Anatase nanoparticles synthesized by the sol-gel method were surface-functionalized with long alkyl chain coupling agents as compatibilizers for a nonpolar environment, containing different anchor groups for surface interaction namely phosphonate (dodecyl phosphonate), carboxylate (dodecanoic acid), sulfate (sodium dodecyl sulphate), and amine (dodecyl amine). It was shown that the surface of the nanoparticles can be functionalized with the various surface groups applying similar reaction conditions. The kind of surface interaction was analyzed applying FTIR spectroscopy. The phosphonate and the carboxylate groups interact with the surface via quite strong covalent or coordinative interactions, respectively. The sulfate and amine based coupling agents on the other hand exhibit electrostatic interactions. UV stability studies of the surface bound groups revealed different degradation mechanisms for the various functionalities and moreover showed that phosphonates are the most stable among the investigated surface capping groups. TitanThe modification of metal oxide nanoparticles with organic ligands, coupling agents and surfactants is also a hot topic these days . A largeIt is known that the anatase surface exhibits amphoteric properties –17. The 2 surface . Th. Th2 . For the particles modified with DDAmine, a signal at 1520 cm−1 is arising, which can be assigned to the NH3+ bending vibration [2 stretching signal is observed at 3374 cm−1 in the spectrum of DDAmine, the modified particles exhibit various broad signals between 3100 and 3500 cm−1 indicating the presence of ammonium species and the formation of hydrogen bonds [It is known that amines can adsorb to titania surfaces at Lewis-acid sites, via hydrogen-bonds, or electrostatic interaction ,16. The ine@TiO2  and 8 reen bonds . Hence, en bonds   [At pH 2 the highest surface coverage was obtained for SDS as observed from or TiO2) . However2 revealed one onset temperature at 300°C, which is assigned to the oxidation of the alkyl chain. The surface coverage was calculated from the mass loss between 200 and 700°C.Thermogravimetric analysis of DDA@TiOHighest surface coverage for DDA could be achieved by modification at pH 3 . The sur2 thermogravimetric analysis exhibited one onset temperature at 280°C. Owing to the weak electrostatic interaction detected for the amine, the mass loss might be ascribed to the desorption of DDAmine. However, the onset temperature is in the range of the alkyl chain oxidation temperature (compare with DDA and SDS). Hence, it is assumed that the alkyl chain is rather degraded than desorbed. The surface coverage was calculated from the mass loss between 200 and 700°C.For DDAmine@TiOHighest surface coverage was detected for particles modified at pH 2 . The cov2 revealed one onset temperature at 300°C, which can be assigned to the oxidation of the alkyl chain. The surface coverage calculated from the mass loss between 200 and 700°C was highest for particles modified at pH 3  were prepared and illuminated for 2 days in a water-cooled chamber equipped with two 9 W UVA black light lamps (Sylvana UVA BLB Lynx) as presented in It is assumed that the different interactions of the coupling agents with the titania surface can lead to unequal photodegradation mechanisms and kinetics. For this reason, anatase nanoparticles modified with DPA, DDA, SDS and DDAmine were illuminated under the same conditions. 0.2 wt% dispersions of DPA@TiO−1 disappeared in all cases. Exemplarily we also modified TiO2 synthesized with HCl as acid for the weakest bonding surface groups, namely SDS and the FT-IR spectra after modification were identical  can be taken as a measure for the amount of alkyl chains present on the particles’ surface. Thus, the absorbance intensity (A) change of this signal under UV illumination was tracked. Assuming 1st order kinetics the rate law is:The absorbance maximum at 2921 cmAfter integration the following equation is obtained:A0/A) is plotted versus time to determine the reaction rate constant k. The kinetic plots and reaction rate constants obtained for the different capping agents are presented in Thus, ln was decreasing while the broad signal at 1050 cm−1, which is assigned to the P–O–Ti bond, did not lose intensity during illumination. Hence, it is concluded that the P–O–Ti bond was stable against UV-irradiation, while the alkyl chain was degraded. It is assumed that a phosphate species was left on the particles’ surface after illumination. In fact, after prolonged illumination for 16 days 31P MAS NMR investigations revealed the presence of a phosphate species . Furthermore, the bonding mode did not change as no shifting of the carboxylate bands was observed. Unfortunately, 13C CPMAS NMR could not confirm this assumption, because such species cannot be distinguished from the chemisorbed dodecanoic acid. The UV-stability of the C–O–Ti bond confirmed the assumption that the interaction is a chemical one. An electrostatic interaction between COO− and a positively charged titania surface would be weakened upon illumination due to the extensive formation of radicals, which react with ions present on the surface of the particles. The fast degradation of DDA compared to DPA can be assigned to the effective charge transfer over the carboxy-group as found by Kaletas et al. [From the FT-IR spectra of DDA@TiO2  it can by effect ) were ovs et al. . Howevers et al. .2 the degradation was measured observing the CH signal at 1425 cm−1 as well as the OSO3− bands at 1248 and 1178 cm−1. The infrared spectra for various illumination times are presented in the 3− bands disappeared. Thus, the radicals formed upon illumination reacted with the ionic species present on the titania surface and the electrostatic interaction was broken.For SDS@TiO2 the illumination experiments were carried out with the sample modified at pH 1, which showed mainly electrostatic interaction. FT-IR spectra of DDAmine@TiO2 after different times of illumination   see  revealedcreasing . Thus, i3.3.1.et al. [Titanium isopropoxide (TIP), sodium dodecylsulphate (SDS), dodecanoic acid (DDA) and dodecylamine (DDAmine) were purchased from Sigma Aldrich and used as received. Dodecylphosphonic acid was prepared by a procedure according to Kosolapoff et al. ,56. Meth3.2.−1. The software used for analysis was OPUS™ version 4.0. For kinetic studies, a baseline correction was carried out. Thermogravimetric analyses were performed on a Netzsch Iris TG 209 C  with a 414 TASC controller in a platinum crucible with a heating rate of 10 K/min under synthetic air. High-resolution transmission electron microscopy (HR-TEM) micrographs were performed on a TECNAI F20 FEGTEM  at the University Services Centre for Transmission Electron Microscopy (USTEM), Vienna University of Technology. DLS measurements were performed with an ALV/CGS-3 compact goniometer system  controlled by an ALV/LSE-5003 Multiple Tau Digital Correlator (ALV-GmbH) at a scattering angle of 90° and a temperature of 25°C. The light source was a Uniphase cylindrical 22-mW HeNe-laser  operating at a wavelength of 632.8 nm with vertically polarized light. Elemental analysis was carried out at the Microanalytical Laboratory at the University of Vienna. NMR spectra in solution were recorded on a Bruker Advance 300 instrument  equipped with a 5 mm broadband probe head and a z-gradient unit. Solid state NMR spectra were recorded on a Bruker AVANCE DPX300 equipped with a 4 mm broadband MAS probe head . Spectra were acquired using magic angle spinning (MAS) and high power proton decoupling at a rotor spinning rate of 4–9 kHz. Nitrogen sorption measurements were carried out on a Micrometric ASAP 2000 or ASAP 2010 instrument . The samples were degassed for 5 h at 40°C prior to measurement. For the interpretation of the data, the Brunauer, Emmett and Teller (BET) model was applied. XRD experiments of powders were carried out on a Philips X’Pert diffractometer  equipped with an XCelerator multi-channel detector, Bragg Brentano geometry and a silicon single crystal sample holder. The diffraction pattern was recorded between 5° and 90° (2θ) with 1 s/step and a step size of 0.02°.Infrared spectra were recorded on a Bruker Tensor 27 instrument  either working in ATR Micro Focusing MVP-QL with a diamond crystal or in transmission mode using KBr pellets as a sample matrix. Spectra were recorded with 32 scans at a resolution of 4 cm3.3.3.3.1.et al. [iPr)4 were dissolved in 50 mL absolute ethanol in a dropping funnel and added drop-wise to acidified water under ice-cooling and vigorous stirring. The mixture was stirred overnight. The solvent was removed under reduced pressure at 40°C and the particles were dried under vacuum to collect a white powder. 0.6 g (94%) of titania nanoparticles were obtained.Titania nanoparticles were synthesized by slight modification of a procedure by Choi et al. . 500 mL −1): 3244, 1638, 1413, 1310, 795. TGA : 30–400°C: 15.3%, 400–700°C: 0.5%. BET: 144 m2/g.DLS: 5 nm ± 0.2 nm. TEM: 5 nm. XRD: Anatase 100%, a (Å) = 3.79959, c (Å) = 9.4778; crystallite size: 3.9 nm. FT-IR  SDS were dissolved in a mixture of 5 mL water and 5 mL ethanol. This solution was added drop-wise to the dispersion of titania nanoparticles under vigorous stirring. The corresponding pH was adjusted using a 1 M solution of HNO−1 (νas −CH3); 2922 cm−1 (νas −CH2−); 2872 cm−1 (νs −CH3); 2852cm−1 (νs CH2−); 1248 cm−1 (νas OSO3−), 1178 cm−1 (νas OSO3−); 1064 cm−1 (νs OSO3−), below 800 cm−1 (ν Ti−O).FTIR: 2951 cm3), mass loss (220°C to 650°C): pH 1: 15.00%; pH 2: 14.45%; pH 3: 14.05%; pH 4: 13.80%; pH 5: 8.58%.TGA (HNOTGA (HCl), mass loss (220°C to 650°C): pH 1: 15.95%; pH 2: 15.06%; pH 3: 14.15%; pH 4: 13.84%; pH 5: 8.60%.To investigate the effect of concentration of SDS on the surface coverage, a set of experiments was conducted at pH 2, adjusted using a 1 M solution of HNO3. For 0.1 g of titania nanoparticles 0.13 g (0.5 mmol) of SDS were used for Sample-1, 0.1 g (0.375 mmol) for Sample-2, 0.067 g (0.25 mmol) for Sample-3 and 0.033 g (0.125mmoles) of SDS were used for Sample-4.TGA, mass losses (220°C to 650°C): Sample-1: 21%; Sample-2: 20%; Sample-3: 15%; Sample-4: 12%.3.3.3.3 or NaOH.0.1 g of titanium dioxide nanoparticles were dispersed in 30 mL of water in a 100 mL round bottom flask. 0.1 g (0.5 mmol) of DDA were dissolved in 5 mL of ethanol prior to its drop-wise addition in titania suspension. The functionalization experiments were performed at pH 1, 2, 3, 4 and 5. The pH was adjusted using a 1 M solution of HNOThe suspension was treated in an ultrasonic bath for one hour and stirred at room temperature for two days. The particles were separated by centrifugation, washed several times with ethanol and water and dried under reduced pressure at room temperature.−1 (νas −CH3); 2922 cm−1 (νas −CH2−); 2872 cm−1 (νs −CH3); 2852cm−1 (νs −CH2−); 1521 cm−1 (νas COO); 1412 cm−1 (νs COO); below 800 cm−1 (ν Ti−O).FTIR: 2951 cmTGA, mass loss (220°C to 550°C): pH 1: 19%; pH 2: 20%; pH 3: 21%; pH 4: 12%; pH 5: 11%.Four samples of titania functionalized with different amounts of DDA were prepared at pH 3 to investigate the effect of concentration of DDA on the surface coverage. For each experiment, 0.1 g of titania nanoparticles were dispersed in 30 mL of water. 0.1 g (0.50 mmol) of DDA was used for Sample-1; 0.075 g (0.375 mmol) for Sample-2; 0.05 g (0.25 mmol) for Sample-3 and 0.025 g (0.125 mmol) of DDA were used for Sample-4.TGA, mass loss (220°C to 550°C): Sample-1: 19%; Sample-2: 18%; Sample-3: 14%; Sample-4: 12%.3.3.4.3 or NaOH. The suspension was treated in an ultrasonic bath for one hour and subsequently stirred at room temperature for two days. The particles were separated by centrifugation and washed repeatedly with ethanol and dichloromethane. The samples were dried under vacuum overnight at room temperature.0.1 g of titania nanoparticles were dispersed in 30 mL deionized water in a 100 mL round bottom flask. 0.093 g (0.5 mmol) of DDAmine were dissolved in 5 mL of ethanol and added drop-wise to the titania suspension. The functionalization experiments were performed at pH 1, 2, 3, 4 and 5. The pH was adjusted using 1 M solution of HNO−1 (νas −CH3); 2922 cm−1 (νas −CH2−); 2872 cm−1 (νs −CH3); 2852cm−1 (νs CH2−); 1610 cm−1 (H2O); 1510 cm−1 (νasNH2); 1600 cm−1 (νsNH2); below 800 cm−1 (ν Ti−O).FTIR: 2951 cmTGA, mass loss (220°C to 550°C): pH 1: 15%; pH 2: 16%; pH 3: 15%; pH 4: 14%; pH 5: 10%.Elemental Analysis: pH 1: 11.10% C, 2.5% H, 0.95% N; pH 2: 12.13% C, 2.91% H, 1.18% N; pH 3: 11.52% C, 2.32% H, 0.89% N; pH 4: 10.6% C, 2.40% H, 1.09% N; pH 5: 7.33% C, 1.81% H, 0.81% N.Four samples of anatase nanoparticles functionalized with different amounts of DDAmine were prepared at pH 2 to investigate the effect of concentration of DDAmine on the surface coverage. For each experiment, 0.1 g of titania nanoparticles were dispersed in 30 mL of water. 0.093 g (0.50 mmol) of DDAmine were used for Sample-1; 0.070 g (0.375 mmol) for Sample-2; 0.046 g (0.25 mmol) for Sample-3 and 0.023 g (0.125 mmol) of DDAmine were used for Sample-4.TGA, mass loss (220°C to 550°C): Sample-1: 16%; Sample-2: 15%; Sample-3: 14%; Sample-4: 13%.3.3.5.3 or NaOH. The particles were separated by centrifugation and washed repeatedly with distilled water and methanol. The samples were dried under reduced pressure at room temperature.0.1 g of titania nanoparticles were suspended in 30 mL water in a single neck round bottom flask. 0.125 g (0.5 mmol) of DPA were dissolved in 5 mL of ethanol and added drop-wise to the suspension. The functionalization experiments were performed at pH 1, 2, 3, 4 and pH 5. The pH was adjusted using 1 M solution of HNO−1(νas −CH3); 2922 cm−1 (νas −CH2−); 2872 cm−1 (νs −CH3); 2852cm−1 (νs CH2−); 1200 to 1000 cm−1 (νas P−O−Ti).FTIR: 2951 cmTGA, mass loss (220°C to 650°C): pH 1: 16%; pH 2: 16%; pH 3: 17%; pH 4: 15%; pH 5: 15%.Elemental Analysis: pH 1: 9.28% C, 2.5% H, 0.85% P; pH 2: 11.13% C, 2.85% H, 1.03% P; pH 3: 11.58% C, 2.91% H, 1.18% P; pH 4: 9.6% C, 2.32% H, 1.08% P; pH 5: 8.33% C, 2.01% H, 0.9% P.Four samples of titania functionalized with different amounts of DPA were prepared at pH 3 to investigate the effect of concentration of DPA on the surface coverage. For each experiment, 0.1 g of titania nanoparticles were dispersed in 30 mL water. 0.125 g (0.50 mmol) of DPA was used for Sample-1; 0.093 g (0.375 mmol) for Sample-2; 0.0625 g (0.25 mmol) for Sample-3 and 0.312 g (0.125 mmol) of DPA was used for Sample-4.TGA, mass loss (220°C to 650°C): Sample-1: 23%; Sample-2: 21%; Sample-3: 17%; Sample-4: 15%.3.3.6.2, DDA@TiO2, SDS@TiO2 and DDAmine @ TiO2 in mixtures of ethanol and water (4:1) were prepared and illuminated for 2 days in a water-cooled chamber equipped with two 9 W UVA black light lamps (Sylvana UVA BLB Lynx). Samples were withdrawn after 2, 4, 8, 24 and 48 h. The particles were centrifuged, washed with ethanol and dried under reduced pressure. The samples were characterized by KBr-IR. 2 mg ± 0.03 of the samples were homogenized and pressed with 150 mg ± 2 of KBr.0.2 wt% dispersions of DPA@TiO4.Anatase titania nanoparticles with a diameter of around 5 nm were functionalized with SDS, DDA, DDAmine and DPA in acidic pH conditions. It was confirmed that SDS and DDAmine interact electrostatically with the surface of titania whereas DDA bonds covalently to the titania surface through bidentate chelating mode and DPA bond covalently to the titania in a tridentate fashion. It has been shown that an optimal pH range exists for the modification with all studied capping molecules. The existence of such optimum is assigned to the dependence of the surface charge of the particles on the pH. For the interaction with acidic and anionic species, a highly positive surface charge is favorable. For interaction with the basic amine, the presence of nitrates on the particles’ surface, which is true at moderately low pH, increases the interaction. At highly acidic conditions, however, the acidic compounds exhibit lower tendency for dissociation, leading to inhibition of the interaction with the particles. For amines on the other hand, low surface coverage at low pH is assigned to the highly positive charged surface.2 and phosphate or carboxylate is stable under UV irradiation. Coupling agents which are adsorbed by electrostatic interaction on the other hand are desorbed from the TiO2 surface owing to the extensive formation of radicals which can combine with the ionic species present on the surface. Moreover, it was found that among the investigated coupling agents phosphonate exhibits the highest stability under photocatalytic conditions.Photocatalytic investigations revealed that coupling agents exhibiting covalent interaction with the titanium dioxide surface show degradation of the organic moiety, while the covalent connection between TiO


We demonstrate that tuning the stereo-hindrance of the phenoxy-alkyl chains at the 4-position of a curcumin scaffold could lead to certain selectivity for soluble Aβs over insoluble Aβs. CRANAD-102 showed a 68-fold higher affinity for sAβ than for insAβ (7.5 ± 10 nM vs. 505.9 ± 275.9 nM). Moreover, our imaging data indicated that CRANAD-102 was indeed capable of detecting sAβ in vivo using 4 month old APP/PS1 mice, in which sAβ is the predominant species in the brain. In addition, we also demonstrated that CRANAD-102 could be used to monitor the increase in sAβ loading from the ages of 4 months old to 12 months old. We believe that CRANAD-102 can be a useful probe for selectively detecting sAβ species in AD and that our probe designing strategy can be applied to other amyloids and will have tremendous impact on AD drug development and other amyloid research.Amyloid peptides and proteins are associated with the pathologies of numerous diseases. In the progression of a disease, amyloids exist in soluble and insoluble forms, which are the dominant species at different stages of the disease and they have different degrees of toxicity. However, differentiating between the soluble and insoluble forms is very challenging with small molecule probes due to multiple obstacles that need to be overcome. Inspired by the recognition principle of antibodies for sAβ, we hypothesized that the accessibility/tightness of soluble and insoluble amyloids could be utilized to design imaging probes to recognize different amyloid forms and the stereo-hindrance tuning strategy could be used to design imaging probes for selectively detecting the soluble amyloid beta (sAβ) species in Alzheimer’s disease (AD). Herein, we demonstrated that tuning the stereo-hindrance of the phenoxy-alkyl chains at the 4-position of a curcumin scaffold could lead to certain selectivity for sAβ over insoluble Aβs (insAβ). Among the designed compounds,  Amyloids are a large family of proteins/peptides that are prone to aggregate into large assemblies, which are usually harmful to human health. The most studied amyloids include amyloid beta (Aβ) and tau in Alzheimer’s disease (AD), α-synuclein in Parkinson’s disease, amylin in type II diabetes, and TDP-43 in amyotrophic lateral sclerosis.There are multiple obstacles that need to be overcome in designing small molecules to differentiate between the soluble and insoluble forms of amyloids. First, rare crystal structures of amyloids are available for conventional computer aided design (CAD). Secondly, the basic units of the soluble and insoluble amyloids are the same peptides or proteins. Thirdly, prior knowledge about what the design principles are is rare.et al. reported that the experimental antibody drug Solanezumab could probably be beneficial for AD patients in their early disease stages.21For Aβs, the soluble species include monomers, dimers, and oligomers and the insoluble species include fibrils/aggregates and plaques.sAβ species are believed to be the dominant biomarkers in early/presymptomatic stages of AD,via tuning the stereo-hindrance of the curcumin scaffold, it is feasible to develop NIRF imaging probes for the selective imaging of sAβ species.Numerous fluorescence imaging probes for Aβ species have been reported in the past few decades and most of the probes are responsive to insAβ species.1.CRANAD-65, -75, and -102 . The introduction of a phenoxy-alkyl chain into the curcumin scaffold likely increases the hydrophobicity of the probe, which may lead to strong non-specific binding to blood serum. To investigate whether the probes have a strong interaction with serum, we incubated the probes with different concentrations of bovine serum albumin (BSA) and found that there were no apparent changes in the fluorescence properties   . Again, 3.CRANAD-102 can differentiate between soluble and insoluble Aβs in a relevant biological environment, we used phantom imaging with soluble Aβ40 monomers, Aβ40 dimers, Aβ42 oligomers, and insoluble Aβ40 aggregates. In this experiment, we added the same amount of Aβ species to mouse brain homogenates and then imaged the brain homogenates after the addition of CRANAD-102. CRANAD-102 had significant selectivity for sAβ over insAβ in brain-relevant biological environments.To investigate whether 4.CRANAD-102 can stain insoluble Aβ plaques in brain slices, we incubated it with a brain slice from a 14 month old APP/PS1 mouse. No apparent plaque staining was observed  penetration is a crucial requirement for a brain-imaging probe. To validate whether 6.CRANAD-102 was able to detect soluble species in vivo by NIRF imaging, we first tested it with 4 month old APP/PS1 mice. At this age sAβ species are the predominant species in the brain.CRANAD-102 was indeed capable of detecting soluble Aβ species in vivo.To test whether CRANAD-102 can be used to detect the changes in sAβ concentration with the progression of AD, we performed NIRF imaging with 12 month old APP/PS1 mice. Our data indicated that the difference between APP/PS1 mice and age-matched WT mice was about 1.89-fold at 30 min after the injection . Mass spectra were obtained at the Department of Pharmaceutical Analysis of China Pharmaceutical University. Transgenic female APP-PS1 mice and age-matched wild type female mice were purchased from Jackson Laboratory. All animal experiments were performed in compliance with institutional guidelines and were approved by the IACUC Committee at Massachusetts General Hospital.The reagents used for the synthesis were purchased from Aldrich and used without further purification. Column chromatography was performed on silica gel  slurry packed into glass columns. Synthetic Aβ peptide (1–40/42) is from rPeptide . Aβ dimers of S26C Aβ40 were purchased from AnaSpec . 1.3c was performed according to a modified protocol of our previously reported procedure.333c  was dissolved in acetonitrile (3.0 mL), followed by the addition of acetic acid , tetrahydroisoquinoline , and 6-(diethylamino) nicotinaldehyde . The resulting solution was stirred at room temperature for 4 h. A black residue, which was obtained after removing the solvent, was subjected to flash column chromatography with methylene chloride to give a black powder . m.p.: 210–215 °C. 1H NMR  δ (ppm): 8.35 , 8.03 , 7.68 , 7.06 , 6.96 , 6.40 , 3.89 , 3.55 CH2CH3), 2.89 , 2.31 , 2.03 , 1.20 CH2CH3). ESI-MS (M + H) m/z: 631.4. HRMS calculated for C36H46BF2N4O3, m/z, 630.3662 [(M + H)]+; found, 630.3662. 13C NMR (CDCl3) δ (ppm): 12.92, 16.47, 22.31, 32.16, 43.06, 70.93, 106.82, 110.27, 112.26, 118.84, 123.87, 128.92, 130.93, 135.56, 145.09, 153.02, 155.93, 158.57, 176.49. HPLC purity: 98.83%.The synthesis of compound CRANAD-65, -75 and -102 can be found in the ESI.The details of the synthesis of 2.The measurements were conducted by following the reported protocol,3.3 cells per well in 96-well microliter plates. After 24 h, cells were exposed to CRANAD-65 (5.0 μM) for 48 hours. Then, cell survival was determined by the addition of an MTT solution (20 μL of 5 mg mL–1 MTT in PBS). After 6 h, the medium was removed by aspiration. The cells were dissolved in 150 mL DMSO and optical absorbance was measured at 570 nm using a SpectraMax M2 Microplate Reader (Molecular Devices). The survival ratios were expressed in percentages with respect to the untreated cells.HEK293 cell lines were seeded at 5 × 104.The preparations were performed according to previous reports.5.X with Aβs, the following procedure was followed. Step 1: 1.0 mL of PBS buffer was added to a quartz cuvette as a blank control and its fluorescence was recorded using the same parameters as for CRANAD-X. Step 2: the fluorescence of a CRANAD-X solution  was recorded with excitation at 610 nm and emission from 630 nm to 900 nm. Step 3: to the above CRANAD-X solution, 30 μL of Aβs  was added to make a final Aβ concentration of 750 nM. The fluorescence readings from this solution were recorded as described in step 2. The final spectra from steps 2 and 3 were corrected using the blank control from step 1.To test the interactions of CRANAD-6.CRANAD-102  and the Aβ monomers, oligomers, and aggregates . The resulting brain homogenates were imaged with Ex/Em = 640/700 nm using an IVIS®Spectrum imaging system.A 4 month old wild type (B6C3F1/J) mouse was sacrificed and its brain was dissected. The brain was then homogenized with 2.0 mL of PBS and 0.1 mL of homogenate was added to 15 wells of a 96-well plate, followed by the addition of 7.CRANAD-102  was incubated with 300 μL of mouse serum at 37 °C for 0, 30, and 60 min. The proteins in the serum were precipitated by adding 500 μL of acetonitrile after centrifugation at 5000 rpm for 15 min at 4 °C, and the supernatant was collected. Approximately 0.1 mL of the supernatant solution was analyzed using HPLC .8.In vivo NIRF imaging was performed using an IVIS®Spectrum animal imaging system . The images were acquired with a 640 nm excitation filter and a 700 nm emission filter. Data analysis was performed using LivingImage® 4.2.1 software.n = 3–4 and age-matched female wild type control mice, n = 3–4) were shaved before background imaging. An injection solution of CRANAD-102 (2 mg kg–1) was freshly prepared in 15% DMSO, 15% cremorphor, and 70% PBS and the solution was stabilized for 20 min before injection. Each mouse was injected intravenously with 100 μL of CRANAD-102. Fluorescence signals from the brain were recorded before and 15, 30, 60, 120, and 240 min after intravenous injection of the probe. To evaluate our imaging results, an ROI was drawn around the brain region. The P values were calculated using Student’s t-test.Five-month old mice  for 15 minutes and then washed with 20% ethanol followed by washing with dd water. The slice was covered with VectaShield mounting media. A consecutive slide was stained with 1% Thioflavin S solution (20% ethanol and 80% dd water). The fluorescence images were taken using a Nikon Eclipse 50i microscope.A 30-micron brain slice from a 14 month old APP/PS1 mouse was incubated with 1% There are no conflicts to declare.Supplementary informationClick here for additional data file.


Cenchrus pauciflorus) is a globally widespread invasive species capable of adapting to broad environmental conditions. However, its germination response to water and temperature still remains unclear. C. pauciflorus contains two seeds in the same bur that differ in size: big seeds (M) and small seeds (P). Separate greenhouse experiments were conducted under different temperature regimes  and water potentials  for M and P seeds. The results support the hypothesis that germination of C. pauciflorus is significantly influenced by seed type, temperature and water potential. M and P seeds responded differently to varied alternative temperatures and water potentials. However, M and P seeds were more sensitive to water potential than to temperature. Optimal conditions for M and P seed germination were measured at 25/35°C (night temperature/day temperature) and 20/30°C, respectively. In contrast, the highest germination rate was observed for the 0Mpa of the water potential treatment. Additionally, base temperature (Tbase) and base water potential (Wbase) were lower for M  than for P . These different germination strategies of M and P seeds with respect to temperature and water potential increased overall plant propagation. These results indicate that tropical and subtropical regions water potentials beyond -0.50Mpa (10/20°C) or -1.00Mpa (20/30°C) face a potential risk of C. pauciflorus invasion.The success of a biological invasion relies on the environment and is closely linked to factors such as water and temperature. Invasive plant species display different seed characteristics, including shape. Field sandbur ( Due to climate change, ecosystems are facing an increasing risk of exotic plants invasion , 2. MoreInvasive plant species show strong adaptability and anti-adversity in a range of environments beyond their native habitats. This has been considered an important trait for successful establishment in new habitats . Plants Ceratocarpus arenarius, Piper aduncum and Tithonia diversifolia has been reported. These reports have identified a system for invasion risk estimation, illuminating the importance of adaptation and possible mechanisms of invasion [In the agro-ecosystem, germination is a key step in the process of successful population establishment for any weed. Each species has its own requirements for germination in an unpredictable environment and a strategy to maximize survival . The difinvasion –20. In tinvasion .Aegilops cylindrical) produces larger seed in the second or third positioned floret, and smaller seed in the primary positioned floret, larger seed consistently germinates first [Variation in seed size and shape is an important phenomenon of heteromorphism. This characteristic was also deemed a possible reason for invasiveness in favor of adapting to a diverse environment , 23. Difes first .Cenchrus pauciflorus)  , is know nettles ) and is  nettles . It is e nettles , 29. The nettles , which c nettles . Current nettles –34. In C nettles , and the nettles . This ha nettles . The pla nettles . C. pauc spacing , 36. HowC. pauciflorus is limited. Therefore, the objectives of this study were to: 1) Evaluate the effect of temperature and water potential on the germination rate of C. pauciflorus seeds (M and P) in the same bur; 2) Quantify the base thermal time and base water potential for successful germination of C. pauciflorus seeds.However, current knowledge of how temperature and water potential affect germination of the two different seeds types of C. pauciflorus were collected from Horqin sandy land in August 2010 (43.52° N 122.32° E) and stored in paper bags below 4°C in darkness before commencement of experiment (The owner of the land gave permission to conduct the study on this site). The mean bur dry weight per 100 burs was 3809 ± 716 g. All seeds were less than eight months old when the experiment was conducted. In each bur, there are two distinct seeds in size and mass due to the cryptic heteromorphism, which can be recognized visually. We defined the big seeds  as Mango-type (M) and small seeds  as Plum-type (P) based on their size and shape (Mature burs of nd shape  (C). Mea6000) at -1.50 Mpa, -1.00 Mpa, -0.75 Mpa, -0.50 Mpa, -0.25 Mpa and 0 Mpa at 10/20°C and 20/30°C. Control refers to 0Mpa with ample water is available. All treatments were subjected to a 12 h night and 12 h day cycle. The temperatures in experiment 2 were selected based on the optimal temperatures found in experiment 1.Two experiments were carried out at different levels of temperature and water stress. Seeds in experiment 1 were set at different alternating (night time/day time) temperatures of 0/10°C, 5/15°C, 10/20°C, 15/25°C, 18/28°C, 20/30°C and 25/35°C to simulate field temperatures in different months. Seeds in experiment 2 were placed on a polyethylene glycol solution (PEG50). The average temperature was used for alternative temperature treatments. The germination rate was calculated as 1/T50. Data outside the linear range effect of temperature  extremes were dropped [Twelve seeds were placed into one Petri dish each (diameter of 100 mm), fitted with a double layer of humid filter paper. Six replications were conducted per treatment in a completely randomized design and dampened with deionized water  to evaluate germination. All Petri dishes were kept in sealed plastic bags to prevent the seeds from desiccating. The filter paper used in the experiment was folded in a cross-shape ridge for easy data recording . Prior t dropped .P < 0.05 via the ANOVA packages of SPSS 22.0 . Data of all germination percentages were subjected to an arcsine transformation subsequent to ANOVA to ensure homogeneity of variance . Mean comparisons were performed via least significant difference (LSD) to determine whether observed differences among means were significant between treatments and within each temperature, water potential and seed type (P < 0.05). To avoid distortion of the final results, outlier values were dropped from the analysis. Outliers were defined via a comparison of the differences between values and the 3.0×interquartile range [All data are expressed as mean values with standard error (in bracket) in tables and the mean values in figures. Treatment effects of accumulated germination rates from all experiments were separately analyzed using one-way , two-way  or three-way  analysis of variance (ANOVA) and a significance level of le range .FG) and final germination percentage (FGP) of C. pauciflorus (P < 0.05). Specifically, each increment in temperature shortened TFG of both M and P seeds, although we only observed a significant difference in M seeds at 10/20°C. Final germination percentage was relatively stable among different temperature treatments for M. However, germination percentage of P seeds increased with temperature until 30/40°C, beyond which it slightly declined. A significant increment could be observed between 10/20°C and 15/25°C, as well as between both treatments and other treatments  for P, while no significant differences were found in M. Therefore, treatments from 10/20°C to 30/40°C and from 18/28°C to 30/40°C were revealed as an optimal temperature for the germination of M and P seeds of C. pauciflorus, respectively. The maximum value of germination for M seeds was observed in the 25/35°C treatment, while P seeds had a maximal germination percentage at 20/30°C , whereas M and P seeds performed similarly under varied alternative temperature treatments towards TFG. A strong interaction was observed between temperature and seed type (P < 0.001), indicating that M and P seeds showed a different response among the temperature treatments at FGP   , Fig 3.FG was significantly shorter compared to other germinated treatments with one except for the -0.25Mpa treatment at 20/30°C (P < 0.05). No significant variation was observed for different water potentials in the other germination treatments. However, FGP of M seeds at -0.75Mpa (12.5%) and P seeds at -0.25Mpa (18.1%) varied significantly compared to the treatments with lower water potential and the 0Mpa treatment at 10/20°C (P < 0.05). Similarly, for M seeds, FGP at 20/30°C significantly differed between treatments of -0.75Mpa and -1.00Mpa, FGP was also significantly lower for -1.00Mpa compared to other treatments . For P seeds, FGP differed significantly at equal temperatures between -0.50Mpa, -0.75Mpa, and -1.00Mpa treatments and control, and -0.25Mpa treatment (P < 0.05)  , Fig 3.P < 0.05) differences for TFG were only observed between M and P seeds for 0Mpa and -0.25Mpa treatments at 20/30°C among all levels of water potentials. In contrast, significant differences of FGP were detected for all germinated treatments under 10/20°C and 20/30°C conditions (P < 0.05). Furthermore, germination activity of M and P seeds stopped at -1.00Mpa and -0.50Mpa for the 10/20°C treatment, and both disappeared at -1.50Mpa for 20/30°C. The highest final germination percentage occurred in the control treatment for both temperatures for M and P seeds. On average, M seeds germinated quicker  and with higher germination rate  at 10/20°C and 20/30°C treatments and compared to P seeds  and base water potential (Wbase) of M and P seeds under greenhouse conditions. The effect of temperature could be described by functions of y = -0.0185 + 0.0024x (R2 = 0.944) (M seed), y = -0.0235 + 0.0025x (R2 = 0.970) (P seed), respectively. In contrast, the influence of water potential at 10/20°C followed functions of y = 0.0151 + 0.0164x (R2 = 0.999) (M seed), y = 0.0101 + 0.0193x (R2 = 0.986) (P seed). Under 20/30°C, the influences of water potential were y = 0.0451 + 0.0408x (R2 = 0.997) (M seed), y = 0.0303 +0.0282x (R2 = 0.976) (P seed), respectively. The results calculated from the x interceptions revealed Tbase of M and P seeds to be 7.7 and 9.4°C, respectively. However, Wbase at 10/20°C and 20/30°C treatments were -1.11Mpa and -0.92 Mpa for M seeds and -1.07Mpa and -0.52 Mpa for P seeds  in all tested variable day and night temperatures , showing a germination potential for constant temperatures from 5°C to 30°C [C. pauciflorus seeds germinate within a broad range of alternating temperatures . Optimal germination occurred from 10/20°C to 30/40°C for M seeds and from 18/28°C to 30/40°C for P seeds  to 27°C (July) before dropping back to 9°C (October). Accompanying species  typically germinate from mid-April or early-May [C. pauciflorus compared to native species. Qu et al. [C. pauciflorus at 25°C, while maximal germination performances of other invasive weeds (Piper aduncum and Iresine diffusa) were observed at 25°C and 20°C, respectively [C. pauciflorus during the cold months or in cold regionsand minimizing their invasion capability accordingly.Invasive weeds generally germinate in variable conditions. E.g.  to 35°C . Iresine to 30°C . Our res P seeds , reflectarly-May , indicatu et al.  reportedectively , 38. In C. pauciflorus can be outperformed with irrigation than without irrigation in biomass and many morphological characteristics such as height, seed number, tiller number, and leaf area [C. pauciflorus germination [C. pauciflorus is sensitive to the water condition during the whole growing season. Our results reveal that exposure to an increasing water potential substantially decreased germination percentage and extend germination time for C. pauciflorus. Similar results were obtained by Acosta et al. [Iresine diffusa: germination reduced significantly for an osmotic potential of -0.4 Mpa. C. pauciflorus in our experiment was sensitive to increasing osmotic stress. Its occurrence is rare under extreme drought conditions. Rainfall in Horqin is typically low during April and May [C. pauciflorus germination. Moreover, C. pauciflorus germination was more sensitive at 10/20°C than at the optimal treatment (20/30°C), indicating C. pauciflorus is relatively vulnerable in spring and autumn, suggesting this period to be ideal to control further development.Previous studies reported that eaf area , 44. A fmination . This ina et al.  in a ger and May , but stiBidens frondosa showed different dormancy and germination. Xu et al. [C. pauciflorus. Correspondingly, in our experiment, quicker and improved germination was observed in M seeds compared to P seeds. The difference in mass might be one of the factors that contribute to the different germination behavior of M and P seeds. It represents the reproductive strategy of varying allocation of storage reserves within each seed, guaranteeing that at least one seed per bur germinates and contributes to successful seedling establishment the following season. Besides, P was more sensitive under both 10/20°C and 20/30°C conditions. Also, M and P seeds had different Tbase and Wbase  and seed type on seed germination as well as on possible mechanisms of S1 Fig(A) seed, (B) seedling, (C) population, (D) spiking in the field,  spiking in different stages, (G) maturity,  whole single plant.(TIF)Click here for additional data file.S2 FigSeed length, width and thickness are marked with line AB, line CD and line EF, respectively.(TIF)Click here for additional data file.S1 TableLowercase letter indicates significant difference between M and P seed at P< 0.05.(DOCX)Click here for additional data file.


The motor features of Parkinson's disease (PD) primarily result from a lesion to the nigrostriatal dopamine system. Numerous non-motor symptoms occur in PD, many of which are postulated to stem from pathology outside of the nigrostriatal dopamine system. Perturbations to protein trafficking, disruption of mitochondrial integrity, and impaired autophagy have repeatedly been implicated in dopaminergic neuron cell death. Previously, we demonstrated that multiple markers of autophagy are disrupted in a rotenone model of PD, with alterations occurring prior to an overt lesion to the nigrostriatal dopamine system. Whether these events occur in extra-nigral nuclei in PD and when relative to a lesion in the nigrostriatal dopamine system are generally unknown. The primary goal of these studies was to determine whether autophagy disruptions, in non-dopaminergic neuronal populations occur in an environmental model of PD utilizing a mitochondrial toxin. Here, we utilized the rat rotenone PD model, with sampling time-points before and after an overt lesion to the nigrostriatal dopamine system. In analyzing autophagy changes, we focused on optineurin (OPTN) and the autophagy marker, LC3. OPTN is an autophagy cargo adapter protein genetically linked to amyotrophic lateral sclerosis and glaucoma. In the present study, we observed OPTN enrichment in all PD-relevant brain regions examined. Further, alterations in OPTN and LC3 expression and colocalized puncta suggest specific impairments to autophagy that will inform future mechanistic studies. Thus, our data suggest that autophagy disruptions may be critical to PD pathogenesis in non-dopaminergic neurons and the onset of non-motor symptoms. Parkinson's disease is a progressive debilitating neurological disease, where only a small proportion of cases have a known genetic link (<10%). The vast majority of cases have an unknown sporadic incidence which are thought to be caused by gene-environment interactions  and the United States Public Health Service (USPHS) in accordance with the Animal Welfare Act and Purdue's Animal Welfare Assurance The protocol was approved by the Purdue University Animal Care and Use Committee.All animals were male wild-type Lewis rats purchased from Harlan  and were between the ages of 31–42 weeks when euthanized. Rats were housed under standard 12 h light cycle, with free access to water and fed ®; 647 anti-sheep (713-605-147), Alexa Fluor®; 488 anti-rabbit (711-545-152), and CyTM3 anti-mouse (715-165-151) were purchased from Jackson ImmunoResearch Laboratories, Inc. . Rotenone (R8875) and glycerol (G5516) were purchased from Sigma-Aldrich . Triton X-100 was purchased from Fisher BioReagents .Primary antibodies for rabbit anti-OPTN (ab23666) and rabbit anti-beclin-1 (ab62557) were purchased from abcam ; mouse anti-LC3 was purchased from MBL International Corporation ; mouse anti-alpha-synuclein was purchased from BD Transduction Labs ; chicken anti-MAP2 (AB5543), sheep anti-tyrosine hydroxylase (AB1542), anti-tryptophan hydroxylase (AB1541), and anti-choline acetyltransferase (AB1582) were purchased from EMD Millipore . Normal donkey serum (017-000-121) and secondary antibodies, Alexa Fluorn = 3), 5 days (n = 3), or at end-stage (n = 3) . In this model, overt behavioral deficits are associated with a lesion to the nigrostriatal dopamine system , magnocellular raphe (RMg), locus coeruleus (LC), pedunculopontine tegmentum nucleus (PTg). We also considered changes to expression of alpha-synuclein and beclin-1 in the substantia nigra pars compacta (SNpc) because we previously showed that OPTN and LC3 expression and puncta were altered in this brain region . In pilot studies, we optimized antibody titers using graded dilutions and columetric staining; then we performed staining for MAP2A  + staining for autophagy markers to verify that the majority of total puncta were observed within neurons and not extracellular (we expect the few outside the MAP2A stain were present in glia).Rats were placed under deep anesthesia with pentobarbital (>50 mg/kg) , then transcardially perfused with 100–150 mL PBS, followed by 250–300 mL 4% buffered paraformaldehyde (PFA). Brains were surgically removed, post-fixed in 4% PFA for ~24 h, and then saturated with 30% sucrose at 4°C for at least 5–7 days until sinking. Each brain was coronally sectioned on a frozen sliding microtome  at a 35 μm thickness and stored in cryoprotectant at −20°C until used for staining. Brain sections containing the desired regions were randomly selected, rinsed in 10x PBS for 10 min, six times, at room temperature (RT) on an open-air platform shaker; blocked in 10% normal donkey serum  in PBS with 0.3% Triton X-100 (PBS-T) for 90 min at RT; incubated with primary antibodies for ~48 h in PBS-T with 1% NDS at 4°C. The sections were then rinsed three times with 10x PBS at RT for 10 min each time; incubated with secondary antibodies in PBS-T with 1% NDS for 90 min at RT; and rinsed six times with 10x PBS at RT for 10 min each time before being mounted on slides and coverslipped as a wet mount using 50/50 glycerol:PBS solution. Primary antibody dilutions were: mouse anti-LC3 (1:1000); rabbit anti-OPTN (1:5000); sheep anti-tyrosine hydroxylase (1:1000); sheep anti-tryptophan hydroxylase (1:4000); sheep anti-choline acetyltransferase (1:1500). Secondary antibody dilutions were: Alexa FluorConfocal images were captured on an inverted Nikon Eclipse TE 2000-U microscope with an EZ-C1 confocal, equipped with 10x, 20x, and 60x Plan Fluor objectives. Region of interest (ROI) analysis was then conducted on images using the NIS-Elements software ver. 4.30 to measure signal intensity. High magnification images (60x) were analyzed for puncta analysis; puncta were considered bright circular dots with diameters ranging from 0.5 to 2.0 μm, as has been previously described for autophagosome diameters , while 5 d and end-stage expression were significantly increased . Mean LC3 expression (relative to control) was significantly increased across all stages . Punctate expression of OPTN, LC3 and colocalized puncta were also quantified; overtly identifiable puncta with a diameter of 0.5-2 um were counted. Figures p < 0.001). We then considered changes in the percent of OPTN or LC3 puncta that were colocalized by PD stage. Results show decreased percent of OPTN puncta and LC3 puncta were found colocalized only after 24 h rotenone when compared to control; 13.7 ± 0.8% and 10.8 ± 0.9% for OPTN (p < 0.01), 33.5 ± 2.0% and 23.1 ± 1.7% for LC3 (p < 0.001), respectively .ROI analysis was used to evaluate changes in OPTN and LC3 expression across stages of PD in our rat models: control (145 neurons), 24 h (123 neurons), 5 d (91 neurons), and end-stage (186 neurons); representative images are shown in Figure p < 0.001). Figure p < 0.001), a significantly decreased expression after 5 d rotenone  and in end-stage rats . Analysis of OPTN puncta , for control, 24 h, 5 d, and end-stage, respectively. Mean LC3 puncta were increased across all stages, but only reached significance after 24 h rotenone  when compared to control (35.6 ± 1.2). Colocalized puncta per cell exhibited no differences after 24 h (23.5 ± 1.0), but significantly reduced number of puncta at 5 d and end-stage  when compared to control , 14.3 ± 0.8% (p < 0.001) and 13.9 ± 0.9% (p < 0.001) for OPTN, 65.8 ± 2.2%, 48.3 ± 1.7%, 41.7 ± 1.9%, and 43.7 ± 2.2% for LC3 , respectively .Representative images of RMg staining are shown in Figure a Figure  exhibitep < 0.05), and 124.19 ± 2.65% (p < 0.001) in 24 h, 5 d, and end-stage models, respectively. Figure p < 0.001), and significantly increased expression after 5 d rotenone  and in end-stage rats . Analysis of OPTN puncta  for control, 24 h, 5 d, and end-stage, respectively. Figure p < 0.05). Figure p < 0.01), 5 ± 0.4 (p < 0.001), and 11 ± 0.6 for control, 24 h, 5 d, and end-stage, respectively. We then considered changes in the percent of OPTN or LC3 puncta that were colocalized by PD stage. Results show decreased percent of OPTN puncta and LC3 puncta across all stages when compared to control; 40.8 ± 2.3%, 25.1 ± 1.7% (p < 0.001), 12.6 ± 0.9% (p < 0.001) and 28.3 ± 2.2% (p < 0.01) for OPTN, 65.8 ± 2.1%, 51.7 ± 1.9%, 25.9 ± 1.4% and 48.4 ± 2.0% for LC3 , respectively .Representative images of LC staining are shown in Figure a Figure  exhibitep < 0.001) and 84.37 ± 1.63% (p < 0.001) respectively; while OPTN expression was increased after 5 d rotenone . Figure p < 0.001, respectively) and significantly decreased expression in end-stage rats . Analysis of OPTN puncta  for control, 24 h, 5 d, and end-stage, respectively. Figure p < 0.001). Slightly increased values were observed in 5 d and end-stage PD models when compared to control, but were not statistically significant . Figure p < 0.001), and 16.8 ± 1.0 (p < 0.001) for control, 24 h, 5 d, and end-stage, respectively. We then considered changes in the percent of OPTN or LC3 puncta that were colocalized by PD stage. Results show decreased percent of OPTN puncta only after 5 d rotenone when compared to control: 28.3 ± 1.1% and 17.8 ± 0.7% (p < 0.001), respectively. Results show decreased percent of LC3 puncta colocalized after 24h and 5 d rotenone, but increased at end-stage: 38.5 ± 1.8%, 30.9 ± 1.8% (p < 0.05), 30.5 ± 1.3% (p < 0.01) and 42.8 ± 1.7% (p < 0.05) for LC3, respectively  and 73.68 ± 2.85% (p < 0.001) respectively. Figure p < 0.001, respectively), and slightly decreased expression in end-stage rats .Representative images of SNpc staining are shown in Figure Finally, we compared the mean OPTN and LC3 puncta in control animals across all brainstem regions considered here Figure . We alsoAutophagy disruptions have been repeatedly implicated in the pathogenesis of PD and other neurodegenerative diseases. However, the temporal development of autophagic changes is poorly characterized with respect to phenotype onset. Further, there is a lack of data on how brain regions outside the substantia nigra may be affected. In this study, we considered OPTN's autophagic role in four extranigral regions during PD pathogenesis by analyzing the expression and interaction of OPTN and LC3 using a rat rotenone model of PD. Collectively, we show that autophagy is affected in multiple brain nuclei important in PD, and that changes in these regions precede a lesion to the nigrostriatal dopamine system. Taking these data together, we can assess impairments to autophagy that may result in autophagic stress. While autophagy is ideally assessed dynamically , our approach to assess multiple endpoints and assessing populations of neurons improves our ability to assess its activity at a single time point. Finally, our data suggest dopaminergic neurons exhibit lowered basal autophagic activity, and may be limited in their autophagic capacity—especially those in the SNpc , impaired induction (reducing LC3 expression and number of autophagosomes), impaired cargo recruitment , or impaired autophagosome-lysosome fusion (resulting in increased LC3 expression and number of autophagosomes). Another potential impairment in autophagy lies in the activity of cargo adaptors , though this is much more difficult to detect in vivo as many of these proteins require post-translational modifications to encourage binding to cargo or LC3  impaired induction, (2) impaired OPTN-LC3 binding, and (3) impaired autophagosome-lysosome fusion. We also considered the possibilities of OPTN failing to serve as a cargo adaptor protein and of depleted autophagosome pool. Interestingly, our results suggest different types of impairment in different regions  quantifying expression of LC3 and OPTN (a known autophagy cargo-adaptor), (2) quantifying puncta formation of each , and (3) by assessing changes to the percent of total LC3 and OPTN puncta that are colocalized. This novel approach to in vivo autophagy analysis enables the evaluation of several critical steps in autophagy flux. Here, we use this approach to asses changes in autophagy induction, interaction between LC3 and OPTN (an autophagy cargo adaptor), autophagosome-lysosome fusion, and exhaustion or depletion of OPTN serving a cargo adaptor function or availability of autophagosomes. Given that autophagy is a complex process, the best approach to analyze its function or dysfunction utilizes live-cell imaging and pharmacological inhibition of specific junctions . To address the challenge of autophagy changes over time and to assess autophagic activity changes over the course of PD progression, we considered these endpoints using a thoroughly characterized dosing scheme for the PD neurotoxin rotenone. Our results demonstrate variable types of impairment in extranigral regions implicated in preclinical PD, which supports our hypothesis that autophagic dysfunction may precede the spread of Lewy bodies through the PD brain. Further investigation is needed to understand the complexity of autophagic dysfunction in these regions and how it contributes to PD progression. Importantly, if autophagic dysfunction precedes LBP, it may become an important target for future PD therapeutics to help slow the progression of the disease.Assessing autophagic dysfunction JW Designed, performed, and analyzed experiments; wrote the 1st draft, and subsequent drafts. CP Performed and analyzed experiments. JA Performed and analyzed experiments. JC Responsible for all aspects of the research design, execution and interpretation; review and refinement of initial and subsequent manuscript drafts. JC also funded the work through internal and extramural awards.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bacillus coagulans LA204 using fed-batch simultaneous saccharification and fermentation under non-sterile conditions. The lactic acid titer, yield, and productivity from 16% (w/w) NaOH-pretreated and washed corncob were 122.99 g/L, 0.77 g/g corncob, and 1.37 g/L/h, respectively, and from 16% NH3-H2O2-pretreated and washed corncob were 118.60 g/L, 0.74 g/g corncob, and 1.32 g/L/h, respectively. Importantly, the lactic acid titer, yield, and productivity from 18.4% NH3-H2O2-pretreated and unwashed corncob by using fed-batch simultaneous saccharification and fermentation reached 79.47 g/L, 0.43 g/g corncob, and 1.10 g/L/h, respectively, demonstrating that this method is possible for industrial applications and saves washing water.Lignocellulose is one of the most abundant renewable feedstocks that has attracted considerable attention as a substrate for biofuel and biochemical production. One such biochemical product, lactic acid, is an important fermentation product because of its great potential for the production of biodegradable and biocompatible polylactic acid. High-titer lactic acid production from lignocellulosic materials has been achieved recently; however, it requires biodetoxification or results in large amounts of waste washing water. In this study, we employed two alkaline pretreatment methods and compared their effects on lactic acid fermentation of pretreated corncob by  Lignocellulose, the most abundant global source of biomass, has been largely unutilized for biofuel and biochemical production. Over 800 million tons of lignocellulose have been produced in China since 2008, with approximately 505.5 million tons of primary biomass being available for further utilization37l-lactic acid (l-LA), and cheap renewable substrates such as lignocellulosic biomassLactic acid (LA) is an important biochemical product that has attracted increasing attention because of its widespread application in the food, chemical, cosmetic, and pharmaceutical industries. Furthermore, LA has great potential for the production of biodegradable and biocompatible polylactic acid (PLA) polymers, which has driven the current market development for LA. Fermentative production is the main route for producing LA; the advantages of this method include low production temperature and energy consumption, production of optically pure D- or 2O2) post-treatment was investigated using corn stover as the substrate. It was found that NaOH first solubilized and removed the easily-extracted lignin and xylan and the oxidizing post-treatment then removed the more recalcitrant lignin from the cell wallsThe crystalline structure of lignocellulosic biomass results in two major technical obstacles to LA production: biomass pretreatment and hydrolysis and efficient fermentation of pentose from lignocellulosic hydrolysates. The pretreatments applied to agro residues include physical (size reduction), physicochemical , chemical , and biological methods121314153) or NaOH are required to maintain the neutral or mildly acidic conditions favorable for LA fermentation. The resulting accumulation of sodium lactate or calcium lactate in the fermentation broth can have various stress effects on lactic acid bacteriaLactobacillus strains and Bacillus coagulans, have been reported to produce high-titer LA from lignocellulosic materials. B. coagulans strains, possessing robust inhibitor tolerance, were shown to be suitable for lignocellulosic LA production and were engineered for ethanol production because of their thermophilic growth characteristics and strong pentose homofermentative activity16B. coagulans DSM 2314 reached 0.26 g/g lime-pretreated wheat stover and 40.7 g/L, respectivelyB. coagulans reached 0.97 g/g and 59.2 g/L, respectivelyB. coagulans IPE22 reached 0.46 g/g acid-pretreated wheat stover and 38.73 g/L, respectivelyB. coagulans LA204 reached 0.68 g/g substrate and 97.6 g/L, respectively, for fed-batch fermentation with 14.4% solid content using NaOH-pretreated and washed corn stoverPediococcus acidilactici DQ2. The LA titer reached 101.9 g/L, however, the yield only reached 0.38 g/g stover and P. acidilactici DQ2 cannot utilize xylosel-LA was obtained from dilute acid-pretreated and biodetoxified corn stover with <30% solid content using an engineered P. acidilactici TY112 (CGMCC 8664) strain. The yield reached 0.72 g/g glucose from total corn stover without considering xylose unavailabilityHowever, pretreatments generate inhibitors  that repress LA fermentation. Thus, efficient LA production from pretreated biomass requires the removal of these inhibitors prior to fermentation or the use of inhibitor-tolerant bacteria. Moreover, calcium carbonate (CaCO3-H2O2)-pretreated corncob via SSF with strain B. coagulans LA204. The LA yield, titer, and productivity reached 0.43 g/g corncob, 79.47 g/L, and 1.10 g/L/h, respectively, using NH3-H2O2-pretreated and unwashed corncob. This study provides a useful industrial application to avoid the generation of waste washing water.In order to improve lignocellulosic LA production from both cellulose and hemicellulose hydrolysates and reduce the volume of washing water, we compared LA fermentation efficiency using NaOH-pretreated and ammonium-hydrogen peroxide .Acid and alkaline pretreatments are commonly used to remove lignin from lignocellulosic materials. However, acid pretreatment results in loss of hemicellulose, while alkaline pretreatment maintains most of the hemicelluloses in the solid content and is thus more feasible for biochemical or biofuel fermentation using SSFreatment . Subsequ corncob . The ligeatments . SubsequB. coagulans LA204 demonstrated remarkably efficient lignocellulosic LA production, with high LA yield and titer and low byproduct generation3-H2O2 were used to pretreat corncob, one of the most abundant agro biomasses in the world, and the LA fermentation ability of B. coagulans LA204 using these materials was compared. The use of 8% NaOH-pretreated and washed corncob as the carbon source, 10 g/L yeast extract as the nitrogen source, and 10 M NaOH solution as the neutralizer resulted in an LA yield of 0.79 g/g total corncob and an LA titer of 62.91 g/L. LA was produced rapidly initially and LA fermentation was nearly complete by 18 h  with CaCO3 as the neutralizer, LA productivity was 2.66 g/L/h, which was slightly lower than that obtained when NaOH was used as the neutralizer; however, LA was continuously produced during the period from 18 to 36 h with a productivity of 1.26 g/L/h . Because CaCO3 was sufficient for enhancing LA yield, we performed fed-batch fermentation for high-titer LA production from corncob using CaCO3 as the neutralizer. A similar fermentation curve as shown in l-LA optical purity was 98%. These impressive results represent one of the highest levels of LA production from agro biomass reported to date. However, the key issue of efficient inhibitor removal without washing water requires resolution prior to feasible industrial application. Previously, we reported that B. coagulans is sensitive to inhibitors in corn stover created by NaOH pretreatment and that removal of the inhibitors by simple washing enhanced LA yield, titer, and productivityIn our previous study,  by 18 h ; the LA 26 g/L/h . These rB. coagulans reached relative high titer and yield3 has been shown to remove lignin from lignocellulosic materials3 residues can be collected by volatilization, eliminating the neutralization step post pretreatment. Furthermore, H2O2 is able to oxidize the phenolic compounds generated by alkaline pretreatment, which may reduce the use of washing water to remove these inhibitors3-H2O2-pretreated corncob. For the first experiment, LA fermentation was performed using corncob pretreated for 1 day with NH3 and then further treated for 1 day with H2O2 with a NaOH solution as the neutralizer. LA yield, titer, and productivity were 0.42 g/g corncob, 33.70 g/L, and 0.70 g/L/h, respectively . The total phenolic concentration in both experiments was similar; however, we propose that the phenolic compounds from the NH3 pretreatment experiment could be oxidized following extended H2O2 treatment and thus their inhibitory effects were reduced. One significant difference between these fermentations was that a higher amount of LA was produced at 12 h, and importantly, more xylose was liberated using corncob with extended H2O2 pretreatment  was lower using CaCO3 as the neutralizer; additionally, more glucose was liberated from the biomass and accumulated in the fermentation culture  of fermentation using 8% NH3-H2O2-pretreated and washed corncob were similar to those in l-LA optical purity was 98%. These results indicate that NH3-H2O2 pretreatment efficiently promotes sugar liberation by cellulase and hemicellulase for subsequent LA fermentation, although higher levels of lignin were detected in the pretreated corncob  and unwashed corncob as the substrate. However, in these experiments, we used a NaOH solution as the neutralizer, because the addition of CaCO3 to a bioreactor with high solid loading of NH3-H2O2-pretreated corncob led to a viscous fermentation culture, which was difficult to agitate. Fermentation was initiated with 8% unwashed corncob and the substrate was fed to 18.4% (w/w) at 24 h. The total phenolic concentration was 3.0 g/L during the initial stage (0 to 24 h) and increased following substrate feeding to 6.0 g/L. Liberated sugars were consumed rapidly during the initial stage; however, glucose and xylose accumulated in the culture post substrate feeding , and inhibitor concentrations were low . Following the feeding, LA was still produced rapidly (12 to 36 h) and the productivity was 1.47 g/L/h during this period. However, once the total phenolic concentration reached a maximum of 4.8 g/L at 36 h and was maintained at that level, LA productivity suddenly decreased to 0.39 g/L/h (36 to 84 h) and xylose accumulated in the culture to a concentration of 8 g/L. Fermentation was continued for 84 h with an LA titer, yield, and productivity of 84.46 g/L, 0.53 g/g corncob, and 1.01 g/L/h, respectively. LA titer and yield could be increased by extending fermentation time; however, this may result in lower overall productivity. In contrast, high-titer LA production was obtained from sulfuric acid-pretreated and biodetoxified corn stover by P. acidilactici DQ2. In their studies, acid-pretreated corn stover was subsequently detoxified by inoculation of Amorphotheca resinae ZN1 for 5 days in a separated fermentation process and then the biodetoxified corn stover was used for LA fermentation243-H2O2 pretreatment with biodetoxification to enhance LA fermentation by B. coagulans FL204 in our further study. It also should be noted that several additives can enhance fermentation performance. For example, 30 mM citrate buffer greatly influenced acetone-butanol-ethanol fermentation by Clostridium beijerinckii when corn stover hydrolysate was used as the substrate3-H2O2 pretreatment might completely eliminate the need for washing water, at least when corncob is used as the substrate. However, it should also be noted that the LA titer and yield from NH3-H2O2-pretreated corncob are still very low compared with those from NH3-H2O2-pretreated and washed corncob. Moreover, NH3-H2O2 pretreatment is lengthier than the NaOH method, which will impact equipment size, throughput, and process economics.In order to fully eliminate the need for washing water, we performed fed-batch fermentation using NH feeding . These r feeding . We hypowere low . The fir3·H2O) for 1 day and then treated with 5% (w/w) H2O2 solution for 1 day or 7 days using 20% (w/w) corncob loading at room temperature. A portion of the NH3-H2O2-pretreated corncob was washed with water until the pH decreased to 8.0 and then filtered to a moisture content of 25% (w/w). The pH value of the remaining substrate was adjusted to 8.0 using a 50% (w/w) H2SO4 solution. The cellulase used in this study was Cellic CTec2 , which contains cellulase, β-glucosidase, and xylanase activity. YEX medium (10 g/L xylose and 10 g/L yeast extract) was used for seed culturing. The analytical methods of the National Renewable Energy Laboratory (NREL) were used to determine the raw and pretreated material composition in terms of structural carbohydrates and ligninCorncob was harvested in 2014 in the Hubei province of China. After harvest, the corncob was cleaned, dried, and sieved using a 200-mesh. The raw corncob consisted of 37.26 ± 0.56% cellulose, 29.05 ± 0.04% hemicellulose, 19.60 ± 0.64% lignin, and 11.17 ± 0.01% ash. Two pretreatment methods were used in this study. The first method involved pretreating the corncob with 5% NaOH solution at 75 °C for 3 h using 20% (w/w) corncob loading. The resulting slurry was then washed with water until the pH decreased to 8.0 and then filtered to a moisture content of 25% (w/w). In the second method, the corncob was pretreated with 3% (w/w) ammonium hydroxide /mL. The SSF process was established by inoculating 3 L of 8% (w/w) NaOH-pretreated and washed corncob, 10 g/L yeast extract, and 30 filter paper cellulase units (FPU)/g corncob with 300 mL seed culture in a 5-L bioreactor . LA fermentation by B. coagulans LA204 was performed at optimal conditions (50 °C and pH 6.0 [maintained by an automatic feed of 10 M NaOH solution or using excess CaCO3]), as previously reportedThe 3-H2O2-pretreated and unwashed corncob, 10 g/L yeast extract, and 30 FPU/g substrate of cellulase were inoculated with 300 mL seed culture in a 5-L bioreactor. Fermentations were carried out at 50 °C for 48 h with agitation at 200 rpm; the pH was maintained at 6.0 using an automatic feed of 10 M NaOH solution or using excess CaCO3. Two fed-batch fermentations were carried out to determine LA production ability. In the first fed-batch experiment, 8% NH3-H2O2-pretreated and unwashed corncob, 30 FPU/g substrate of cellulase, and 150 mL seed culture were used to initiate fermentation under the same fermentation conditions in a 1.5-L volume. The NH3-H2O2-pretreated and unwashed corncob was fed to 18.4% (w/w) and cellulase was fed to maintain 30 FPU/g substrate at 24 h; the final volume was 3 L. In the second fed-batch experiment, 4% (w/w) NH3-H2O2-pretreated and washed corncob, 30 FPU/g substrate of cellulase, and 150 mL seed culture were used to initiate fermentation under the same fermentation conditions in a 1.5-L volume. The NH3-H2O2-pretreated and unwashed corncob was fed to 8% (w/w) at 12 h and to 16% at 24 h and cellulase was fed to maintain 30 FPU/g substrate; the final volume was 3 L. In addition, fed-batch fermentations using NH3-H2O2-pretreated and washed corncob were also conducted using CaCO3 as the pH neutralizer; 8% NH3-H2O2-pretreated and washed corncob, 30 FPU/g substrate of cellulase, and 150 mL seed culture were used to initiate fermentation under the same fermentation conditions in a 1.5-L volume. The NH3-H2O2 pretreated and washed corncob was fed to 16% (w/w) and cellulase was fed to maintain 30 FPU/g substrate from 18 h to 24 h; the final volume was 3 L. Samples were collected every 6 or 12 h during fermentation and the concentrations of LA, acetic acid, formic acid, glucose, xylose, and total phenolic compounds were determined and the yields and productivities were calculated. These fermentations were conducted in duplicate under non-sterile condition. The crude HPLC data were included in To establish the SSF process, a 3-L volume of 8% (w/w) NH2SO4 at a flow rate of 0.6 mL/min at 40 °C. All samples were centrifuged at 12,000 rpm for 2 min and filtered using nylon syringe filters (pore size 0.22 μm) prior to loading. All standards for HPLC analysis  were obtained from Sigma-Aldrich. The concentration of l-LA was determined using an SBA-40X biosensor . The total content of phenolic compounds in the samples was determined using the Folin-Ciocalteu methodThe levels of glucose, xylose, LA, acetic acid, and formic acid in the samples were measured using high performance liquid chromatography (HPLC) with a Bio-Rad HPX-87H ion-exclusion column equipped with an Agilent 1200 and a RID-10A or SPD-20A detector. The mobile phase was 5 mM HHow to cite this article: Zhang, Z. et al. Comparison of high-titer lactic acid fermentation from NaOH- and NH3-H2O2-pretreated corncob by Bacillus coagulans using simultaneous saccharification and fermentation. Sci. Rep.6, 37245; doi: 10.1038/srep37245 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Preclinical studies have demonstrated that docetaxel and bevacizumab may act synergistically by decreasing endothelial cell proliferation and preventing circulating endothelial progenitor mobilization. The objective of this study was to assess the efficacy and safety of a combination therapy of bevacizumab, cisplatin, and docetaxel in chemotherapy-naive Japanese patients with advanced non-squamous non-small-cell lung cancer (NSCLC).2), cisplatin (80 mg/m2), and bevacizumab (15 mg/kg) once every 3 weeks, followed by bevacizumab as maintenance therapy, every 3 weeks until disease progression or attainment of unacceptable toxicity level. The primary endpoint was objective response rate (ORR). The numbers of circulating endothelial cells (CEC) were also estimated on days 1 and 8 of the first cycle for the exploratory analysis of efficacy prediction.Eligible patients were chemotherapy-naive and had advanced/recurrent non-squamous NSCLC. The patients received 4 cycles of docetaxel (60 mg/mp = 0.074).A total of 47 patients were enrolled from October 2010 to April 2012. Bevacizumab as maintenance therapy was administered to 41 patients (87.2%), and the median number of total treatment cycles was 9 (range: 1–36). ORR, median progression-free survival (PFS), and median overall survival of the patients were 74.5%, 9.0 months, and 27.5 months, respectively. The most common grade 3/4 adverse event was neutropenia (95.7%), followed by leukopenia (59.6%) and hypertension (46.8%). PFS was longer in patients with ≥10 count increase in CECs than that in patients with < 10 count increase in CECs (respective median PFS of 11.0 months versus 6.90 months) although the difference was not statistically significant .UMIN Clinical Trial Registry; The online version of this article (10.1186/s12885-018-4150-y) contains supplementary material, which is available to authorized users. The median number of total treatment cycles was 9 (range: 1–21). Cisplatin dose reduction was required in 13 patients (27.7%), and docetaxel dose reduction was required in 12 patients (25.5%). A total of 35 patients (74.5%) achieved partial responses with an ORR of 74.5% [95% confidence interval (CI): 59.7–86.1%]. Eleven patients (23.4%) archived a stable disease status with a disease control rate of 97.9% (95% CI: 88.7–99.9%). Median PFS was 9.0 (95% CI: 7.0–11.3) months , the grade ≥ 3 AE was rarely observed, except for hypertension (34.1%).Hematological and non-hematological toxicities of all patients are summarized in Table p = 0.074).In 35 patients, CEC count was measured on days 1 and 8  and cisplatin (75 or 80 mg/m2) as the first-line treatment of patients with metastatic non-squamous NSCLC have revealed favorable anti-tumor effect, with an ORR of 33.3–63.0%, a median PFS of 4.4–7.8 months, and a median OS of 13.3–13.5 months [2), the present study revealed much higher ORR and much longer PFS and OS than in the above-mentioned past two studies and the past studies using other third generation chemotherapy plus platinum with bevacizumab, such as E4599, AVAiL, AVAPERL and PointBreak trials [The past two single-arm phase II studies of bevacizumab (15 mg/kg) in combination with docetaxel . According to the past phase III trials of docetaxel monotherapy, grade ≥ 3 neutropenia was more frequently observed in Japanese patients treated with 60 mg/m2 (73.3–85.9%) than in the patients from the Western countries treated with 75 mg/m2 dose (21–37%) although the efficacy was almost equivalent in both the cases [2) plus cisplatin in Japanese patients with advanced NSCLC have revealed that the incidence of grade ≥ 3 neutropenia and leukopenia was 73.4–74.2% and 45.7–55.2%, respectively [In terms of toxicity, neutropenia and leukopenia were the most common AEs among grade ≥ 3 AEs in the past phase II studies of cisplatin, docetaxel, and bevacizumab  , 12. How/m2 dose –37% althhe cases . The resectively , 31. Morectively . These rTo date, no useful biomarker has been established for predicting the efficacy of bevacizumab. CECs are mature endothelial cells that are sloughed from the vessel wall, and increase in CECs in the peripheral blood is expected to serve as a potential biomarker for predicting response to bevacizumab , 33–35. This study had some limitations. First, the lack of a standard assay for CEC counting may hinder the clinical application of the proposed concept in clinical practice. However, the variability in CEC values is smaller among studies that measure CECs by CellSearch® than those that use other systems. Second, 11 of 13 patients with EGFR-sensitive mutations received EGFR-tyrosine kinase inhibitor as a second- or third-line treatment, which warrants a careful interpretation of the evaluated OS values.Bevacizumab, cisplatin, and docetaxel in combination, followed by bevacizumab alone as a maintenance treatment was highly effective in patients with non-squamous NSCLC despite the high incidence of grade 3/4 neutropenia. The increases in CECs count between days 1 and 8 may predict the efficacy of our bevacizumab-contained treatment regimen.Additional file 1:Figure S1. Trial profile. Abbreviations: CEC, circulating endothelial cell. (JPEG 147 kb)Additional file 2:Table S1. CEC data and treatment efficacy of 35 patients whose CEC count was measured on days 1 and 8. Abbreviations: EGFR, epidermal growth factor receptor; CEC, circulating endothelial cell; PR, partial response; SD, stable disease; PFS, progression-free survival; OS, overall survival. (DOCX 22 kb)


Furthermore, the potential recovery through 2 weeks of T4 treatment discontinuation was also investigated. PTU and T4 recovery partially reduced the T4-prompted increase in BP. Alternatively, PTU significantly improved the in vivo left ventricular (LV) function with no considerable effects on cardiac hypertrophy or ex vivo right ventricular (RV) contractile alterations subsequent to T4 treatment. Conversely, T4 recovery considerably enhanced the T4-provoked cardiac changes both in vivo and ex vivo. Altogether, our data is in agreement with the proposal that hyperthyroidism-induced cardiovascular pathology could persevere even with antithyroid treatments, such as PTU. However, this cannot be generalized and further investigation with different antithyroid treatments should be executed. Moreover, we reveal that recovery following experimental hyperthyroidism could potentially ameliorate cardiac function and decrease the risk for additional cardiac complications, yet, this appears to be model-dependent and should be cautiously construed.Persistent cardiovascular pathology has been described in hyperthyroid patients even with effective antithyroid treatment. Here, we studied the effect of a well-known antithyroid drug, propylthiouracil , on thyroxine (T4; 500  The heart is a well-known target of thyroid hormones. Hyperthyroidism may intensify antecedent heart disorder; nonetheless, it could also induce new cardiovascular diseases such as pulmonary hypertension , atrial Cardiovascular complications have been revealed to significantly contribute to the death of hyperthyroid patients. Thus, early, efficient management of cardiac manifestations is essential for these patients. Yet, the heart failure treatment in hyperthyroid patients is challenging as it may be related to various entities . Additioµg/kg/day in the adult FVB/N male mice for 2 weeks leads to hypertension, cardiac hypertrophy, LV systolic dysfunction, and contractile changes in the isolated RV papillary muscles [So far, inconsistent data has been conveyed on the cardiovascular outcomes following euthyroidism restoration in hyperthyroid patients either by effective antithyroid therapy , 10, 11  muscles –24. TherAdult (9-10 months old) FVB/N male mice were purchased from the Jackson Laboratory  and kept at the Research Animal Facility of The Ohio State University for the whole duration of the experimental procedures. The experimental procedures and protocols used in this study were approved by the Animal Care and Use Committee of the Ohio State University, conforming to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health . A total of 38 mice were used for all experiments as described below.µg/kg/day for 2 weeks [Basal BP as well as in vivo LV dimension and contractile function were assessed in 19 untreated mice. Sodium-L-thyroxine, T4, from Sigma-Aldrich  was prepared as described previously  and inje 2 weeks –24, 26.  2 weeks . At the  2 weeks , 24 showBP was measured noninvasively in conscious untrained mice by the tail cuff method using a six-Channel CODA High Throughput Acquisition system  as described before . Each exBy using a high-frequency ultrasound imaging system  the in vivo LV dimension and contractile function in mice were evaluated as described before –25. Expe4, 1.2 NaH2PO4, 20 NaHCO3, 0.25 Ca2+, and 10 glucose, equilibrated with 95% O2- 5% CO2, resulting in a pH of 7.4. Additionally, 20 mmol/L 2,3-butanedione monoxime (BDM) was added to the dissection buffer to prevent cutting injury [µm). The cross-sectional areas were calculated assuming ellipsoid cross-sectional shapes. There was no significant difference between average dimensions (width × thickness × length) of the Basal (0.31 ± 0.04 × 0.21 ± 0.03 × 0.87 ± 0.14 mm), T4 (0.28 ± 0.02 × 0.19 ± 0.01 × 0.68 ± 0.11 mm), PTU + T4 (0.27 ± 0.02 × 0.18 ± 0.01 × 0.81 ± 0.11 mm), and recovery (0.28 ± 0.02 × 0.19 ± 0.01 × 0.87 ± 0.10 mm) groups. P values are 0.9760 , 0.9760 , and 0.6568 , respectively.First, mice were weighed, and five minutes after IP heparin injection mice were euthanized by cervical dislocation. After bilateral thoracotomy, hearts were rapidly excised and placed in Krebs–Henseleit buffer containing (in mmol/L): 120 NaCl, 5 KCl, 2 MgSOg injury –24. Extr2+ concentration was raised to 2 mmol/L and muscles were allowed to stabilize for at least 30 minutes before the experimental protocol was initiated. The 4 Hz baseline was selected rather than a more physiological 12 Hz, as in our previous reports [With the use of the dissection microscope, muscles were mounted between basket-shaped extension of a force transducer  and a hook  connected to a micromanipulator as previously mentioned –24. Musc reports –24. Howe reports .β-adrenergic stimulation were assessed in mouse papillary muscles under near physiological conditions as previously mentioned [β-adrenergic stimulation were assessed by a concentration–response curve with isoproterenol (10−9–10−6 mol/l) at a baseline stimulation frequency of 4 Hz.To obtain a broad scope of quantitative data to dissect contractile function and dysfunction, two of the three main mechanisms utilized in vivo to physiologically modify force of contraction, frequency-dependent activation, and entioned –24. We aFdev) was determined and normalized to the cross-sectional area of the muscle. Additionally, as a force-independent parameter of force decay kinetics, time to peak force (TTP) and time from peak force to 50% relaxation (RT50) were determined. Muscles with an initial Fdev or a Fdev after restabilization following frequency-dependent activation < 5 mN/mm2 were excluded from the analysis of all experimental parameters. Additionally, muscles that displayed arrhythmia early at the initial isoproterenol concentrations were excluded from the final isoproterenol-Fdev data analysis.In all experiments performed peak isometric developed force  was applied followed by Dunn's post hoc multiple comparison test. On the other hand, ordinary repeated measures ANOVA assumes effective matching among means. If the matching appears not to be effective, Friedman test (nonparametric repeated measures ANOVA) was applied followed by Dunn's post hoc multiple comparison test.Generally, a two-tailed value of P < 0.001) (P = 0.2119), SBP (P = 0.0678), and MAP (P = 0.1446) to levels that also are not significantly different from those of the basal group  (P = 0.1634), SBP (P = 0.0647), and MAP (P = 0.1152) to levels that are insignificant compared to those of the basal group too   . PTU tre, resp.) . Similar, resp.) .P < 0.001) (P < 0.001) (P < 0.001) (P < 0.001) (P < 0.001) (P < 0.001) . Conversely, the 2-week T4 recovery clearly attenuated the T4-provoked increase in LV mass (124 ± 5 mg) (P < 0.05)    and frac< 0.001)  compared< 0.001)  and FS (< 0.001) . Besides< 0.001)  and LV m< 0.001)  compared< 0.001)  or LV ma< 0.001)  compared ± 5 mg) , but it  < 0.05)  compared < 0.05) .P < 0.001) and HW/BW ratio (P < 0.001) compared to the basal group, which confirm the cardiac hypertrophy development in these mice  (P < 0.001 and P < 0.01) and HW/ BW ratio (P < 0.001 and P < 0.001) compared to both T4-treated mice and (PTU + T4)-treated mice, respectively,  . Oppositctively, .μm [Fdev of RV papillary muscles was sustained (P = 0.3039) among the groups at a stimulation frequency of 4 Hz  (P < 0.001) (P < 0.05) (P < 0.001 and RT50: P < 0.01). However, 2-week T4 recovery returned the T4-induced decrease in TTP  (P < 0.001 versus T4 and PTU + T4 groups)  is well-maintained in the hearts of these T4-treated mice as we have lately defined . Thus, wμm , Fdev of mN/mm2) . Contrar< 0.001)  and RT50 < 0.05)  compared < 0.05)  or the R < 0.05) , and the groups)  and RT50 groups)  back to Fdev was estimated within the murine in vivo physiological range (8–12 Hz) in addition to the baseline stimulation frequency of 4 Hz. Unexpectedly, one-way ANOVA showed insignificant differences among basal, T4, or recovery groups in response to increasing frequencies at all frequency range. Nevertheless, PTU treatment along with T4 resulted in a significantly negative response to increasing frequency at 12 Hz and 14 Hz (P < 0.01) compared to the basal group (P < 0.001) and frequency (P < 0.001) variations on the Fdev. However, there was no significant interaction between the group and frequency variations. Additionally, the β-adrenergic stimulation effect was evaluated by a concentration–response curve with isoproterenol (10−9–10−6 mol/l) at a baseline stimulation frequency of 4 Hz. As we previously demonstrated [β-adrenergic stimulation (1 μmol/l isoproterenol), muscles from T4-treated mice displayed markedly depressed responses (P < 0.05) versus those from the basal group (P < 0.01) and PTU + T4 (P < 0.05) groups (P < 0.001) and isoproterenol concentration (P < 0.001) variations on the Fdev. Likewise, there was a significant interaction between the 2 variants (P < 0.001), as we previously verified [β-adrenergic stimulation (1 μmol/l isoproterenol): 7 out of 8 muscles from T4 group and 7 out of 10 muscles from PTU + T4 group showed arrhythmic behavior versus 1 out of 9 muscles from the recovery group and none out of 8 muscles in the basal group  groups . In addiverified –24. Moreal group . Hcn2, β2-adrenergic receptor, and Bnf, a thyroid hormone-dependent ventricular hypertrophy marker, were raised, along with severe bradycardia, concluding that thyrotoxicosis may have pathological consequences that could continue ahead of the serum T4 level recovery [Overt and subclinical hyperthyroid patients are at increased cardiac mortality risk. This excessive risk could be due to increased atrial arrhythmias as well as heart failure in these patients . Actuallrecovery . Howeverrecovery –24. To tPTU is a thiouracil derivative that is considered as one of the most commonly used antithyroid drugs in the hyperthyroidism management . This isP = 0.0678 and 0.0647, resp.), could be due to interaction with several pathways that have been reported to be involved in the development of hypertension, such as increased nitric oxide synthase [In accordance with our previous data –24 alongsynthase , 39–41 asynthase , 43. PTUsynthase . Also, isynthase  that is Noteworthy, our data exhibited that PTU significantly prevented the T4-induced in vivo LV dysfunction and the T4 usage recovery markedly inhibited all T4-prompted cardiac pathologies, including cardiac hypertrophy, in vivo LV dysfunction, and ex vivo RV contractile changes; however, they could not distinctly avert T4-stimulated hypertension in these mice. We have previously revealed that T4-induced cardiac changes in this model are independent of hemodynamic changes, that is, increased BP, which means that T4-induced cardiac pathology and hemodynamic alterations could be produced through different signaling pathways . A recenβ-adrenergic response together with increased arrhythmia. The difference among the basal and T4 groups on the frequency-response was similar, not statistically significant. This response  was smaller than in our previous studies, where the difference reached a statistical significance [T4-induced cardiac hypertrophy is well recognized and occasionally associated with LV dysfunction , 22–24 iificance . Only, Pβ-adrenergic response, or increased development of arrhythmia. Indeed, this is consistent with our previous data showing that apocynin, a NADPH oxidase inhibitor, only prevented the T4-induced LV dysfunction with no positive effects on either the cardiac hypertrophy or the ex vivo RV contractile changes [Here, we showed that PTU could not significantly attenuate the T4-induced cardiac hypertrophy, but it significantly improved the in vivo LV function as indicated by increased EF and FS compared to T4-treated mice. In agreement with our results, Wang et al. reported that PTU improved the cardiac function and life expectancy by increasing LV EF in dilated cardiomyopathy mice linked to local hyperthyroidism . In cont changes . PTU has changes . Overall changes . Still,  changes , was abl changes .On the other hand, we showed that T4 recovery due to cessation of T4 treatment for equal period of 2 weeks in the adult mice used in this study almost prevented all T4-induced cardiac pathologies, including cardiac hypertrophy, in vivo LV dysfunction, and ex vivo RV contractile changes. Importantly, T4 treatment in mice for 2 weeks followed by 2 weeks  or 10 daFinally, the heart primarily depends on serum T3 due to the absence of a major myocyte intracellular activity of deiodinase, which converts T4 to T3, and it looks that T3, but not T4, is conveyed into the myocyte. T3 binds to thyroid hormone nuclear receptors and exerts its cellular actions . But, itIn summary, our data is in agreement with the proposal that hyperthyroidism-induced cardiovascular pathology could persevere even with antithyroid treatments, such as PTU. However, this cannot be generalized and further investigation with different antithyroid treatments are warranted. Moreover, our data shows that recovery of experimental hyperthyroidism could potentially ameliorate cardiac function and decrease the risk for further cardiac complications. Yet, this appears to be model-dependent and should be cautiously construed. Finally, we propose that the mechanisms responsible for T4-promoted cardiovascular changes are possibly due to mutual thyroid hormone effects on certain molecular pathways, which require further exploration.


Allelic imbalance or loss of heterozygosity (LOH) studies have been used extensively to identify regions on chromosomes that may contain putative tumour-suppressor genes. We have undertaken an extensive allelotype of 80 specimens of squamous cell carcinoma of the head and neck (SCCHN) using 145 polymorphic microsatellite markers on 39 chromosome arms. Allelic imbalances were found most frequently on chromosome arms 3p, 9p, 17p and 18q with over 45% LOH and imbalances on 1p, 1q, 2p, 5q, 6p, 6q, 8p, 8q, 9q, 11q, 13q, 17q and 19q were found in more than 20% of SCCHN. These LOH data were analysed against a range of clinicopathological parameters which included previously untreated and previously treated tumours; correlations were found between LOH on 9q and nodes at pathology (P = 0.02) and between histopathological grade and LOH on 12q (P = 0.02) and 13q (P = 0.01). In the group of previously untreated tumours, a correlation was found between site of tumour and LOH on 3p (P = 0.019), and 8p (P = 0.029), while TNM staging correlated with LOH on 3p (P = 0.019) and 17p (P = 0.016). Fractional allele loss (FAL) was calculated for 52 tumours with LOH data on nine or more chromosomal arms and found to have a median value of 0.22 (range 0.0-0.80). Correlations were found between FAL > median value and nodes at pathology (P = 0.01) and tumour grade (P = 0.06), demonstrating that advanced tumours with lymph node metastasis often had LOH at multiple sites. FAL > median value was found to correlate with a poor survival (P < 0.03) and, furthermore, FAL > median value correlated with poor survival in the previously untreated patients (P < 0.019). These results indicate that assessment of the accumulation of genetic damage, as provided by allelotype data, provides a useful molecular indicator of the tumour behaviour and clinical outcome.