperature for 1 h. The aspiration of the solution was continued in wells and rinsed with wash buffer, then the test plate preparation was completed. An amount of 100 μL medium was added to the test plate and reacted at room temperature for 2 h, the medium in the well was aspirated and washed with wash buffer. An amount of 100 μL detection antibody was added in and reacted for 1 h under room temperature.After the solution in each well was aspirated and washed with wash buffer, 100 μL horseradish peroxidase-conjugated streptavidin was added and further reacted at room temperature for 30 min. The solution in each well was aspirated and washed with the wash buffer, then 100 μL substrate solution was added into the test plate. The test solution was reacted at room temperature for 20 min in the dark, and 100 μL of the stop solution was added for terminating the reaction. The absorbance of each well was measured by using an ELISA reader at a of wavelength 450 nm.p < 0.05), then Duncan’s new multiple range test was used to compare treatment means.Experimental tests and treatments were carried out in triplicate. Data was reported as a mean (SD). All data were analyzed by using single-factor of ANOVA, with the F-value significant at G. coronopifolia as a red seaweed prebiotic, mixed with five probiotics of Lactobacilli and Bifidobacteria, and supplemented with 15% skim milk powder as a protective agent, with the probiotics and prebiotics blending ratio of GS1 (30:70), GS2 (50:50), and GS3 (70:30). The number of bacteria of probiotic products containing live bacteria needs to be ≥106 cfu/g to exert their activity; thus 106 cfu/g is used as a screening condition for storage [G. coronopifolia synbiotics  was performed at different temperatures (4 °C and −20 °C). With storage temperature 4 °C for G. coronopifolia synbiotics for 60 days, the number of bacteria remained at 109 cfu/g  was measured. The value should be ≥600 Ω·cm85 Ω·cm2 a; the reTHP-1 cells are established from monocytes in the blood of leukemia patients. They secreted inflammatory cytokines and are appropriately induced to transform into macrophages. They are a good source to substitute cells for in vitro human mononuclear macrophages. We used cultured THP-1 human monocytes to establish the in vitro pattern of differentiation into macrophages. THP-1 cells were differentiated with 200 nM phorbol-12-myristate-13-acetate under microscopy observation. After 4 days, the cell morphology was transformed into a macrophage-like mode. The two cell types were co-cultured on a plug-in cell culture dish with the upper layer of Caco-2 cells and the lower layer of THP-1 macrophages for subsequent intestinal inflammatory evaluation.G. coronopifolia synbiotics were added to the upper layer of cells. At certain time intervals, the lower cell culture medium was sampled, and the permeability of the samples toward intestinal cells was observed. THP-1 cells were induced to secretes TNF-α, causing Caco-2 cell-surface damage after 24 h and TNF-α secretion was increased significantly. The production of TNF-α induced the Caco-2 cells to secrete interleukin-8 (IL-8) (an inflammatory factor that can induce an inflammatory reaction), which verified that inflammatory intestinal cells were induced by LPS and TNF-α and could promote the secretion of inflammatory factors with the co-culture system. Co-culture was feasible as an experimental model of intestinal inflammation. Co-cultured cells were treated with G. coronopifolia synbiotics at 0.001–0.1 mg/mL. The TEER value for 0.1 mg/mL GS1 synbiotic relative to FD-4 (100%) was 86% at the beginning of the test and increased to 95% after 180 min. Concurrently, TEER values at 0.001, 0.005 and 0.05 mg/mL were 105%, 103% and 101%, respectively, after 180 min . Cell viability was verified after 24-h co-culture (2O2 concentration (2O2), 83.3% (0.25 mM H2O2) to 79.0% (0.5 mM H2O2). After 1 mM H2O2 treatment, the cell survival rate was 67.4%, which produced oxidative stress on cells but did not kill them. 2O2 and 20 ng/mL TNF-α were used to induce oxidative stress.The oxidative stress of THP-1 cells was induced by H-culture . Cell vintration a. The ceG. coronopifolia synbiotics and THP-1 cells was used with of 1 mM H2O2 and 20 ng/mL TNF-α to induce oxidative stress formation, followed by cell viability tests. The viability of THP-1 cells after 1 mM H2O2 treatments was reduced to 70.7% , slightly decreased to 85.2% (0.05 mg/mL) and increased to 100.3%. Likewise, the GS2 synbiotic also slightly changed THP-1 cell viability. GS2 synbiotic at 0.05 mg/mL conferred a higher cell viability rate (105.5%) and 103.9% at 0.1 mg/mL. However, 0.5 mg/mL of GS3 synbiotic increased cell viability to 81.2% (0.001 mg/mL), which steadily decreased to 70.7% (0.05 mg/mL) and increased to 75.4%. Concurrently, cell viability decreased to 74.4% under 20 ng/mL THF-α induction (G. coronopifolia synbiotics effectively reduced the cell damage caused by oxidative stress (H2O2 and TNF-α) and increased the cell survival rate. GS1, GS2, and GS3 at 0.1, 0.05, and 0.005 mg/mL were used for subsequent tests.The culture system of to 70.7% c. Also, nduction d. The ce2O2 (0.5–5 mM). The cell viability of Caco-2 cells gradually decreased from 94.1% (0.5 mM H2O2) to 84.9% 1 mM H2O2 and greatly decreased after induction with 2 mM H2O2 (48.7%). Hence, 0.5 mM H2O2 was used for the following ROS production test (Caco-2 cells) instead of 1 mM H2O2 because Caco-2 cells were suspended at the higher dose of H2O2. ROS production in Caco-2 cells with 0.5 mM H2O2/ 20 ng TNF-α, then treatment with G. coronopifolia synbiotic, is presented in 2O2. After G. coronopifolia synbiotic  treatment, the amount of ROS produced was decreased greatly. At 0.05 mg/mL, ROS production decreased sharply to 63.3% and 48.8% with GS1 and GS2 synbiotics, respectively. In addition, 0.01 mg/mL GS3 synbiotic greatly decreased ROS production to 56.2%. Yet, after induction with 20 ng/mL of TNF-α, ROS production increased to 124.5% (100% for the control group). The amount of ROS produced decreased significantly with 0.005 mg/mL GS3 synbiotic (57.7%). Moreover, 0.01 mg/mL GS1 and 0.001 mg/mL GS2 also successfully decreased the ROS production to 93.0% and 60.6%, respectively. G. coronopifolia synbiotics capably slowed the production of ROS induced by H2O2 and TNF-α and reduced the oxidative stress of Caco-2 cells, which is expected to prevent the inflammatory reaction caused by oxidative stress. 4.0 mg/kg marine seaweed (G. verrucosa) successfully enhanced the immunity system of the rats and stimulated phagocytosis of the cells [G. coronopifolia, G. parvispora, G. salicornia and G. tikvahiae was determined to be 27.0, 26.4, 35.8 and 28.4, respectively [Gracilaria seaweed, is equivalent to 5–10 g total dietary fibre. Furthermore, rats fed a 0.5 mg/kg dose of water extract of G. verrucose and G. chorda showed alleviated gastrointestinal dysfunction [he cells . The totectively . For exafunction .G. coronopifolia synbiotic treatments (0.001–0.05 mg/mL). The IL-8 level decreased significantly to 1.13 and 1.10 ng/mL with 0.001 and 0.005 mg/mL GS1, respectively. Simultaneously, the IL-8 level decreased greatly to 0.56 and 1.08 ng/mL with 0.005 and 0.01 mg/mL GS2 treatments, respectively, and IL-8 sharply to 1.11 ng/mL with 0.01 mg/mL GS3 treatment. Thus, G. coronopifolia synbiotics efficiently reduced IL-8 production induced by TNF-α, diminishing the inflammatory response, and thereby preventing an intestinal cell inflammatory response. The results agree with treatment with 10 µg/mL methanolic extract of red seaweed, G. changii during differentiation of U937 cells, which intensely inhibited the TNF-α response level [G. coronopifolia synbiotics may be effective for gastrointestinal disorders.se level . Many dise level . Hence, G. coronopifolia extract as marine-based seaweed prebiotics and mixed with several probiotics (Lactobacili and Bifidobacteria) to form natural G. coronopifolia synbiotics, which was verified and suitable for maintaining the health of intestinal tract. G. coronopifolia synbiotics are nontoxic and effectively inhibited the production of ROS under the oxidative stress of intestinal cells, reduced the damage of cells with oxidative stress, and inhibited the inflammatory response in cells. They also protected and upheld the function of intestinal cells. The development of a combination of marine-based G. coronopifolia synbiotics could be used to treat gastrointestinal diseases caused by improper diet or aging of the human body to promote intestinal health. We describe a fundamental, in vitro G. coronopifolia synbiotic study that is appropriate for the assessment of intestinal health functions. However, further examinations are required to analyse the several unidentified phytochemical and bioactive compounds of G. coronopifolia synbiotics and their biological mechanisms.We still lack healthy food products that combine prebiotics and probiotics. In this study, we used