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As pointed above, sulfuric acid hydrolysis leads to reduced thermal stability of acid hydrolyzed CNC compared to that of neat cellulosic feedstock. However, this effect can be diminished by neutralizing the CNCH2SO4 with NaOH solution (~1% NaOH). After neutralization with NaOH, no acid sulfate groups remained, and the thermal stability of neutralized CNCCr(NO3)3 was shifted to a higher temperature. However, the presently used method for isolating the CNCCr(NO3)3 has some restrictions, such as prolonged production time and capital cost in order to remove the free sulfuric acid remaining on the CNCCr(NO3)3 surface after hydrolysis process, for their use in industrial scale. Thus far, the residual sulfuric acid in the cellulosic fiber is usually eliminated by the dialysis process against water until neutral pH is achieved, which is a costly procedure and usually takes a long time (3–7 days) . Some studies reported that the presence of the divalent group (SO42−) or anionic (–OSO2O–) of HSO4 and sulfates on the surface of nanocellulose may significantly reduce the interaction of cellulose fibrils with other polymer and biopolymer materials . Even if the chemical neutralization method is simpler with less processing steps to produce unsulfated CNCH2SO4 than time-consuming dialysis process, the large amount of waste effluents (salts) produced during the neutralization process needs to be further recycled before being discharged into the local waterway or ecological system. In fact, the sulfur content is only able to affect the thermal behavior of the nanocellulose, but maybe not influence or cause a negative effect on the crystallinity of the H2SO4 hydrolyzed CNC . In this study, the thermal degradation pattern of neutralized CNCH2SO4 was similar as native cellulose and CNCCr(NO3)3, in which the low-temperature degradation peak in the DTG curve disappeared completely after neutralization. The neutralized CNCH2SO4 started to degrade at the temperature of ca. 237 °C. As expected, the Tmax value of neutralized CNCH2SO4 profoundly increased to 349 °C, which can be explained by two main reasons. First, the absence of amorphous regions in the treated cellulose matrix and the highly structured crystals segments rendered the higher-temperature degradation. Second, the replacement of hydrogen ions (H+) from acid hydrolysis by alkaline ion (Na+) inhibited the dehydration catalyzed by H2SO4 .
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After 500 °C, the thermal deposition temperature of all the cellulosic samples leveled off, and a slow thermal degradation profile was attained. This can be accredited to the further carbonization of polysaccharide chains initiated by the cleavage of C–C and C–H bonds. According to Figure 6, it is interesting to note that the sulfated-CNCH2SO4 possesses the highest residues (black char) at 600 °C as compared to that of other cellulosic sample. This behavior was attributed to that the sulfate groups in the CNCH2SO4 tend to play the role of flame retardant by promoting the dehydration reactions, which is conducive to the production of anhydrocellulose and leads to its decomposition to carbonaceous residues, as reported by other authors as Mohamed and his group . Moreover, the CNCH2SO4 product with the acid sulfate group might be decomposed by more pathways by the action of acid, and, eventually, the intermediate products obtained, which could be further decomposed into the char residue, were more complex compared to the cellulose with acid content. Therefore, it is expected that char yield of neutralized CNCH2SO4 (6.57%) is remarkably lower than sulfated CNCH2SO4 (17.4%), considering the lack of remnant surface sulfate in the CNCH2SO4. It is quite interesting to note that the char level of neutralized CNCH2SO4 and CNCCr(NO3)3 (6.57%–9.03%) was much lower than native cellulose (10.2%). The decreased char residue for the nanocellulose sample may be mostly related to the increased split hydrogen bonds and/or due to the absence of non-cellulosic components in the treated nanocellulose after hydrolysis treatment . Furthermore, the presence of alkaline ions with highly polar field might act as the catalyst for complete degradation of glycosidic linkages via the hemolytic mechanism, which resulted in the decrement of char yield .
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In view of the above thermogravimetric analysis, it was concluded that the produced CNCCr(NO3)3 product from cellulosic feedstock exhibits better thermal stability than sulfated CNCH2SO4, and it will be more suitable for the production of green bio-nanocomposites as the typical processing temperature for polymer synthesis (i.e., thermoplastics) is normally higher than 200 °C. In this sense, CNCCr(NO3)3 with more thermal stability than that of MCC and sulfated CNCH2SO4 provide better-reinforcing capability for different applications such as cosmetics, thermal sensitive papers, and disposable products, owing to the high elastic modulus of its crystal domains . Table 2 summarizes the values of Tmax for cellulose and its nanomaterials derived from different species and collected from the literature.
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Figure 7 shows photographs illustrating the visual appearance of the aqueous suspension of CNCH2SO4 and CNCCr(NO3)3 samples with a white light source. For comparison, all nanocellulose samples were dispersed in water at the same concentration. After the ultrasonication process, the electronic photos of CNCH2SO4 and CNCCr(NO3)3 suspensions in the sample bottle are taken at 0 h, 24 h and 168 h. As can be seen in Figure 7a, two milk-like turbid suspensions were produced, suggesting that CNCH2SO4 and CNCCr(NO3)3 both formed stable dispersions in water, with the latter being whiter than the former. After 24 h standing, the turbid phenomena continued for CNCH2SO4 colloidal suspensions, as shown in Figure 7b. As the time prolonged, however, some sedimentation from CNCCr(NO3)3 were flocculated, whereas the good stability of CNCH2SO4 suspension could be kept at ambient temperature condition. For CNCCr(NO3)3, the original suspension had separated into a two-layer mixture, which consists of a clear layer and a turbid layer that can be visibly observed. This could be attributed to the fact that no surface groups have been attached to CNCCr(NO3)3 when prepared from Cr(NO3)3 metal salt catalyst, and thus the colloidal dispersion was expected to be less stable than CNCH2SO4 hydrolyzed by sulfuric acid. The high stability of sulfated CNCH2SO4 suspension can be achieved due to the sulfate negative charges (SO42−) on the CNCH2SO4 surface being able to create a strong electrostatic repulsion between the cellulose surfaces, and these charged groups limit their ability to flocculate . For this reason, CNCCr(NO3)3 showed a higher risk for agglomerate in a water medium.
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Due to the unmodified surface of CNCCr(NO3)3 with the high aspect ratio and high content of hydroxyl groups on the fiber surface, the CNCCr(NO3)3 tends to intertwine with their neighbor fibers and these networks were caused by the physical entanglement lock between the fibers. Furthermore, the lack of imparting electrostatic stability on CNCCr(NO3)3 resulted in poor stability, leading to agglomeration as compared to the sulfated CNCH2SO4 which remained individually dispersed. A similar observation has been reported by others . As a result, the CNCCr(NO3)3 suspension was unable to maintain its stability after being stored for seven days and obvious precipitation can be seen, while the CNCH2SO4 still remained a milky colloidal suspension with negligible precipitation, as displayed in Figure 7c. In summary, the excellent stability of CNCH2SO4 suspensions was beneficial to reinforce the hydrophilic polymers (such as polyvinyl alcohol). Further studies on the potential reinforcement of composites by the CNCH2SO4 and CNCCr(NO3)3 manufactured in this study are underway.
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When comparing the two nanocellulose production procedures, the important differences are noticed in terms of environmental impact, productivity, fiber quality, and economic point of view. For CNCH2SO4 production, concentrated sulfuric acid (64 wt %) is required; however, the strong acidic waste effluent is a potentially harmful hazard to the biological system or nearby environment if the acidic wastewater is being discharged without any purification treatment. Therefore, it is a necessary step to neutralize this acid waste with base chemicals (i.e., KOH, NaOH, or NaHCO3) in the waste neutralization system before it can be safely released into the environment. In contrast, the Cr3+-containing solution produced from the Cr(NO3)3 catalytic system can be treated using several effective methods . Among these techniques, chemical precipitation is worth mentioning as it is the most effective way for converting the Cr3+ to Cr(OH)3 via precipitation process with the presence of aqueous ammonia or sodium hydroxide. For commercial use, Cr(OH)3 is widely used as a pigment, mordant, and catalyst for organic reactions. This means that Cr(NO3)3 catalyst can not only produce value-added nanocellulose (solid residue) from the low-cost cellulosic feedstock, the generated supernatant may also be transferred into another useful byproduct, rather than discharged into the wastewater treatment plant without any profit. Approximately 9 kg of H2SO4 is required to produce 1 kg of CNCH2SO4 and the industry facing the difficulties in acid recovery economically. Therefore, the Cr(NO3)3 hydrolysis system could be sustainable and economically feasible. This will be the objective of further studies.
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Additionally, the CNCCr(NO3)3 produced via Cr(NO3)3 system rendered a better thermal stability than that of sulfated CNCH2SO4. This could make the CNCCr(NO3)3 a promising candidate for reinforcing agent and gas-barrier material, as well as providing convenience for polymer applications and processing performed under high temperatures, especially thermoplastics. Oppositely, the introduction of ester groups on the surface CNCH2SO4 could enhance its stability in suspension, which is beneficial for the manufacture of nanocomposites. Unfortunately, the uncontrollable degradation of cellulose into oligomers liquid fraction is a common phenomenon observed from H2SO4 hydrolysis, resulting in a very low yield (~55%). In other words, extra attention is necessary during the process not only due to the highly corrosive acid treatment, most importantly to prevent the acid hydrolysis process going too far. Table 3 displays an exhaustive list of nanocellulose obtained from numerous lignocellulosic biomasses by different hydrolysis techniques, detailing the reaction conditions and presenting the physicochemical properties.
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Microcrystalline cellulose (MCC) was purchased from Sigma-Aldrich (Saint Louis, MO, USA). The chemical compositions of the native cellulose determined according to the ASTM/TAPPI standard protocols are shown in Table 4. The hydrolyzing catalysts used in this study, namely sulfuric acid (H2SO4, 95%–97% purity) and chromium (III) nitrate (Cr(NO3)3), were bought from Merck (Kuala Lumpur, Malaysia). All chemicals were of analytical grade and used as received without further purification.
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In order to optimize the operating hydrolysis conditions, one-variable-at-a-time (OVAT) method was applied in this study to investigate the significance of every hydrolysis parameters in nanocellulose production. The catalytic hydrolysis reaction was initiated by reacting the native cellulosic material (1.0 g, oven dry weight) with Cr(NO3)3 solution (0.1–1.2 M) in a round-bottom flask with constant mechanical agitation using a heating mantle. The reaction was conducted at different temperatures (20–100 °C) and for different hydrolysis times (0.5–2.5 h). The solid–liquid ratio varied from 1:10 to 1:50. After the course of the reaction, the solid residue was separated from the reaction suspension via high-speed centrifugation (higher centrifuge speed can be used to improve separation) and washed thoroughly with cold deionized water until the wash water was maintained at constant pH. Afterward, the collected solid residue was subjected to lyophilization process and finally the fluffy white fiber product was obtained. Three replications were applied in each group of experiments in order to confirm the good reproducibility. The yield of CNCCr(NO3)3 was determined by gravimetric analysis, as described elsewhere .
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The procedure for isolating of CNCH2SO4 was adapted from with slight modification. Briefly, 1 g of cellulose material was dispersed in 100 mL deionized water through continuous mechanical stirring before the dropwise addition of 60 mL concentrated H2SO4 under an ice bath. This was to minimize a sudden temperature spike due to the exothermic reaction caused by the ion interaction between water and sulfuric acid. The suspension then heating up to 45 °C for 135 min. The reaction was terminated by diluting it with 10-fold cold water to avoid crystallization (iced water can be used to minimize dilution water usage while effectively terminating the hydrolysis reaction). The diluted suspension was centrifuged repeatedly to get the precipitate while removed the excess acid simultaneously by washing with deionized water. The supernatant was decanted off; fresh deionized water was added to the solids and mixed it well. This washing and centrifuging procedure were repeated until the supernatant was turbid, suggesting that CNCH2SO4 were being dispersed in solution. The turbid supernatant and settled solids were then mixed together and this slurry dialyzed against distilled water for 3–4 days until a constant pH was achieved, and freeze-dried to yield a white sulfate ester nanocellulose product. The dried product was stored in a vacuum for further product characterization.
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Fourier transform-infrared spectroscopy (FTIR) of native cellulose and yielded nanocellulose were analyzed using a FTIR spectrometer (Bruker IFX 66/S, (PerkinElmer, Germany)) in order to determine the functional groups of the cellulosic product. In fact, the effects of the hydrolysis treatment on the chemical composition changes can be tracked from the FTIR spectra. The FTIR analysis was carried out in the transmittance mode with a wavelength range of 4000–400 cm−1 with a resolution of 4 cm∓1 at 32 scans. The samples were ground and mixed with KBr powder with a ratio of 1:100 (w/w), then pressed into an ultra-thin pellet before the analysis.
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The impacts of the hydrolysis treatments on the crystallinity index of treated products can be investigated from the X-ray diffractograms. The crystallinities of native cellulose and isolated nanocellulose products were characterized by X-ray diffraction (XRD) study. The XRD patterns of the specimens were obtained from an X-ray diffractometer (PANalytical Empyrean) with a Cu-Kα radiation source which operated at 40 mA and 40 kV. The diffractograms were recorded from 5° to 60°. Crystallinity index (CrI) of samples was calculated by the peak deconvolution method as described by . Generally, the crystallinity of samples was calculated by dividing the total peak area of all the crystalline peaks by the total peak of all non-crystalline plus crystalline peaks according to the Equation (5): (5)CrI (%)AcAc+An×100 where Ac and Am are the area under the crystalline and non-crystalline regions, respectively.
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The surface morphology analysis of cellulose and nanocellulose products was studied with a field emission scanning electron microscope (FESEM; Hitachi SU8030, (Hitachi HTA, Schaumburg, IL, USA)) at an accelerating voltage of 5 kV. The samples were mounted on double-sided adhesive carbon tapes that stick on aluminum stubs, and coated with platinum using an auto-fine coater (JFC-1600, (JEOL, Ltd., Tokyo, Japan)) to improve conductivity and avoid over-charging. The elemental analysis of each sample was determined using EDX coupled with FESEM unit. The size and dimensions of yielded nanocellulose were further investigated by a transmission electron microscope (TEM; Tecnai G2 F20 Series, (FEI company, Hillsboro, OR, USA)) performed at a 200 kV acceleration voltage. To perform the TEM analysis, a nanocellulose suspension was treated with ultrasound for 3 min to separate agglomerated fibers. A droplet of the diluted suspension was deposited on a copper grid coated with a thin carbon film, and dried in a vacuum desiccator prior to analysis to ensure the samples were completely dry. The morphology and diameter of the yielded nanocellulose fibers were determined by analyzing the micrographs with ImageJ software (National Institutes of Health, New York, NY, USA).
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The thermal stability of samples was determined by a thermogravimetric analyzer (Model Q500 brand TA) under a nitrogen atmosphere. The sample was put in an aluminum cup and heated from 25 to 600 °C at a constant heating rate of 10 °C·min−1 in order to prevent any thermo-oxidative degradation.
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The changes in dispersion of the CNCH2SO4 and CNCCr(NO3)3 suspension after the hydrolysis process were studied through visual appearance in the aqueous medium (water). For this purpose, each nanocellulose aqueous suspension was prepared with a ratio of 2 mg/mL and the cellulose–water slurries were simultaneously treated by ultrasonication for 10 min in order to disperse CNCH2SO4 and CNCCr(NO3)3 thoroughly. The pictures of samples were taken immediately after the ultrasonic treatment and then allowed to stand motionlessly for 1 day and 7 days at room temperature and atmospheric pressure before further photographing.
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A comparative study on two types of nanocellulose products, namely CNCH2SO4 and CNCCr(NO3)3, derived from native MCC feedstock using concentrated H2SO4 and Cr(NO3)3 hydrolysis system, respectively, was conducted in this work. The optimal conditions for production of CNCCr(NO3)3 given by RSM methodology were 80 °C, 1.5 h, 0.8 M Cr(NO3)3 catalyst and solid–liquid ratio of 1:30. Under these experimental conditions, the manufactured CNCCr(NO3)3 displayed a spider-web-like fibrous morphology with a higher crystallinity of 86.5% ± 0.3%, yield of 83.6% ± 0.6%, and Tmax value of 344 °C. For comparison, the CNCH2SO4 hydrolyzed by H2SO4 exhibited a rice-shaped structure with the crystallinity index of 81.4% ± 0.1%, yield of 54.7% ± 0.3%, and Tmax value of 273 °C. Due to their crystallinity and aspect ratio differences, the isolated CNCCr(NO3)3 with higher crystallinity, higher aspect ratio (about 15.7) and more thermal stability could potentially be used as reinforcement material for application in various fields targeting specific properties. In conclusion, the proposed Cr(NO3)3 hydrolytic system is easy to implement and permits producing more thermally-stable nanocellulose with high yield under milder hydrolysis conditions, thus eluding the customary usage of conventional concentrated H2SO4 treatment for producing better quality nanocellulose.
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During many years the problem of the Apollonius tiling has interested a great number of scientists. Many well known mathematicians and physicists have worked on this for several hundred years. These include W. L. Bragg , F. Soddy , H.S.M. Coxeter , R. Descartes , P. Beecroft , and more recently P. G. De Gennes , whose works have a great importance in Soft Matter Physics. René Descartes, in a letter of November 1643 to Princess Elisabeth of Bohemia, developed a formula relating the radii of four mutually tangent circles (Descartes’s theorem), which is: ddeeff+ddeexx+ddffxx+eeffxx=2deffxx+2deeffx+2deefxx+2ddeffx+2ddefxx+2ddeefx where d, e, f are the radii of the three externally tangent circles and where x is the radius of the fourth circle. These notations have been simplified, demonstrated by Coxeter and generalized to the case of internal tangent circles in order to obtain:(1)1R4=1R1+1R2+1R3±21R1R2+1R1R3+1R2R3 where R1, R2, R3 denote the radii of the three initial circles and R4 the radii of the fourth circles (see Figure 1a).
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In has been recalled that the radius of the m′th circle in a subseries of circles is of the form (2)Rm≅L(m+m0)2 where m0 is a numerical constant, depending on the choice of the radius of the first circle and L is some macroscopic distance associated with the dimension of the largest circle. The problem of Apollonius has been also examined by Mathematicians like Marcel Berger in the frame of geometrical constructions. Nowadays, some papers continue to examine the high dimensional Apollonius Networks .
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The aim of this paper is to show the importance in soft matter physics of such well ordered structures. J.-B. Fournier and G. Durand have studied the equilibrium shapes of SmA nucleated inside the isotropic phase of some liquid crystal. A particular part of their paper (part 6) concerns asymptotic equilibrium shapes in the limit of large SmA volumes and they have shown that it must correspond to spheres with a radial Apollonius network of conics. The same Apollonius tiling of FCD has been shown by O. D. Lavrentovich and V. M. Pergamenshchik in SmA films made with octyloxycyanobiphenyl/glycerin (8OCB/glycerin). The temperature is decreased from the nematic phase. In the simplest case of toroidal domains (TFCD), the layers are folded around the circle which bounds the domain base and a straight line passing through the center of the circle. The deformation region is restricted by the cylinder (see figure 10b of reference ). A very interesting review of the different organizations of FCDs in smectic liquid crystals has been detailed by Y. Bouligand . The geometric constructions are explained, allowing one to better understand the different rules of FCD’s association (for instance, the law of corresponding cones (l.c.c.) mentioned in the suite of our paper, polygonal textures, smectic layers as Dupin cyclides,...). Recently, C. Blanc and collaborators have published some beautiful observations in lyotropic liquid crystals in the system CPCl/hexanol/brine inside the lamellar phase [12, 13]. The textures in the lamellar phase made by focal conics show different generations of focal conics as a function of the sample thickness. Using capillaries of about 100 microns of thickness, they have obtained evidence for three different generations of focal conics. The first generation is made by focal conics at the apices of some hexagonal tiling. The second generation fill the interspacing between focal conics again by some hexagonal tiling of focal conics of smaller sizes. The third generation is made with the decoration of the previous generations by three focal conics of smaller size. This experimental situation can be compared to the Apollonius tiling with three generations. Note that these FCDs do not completely tile the space with these defects and it is this lack of completeness that has been investigated in [6, 11, 14].
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(a): three circles of radii R1, R2, R3 are tangent. The figure has been made in the particular case R1=R2=R3. Two circles of Radii R4 and R4′ are tangent to the three initial circles; (b): coordinates system with the two angles, longitude θ and latitude ϕ.
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| 99.9 |
Experiments on TFCDs have been recently carried out in order to explore the internal structure of TFCDs. TFCDs have been confined within a microchannel by Kim and coworkers . They have shown in particular that the formation of TFCDs is very much influenced by the channel depth. Another way to investigate the internal structure of TFCDs consists of the use of AFM study on Smectic A droplets either on coated silicon substrates or on MoS2 substrates both with hybrid anchoring conditions (homeotropic anchoring with the air-interface and planar anchoring at the solid substrate) [17, 18]. Optical measurements of the birefringence permit the description of TFCDs in thin films of smectic A deposited on mica in air .
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| 100.0 |
In this paper, two different thermotropic liquid crystals have been used and observed in the smectic phases under a polarizing microscope. Mainly two different very regular textures have been obtained: a texture that we decided to call the flower texture and another one involving the generation of circles, the generation texture. The coordinate system for the generation texture is indicated in Figure 1b.
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| 99.8 |
For both textures, the experimental situations will be described in detail. For the flower texture, some visualization of the geometry of the different ellipses and confocal hyperbolae is presented. For the generation texture, some simulation has been made in order to understand how the layers are inside the sample. The aim is to visualize clearly the organization of the smectic layers which take the form of Dupin cyclides. A precise examination of the association of focal conic domains is needed. Note that these Dupin cyclides are useful in a great number of physical situations i.e. not only in the physics of liquid crystals, for more information, see .
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| 98.5 |
Two smectic A phases from two different materials have been used. One of them belongs to the cyano-biphenyl series: 8CB (4-n-octyl-4’-cyanobiphenyl). The second one, called TBDA (terephthalylidene-bis-4-n-decylaniline) also possesses a smectic A phase. 8CB is a nematogenic compound whereas TBDA does not possess any nematic phase; TBDA transits directly into the smectic A phase from the isotropic phase when the temperature is decreased (TI/SmA∼190∘C). Optical measurements were performed with a polarizing microscope (Olympus CX60) coupled to a digital camera which provides images on a computer via an acquisition card. Image Pro Plus (IPP) software permits the recording of the photographs. The temperature control is performed using a Hot Stage HS-2 from Instec which provides a thermo-stabilization accuracy better than ±0.01∘ C and a temperature change at a controlled rate, which can be as low as 0.005∘ C/min. Some droplets of liquid crystal have been deposited between two untreated slides. The liquid crystal is surrounded by a glycerol matrix.
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| 100.0 |
The sample has been made with some droplets of TBDA easily deposited on a glass slide without any surface treatment. When the temperature is decreased from the isotropic phase, the smectic A texture appears below 188∘C. The texture is not well ordered as it possesses focal conics made up of ellipses and confocal hyperbolae. The temperature has been changed and after several temperature increases and decreases, the sample shows some very regular focal conics texture, the flower texture shown in Figure 2a. What is surprising is the "quasi-isotropic" orientation of the focal conics with some source at the center of the picture. When the temperature is increased or decreased, a focal conic appears from this point and nucleates toward the boundary of the droplet. The untreated glass provides a quasi-homeotropic anchoring but the successive temperature variations induce a specific texture, i.e. the flower texture, which exists in the bulk of the sample far from the boundaries. Therefore, the untreated glass doesn’t seem to play any fundamental role in the generation of this texture. In another part of the sample, the texture of Figure 2b is visible: we cannot see the boundaries of the sample keeping the same magnification (objective of the microscope times 50).
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| 100.0 |
8CB smectic liquid crystal has been used surrounded by a glycerol matrix. This glycerol will not mix with 8CB but is used to provide some planar degenerate boundary conditions. Figure 3a represents the generation texture observed under crossed polars microscope which shows the presence of some hexagonal tiling of focal conic domains in the particular case where the ellipses are degenerated into circles and the hyperbolae into straight lines. In this situation, the Dupin cyclide layers become simple torii (see figure 4b). In Figure 3b, some simulation with the Apollonius tiling has been done with 4 generations of circles.
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| 99.94 |
Note that in the particular case where the ellipse is degenerated into a circle, the Dupin cyclides are simple torii and therefore only two kinds of layers exist: layers of type 2 and layers of type 3. A schematic representation (Figure 4b) recalls two important characteristics of a given torus: the radius R of the directing circle and the radius μ of the generating circle.
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(a): three kind of layers for FCD of the first species; (b): representation of a torus: R being the radius of the directing circle and μ the radius of the generating circle; (c): representation of a TFCD (Toric Focal Conic Domain) with some negative Gaussian curvature.
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| 99.9 |
In the smectic A phase, the director field should also satisfy to the association rules of focal conic domains (called law of corresponding cones (l.c.c)) first established by G. Friedel . They have been illustrated in particular in the caption of Figure 4 in the paper where the authors wrote that "the cyclides of Dupin mesh smoothly onto spheres. The interface is along a cone of generators; the apex of the cone lies on the hyperbola and is the center of the sphere. The apex of the cones of two adjacent focal domains must coincide at this point." These laws have been recently extended . Two adjacent FCDs should have in common a pair of common generatrices as illustrated in the Figure 5a. The branches of the two hyperbolae intersect in two points corresponding to the "poles" of the hyperbolae (see ). These poles both project in the plane of the ellipses onto the same point of the tangent Mt common to the ellipses at M. One can see that the two cones with the two poles as apices and which lies on the ellipses have one common generatrix (blue cones). Also, the two physical branches of the hyperbolae belong to the same cone of revolution (red cone) with apex M, axis Mt. Note that one can go from one layer belonging to FCD1 to one corresponding layer belonging to FCD2 continuously. Figure 5b shows a 2D representation of the l.c.c. Each ellipse possess two focii: one physical called Fi, i=1,2 and one non physical called Fi′. The l.c.c reads as follows: the line F1F2′ goes exactly through the tangency point M of the two ellipses.
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| 99.9 |
Illustration of the law of corresponding cones (l.c.c) for two tangent ellipses and their confocal hyperbolae ; (a): 3D representation: the two cones which lie on the two ellipses (blue cones) have one common generatrix; likewise for the two cones, which lie on the two hyperbolae (red cones); adapted from ; (b): 2D representation: the line joining the visible focus F1 of one ellipse to the invisible focus (not physical) of the other F2′ goes exactly through the tangential point M between the two ellipses; adapted from .
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| 99.9 |
The flower texture is particularly symmetric. The ellipses are easily visible. Their confocal hyperbolae appear as straight lines so that they lie in some vertical plane (perpendicular to the horizontal 2D observation plane). The focus with crossed polars optical microscope has been made at the apex of the droplet that we called S in the description. The boundaries of the droplet (see Figure 2a) are completely blurred because of the absence of any cover glass slide. The organization of the ellipses inside the droplet is relatively complicated. The flower texture does not seem to be a classical clustering of FCDs. In fact, the physical hyperbolae seem to be directed outwards of the droplet so that the habit cone of revolution with apex M, axis Mt that lies on the physical branches of the hyperbolae is not relevant. On the contrary, we hypothesize that the virtual branches of the hyperbolae have one point of intersection at the apex S of the droplet (see Figure 6). Note that in our case, the physical part of the hyperbolae have not been represented; otherwise Figure 6 would have been too much cluttered.
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study
| 99.94 |
Coming back to the experimental flower texture (Figure 2), if we move from the droplet center to the right along a radius, we see one ellipse then a second one then a third one. Approximately, three ellipses (or four at a few locations) are visible whatever the radius that we consider so that we can imagine three circles relating all the apices of the ellipses located at a constant distance from the center. These circles correspond to cones in the 3D space. Therefore for the model, three cones have been represented: one blue cone corresponds to the ellipses which are located close to the center, a green cone for intermediate ellipse positions and a red cone for the ellipses much closer to the boundary of the droplet. The simulation has been made in the Figure 6a,b with three cones of semi-apex angles of 30∘, 45∘ and 60∘, respectively. These values are in reality close to 90∘ but this drawing permits us to explain the model we have in mind. Note also that the differences between the semi-apex angles of the cones are certainly rather small. Looking now to one single cone, all the ellipses which lie on the Figure 6Three cones have been drawn; each cone contains some assembly of mutually tangent ellipses. The virtual branches of the hyperbolae have one point of intersection at the apex S of the droplet (a): top view; (b): side view. generatrices of the cone are mutually tangent. These ellipses respect the law of corresponding cones (l.c.c.). To check this point, let us consider two adjacent tangent ellipses. We check that the line joining the visible focus of one ellipse to the invisible focus (not physical) of the other goes exactly through the tangential point M between the two ellipses. This argument has been used in the book of P. G. De Gennes and J. Prost in the case of the well known "fan-shaped" texture of C. Williams whose associate drawing is given in Figure 1 of reference . We emphasize that in the "fan-shaped" texture, the physical hyperbolae are merging outwards instead of inwards as in the case of the flower texture. Such analysis in terms of application of the l.c.c has been also checked on a very large number of examples .
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| 100.0 |
So the question is: how do the layers fill the space between some hexagonal tiling of circles of some given radius of the first generation of circles. The two parameters of a torus R and μ (see figure 4b,c) can vary simultaneously but for the sake of clarity, let us first consider the variation of only one of them; the second being fixed. We recall that R denotes the radius of the directing circle and that each value of μ corresponds to one particular smectic layer.
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other
| 99.25 |
(a): torii with R=2 for different increasing values of μ=1,2,3 from the left to the right. When μ>R, two sheets exist: layer of type 3 takes place instead of type 2; (b): torii with μ=2 for different increasing values of R=1,2,3 from the left to the right. When R>μ, only one sheet exist: layer of type 2 takes place instead of type 3.
|
other
| 99.8 |
When R is fixed, the generation number is fixed because all the radii of circles belonging to a same generation are identical. In the figure 7a where R=2, we see clearly that when μ<R the torii are of type 2, and that when μ>R the torii are of type 3. Therefore, increasing the value of μ for a given value of R permits to pass from layers of type 2 to layers of type 3 with two sheets. The limit between these two kind of layers is μ=R as represented in Figure 7a, in which R=2 for different varying values of μ (μ=1, μ=2 and μ=3). In fact, when one moves in a vertical plane perpendicular to the Apollonius tiling, the layers of type 2 become of type 3 with two sheets (for increasing values of μ).
|
study
| 100.0 |
When μ is fixed, a decrease of R corresponds to a change (increase) in the generation number. In the figure 7b where μ=2, we see clearly that for R<μ the torus are of type 3 and that for R>μ, the torus are of type 2. Therefore, increasing the value of R for a given value of μ permits to pass from layers of type 3 with two sheets to layers of type 2. The limit between these two kinds of layers is R=μ as represented in Figure 7b, in which μ=2 for varying values of R (R=1, R=2 and R=3). In fact, when one moves in the plane of the Apollonius tiling from the biggest circles towards the smaller circles, the layers of type 2 become of types 3 with two sheets. These two tendencies are summarized in Figure 8a,b when one moves in the two directions indicated by the arrows.
|
study
| 100.0 |
In the experiments on thermotropic liquid crystals in the SmA phase, four generations of FCDs have been observed under optical crossed polars; this is the reason why the simulation has been stopped with four generations although the simulation allows to show up to 10 generations. We do not see higher generations: there is some critical size under which the FCDs do not exist . The macroscopic observations are done when the sample is observed from the top (it means θ=0∘ and φ=90∘). Figure 9a illustrates the simulation corresponding to this experimental situation. Figure 9b corresponds to the visualization of the smectic layers with θ=45∘ and φ=45∘.
|
study
| 100.0 |
Each generation corresponds to some fixed value of R, so let us try the simulation with some small radius. Our previous discussion shows that if R is small enough, only layers of type 3 occur; this situation has been represented in Figure 10a (only one sheet of each layer is visible) and Figure 10b (the two sheets are visible). Now increasing the starting value of the radius of the first generation of circles R, some FCD of type 2 are visible for the first generation (see Figure 10b).
|
study
| 98.3 |
This point leads to a first important result: we can now understand how the continuity between layers of type 2 and 3 occurs: the two sheets of each layer create two hexagonal tilings of the layers: one for the upper sheet and another for the lower sheet (see Figure 11).
|
other
| 99.75 |
These two hexagonal tilings are continuously attached with FCDs of type 2 existing for lower generations. Showing now not one value of μ for each radius (one layer) but three different values of μ, one obtains Figure 12 which allows us to better understand how are the smectic layers close to the singular Figure 10(a): simulation of the smectic layers for one single value of μ (μ=3) corresponding to layers of type 3 with two sheets (only one sheet is visible with this point of view); R=0.9, θ=10∘, φ=80∘; (b): the same simulation with another point of view (θ=45∘, φ=45∘). Figure 11simulation of the smectic layers for one single value of μ (μ=3) corresponding to layers of type 3 with two sheets from the second generation of circles; layers are of type 2 for the first generation of circles; R=0.9. lines that are the circles and their cofocal straight lines. The Apollonius tiling corresponding to these singular lines appears always in black color. Finally, some picture of the smectic layers with some visual Figure 12simulation of the smectic layers for three values of μ (μ=1; μ=2; μ=3) corresponding to layers of type 3 with two sheets; R=0.9, θ=80∘, φ=10∘. Figure 13simulation of the smectic layers for different values of μ. effect is presented (see Figure 13).
|
study
| 100.0 |
(a): simulation of the smectic layers for one single value of μ (μ=3) corresponding to layers of type 3 with two sheets (only one sheet is visible with this point of view); R=0.9, θ=10∘, φ=80∘; (b): the same simulation with another point of view (θ=45∘, φ=45∘).
|
other
| 99.25 |
Some textures of liquid crystals have been reported in this paper: the flower texture and the generation texture. In the case of the second texture, we successfully represented the smectic layers at the vicinity of the focal conic domains that are circles and straight lines perpendicular to the circles and merging the centers of the circles. The layers of FCD are in general difficult to visualize close to singularities. In the case of a very particular situation (hexagonal tiling of circles), the visualization of the smectic layers allows a better understanding of the topological continuity of the layers. Recently, some imperfections on FCD have been reported . These imperfections are interpreted in terms of interactions of these FCD and dislocations. The visualization of the smectic layers in such a case could be interesting to investigate. Furthermore, this has some great interest in the case of mixture of liquid crystal doped with nanoparticules. The location of the nanoparticules in the core of the FCD as shown recently is another reason to continue the investigation for the representation of the deformed smectic layers at the vicinity of the defects.
|
study
| 100.0 |
Microalbuminuria is considered to be a marker of endothelial dysfunction and is a predictor of cardiovascular disease and mortality.1,2 Studies have implicated systemic vascular damage, extensive endothelial dysfunction, a glomerular haemodynamic state of hyperperfusion and hyperfiltration, a prothrombotic state, and a low-grade chronic inflammatory state.3 Microalbuminuria is also associated with several cardiovascular disease risk factors, such as hyperglycaemia, hypertension, dyslipidaemia, renal dysfunction, obesity and smoking.4 All of these factors contribute to the genesis of atherosclerosis.
|
review
| 99.9 |
Proteinuria is also an early marker for potentially serious renal disease in diabetics. It refers to an abnormally increased excretion rate of albumin in the urine, and is a sensitive indicator of generalised microvascular disease and a marker for vascular endothelial injury and multi-organ damage.5 Reduction of microalbuminuria in diabetics may retard its progression to overt diabetic nephropathy.5
|
other
| 99.3 |
Once microalbuminuria is present, the rate of progression to end-stage renal disease can be delayed by inhibition of the renin– angiotensin system.6 There is evidence that the use of agents that block the renin–angiotensin–aldosterone system, notably angiotensin receptor antagonists, may provide cardiovascular protection to diabetic patients with microalbuminuria.
|
review
| 98.4 |
Microalbuminuria increases following open-heart surgery where coronary artery bypass grafting (CABG) is utilised.7 CABG activates an inflammatory cascade, which may increase capillary permeability and cause microalbuminuria. The increase in capillary permeability may induce exudation of proteins from the lung capillaries into the capillary–alveolar interspace and alveoli, causing the so-called postperfusion lung, which resembles pulmonary oedema. In a recent study, Loef et al. demonstrated that CABG potentiates transient renal failure and microalbuminuria.8
|
study
| 99.1 |
This observational study was approved by the local institutional review board (LUT/05/38/2006) and conducted in accordance with the amended Declaration of Helsinki and Good Clinical Practice regulations. Written informed consent was obtained from all subjects. Patients admitted to the Department ofCardiovascular Surgery of our tertiary centre between June 2006 and February 2007 who had type 2 diabetes mellitus and had undergone CABG surgery constituted the study group.
|
study
| 99.94 |
Patients were divided into two groups with block randomisation, using the sealed envelope technique: group T (telmisartan group) consisted of patients who received the angiotensin receptor blocking agent, telmisartan (Micardis®, Boehringer Ingelheim, Istanbul, Turkey) 80 mg daily for at least six months in the pre-operative period; group N-T (non-telmisartan group) consisted of patients who received neither telmisartan nor any other angiotensin receptor blockers. In both groups, no patients were using angiotensin converting enzyme inhibitors for at least six months prior to the study.
|
other
| 99.6 |
Cases with severely impaired left ventricular function, chronic pulmonary obstructive disease, severe systemic non-cardiac disease, severe renal or liver impairment, infectious diseases before surgery, malignancy, those receiving corticosteroids or other immunosuppressive treatment, and patients with stroke, inflammatory disease, and/or previous cardiac surgery, and valvular heart disease were excluded from the study.
|
study
| 99.9 |
Cardiac medication, including beta-adrenergic blocking agents, calcium channel blocking agents and nitrates, was continued until the morning of surgery. The same general anaesthetic drugs were used in all patients. A standard median sternotomy incision was used to expose the heart and place the internal mammary artery and saphenous vein grafts used for coronary anastomosis.
|
other
| 99.75 |
In each group, routine surgery was performed using a membrane oxygenator (Edwards Vital, Edwards Lifesciences LLC, Irvine, CA, USA), a 3-mg/kg dose of sodium heparin, 2 000 ml of Ringer’s lactate primer and a roller pump at a body temperature of 28°C. Cardiopulmonary bypass was instituted via the ascending aorta and single two-stage venous cannulation (maintained at 2.2–2.4 l/min/m2).
|
other
| 99.5 |
Following cross-clamping of the aorta, the heart was arrested using 10–15 cm3/kg cold blood cardioplegia through the aortic root and topical ice slush was continued every 20 minutes for myocardial protection. Heparin was neutralised with protamine hydrochloride (Protamin 1000; Roche, Istanbul, Turkey). The circuit was primed with 2 000 ml Ringer’s lactate.
|
clinical case
| 99.5 |
After completion of the surgery, patients were transferred to the intensive care unit (ICU), where standard care and processes were followed until discharge. Patients were weaned from mechanical ventilation when they were haemodynamically stable, responding to verbal stimulation, and had been fully rewarmed. Patients were discharged from the ICU if they were haemodynamically stable, had normal blood gasses during spontaneous breathing, and had a satisfactory renal function.
|
other
| 99.6 |
Smoking, obesity, hypertension, duration of diabetes, family history of coronary artery disease, pre-operative myocardial infarction, and pre-operative haemodynamic data were recorded. During the surgical procedure, haemodynamic parameters, including heart rate, mean arterial pressure, central venous pressure, arterial blood gasses and urine output were monitored. In the postoperative period in the ICU, cardiovascular and respiratory values and temperature were recorded every 15 minutes before extubation and then hourly until discharge from the ICU. The length of stay in the ICU was also recorded.
|
study
| 99.94 |
Microalbuminuria levels were studied pre-operatively, on the first hour postoperatively, and on postoperative days (POD) one and five. High-sensitivity C-reactive protein (hsCRP) levels were studied pre-operatively, and on POD 1 and 5. Patients who were considered to be in a low-cardiac output state received positive inotropic agents (dopamine or adrenaline or both). They were assessed for persistent systemic blood pressure below 90 mmHg, urinary output lower than 20 cm3/h, and the state of peripheral circulation was evaluated for adequate preload and optimal afterload. Urine samples were measured for microalbuminuria using Micral test sticks (Roche).
|
study
| 99.94 |
Categorical variables were analysed with chi-squared and Fisher’s exact tests, as appropriate, in contingency tables, whereas the unpaired t-test and Mann–Whitney U-test were performed, as appropriate, for comparison of continuous variables. Comparisons for microalbuminuria and hsCRP levels in the groups were done with repeated measures of ANOVA and the Bonferroni test.
|
study
| 99.94 |
Data are expressed as means ± standard deviation. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed with the Statistical Package for Social Sciences (SPSS 10.0 for Windows, SPSS, Inc., Chicago, IL). The calculation of sample size was based on a power analysis. At a power of 80% using a significance level of p < 0.05, the sample size required was 20 subjects per study group.
|
study
| 99.94 |
Forty patients met the eligibility criteria for the study. Of the 40 patients (29 males, 11 females) whose charts were reviewed, the average age was 65.0 ± 8.6 (range 40–79) years. Group T included 20 patients (15 males, 5 females) with a mean age of 65.6 ± 7.8 years, who had been using telmisartan 80 mg daily for at least six months. Group N-T included 20 patients (14 males, 6 females) with a mean age of 64.4 ± 9.5 years, who used no angiotensin receptor blocking agent prior to the operation. The groups were similar with regard to age and gender (p = 0.680 and p = 0.723, respectively).
|
study
| 100.0 |
With regard to clinical characteristics such as body mass index, smoking habit, hypertension, hyperlipidaemia, and history of myocardial infarct, the two groups did not show significant differences and were comparable (Table 1). The groups were also similar with regard to number of bypass grafts, cardiopulmonary bypass time, cross-clamp time, flow, atrial fibrillation, inotrope usage, time of endotracheal intubation and mortality rate (Table 2).
|
study
| 100.0 |
Pre-operative, first hour postoperative, POD 1 and POD 5 microalbuminuria levels were 16.5 ± 17.2, 28.5 ± 17.2, 59.0 ± 29.8 and 23.0 ± 20.0 mg/l in group T, and 30.0 ± 17.7, 51.0 ± 28.4, 75.0 ± 25.6 and 52.5 ± 27.5 mg/l in Group N-T, respectively, and there were statistically significant differences between four microalbuminiria levels in each group (p < 0.001) (Table 3). Pre-operative, first hour postoperative and POD 5 values were statistically significantly different between the groups (p = 0.018, p = 0.008 and p = 0.001, respectively) (Table 3). However, the difference in POD 1 values between the groups was at the threshold of significance (p = 0.071).
|
study
| 100.0 |
Pre-operative plasma levels of hsCRP (0.35 ± 0.17 vs 0.50 ± 0.32 mg/l) showed a trend towards significance (p = 0.069). Although POD 1 hsCRP levels (10.0 ± 2.0 vs 17.8 ± 3.9 mg/l) did not differ (p = 0.405) between the groups, a decrease in POD 5 hsCRP levels in group T (8.6 ± 2.9 vs 10.9 ± 3.2 mg/l) was statistically significant between the groups (p = 0.024) (Table 4).
|
study
| 100.0 |
All CABG surgeries were performed successfully. There was no repeat surgery for bleeding or peri-operative myocardial infarction in either group. The only complication was one cerebrovascular accident in the N-T group. There was no clinical or laboratory evidence of postoperative renal dysfunction in either group. Urine output during surgery and in the postoperative period did not differ between the groups. No wound infection was observed for any patient.
|
other
| 63.1 |
Coronary artery bypass grafting is often followed by a systemic inflammatory response. The clinical relevance of CABGrelatedsystemic inflammation varies with patients and such inflammation may be accompanied by intermittent organ dysfunction and finally, multi-organ failure, including renal and pulmonary dysfunction.9,10
|
review
| 99.7 |
In some patient groups, the effect of extracorporeal circulation is serious after open-heart surgery and it is well known that diabetic patients are frequently associated with renal and cardiovascular disease, requiring surgical and medical intensive care. Some pathophysiological mechanisms such as microalbumiuria and urinary protein over-excretion are responsible for these damaging effects in this particular group of patients.
|
other
| 99.9 |
In patients with diabetes, angiotensin II is believed to play a main role in the progression of renal damage, not only through haemodynamic effects but also non-haemodynamic effects, including stimulation of growth factors and cytokines and changes in extracellular matrix metabolism.11 Angiotensin II gives rise to glomerular hypertension and can alter the filtration properties of the glomerular basement membrane, leading to proteinuria.12-13 Angiotensin receptor antagonists have been shown to consistently produce favourable mortality and morbidity outcomes in endpoint trials in patients with type 2 diabetes and diabetic nephropathy.14-16
|
review
| 99.9 |
Microalbuminuria refers to the increased excretion of albumin into the urine, which is so slight that it can be detected only by sensitive immunological analysis. Microalbuminuria is measured in diabetic patients to predict incipient nephropathy. The predictive value of microalbuminuria for the expression of cardiovascular diseases has also been investigated and, in fact, is as powerful for predicting hyperlipidaemia or hypertension.17 Microalbuminuria also occurs in acute conditions where capillary permeability increases.
|
review
| 99.7 |
Microalbuminuria increases during major surgery such as CABG, and extracorporeal circulation activates an inflammatory cascade, which may increase capillary permeability and cause microalbuminuria. The increase in capillary permeability may induce exudation of proteins from the lung capillaries into the capillary–alveolar interspace and alveoli, causing the so-called post-perfusion lung, which resembles pulmonary oedema.
|
other
| 97.5 |
We found that telmisartan, as an angiotensin II receptor antagonist, had a significant lessening effect on microalbuminuria in type 2 diabetes patients undergoing coronary bypass surgery in our study. A significant decrease in hsCRP levels on day 5 was also noticed between the groups.
|
study
| 100.0 |
Several previous studies have shown that angiotensin receptor antagonists are effective anti-inflammatory agents, and our patients receiving telmisartan revealed decreased levels of systemic inflammation after CABG. This anti-inflammatory effect of telmisartan may help preserve postoperative renal function and also vascular endothelial function, which may also be seen after bypass surgery.
|
other
| 44.38 |
We know that renal dysfunction is a serious complication of coronary revascularisation with CABG and results in increased morbidity and mortality rates and prolonged hospital stay.18 The injurious action of CABG on renal function is caused by several mechanisms, including non-pulsatile perfusion and increased levels of circulating catecholamines, cytokines and free haemoglobin.19 These effects result in damage to the glomerular as well as tubular structures, which, in turn, may cause renal dysfunction, especially in the presence of additional risk factors.20-21
|
study
| 98.6 |
Microalbuminuria is one of the sensitive markers of increased capillary permeability and may be useful to study the systemic inflammatory response after CABG.6,22,23 According to previous investigations, urinary microalbuminuria increased significantly in the early postoperative period and one day after CABG.
|
study
| 100.0 |
In our study, peak increase in microalbuminuria was observed in both groups but there was no statistically significant difference (p = 0.071). These levels decreased, particularly on the fifth day in our cases, and the decrease was statistically significantly different in group T. In both groups, hsCRP increased and peaked on the first postoperative day in both groups. However, in group T, hsCRP, as one of the pro-inflammatory agents, decreased significantly on the fifth day. Therefore, the increase in acute inflammatory response was similar in both groups on the first postoperative day, and in group T, both markers had decreased by the fifth day.
|
study
| 100.0 |
Borch-Johnsen et al. showed the direct relationship between proteinuria and cardiovascular mortality rate in insulindependent diabetic patients after open-heart surgery in patients undergoing CABG.24 Telmisartan was also shown to reduce or normalise microalbuminuria in 34% of patients with diabetes, and in a second, smaller study including 64 hypertensive and 60 normotensive patients, to reduce the incidence of renal dysfunction. This confirmed that telmisartan reduced microalbuminuria independently of its blood pressure-lowering effects. Restoration of normal urine albumin levels has also been demonstrated by telmisartan.25
|
review
| 99.25 |
Our study showed that telmisartan reduced microalbuminuria, not only pre-operatively, but also after open-heart surgery. The return to baseline levels was also faster than in group N-T. Angiotensin receptor blocking agents decrease some of the postoperative acute inflammatory agents in on-pump CABG patients with diabetes mellitus by lessening the systemic consequences of renal dysfunction, and may have additional cardiovascular effects by exerting beneficial effects on endothelial tissue elsewhere in the body and within the heart in this patients group. The cardiovascular benefits of angiotensin receptor antagonists have been evaluated, not only in terms of their ability to lower blood pressure, but also on their ability to prevent strokes, cardiac events and target-organ damage.14,16
|
study
| 99.94 |
Limitations of our study are the relatively small size of our series and the lack of definite criteria for selection of patients for this study. As most coronary patients are already being treated with angiotensin receptor blocking agents, the results of our study will not have a major impact on clinical practice. Furthermore, it would have been better to test the predictivevalue of microalbuminuria on prognosis in this category of patients. However, we hope that this study will pioneer further studies on this method.
|
study
| 99.9 |
Our results showed that telmisartan decreased systemic inflammation and urinary albumin excretion in diabetic patients after CABG surgery, compared to those not taking angiotensin receptor antagonists. These beneficial effects of telmisartan treatment on diabetic patients after CABG should be investigated further in prospective, randomised studies.
|
study
| 99.94 |
Hypoxia, or reduced oxygen availability, is an important developmental cue in multicellular organisms, but it is also involved in a number of human pathologies . In response to hypoxia, cells orchestrate a tightly controlled and coordinated response, mostly dependent on transcriptional changes . In the centre stage of such a response, stands the transcription factor family, hypoxia inducible factor (HIF).
|
review
| 80.75 |
HIF is a heterodimer consisting of a α and a β subunits, also known as aryl hydrocarbon nuclear translocator . While HIF-1β protein levels remain largely unchanged in hypoxia, HIF-α levels are extremely sensitive to change in oxygen availability. Oxygen sensitivity is conferred to the HIF system, via the action of a class of dioxygenases called prolyl hydroxylases (PHDs). PHDs require molecular oxygen, 2-oxoglutarate, and iron as cofactors to perform proline hydroxylation of their targets . Proline hydroxylation of two conserved residues within HIF-α creates a high-affinity binding site for a ubiquitin E3 ligase complex containing the tumour suppressor, von Hippel Lindau (VHL). VHL thus promotes the ubiquitination and proteasome-dependent degradation of HIF-α under normal oxygen conditions .
|
study
| 100.0 |
In addition to changes in HIF-α protein stability, an additional level of oxygen control is mediated by another dioxygenase called factor inhibiting HIF (FIH). FIH mediates asparagine hydroxylation in the transactivation domain of HIF-α, a modification that prevents efficient recruitment of coactivators such as p300/CBP to HIF-α, thus limiting the transcriptional output of HIF . It has been shown that FIH is still functional at oxygen concentrations where PHDs are already inhibited . Furthermore, genetic studies have shown that FIH regulates 40% of HIF-dependent genes . However, FIH knockout mice develop normally, suggesting that FIH is not involved in controlling HIF-dependent transcription during developmental hypoxia .
|
study
| 100.0 |
Structural studies of the FIH enzyme revealed an interesting fold, identical to a domain called Jumonji C (JmjC) . JmjC enzymes are protein demethylases, and a number of them possess histone demethylase activity . As such, these enzymes have the potential of sensing and being controlled by oxygen levels in the cell. Changes in histone methylation have been described in hypoxia in a number of cellular systems . One of the JmjC enzymes, lysine (K)-specific demethylase (KDM)4E, has been analysed in terms of its oxygen dependency, revealing a similar response to oxygen to that of PHDs . Interestingly, some of the JmjC enzymes have been shown to be induced in hypoxia, and some in a HIF-dependent manner (reviewed in ). These include KDM3A, KDM4B and C, and KDM5C .
|
review
| 99.8 |
One interesting family of JmjC enzymes that is deregulated in human disease is KDM2. KDM2A and KDM2B demethylate histone 3 lysine 36 when mono- and dimethylated . The KDM2 family possesses additional functional domains to JmjC, including an Fbox domain, a PHD domain, a CXXC-zinc finger domain, and a leucine-rich repeat domain . Apart from demethylating histone H3 tails, these proteins have also been shown to have non-histone targets such as RelA and β-catenin . Furthermore, these enzymes have been shown to be deregulated in cancers such as breast and pancreatic .
|
review
| 99.1 |
Here, we demonstrate that KDM2A is hypoxia-inducible in a HIF-dependent manner. We find that HIF-1α regulates the KDM2A promoter and that HIF-1 is required for the recruitment of RNA polymerase II to the KDM2A promoter. Interestingly, although KDM2B mRNA is induced in a HIF-dependent manner in hypoxia, KDM2B protein level changes in hypoxia are dependent on the cell type analysed.
|
study
| 100.0 |
Cells and cell culture: Human embryonic kidney cells HEK293, U2OS osteosarcoma, HeLa cervix carcinoma, and MCF-7 breast cancer cells were obtained from the European Collection of Cell Cultures and grown in Dulbecco’s Modified Eagle Medium (Lonza) supplemented with 10% foetal bovine serum (Gibco), 50 units/mL penicillin (Lonza), and 50 µg/mL streptomycin (Lonza) for no more than 30 passages at 37 °C and 5% CO2. Cells were routinely tested for contamination.
|
other
| 62.78 |
Hypoxia treatments: All hypoxia exposures were performed at 1% O2 in an in vivo 300 hypoxia workstation (Ruskin, Pencoe, UK). Cells were lysed for protein extracts, RNA extraction, immunofluorescence, and chromatin immunoprecipitation fixation in the workstation to avoid reoxygenation.
|
study
| 82.5 |
Antibodies: HIF-1α (610958, BD Biosciences San Jose, CA, USA, and sc-53546 Santa Cruz Biotechnology, Dallas, TX, USA), HIF-2α (sc-13596, Santa Cruz Biotechnology ), HIF-1β (#5537, Cell Signaling, Danvers, MA, USA), β-actin (3700, Cell Signaling), RNA polymerase II (sc-47701, Santa Cruz Biotechnology), KDM2A (A301-476A, Bethyl laboratories, Montgomery, TX, USA), and KDM2B (09-862, Millipore; GTX104868. Genetex, Zeeland, MI, USA).
|
other
| 99.9 |
Immunoblot: Cells were lysed in RIPA buffer, 50 mM Tris-HCl (pH 8), 150 mM NaCl, 1% (v/v) NP40, 0.5% (v/v) Na-deoxycholate, 0.1% (v/v) SDS, and 1 tablet/10 mL Complete, Mini, EDTA-free protease inhibitors (Roche). SDS-PAGE and immunoblots were carried out using standard protocols.
|
study
| 97.75 |
mRNA analysis: RNA was extracted using peqGOLD total RNA kit (Peqlab, Erlangen, Germany) or Direct-Zol RNA kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions, and reverse transcribed using QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany). For quantitative PCR, Brilliant II Sybr Green kit (Statagene/Agilent, Santa Clara, CA, USA), including specific MX3005P 96-well semi-skirted plates, were used to analyse samples on the MX3005P qPCR platform (Stratagene/Agilent). Actin was used as a normalising agent in all experiments. qPCR results were analysed by the ΔΔCt method. Control normoxia or hypoxia were used as calibrators in the different experiments, and set to 100.
|
study
| 99.94 |
siRNA: siRNA oligonucleotides were purchased from MWG and used in a final concentration of 27 nM. They were transfected using Interferin from Polyplus according to manufacturer’s instructions. The following oligonucleotides sequences were used for siRNA knockdown:
|
other
| 99.9 |
Chromatin immunoprecipitation: Following the different treatments, proteins and chromatin were cross-linked with 1% formaldehyde at room temperature for 10 min. Glycine was added to a final concentration of 0.125 M for 5 min to quench the reaction. Cells were harvested into 400 µL of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1, 1 mM PMSF, 1 μg/mL leupeptin, 1 μg/mL aprotinin) and left on ice for 10 min. Samples were then sonicated at 4 °C eight times for 15 s with a 30 s gap between each sonication at 50% amplitude (Sonics Vibra-Cell # VCX130). Supernatants were recovered by centrifugation (12,000 rpm for 10 min at 4 °C) before 10% of each sample was stored as input. Remaining samples were split into 120 µL aliquots before being diluted 10-fold in dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl pH 8.1). Diluted samples were precleared for 2 h at 4 °C by incubating with 2 µg of sheared salmon sperm DNA and 20 µL of protein G-Sepharose (50% slurry). Immunoprecipitations were performed overnight with 2 µg of specific antibody or IgG control, with the addition of Brij 35 detergent to a final concentration of 0.1%. Immune complexes were captured by incubation with 40 µL of protein G-Sepharose (50% slurry) and 2 µg salmon sperm DNA for 1 h at 4 °C. The immunoprecipitates were washed sequentially for 5 min each at 4 °C in Wash Buffer 1 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), Wash Buffer 2 (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), and Wash Buffer 3 (0.25 M LiCl, 1% Nonidet P-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1). Beads were washed twice with Tris-EDTA buffer and eluted with 120 µL of Elution Buffer (1% SDS, 0.1 M NaHCO3). Cross-links were reversed by incubation with 0.2 M NaCl at 65 °C overnight and Proteinase K (20 µg each), 40 mM Tris-HCl pH 6.5, and 10 mM EDTA for 1 h at 45 °C was used to remove protein. DNA was purified using a PCR-product purification kit according to manufacturer’s instructions (NBS Biologicals #NBS363). DNA (3 µL) was used for qPCR with the following primers for the putative hypoxia response element (HRE) sites at the KDM2A and KDM2B promoters:
|
study
| 100.0 |
Immunofluorescence: Cells were plated onto sterilised glass coverslips (VWR 19 mm) in 35 mm plates 24 h before transfection at a density of 1.2 × 105 cells in a total volume of 2 mL media. Forty-eight hours following transfection, cells were fixed to coverslips in 100% methanol for 5 mins at −20 °C. Cells were then washed with PBS and blocked with 1% donkey serum in PBS/0.05% Tween for 30 min at room temperature. Cells were placed in a humidified chamber, incubated with primary antibody for 1 h, washed three times (5 min each) in PBS, incubated for 1 h with secondary antibody, and washed three times (5 min each) in PBS. Nuclear staining was performed by incubation with 33.3 μM Hoechst (Life Technologies) for 2 min. Coverslips were then washed in dH2O and mounted onto VWR SuperFrost slides using mounting medium (H-1000; Vector labs, Peterborough, UK) and sealed with nail varnish. Cell images were acquired using a DeltaVision microscope. Images were deconvolved and analysed using OMERO client software (5.2.7, Open Microscopy Environment, Dundee, UK) .
|
study
| 99.75 |
Luciferase reporter assay: Cells (2 × 105) were seeded in six-well plates and transfected with 1 µg DNA. At 48 h post-transfection, cells were lysed in 400 µL passive lysis buffer (Promega). Luciferase assays were performed according to the manufacturer's instructions (Luciferase Assay System, Promega). Results were normalised for protein concentration with all experiments being performed a minimum of three times before calculating means and standard error of the means.
|
study
| 99.94 |
In order to determine if KDM2 family members are transcriptionally regulated in hypoxia, U2OS and HeLa cells were exposed to 1% O2 for 24 h prior to mRNA extraction and qPCR analysis. Levels of KDM2A and KDM2B were analysed (Figure 1A,B) and CA9, a known HIF-dependent target, was used as a control (Figure 1C). Both KDM2A and KDM2B mRNA were induced by hypoxia (Figure 1A,B). As expected, CA9 mRNA was robustly induced in both cell lines following hypoxia (Figure 1C). In addition, we also investigated if hypoxia was able to induce KDM2 in the model organism Drosophila melanogaster, where only one gene is present. Here, whole flies were exposed to 5% oxygen for 24 h prior to mRNA extraction and gene expression analysis. As it can be seen in Figure 1D, hypoxia induces the levels of KDM2 mRNA in this organism as well.
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Next, we assessed if hypoxia could induce KDM2A and KDM2B protein levels. Western blot analysis revealed that, indeed, hypoxia induces KDM2A protein (Figure 2A) in the cell lines analysed. While numerous attempts were made to investigate KDM2B protein levels by Western blot, none of the antibodies tested were specific for KDM2B when an siRNA depletion control was included (Figure S1A), despite all the antibodies presenting a band of the expected size (Figure S1B–D). However, we could determine that one particular antibody was specific when used in immunofluorescence, accessed by siRNA depletion, overexpression of KDM2B, and specific controls for antibody staining (Figure S2A–C).
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Using immunofluorescence (IF), we find that hypoxia exposure results in reduced expression of KDM2B protein in U2OS cells and in a small statistical upregulation in HeLa cells following 24 h of hypoxia, assessed by analysing the intensity of antibody staining across several cells (Figure 2B).
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Given that KDM2B possesses two different isoforms , we investigated the possibility that our mRNA analysis was only measuring one specific isoform, not recognised by the antibodies we have tested. As such, we designed specific PCR primers directed at each of the KDM2B isoforms and repeated our qPCR analysis in cells exposed or not to hypoxia. This analysis revealed that hypoxia induces the expression of both KDM2B transcripts significantly in both HeLa and U2OS (Figure 2C). This confirmed our initial mRNA analysis for total KDM2B mRNA, and suggests that hypoxia induces changes in KDM2B protein at the post-transcriptional level.
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Given that hypoxia induces changes to KDM2 proteins, we next determined if these were HIF dependent. First, we analysed KDM2A protein levels in response to hypoxia in the presence or absence of HIF subunits. Western blot analysis revealed that KDM2A protein levels were induced in hypoxia in a HIF-1α-, HIF-1β-dependent manner, while HIF-2α did not contribute to these effects (Figure 3A). Interestingly, KDM2B protein levels are also HIF-1α dependent, as HIF-1α depletion resulted in higher KDM2B protein levels both in normoxia and especially in hypoxia in U2OS cells (Figure 3B). This data was also observed when additional siRNA oligonucleotides were used in HeLa cells (Figure S3A).
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As HIF is a transcription factor, we next analysed the levels of KDM2A and KDM2B mRNAs in the presence or absence of HIF subunits (Figure 4). Both KDM2A and KDM2B mRNA levels in HeLa and U2OS cells were dependent on the presence of HIF-1α and HIF-1β in hypoxia (Figure 4A,B, Figure S4A). These results suggest that KDM2 family is transcriptionally regulated by HIF-1, which results in changes in protein levels in hypoxia. Similarly, overexpression of HIF-1α could induce KDM2A mRNA levels in both cells lines in normoxia and hypoxia (Figure 4C, Figure S4B). Interestingly, HIF-1α overexpression also resulted in increased levels of KDM2B, but this was only observed under normoxic conditions (Figure 4C, Figure S4B).
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Given our results, we next determined if HIF-mediated regulation of KDM2 was occurring at the promoter level. Bioinformatic analysis of the KDM2A and KDM2B promoters revealed putative hypoxia response elements (HREs) in both promoters, some quite close to the transcriptional start site (Figure 5A, Figure S5). We thus designed primer sets for the putative HRE sites present in the KDM2A and KDM2B promoters nearer the transcription start and performed chromatin immunoprecipitation (ChIP) analysis in hypoxia, for the levels of HIF-1α and HIF-1β present at these sites using qPCR (Figure 5A). We could detect significant levels of both HIF-1α and HIF-1β present at the KDM2A promoter when cells were exposed to hypoxia (Figure 5A). Interestingly, HIF-1α and HIF-1β were also present at the KDM2B promoter, under the same conditions.
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As we had observed HIF-1-dependent increases in mRNA and protein of KDM2A in hypoxia, we next investigated the functional significance of HIF-1 presence at the KDM2A promoter. To this end, HIF-1 subunits were depleted by siRNA from cells and levels of RNA polymerase II present at the KDM2A promoter were analysed by ChIP-qPCR. This analysis was performed following 24 h of hypoxia, where we had observed the highest mRNA induction, in a HIF-dependent manner. Levels of RNA polymerase II were robustly detected in control cells exposed to hypoxia, when the KDM2A promoter was analysed (Figure 5B). However, when either HIF-1α or HIF-1β were depleted by siRNA from cells, levels of RNA polymerase II present at the KDM2A promoter dropped significantly and to almost background levels (Figure 5B). These results demonstrate the importance of HIF-1 subunits in the regulation of the KDM2A promoter, indicating that HIF-1 is required for transcriptional activation of this gene. To further determine the functionality of the HRE sites present at the KDM2A promoter, we created luciferase constructs expressing the two putative HRE sites in tandem, as well as mutant versions of the HRE sites. Analysis of luciferase activity in cells revealed that indeed these HREs confirm promoter activity (Figure 5D). Furthermore, this analysis revealed that both HREs are required for full luciferase activity, since, when mutated, luciferase output reverts to background level, while mutation of the −186 HRE site gives an intermediate activity level (Figure 5D).
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In this report, we have identified that the KDM2 family of JmjC demethylases is responsive to hypoxia, and that this is conserved in the model organism Drosophila melanogaster. Furthermore, we show that both KDM2A and KDM2B protein levels change in response to hypoxia in a HIF-1 dependent manner. KDM2A mRNA and protein are increased in response to hypoxia by a mechanism dependent on the HIF-1 heterodimer. However, while KDM2B mRNA is also increased in response to hypoxia, KDM2B protein is reduced in a HIF-1-dependent manner by a post-transcriptional mechanism in U2OS cells, while KDM2B protein levels are slightly higher in HeLa cells exposed to hypoxia.
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