In contrast, the absolute oral bioavailabilities of ethoxycarbonyl 1-ethyl hemiacetal ester (EHE) prodrugs of LFX and CFX , which are non-chelating prodrugs at least in in vitro condition, were comparable between control and EDTA-treated rabbits irrespective of the presence of Al(OH)3, although some variation was observed among rabbits. The oral bioavailabilities of LFX and CFX from their EHE prodrugs were comparable with LFX and CFX alone, respectively, even in the presence of Al(OH)3. In conclusion, LFX, CFX and TC exhibited higher oral bioavailabilities in EDTA-treated rabbits than in control rabbits, indicating that the oral bioavailabilities of these chelating drugs are reduced in untreated rabbits. In conclusion, EDTA-treated rabbits were found to exhibit low gastric contents including metals and low gastric pH, without causing mucosal damage. Ester prodrug of CFX was effective in preventing chelate formation with Al(OH)3 in vitro and in vivo, as well as in the case of ester prodrugs of LFX. EDTA-treated rabbits are expected to provide great advantages in preclinical oral bioavailability studies of various drugs and dosage formulations. However, a marked interspecies difference was still observed in the oral bioavailability of CFX and TC between EDTA-treated rabbits and humans, possibly due to the contribution of adsorptive interaction in rabbits. Further study is necessary to seek out the usefulness of the EDTA-treated rabbit with less gastric contents and metals as an experimental animal.Laboratory rabbits are fed foods rich with cationic metals, and while fasting cannot empty gastric contents because of their coprophagic habits. This implies that, in rabbits, the oral bioavailability of chelating drugs could be modulated by the slow gastric emptying rates and the interaction  with gastric metals. In the present study, we tried to develop a rabbit model with low amounts of cationic metals in the stomach for preclinical oral bioavailability studies of chelating drugs. The elimination of gastric metals was achieved by preventing food intake and coprophagy and administering a low concentration of EDTA 2Na solution one day before experiments. Control rabbits were fasted but coprophagy was not prevented. The efficacy of rabbits treated with EDTA 2Na was evaluated by comparing the gastric contents, gastric metal contents and gastric pH between EDTA-treated and control rabbits. The treatment with more than 10 mL of 1 mg/mL EDTA 2Na solution decreased the amounts of gastric contents, cationic metals and gastric pH, without causing mucosal damage. The absolute oral bioavailabilities  of levofloxacin (LFX), ciprofloxacin (CFX) and tetracycline hydrochloride (TC), chelating antibiotics, were significantly higher in EDTA-treated rabbits than those in control rabbits as follows: 119.0 vs. 87.2%, 9.37 vs. 13.7%, and 4.90 vs. 2.59%, respectively. The oral bioavailabilities of these drugs were significantly decreased when Al(OH) Rabbits have significant advantages as experimental animals in preclinical pharmacokinetic studies, as compared with small animals such as mice, rats and guinea pigs. For example, rabbits are low-cost, have a rapid growth rate, are easy to manage, and repeated blood sampling can be made in rabbits without anesthesia for a crossover study. In addition, most of the dosage formulations for human use can be examined in rabbits. In contrast, however, rabbits perform coprophagy directly from the anus, and one or two days’ fasting cannot empty gastric contents. The stomach of rabbits is always full of contents such as feces, food, hair, and so on, in contrast to the case of rats and humans ,2,3. CopIn general, laboratory rabbits are fed foods rich in cationic metals in addition to various essential nutrients, suggesting that the gastrointestinal contents of rabbits contain various cationic metals at higher concentrations due to coprophagy and fullness. Thus, it is speculated that the oral bioavailability of chelating drugs could be reduced by the interaction with various cationic metals in the gastrointestinal lumen, in addition to the delayed GER in rabbits. Various antibiotics such as fluoroquinolones, macrolides, polypeptide antibacterial agents, sulfonamides, and tetracyclines are known to form chelate complexes with metal cations in water and physicochemical adsorption to metal cation-containing solid materials ,15,16. I3), a widely used antacid, in control and EDTA-treated rabbits. The establishment of EDTA-treated rabbits with less gastric contents including metal contents is expected to provide great advantages in preclinical oral bioavailability studies of various drugs, and formulations. The chemical structures of LFX, LFX-EHE, CFX, CFX-EHE, and TC used in the present study are shown in In the present study, we tried to develop a rabbit model with a low amount of cationic metals in the stomach for preclinical oral bioavailability studies of chelating drugs. Elimination of gastric metals was achieved by administering a low concentration of disodium ethylenediamine tetraacetic acid (EDTA 2Na) orally. EDTA 2Na, a typical chelating agent, can chelate various polyvalent metal cations and form water-soluble chelate complexes in water. The low toxicity of EDTA 2Na is also recognized. For example, in EDTA chelation therapy, 3 g of EDTA 2Na or EDTA-Ca is infused intravenously to eliminate toxic metals such as lead (Pb), cadmium (Cd), gadolinium (Gd), mercury (Hg), and so on in the body into urine ,18. In t3 gel , anhydrous aluminum chloride , Mueller–Hinton broth , deuterated chloroform , hexadeuterodimethyl sulfoxide , filter paper , and syringe filter with 0.22 μm of pore size . Bacteria such as Staphylococcus (S.) aureus NBRC 15035 (ATCC 29213) and Escherichia (E.) coli NBRC 15034 (ATCC 25922) were provided by Biological Resource Center Department of Biotechnology, National Institute of Technology and Evaluation . Pseudomonas (P.) aeruginosa JCM 6119 (ATCC 27853) were provided by the RIKEN BRC through the National BioResource Project of the MEXT . The high-pressure liquid chromatography (HPLC) column  was purchased from CERI Co., Ltd. . Other materials including organic solvents for HPLC used were of the highest grade available. The following materials  were used: LFX , CFX , TC hydrochloride , 1-chloroethyl ethyl carbonate , dried Al(OH)®, Tokyo, Japan) and dried at 40 °C for 5 h. Melting point (MP) was measured on the Yanagimoto micro-melting point apparatus  and was uncorrected. Proton nuclear magnetic resonance (1H-NMR) spectra were recorded on a JEOL JNM-400S at 400 MHz , and chemical shifts relative to tetramethylsilane  were estimated. NMR spectra were measured in CDCl3 (δ 7.26 ppm). The carbon nuclear magnetic resonance (13C-NMR) spectroscopic data were recorded with a JEOL JNM-400S at 101 MHz , and chemical shifts relative to CDCl3 (δ = 77.0 ppm) and DMSO-d6 (δ 39.7) were estimated. The mass spectra of the prodrugs were recorded on a JEOL JMS-700 spectrometer  through the direct inlet system.LFX-EHE was synthesized in the same manner as reported previously according to the reported modified method ,34. Briev/v) as a developing solvent. Detection of CFX, Boc-CFX, Boc-CFX-EHE, and CFX-EHE was made by short-wave ultraviolet light at 254 nm. The Rf values of CFX, Boc-CFX, Boc-CFX-EHE, and CFX-EHE were 0.08, 0.35, 0.78, and 0.24, respectively. The synthetic process of CFX-EHE is summarized in CFX-EHE was newly synthesized in the present study via Boc-CFX and Boc-CFX-EHE, according to the reported modified method for ampicillin prodrugs . The pur2O, 360 mg/1.66 mmol) in THF (10 mL) and stirred at room temperature for 16 h. The solvent was removed under reduced pressure and the resulting material was diluted in H2O and neutralized with a saturated NH4Cl solution. The precipitate was collected by vacuum filtration and washed with H2O to afford the product as a white amorphous solid . 1H NMR (CDCl3) δ: 1.19–1.23 , 1.37–1.43 , 1.50 , 3.28–3.30 , 3.51–3.56 , 3.66–3.68 , 7.36 , 8.03 , 8.77 . 13C-NMR (CDCl3) δ: 8.2, 28.4, 35.3, 49.7, 80.4, 105.0, 108.1, 112.5, 112.8, 120.3, 120.3, 139.0, 145.7, 145.8, 147.6, 152.4, 154.6, 154.9, 166.8, 177.1. MS m/z: 431(M+). Chemical formula: C22H26FN3O5. Molecular weight: 431.4644.Boc-CFX was synthesized by the reported method . Briefly2O (120 mL) were slowly added to the mixture after 2 h-stirring and cooling. Crystal precipitated was filtrated with filter paper and was dried at 40 °C for 5 h. The synthesized yield of Boc-CFX-EHE was 82% (632 mg). MP. 196–198 °C (dec.). 1H-NMR (CDCl3) δ: 1.10–1.14 , 1.29–1.35 , 1.49 , 1.66 , 3.21 , 3.40–3.43 , 3.65 , 4.22 , 7.00 , 7.26 , 8.03 , 8.51 . 13C-NMR (CDCl3) δ: 8.2, 14.1, 19.8, 28.4, 34.6, 49.9, 64.4, 80.2, 91.6, 105.0, 109.1, 113.4, 113.6, 123.2, 123.3, 137.9, 144.5, 144.6, 148.6, 152.2, 153.1, 154.6, 154.7, 163.1, 172.9. MS m/z: 547(M+). Chemical formula: C27H34FN3O8. Molecular weight: 547.5804.Boc-CFX-EHE was synthesized by the reported modified method . Anhydro2) and the resulting gum was triturated with ice-cold ethyl acetate. The precipitate was collected, diluted in H2O and DCM, and basified to pH 9 with a 3% NaHCO3 solution. The product was extracted with chloroform, washed with distilled water, dehydrated with anhydrous sodium sulfate, and the organic solvent was evaporated to dryness. Recrystallization of CFX-EHE was made from ethyl acetate. The synthesized yield of CFX-EHE was 56% (291 mg). MP. > 300 °C (dec.). 1H-NMR (DMSO-d6) δ: 1.05–1.15 , 1.21 , 1.25–1.27 , 1.53 , 3.25 , 3.39–3.40 , 3.65–3.71 , 4.15 , 6.78 , 7.46 , 7.77 , 8.44 . 13C-NMR (CDCl3) δ: 8.1, 8.1, 14.4, 19.9, 35.6, 43.5, 47.6, 64.6, 91.7, 107.3, 108.0, 112.2, 112.4, 116.2, 119.1, 122.8, 122.8, 138.5, 143.6, 143.7, 149.6, 151.9, 153.1, 154.3, 158.7, 159.0, 162.7, 172.3. MS m/z: 447(M+). Chemical formula: C22H26FN3O6. Molecular weight: 447.4634. Spectrum of 1H-NMR (A), and 13C-NMR (B) of CFX; spectrum of 1H-NMR (A), and Mass (B) of Boc-CFX; spectrum of 1H-NMR (A), 13C-NMR (B) and Mass (C) of Boc-CFX-EHE; and spectrum of 1H-NMR (A), 13C-NMR (B) and Mass (C) of CFX-EHE are shown in Boc-CFX-EHE (500 mg/0.91 mmol) was dissolved in anhydrous dichloromethane  and cooled to 0 °C. Anhydrous anisole (100 μL) was added followed by the dropwise addition of trifluoroacetic acid . The reaction mixture was stirred at 0 °C for 30 min, warmed to room temperature, and stirred for a further 40 min. The solvent was removed under a stream of nitrogen gas  in the same manner as reported previously ,33. An e3 was also dissolved in the same buffer at a concentration of 50 μM, 100 μM, 200 μM, 1 mM, or 2 mM, and each drug solution and AlCl3 solution were mixed at an equal volume. The mixture solution was shaken, stood for 30 min at 24 °C and centrifuged. The supernatant was filtrated through a syringe filter with 0.22 μm of pore size . The concentrations of CFX in each filtrate were determined by HPLC after hydrolyzing CFX-EHE with 1 mol/L NaOH containing 1.0 mg/mL EDTA-2Na.Chelating potencies of CFX, CFX-EHE and TC were determined in the same manner as reported previously ,33. BrieThe chemical and enzymatic stability of CFX-EHE, an ester prodrug of CFX, was evaluated in the same manner as reported previously ,33. BrieS. aureus, E. coli, and P. aeruginosa were adjusted to McFarland 0.5 (1.5 × 106 cells/mL) with the sterilized isotonic NaCl solution. These bacterial solutions were inoculated in Mueller–Hinton broth containing CFX or CFX-EHE. Facultative anaerobic and aerobic bacteria were cultured under the aerobic condition at 37 °C for 20 h.MIC values of CFX and CFX-EHE were determined by agar plate dilution method using Mueller–Hinton broth in the same manner as reported previously ,33. Brie2+, Fe3+) in addition to various energy sources. One day before experiments, control rabbits were fasted with free access to water by keeping them in the stainless-steel cages as described above. Coprophagy was not prevented in control rabbits. In the case of EDTA-treated rabbits, rabbits were moved to wooden rabbit fixing boxes  one day before experiments, in which the neck is fixed to prevent food intake and coprophagy but with free access to 1 mg/mL EDTA 2Na aqueous solution. In addition, 1 h before the bioavailability study of antibiotics, control rabbits received 10 mL of water orally and EDTA-treated rabbits received 10 mL of water containing 1 mg/mL EDTA 2Na by stomach intubation, respectively.Male albino rabbits weighing 2.5 ± 0.5 kg were purchased from Japan SLC, Inc. , and kept in stainless steel cages equipped with an automatic water supply and excrement flushing device. The room was maintained in a 12:12-h dark-light cycle and kept at a constant temperature of 22 ± 0.5 °C. Rabbits were supplied solid food for rabbits , and the average amount of CR-3M that each rabbit ingested was 100–110 g/day. CR-3M contains various vitamins and cationic metals including Ca, Mg, and iron , control (fasted) and EDTA-treated rabbits, rabbits were sacrificed by intraperitoneal injection of an excess amount of pentobarbital. Rabbits were opened along the midline and their stomach was isolated. The stomach contents were taken out into a test tube and weighed, and the suspension was centrifuged at 3000 rpm for 10 min to measure the amount of gastric water. Then, concentrations of the Ca and Mg in the supernatant were measured by using L type Wako Ca  and L type Wako Mg N , respectively, and an automated clinical chemistry analyzer, TBA-nx360 . The pH of gastric water was also determined.g for 5 min at 4 °C. The concentrations of FD-4 in plasma were determined by Spectro fluorophotometer RF-1500  using an excitation and emission wavelength of 480 nm and 520 nm, respectively [To evaluate the plausible mucosal damage or change in membrane permeability after treatment with EDTA 2Na, the oral absorption of fluorescein isothiocyanate (FITC) dextran with an average molecular weight of 4400 (FD-4), a poorly absorbable marker compound due to the hydrophilic macromolecule , was comectively .3 (100 mg/kg) in control and EDTA-treated rabbits as follows; administration of Al(OH)3 suspension (approximately 3 mL) and water (approximately 2 mL) to wash the tube line, and then drug solution (approximately 3 mL) followed by water (approximately 2 mL). The total amount of water ingested was 10 mL. After administration of each drug, blood (100 μL each) was taken periodically from ear veins. Blood samples were centrifuged at 3000× g for 10 min, and plasma samples were taken and kept in a deep freezer until analysis.Either LFX 20 mg/kg (0.055 mmol/kg), LFX-EHE 26.4 mg/kg (0.055 mmol/kg), CFX 30 mg/kg (0.091 mmol/kg), or CFX-EHE 40.5 mg/kg (0.091 mmol/kg) was administered orally by stomach intubation without (control) or with Al(OH)g for 10 min to obtain plasma samples.TC hydrochloride was administered orally at a dose of 150 mg/kg together with 10 mL water to control and EDTA-treated rabbits. In a separate experiment, 1 mg/mL EDTA 2Na aqueous solution was administered 1 day before administration of each drug at a volume of 40 mL  for treatment, in addition to the free access to the drinking water containing 1 mg/mL EDTA 2Na. After administration orally by stomach intubation, blood samples (100 μL each) were taken periodically from the ear vein at 0.25, 0.5, 1, 2, 4 and 6 h. Blood samples were centrifuged at 3000× 0–∞) values of each drug in plasma after intravenous  and oral  administrations were estimated as follows: AUC0–∞ = AUC0–xh + C(xh)/β, where AUC0–xh  was estimated by trapezoidal rule and AUC after blood sampling (AUCxh–∞) was estimated by dividing the blood concentration at x h, C(xh), with the slope (β) of the terminal phase. The calculation of AUC was made by using Excel. The plasma concentrations of LFX were determined for 6 h after intravenous and oral administrations, those of CFX were determined for 6 h after intravenous and for 4 h after oral administration, respectively, and those of TC were determined for 4 h after intravenous and for 6 h after oral administration, respectively. The absolute oral bioavailability (F) was estimated by using the following equation: F = AUC0–∞,po × Doseiv/, where Dosepo and Doseiv represent the dose of each drug for oral administration and intravenous infusion, respectively.To estimate the absolute oral bioavailability (F) of LFX (20 mg/kg), CFX (30 mg/kg) and TC (10 mg/kg), respectively, antibiotics were administered intravenously by a constant-rate infusion for 2 min 50 s into the ear vein. Blood was taken periodically at 3 min, 15 min, 30 min, 1 h, 2 h, 4 h and 6 h after the start of constant-rate infusion of each antibiotic from the opposite ear vein. Dosing solutions of LFX, CFX and TC were prepared by dissolving in saline at a concentration of 4 mg/kg/mL, 6 mg/kg/mL and 2 mg/kg/mL, respectively. The area under the concentration-time curve (AUC) from 0 to infinity (AUCv/v). The flow rate of the mobile phase was 1 mL/min. The detection was made at UV 295 nm.Concentrations of LFX in plasma samples were determined by HPLC using CFX as an internal standard (IS) in the same manner as reported previously ,33. In tg for 5 min at 4 °C, and a 20 μL aliquot of the supernatant was injected into the HPLC column . In the case of CFX-EHE aqueous samples (each 1.0 mL) obtained from chelation, partition coefficients and solubility studies, 0.5 mL of 1.0 mol/L NaOH was added, incubated for 30 min and then neutralized with 0.5 mL of 1.0 mol/L HCl solution. The solution was filtered with a syringe-type filter to obtain a filtrate. The mobile phase of HPLC was a mixture of acetonitrile and water containing 0.3% triethylamine (pH 3.3 adjusted with phosphoric acid) (16:84 v/v). The flow rate of the mobile phase was 1 mL/min. Detection was made by a fluorescence detector at 278 nm and 450 nm for an excitation and emission wavelength, respectively [Concentrations of CFX in aqueous solution and plasma samples were determined by HPLC using LFX as IS according to the reported modified method . Brieflyectively .g for 5 min at 4 °C, and a 20 μL aliquot of the supernatant was injected into the HPLC column . The mobile phase was a mixture of acetonitrile and 0.3% triethylamine adjusted at pH 3.3 with phosphoric acid (16:84 v/v). The flow rate of the mobile phase was 1 mL/min. The detection was made at 273 nm.The concentrations of TC in aqueous solution and plasma were determined by HPLC using LFX as IS according to the modified method . BrieflyXL). Data acquisition was performed with the Sept 3000’s processor . Examples of chromatograms of LFX, CFX, and TC in rabbits’ plasm are shown in The HPLC system  consisted of a model LC-20AD pump, a fixed injection loop of 20 μL, and a fluorescence spectrophotometer . Statistical analysis was performed using the unpaired Student’s lH-NMR spectral data displayed only specific signals of CFX-EHE. The purity of CFX-EHE was also evaluated by TLC using a mixture of ethanol-ammonia water  as a developing solvent. The spot detected using shortwave ultraviolet light (254 nm) was one spot, and the Rf values of CFX and CFX-EHF were 0.08 and 0.24, respectively. Data: 1H-NMR (DMSO-d6) δ: 1.05–1.15 , 1.21 , 1.25–1.27 , 1.53 , 3.25 , 3.39–3.40 , 3.65–3.71 , 4.15 , 6.78 , 7.46 , 7.77 , 8.44 . 13C-NMR (CDCl3) δ: 8.1, 8.1, 14.4, 19.9, 35.6, 43.5, 47.6, 64.6, 91.7, 107.3, 108.0, 112.2, 112.4, 116.2, 119.1, 122.8, 122.8, 138.5, 143.6, 143.7, 149.6, 151.9, 153.1, 154.3, 158.7, 159.0, 162.7, 172.3. MS m/z: 447(M+). Chemical formula: C22H26FN3O6. Molecular weight: 447.4634.CFX-EHE d was newp values are listed in To evaluate the lipophilicity of CFX and CFX-EHE, the apparent partition coefficients (PCs) of CFX and CFX-EHE were determined in a partition system of chloroform and pH 6.5 buffer. Estimated PCs and calculated log 3 solution . The precipitation percentages of TC were greater than those of CFX. CFX-EHE showed no precipitation for 30 min , as well as in the case of LFX-EHE [3, the precipitation was completely inhibited.To evaluate the chelating ability of CFX, CFX-EHE and TC, drug (100 μM) solution of each drug was mixed with AlClr 30 min . Previou LFX-EHE . When EDThe stability of orally administered CFX-EHE in the small intestine, intestinal membrane, plasma and liver was evaluated in vitro. CFX-EHE was stable chemically in pH 6.5 and pH 7.4 phosphate-buffered saline (PBS), but rapidly hydrolyzed enzymatically in 10% plasma, 2% intestinal mucosa homogenates and 2% liver homogenates . The aveS. aureus, E. coli, and P. aeruginosa were determined. In the prodrug concept, prodrugs should have lower pharmacological action as compared with their parent drug [S. aureus, E. coli, and P. aeruginosa were more than 10 times higher than that of CFX . On one hand, the intravenous dose of TC was reduced to 10 mg/kg, although the oral dose was 150 mg/kg, based on the results of the preliminary study. Plasma disposition-time courses of LFX, CFX and TC are shown in 0–6h for LFX and CFX or AUC0–4h for TC, and AUC0−∞ are listed in iv was compared among three antibiotics, the order was as follows; LFX = CFX < TC, indicating that the tissue distribution volume of TC is smaller than those of LFX and CFX, and the distribution volume of LFX and CFX are comparable  estimated by Doseiv/AUC0–∞ were in the following order: CFX (1273 mL/h/kg) = TC (1130 mL/h/kg) > LFX (823 mL/h/kg).To estimate the absolute oral bioavailability (F) of LFX, CFX, and TC in rabbits, the AUCmparable . Similar3 was 100 mg/kg in both control and EDTA-treated rabbits. The time courses of LFX concentrations in plasma after oral administration of LFX and LFX-EHE in the absence and presence of Al(OH)3 are shown in 0–6, AUC0–∞ of plasma LFX, and oral bioavailability (F) of LFX are listed in 3, both the rate of oral bioavailability (Cmax and Tmax) and the extent of oral bioavailability (AUC) of LFX in EDTA-treated rabbits were higher than in control rabbits. This indicated that both the rate and extent of oral bioavailability of LFX in control rabbits are significantly lower in control rabbits than in EDTA-treated rabbits. When Al(OH)3 was administered concomitantly with LFX, the Cmax, and AUC0–6 of LFX were significantly lower than those in the absence of Al(OH)3 in both control and EDTA-treated rabbits. In contrast, the rate and extent of oral bioavailability of LFX from LFX-EHE were comparable between control and EDTA-treated rabbits and co-administration of Al(OH)3 exerted no significant effects on the rate and extent of oral bioavailability of LFX from LFX-EH. This would indicate that LFX-EHE can prevent chelate formation in vitro and in vivo. Although there was no significant difference in the oral bioavailability between the absence and presence of Al(OH)3, however, the average Cmax and AUC of LFX from LFX-EHE in the presence of Al(OH)3 appeared to be lower as compared with the results of LFX without Al(OH)3 in EDTA-treated rabbits  rabbits .3 was 100 mg/kg in both control and EDTA-treated rabbits. Both the rate and extent of oral bioavailability of CFX in EDTA-treated rabbits were significantly higher than in control rabbits, as well as in the case of LFX. These data will indicate that the rate and extent of oral bioavailability of CFX in control rabbits are suppressed by gastric cationic metals, by forming a poorly soluble chelate complex. By co-administrating Al(OH)3 with CFX to control and EDTA-treated rabbits, the rate and extent of oral bioavailability of CFX were suppressed. In contrast, the rate and extent of oral bioavailability of CFX-EHE were comparable between control and EDTA-treated rabbits. When the oral bioavailability of CFX from CFX-EHE was compared with the oral bioavailability of CFX itself, there was no significant difference between them in both control and EDTA-treated rabbits, as well as the cases of LFX-EHE. The co-administration of Al(OH)3 exerted no significant effects on the rate and extent of oral bioavailability of CFX from CFX-EHE, indicating that CFX-EHE can prevent chelate formation in vivo. However, it would be considered that there may be some interaction such as adsorption between CFX-EHE and Al(OH)3, especially in EDTA-treated rabbits  rabbits .0–6h of TC were significantly greater than those in control rabbits, in which the value of Tmax was comparable between both rabbits. In a separate experiment, 20 mL (10 mL on one day before the experiment and 10 mL on the experiment day) of 1 mg/mL EDTA Na was administered orally for treatment, in addition to the free access to the drinking water containing 1 mg/mL EDTA 2Na. The dose of TC hydrochloride was the same (150 mg/kg). In this case, the value of standard deviation (SD) of plasma TC concentrations became smaller than 10 mL single ingestion of 1 mg/mL EDTA 2Na, although there was no significant difference in the average plasma concentrations of TC between 10 mL and 20 mL of EDTA 2Na. This may suggest that the dose of 20 mL of 1 mg/mL EDTA 2Na ingestion is preferred to eliminate the cationic metals from the stomach completely.  to prevent coprophagy, fasting for one day with free access to 1 mg/mL EDTA 2Na, and ingesting 10 mL EDTA 2Na 1 h before the experiment. The EDTA treatment decreased the gastric Ca contents to <1% of untreated control rabbits , and theLaboratory rabbits are fed foods rich in cationic metals in addition to various essential nutrients. In the present study, CR-3M  was used as rabbits’ food and this food contains the following cationic metals: Ca, 2.10–2.13 g; Mg, 0.23–0.26 g; Fe, 38.1–39.1 mg; Mn, 7.90–9.12 mg; Zn, 4.72–5.08 mg; Cu, 0.78–0.96 mg; and so on/100 g food (information from CLEA Japan Inc.). Similarly, another commercially available food for rabbits also contains cationic metals as follows: Ca, 1.2 g; Mg, 0.32 g; Fe, 51.9 mg; Mn, 7.7 mg; Zn, 5 mg; Cu, 1.05 mg; and so on/100 g food . Rabbits ate 100–110 g of food per day. It was reported that 47 % of the total excreted amount of Ca was excreted in the urine and 53% was in the feces in female New Zealand White rabbits . The stoIn developing EDTA-treated rabbits, the concentration of EDTA 2Na was fixed at 1 mg/mL  in water, since higher concentrations of EDTA 2Na are known to modify the membrane permeability of the paracellular route by opening the cellular tight junctions between enterocytes. For example, in Caco-2 cells, the coadministration of 2.5 mM EDTA 2Na  increased the permeability of fluorescein isothiocyanate (FITC) and FITC-dextrans, and produced changes in ZO-1, claudin-1, occludin, and E-cadherin distribution, indicating a tight junction effect. The copresence of 0.25% EDTA 2Na increased the permeability of PEG 4000 to 14 times the control ,45. In c3 in control and EDTA-treated rabbits. LFX, CFX and TC are known to form chelate complexes with various cationic metals. In addition, CFX and TC are also known to adsorb various metal-containing solid materials [3 was reported to be weak among quinolones [As shown in aterials ,53,54,55inolones .In Biopharmaceutics Classification System (BCS), LFX was classified as a BCS Class 1 drug with high solubility and high permeability . The ora3+ was highest, whereas that of Mg2+ was lowest. It was also found that quinolones have a similar affinity for the metal ions, forming chelates which are more stable with hard Lewis acids such as the trivalent cations  [2+, Ca2+, Cu2+, Zn2+, Fe2+, Co2+, etc.) bind to carbonyl and carboxyl groups in neighboring positions and form chelates with 1:1 or 1:2  stoichiometry, and trivalent cations  form chelates with 1:1, 1:2 or 1:3  stoichiometry [3 (2.5 mg/mL) was in the following order: norfloxacin  > enoxacin  > LFX (48.1%) = OFX, (47.2%). In addition, the elution rate of adsorbed quinolones with water was in the following order: LFX (17.9%) approximately OFX (20.9%) approximately ENX (18.3%) > NLX (11.9%) [Regarding the chelate formation of LFX, many articles are available. For example, when the chelating ability was compared among cationic metals using four fluoroquinolones, LFX, CFX, sparfloxacin and enrofloxacin, the chelation potential of Al+, Fe3+) . In quinhiometry . Chelatehiometry ,71,72,73hiometry ,32,33,74hiometry ,75,76,77 (11.9%) . These d (11.9%) ,78.3 reduced the oral bioavailability of LFX in both control and EDTA-treated rabbits. This would also indicate that EDTA 2Na used for treatment in the present study did not disturb chelate formation significantly. There was no difference in the oral bioavailability of LFX between LFX-EHE and LFX itself in both control and EDTA-treated rabbits 3, the average oral bioavailability of LFX in the presence of AL(OH)3 appeared to be reduced by 18.6% in the control rabbits and 28.5% in EDTA-treated rabbits, respectively  and low permeability, and CFX hydrochloride is classifieds as a Class III drug with high solubility (approximately 36 mg/mL at 25 °C) and low permeability ,80,81,823, Mg(OH)2, magnesium carbonate, magnesium trisilicate and magaldrate, resulting in decreased oral bioavailability of CFX [w/v % soil), β-lactams and florfenicol antibiotics remain bioactive in soils while CFX, neomycin, and TC were neutralized [Regarding the interaction of CFX with cationic metals, many articles are available. For example, it was reported that the dissolution rate of CFX-containing tablets was markedly retarded in the presence of antacids, such as sodium bicarbonate, calcium hydroxide, calcium carbonate, Al(OH)y of CFX ,93,94,95y of CFX ,97,98. Sy of CFX ,70,71,72y of CFX ,33,70,74tralized . Among v3 decreased the oral bioavailability of CFX in both control and EDTA-treated rabbits 3 and/or substrate specificities of CFX-EHE for P-gp. Further study is necessary to clarify the mechanism of the scattering oral bioavailability of CFX from CFX-EHE in the presence of Al(OH)3.In the present study, in rabbits, the absolute oral bioavailability of CFX was 13.73 ± 2.48% in control rabbits and 19.37 ± 2.28% in EDTA-treated rabbits, in which EDTA-treated rabbits exhibited significantly higher oral bioavailability of CFX than control rabbits. Coadministration of Al(OH) rabbits . In cont in vivo . There w in vivo . In this3, Fe2+, Al3+, Mg2+ and Ca2+. Many of these tetracycline-metal complexes are either insoluble or otherwise poorly absorbable from the gastrointestinal tract [The absolute oral bioavailability of TC, a chelating/adsorptive drug, in humans, is reported to be 77–88% at a dose of 250 mg, in which 5% of TC was metabolized to a less active metabolite and the remainder was eliminated by renal excretion ,100. In al tract .2+ was found to form a 2:1 metal-ion complex with TC, and Mg2+ formed the complex at a 1:1 ratio. In the Ca2+-TC complex, Ca2+ binds to the C-10 and C-11 sites of TC with the subsequent addition of a second metal ion at the C-12 and C-1 sites. The Mg2+ chelation occurred at the C-11 and C-12 β-diketone sites of TC [1 (3.3), the TC was dominant with species of TC3+. With the pH ranging between pKa1 and pKa2 (3.3 < pH < 7.7), the TC was presented with TC2+. At pH further increasing to a range of (7.7 < pH < 9.7) and (pH > 9.7), the dominant species of TC were TC− and TC2−, respectively [2+, Zn2+ and Al3+. Separately, the adsorption process on discarded natural coal gangue was classified as a thermodynamic endothermic and spontaneous reaction, which was controlled by chemical and physical adsorption, including electrostatic interaction between negatively charged TC and cationic metals and cation exchange between positively charged TC and cationic metals, in addition to physical adsorption [When analyzed with circular dichroism (CD) spectrum, Caes of TC ,111,112.es of TC ,25,26,27ectively . Maximumsorption ,114. Liksorption . In clinsorption .In the present study, TC was also administered at a dose of 150 mg/kg, because TC was not detected in plasma at a lower dose than 30 mg/kg. The absolute oral bioavailability of TC in control rabbits was 2.56 ± 0.95%, and it significantly increased to 4.90 ± 0.81% in EDTA-treated rabbits. These oral bioavailabilities (<5% of dose) of TC in rabbits, however, are far lower than those reported (77–88% at a dose of 250 mg) in humans ,100. TheThe oral bioavailabilities of LFX, CFX and TC were compared between humans, control rabbits, and EDTA-treated rabbits .3+ in water was in the following order: TC > CFX > FLX . In contrast, the oral bioavailability of CFX and TC, especially TC, in rabbits were markedly lower as compared with those in humans, The chelating ability of these antibiotics to AlFX > FLX . HoweverFX > FLX . The adsEDTA-treated rabbits with low amounts of gastric contents including metals and low gastric pH were developed without causing mucosal damage as experimental animals for preclinical bioavailability studies. The treatment of rabbits with >10 mL of 1 mg/mg EDTA 2Na decreased the gastric metal contents to less than 1% of untreated control rabbits and gastric pH to physiologically normal levels. The oral bioavailabilities of LFX, CFX and TC in EDTA-treated rabbits were significantly higher than those in control rabbits, indicating that, in general, the oral bioavailabilities of chelating drugs are reduced by the interaction with gastric metals in rabbits. Ester prodrugs of fluoroquinolones such as LFX-EHE and CFX-EHE were effective in preventing chelate formation in gastrointestinal water but may not be effective in preventing physicochemical adsorption to metal-containing materials, because the oral bioavailabilities of TC and CFX, which are both potent adsorptive antibiotics to metal-containing materials, in EDTA-treated rabbits were far lower as compared with reported oral bioavailabilities. In this study, it was found that EDTA-treated rabbits exhibit quite low gastric contents, low gastric metals and low gastric pH, as well as fasted rats and humans. Thus, EDTA-treated rabbits may be expected to be useful preclinical experimental animal models in studying various drugs and dosage formulations. Further study is necessary to empty the gastrointestinal contents of EDTA-treated rabbits completely, to clarify the contribution of adsorptive interaction with gastrointestinal metals, and to seek out the usefulness of the EDTA-treated rabbit as an experimental animal model.


The high-osmolarity glycerol (HOG) mitogen-activated protein kinase (MAPK) pathway responsible for signal integration and transduction plays crucial roles in environmental adaptation, especially in the response to osmotic stress. Hog1 is activated by transient Ca2+ increase in yeast, but the functions of the HOG pathway in Ca2+ homeostasis are largely unknown. We found that the HOG pathway was involved in the regulation of Ca2+ homeostasis in Fusarium graminearum, a devastating fungal pathogen of cereal crops. The deletion mutants of HOG pathway displayed increased sensitivity to Ca2+ and FK506, and elevated intracellular Ca2+ content. Ca2+ treatment induced the phosphorylation of FgHog1, and the phosphorylated FgHog1 was transported into the nucleus by importin β FgNmd5. Moreover, the increased phosphorylation and nuclear accumulation of FgHog1 upon Ca2+ treatment is independent of the calcineurin pathway that is conserved and downstream of the Ca2+ signal. Taken together, this study reported the novel function of FgHog1 in the regulation of Ca2+ homeostasis in F. graminearum, which advance the understanding of the HOG pathway and the association between the HOG and calcineurin pathways in fungi.Maintaining cellular calcium (Ca Saccharomyces cerevisiae, Hog1 MAPK, a central component of the HOG pathway, is rapidly activated by two branches of upstream osmosensors that converge at Pbs2 MAPK kinase [2+) concentration in eukaryotic cells [2+ signaling pathways. In S. cerevisiae, Hog1 is immediately activated and accumulated in the nucleus upon transient Ca2+ elevation, and required for the Ca2+-induced expression of the glyoxalase 1 encoding gene GLO1 through Msn2/4 transcription factors [2+ signal was reported to dysregulate the HOG pathway in S. cerevisiae [2+ accumulation and the cross-talk between the HOG and calcineurin pathways are largely unknown in fungi.In eukaryotic cells, mitogen-activated protein kinase (MAPK) pathways are responsible for numerous signal integrations, and transducing extracellular signals into the nucleus in response to a wide range of environmental stresses. Among them, the high-osmolarity glycerol (HOG) pathway has been widely studied to play a key role in cell response to hyperosmotic stress . In SaccK kinase . In addiK kinase ,5, DNA dK kinase , cadmiumK kinase ,8, tempeK kinase ,10, fludK kinase  and tebuK kinase . Hyperosic cells ,14,15,16 factors . Additiorevisiae ,18. HoweS. cerevisiae, importin β Nmd5 is responsible for nuclear translocation of phosphorylated Hog1 under osmotic stress, and exportin XPO1 mediates the export of dephosphorylated Hog1 from nucleus to cytoplasm [F. graminearum, while Ca2+ transient elevation and the late phase of the endoplasmic reticulum stress response all can lead to the accumulation of activated Hog1 in the nucleus of S. cerevisiae [The nucleus translocation of phosphorylated Hog1 is required for its function in stimulating stress-induced gene expression. In general, the nucleoplasmic transport of biomacromolecules (more than 30 kDa) is mediated by karyopherins (importin/exportin/transportin), which are a superfamily of transport receptors through the nuclear pore complex (NPC) . The nucytoplasm . Tebuconrevisiae ,17,25. HF. graminearum causes Fusarium head blight (FHB) on triticeae crops and other important cereal crops, which is devastating to cereal crops worldwide [2+ and the calcineurin inhibitor FK506 in F. graminearum. Further, we explored the molecular mechanism of Ca2+ homeostasis regulation mediated by FgHog1, which provides a novel insight into the functions of the HOG pathway and the association between the HOG and calcineurin pathways in fungi.The plant pathogen orldwide . In addiorldwide ,28. In tFusarium graminearum wild-type strain PH-1 (NRRL 31084) was used as a parental strain for constructing gene deletion mutants in this study. PH-1 and the resulting transformants were cultured at 25 °C on minimal medium  for mycelia growth analysis. Fresh mycelia used for protein extraction were inoculated in yeast extract peptone dextrose  liquid medium at 25 °C for 12 h in a shaker (180 rpm). The experiment was repeated three times.2 or calcineurin inhibitor FK506, and then incubated at 25 °C for 3 days. Three biological replicates were used for each strain and each experiment was repeated three times independently.To determine sensitivity phenotypes, 5 mm (diameter) mycelial plugs of each strain taken from the edge of a 3-day-old colony were inoculated on MM or complete medium  supplemented with or without CaClHPH) driven by the constitutive trpC promoter, which was amplified from the pBS-HPH1 vector [F. graminearum protoplast transformation based on the previous study [2). The protoplasts were resuspended in 800 μL STC buffer and added to 35 μg construct, 20 μL spermidine and 200 μL SPTC for transformation. After 45 min, 400 μL SPTC was added into the suspension and incubated at 25 °C for 20 min. Protoplasts were transferred into 20 mL RM liquid medium  and incubated at 25 °C for 16 h, and then RM liquid medium containing protoplasts was mixed into PDA containing 100 μg/mL hygromycin B, and poured into plates. After incubation at 25 °C for 4 days, the importin β deletion mutants ΔFgNmd5, ΔFgKap123, ΔFgPse1, ΔFgKap114, ΔFgKap104 and ΔFgKap95 were screened by PCR assays with relevant primers  cassette, the double-joint PCR approach was used to amplify FgHog1 containing the promoter region and open-reading frame (without the stop codon). The resulting PCR products were co-transformed with XhoI-digested pYF11 into the yeast strain XK1-25 using the Alkali-CationTM Yeast Transformation Kit  to generate the recombined FgHog1-GFP fusion vector . Subsequ2+ imaging, mycelia of each strain were inoculated into YEPD for 12 h at 25 °C in a shaker with 180 rpm, and then were harvested after washing 3 times with PBS. A green fluorescent Ca2+ binding dye Fluo-8 AM  was used to detect the intracellular Ca2+. After PBS washing, mycelia were incubated with Fluo-8 AM at the concentration of 2 μM in PBS for 30 min at 37 °C and then washed with PBS three times. Subsequently, the treated mycelia were incubated with PBS without Fluo-8 AM for 20 min at 37 °C to ensure adequate conversion of Fluo-8 AM to Fluo-8. Fluorescent signals were excited at a wavelength of 488 nm using a Zeiss LSM780 confocal microscope . Fluorescence intensity was quantified using ImageJ software . The experiment was repeated three times.For intracellular Ca2+ concentration, fresh mycelia of each strain were inoculated in 20 mL of CMC  liquid medium at 25 °C for 4 days in a shaker with 180 rpm to obtain conidia. The number of conidia in each flask was determined using a hemacytometer . A total of 105 conidia of each strain was loaded with Fluo-8 AM and the procedures were performed as described above and then plated in clear-bottomed 96-well plates, respectively. BioTek Cytation 5 cell imaging multi-mode readers were used to evaluate Fluo-8 fluorescence intensity. The experiment was repeated three times with similar results.For quantitative assessment of Ca2O. Protein samples were extracted as described previously [g to obtain protein samples. The protein samples separated on the 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels were transferred onto Immobilon-P transfer membrane  with a Bio-Rad electroblotting apparatus. Total protein of FgHog1 and the phosphorylated FgHog1 were detected using the anti-Hog1 antibody  and anti-phospho-p38 antibody . The monoclonal anti-GFP  was used to detect GFP-tagged proteins. The samples were also detected with the monoclonal anti-GAPDH antibody  or monoclonal anti-H3 antibody  as a reference. The experiment was conducted three times independently.Mycelial plugs of each strain were incubated in 150 mL YEPD liquid medium for 36 h at 25 °C with 180 rpm. Mycelia were harvested after washing 3 times with ddHeviously . After b2, 25% glycerol, 250 mM sucrose and 5 mM DTT) containing 1:100 v/v protease inhibitor cocktail  for 15 min at 4 °C. The homogenate was filtered through cell strainers  and then centrifuged at 1500× g for 10 min at 4 °C. The supernatant consisting of the cytoplasmic fraction was centrifuged at 10,000× g for 15 min at 4 °C and collected. The precipitate consisting of the nuclear fraction was gently washed five times with 5 mL of nuclear resuspension buffer  and centrifuged at 1500× g for 10 min at 4 °C. The final nuclear pellet was resuspended in 800 μL lysis buffer. Protein samples were analyzed with Western blotting assay with the anti-Hog1 antibody. As quality controls for the fractionation, GAPDH was detected by using monoclonal anti-GAPDH antibody as cytoplasmic markers and histone H3 was detected by using monoclonal anti-H3 antibody as nuclear markers.The wild-type PH-1, ΔFgNmd5 and ΔFgSsk2 strains were incubated and harvested for nuclear–cytoplasmic fractionation experiment as described previously . About 2S. cerevisiae strain Y2H Gold following the lithium acetate/single-stranded DNA/polyethylene glycol transformation protocol. Next, 2 mL Y2H Gold liquid was centrifuged for 1 min at 12,000× g and washed twice with ddH2O. The precipitate was resuspended in 1 mL 0.1 M LiAc and incubated for 5 min at 30 °C. Subsequently, pairs of Y2H plasmids were added into the precipitate and incubated for 20 min at 42 °C. In addition, a pair of plasmids, pGBKT7-53 and pGADT7-T, served as a positive control, and another pair of plasmids, pGBKT7-Lam and pGADT7-T, served as a negative control. Transformants were grown at 30 °C for 3 days on synthetic medium (SD) lacking Leu and Trp, and subsequently transferred to SD stripped of Leu, Trp, His and Ade (SD-Leu-Trp-His-Ade) to assess binding activity. The experiment was conducted three times independently.To construct plasmids for Y2H analyses, the coding sequence of each tested gene was amplified from cDNA of the wild type, with primer pairs indicated in For Co-IP assays, the extracted total proteins were incubated with the GFP-trap agarose  for 4-6 h at 4 °C with rotation. After being washed six times with 1 × TBS buffer  at 4 °C, proteins binding to GFP-trap agarose were eluted and analyzed with Western blotting assay with the anti-Hog1 antibody and the monoclonal anti-GFP as described in the previous section. The monoclonal anti-GAPDH antibody was also used as a reference. The experiment was conducted three times independently.For the BiFC assay, the PHZ65 vector carrying NYFP  and PHZ68 vector carrying CYFP  were used for generating the protein expression construct. The full lengths of FgNmd5 and FgHog1 were amplified and co-transformed with XhoI-digested PHZ65 into the yeast strain XK1-25 using the Alkali-CationTM Yeast Transformation Kit. The full length of FgHog1 was amplified and co-transformed with XhoI-digested PHZ68 into the yeast strain XK1-25. Then, the FgNmd5-NYFP and FgHog1-CYFP fusion constructs were recovered from the yeast-positive transformant using the Yeast Plasmid Kit and transferred into Escherichia coli strain DH5α for amplification. Subsequently, construct pairs of FgNmd5-NYFP and FgHog1-CYFP were co-transformed into PH-1::H1-RFP (red fluorescent protein) strain , respectively. As negative controls, construct pairs of FgNmd5-NYFP and CYFP, and NYFP and FgHog1-CYFP, were co-transformed into PH-1::H1-RFP strain. Transformants resistant to both hygromycin and neomycin were isolated and confirmed with PCR. YFP signals were examined with a Zeiss LSM780 confocal microscope. The experiment was repeated three times.For conidiation assays, fresh mycelia of each strain were inoculated in a 50 mL flask containing 20 mL of CMC liquid medium. The flasks were incubated at 25 °C for 4 days in a shaker running at 180 rpm. The number of conidia in each flask was determined using a hemocytometer. In addition, septum was examined after conidia of each strain were stained with calcofluor white (CFW). Conidial morphology was observed with a Leica TCS SP5 imaging system. Three biological replicates were used for each strain and each experiment was repeated three times independently.5 mL−1) of each strain was injected into one spikelet in flowering wheat heads of the FHB susceptible variety Jimai 22, while the control treatments were inoculated with 10 μL of sterile ddH2O. There were 20 replicates for each strain. After 15 days post inoculation, the infected spikelets in each inoculated wheat head were recorded. The experiment was repeated three times, and typical symptoms were shown. Wheat (Triticum aestivum) cultivar Jimai22 was used for coleoptile infection. Three days after sowing, the top 2 mm to 3 mm of the coleoptiles was removed. Then, 5 mm (diameter) mycelial plugs of each strain taken from the edge of a 3-day-old colony were inoculated on the top of wounded coleoptiles. After inoculation, the seedlings were grown in a growth chamber at 25 °C and 95% humidity for 3 days before examination for the infection. The experiment was repeated three times.To assess virulence on flowering wheat heads, 10 μL spore suspension  in the dark. DON Quantification Kit Wis008  was used to quantify the DON production for each sample. The experiment was repeated three times.S. cerevisiae. The external hypertonic signal is transduced to MAPK kinase kinases Ssk2 and Ssk22, which further activates the MAPK kinase Pbs2 and phosphorylates Hog1 by Pbs2 [2+ homeostasis in Fg, we determined the sensitivity of the pathway mutants to Ca2+ and calcineurin inhibitor FK506. The MAPK kinase kinase FgSsk2, MAPK kinase FgPbs2 and MAPK FgHog1 deletion mutants were obtained in our previous study [2+ and FK506. The levels of free intracellular Ca2+ in these three mutants were higher than the wild-type strain PH-1 using green fluorescent Ca2+ binding dye Fluo-8 AM  signaling pathway, is mainly associated with regulation of cellular responses to hypertonic stress in  by Pbs2 . To inveus study . As showluo-8 AM B,C. To fnd FK506 A. Furthereatment D. In add2+ treatment. The FgHog1-GFP (green fluorescent protein) construct was generated and transformed into PH-1 and the ΔFgSsk2 mutant strain. Confocal microscopic examination showed that FgHog1-GFP entered into the nucleus in PH-1, but not in ΔFgSsk2 upon Ca2+ treatment ) with importin homologs from S. cerevisiae as queries  assays showed that FgHog1 binds to FgImportin α, but not FgNmd5 D, indicaf FgHog1 E,F. In areatment G and thereatment B. These F. graminearum [F. graminearum.Given that FgNmd5 and FgHog1 are all involved in calcium homeostasis, we test the phenotypes of their mutants to explore the functions of calcium homeostasis. As shown in minearum , we ther2+ are important second messengers in all organisms. In fungi, the regulation of the Ca2+ homeostasis system maintains the cytoplasmic Ca2+ concentration in a range from 50 to 200 nM [2+ influx system is activated in response to various external stresses, and increased Ca2+ concentration affects a wide range of cellular processes [2+ concentration is detrimental to fungal cells. The increased Ca2+ concentration not only modulates signaling cascades, but also activates the calcineurin pathway to reduce the Ca2+ concentration to the basal level [S. cerevisiae, the HOG pathway has been found to be activated by Ca2+ treatment [Magnaporthe oryzae, putative type 2C protein phosphatases MoPtc1 and MoPtc2 are induced by Ca2+ and also participate in the regulation of calcium homeostasis. Deletion of both MoPtc1 and MoPtc2 leads to overstimulation of Hog1 [2+ homeostasis regulation; moreover, this role of the HOG pathway is independent of the calcineurin pathway in F. graminearum  can directly bind to the promoter of SSKB (the MAPKKK kinase of the HOG pathway), thereby inducing the SSKB mRNA accumulation, and subsequently modulating SakA (Hog1 ortholog) phosphorylation upon osmotic stress [S. cerevisiae, methylglyoxal, a metabolite derived from glycolysis, was reported to have the ability to activate these two pathways in parallel [Intracellular Cao 200 nM . In generocesses ,39,40. Hal level ,42. In Sreatment . In Magn of Hog1 . In thisminearum , which irevisiae . In A. fc stress . In S. cparallel . Taken tS. cerevisiae Hog1 has been reported to be transported into the nucleus by importin β Nmd5 upon osmotic stress [2+ treatment  paralogues in Beauveria bassiana are critical for different multiple stress responses and virulence [2+/Mn2+-ATPase Spf1 supplies ER lumen with Ca2+/Mn2+ and regulates hyphal growth, conidiation, host penetration and pathogenicity in M. oryzae [2+/Mn2+ transporters, FgGdt1 and FgPmr1 are essential in various development and infection processes of F. graminearum by maintaining the homeostasis of Ca2+ and manganese in the Golgi [2+-calcineurin pathway was reported to regulate development, pathogenicity and stress responses in various fungal species [2+ homeostasis regulators FgHog1 or FgNmd5 led to reduced conidiation and pathogenicity (2+ homeostasis is critical for multiple biological processes in fungi.Carocesses ,53,54. IA damage . The Ca2sistance . Five Vcirulence . One Ca2. oryzae . As Ca2+he Golgi . The Ca2 species ,61. In ogenicity . Taken tF. graminearum, a predominant causal agent of FHB. Deletion of the HOG pathway components FgSsk2, FgPbs2 or FgHog1 caused increased sensitivity to calcium and FK506 and elevated levels of free intracellular Ca2+. The treatment with Ca2+ induced the phosphorylation of FgHog1, which was subsequently translocated to the nucleus, mediated by importin β FgNmd5. Collectively, our data uncover a novel role of FgHog1 in the regulation of calcium homeostasis in F. graminearum.The HOG MAPK pathway plays a crucial role in signal transduction, which is responsible for environmental adaptation in fungi. Previous reports detailed the function of the HOG pathway in overcoming osmotic stress. However, the regulatory mechanisms of the HOG pathway in calcium homeostasis are not well characterized. In this study, we found that the HOG pathway is involved in the regulation of calcium homeostasis in


This study was undertaken to investigate the interaction between the sodium channel blocker amiloride (AML) and human serum albumin (HSA). A combination of multi-spectroscopic techniques and computational methods were employed to identify the AML binding site on HSA and the forces responsible for the formation of the HSA–AML complex. Our findings revealed that AML specifically binds to Sudlow’s site II, located in subdomain IIIA of HSA, and that the complex formed is stabilized using van der Waals hydrogen-bonding and hydrophobic interactions. FRET analysis showed that the distance between AML and Trp214 was optimal for efficient quenching. UV-Vis spectroscopy and circular dichroism indicated minor changes in the structure of HSA after AML binding, and molecular dynamics simulations (MDS) conducted over 100 ns provided additional evidence of stable HSA–AML-complex formation. This study enhances understanding of the interaction between AML and HSA and the mechanism responsible. Human serum albumin (HSA) (molecular weight-66 kDa) is the most abundant protein in human plasma. This heart-shaped transporter plasma protein is produced in the liver, is soluble in water, and consists of 585 amino acids arranged in a single polypeptide chain folded into three structurally homologous domains , each composed of two subdomains (A and B) . The phy+/H+ antiporter and Na+/Ca+ exchanger in cardiac myocytes and other epithelial cells  represents the quencher concentration . SVK is the Stern–Volmer quenching constant; oτ is the fluorescence lifetime of HSA in the excited state in the absence of a quencher ; and qk is the protein bimolecular-quenching-rate constant. o/FF versus [Q]) at different temperatures, which were used to obtain SVK values. SVK and other parameters. The magnitude of SVK obtained at 298 K was 3.82 × 104 M−1, which is in excellent agreement with earlier reports for HSA drug interactions for many drugs  calculated from SVK, i.e., qk = SVK/oτ. The obtained qk values fell in the range of 6.6–3.75 × 10−12 which were much higher than the diffusion constant , further confirming that the quenching mechanism is static during complex formation. The qk and SVK values suggest that the quenching mechanism was static, involving ground-state complex formation between HSA and AML at equilibrium.In this equation,  106 M−1 ,28. Furtn) and (ii) the binding constant for static quenching (bK).The binding between HSA and ligands is stabilized through diverse non-covalent interactions, such as hydrophobic forces, hydrogen bonds, electrostatic interaction, and van der Waal interactions. The nature of forces associated with HSA–AML complex formation can be predicted from the thermodynamic parameters of the binding process. So, it is essential to understand the thermodynamic parameters of binding between HSA and AML. In particular, the binding constant, which represents equilibrium between free and bound molecules, is essential for calculating thermodynamic parameters. The modified Stern–Volmer equation was used to determine two parameters of AML to HSA binding, namely, (i) the number of binding sites (oF − F)/F] vs. log [Q] in 4 M−1 at 298 K to 0.99 × 104 M−1 at 315 K. From 2 and 106 M−1, indicating weak to strong binding  is mean residual ellipticity (in degcm2mol−1); Mo is mean residue weight of the protein; c is protein concentration (mg/cm3); and l is path-length (cm). Further, the percentage of α-helix present in HSA under different experimental conditions was calculated using Equation (3)θ]222 represents mean residual ellipticity at a wavelength of 222 nm.A Jasco J-815 spectropolarimeter , a circulating water bath, and a Peltier-type temperature controller were employed to assess the far-UV CD spectra of AML and HSA. The spectropolarimeter was periodically calibrated using D-(10)-camphor sulfonic acid. The far-UV CD spectra of HSA in the presence of AML at molar ratios of 1:5 and 1:10 were obtained using a cuvette of path length 0.1 cm, at a fixed HSA concentration of 4 μM. Measurements were taken in triplicate at a scan speed of 50 nm minhttps://www.rcsb.org/structure/2BXF (accessed on 18 April 2019)) [A computational check of the possible interaction between AML and HSA was performed using MD, and using “Glide”  as previously described . The cryl 2019))  and pre-l 2019)) . After al 2019)) , the prol 2019))  with 0.3https://pubchem.ncbi.nlm.nih.gov/compound/16230 (accessed on 18 April 2019))) and prepared using “LigPrep”  before conducting MD. The ionization states of AML in the pH range 7.0 ± 2.0 were generated by “Epik” , and, subsequently, the ligand was desalted [The structure of AML (CID: 16230) was downloaded from the PubChem database  with periodic boundary conditions were used to check the stability of the HSA–AML complex . The simSVK) and binding constant (bK) at elevated temperatures suggested that the HSA–AML complex exhibits static quenching.In this study, we conducted a comprehensive investigation on the binding of AML to HSA. A variety of techniques were employed, including spectroscopy, thermodynamics, MDS, and MD, to characterize the interaction between the two. This study aimed to identify the binding site on HSA responsible for its interaction with AML and elucidate the mechanism responsible for HSA–AML complex formation. Our findings demonstrate that AML efficiently quenches the fluorescence of HSA by forming a 1:1 stoichiometric complex. The reduction in quenching (Furthermore, thermodynamic analysis of fluorescence-quenching experiments at varying temperatures showed that HSA–AML complex formation is spontaneous and driven by entropy changes. MD showed AML specifically binds to HSA subdomain IIIA, also known as Sudlow’s site I, through hydrogen-bonding and hydrophobic interactions. FRET analysis provided further evidence of this binding by confirming the proximity of AML and Trp214, which also aligns with the observed quenching effect of HSA. Overall, our findings strongly support efficient binding between AML and HSA and suggest that HSA plays a crucial role in the systemic transport of AML via the plasma. This study provides valuable insights into the binding mechanisms of AML to HSA, enhancing our understanding of the behavior of AML during transport and distribution in vivo and should aid the development of appropriate AML delivery systems.


Eimeria stiedae. It may be fatal for rabbits and leads to important economic losses. The disease is well described in domestic rabbits, but little is known about infection with E. stiedae in wild rabbits. In this study, we investigated (a) whether wild rabbits from the island of Lemnos, Greece, where this species is overpopulated, are infected with E. stiedae and (b) what the effects are of this infection on their liver function. Based on our findings, 13.3% of the wild rabbits included in this study were infected with E. stiedae. Moreover, we observed several alterations in the liver variables in infected individuals compared to the non-infected ones, which were indicative of the negative effects of E. stiedae infection on the liver function of wild rabbits. This study adds to the current knowledge on the pathogens affecting wild rabbits and those circulating in this population on the island of Lemnos, Greece, and shows that E. stiedae infection impairs liver function in wild rabbits. Hepatic coccidiosis is a severe rabbit parasitic disease caused by Eimeria stiedae, is a devastating disease with high morbidity and mortality rates. The disease is well described in rabbits, but little is known about E. stiedae infection in wild rabbits. In this study, we investigated the presence of E. stiedae infection in wild rabbits from the island of Lemnos, Greece, where this species is overpopulated, and the effects of infection on common hepatic biomarkers. (2) Methods: We used liver impression smears to detect the coccidian oocysts, and we defined the liver biochemical profile of the infected individuals. (3) Results: Overall, 13.3% of the liver imprints examined were positive for the presence of coccidial oocysts. The activities of liver enzymes, that is, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and glutamyltransferase (GGT), as well as globulins (GLOB), were increased while the concentrations of albumins (ALB), total proteins (TP) and the albumin to globulin (A/G) ratio were decreased in the infected individuals compared to the non-infected ones. (4) Conclusions: This study adds to the current knowledge on the pathogens affecting wild rabbits and those circulating in this population on the island of Lemnos, Greece. Moreover, we showed that E. stiedae infection exerts pathological effects on the hepatocyte integrity and liver function of wild rabbits, as reflected by the abnormal values of liver injury and dysfunction biomarkers.(1) Background: Rabbit hepatic coccidiosis, caused by  Eimeria Schneider, 1875. Eleven coccidian species infected domestic and wild rabbits but only a few of them were implicated in clinical disease. Two types of coccidiosis were described: the intestinal form—caused by the coccidian species classified as non-pathogenic (Eimeria coecicola), slightly pathogenic , mildly pathogenic or pathogenic , and highly pathogenic , and the hepatic form caused by E. stiedae. The latter species has the highest pathogenicity and causes the most severe form of disease [Coccidiosis is a highly contagious parasitic disease caused by monoxenous coccidia of the genus  disease . Hepatic coccidiosis is a devastating disease with high morbidity and mortality rates that results in important economic losses in rabbit farms ,4. YoungE. stiedae occurs through the fecal-oral route [The transmission of al route . Once inal route . The paral route . The bilal route .E. stiedae may predispose infected animals to other diseases [Experimental studies showed that rabbits developed clinical signs at 20 days post-infection. The clinical manifestations include anorexia, diarrhea, lethargy, rough and dull hair and glazed eyes. The disease results in poor weight gain, liver enlargement, ascites, icterus, distended abdomen and finally, death. On top of the premature loss of rabbits, diseases ,8,9. The post-mortem findings confirmed that hepatic eimeriosis significantly disturbed the liver function: hepatomegaly, multifocal yellowish nodules diffusely spread over the liver surface and in the parenchyma, and considerably dilated bile ducts, biliary hyperplasia and enlarged gallbladder were commonly reported ,10,11,12Liver damage and/or dysfunction were further reflected in the alterations of the blood and biochemical variables, as shown previously in experimental studies. A significant increase in the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutamyltransferase (GGT) and lactate dehydrogenase (LDH) activities were recorded while albumin levels, alkaline phosphatase (ALP) activity and cholesterol levels appeared to decrease ,8,14,15.E. stiedae oocysts in faeces using faecal flotation [E. stiedae have a characteristic extra membrane compared to other coccidian species, called a veil, which, however, can only be visualized using electron microscopy [E. stiedae DNA using PCR in liver samples, as early as 12 days post-infection, has been shown previously in an experimental study [The routine diagnosis of coccidiosis is based on clinical manifestations and the detection of lotation . Oocystscroscopy . Other lcroscopy ,19,20,21al study ,8. In pral study . The impression of smear examinations following necropsy is a traditional method for the diagnosis of hepatic coccidiosis that presents several advantages; it is simple and quick, and usually, the smears are of high cellularity and good diagnostic quality. The liver samples contain high numbers of coccidial organisms in various stages of development, from early gametogonous stages to fully formed oocysts ,22. Eimeria spp. have been found to infect wild rabbits [E. stiedae was the causative agent of the majority of white-spotted liver lesions in wild rabbits [E. stiedae infection in wild rabbits and its pathological effects. In this study, we investigated the presence of E. stiedae infection in a population of wild rabbits (Oryctolagus cuniculus) from the island of Lemnos, Greece, where this species is overpopulated, using liver impression smears to detect the coccidian oocysts. Moreover, we defined the biochemical profile of the infected individuals based on the analysis of liver biomarkers and their alterations in hepatic coccidiosis in wild rabbits. Several  rabbits ,24. In a rabbits . Althoug rabbits , little Hunting harvested wild rabbits were sampled during the hunting season 2019–2020 on the island of Lemnos, which was set by the competent authorities . Hunting on this island takes place usually as a population control measure to prevent crop damage in compliance with Greek Legislation  . The autLeishmania infantum, Toxoplasma gondii, Anaplasma phagocytophilum and Babesia caballi. For the purpose of a previous study, blood samples were taken from the heart of wild rabbits within 3 h after their death, and sera had been recovered after blood clotting and consequent centrifugation. A total of 60 sera samples were selected from these samples using the following criteria: (a) the absence of hemolysis, (b) an adequate volume for biochemical analyses, and (c) a negative result for the presence of antibodies against each pathogen tested in the previous study namely The serum total protein (TP) concentration was immediately measured using a temperature-compensated refractometer  . AlbuminThe liver tissue from each animal was imprinted on a slide, fixed in methanol, and Hemacolor stained. A microscopical examination of stained imprints was used to reveal the presence of coccidia.p ≤ 0.05 was considered significant in all comparisons. To maximize the statistical information provided, a raincloud plot was created by combining a cloud of points with a box plot and a one-sided violin plot [Data were analyzed using the statistical program JASP 16.1. The normality of data distribution was assessed with the Shapiro–Wilk test; depending on the normality results, the Student t-test or Mann–Whitney test was run to determine the significance of the differences between groups for each variable evaluated. A value of lin plot . Coccidia was retrieved in 8 out of the 60 liver imprints (13.3%)  in high The mean values for ALT, AST and GGT activities, the TP, ALB and GLOB concentrations, and the A/G ratio for Group A and Group B are shown in In Group A, the ALT activities ranged from 21 to 83 U/L and exceeded the upper reference limit for domestic rabbits in only one case . In Group B, they ranged from 16 to 49 U/L and did not exceed the reference interval in all cases . MoreoveRegarding the AST activities in Group A, they ranged widely from 52 to 143 U/L and exceeded the reference values in 75% (6/8) of the wild rabbits. On the contrary, in Group B, none of the animals had AST activities above the upper reference limit (range 14–55 U/L) . A signiThe GGT activities were above the reference values in 100% (8/8) of the animals in Group A, ranging from 11 to 40 U/L. Similarly, the animals in Group B had GGT activities ranging from 7 to 19 U/L, which also exceeded the reference values but presented a much narrower distribution, as it is depicted in As for TP concentrations, they were within the reference intervals for all the animals in Group A except for the two cases with higher values (range 6.0–7.7 g/dL). In Group B, none of the animals had TP concentrations below the reference values, but 67.3% had values greater than 7.2 g/dL  . MoreoveThe albumin concentrations were below the lower reference limit in 50% of the animals in Group A, ranging from 2.1 to 3.1 g/dL. In Group B, 98% of the individuals had ALB values within the reference interval (3.4–5.0 g/dL), with only one case (1/52) experiencing an elevated ALB concentration . The meaThe GLOB concentrations were above the upper reference limit in all cases in Group A and in the majority (88%) of cases in Group B . The meaThe A/G ratio was below one in all Group A wild rabbits and above one in all Group B individuals . MoreoveE. stiedae. We also defined the biochemical profile of the infected individuals based on the analysis of specific variables that reflect liver injury and/or function.In this study, we showed that wild rabbits on the island of Lemnos, Greece, are naturally infected with E. stiedae oocysts. In addition, the different rabbit intestinal coccidian species parasitize distinct parts of the intestine and different parts of the mucosa, while E. stiedae is the only Eimeria sp that localizes the epithelium of biliary ducts in the liver [Overall, 13.3% (8/60) of the liver imprints examined were positive for the presence of coccidial oocysts. Although not identified at a species level using a specific diagnostic tool, based on the current knowledge, we expect that they were he liver .E. stiedae was detected in 24% of examined wild rabbits [Eimeria spp. were identified in wild rabbit feces with prevalence ranging from 7.4% for E. coecicola to 84.2% for E. perforans while the prevalence for E. stiedae was 44.7% [Eimeria spp. in wild rabbits, with prevalence ranging from 0 to 100% for different species and locations; the prevalence of E. stiedae ranged from 4 to 21% in adult individuals [Eimeria spp. by floatation technique and six Eimeria species, including E. perforans (18.3%), E. magna (16.9%), E. media (14.1), E. irresidua (11.2%), E. flavescens (4.2%), and E. coecicola (2.8%) were identified [Our results are in agreement with the limited number of studies that have been conducted in wild rabbit populations to date. In the UK,  rabbits . In Austas 44.7% . In a 20ividuals . Anotherividuals . Researcentified .Eimeria spp. in pet rabbits worldwide. The detection rates were constantly high in the different studies, i.e., 41.9–56.4% in China [Eimeria spp., Passalurus ambiguus and Cyniclomyces guttulatus [Eimeria spp. detection in other animal species: poultry (85.7%) [A larger number of studies reported the detection of in China ,35, 65% in China , 100% ofin China , 21.2% iin China , 28–67% in China ,39, 70.3in China , 47.6% iin China . Recentlttulatus . Previou (85.7%) , small r (85.7%)  and fish (85.7%) .Eimeria oocysts in the liver of wild rabbits; this technique is easy, time-saving, and it does not require specific equipment and trained personnel. Researchers in previous studies have successfully used the impression smear method for the diagnosis of hepatic coccidiosis following post-mortem examination and showed its utility and reliability for the detection of E. stiedae oocysts [In the majority of the above-mentioned studies in domestic and wild rabbits or other animal species, the diagnostic tools used were fecal examination with flotation techniques and/or molecular methods. In this study, we used liver impression smears which were Hemacolor stained, for the detection of  oocysts ,22.E. stiedae infection on liver function. For instance, the experimental study of Jing et al. showed that ALT, AST and GGT activities increased significantly and were indicative of the liver injury caused by E. stiedae infection and the parasite reproduction in liver parenchymal cells. In particular, the AST levels were seven-fold compared to the non-infected individuals [We also defined the serum biochemical profile of naturally infected wild rabbits based on the analysis of variables that reflected liver damage and dysfunction; the liver enzyme activities, that is, ALT, AST and GGT, were increased while the concentrations of ALB, GLOB and TP were decreased in the infected individuals compared to the non-infected ones. Statistically significant differences were found for all the above-mentioned biochemical variables between the infected and non-infected animals. Our findings are well conformed to the observations of previous experimental studies in domestic rabbits ,8,14,15 ividuals . Howeverividuals  and the The increased activity of ALT, an enzyme that is primarily found in the cytosol of hepatocytes, is an important biomarker for liver damage due to hepatocyte injury and inflammation in humans, dogs and cats. In rabbits, though, similar to other herbivores, ALT activity is of lesser value due to little tissue specificity and a shorter half-life. However, a correlation between the degree of liver necrosis to serum ALT activity has been shown for rabbits . Raised Regarding AST activity, this enzyme was widely distributed in many tissues in rabbits, including the cardiac tissue and muscle, as well as the liver—and it has a short half-life as well. Similarly to ALT, elevated AST levels may be found in rabbits diagnosed with liver damage ,49. In tThe activity of GGT is generally low in rabbits. The elevated GGT activities in this species are associated with obstructive lesions of the bile ducts, and it is considered an important enzyme with which to estimate liver disease . SimilarE. stiedae, along with their combined effect on the A/G ratio [TP and albumin concentrations were decreased in infected individuals, while the globulin concentrations were increased . The obs/G ratio . Regardi/G ratio .Our findings clearly support that, in terms of the comparison between infected and non-infected wild rabbits, the liver enzymes activities, that is, ALT, AST and GGT, were increased while the concentrations of ALB, GLOB and TP were decreased in the infected individuals compared to the non-infected ones. However, the clinical interpretation of biochemical parameters based on the reference intervals for domestic rabbits was quite challenging and inconclusive for hepatic coccidiosis in wild rabbits. This observation could be attributed to the validity of the reference intervals. Foremost, the reference intervals used in this study were for domestic rabbits because no reference interval studies have been conducted up to now for wild rabbits. In addition, by definition, the reference intervals do not reflect the whole population, and it is expected that 5% of all results from “healthy” individuals fall outside the reference intervals . MoreoveE. stiedae infection which stood at the top of a prioritized differential list.Except for hepatic coccidiosis, elevations in liver enzymes and hypoproteinemia are expected in cases of hepatic damage, necrosis, biliary obstruction and cholestasis . When liIn Greece, the geographical distribution of the wild rabbit population is limited to the island ecosystems, while this species is absent from the mainland. Over the last two decades, the wild rabbit has overpopulated Lemnos Island, causing devastating effects on the rural economy and is considered a threat to biodiversity, ecosystems, and crops on the island ,57. ConsL. infantum [L. infantum, T. gondii, A. phagocytophilum and B. caballi, respectively [E. stiedae infection exerts pathological effects on the liver function of wild rabbits, which are reflected in the liver variables.To the best of our knowledge, only two studies have been conducted up to this point to investigate the exposure of this population to important pathogens. The 2019 study by Tsakmakidis et al. showed that 1% of the wild rabbits sampled during two hunting seasons were exposed to infantum . Later, ectively . This st


The initiation and amplification of endothelial cell IP3R-mediated Ca2+ signals requires an intact microtubule cytoskeleton. Since tau accumulation in endothelial cells disrupts native microtubule stability, we reasoned that tau-induced microtubule destabilization would impair endothelial IP3-evoked Ca2+ signaling. We tested the hypothesis that tau disrupts the regulation of local cerebral blood flow by reducing endothelial cell Ca2+ signals and endothelial-dependent vasodilation. We used a pathogenic soluble tau peptide (T-peptide) model of tau aggregation and mice with genetically encoded endothelial Ca2+ sensors to measure cerebrovascular endothelial responses to tau exposure. T-peptide significantly attenuated endothelial Ca2+ activity and cortical capillary blood flow in vivo within 120 seconds. Further, T-peptide application constricted pressurized cerebral arteries and inhibited endothelium-dependent vasodilation. This study demonstrates that pathogenic tau alters cerebrovascular function through direct attenuation of endothelial Ca2+ signaling and endothelium-dependent vasodilation.The accumulation of the microtubule-associated tau protein in and around blood vessels contributes to brain microvascular dysfunction through mechanisms that are incompletely understood. Delivery of nutrients to active neurons in the brain relies on capillary inositol 1,4,5-triphosphate receptor (IP Tau disrupts cerebral blood flow regulation, but mechanisms are incompletely understood 7. The precise and efficient delivery of nutrients to active neurons in the brain relies on the intricate network of capillaries in which local inositol 1,4,5-triphosphate IP3 receptor (IP3R)–mediated calcium (Ca2+) signals through nitric oxide production direct blood flow 8. These events range from small, brief proto-events, which represent the release of Ca2+ through a few IP3Rs channels, to larger, sustained compound events lasting up to approximately one minute, involving clusters of channels 8. As a result, intracellular Ca2+ activates nitric oxide synthesis, which enhances local blood flow to active neurons.Impaired cerebral blood flow plays a crucial role in the development of degenerative neurological diseases such as Alzheimer’s disease (AD) and Chronic Traumatic Encephalopathy (CTE). Both AD and CTE are characterized by accumulation of hyperphosphorylated forms of the microtubule-associated protein tau around blood vessels 7. Endothelial cell internalization of soluble tau triggers phosphorylation of endogenous tau at threonine 231, which promotes destabilization of microtubules and reduces microtubule density 7. This has important implications, because microtubule function is required for IP3R clustering and Ca2+ signaling in endothelial cells. Prior work has shown that perturbation of the microtubule cytoskeleton by the drugs colchicine and colcemid prevents the initiation of IP3-evoked Ca2+ signals in endothelial cells 10. Furthermore, disruption of microtubules with nocodazole impairs the microtubule-facilitated motility of IP3Rs, preventing the localization of the channel which is needed for efficient and graded recruitment of channels with increasing stimulus strength 11. Microtubules also have direct interactions with the consensus motif for EB binding (TxIP) of IP3Rs through the microtubule end-binding protein 3 (EB3); depletion or deletion of EB3, or mutation of the TxIP motif on IP3Rs, prevents clustering and functional coupling between IP3Rs 12. Since tau accumulation in endothelial cells disrupts native microtubule stability, we reasoned that tau-induced endothelial microtubule destabilization would impair endothelial IP3-evoked Ca2+ signaling.It was recently demonstrated that brain microvascular endothelial cells internalize soluble tau oligomers in pathological conditions through a process that depends on heparan sulfate proteoglycans 2+ signals and disrupting endothelium-dependent vasodilation. We used a soluble pathogenic tau peptide (T-peptide) model of tau aggregation. This cell-permeable hexameric peptide derived from the third microtubule-binding repeat of tau self-assembles in vitro into paired helical filament-like aggregates providing an established paradigm for studying mechanisms underlying tauopathy 14. To isolate and measure Ca2+ activity from endothelial cells, we studied mice with endothelial-specific expression of the Ca2+ biosensor GCamP8 driven by the cadherin 5 promotor (GCaMP8-Cdh5 mice) 8. We utilized two-photon laser scanning microscopy via a cranial window to record microvascular endothelial Ca2+ signals in vivo; the addition of intravenous FITC-dextran allowed measurement of regional cerebral blood flow. We also isolated small cerebral arteries to study direct vascular effects of T-peptide with pressure myography. Together, these experiments demonstrate that T-peptide has rapid and direct effects on brain microvascular endothelium, reducing Ca2+ signaling and impairing the regional regulation of cerebral blood flow.Here, we tested the hypothesis that tau aggregates impair the regulation of regional cerebral blood flow by acutely reducing endothelial cell CaCdh5-GCaMP8 mice  were used for in vivo Ca2+ imaging studies. Mice were kept on a 12 h light/dark cycle, with ad libitum access to food and water and were housed in groups of five. All studies were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals , Guidelines for Survival Rodent Surgery  and approved by the Institutional Animal Care and Use Committee of the University of Vermont. All procedures were conducted and reported in accordance with the ARRIVE guidelines  (https://www.nc3rs.org.uk/arrive-guidelines).Male C57BL/6j mice , aged 8–12 weeks, were assigned at random to either experimental or control treatment groups. 2, 2 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 4 glucose (pH 7.4). A pipette was introduced into the cortex, maneuvered adjacent to the capillary under study and pressurized injections  of 100 nM T-peptide  with tetramethylrhodamine isothiocyanate -labeled dextran were ejected directly onto the capillary. RBC flux data was collected by line scanning the capillary of interest at 15 kHz. Single-plane imaging experiments involved imaging a 425 μm × 425 μm field at ~2.5 Hz for 360 s. Images were acquired through a Zeiss 20x Plan Apochromat 1.0 NA DIC VIS-IR water-immersion objective mounted on a Zeiss LSM-7 multiphoton microscope  coupled to a Coherent Chameleon Vision II Titanium-Sapphire pulsed infrared laser . Cdh5-GCaMP8 and TRITC-dextran were excited at 920 nm, and emitted fluorescence was separated through 500- to 550- and 570- to 610-nm bandpass filters, respectively.Mice were anesthetized with isoflurane . The skull was exposed, a stainless-steel head plate was attached, and a small circular cranial window was created over the somatosensory cortex. Approximately 150 μL of a 3-mg/mL solution of FITC or TRITC-dextran (MW 150 kDa) in sterile saline was injected intravenously into the retro-orbital sinus to allow visualization of the cerebral vasculature and contrast imaging of red blood cells (RBCs). Upon conclusion of surgery, isoflurane anesthesia was replaced with α-chloralose (50 mg/kg) and urethane (750 mg/kg). Body temperature was maintained at 37°C throughout the experiment using an electric heating pad. The brain was continuously perfused with artificial cerebrospinal fluid (aCSF) containing (in mM): 124 NaCl, 3 KCl, 2 CaCl15. Briefly, mice were euthanized and a full craniectomy was performed to expose the brain, which was then dissected and placed into cold (4°C) HEPES-buffered physiological saline solution (HEPES-PSS) with the following composition (in mM): 134 NaCl, 6 KCl, 2 mM CaCl2, 1 MgCl2, and 7 glucose (pH 7.4). The posterior cerebral artery (PCA) was then excised and cannulated in a pressure myograph chamber  as previously described.15 Briefly, arteries were maintained in aCSF solution at 37° C and allowed to equilibrate for 10 minutes at 10 mm Hg before experimentation. Intraluminal pressure was controlled using a pressure servo system  and blood vessel diameters were continuously recorded using edge-detection software . Intraluminal pressure was increased to 80 mm Hg and vessels were allowed to develop spontaneous myogenic tone. Endothelial viability was tested using 1 μM NS309  and a dilatory response > 85 % was considered “intact.” Experimental interventions were then carried out using T-peptide , bradykinin , NS309 (1 μM), or spermine NONOate . The bath solution was then exchanged with 0-Ca2+ saline solution with 100 μM diltiazem  to elicit maximal passive diameter. The percentage change in diameter to pharmacological intervention was measured at the point of peak dilation or constriction and calculated as: change in diameter (%) = [(diameter after drug administration – baseline diameter)/(passive diameter – baseline diameter) × 100].Cerebral vessels were harvested and pressurized in a myograph system as previously described 0 methods and accurately characterize and analyze Ca2+ events in brain capillaries, we used our custom autonomous image analysis program ‘VolumetryG9’ 8. This method uses signal-to-noise ratios and statistical z-scores (Zscrs) combined with spatiotemporal (ST) mapping to accurately quantify the full dimensional characteristics of Ca2+ events, combining amplitude (Zscr), size (μm) and duration (s) as the conglomerate variable ZUMS (Zscr·μm·second) (8). Each animal or vessel served as its own control and experiments were analyzed in a pairwise fashion; therefore anonymizing was not feasible. Sample sizes for all experimental preparations were estimated using our previous experience 16.GraphPad Prism software  was used for X–Y graphing and analysis; values are presented as means ± standard deviation (SD). Data were tested for normality and appropriate parametric or non-parametric statistical tests were subsequently applied. Differences were considered significant if P <0.05. RBC flux was analyzed offline using custom software . Flux data were binned at 1-second intervals. Mean baseline flux data for summary figures were obtained by averaging the baseline (6 second) for each measurement before pressure ejection of 100 nM T-peptide. The peak response was defined as the peak 1-s flux bin after delivery of T-peptide within the remaining scanning (240–300 seconds). The depth of capillaries below the surface was estimated from z-stack series acquired before pipette placement. To avoid limitations of standard F/F3R)–mediated Ca2+ signals. Since tau accumulation in microvascular brain endothelial cells disrupts the native microtubule function 7, and these microtubules are needed for initiation and amplification of the IP3R-mediated Ca2+ signals, we postulated that tau exposure would lead to a rapid reduction in endothelial Ca2+ signaling. To test this hypothesis, we leveraged our recently developed Cdh5-GCaMP8 transgenic mice that express the Ca2+ indicator, GCaMP8, under the transcriptional control of the Cdh5 (cadherin 5) promoter for targeted expression in vascular endothelial cells 8. For these experiments, we injected TRITC-dextran and used multi-photon microscopy to visualize brain capillaries through a cranial window over the somatosensory cortex. This provided a map of the cortical vasculature using 3-dimension z-stacks in a 425 × 425 μm field of view before and after low pressure ejection of T-peptide  on the cortical surface. We defined capillaries as vessels located between penetrating arterioles and ascending venule and assigned them branch orders as previously described 17. We then recorded the capillary endothelial cells (cECs) Ca2+ activity in layer II-III of the somatosensory cortex before and after cortical application of T-peptide (100 nM)  of T-peptide exposure, we observed a significant reduction in Ca2+ activity from baseline  . This indicates that T-peptide decreases the Ca2+ output generated from individual active sites without impacting the number of sites themselves. This observation is consistent with a mechanism in which T-peptide disrupts microtubule function needed for IP3R clustering and efficient intracellular Ca2+ release. T-peptide exposure also resulted in a decrease in regional blood flow in 3rd-4th order branches capillaries  , A–D. Un P<0.05) –D. Of noillaries –H. We ob2+-activated K+ channels, SKCa and IKCa, by NS309 (1 μM)   was unaffected by T-peptide application   application, vessels began to constriction. The endothelium-dependent vasodilator bradykinin was then used to test the effect of T-peptide on endogenous nitric oxide signaling. Bradykinin (10 μM) was administered before and 20 minutes after bath perfusion of T-peptide (100 nM). Time control experiments show that repeat application of bradykinin after 20 minutes produced a vasodilatory response that was not different from baseline . In contrast, after 20 minutes of T-peptide exposure, bradykinin-induced dilation was significantly attenuated  –B. T-pep8; n.s.) –D. These3, n.s.) –F. This 19. Application of tau through a cranial window impairs local blood flow, reducing the endothelium-dependent vasodilation caused by acetylcholine 6. Similarly, mice with tauopathy produced through expression of aggregation-prone P301S-mutant human tau also exhibit impaired endothelium-dependent vasodilation in vivo7. Administration of tau oligomer-specific monoclonal antibody (TOMA) rescued endothelium-dependent brain microvascular responses in this mouse model of tauopathy 7. Our results showing increased myogenic tone, impaired endothelium-dependent vasodilation, and reduced blood flow after t-peptide application are consistent with these prior reports. Together, these studies support a model in which tau protein accumulation in the brain leads to dysfunction of cerebral blood vessels, affecting their ability to regulate blood flow and maintain proper vascular tone. This dysfunction can impair blood flow to brain cells, limiting the delivery of oxygen and nutrients and the removal of waste products, thereby contributing to neurodegenerative processes.Tauopathy disrupts the targeted delivery of nutrients to active neurons in the brain through regional control of cerebral blood flow; this impairment in local functional hyperemia is an early manifestation of degenerative neurological diseases 6. In another study, it was reported that soluble pathogenic tau enters brain vascular endothelial cells and drives cellular senescence with impaired cerebrovascular function. Specifically, cultured brain microvascular endothelial cells were shown to internalize soluble tau aggregates 7. The misfolded forms of tau protein contained in these aggregates promoted microtubule destabilization in cultured cells, as indicated by reduced levels of acetyl-alpha-tubulin and overall microtubule density 7. This impaired the microtubule-dependent transport required for endothelial nitric oxide synthase (eNOS) translocation to the cell surface and subsequent nitric oxide production and induced cellular senescence. These competing models of tauopathy-induced cerebrovascular dysfunction are not mutually exclusive, however, cellular senescence would not explain the acute vasoconstriction or reduction in endothelium-dependent vasodilation we and other have observed within minutes of application of tau to brain microvessels. Instead, we propose the novel concept that tau-induced microtubule reorganization disrupts IP3R clustering, thereby diminishing the ability of endothelial cells to produce the Ca2+ signals needed for nitric oxide production and vasodilation. It has been well established that the microtubule cytoskeleton is needed for IP3-evoked Ca2+ signals in endothelial cells 10. Microtubules support the motility of IP3Rs, which is important because the diffusion and subsequent localization of the channel which is needed for efficient and graded recruitment of channels with increasing stimulus strength 11. Microtubules interact with the TxIP motif of IP3Rs through the microtubule end-binding protein 3 (EB3), which is critical for clustering and functional coupling between IP3Rs 12. Any effects on microtubule structure produced by soluble pathogenic tau aggregates would therefore be likely to influence IP3R-evoked Ca2+ signals. This model is supported by our results showing that when T-peptide was applied directly to the brain vasculature of mice, we observed a decrease in total cerebrovascular endothelial Ca2+ activity. This novel observation depended on the use of genetically encoded Ca2+ biosensor mice (Cdh5-GCaMP8) and also advanced spatiotemporal mapping techniques to precisely quantify the mass of individual Ca2+ transients in brain capillary endothelial cells. The resulting statistical z scores (zscr·μm·s) encompass the pixel intensity across the entire spatial area of an event over time. This provides reliable quantification of event amplitude for detection of even minute fluorescence changes with high reliability. 20 Of note, T-peptide decreased the Ca2+ output generated from individual active sites without reducing the number of active sites themselves, suggesting a mechanism by which T-peptide disrupts microtubule-dependent IP3R clustering needed to amplify local Ca2+ release events. This decrease in Ca2+ activity was associated with a significant reduction in local cerebral blood flow.Previously, the deranged cerebrovascular responses to tau were attributed to alterations in neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) function. In one study, it was shown that tau-induced dissociation of neuronal nitric oxide synthase (nNOS) from postsynaptic density 95 reduces nitric oxide production during glutamatergic synaptic activity, leading to failure of neurovascular coupling responses required for the regulation regional blood flow 5. Tau is extruded to the extracellular space from damaged neurons where it oligomerizes in a process accelerated by tau phosphorylation. Pathogenic tau oligomers can transfer across synaptic clefts in brain circuits, to promote phosphorylation, misfolding and aggregation of native tau in adjacent cells through a prion-like process. This prion-like transmission through which tau proteins adopt pathogenic self-propagating conformations characteristic of prions has been well documented 23. Interestingly, it has been shown that lung microvascular endothelial cells themselves can release extracellular tau into the blood stream during bacterial pneumonia, causing cerebral tau aggregation and cognitive deficits 25. Of note, while the T-peptide portion of tau we utilized includes the C-terminal repeat region that is known to be important in tau aggregation, recent reports have demonstrated that mutations in the N-terminal region also impact the ability of tau to form larger-order complexes 26. These findings warrant further effort to elucidate the potential vasoactive effects of tau protein in its full-length form, and also, in the presence and absence of post-translational modifications such as hyper-phosphorylation and acetylation that play a role in neurodegeneration 27.Our findings are important because in Alzheimer’s disease (AD), Chronic Traumatic Encephalopathy (CTE) and other degenerative neurological diseases, phosphorylated tau protein aggregates in cortical sulcus around small blood vessels 2+ signaling, causes vasoconstriction, impairs endothelial-dependent vasodilation, and disrupts local cerebral blood flow. These findings are significant because they support the strategy of protecting endothelial cells from tau to prevent the loss of cerebrovascular function in dementia.In conclusion, our results support a novel paradigm in which tau accumulation contributes to abnormal cerebrovascular function through a mechanism specifically involving endothelial cells. We demonstrated that T-peptide reduces endothelial Ca