Stability of fatty acyl-coenzyme a thioester ligands of hepatocyte nuclear factor-4α and peroxisome proliferator-activated receptor-α

Although long-chain fatty acyl-coenzyme A (LCFA-CoA) thioesters are specific high-affinity ligands for hepatocyte nuclear factor-4α (HNF-4α) and peroxisome proliferator-activated receptor-α (PPARα), X-ray crystals of the respective purified recombinant ligand-binding domains (LBD) do not contain LCFA-CoA, but instead exhibit bound LCFA or have lost all ligands during the purification process, respectively. As shown herein: (i) The acyl chain composition of LCFA bound to recombinant HNF-4α reflected that of the bacterial LCFA-CoA pool, rather than the bacterial LCFA pool. (ii) Bacteria used to produce the respective HNF-4α and PPARα contained nearly 100-fold less LCFA-CoA than LCFA. (iii) Under conditions used to crystallize LBD (at least 3 wk at room temperature in aqueous buffer), 16∶1-CoA was very unstable in buffer alone. (iv) In the presence of the respective nuclear receptor (i.e., HNF-4α and PPARα), LBD 70–75% of 16∶1-CoA was degraded after 1 d at room temperature in the crystallization buffer, whereas as much as 94–97% of 16∶1-CoA was degraded by 3 wk. (v) Cytoplasmic LCFA-CoA binding proteins such as acyl-CoA binding protein, sterol carrier protein-2, and liver-FA binding protein slowed the process of 16∶1-CoA degradation proportional to their respective affinities for this ligand. Taken together, these data for the first time indicated that the absence of LCFA-CoA in the crystallized HNF-4α and PPARα was due to the paucity of LCFA-CoA in bacteria as well as to the instability of LCFA-CoA in aqueous buffers and the conditions used for LBD crystallization. Furthermore, instead of protecting bound LCFA-CoA from autohydrolysis like several cytoplasmic LCFA-CoA binding proteins, these nuclear receptors facilitated LCFA-CoA degradation.     

Comparative effect of fenofibrate on hepatic desaturases in wild-type and peroxisome proliferator-activated receptor α-deficient mice

In this study is presented the effect of fenofibrate, a prototypical peroxisome proliferator of the fibrate class, on wild-type and peroxisome proliferator-activated receptor α (PPARα)-/-mouse liver FA profile, desaturase mRNA levels, and activities. We established that, following peroxisome proliferator exposure, the hepatic FA profile was greatly modified. These modifications in hepatic FA content required the expression of PPARα, as they are suppressed in transgenic mice deficient in this nuclear receptor. Following peroxisome proliferator exposure, Δ6- and Δ5-desaturase mRNA levels and activities were increased in wild-type but not in PPARα-deficient mouse liver. These results suggest the involvement of PPARα in the control of hepatic Δ6- and Δ5-desaturases in mice. Their roles in minimizing long-chain PUFA depletion in the liver during peroxisome proliferator exposure are discussed.     

Effect of p-aminobenzoic acid on synthesizing ordered mesoporous alumina via the sol–gel method

Ordered mesoporous alumina (OMA) samples were prepared via the sol–gel method. The triblock copolymer P123 and aluminum isopropoxide were chose as the template and aluminum source, respectively. The p-aminobenzoic acid (PABA) worked as both the interfacial protector and the assisted template. Different techniques, including X-ray diffractions, N₂ physisorption measurement and transmission electron microscope, were employed to study the structural properties of the prepared samples. The results showed that the mesostructure ordering and specific surface area of the samples highly depend on the quantity of the added PABA. The samples after calcinated at 800 °C with the n(PABA)/n(Al³⁺) molar ratio lying in 0.30 exhibited high specific surface areas (301 m² g^?1) and retained an ordered mesostructures. It was indicated that the carboxyl groups of PABA have multiple functions in the formation of OMA with a long-range ordering and high specific surface area. The carboxyl groups of PABA can slow down the hydrolysis-condensation reaction of aluminum isopropoxide, reduce the influence of chloride ions on self-assembly process. The amino groups of PABA can also react with the hydrogen ions to form quaternary ammonium salt, which can neutralize the charges of the chloride ions, protect the balance of inorganic–organic interface, further to improve the mesostructure ordering and specific surface area of the prepared OMA.     

Effect of heating rate on microstructure and properties of spark plasma sintered Li-α-sialon

Li-α-sialon ceramics with low oxygen content were prepared by spark plasma sintering at 1750 °C, using three heating rates of 100 °C/min, 200 °C/min and 300 °C/min. In all cases, the densification of Li-α-sialon ceramics is effectively promoted. The rapidly anisotropic growth of grains, either α-sialon or β-sialon, is significantly enhanced with a heating rate above 200 °C/min. This could be attributed to a large amount of low viscosity oxygen-rich liquid formed at elevated temperatures, which gives rise to a so-called dynamic ripening mechanism at the early stage of sintering. Furthermore, for the composition near α-sialon boundary, the oxygen-rich liquid results in the formation of β-sialon together with a large amount of intergranular glassy phase. Only the Li-doped sialon with low lithium and oxygen content possesses relatively high infrared transmittance when sintered at a rate of 100 °C/min.     

Effect of the composition of α-MSAN copolymer on the miscibility of PVC/α-MSAN blends

A series of α-methylstyrene, styrene, and acrylonitrile (α-MSAN) copolymers with different acrylonitrile (AN) contents were synthesized by altering α-MSt, St, and AN ratios with emulsion copolymerization method. By melt-blending these copolymers with PVC resin and di-isooctyl phthalate (DOP), PVC/α-MSAN, and PVC/α-MSAN/DOP blends were prepared. The miscibility and morphology of the blends were investigated by dynamic mechanical analysis (DMA) and scanning electron microscopy. The PVC is immiscible with SAN by melt-mixing, whereas PVC is miscible with α-MSAN (α-MSt/St = 1/1) if AN weight percent is within the window range of 20–25 wt %, and α-MSAN (not containing St) with 35 wt % AN is miscible with PVC even when they are blended by melt-mixing. Replacement of styrene with α-methylstyrene widens the miscibility window with PVC. The miscibility of PVC/α-MSAN blends is substantially improved with the increasing α-MSt content in α-MSAN copolymer containing identical AN content. When DOP was introduced into the PVC/α-MSAN (α-MSt/St = 1/1) blends, a single tan δ peak over room temperature in DMA detection is found as AN content in α-MSAN copolymer is within the range of 15–25 wt %, and SEM observation also shows that the blends are homogeneous. When the AN content in α-MSAN copolymer is over 35 wt %, the presence of DOP causes the phase domain extended. The phase domain size of the PVC/α-MSAN/DOP blends intensively depends on AN content in α-MSAN copolymer. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Effect of γ-Cyclodextrin Inclusion Complex on the Absorption of R-α-Lipoic Acid in Rats

R-α-lipoic acid (RLA) is an endogenous organic acid, and works as a cofactor for mitochondrial enzymes and as a kind of antioxidant. Inclusion complexes of RLA with α-, β- or γ-cyclodextrins (CD) were prepared and orally administered as a suspension to rats. Among them, RLA/γ-CD showed the highest plasma exposure, and its area under the plasma concentration-time curve (AUC) of RLA was 2.2 times higher than that after oral administration of non-inclusion RLA. On the other hand, the AUC after oral administration of non-inclusion RLA and RLA/γ-CD to pylorus-ligated rats did not differ. However, the AUC after intraduodenal administration of RLA/γ-CD was 5.1 times higher than that of non-inclusion RLA, and was almost comparable to the AUC after intraduodenal administration of RLA-Na solution. Furthermore, the AUC after intraduodenal administration of RLA/γ-CD was not affected by biliary ligation or co-administration of an amylase inhibitor. These findings demonstrated that RLA was absorbed from the small intestine effectively when orally administered as a γ-CD inclusion complex, which could be easily dissolved in the lumen of the intestine. In conclusion, γ-CD inclusion complex is an appropriate formulation for supplying RLA as a drug or nutritional supplement with respect to absorption.     

Effect of NaCl on the properties of Eu-doped Ca-α-SiAlON phosphors prepared by combustion synthesis

Eu-doped Ca-α-SiAlON phosphors, featuring high phase purity, uniform particle size of 3–5 μm and good luminescent properties with a yellow emission spectrum under blue light excitation, were prepared by a highly efficient combustion synthesis (CS) method. A certain amount of NaCl was applied as an innovative additive to regulate and control the properties of synthesized phosphors. Further, the effects of NaCl additive in the CS system were systematically investigated and rational proposed. It was found that the effect on accelerating nitridation and crystallization played a dominant role in the reaction as the content of NaCl was less than 6 wt%, while the effect of absorbing reaction heat through vaporization was dominant with the further-increased content of NaCl. The intensity of the emission spectrum for the sample doped with 6 wt% of NaCl was remarkably enhanced, nearly 40% more than the sample which was not NaCl-doped. Moreover, a continuous blue-shift phenomenon in emission spectra was observed with the increased content of NaCl.     

Effect of dopant on ferroelectric,dielectric and photocatalytic properties of Co–La-doped dysprosium chromite prepared via microemulsion route

In the current study, pristine and a series of La and Co-doped dysprosium chromite (Dy_1-yLa_yCr_1-xCo_xO₃) nanoparticles have been fabricated via a facile microemulsion technique. The influence of doping was evaluated based on structural, ferroelectric, dielectric, and photocatalytic properties. The prepared nanoparticles were characterized by XRD, SEM, Raman, and UV–Vis techniques. XRD patterns confirm the synthesis of a monophase orthorhombic structure with space group Pbnm with an average crystalline size in the 18–37 nm range. The saturation polarization (Ps), remanence (Pr), and coercivity (Hc) were determined using a hysteresis loop, and it was observed that by increasing the concentration of dopants, the value of Ps and Pr were improved. According to the PL spectra, highly substituted materials had a low recombination rate and higher charge separation (e^? - h⁺), which was ultimately accountable for higher photocatalytic activity. The dielectric loss decreases with frequency and dopant concentration. The photocatalytic activity of Dy_1-yLa_yCr_1-xCo_xO₃ was investigated against Crystal Violet (CV) dye under sunlight irradiation. The Dy_1-yLa_yCr_1-xCo_xO₃ furnished a 70% dye degradation in 90 min, which is attributed to the tunned bandgap and efficient electron-hole pair separation and the photocatalytic activity under visible light making Dy_1-yLa_yCr_1-xCo_xO₃ a promising photocatalyst for dye removal from wastewater.     

Effect of Thyrotropin on Osteopontin,Integrin αvβ3, and VCAM-1 in the Endothelium via Activation of Akt

Numerous epidemiological studies have shown that subclinical hypothyroidism (SCH) can impair endothelial function and cause dyslipidemia. Studies have evaluated the effects of thyroid stimulating hormone (TSH) on endothelial cells, but the mechanism underlying the proatherosclerotic effect of increased TSH levels remains unclear. In the present study, SCH rat models were established in thyroidectomized Wistar rats that were given l-T₄ daily. The results showed that in vivo, the expression of osteopontin (OPN) vascular cell adhesion molecule (VCAM-1), and levels of integrin α_vβ₃ in the aortic tissue in SCH and Hypothyroidism (CH) groups was higher than in the control group. However, the effect in the SCH group was higher than in the CH group. In vitro, results showed that different concentration and time gradients of TSH stimulation could increase the expression of OPN, VCAM-1, and integrin α_vβ₃, and this was accompanied by extracellular signal regulated kinase 1/2 (Erk1/2) and Akt activation in human umbilical vein endothelial cells (HUVECs). TSH induced elevation of these proatherosclerotic factors was partially suppressed by a specific Akt inhibitor but not by a specific Erk inhibitor. Findings suggested that the endothelial dysfunction caused by SCH was related to increased proatherosclerotic factors induced by TSH via Akt activation.     

Effect of compatibilizer on interfacial tension of SAN/EPDM blend as measured via relaxation spectrums calculated from Palierne and Choi–Schowalter models

Morphology, interfacial tension, and stress relaxation spectra of immiscible SAN/EPDM blend and its compatibilized blend with SAN-g-EPDM (Centrex) was studied. The results showed that the morphology of the blend had a quick response to added Centrex. In the compatibilized blend with 20-wt% compatibilizer (optimized blend) having a droplet-in-matrix type of morphology, the particle sizes were reduced by a factor of 4. The power-law index of EPDM and SAN obtained 0.33 and 0.53, respectively. With increasing of compatibilizer the power-law index decreased. It meant that at the same amount of EPDM its influence in the blend was increased. Also the cross-over point of G′ and G″ curves in the melt of optimized blend decreased which was attributed to increased elasticity. These observations were in good correspondence with the morphological observations. In optimized blend, the number average diameter of EPDM dispersed particles had the lowest value of about 1.8 μm. The interfacial tension of the compatibilized SAN/EPDM blend was determined from the morphological studies and the relaxation time was calculated using the Palierne and Choi-Schowalter models. The optimized blend showed the least interfacial tension about 0.306 (N/m) which was in agreement with the morphological observations.     

Effect of specific surface area on capacitance in asymmetric carbon/α-MnO2 supercapacitors

α-MnO₂ has been made using a solid state synthesis and the specific surface area then modified through milling. The formation of α-MnO₂ (specific surface area 96 m² g⁻¹) has been studied by SEM and powder XRD prior to milling. Electrode films (cast using MnO₂, graphite and PVDF) have been investigated using N₂ sorption at 77 K and show a more complex relationship than their parent oxides. Specific capacitances of 235 F g⁻¹ were observed in cyclic voltammetry studies in (NH₄)₂SO₄ (aq.) electrolyte. Good cyclability was observed in hybrid C/MnO₂ cells investigated through both galvanostatic and electrochemical impedance techniques. The specific capacitances of the cells were found to correlate with S_BET of the electrode films and not that of the parent MnO₂ powders.     

Effect of calcination temperature on friction and wear behavior of α–alumina (α-Al2O3) for biomedical applications

In this study, structural evolution, sliding friction, and wear behavior of the nano-sized alpha-alumina (α-Al₂O₃), synthesized by calcination of gibbsite (Al₂(OH)₆) at different temperatures (273-1473 K) are reported using X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Scanning electron microscopy (SEM), and ball-on-disk wear tests. The effect of calcination temperature and the correlation between grain size, mechanical properties, and tribological behavior of the compounds are investigated. After calcination at 1473 K, the crystallite size of calcined and sintered alumina (α-Al₂O₃) compacts is as small as 10 nm. Also, it is observed that the average particle and grain size significantly affect the tribological properties of the compounds. At all the applied loads of 2, 8, and 16 N, respectively, the sliding wear rate and coefficient of friction were lower in the alumina compacts calcined at 1473 K. The improved tribological properties are attributed to the finer microstructure resulting in enhanced hardness of the sintered compacts calcined at 1473 K.     

Effect of yttria doping on the microstructure and mechanical properties of Al2O3–FeAl2O4 nanocomposites developed via solid state precipitation

We have recently reported the production of Al₂O₃-matrix nanocomposites via solid state precipitation of nanosized FeAl₂O₄ particles within the matrix grains during aging of Al₂O₃–10 wt.% Fe₂O₃ solid solutions in a reducing atmosphere (N₂ + 4% H₂). In addition to these nanoparticles, however, coarse micron-sized FeAl₂O₄ particles were present along the matrix grain boundaries. In the present work, we show that the addition of ～250 ppm yttria to the solid solutions suppressed the development of these intergranular particles, reducing their size by a factor of ～2 with optimum aging. A fracture toughness improvement by 45% and flexural strength improvement by 50% with respect to monolithic Al₂O₃ were recorded with the yttria-containing nanocomposite developed by aging for 20 h at 1450 °C. Aging also improved the hardness with respect to the solid solution. The change in fracture mode in the presence of the nanosized intragranular particles was believed to be the major contributing factor towards the improvement in toughness and therefore the strength. The higher strengths obtainable in the presence of yttria were attributed to the reduction of intergranular precipitate size relative to yttria-free nanocomposites.     

Effect of Fe2O3 additive on the preparation of Si3N4−Si2N2O composite ceramics via diamond-wire saw silicon waste

In this study, Si₃N₄−Si₂N₂O composite ceramics were prepared through a two-step combination of the direct nitriding method and spark plasma sintering method with diamond-wire saw silicon waste in the photovoltaic industry as raw material. The whole holding time at the constant temperature of the preparing process only lasts 35 min, in which the nitriding time only takes 30 min and the sintering time only takes 5 min. The effects of the Fe₂O₃ additive on the nitriding and sintering process were investigated in detail. The results show that the Fe₂O₃ additive accelerates the nitriding process by promoting the formation and diffusion of the gas phase and reducing the apparent activation energy of the reaction between the gas phase containing silicon and N₂. Meanwhile, the Fe₂O₃ additive also promotes the sintering process by lowering the eutectic temperature of the SiO₂−Al₂O₃ and the SiO₂−Y₂O₃ binary system. The optimal technical parameters follow the nitriding temperature of 1400°C, the nitriding time of 30 min, the sintering temperature of 1800°C, and the sintering time of 5 min. The Si₃N₄−Si₂N₂O composite ceramic prepared under the above conditions exhibits good performance with an apparent porosity of 2.7%, Vickers hardness of 16.54 GPa, and fracture toughness of 4.1 MPa·m^1/2.     

Effect of Calcination Temperature on Structure and Properties of Sn–Nb2O5/α-Al2O3 Catalyst for Ethylene Oxide Hydration

Sn–Nb₂O₅/α-Al₂O₃ catalysts were prepared by impregnation and tested for ethylene oxide (EO) hydration to form ethylene glycol. The effect of the calcination temperature on the structure, acidity, H₂O and EO adsorption properties and catalytic performance of the catalyst were investigated by using XRD, TG-DTA, IR, NH₃-TPD and EO-TPD. It was found that the phase compositions, acidity, the EO adsorption strength and water adsorption capacity of the Sn–Nb₂O₅/α-Al₂O₃ catalyst were markedly influenced by the calcination temperature. The catalyst calcined at a temperature between 350 and 400 °C showed the best catalytic performance. A correlation between catalytic performance and characterization was proposed.     

The influence of sintering temperature on the structure,optical and magnetic properties of Yttrium iron oxide YFeO3 prepared via Lα-alanine assisted combustion method

Single-crystalline YFeO₃ nanoparticles with orthorhombic perovskite structure were synthesized via L_α-alanine assisted combustion approach. The effect of sintering temperature on the lattice and distortion parameters of the YFeO₃ was discussed. The increase of the sintering temperature increased the lattice parameters and crystallite size, while decreased spontaneous strain, cell distortion, orthorhombic distortion and global instability factors. The sintering temperature did not show any significant effect of the atomic percentage of the elemental compositions of the YFeO₃, where the powders showed excellent stoichiometry preserving the Y:Fe ratio (1:1). SEM micrographs showed that the YFeO₃ powders exhibited quasi-rectangular shape. The magnetic properties showed an improvement of the remnant magnetization and coercivity with increasing the sintering temperature, owing to the decrease of distortion of orthorhombic perovskite structure. The diffuse reflectance UV–vis spectrophotometer was employed to investigate the optical behavior and band gap of the sintered YFeO₃ powders at different temperatures. It was noticed that the reflectance bands are blue shifted and the optical band gap is decreased from 2.02 to 1.44 eV. This will gave rise to the possibility to use the developed YFeO₃ orthoferrites as magnetic recoverable catalyst.     

Effect of Alkanediyl-α,ω-Type Cationic Dimeric (Gemini) Surfactants on the Reaction Rate of Ninhydrin with [Cu(II)-Gly-Tyr]+ Complex

The effect of gemini (16‐s‐16, s = 4, 5, 6) surfactants on the reaction rate of ninhydrin with [Cu(II)‐Gly‐Tyr]⁺ complex was determined using a spectrophotometric technique. The ninhydrin concentration was kept in excess in order to maintain pseudo‐first‐order conditions. The reaction followed irreversible first‐ and fractional‐order kinetics with respect to [Cu(II)‐Gly‐Tyr]⁺ and [ninhydrin], respectively. It is found that gemini surfactants effectively catalyze the reaction. The rate constants (k_ψ) first increase and then become relatively constant with increasing gemini surfactant concentration similar to conventional cetyltrimethylammonium bromide. At higher gemini surfactant concentration a third region of increasing k_ψ is observed. The unusual third region is ascribed to changes in micellar morphology. The kinetic data has been analyzed using a micellar pseudo‐phase model.     

Effect of a natural extract of chicken combs with a high content of hyaluronic acid (Hyal-Joint<Superscript>®</Superscript>) on pain relief and quality of life in subjects with knee osteoarthritis: a pilot randomized double-blind placebo-controlled trial

Background

Intra-articular hyaluronic acid represents a substantive addition to the therapeutic armamentarium in knee osteoarthritis. We examined the effect of dietary supplementation with a natural extract of chicken combs with a high content of hyaluronic acid (60%) (Hyal-Joint^®) (active test product, AP) on pain and quality of life in subjects with osteoarthritis of the knee.

Methods

Twenty subjects aged ≥40 years with knee osteoarthritis (pain for at least 15 days in the previous month, symptoms present for ≥6 months, Kellgren/Lawrence score ≥2) participated in a randomized double-blind controlled trial. Ten subjects received AP (80 mg/day) and 10 placebo for 8 weeks. The Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and quality of life by the Short Form-36 (SF-36v2) were administered at baseline and after 4 and 8 weeks of treatment.

Results

WOMAC pain (primary efficacy variable) was similar in both study groups (mean [SD]) with 6.6 (4.0) points in the AP group and 6.4 (2.7) in the placebo group (P = 0.943). As compared with baseline, subjects in both groups showed statistically significant improvements in WOMAC pain, stiffness, physical function subscales, and in the aggregate score, but the magnitude of changes was higher in the AP group for WOMAC physical function (-13.1 [12.0] vs. -10.1 [8.6], P = 0.575) and total symptoms (-18.6 [16.8] vs. -15.8 [11.4], P = 0.694). At 4 weeks, statistically significant mean changes compared with baseline were observed in the SF-36v2 scales of role-physical, bodily pain, social functioning and role-emotional among subjects in the AP group, and in physical functioning, bodily pain, and social functioning in the placebo group. At 8 weeks, changes were significant for role-physical, bodily pain, and physical component summary in the AP group, and for physical functioning and role-emotional in the placebo arm. Changes in bodily pain and social functioning were of greater magnitude in subjects given AP.

Conclusion

This pilot clinical trial showed that daily supplementation with oral hyaluronic acid from a natural extract of chicken combs (Hyal-Joint^®) was useful to enhance several markers of quality of life in adults with osteoarthritis of the knee. The results warrant further study in larger sample sizes.

     

Protective Effect of Resveratrol against IL-1β-Induced Inflammatory Response on Human Osteoarthritic Chondrocytes Partly via the TLR4/MyD88/NF-κB Signaling Pathway: An “in Vitro Study”

Resveratrol is a natural polyphenolic compound that prevents inflammation in chondrocytes and animal models of osteoarthritis (OA) via yet to be defined mechanisms. The purpose of this study was to determine whether the protective effect of resveratrol on IL-1β-induced human articular chondrocytes was associated with the TLR4/MyD88/NF-κB signaling pathway by incubating human articular chondrocytes (harvested from osteoarthritis patients) with IL-1β before treatment with resveratrol. Cell viability was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and TNFα levels in culture supernatants were measured by ELISA(Enzymelinked immunosorbent assay). The levels of TLR4 and its downstream signaling targets (MyD88 and TRAF6) and IL-1β were assessed by measuring the levels of mRNA and protein expression by real-time RT-PCR and western blot analysis, respectively, in addition to assessing NF-κB activation. In addition, TLR4 siRNA was used to block TLR4 expression in chondrocytes further demonstrating that resveratrol prevented IL-1β-mediated inflammation by TLR4 inhibition. We found that resveratrol prevented IL-1β-induced reduction in cell viability. Stimulation of chondrocytes with IL-1β caused a significant up-regulation of TLR4 and its downstream targets MyD88 and TRAF6 resulting in NF-κB activation associated with the synthesis of IL-1β and TNFα. These IL-1β-induced inflammatory responses were all effectively reversed by resveratrol. Furthermore, activation of NF-κB in chondrocytes treated with TLR4 siRNA was significantly attenuated, but not abolished, and exposure to resveratrol further reduced NF-κB translocation. These data suggested that resveratrol prevented IL-1β-induced inflammation in human articular chondrocytes at least in part by inhibiting the TLR4/MyD88/NF-κB signaling pathway suggesting that resveratrol has the potential to be used as a nutritional supplement to counteract OA symptoms.