The purpose of this paper was to update the knowledge concerning the wear, translucency, as well as clinical performance of monolithic zirconia ceramics, aiming at highlighting their advantages and weaknesses through data presented in recent literature. New ultra-translucent and multicolor monolithic zirconia ceramics present considerably improved aesthetics and translucency, which, according to the literature reviewed, is similar to those of the more translucent lithium disilicate ceramics. A profound advantage is their high strength at thin geometries preserving their mechanical integrity. Based on the reviewed articles, monolithic zirconia ceramics cause minimal wear of antagonists, especially if appropriately polished, although no evidence still exists regarding the ultra-translucent compositions. Concerning the survival of monolithic zirconia restorations, the present review demonstrates the findings of the existing short-term studies, which reveal promising results after evaluating their performance for up to 5 or 7 years. Although a significant increase in translucency has been achieved, new translucent monolithic zirconia ceramics have to be further evaluated both in vitro and in vivo for their long-term potential to preserve their outstanding properties. Due to limited studies evaluating the wear properties of ultra-translucent material, no sound conclusions can be made, whereas well-designed clinical studies are urgently needed to enlighten issues of prognosis and long-term survival. Monolithic zirconia restorations, manufactured exclusively by the CAD/CAM technology, have considerable advantages: they exhibit high flexural strength, require more conservative dental preparation, minimize wear on the antagonists, exhibit satisfactory aesthetics, require less laboratory time and fewer dental sessions, and as monolithic, they lack the unwanted complication of chipping ,2,3. TheThe purpose of this paper was to update the current knowledge concerning the wear, the optical properties and clinical performance of monolithic zirconia ceramics, aiming at highlighting the advantages and weaknesses of these materials, through the data presented in recent literature.zI) of the light beam after transpassing through a material with z thickness from Beer’s law [I0 is the initial intensity of the beam and α the absorption coefficient.When a light beam falls on a polycrystalline material, a part of it is reflected from its surface, depending on its surface roughness, a part is transmitted through its mass and a part is scattered or absorbed into its bulk. The amount of light transmitted is therefore related to the reflectivity of the surface and also to the way the light propagates through the medium. During light propagation, absorption occurs when the frequency of the light is resonant with the transition frequencies of the atoms in the material. The light transmitted is clearly related to the absorption, because only unabsorbed light will be transmitted. The absorption of light by an optical medium is quantified by its absorption coefficient α. This can be integrated to obtain the decreased intensity  [R is the reflection depending on the refractive index and A the absorbance depending on the thickness and the absorption coefficient. For strongly absorbing materials  multiple reflections are negligible and T is defined by:The coefficient of transmission or transmittance tion (2) :(2)T = 1N is the number of scattering centers per unit volume, and σs is the scattering cross-section of the scattering center.Scattering is the phenomenon in which as the light passes through the object it changes direction and possibly also its frequency after interacting with the material. This could be caused by the presence of impurities, defects, or inhomogeneities. The intensity of the light beam decreases exponentially as it propagates into the material according to:E*) between a uniform thickness material measured over white and black background [L (lightness), a* (red/green coordinate), and b* (yellow/blue coordinate) are the color coordinates of the CIE Lab coloring system and the subscript (b) refers to the color co-ordinate against black background while the subscript (w) refers to the color co-ordinate against white background.The most common optical properties that are investigated for monolithic zirconia ceramics are the translucent parameter (TP) and the contrast ratio (CR). CR is the ratio of the reflectance of a specimen over a black background to that over a white background of a known reflectance and is an estimate of opacity . CR rangckground . TransluΥb is the spectral reflectance of light measured over a black background and Υw the luminance measured over a white background.CR is measured according to the following formula :(6)CR = In order to make monolithic zirconia more aesthetic and translucent, the light scattering from the bulk of the material has to be significantly eliminated. Internal light scattering may result from several sources, such as pores, different crystalline phases, incomplete sintering, impurities, defects, and grain boundaries. A presentation of the major factors affecting light scattering in Y-TZP ceramics is following.2O3) are known to enhance densification of Y-TZP ceramics creating fine-grained and homogeneous microstructures while decreasing the sintering temperature and time required [n = 1.76 and n = 2.21 respectively, alumina inclusions were considered significant scattering centers. It has been reported that lowering the amount of alumina addition from 0.25 to 0.1 or 0.05 wt.% can significantly increase the translucency. Although alumina elimination improves translucency [4+, that segregate at grain boundaries. The introduction of 0.2 mol% La2O3 in conventional 0.1–0.25 wt.% Al2O3-doped 3Y-TZP resulted in an excellent combination of high translucency and superior hydrothermal stability, while retaining excellent mechanical properties. Alumina (Al3+) and lanthanum (La3+) preferable segregation at grain boundaries eliminates the presence of secondary phases and reduces porosity and birefringence at the grain boundaries [Composition: Towards light transmittance, the first step in the manufacture of translucent monolithic zirconia was the reduction of the amount of alumina sintering aids. Small quantities of alumina  and zirconia (n = 2.1–2.2) [2O3–ZrO2 (8Y) ceramics fabricated by the hot isostatic pressing method presented submicron intragranular pores that were solely responsible for light scattering as cubic structures do not present birefringence. Furthermore, it was pointed out that small intergranular pores are important to achieve high transparency [Porosity: Pores are the main cause of light scattering, especially when they are of a size similar to that of the wavelength of visible light (400 to 700 nm) . They pl2.1–2.2) ,39. Pore2.1–2.2)  demonstrsparency . Howeversparency ,42,43,442O3.A few studies have investigated the optical properties of monolithic zirconia and especially its TP and CR but the results are very inhomogeneous due to variations in instrumentation, zirconia brand, the thickness of the specimens and parameters evaluated  45,46,4,67,68,69203) > EI  > NT  > LD. However, the differences in CR between ST and EI (0.05) and between EI and NT (0.04) are not considered visible by the human eye. The high translucency of zirconia ceramics containing a high amount of yttrium is a common finding in many studies [2.Carrabba et al.  resulted studies ,57,61,63 studies . A high ab > 3.7) regardless of the coloring liquid applications. For the majority of the rest subgroups, differences were within the range of perceptibility threshold (ΔΕab < 3.7). TP values ranged between 2.27 and 5.34 and increased as the thickness reduced in all groups with highly significant correlations . Subaşı et al. [Sulaiman et al.  evaluateı et al.  investigı et al.  and Nassı et al.  the monoa*, b*, L* and TP values among different brands. Specimens of pre-colored monolithic zirconia with higher yttria contents had significantly higher TP values. Elsaka et al. [Tuncel et al.  evaluatea et al.  investiga et al. , when laThe optical properties of from cubic ultra-translucent (UT) zirconia crowns and super translucent (ST) zirconia crowns were compared to lithium disilicate (L-DIS) glass-ceramic crowns . The valp < 0.001). Conventional sintering had higher TP values at 0.5 mm (p = 0.002) and 1.0 mm (p < 0.001), but the sintering method did not have an impact on TP values at 1.5 mm (p = 0.357). Microwave sintering resulted in larger grain size (both produced nano-sized grains), smoother surfaces and higher color values, while conventional sintering led to a slight increase in translucency.The effect of sintering method  on the optical properties of pre-colored dental monolithic zirconia ceramics of various thicknesses was investigated by Kim et al. . TP valuThe color of monolithic zirconia after cementation was investigated by Malkondu et al. , who evaThe effect of hydrothermal treatment of translucent monolithic zirconia on the light transmission was investigated only in a few studies ,65,66,67Summarizing the results of the aforementioned studies, new generations of cubic zirconia ceramics present higher translucency compared to the conventional tetragonal but lower compared to lithium disilicate . In agreThe surface roughness of a material greatly affects its abrasion, as well as the wear of opposing teeth . AccordiThe wear properties of monolithic zirconia were evaluated in many in vitro studies  73,74,7,109,110.Resulting from the above studies, it appears that monolithic zirconia, compared to the other restorative materials used, causes the smallest abrasion to the antagonists and that the surface treatment affects its abrasive ability. All but one studies  showed tWith regard to the wear caused to various materials by monolithic zirconia, low abrasiveness against steatite compared to glass ceramics has been reported by Kaizer et al. , whereasA few clinical studies have investigated the wear of monolithic zirconia crowns to antagonist enamel and other ceramic/metal-ceramic crowns after an observation period of up to two years. Mundhe et al.  studied Zirconia restorations present almost comparable results with other types of dental restorations. Porcelain veneered single crowns present success rates ranging from 88.8% to 100% for a follow-up period of 58.7 to 60 months ,115. AccLiterature search strategy for clinical studies included electronic search in PubMed and Scopus databases by using combinations of the terms “monolithic zirconia”, “clinical performance”, “survival”. All potentially relevant abstracts and titles were read and those included were clinical trials and case reports involving humans, without any other inclusion or exclusion criteria, as the concept was a general and not a systematic review of the literature. The results of only 10 clinical studies evaluating monolithic zirconia restorations on teeth are available, as of April 2019 . BömickeNewly introduced ultra-translucent and multicolor monolithic zirconia ceramics present considerably improved aesthetics and translucency, but they have to be further evaluated both in vitro and in vivo for their long-term potential to preserve their outstanding properties. Compared to other ceramic materials, monolithic zirconia causes minimal wear of antagonists, especially if appropriately polished, so the initial concerns that zirconia, as a hard polycrystalline material, would cause significant tooth structure loss have been significantly overcome. Unfortunately, no study exists, either in vitro or clinical, to evaluate the wear properties of new ultra-translucent zirconia ceramics. Concerning zirconia restorations survival, few short-term studies reveal promising results, especially for implant-retained monolithic zirconia crown and FDPs restorations. Due to limited evidence available, well-designed clinical studies are urgently needed to enlighten issues of prognosis and long-term survival.


Microcrystalline cellulose (MCC) aerogels were synthesized, blendingwith high amylose corn starch of different contents based on a NaOH–urea solution, and following by vacuum freeze-drying technology. The microstructure of the aerogel was observed by scanning electron microscopy (SEM) as an interconnected, porous three-dimensional structure, while X-ray diffractogram (XRD) measurements showed that the crystalline form was converted from cellulose I to cellulose II during dissolution and regeneration. Thermogravimetric analysis (TGA) showed that the content of starch had little effect on the thermal stability of the aerogel, whereas the content of starch had great influences on absorption and viscoelastic properties. When the ratio of starch was 10% and 15%, the prepared aerogels presented a low density and abundant pores, which endowed the aerogels, not only with the highest absorption ratio of pump oil and linseed oil , but also with better dynamic viscoelastic properties. The removal of pollution from water is; therefore, highly desirable. To date, the most commonly used methods to deal with water pollution are physical, chemical, and biological treatments, among which absorption is the simplest and most commonly used physical treatment for its efficiency and cost effectiveness. Among natural materials, the most common absorbents include wheat straw , walnut  aerogel , graphen aerogel  and otheThese materials constitute a new generation of aerogels, which have not only inherited the high porosity and large surface area of preceding aerogels , but alsStarch is a polysaccharide that can be extracted directly from plants. High amylose starch always has a high resistance to gelatinization and hydrolysis ,23. In aIn this paper, we present a new composite aerogel based on microcrystalline cellulose and high amylose corn starch dissolved in a NaOH–urea solution, produced by using vacuum freeze-drying technology. The objective of the study is to investigate the influence on structural characteristics, thermodynamic, absorption, and viscoelastic properties, of charging cellulose-based aerogels with different starch content.Microcrystalline cellulose  was provided by Sigma-Aldrich (Shanghai) Trading Co., Ltd. . High amylose corn starch  was purchased by Yujing Food Co., Ltd.  Sodium hydroxide (NaOH) and urea were received from Beijing Chemical Works . Hydrochloric acid (HCl) was supplied by Sinopharm chemical reagent co. Ltd .2 balls (6 mm in diameter) in a volume ratio of 1:10 or 1:6 for 1 h, which is more conducive to chemical reaction and promotes structural recombination of new materials.Firstly, a ball mill  was used to produced microcrystalline cellulose and high amylose corn starch in smaller particle sizes, about 20 and 11 µm, respectively. This was done by mixing microcrystalline cellulose or high amylose corn starch and ZrO0CA, S5CA, S10CA, S15CA, S20CA, S25CA, which corresponded to additions of 0%, 5%, 10%, 15%, 20%, and 25% of starch, respectively. Then 14 g NaOH and 24 g urea were added to make the emulsion transparent. After freezing the samples for 24 h, the transparent solid was defrosted and stirred vigorously at atmospheric conditions for 30 min. The white hydrogel was obtained by washing with 5% hydrochloric acid (HCl) and distilled water several times until the leaching solution was neutral. Then the prepared hydrogel was transferred into a freeze-drying machine  to freeze-dry at a temperature of −60 °C and a pressure of 15 Pa for 48 h to fabricate aerogels.High amylose corn starch–microcrystalline cellulose aerogel samples were prepared utilizing the following methods . Total mwetV. The diameter and height of aerogels after drying were measured, and the volume was calculated, denoted as V. Each sample was measured three times and the average value was taken. Volumetric shrinkage M (%) was expressed in the following Equation (1):The volumetric shrinkage of aerogels was measured by the volume change of samples before and after vacuum freeze-drying. The diameter and height of hydrogels before drying were measured, and the volume was calculated, denoted as 0 of aerogels was accurately measured by electronic balance (AB204-S), and the volume after drying was calculated. Each sample was measured three times and the average value was taken. The skeletal density of aerogel was calculated by the following Equation (2):The mass m0 is the bulk density. The porosity P (%) was expressed in the following Equation (3):Ignoring the internal air density of aerogels, The morphologies of the aerogels were characterized using a scanning electron microscope (SEM) , operated at an accelerating voltage of 15 kV after being coated with Au.CI (%) was shown in the following Equation (4):002 is the maximum intensity of the (002) lattice diffraction and Iamorph is the intensity diffraction of the amorphous band [Aerogel crystal structures were identified by X-ray diffraction techniques (XRD) . Scattered radiation was detected in the range of 2θ, from 4° to 40°, with a scanning rate of 2°/min. Crystallinity index Ious band .Aerogel samples were subjected to thermogravimetric analysis (TGA) in a thermogravimetric (TG) analyzer . All samples were analyzed under a nitrogen atmosphere with flow rate of 100 mL/ min, heating rate of 20 °C/ min, and temperature range of 50–700 °C.W0 and W are weights of aerogel sample before and after absorption, respectively.Aerogel samples of about 20 mg were immersed into plenty of linseed oil or pump oil. After placing it in the beaker for 24 h to ensure the absorption equilibrium, then the aerogel was put on the sieve for 30 s to remove surface oil, and then was weighed. Each sample was measured three times with an average value, and the adsorption ratio C (%) was measured in the following Equation (5):Aerogel samples was placed on the dynamic mechanical analyzer  to determine the creep curves. The aerogels were cut into 1 cubic centimeter cubes using compression. The preload (0.01N) and the force track (125%) was set and after equilibrating at 30 °C, a stress of 0.0001 MPa was supplied to the aerogels and then the applied stress was removed. The creep time and recovery time were both 10 min. The data of strain was recorded as a function of time.ε(t) represents the creep strain of the aerogel sample; σ0 represents the loaded stress, 0.0001 MPa; ME and Mη represent the modulus and viscosity of the Maxwell spring and dashpot, respectively; KE and Kη represent the modulus and viscosity of the Kelvin spring and dashpot, respectively, and τ = Kη/KE; t denotes the time (s) after loading.Burger’s model was used to explain the creep curves. Creep equation of Burger’s model was in the following Equation (6):The mechanical characteristic of the aerogels was measured using a dynamic mechanical analyzer . Aerogel samples were pretreated as the creep-recovery measurements. The temperature and the force track were set at 30 °C and 125%. The frequency ranged from 0.1 to 50 Hz and the controlled strain was set at 0.15%. The storage modulus G′, loss modulus G″, and tan δ of the samples were recorded to create the corresponding frequency sweep curves.The mechanical characteristic of the aerogels was measured using a dynamic mechanical analyzer . Aerogel samples were pretreated as the creep-recovery measurements. A constant strain of 0.15% (within liner elastic limits) was applied to the samples and the storage modulus G′, loss modulus G″, and tan δ as a function of temperature. The test temperature was increased from 30 to 100 °C at a heating rate of 3 °C/ min with frequency of 1 Hz. The temperature sweep tests were all replicated three times.A summary of the density, porosity volume shrinkage, and absorption rate of the aerogel samples with different starch content are given in −3, and porosity ranged from 90.1% to 91.8%, which were much better than what was reported in the literature, whereby the cellulose derivatives -based aerogels had high densities (0.25–0.85 g/cm3) and low porosities (41%–85%) [−3, which equaled that of the aerogels produced in the present study. As the table showed, there were no significant differences between S0CA and S5CA of the density and porosity (P > 0.05). However, as a greater amount of starch was added, the density reduced greatly and porosity increased significantly (P < 0.05), compared with S0CA, perhaps because of the decrease of the content of microcrystalline cellulose. At the same time, high amylose corn starch can form porous three-dimensional structure to avoid the collapse of microcrystalline cellulose aerogel skeleton.The principle of vacuum freeze-drying is to utilize sublimation to makes the material solvent change from solid to gas directly. Through vacuum freeze-drying, the volume shrinkage rate of S0CA was the largest, and compared to the volume shrinkage rate of aerogels with starch were significantly reduced (p < 0.05), indicating that starch can effectively prevent the collapse of aerogels’ skeleton.Their density was in a range of 0.125 to 0.151 g cm41%–85%) . The den41%–85%) , and new41%–85%) , were alWhen the starch content reached 25%, the density and volume shrinkage rate of aerogels increased, and the porosity decreased, which may be related to the excessive amount of starch, as well as the agglomeration aggravating the collapse of the skeleton.Oil viscosity is an important factor affecting oil absorbing ability. It has been found that the viscosity of oil could have two different effects on adsorbent adsorption: First, the increase of oil viscosity is more conducive to oil adsorption on the absorbent surface, which increases the absorption ratio; secondly, the increase of oil viscosity will prevent the oil molecules from entering the absorbent, so as to reduce the absorption ratio . The visCompared with non-added starch aerogel, the adsorption ratio of the aerogels with different starch all increased significantly (p < 0.05). When the starch content was 15%, the absorption ratio of aerogels to pump oil and linseed oil both reached to the maximum, while the density of aerogels was the minimum and the porosity reached to the maximum according to the 0CA  patterns of obtained aerogels with different starch additions, as well as of starch and of microcrystalline cellulose ball-milled for 1 h are shown in CI, refined and calculated by JADE software  showed that with the increasing starch content, the crystallinity of the aerogels first decreased and then increased. When the addition of starch was 15%, the crystallinity reached a minimum of 32.57%. The crystallinity of the sample decreases due to the addition, within aerogels, of starch, with a high content of amorphous region [For the XRD patterns of the aerogels, the height of the peak observed at 12°, 20°, and 22° all decreased at first, but once the starch content was over 15%, the height began to increase. According to s region . The cel0CA was the highest, about 39.73%, while S15CA was the lowest, about 34.15%. This may be because when the content of starch was 15%, the network of aerogels through hydrogen bonds was more orderly and the heat transfer was more uniform, attributing more thorough combustion. The onset temperature of all aerogels was much lower than MCC and starch, indicating that the dissolution and regeneration of MCC and starch contributed to reducing the thermal stability. However, there were no obvious differences between pure cellulose-based aerogel and the aerogels with different starch content, revealing that the introduction of starch did not enhance the cellulose skeleton stability to some degree.Thermogravimetric (TG) curves and derivative thermogravimetric (DTG) curves of microcrystalline cellulose, high amylose corn starch, and the aerogels with different additions of starch are presented in 0CA, indicating that the starch was distributed well in the cellulose-base aerogels and the addition of starch enhanced the creep resistance. S10CA presented the lowest creep strain, demonstrating that the aerogels reached the best creep resistance. However, when the addition of starch exceeded 20%, the creep strain curves were significantly higher than the other aerogels. This was probably due to excess starch that could not be distributed in the cellulose-based aerogels uniformly; it was confirmed that some severe agglomeration of starch attached to the surface from The creep and creep-recovery strains as a function of time of the aerogels prepared with different starch content were presented in M, EK, τ, and ηM) and the determination coefficient R2 obtained from the Burgers model were fitted as shown in 2) ≥ 0.989, suggesting that the four-element Burgers model can represent the creep behavior of the aerogels well.The creep behavior can be described by a four-element Burgers model . The fouM represents an instantaneous creep deformation that can be restored immediately when the external force is withdrawn. The greater the value, the better the elasticity of the material [M increased when the starch content increased from 0% to 15%, whereas it decreased within 20% to 25%, indicating that excessive starch could destroy the viscosity of cellulose-based aerogels. When the content of starch reached 15%, the elasticity of aerogels was the best. EK represents the retarded elastic modulus and τ is the retardation time of the Kelvin component. The greater the values, the stickier the material. For S10CA, the value of EK reached to maximum; however, τ reached to minimum. When the starch content reached 15%, the value of EK and τ were both high relatively. ηM represents the viscous component of the dashpot. S15CA showed the maximum ηM value of 1.777 ± 0.359MPa·s−1, which meant that S15CA had the least irreversible variables and better viscoelasticity. However, once the starch content reached 20% or more, the value of ηM decreased significantly and even was lower than S0CA.Ematerial . The tabThrough the above chart analysis, it shows that the incorporation of high amylose corn starch could improve the viscoelasticity and creep resistance of cellulose-based aerogels. Similarly, the cellulose-based aerogels blended with the starch content of 15% presented the best viscoelasticity.The variation of the storage modulus (G′), the loss modulus (G″), and tan δ as a function of frequency with the different starch content range is represented in The temperature sweep curves of the aerogel samples showed similar profiles in different starch content, all of which first increased and then decreased, and reached the maximum value between 50–70 °C. The softening of materials can be explained by molecular motion in free volume theory . The molIn this study, a low density, high porosity, and low-cost aerogel with high absorption capability has been successfully synthesized using MCC and starch, through a vacuum freeze-drying process. SEM analysis revealed an interconnected, highly porous three-dimensional structure of the obtained aerogel, while XRD measurements showed a transformation of the crystal form from cellulose I to cellulose II during the dissolution and regeneration process. The weight ratio of starch had a great influence on the aerogel’s properties. When the ratio was 10% and 15%, the prepared aerogel presented a low density and abundant pores, which endowed the material not only with the highest absorption ratio of pump oil and linseed oil , but also with excellent dynamic viscoelastic properties. In conclusion, the study has identified the suitable starch content range for fabricating operable, efficient, and cost-efficient aerogels.


Protein degradation mechanisms are integral to protein homeostasis. Their reduced efficiency during aging leads to accumulation of misfolded and aggregated proteins which potentiate proteotoxic disorders. Paradoxically, our lab reported that the Caenorhabditis elegans rpn-10(ok1865) proteasome mutant possesses enhanced proteostasis and extended lifespan. RPN-10/PSMD4 is a ubiquitin receptor of the 26S proteasome that targets polyubiquitinated substrates to its catalytic core for degradation. Proteasome dysfunction of the rpn-10 mutant is characterized by reduced, not inhibited, ubiquitin fusion degradation. We ascertained that upregulated autophagy and SKN-1/Nrf-mediated responses partially contribute to the robust rpn-10 mutant phenotype. Further investigation of its underlying mechanism revealed that several ERQC genes are transcriptionally upregulated in the rpn-10 mutant. Thus, we hypothesized that the rpn-10 mutant exhibits improved ER proteostasis which mediates its elevated cellular stress resistance. Accordingly, the rpn-10 mutant shows increased ER stress resistance and altered ER homeostasis. Complementarily, attenuated expression of the aggregation-prone α-1 antitrypsin (ATZ) reporter proves that ER proteostasis is ameliorated in the rpn-10 mutant. Via a genetic screen for suppressors of decreased ATZ aggregation in the rpn-10 mutant, we identified novel player H04D03.3, which is a homolog of the proteasome adaptor ECM29. This suggests that assembly of the rpn-10 mutant proteasome itself critically regulates its ER proteostasis. Moreover, we observed that cytosolic proteostasis and longevity depend on ER master chaperone hsp-3/-4(BiP) and ER ATPase cdc-48.2(p97/VCP), further highlighting ERQC significance in the rpn-10 mutant. Altogether, it appears that mild proteasomal dysfunction induces ERQC adaptation that underlies proteostasis and longevity benefits of the rpn-10 mutant.


SEX4 and LIKE SEX FOUR2 (LSF2) were repressed using RNAi constructs. Although repression of either SEX4 or LSF2 inhibited leaf starch degradation, it had no effect on cold-induced sweetening in tubers. Starch amounts were unchanged in the tubers, but the amount of phosphate bound to the starch was significantly increased in all the lines, with phosphate bound at the C6 position of the glucosyl units increased in lines repressed in StSEX4 and in the C3 position in lines repressed in StLSF2 expression. This was accompanied by a reduction in starch granule size and an alteration in the constituent glucan chain lengths within the starch molecule, although no obvious alteration in granule morphology was observed. Starch from the transgenic lines contained fewer chains with a degree of polymerization (DP) of less than 17 and more with a DP between 17 and 38. There were also changes in the physical properties of the starches. Rapid viscoanalysis demonstrated that both the holding strength and the final viscosity of the high phosphate starches were increased indicating that the starches have increased swelling power due to an enhanced capacity for hydration.To examine the roles of starch phosphatases in potatoes, transgenic lines were produced where orthologs of  It is a polyglucan consisting of two fractions; branched amylopectin and lesser branched amylose. Most starch used in industry is isolated from seeds of cereals, such as maize or wheat, with smaller, yet significant amounts coming from plants with underground storage organs, such as potato or cassava  and phosphoglucan, water dikinase (PWD). GWD specifically phosphorylates the C6-position while PWD is responsible for incorporation at the C3-position , 2006, bLSF1), that shows similarity to both SEX4 and LSF2, is not thought to encode a starch phosphatase . SEX4 was originally identified as being involved in starch metabolism through study of a mutation that impairs starch mobilization in Arabidopsis leaves , while Lsphatase  as seversphatase . Interessphatase , increassphatase .AtLSF2, but not AtSEX4, led to increases in starch bound phosphate in Arabidopsis leaves  using the tBLASTn algorithm at the NCBI2. Plasmids containing EST’s corresponding to each ortholog were obtained from the French Plant Genomic Resource Center and 300 bp fragments were amplified by PCR using the following primers: . The NtSEX4 and NtLSF2 sequences are sufficiently similar in sequence to be effective in strategies designed to silence their putative potato orthologs [Accession numbers DQ241838 (StSEX4) and XM_006361255 (StLSF2)]. Amplicons were restricted with SmaI and SalI or EcoRV and BamHI before being ligated into the same sites of pBluescript-SK+ (Agilent Technologies Inc.). RNAi constructs were produced in the plant transformation vector pHellsgate2 (GACAAGTTTGTACAAAAAAGCAGGCTGTAATACGACTCACTATACGGC3′ (AttB1 site underlined) and T3AttB2 5′GGGGACCACTTTGTACAAGAAAGCTGGGTAATTAACCCTCACTAAAGGG3′ (AttB2 site underlined)]. The silencing vectors were transformed into Agrobacterium tumefaciens GV2260 by the freeze-thaw method (Solanum tuberosum L. var Désirée) plants by the method of Arabidopsis SEX4 (UniprotKB/Swiss-Prot: Q9FEB5.1) and LSF2 (UniprotKB/Swiss-Prot: Q9SRK5.1) peptide sequences were used to identify tobacco expressed sequence tags (EST’s) . Potato sequences were obtained from the Potato Genome Sequencing Consortium4 . The aligned matrix contained 71 taxa and 155 amino acid characters, of which 148 were parsimony-informative. Parsimony analysis was performed using the PAUP∗ V 4.0 10 computer program  sand 50% (v/v) potting mix and were grown in a glasshouse. Leaf samples for starch and sugar analysis were taken from 10-week-old plants over a diel cycle using a cork borer and were immediately frozen in liquid Nvia reverse transcription of 1 μg of total RNA, using an oligo (dT15) primer and M-MLV (H-) reverse transcriptase  according to the manufacturer’s instructions. The semi quantitative-PCRs (sq-PCR) were conducted with GoTaq® DNA polymerase (Promega) in a 50-μl reaction  for 24 cycles, with a primer annealing temperature optimum of 58°C for all respective sq-PCR primer pairs. The constitutively expressed gene, β-tubulin2 (609267), was used to determine the number of cycles used for the sqRT-PCR, where expression occurred in the linear range. The Stβ-tubulin2, StLSF1, StLSF2, and StSEX4 primer pairs  were designed to amplify fragments of 0.5 kb from the cDNA.Total RNA was isolated from tuber and leaf material using the Plant RNeasy Mini kit , as described by the manufacturer. The complementary DNA (cDNA) template was obtained Starch and soluble sugar amounts were determined enzymatically from tuber or leaf tissues by the method of Amylose was estimated using an iodine binding assay  while th31P NMR. NMR samples contained 50 mg starch digested with α-amylase and amyloglucosidase, D2O was added (10% v/v) and the pH adjusted to 6.0 with 0.2 M NaOH as described by 1H and 31P spectra were measured for each sample. 31P 1D spectra were accumulated in multiple experiments for a total acquisition times of 1.2 h per sample.Glucose 3-phosphate content was calculated from that figure using the ratio of C6:C3 bound phosphate in each of the transgenic lines determined by g. Linear glucans in the supernatant were separated and measured by HPAEC-PAD using a Dionex PA-200 column.Chain length distributions were analyzed by boiling 20 μg of purified starch in water followed by debranching using isoamylase, as described by Swelling power was determined by the method of Starch granule size was determined using a Saturn DigiSizer 5200 particle size analyzer. Five grams of starch was suspended in MilliQ water within the machine and directed toward a 30 mW, 687 nm solid-state laser. Starch granule size distributions were determined based upon the amount of light scattered by each particle using a refractive index value of 1.54, as outlined by Isolated starch granules were sprinkled onto double sided tape and coated in gold using a sputter coater . Micrographs were taken in a scanning electron microscope (Zeiss Evo MA15VP) at an accelerating voltage of 25 kV.2, 1 mM β-mercapoethanol, 0.1% (v/v) TWEEN-20 and 1 mM PMSF. Following centrifugation, the supernatant was isolated, and the amount of protein determined using a Bradford assay kit (Bio-Rad).Proteins were extracted from tuber material by grinding in 50 mM TRIS-HCl (pH7.0), 5 mM EDTA, 5 mM MgClFor immunoblots 25 μg of crude protein extract was denatured by heating at 95°C for 5 min in 2% (w/v) SDS, 10% (v/v) glycerol, 60 mM TRIS-HCl (pH 6.8) before being separated by either 8 or 10% (w/v) SDS-PAGE. Separated proteins were blotted onto nitrocellulose membranes using a semi-dry blotter (Bio-Rad). Immunoblots were performed using antibodies that specifically detect GWD , PWD Kö, or SEX4-1. For starch synthase the gel was incubated in 50 mM Tricine-NaOH (pH 8.5), 0.5 M trisodium citrate, 2 mM EDTA and 2 mM DTT for 30 min at 4°C before the buffer was removed and replaced with the same buffer, but containing 1 mM ADP-glucose in addition. This was left at room temperature overnight and starch synthase activity revealed through staining with Lugol’s solution. For starch branching enzyme and starch phosphorylase two gels were incubated in 50 mM HEPES-NaOH (pH7.0) 10% (v/v) glycerol of 30 min at 4°C. This buffer was removed and replaced with the same buffer containing 2.5 mM AMP and 50 mM glucose 1-phosphate for one gel and 2.5 mM AMP, 50 mM G1P and 28 units of phosphorylase a (from rabbit muscle) for the other. Both the gels were incubated at room temperature overnight and activities were revealed by staining with Lugol’s solution. Bands appearing in both gels are formed by starch phosphorylase, while bands appearing only in their gel incubated with phosphorylase a are formed by starch branching enzyme.Enzyme activities were examined by separating 100 μg of crude protein extract on 8% (w/v) polyacrylamide gels containing either 0.3% (w/v) oyster glycogen (for starch synthase) or 0.02% (w/v) oyster glycogen (for starch branching enzyme and starch phosphorylase) for 3 h at 4°C at 10 V cmpost hoc Bonferroni–Holm partitioning were determined in Microsoft Excel 2010 using Daniel’s XL Toolbox add-in . The potato proteins fell into each of the clades indicating that the three proteins identified in Arabidopsis are conserved in potato and are likely to have the same biochemical function. The predicted protein sequences from the potato genes were similar in structure to the equivalent Arabidopsis proteins . Repression of StSEX4 appeared to also repress StLSF2 in one line (SEX4-1), however we were unable to detect expression of StLSF1 in growing tubers. Immunoblots showed that repression of StSEX4 led to a decrease in StSEX4 protein accumulation, but we were unable to examine StLSF2 protein abundance due to the lack of an appropriate antibody. Repression of neither StSEX4 nor StLSF2 genes altered the amounts of the starch phosphate incorporating enzyme, GWD . However, PWD amounts appeared to be slightly decreased in all the transgenic lines . In gel assays were used to examine the activities of enzymes known to affect starch polymerization. There were no consistent differences in starch synthase, starch branching enzyme or starch phosphorylase activities in crude extracts from tubers of the transgenics lines, compared with the controls . The transgenic plants were phenotypically unaltered from each other or from the untransformed control . Although there were some differences in tuber yield between the transgenic lines, this was not consistent within different lines where the same gene was repressed , however, the tubers in three of the four transgenic lines were smaller than the untransformed control .RNAi constructs designed to repress either Figure 3A). Quantitative analysis over a diel cycle demonstrated that leaves of three of the four transgenic lines contained significantly increased starch at the end of the dark period compared with the controls . Tubers harvested from the plants were kept at 4°C for 8 weeks before glucose, fructose, and sucrose concentrations were determined. Although soluble sugars increased in tubers stored at 4°C compared with those stored at room temperature, no significant differences were found between the amounts of these sugars present in any of the lines .Staining of starch using Lugol’s solution in leaves that had been darkened for 72 h showed that the polymer was still present in all the transgenic lines, but not the control . This analysis demonstrated that the majority of granules from all lines were roughly ovoid, without any obvious morphological change between samples.Starch granules isolated from all the lines were visualized by scanning electron microscopy  demonstrated that the transgenic lines had fewer chains with degrees of polymerization (DP) between 5 and 20 and more of between DP20 and 40.To examine if the genetic alteration also affected the branching distribution within starch, isoamylase digested samples were separated by HPAEC-PAD. It was found that starch from all the transgenic lines had significantly altered chain length distribution. Difference plots (Table 1). None of the transgenic lines contained significantly different amounts compared with the controls and the ratio of amylose to amylopectin was unaltered. However, 31P-NMR showed that the ratio of C6- to C3-bound phosphate was affected in the transgenic lines, with the amount of C6-bound phosphate being increased relative to C3-bound phosphate in the StSEX4 repressed lines, while C3-bound phosphate increased in StLSF2 repressed lines (Table 1). Measured amounts of C6-bound phosphate were also consistently and significantly increased in the StSEX4 lines (Table 1). The ratios of phosphate bound at the C6 and C3 positions, determined by NMR, were used to calculate the amount of C3 bound phosphate in the different lines based on the measured amounts at the C6 position. In this case, a significant increase in C3-bound phosphate was found in both LSF2 lines (Table 1). This led to an increase in total phosphate (combined C3 and C6) in all lines except LSF2-2 (Table 1). All the transgenic lines contained starch granules with a significantly reduced average diameter (Table 1).Some genetic manipulations affecting the structure of potato starch have been demonstrated to reduce starch accumulation, so we examined if starch content was altered in the transgenic tubers  demonstrated significantly increased swelling power compared with the control (Table 1). To examine gelling in more detail we subjected the starches to analysis in a rapid viscoanalyzer (RVA). Peak viscosity was decreased in the LSF2 lines, while trough viscosity and breakdown were both decreased in starches from all the transgenic lines. On the other hand, final viscosity was significantly increased in all lines (Table 2).Because of these alterations in starch bound phosphate, chain length and granule size, we examined the swelling power of the starches. Three out of the four starches . There were no obvious differences in plant morphology  and total tuber production was not consistently altered in the transgenic lines . However, there was a significant decrease in average tuber weight in both SEX-4 lines and one of the LSF2 lines  indicating that repression of StSEX4 or StLSF2 affect tuber initiation. Leaf starch degradation was affected in both StSEX4 and StLSF2 repressed lines. Qualitative iodine-staining showed that leaves kept in the dark for 3 days still contained significant amounts of starch . Quantitative analysis demonstrated that leaves from both StSEX4 and StLSF2 repressed lines contained significantly more starch at the end of the night than wild type plants . Thus, both genes are involved in leaf starch degradation in potato. Furthermore, StLSF2 seems to play a bigger role in leaf starch degradation in potatoes than the orthologous gene does in Arabidopsis, since Atlsf2 knockout mutations have nearly normal rates of starch degradation . One potential explanation for this would be that starch degradation occurs continuously at a lower rate in cold-stored tubers than in leaves, where almost all starch present needs to be degraded during the night period prior to the commencement of starch synthesis the next morning. It is possible that removing SEX4 or LSF2 may, therefore, be less effective at repressing this process in tubers simply due to the rate of degradation within these different tissues. Alternatively it may indicate that functional redundancy is more important in this step of the starch degradation pathway in potato tubers than in leaves. A final possibility for the lack of effect on cold-induced sweetening is that the pathway of starch degradation established in leaves does not represent the one present in tubers. This process needs to be investigated in more detail.To examine this we manufactured transgenic plants repressed in transcription of potato orthologs of either radation . DespiteMany studies have focused on the influence of starch phosphate on alterations in physical properties through analysis of starches from different botanical origins, different plant varieties where phosphate contents have been altered ,d, or geTable 1). Within the StSEX4 repressed lines, the main phosphate increase was at the C6 position, while in the StLSF2 repressed lines it was at the C3 position (Table 1). This corresponds to the known roles of the two enzymes encoded by the orthologous Arabidopsis genes where AtSEX4 and AtLSF2 are thought to have greater specificity for removing phosphate from the C6 and C3 positions, respectively  by 9% in line LSF2-1 and up to 39% in line SEX4-1 . The structural alterations caused by changes in bound starch phosphate may affect the starches suitability as a substrate for enzymes that determine the chain length of the polymer. Although starch branching enzymes are known to be able to use phosphorylated glucans as substrates , it is still possible that the alteration in chain length may also have led to increased phosphate incorporation by GWD as this enzyme has been shown to have a higher preference for chains between DP 28–30 . This would, however, only help to explain the increased phosphate in the SEX4 lines as GWD incorporates phosphate specifically at the C6 position . This was not caused by a reduction in starch accumulation within the tuber (Table 1) and so must be due to an effect either on granule initiation or size determination, processes which are poorly understood . Two recent studies using potato have led to plants synthesizing tuber starch with altered starch phosphate. GWD gene, showed altered starch granule morphology only in lines where starch phosphate was reduced, not where it was increased. In the second study , demonstrating increased water binding. Starch phosphate has also been positively correlated with peak viscosity and breakdown, and negatively correlated with setback (Table 2). The reasons for these differences are unclear. Although starch phosphate is increased in the transgenic lines, we also observed changes in chain lengths within the starch molecules and granule size. As RVA examines the sum of the effects that all these structural changes have on the starches physical properties, it is possible that these other alterations have the opposite effect on peak viscosity and breakdown to that caused by increased starch phosphate.The alterations that we identified in starch isolated from the transgenic plants led us to examine its physical properties. As the presence of covalently bond phosphate is known to improve starches hydration capacity  we exami setback ,c. As woThe alterations that we have identified in starches from the transgenic lines indicate that they may have improved properties for use in specific industries. The observed increased final gel viscosity would be advantageous in the food industry as less starch would be required to achieve the same thickening effect. Alterations in starch granule size has also been shown to help in the manufacture of noodles, with smaller granules – as found in the transgenic line – being preferred . Finally31P NMR. CvdV supervised the plant transformation. JK and JL designed the project while JL and ES wrote the paper together. All authors proofread the paper and provided feeback.ES transformed the plants and performed analysis on immunoblots, RVA and cold-sweetening. JJ and ES analyzed starch phosphate, starch granule size, and cold-sweetening. BL performed the semi-quantitative RT-PCR. KO generated the phylogenetic tree. GG and SZ produced the chain length distributions of debranched starch. FD was responsible for the The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Superabsorbent polymers (SAPs) are crosslinked hydrophilic polymers that are capable of absorbing large amounts of water. Commercial SAPs are mostly produced with acrylic acid that cannot be easily biodegraded. Therefore, in this study, polysaccharide-based SAPs using carboxymethyl cellulose as a major component were prepared. Starch aldehydes and citric acid were selected due to their environment-friendly, non-toxic, and biodegradable properties compared to conventional crosslinking agents. Starch aldehydes were prepared by periodate oxidation, which forms aldehyde groups by taking the places of C–OH groups at C-2 and C-3. Furthermore, starch aldehydes were analyzed through the change in FT-IR spectra, the aldehyde quantitation, and the morphology in FE-SEM images. In the crosslinking of polysaccharide-based SAPs, the acetal bridges from starch aldehydes led to a large amount of water entering the network structure of the SAPs. However, the ester bridges from citric acid interfered with the water penetration. In addition, the swelling behavior of the SAPs was analyzed by the Fickian diffusion model and the Schott’s pseudo second order kinetics model. The relationship between swelling behavior and morphology of the SAPs was analyzed by FE-SEM images. In conclusion, polysaccharide-based SAPs were well prepared and the highest equilibrium swelling ratio was 87.0 g/g. Superabsorbent polymers (SAPs) are crosslinked hydrophilic polymers that are capable of absorbing large amounts of water . It has Polysaccharides are major sources of bio-based SAPs. There have been many studies using starch , carboxyIn SAP preparation, a chemical modification of starch to improve performance has been studied. Although there are many kinds of chemical modification, the most typical method is free radical graft copolymerization of vinyl monomers onto starch ,19,20,21Most starch/acrylic acid copolymers are advantageous in water absorption. However, as mentioned earlier, acrylic acid has poor biodegradability, which leads to environmental problems with their disposal ,20. FromThe addition of a crosslinking agent is also very important for SAP preparation. In this experiment, starch aldehydes and citric acid were selected as crosslinking agents. Starch aldehydes, along with their own absorptivity, were used for effective chemical bonding with the hydroxyl groups of CMC. In previous studies, based on the high functionality and reactivity of aldehyde groups, there have been attempts to use environment-friendly and non-toxic dialdehyde carbohydrates as crosslinking agents to replace glutaraldehyde. The linkages between aldehyde and hydroxyl groups are used to prepare films ,26 whileThe experimental SAP preparation procedure consisting of starch aldehydes, CMC, and citric acid first involves modifying corn and potato starch to starch aldehydes by oxidation. In particular, the oxidation by sodium periodate changes the C-2 and C-3 hydroxyl groups to aldehyde groups ,29 wherew = 700,000) was purchased from Acros Organics, . Methanol used for washing as well as hydroxylamine hydrochloride, methyl orange, and sodium hydroxide used for DS calculation were purchased from Samchun Pure Chemical, Co., Ltd. Citric acid was purchased from Showa Chemical Industry Co., Ltd.  while sulfuric acid was used as the catalyst and purchased from Junsei Chemical Co., Ltd., Tokyo, Japan.Corn and potato starch as well as sodium periodate were purchased from Samchun Pure Chemical, Co., Ltd., . CMC  and 1 g of CMC were added in 100 mL distilled water. The temperature of the flask was maintained at 100 °C for 1 h. After complete dissolution of the polysaccharides, 0.05 mL of sulfuric acid were added to the mixture and stirred for 5 min. At this time, citric acid was added to 10% or 20% molar ratio of anhydroglucose units in some samples. The obtained suspension was poured into a mold and dried in an air oven at 55 °C for 24 h. Finally, the film product was peeled from a mold and pulverized to prepare the powder. The sample code and the detailed ingredients are shown in −1 resolution with 32 scans between 650 and 4000 cm−1.A FT-IR spectrometer  was used to confirm the chemical structure of all samples. Before analysis, the samples were dried in an auto-desiccator  for 24 h. The samples were analyzed at a 4 cmA FE-SEM  was used to observe the morphology of all samples. Before analysis, the samples were dried in an auto-desiccator for 24 h. The specimens were coated with platinum and observed at 1000× magnification.The aldehyde content of modified starch was determined according to a method described in literature . First, STSAPW is the weight of the swollen tea bag containing swollen SAPs, STW is the weight of the swollen tea bag only, and ISAPW is the initial weight of dried SAPs.The tea bag method was used to measure the swelling ratio of the SAPs. A tea bag containing dried SAPs was immersed entirely in distilled water to reach equilibrium at room temperature. The swelling ratio was calculated by the following Equation (1): −1, which are due to hydrogen bonded hydroxyl groups [−1, there were several discernible peaks at 1156, 1081, 1018, and 929 cm−1, which are attributed to C–O bond stretching. The peaks at 1081 and 1018 cm−1 were the characteristics of C–O stretching in anhydroglucose ring [−1 showed a bend of tightly absorbed water present in the starch. The bands at 2930 and 2890 cm−1 were the characteristics of C–H stretching [l groups . In the ose ring . The pearetching ,37.−1, which is the most characteristic peak of C=O vibrations in aldehyde groups [−1 was increased, but the peaks at 1156 and 1081 cm−1 were weakened. In other words, three C–O bond peaks at 1156, 1081, and 1018 cm−1, which are the fingerprint regions of starch, gradually transferred into a broad band. It may be due to the fact that periodate oxidation mainly breaks the C–2 and C–3 bond of anhydroglucose units, followed by the formation of the aldehyde groups and the replacement of the C–OH groups at C–2 and C–3 [−1 was weakened, but the new peak of starch aldehydes at 875 cm−1 was assigned to form hemiacetal bonds between aldehyde groups and neighbor hydroxyl groups [A remarkable change in FT-IR spectra of starch aldehydes was the peak at 1732 cme groups ,38. By i and C–3 ,40. In al groups .In the DS calculation by chemical titration, the key mechanism is a reaction between aldehyde groups and hydroxylamine hydrochloride, which produces oxime compounds and hydrogen chloride ,42. ThisIn previous studies, DS also increased as the amylopectin content increased, because amylopectin is more prone to oxidation than amylose ,45. In tIn The particles of starch aldehydes obviously showed lima bean or hemoglobin-like shapes and showed that the interior of starch granules was easily corroded . Accordi−1 were derived from CMC, which were attributed to COO- stretching and CH2 absorption, respectively [−1 were derived from native starch, which were the characteristics of C–O stretching in anhydroglucose ring.The FT-IR analysis was conducted to investigate the chemical crosslinking of prepared SAPs. First, as shown in ectively ,24. Addi−1 and the carboxyl group peaks of CMC at 1605 cm−1 were detected, respectively. A shift to the peak of 1605 cm−1 compared to the peak of 1630 cm−1 indicated that starch aldehydes and CMC may be involved in the crosslinking to form an acetal bridge. Generally, aldehyde is reacted with the hydroxyl group to form an acetal bridge at acidic pH [−1 was attributed to be the C=C double bond of aldol condensation products between starch aldehydes [The FT-IR spectra of CS10-CA0, CS20-CA0, and CS30-CA0, which were prepared by the chemical crosslinking of starch aldehydes and CMC, showed the following peak characteristics. As expected, the peaks of unreacted starch aldehydes at 1735 cmcidic pH , but it ldehydes .−1 for CS-CA0, CS10-CA0, CS20-CA0, and CS30-CA0 in order to confirm the formation of the acetal bridge. The peaks at 1153, 1080, and 1025 cm−1 for CS-CA0 and CS10-CA0 were no longer present in CS20-CA0 and CS30-CA0. Instead, the new peak appeared at 1140 cm−1. In other words, the C-O stretching at 1153 and 1080 cm−1 were replaced by the new absorption band, which could be attributed to C-O ether and C-O-C acetal bridge formed by CMC crosslinking with starch aldehydes. It was similar to previous studies where polyvinyl alcohol was crosslinked with glutaraldehyde [In the FT-IR analysis, the position of the acetal C-O-C band is similar to that of the fingerprint region for polysaccharides. Thus, it was necessary to analyze the spectra at 1200–1000 cmaldehyde ,52. Ther−1, which could be attributed to the characteristic stretching band of the carbonyl group related to anhydride formation [−1 and the peaks of starch anhydroglucose ring at 1153, 1080, and 1025 cm−1 still remained.Next, the spectra of the SAPs with citric acid were analyzed. As shown in ormation ,53. It d−1 and the peak shift of CMC carboxyl groups at 1604 cm−1. Looking at the peaks associated with the anhydroglucose ring, the peaks at 1153 and 1025 cm−1 shifted to 1137 and 1029 cm−1, respectively, while the peak at 1080 cm−1 weakened. For CS20-CA10 and CS30-CA10, the tendencies were same and these SAPs showed the two bands at 1137–1129 and 1063–1053 cm−1, respectively. Moreover, the C=O peak related to the ester bridging of polysaccharides and citric acid was more strongly expressed than the starch aldehydes-related peaks at 1735–1695 cm−1. The C-O peaks related to the acetal bridge by starch aldehydes and the ester bridge by citric acid were mixed at 1200–1000 cm−1.For CS10-CA10, CS20-CA10, and CS30-CA10 using starch aldehydes, the spectra showed a dramatic change. The spectrum of CS10-CA10 showed the ester linkage peak by citric acid at 1732 cm−1. The positions of other main bands were the same as the results in the above-mentioned analysis. In the FT-IR analysis of all SAPs using potato starch aldehydes  and the PS-CA0 (maximum 58.4 g/g) with only physical interactions showed the highest swelling ratios after 8 h of swelling. It was followed by the gel degradation and the decrease of the swelling ratio.In In The SAPs using PS10 (maximum 59.5 g/g) and PS20 (maximum 77.6 g/g) starch aldehydes had lower equilibrium swelling ratios than the SAPs using CS10 (maximum 83.1 g/g) and CS20 (maximum 87.0 g/g) starch aldehydes. Because of the relatively low DS, the acetal bridges were less formed. Interestingly, the CS20-CA0 (87.0 g/g) showed the highest equilibrium swelling ratio in corn starch series while the PS30-CA0 (80.6 g/g) showed the highest equilibrium swelling ratio in potato starch series. It means that there is a slight difference in reactivity and crosslinking density depending on the starch source. In addition, the SAPs using PS30 starch aldehyde showed the significant equilibrium swelling ratios (from 30.2 to 80.6 g/g) even when citric acid was added. It is considered that the equilibrium swelling ratio is increased when the acetal bridges due to starch aldehydes are higher than the ester bridges due to citric acid.Next, to analyze the swelling behavior in more detail, two swelling kinetic models were applied. The initial diffusion of water molecules into the voids of SAPs could be analyzed by the Fickian diffusion model (Equation (3)). The entire swelling process by chain relaxation was evaluated by the Schott’s pseudo second order kinetics model (Equation (4)) ,55.F is the fractional uptake at the given time t (min) while tW and eqW are the weight (g) of absorbed water at time t and equilibrium, respectively. K is the proportionality constant of SAPs and n describes the type of diffusion mechanism. In the Schott’s pseudo second order kinetics model, tQ (g/g) is the swelling ratio of SAPs at time t, isk ((g/g)/min) is the initial swelling rate constant, and sk ((g/g)/min) is the swelling rate constant.In the Fickian diffusion model, n is between 0.5 and 1.0) while other SAPs followed the Fickian diffusion (n is less than 0.5) [eqQ, from 77.1 to 87.0 g/g). However, the Fickian diffusion SAPs showed the relatively low equilibrium swelling ratios (from 10.9 to 56.5 g/g).Each SAP was slightly different in the detailed swelling behavior, but overall swelling behavior involved the following steps. As shown in han 0.5) . The nonhan 0.5) . It was R2 > 0.9) could be applied to the SAPs using starch aldehydes with low DS. It was because the SAPs using starch aldehydes with low DS had the initial swelling in 15 to 30 min, and after that, it maintained a constant level of swelling less than 2 h. However, the SAPs using starch aldehydes with high DS were swollen additionally from 30 min to 120 min after the initial swelling. As a result, an accurate plot of ln(tW/eqW) as function of ln(t) could not be obtained.The second feature was that the Fickian diffusion model with good correlation coefficient (t/tQ versus t are given in R2 > 0.9). The SAPs plotted with low correlation coefficients were characterized by increased swelling between 8 and 24 h after the start of swelling, so it was difficult to calculate the exact swelling parameters. In spite of this mismatch, the theoretical equilibrium swelling ratios matched the experimental data, which indicated that the swelling process followed the Schott’s pseudo second order kinetics model. In addition, the relationship between the initial swelling rate constant and the equilibrium swelling ratio was analyzed. Although the tendencies of all SAPs were not perfect, the initial swelling rate constant decreased as the DS of starch aldehydes or the content of citric acid increased.In the Schott’s pseudo second order kinetics model, the theoretical equilibrium swelling ratio, the initial swelling rate constant, and the swelling rate constant obtained from the plots of Interestingly, the reduced initial swelling rate constant by adding citric acid resulted in decreased equilibrium swelling ratio. However, the decreased initial swelling rate constant using starch aldehydes increased the equilibrium swelling ratio. The latter case was prominent when measuring the swelling ratio of CS10-CA0, CS20-CA0, CS30-CA0, PS10-CA0, PS20-CA0, and PS30-CA0 SAPs. This phenomenon can be related to the aforementioned Fickian diffusion model. These SAPs are relatively active in water diffusion and polymer chain relaxation through the non-Fickian diffusion behavior. In other words, when a certain period of time has elapsed since the water diffused, it reaches a high equilibrium swelling ratio accompanied by rapid swelling around the acetal bridge. On the other hand, the SAPs added citric acid had low equilibrium swelling ratios, because the crosslinking density was dense and the polysaccharide chains could not freely expand.Prior to obtaining the FE-SEM images, the swollen SAPs lyophilized to eliminate moisture. The surface morphology of the SAPs without citric acid were analyzed first. In In Furthermore, the changes in SAP morphology by adding citric acid were analyzed. In general, the images of SAPs showed various types of particles with irregular distribution. In particular, the addition of starch aldehydes had a significant effect on the surface morphology of the SAPs. In When more citric acid is added into the SAPs, inevitably more crosslinking is formed. Therefore, it was necessary to analyze whether this phenomenon affected the surface morphology. As shown in −1 and the disruption of anhydroglucose ring. The hydroxyl groups of C-2 and C-3 of the anhydroglucose ring were successfully substituted with the aldehyde groups and the DS changed according to the amount of sodium periodate and the source of starch. The DS of corn starch aldehydes was higher than that of potato starch aldehydes and the average maximum DS was about 2.48. The FE-SEM image confirmed that the morphology of starch aldehydes was changed from the elliptical native starch shape to the hemoglobin-like shape.CMC was used as a main material for the production of polysaccharide-based SAPs. Starch aldehydes and citric acid were used as crosslinking agents to make the polymer network structure, which is characteristic of the SAPs. Starch aldehydes were prepared by oxidizing corn and potato starch with sodium periodate. The FT-IR analysis of starch aldehydes revealed the appearance of characteristic aldehyde peak at 1732 cmThe crosslinking reaction between starch aldehydes and CMC formed the network structure of polysaccharide-based SAPs. When citric acid was introduced, an ester linkage was additionally formed in polysaccharide-based SAPs. Through FT-IR analysis and swelling ratio measurement of the SAPs, the acetal bridges by starch aldehydes increased water uptake, but the ester bridges by citric acid had the opposite effect. Among the various SAPs, the highest equilibrium swelling ratio was found in CS20-CA0 and PS30-CA0 depending on the starch source where the values were 87.0 g/g and 80.6 g/g, respectively. In addition, the initial swelling behavior and the entire swelling process for all SAPs was analyzed by the Fickian diffusion model and the Schott’s pseudo second order kinetics model, respectively. Finally, the difference in equilibrium swelling ratio was determined by presence and distribution of the porous structures in FE-SEM images.In summary, the bio-based components were introduced to prepare the SAPs with excellent crosslinking and high water absorption. The materials developed in this experiment may be good candidates for environment-friendly SAP products in the future.


We leave a great year behind us and are looking forward to a very exciting one ahead. The year 2019 is before us, a year that seemed unimaginable 21 years ago until we were right at its border. The phrase “out with the old and in with the new” is very fitting at this time.The rapid succession of years, and now once again a New Year and a new volume are upon us. In 2018 we celebrated the 20With the continued support of our contributing readership worldwide, we and the editorial team will maintain and improve the status of the journal and its position. Therefore, we look forward to your prized contributions to the journal in 2019 and beyond.Iranian Journal of Basic Medical Sciences is proud of being an international journal  and the initial 0.324 of just a few years ago (now 1.514, in agreement with the consistent progress of our journal's profile and influence in the scientific community over the past few years. Due to the use of Ithenticate to check integrity and authenticity, the problem of duplicate publication of a few years ago (Up from the Perhaps due to some upgrades to the IT Department of the University, some e-mails were recently failing to go through. Some Gmail, Hotmail, and Yahoo e-mail addresses were sporadically not receiving notifications, others were being bounced back as undeliverable or sent to junk or spam folders. We were unaware of this problem until notified by some of our members and have been working to fix it. If you use your academic or employer's e-mail address and you are not receiving notifications from IJBMS, please ask your IT department to add MUMS to their safe email list. If you are still not receiving e-mail notifications, especially if you have a Gmail, Hotmail, or Yahoo e-mail address, please contact us to see if the problem can be solved or to provide an alternate e-mail address. We apologize for this inconvenience but look forward to better communications in 2019 and later.We advise our contributors to revise their acceptable manuscripts as quickly as possible. Our readers will notice the speedy editorial handling process and accordingly the turnaround of submissions of recent issues of Iranian Journal of Basic Medical Sciences over the past 12 months, such that papers are now taking as little as 1 month from receipt to appearance. The main delaying factor is the lengthy duration of revision by some of our authors. In most cases, we have achieved our 1-week target for full-length papers from submission to notification of a decision. The number of submissions during 2018 has increased by more than 30% and we are quite confident that this trend will continue.Mashhad will be hosting the Conference of Iranian Medical Journal Editors this year. So there is a great opportunity to exchange experiences and share ideas with our colleagues from across the country and international guests. If you are coming to the meeting this year, we look forward to seeing you there.st volume a success.We are very delighted to take this opportunity to wish our readership and contributors, our editorial team, our independent expert referees, the editorial office, the Vice-chancellor for Research, and the Publications Office of Mashhad University of Medical Sciences a Very Happy New Year, with thanks for all the work they did to make the 21


Arteriovenous grafts (AVG) is a good alternative when native arteriovenous fistula (AVF) is not possible. However, complications are higher and close surveillance is required for successful salvage intervention.We present the case of a 35 year old man with a history of a successful Brachio-Axillary AVG performed in his right arm in 2012. He had a background of multiple previous failed attempts of a native AVF formation. He presented in 2014 with symptoms consistent with AVF stenosis. He underwent a successful fistuloplasty and 2 stent insertion. In June 2015 he presented again with re-stenosis and successful fistuloplasty was performed with balloon dilatation. In October 2015 he presented with AVF thrombosis. This was treated with fistuloplasty and thrombolysis. He presented again in August 2016 with a recurrent thrombosis in his AVG and this was again treated with a successful fistuloplasty and thrombolysis on two separate occasions. He then presented in September 2016 with re-stenosis. This was treated with fistuloplasty and 2 stent insertion. He underwent a successful renal transplant during this time and presented again with a sinus discharge in February 2019 when the plan was made for subtotal graft excision.Incidence of complications is higher when a graft is used over a native AVF. However, close surveillance and prompt intervention can lead to multiple successful salvage procedures thus prolonging the lifespan of the graft. As in our case we were able to prolong the lifespan of the AVG with multiple successful interventions. HoweverComplication rates are high with a one year and two year primary patency rate ranging between 40–50% and 20–30% respectively, and a one year and two year secondary patency rate ranging between 70–90% and 50–70% often with multiple interventions to maintain patency [We report a case of an AVG performed in 2012 that sustained multiple complications but with successful monitoring and timely intervention we were able to salvage the AVG for up to 5 years by which time successful renal transplantation was possible. Our case report is in accordance with the SCARE guidelines for reporting cases .2A 35 year old man with end stage renal disease secondary to IgA nephropathy presented for an elective AVG formation in August 2012. He had previously had multiple attempts at a native AVF formation with a failed left brachio-cephalic AVF in 2009 and a failed right brachio-basilic AVF in March 2012. It was decided based on his previous surgeries and small caliber superficial veins that a brachio-axillary polytetraflouroethylene (PTFE) AVG would be the most suitable option for him.He underwent successful surgery and was discharged. In 2014 there was difficulty noted in using the AVG for dialysis. Subsequent imaging confirmed graft stenosis at two different sites. He underwent successful fistuloplasty with interventional radiology with placement of two stents, an 8 mm stent at the proximal graft and a 7 mm stent at the distal graft Figs. 2 . He agIn 2019 he presented with a sinus discharge over the medial aspect of his right upper arm with pain and swelling. By this point he had a successful renal transplant and the AVG was no longer in use for access. On examination a small sinus was noted over the graft site with active purulent discharge and surrounding erythema. He was however systemically well with normal vitals and with only mildly elevated inflammatory markers. An US scan was performed that suggested possible collection in association with the graft and a DVT was ruled out. At this point it was decided to undergo surgical excision of the AVG.Our patient was taken to theatre. Under general anesthetic a skin incision was made to gain proximal and distal control before a dissection was made into the graft. A note was made of an eroded graft wall with a 1 × 2 cm defect revealing an underlying stent device and intra-luminal thrombosis Figs. 6 .Fig. 4The patient recovered well and was subsequently discharged post-operative day 1 with a course of oral antibiotic and for OPD follow up.3This case demonstrates that although complications are common with a AVG close surveillance and timely intervention can prolong access lifespan. In this case, we were well aware of the difficulty that a failed AVG would pose for this gentleman, given his extensive history of failed AVF formation and co-morbidities. Therefore, stringent monitoring was employed with rapid intervention to treat the various complications that presented over the course of 7 years.AVF infection is an important consideration with incidence ranging between 4–20% with the use of a graft . MultiplStenosis can occur anywhere along the site of graft but often occurs in the juxta-anastomotic areas. Assessment for stenosis can be made in a physical exam. A change in the thrill or pulsatile flow can indicate stenosis. Reduced flow during hemodialysis may also be seen. The recommendation for treatment is percutaneous transluminal angioplasty (PTA) if inflow or outflow stenosis is suspected . StenosiGraft thrombosis often presents secondary to progressive stenosis. Early treatment of thrombosis is recommended to prevent organization of thrombus. Treatment options include thrombectomy and thrombolysis however this is not sufficient as a flow limiting stenosis will also need to be treated to ensure improved patency. Endovascular therapy was shown to have similar patency rates to surgical thrombectomy . The ESVAn important aspect to discuss is the role of surveillance and patient/ staff education in monitoring for graft complications. Patients should be educated in examining their AVG and in identifying any abnormal patterns or signs. Any concerns should be flagged with the hemodialysis unit and the primary team. Examination should also take place during visits for hemodialysis and any concerns for complications should be investigated with further imaging. Monitoring is shown to be cost-effective method in improving patency .In our case, we were fully aware of the challenges associated with a failed graft and our patient was young and fully compliant with education and surveillance measures. We were able to ensure timely interventions that allowed for successful salvage of his AVG until renal transplant was possible.4Complication rates are higher in a AVG with lower patency rates when compared to native AVF. However, close surveillance and prompt intervention can lead to multiple successful salvage procedures thus effectively prolonging the life of the AVG. As in our case we were able to prolong the life of the AVG with 6 successful interventions.There may be role for an individualized approach in managing patients with AVG. Patient education and staff education in recognizing early signs of complications with tailored surveillance programs can help in optimizing outcome.Case report not sponsored.Not applicable as no research on human participants was involved.Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.Tayyaub Mansoor 1st author - Conceptualisation, Methodology, Resources, Writing.Donagh Healy 2nd author - Supervision.Darragh Moneley 3rd author - Supervision.Not applicable as this manuscript does not involve research on human participants.Darragh Moneley.Beaumont Hospital.Dublin Ireland.dmoneley@gmail.com.Not commissioned, externally peer-reviewed.No conflict of interest.


Neural implants offer solutions for a variety of clinical issues. While commercially available devices can record neural signals for short time periods, they fail to do so chronically, partially due to the sustained tissue response around the device. Our objective was to assess the correlation between device stiffness, a function of both material modulus and cross-sectional area, and the severity of immune response. Meta-analysis data were derived from nine previously published studies which reported device material and geometric properties, as well as histological outcomes. Device bending stiffness was calculated by treating the device shank as a cantilevered beam. Immune response was quantified through analysis of immunohistological images from each study, specifically looking at fluorescent markers for neuronal nuclei and astrocytes, to assess neuronal dieback and gliosis. Results demonstrate that the severity of the immune response, within the first 50 µm of the device, is highly correlated with device stiffness, as opposed to device modulus or cross-sectional area independently. In general, commercially available devices are around two to three orders of magnitude higher in stiffness than devices which induced a minimal tissue response. These results have implications for future device designs aiming to decrease chronic tissue response and achieve increased long-term functionality. Paralysis and limb loss pose significant personal, financial, and health burdens. Each year in the U.S. alone, there are over 17,500 cases of spinal cord injury where less than 1% achieve complete recovery . To addrWhile many groups have demonstrated success resolving neural signals with intracortical probes for periods of about one year ,8, thesebk), a function of both the material properties and geometric dimensions of the device.A common goal in the neuroengineering community is the development of more biocompatible implants, which elicit a decreased tissue response, with the intent of increasing their functional lifetime. These efforts are largely divided into two groups: (1) creating devices that are significantly smaller than the state-of-the-art ,17, or  assessments can be made based on calculations incorporating device dimensions to determine relative flexibility as compared to commercially available and other novel devices. The novelty of this study is the recognition that histological outcomes across material and geometric properties may be correlated to a single consolidated variable, bk, as opposed to relating changes in histological outcomes to a single aspect of device design.However, ‘stiffness’ (or ‘flexibility’) is often used synonymously to describe the softness or modulus of the implantable device or device substrate, even though stiffness must consider the contributions of device dimensions. For example, while polymer-based devices may be comprised of inherently soft materials, whether or not the device is highly flexible depends on more than just their material makeup. Instead, stiffness  [tion (1) ).Device tip geometries and shank asymmetries were neglected for the sake of simplicity. It is important to note that many single shank devices do exhibit tapered geometries meaning that cross-sectional area, and area moment of inertia, are not necessarily uniform along the length of the device. However, preliminary computational modeling suggests that using average width values does not have a significant effect on stiffness calculations. Specifically, use of a simplified symmetric model resulted in a 12% difference in maximum tip deflection in the cantilevered device bending simulation, when compared to the original tapered geometries . It is i® v. 5.2.  and the setup mirrored the cantilever-style bend test depicted in h, and width, b, or circular cross sections with diameter, d, affecting the way in which moment of inertia of the cross-sectional area was calculated (Equations (2) and (3) [Bending simulations were performed in COMSOL Multiphysics and (3) ). LengthThe immune response for each study was quantified by analysis of fluorescent immunohistochemical images, from staining with several cell markers commonly associated with the immune response. For the purposes of this analysis, we focused on stains for neurons and astrocytes, specifically neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP), a protein expressed in astrocytes.Images were analyzed using Fiji , an openWhile several of these studies reported their own analyses of fluorescent images, we chose not to include these quantifications in this meta-analysis. This was done in order to ensure that all NeuN density and GFAP intensity values were measured and normalized consistently across all studies, for accurate comparison. However, it is important to note that studies often feature figures that best illustrate the point of the study, i.e., fluorescent images that exemplify a reduced immune response. Therefore, our results likely reflect a conservative estimate of correlations between immune markers and device parameters.ρ values closest to 1 or 1 for positive and negative correlations respectively, with a high correlation being between 0.70 to 1.00 (or −0.70 to −1.0) [p-value less than 0.05 was taken as indicative of a significant correlation.To examine the possible relationships between material properties, dimensions, and device flexibility with both neuronal density and GFAP intensity, a Spearman rank correlation coefficient was calculated for each data set using functions available in MATLAB R2017a . Spearman correlation is a nonparametric test which assesses the monotonic relationship between ranked datasets. Good correlation is indicated by to −1.0) . A p-val3 to 1.6 × 10−4 N/m. Statistical analysis across multiple studies showed a high positive correlation  between device stiffness and normalized GFAP intensity, within a 50-µm band of the device perimeter, indicating that gliosis is more severe when using a stiffer implant  between device stiffness and normalized NeuN density in the same area, indicating that neuronal loss is increased when using a stiffer implant.Additionally, there was a high negative correlation . This can be done through a reduction of material modulus (E) or a cross-sectional moment of inertia (I), with the goal of better matching stiffness to that of brain tissue, and subsequently improving chronic integration with surrounding tissue. Additionally, the possibility remains that the immune response may be a result of cells responding to stress concentrations due to material mismatch, as opposed to stiffness of the device itself. This hypothesis could be tested directly using an approach in which probe geometries are kept constant while varying material stiffness, or similarly, maintaining stiffness but using varied cross-sectional geometries.The results of this meta-analysis should however encourage further exploration of materials for devices which can be fabricated in a way that limits overall stiffness (Intracortical device stiffness may influence the severity of the chronic immune response, more than size or material properties of the device independently. Our novel results, which draw upon findings from multiple studies, indicate that device stiffness is especially important in close proximity to the device perimeter, which may profoundly affect the ability of devices to record from nearby neurons.