ZmES22, encoding a MADS-type transcription factor, was modestly characterized from maize inbred line B73. ZmES22 exhibited high expression level in endosperm at 10 days after pollination (DAP) and peaked in endosperm at 20 DAP, indicating that ZmES22 was preferentially expressed in maize endosperm during active starch synthesis. Transient expression of ZmES22 in tobacco leaf revealed that ZmES22 protein located in nucleus. No transactivation activity could be detected for ZmES22 protein via yeast one-hybrid assay. Transformation of overexpressing plasmid 35S::ZmES22 into rice remarkedly reduced 1000-grain weight as well as the total starch content, while the soluble sugar was significantly higher in transgenic rice lines. Moreover, overexpressing ZmES22 reduced fractions of long branched starch. Scanning electron microscopy images of transverse sections of rice grains revealed that altered expression of ZmES22 also changed the morphology of starch granule from densely packed, polyhedral starch granules into loosely packed, spherical granules with larger spaces. Furthermore, RNA-seq results indicated that overexpressing ZmES22 could significantly influence mRNA expression levels of numerous key regulatory genes in starch synthesis pathway. Y1H assay illustrated that ZmES22 protein could bind to the promoter region of OsGIF1 and downregulate its mRNA expression during rice grain filling stages. These findings suggest that ZmES22 was a novel regulator during starch synthesis process in rice endosperm.As major component in cereals grains, starch has been one of the most important carbohydrate consumed by a majority of world’s population. However, the molecular mechanism for regulation of biosynthesis of starch remains elusive. In the present study,  Zea mays L.) is one of the most widely grown crop world-wide, as well as a critical model for various biological researches, especially for endosperm development [Maize , encoding a cell-wall invertase, was of great importance in regulation of sucrose unloading from phloem into cells of reserve organs. Mutant lines of OsGIF1 showed severe defects in grain filling and in turn reduced the grain weight to 70% of wild type rice at 30 days after pollination (DAP) [Starch is composed of two major components, known as amylose and amylopectin. The process of starch biosynthesis has been reported to be under finely regulated by numerous genes, which mainly encoded multiple subunits or isoforms of four enzymes: ADP-glucose pyrophosphorylase (AGPase), starch synthase (SS), starch branching enzyme (SBE), and starch debranching enzyme (DBE) ,3. At thynthesis . Loss-ofild type . Deficier spaces . Grain Ion (DAP) .OsBZIP58 seeds exhibited altered starch composition as well as morphological defects with apparent white belly region [pISA1 gene to regulate its expression, thus affecting the synthesis of starch in barley [ZmbZIP91 was proved to be a key regulator of the starch synthesis by directly binding to ACTCAT elements in the promoters of starch synthesis genes [ZmDof3 led to defects of the kernel phenotype with decreased starch content and a partially patchy aleurone layer [Since starch biosynthesis and accumulation are critical determinants for both grain quality and production, key transcriptional regulators, including several transcription factors (TFs), have also been demonstrated to play an important regulatory role in starch synthesis. Null mutants of y region . SUSIBA2n barley . ZmbZIP9is genes . The inhZmES22), were further confirmed via qRT-PCR assays. To test if this gene was related to starch synthesis, ZmES22 was cloned from maize inbred line B73. Afterwards, molecular properties and biological functions were modestly comprehensively characterized in transgenic rice lines. Overexpressing ZmES22 in rice significantly reduced 1000-grain weight as well as hindered starch accumulation. Besides, altered expression of ZmES22 in transgenic rice also changed the starch structure and morphology of starch granules. Furthermore, RNA-seq analysis demonstrated that numerous key regulatory genes in starch synthesis were differentially expressed compared to that in WT plants. Yeast one hybrid assay revealed that ZmES22 could bind to the promoter of OsGIF1 and downregulated its expression during grain filling process. This study illustrated that ZmES22 could be a newfound transcription factor, which negatively regulated starch synthesis in rice endosperm.In previous studies, a total of 2298 transcription factors were identified and further examined using RNA-seq dataset from 18 representative tissues from maize , which pZmES22 were related to starch synthesis, this gene was firstly cloned from maize inbred line B73. ZmES22 contained an open reading frame (ORF) of 723 bp and encoded a protein of 240 amino acids with a predicted molecular weight (Mw) of 27,903 Da and an isoelectric point (pI) of 8.92. Pfam analysis of ZmES22 revealed that the deduced protein sequence consisted of four conserved domains, namely the MADS-box domain (MADS-box), intervening (I), K-box domain, and the C terminal domain  model, and genes with diversity less than 0.8 were retained according to empirical experience. A total of 16 genes, including 6 from Zea mays, 5 from Oryza sativa, and 5 from Arabidopsis thaliana were identified, respectively . Accordingly, 1000-grain weight of transgenic plants were significantly depleted by 3.88 g than that of WT plants . Additionally, total starch content, apparent amylose content (AAC) and soluble sugar content of both transgenic rice lines and WT plants were measured according to previously reported methods. Surprisingly, compared to WT plants, both total starch content and AAC were significantly reduced , whereas, the content of soluble sugar in transgenic rice lines were significantly increased by 38% than that of WT plants . In particular, content of soluble sugar was two times larger in transgenic line L9 than that in WT plants. These results revealed that overexpression of ZmES22 gene could significantly block starch biosynthesis process in endosperm of rice.To illustrate the function of pression . Therefopression . After mpression A,B. NeveZmES22, BV and kmax of starch from both ZmES22 overexpression rice seeds and WT plants were determined accordingly. As shown in t-test, p-value = 2.2 × 10−16). Furthermore, morphology of starch granules was examined via scanning electron microscopy (SEM) [ZmES22 could change starch structure as well as influence morphology of starch granules in transgenic rice lines.Both amylopectin blue value and the maximum absorption wavelength reflect the ability of amylopectin binding to iodine. Therefore, different BV and kmax can provide indicators for the basic distinction of starch structure . To deteZmES22 in regulation of starch synthesis, expression profiles of 17 genes, which were preferentially expressed in developing endosperms and were demonstrated to be involved in starch synthesis, were compared between transgenic rice lines with WT plants at different developmental stages , which was reported to be a key regulator to rice grain-filling and yield, was significantly enriched in carbohydrate metabolic process as well as starch and sucrose metabolism pathway in KEGG. The gif1 mutant exhibited slower grain-filling rate and showed markedly more grain chalkiness than wild-type plants [GIF1 gene was downregulated as much as 8-fold in overexpression rice lines compared with WT plants . The relative mRNA expression level of OsGIF1 in four different developmental endosperms  were further confirmed by real-time quantitative PCR  to predict if promoter of GIF1 contained conserved element that MADS type transcription factors could bind to [OsGIF1 gene [ZmES22 was inserted into vector containing both activation domain (AD) and was drove by PT7. Two repeated copies of CATGT was synthesized as bait (designated as pGIF1). Simultaneously, a mutant  was also used as a negative control. As was illustrated in T7-ZmES22 and pGIF1-AbAi grows well, indicating that ZmES22 binds to the core element of the promoter of OsGIF1 , starch synthase (SS), starch branching enzyme (SBE), and starch debranching enzyme (DBE) [OsISA1, OsISA2, OsSSI, and OsSSIIa, decreased during the early stages of endosperm development stages compared to that in wild type rice. These results indicated that overexpression of ZmES22 could negatively affect mRNA expression of the majority of starch synthesis related genes in distinct degree during the early endosperm development stages.In the present study, transgenic rice that overexpressed one MADS type transcription factor me (DBE) . InteresOsGIF1, decreased as much as 8-fold in transgenic rice. Previous result demonstrated that grain filling rate of gif1 mutants was slower and accompanied with distinct chalkiness and loosely packed starch granules, while overexpression of GIF1 driven by its native promoter produces larger grains [gif1 mutant was consistent to transgenic rice lines that overexpressed ZmES22 gene from maize. Evidence from qRT-PCR also validated that mRNA expression level of OsGIF1 continually decreased in overexpression rice lines in comparison to wild type plants. These results indicated that overexpression of ZmES22 in rice might inhibit the mRNA expression of OsGIF1. As is reported that typic MADS type transcription factor are plant specific and often contained four functional domains, the MADS-box conserved domain (MADS-box), intervening (I), K-box domain, which is homologous to keratin (K), and the C terminal domain. MADS-box domain could bind to the promoter and regulate the expression of downstream genes. For example, ZmMADS47 directly binds the core motif CATGT of promoter of zein genes and activated its expression [OsGIF1 and repress its expression. KEGG analysis revealed that DEGs was significantly enriched in starch and sucrose metabolism pathway, in which the mRNA expression of one gene, named r grains . The phepression . AdditioZmES22 affect starch synthesis and endosperm development through binding to and downregulating the expression of OsGIF1, and in turn influencing carbon distribution and transportation of sucrose on grain filling in rice plant.The primary results provide evidence that ZmES22 influence the starch synthesis in rice, ZmES22 mutant are being created via CRISPR/Cas9 system.In conclusion, a MADS type transcription factor from maize was modestly characterized via overexpression in rice and the molecular mechanism for its anticipant role in regulation of starch synthesis were also explored by RNA-seq and yeast one hybrid assay in the present study. Starch synthesis is a complicated and sophisticated process, which is regulated by numerous transcription factors via protein–protein and protein–DNA interactions. In order to shed light on how Oryza sativa L. japonica cv. Zhonghua 11) and transgenic rice lines were grown under natural conditions in experimental field plots for Anhui Agricultural University in Anhui Province, China. Rice endosperms at 3, 6, 10, and 20 DAP were harvested for qRT-PCR assay.Ten representative tissues, including root, stem, leaf, tassel, filament, ear, 10 DAP (days after pollination) embryo, 20 DAP embryo, 10 DAP endosperm, and 20 DAP endosperm, were collected from maize inbred line B73 plants, which were grown in a greenhouse with paddy soil at 28 °C under a 14 h light/10 h dark photoperiod. Each tissue was repeatedly sampled from three individual plants as three biological replicates. Both wild type cultivars  according to manufacturer’s instructions. Afterwards, first-strand cDNA was generated using reverse transcription system . The qRT-PCR  was performed using SYBR Green Master  on an ABI 7300 Real Time PCR System , and the reactions were performed according to previous report . In detaZmES22 without the termination codon was amplified and cloned into pCAMBIA1305 vector under the drive of cauliflower mosaic virus (CaMV) 35S promoter. The fusion construct 35S:: ZmES22-GFP and empty vector 35S:: GFP were transformed into leaves for 35-day-old tobacco (Nicotiana bethamiana) via a syringe without needle, respectively. Infiltrated tobacco plants were transferred into dark condition for 12 h, followed by normal illumination for 48 h. Finally, green fluorescent signals were examined using confocal microscope .Full-length open reading frame (ORF) of ZmES22 was amplified and inserted into pGBKT7 vector , which were fused with the GAL4 DNA-binding domain beforehand. Subsequently, both negative control (null pGBKT7 vector) and positive control (co-transformation of pGBKT7-53 with pGADT7-T vectors), together with fusion construct pGBKT7-ZmES22 was transformed into yeast strain AH109, respectively. AH109 strain carries HIS3, ADE2 and MEL1 reporter genes. Transformed yeast cells were then cultured on SD/-Trp medium for 3 days at 30 °C and then transferred into SD/-Trp/-His/-Ade/X-α-GAL medium for 3 days at 30 °C.Full-length ORF of ZmES22 was amplified and inserted into an overexpression vector pCAMBIA1301a under the drive of CaMV 35S promoter and a NOS terminator. Recombination construct pCAMBIA1301a-ZmES22 also harbored a GUS reporter gene and was transformed into japonica rice cultivar Zhonghua 11 via Agrobacterium mediated transformation [Full-length ORF of ormation . Both hiVernier caliper was adopted to measure the length, width and thickness for 100 uniformly mature seeds at the longest, widest, and thickest point, respectively. 1000-grain weight was determined by counting ten independent repeats of 100-grain samples on an electronic balance. Each measurement was repeated for three times. Embryos and follicles were separated from the embryo and ground into powder. The starch content was measured with total starch determination kit  according to manufacturer’s protocol. Apparent amylose content (AAC) of the samples was measured using iodine colorimetry  . Anthron600. While, with respect to amylopectin, 15 mg isolated amylopectin powder was dissolved in 100 mL double distilled water. The absorption spectra were examined ranging from 500 to 700 nm, and the BV was set to A680.The separation of amylose and amylopectin, and measurement of the blue value (BV) and maximum absorption wavelength (kmax) of starch were referred to the modified alkali impregnation method . DetaileAccording to the methods in previous report , HitachiOryza sativa ssp. japonica cv [http://rapdb.dna.affrc.go.jp/) [p value was 0.05 [http://www.ncbi.nlm.nih.gov/geo/) under accession number SRP063765.Seeds for both overexpression transgenic rice lines and Zhonghua 11 at 20 DAP were collected for RNA-seq, each group were repeated twice as two biological replicates. Subsequently, RNA was isolated and then high throughput sequencing was performed on BGISEQ-500 platform in Beijing Genomics Institute . After trimming of low-quality and adaptor sequences from raw sequencing reads, clean data were aligned to onica cv . Nipponb.go.jp/)  using To.go.jp/) . The res.go.jp/) . Differewas 0.05 . The RNAwas 0.05 . The raw® Gold Yeast One-Hybrid Library Screening System User Manual (Clontech). To test the ability of ZmES22 to bind to the core motif CATGT of GIF1 promoter, CATGT and mutant tandem repeats were cloned and inserted into the BamHI and HindIII site of the p53/AbAi vector. Yeast Y1HGlod was transformed with the vector pGADT7-ZmES22 and CATGT or mutant tandem repeats plasmids. To evaluate interaction between ZmES22 and the core motif CATGT of GIF1 promoter, the transformants were screened by plating on SD /-Leu/AbA plates.Yeast one-hybrid assays were implemented originally according to the MatchmakerZmES22, encoding a typical MADS type transcription factor, which were exclusively highly expressed in maize endosperm, indicating its crucial role in endosperm development of maize. When ZmES22 was overexpressed in rice, the 1000-grain weight, together with total starch content were remarkably reduced, whereas, the soluble sugar content was significantly higher when compared to wild type. Moreover, overexpression ZmES22 altered the relative fraction of long branched starch and changed the morphology of starch granule from densely packed, polyhedral starch granules into loosely spherical granules with larger spaces. These results demonstrate that ZmES22 is a negative regulator that could affect the starch biosynthesis process. Moreover, RNA-seq and qRT-PCR results further illustrated that overexpression of ZmES22 could downregulate mRNA expression level of numerous key genes in starch synthesis pathway, particularly in early developmental stages in transgenic rice lines. Furthermore, ZmES22 could bind to the promoter region of the OsGIF1 and downregulate its mRNA expression throughout the endosperm developmental stages. Therefore, we proposed that ZmES22 might affect starch biosynthesis as well as reducing the rate of grain filling by downregulation of OsGIF1 in rice. Whether knock-down or knockout of the ZmES22 gene could contribute to increase of yield in maize remains to be demonstrated.Starch is one of the major components of cereal grains, providing sufficient calories for both human diet and animal feed. Therefore, comprehensive understanding molecular basis of starch synthesis process and its regulatory network is of vital importance. In the present study, we identified a gene


Background. Reliable bonding between resin composite cements and high strength ceramics is difficult to achieve because of their chemical inertness and lack of silica content that makes etching impossible. The purpose of this review is to classify and analyze the existing methods and materials suggested to improve the adhesion of zirconia to dental substrate by using composite resins, in order to explore current trends in surface conditioning methods with predictable results. Methods. The current literature, examining the bond strength of zirconia ceramics, and including in vitro studies, clinical studies, and a systematic review, was analyzed. The research in the literature was carried out using PubMed and Cochrane Library databases, only papers in English, published online from 2013 to 2018. The following keywords and their combinations were used: Zirconia, 3Y-TZP, Adhesion, Adhesive cementation, Bonding, Resin, Composite resin, Composite material, Dentin, Enamel. Results. Research, in PubMed and Cochrane Library databases, provided 390 titles with abstracts. From these, a total of 93 publications were chosen for analysis. After a full text evaluation, seven articles were discarded. Therefore, the final sample was 86, including in vitro, clinical studies, and one systematic review. Various adhesive techniques with different testing methods were examined. Conclusions. Airborne-particle abrasion and tribo-chemical silica coating are the pre-treatment methods with more evidence in the literature. Increased adhesion could be expected after physico-chemical conditioning of zirconia. Surface contamination has a negative effect on adhesion. There is no evidence to support a universal adhesion protocol. In recent decades, the increasing aesthetic needs in dentistry have led to the progressive overcoming of metal-ceramic prosthesis and led to a focus on indirect metal-free restorations. Zirconia has occupied an increasingly important role, thanks to its excellent mechanical  and biocOver the last few years, many adhesion techniques have been studied. Different treatments of the zirconia surface, application of primers or adhesives, and various types of resin cements have been tested. However, a standardized adhesive cementation protocol, that provides univocal and reliable results, has not been identified ,8,9.The data we have available today come mostly from laboratory studies that, although they are useful for guiding subsequent clinical trials, have limitations in terms of clinical evidence. Furthermore, the results obtained from such a large number of tested techniques are not directly comparable. It is difficult to generalize the results in relation to the zirconia sample, or to the materials used, considering the wide range of products available on the market.The aim of the review is, therefore, to compare different treatments of the zirconia surface, in order to determine a valid operative protocol for adhesive cementation. The main zirconia treatments are summarized in To review the literature, the National Library of Medicine database was consulted using PubMed. The research was carried out on 2 January 2019 and the studies published from 1 January 2013 to 31 December 2018 were selected. The Cochrane Library database was also analyzed with a limitation on publication year (2013–2018). It was decided to choose this time interval to get a picture of what is the current knowledge on the subject discussed, regarding the new materials recently put on the market. Studies regarding the evaluation of the bond between zirconia and composite resins have been included in the review. The following terms and their combination were searched: “Zirconia,” “3Y-TZP,” “Adhesion,” “Adhesive cementation,” “Bonding,” “Resin,” “Composite resin,” “Composite material,” “Dentin,” and “Enamel.” The research includes laboratory studies, clinical studies, and systematic reviews. 2O storage are included in the review. Regarding clinical trials, RCTs and observational studies were included, with a follow-up, at least, of 5 years. The examined articles evaluate the clinical performance of adhesively cemented zirconia prostheses, in particular anterior cantilever prostheses, and prosthesis on inlays in the posterior sector. Studies that analyze traditional bridges with full crowns on the abutment teeth have been excluded. Inclusion criteria are listed in Regarding laboratory studies, no exclusion criteria were set in relation to the type of test performed for the evaluation of the bonding strength. However, it is important to evaluate the ability of the adhesive bond to resist over time. In this regard, studies in which samples are subjected to at least 5000 thermocycles or at least one month of HThe research carried out in PubMed  and the The articles found consist mainly in laboratory studies. The clinical studies are in small numbers. Eight systematic reviews were also selected, including seven that were subsequently discarded following their full reading, since they do not meet the exclusion criteria of this review. The selected studies are summarized in The studies examined in this review mainly consist of laboratory studies. Five clinical studies were also found, while most of the initially included systematic reviews were eliminated because their inclusion criteria do not reflect the limits set for this review. It was decided to review various types of articles, in order to have an overall view of the current knowledge regarding the adhesion of zirconia.Different types of tests are performed to estimate the bond strength. The most widely used is the Macro Shear, which is the easiest to set up. However, it must be considered that the type of test can partly influence the result. The preparation of the Macro Test leads to a greater heterogeneity in the distribution of stress, due to the wider adhesive interface . The µShLong-term water storage and thermocycling are commonly used methods of artificial aging that affect the resin bond to ceramic ,73. ThisRegarding in vitro studies, the main zirconia adhesion protocols involve a mechanical conditioning phase and then the application of chemical adhesion promoters. The use of silane is rationally justified where a layer of silica  was created , while, 2O3), emitted by a high-speed source. The impact involves the erosion of the material with the formation of a rough, clean, and wettable surface [Sandblasting is a process that uses the energy released by the impact of alumina particles  is an unconventional method that leads to erosion of material through electrical impulses in a dielectric medium. In terms of Shear Bond Strength (SBS), the EDM technique obtains good results. However, by SEM analysis, surface cracks can be highlighted .Zirconia is considered to be an inert material. The surface cannot be activated with hydrofluoric acid etching because it does not act on the crystalline component. Anyway, various acid solutions have been proposed to etch zirconia, based on hydrofluoric and nitric acid applied at a temperature of 100 °C. Acid etching of the zirconia surface with these modalities is less effective than Tribological-chemical treatment . Other aSandblasting, like other exclusively mechanical treatments, is able to modify the zirconia surface. However, it is essential to associate these treatments with the use of chemical promoters, capable of improving adhesion. Today 10-MDP-based cements and primers are used for this purpose . PrimersTribochemical silica-coating (TBS) is another method used to promote adhesion to zirconia. This is a sandblasting process that is carried out using alumina-particles covered with silica, which impacts against the surface of the ceramic, as well as creates an irregular surface while releasing silica. The presence of this vitreous component allows the use of silane as a coupling agent. It binds both to the composite and to the silica deposited on the zirconia and improves adhesion . TBS is TBS appears to be more resistant to thermocycling than other treatments. According to thermodynamic calculations, the bond between silica and silane is more resistant to hydrolysis than the bond between zirconia and 10-MDP . SeveralSandblasting with feldspathic ceramic powder appears to have promising results in terms of SBS when compared to the use of silica-coated alumina, with a lower t-m transformation rate and stable results after thermocycling . The useZirconia is a polycrystalline ceramic, not conventionally etched with acid . In orde2 deposition on the zirconia surface does not guarantee adhesion results comparable to those obtained with traditional treatments [Silica deposition on zirconia, which allows the use of silane as a coupling agent, is also pursued by Magnetron-sputtering Physical Vapor Deposition (PVD). Sputtering is a technique for realizing thin films, which allows us to deposit both metallic materials and insulating materials on a substrate. This method of SiOeatments ,86.The silicatization of the zirconia surface is also obtained through “pyrochemical” techniques. The Silano-Pen system, for example, consists of a lighter containing a solution of butane and silane. When the butane is burned, the silane compound decomposes into SiOx-C fragments that adhere to zirconia, which can be silanized. This method is not sufficiently effective to promote a stable and lasting bond to the composite ,49.Zirconia can be modified with a technique called Selective Infiltration Etching (SIE): the ceramic is coated with silica-based material, with a thermal expansion coefficient similar to the zirconia one. During the fusion (when the temperature of 960 °C is reached), this material diffuses in the zirconia structure. Then hydrofluoric acid is applied for about 10 minutes in order to dissolve the glass component completely. The surface of the zirconia appears to be irregular ,54.With regard to the cementation phase, the main alternative to composites is the use of a traditional glass ionomer cement, or a CVI modified with resin. In terms of adhesion, the composite cements have better results ,73. Rega2O, H2O2, ethanol, or acetone, the application of orthophosphoric acid, ethyl cellulose-based paints, ultrasonic cleaning, or plasma treatment are all ineffective in removing contaminants [2O3 powder is the most effective method for removing contaminants, even though it can weaken the structure of zirconia if carried out several times on the material. Cleansing with NaOCl-solutions or with the cleaning paste Ivoclean   seem a valid alternative in the consideration of costs and practicality, and the possible deterioration of the zirconia structure [The zirconia prosthesis can be contaminated during the clinical phases: blood, saliva, impression materials, and other contaminants can deposit on the material and interfere with the adhesion mechanism . Cementsaminants ,51,53,62tructure ,51,53. Ttructure . With thtructure . Furthertructure .The introduction of translucent zirconia on the market allowed the realization of monolithic prosthetic products. In relation to this, interest is growing in realizing safe and standardized adhesive cementation protocols of zirconia. It is important to observe the differences on the possibility of conditioning, between traditional and translucent zirconia. Only a few recent studies evaluate the possibility of adhesion of this material. Results show that bonding of highly translucent zirconia exhibits behavior similar to that of traditional 3Y-TZP ,24.To date, there are still few clinical studies on the realization of Resin Bonded Fixed Dental Prosthesis (RBFDP). Only five articles were found that meet our inclusion criteria and no one involves full zirconia restorations. Two clinical studies ,92 conceRegarding the systematic review included, Thammajaruk  collecteIt could also be useful to broaden the search, including the “Scopus” and “Scholar” databases, to have an even wider view on the subject.Clinical recommendations are difficult to give, for two main reasons such as the small number of clinical studies found in the literature and the difficulty in comparing laboratory studies that evaluate a number of techniques and obtain often conflicting results. Further in vitro studies, that investigate promising techniques and own better homogeneity on the test set-up characteristics, as well as further clinical trials, are needed to have more evidence to support an adhesion protocol with certain predictable results.In literature, we find a variety of adhesion protocols, including the use of different zirconia treatment methods, various adhesion media, different tests, and storage times. The results are difficult to compare.The combination of a mechanical and chemical treatment is essential for good adhesion. Protocols with greater evidence in the literature include sandblasting with silica-coated particles  and traditional alumina sandblasting . The latter has less evidence of long-term stability. Other methods involving the silicatization of zirconia obtain promising results that must be validated by further studies.The choice of the composite cement is less relevant. Surface contamination has a negative effect on adhesion.New highly traslucent zirconia shows a similar behavior, in terms of adhesion, to traditional 3Y-TZP.An adhesion protocol that provides unequivocal results has not yet been identified.


AoB PLANTS is 10 years old. To mark this milestone, the founding editor of the Journal sets out the reasons AoB PLANTS was started and charts the highs and lows in the planning process that lead to its opening in October 2009. The Journal’s subsequent progress during a decade of unprecedented change in science publishing is described and the prospects for its future development and success assessed.The peer-reviewed open access plants science journal  AoB PLANTS is a not-for-profit, open access, plant science journal and one of three peer-reviewed journals owned and managed by the Annals of Botany Company. This article explains events and thinking that led to the starting of AoB PLANTS and how the unique features of the Journal came to be formalized prior to its launch in September 2009. The article also describes how the Journal’s management developed over the first 10 years and summarizes the Journal’s achievements in a decade where open access journals have proliferated despite subscription journals continuing to dominate the publishing of peer-reviewed botanical science. AoB PLANTS was founded by the Annals of Botany Company (Website 1), an educational charity hereafter referred to as the Company. This article reviews events leading to the launching of the Journal and the progress made since it opened in late 2009. The Company is much more long-standing. It was created in 1903 with a membership of 10 to own and manage the general plant science subscription journal Annals of Botany founded 16 years earlier at the personal expense of eight prominent British botanists . The present article evaluates the circumstances that led to this departure from past ways and relates something of the uncertainties that inevitably accompanied the decision by a small philanthropic organization to introduce a new journal into a highly commercialized international market. The account also details the initial concept and design of AoB PLANTS and assesses progress made over its first 10 years in the face of a stream of newer open access titles, many of which seem of questionable virtue . By 2017, this had been expanded to include a related electronic newsletter entitled The Week in Botany. More recently, in 2018, a third peer-reviewed journal, In silico Plants, was launched (Website 4). The founding of AoB PLANTS therefore initiated the Company’s transition from a one-journal enterprise into a botanical publishing house owning and managing five titles. This turning point radically affected how the Company would interpret and implement its constitutional remit as it sought to serve and match, in spirit and in enterprise, the kinds of experimental plant science it most wished to support.Despite an initial hesitancy, documented below, the experience gained from starting Annals of Botany had risen more than 4-fold. This strong upward trend was accompanied by a substantial rise in the proportion of papers being turned-down. Over these years, the policy of Annals of Botany’s Chief Editor (M. B. Jackson and author of the present article) had been to increase selectivity as submissions grew. Thus, by 2006, 75 % of submissions were being rejected and, a year later, this figure had risen to almost 80 % . The 2006 Meeting concluded that the mission of the Annals of Botany Company would be better served by trying to publish more papers rather than reject ever-more papers. The choices were either to publish extra issues of Annals of Botany or to start another journal. The second option was decided upon and the Executive Committee  resolved to prepare a concept note for a new web-based, open access journal and think of a suitable title prior to putting their case to the whole Company.In the 10 years from 1996, manuscript submissions to ost 80 % . This noPLOS Biology, PLOS ONE, BMC Plant Biology, BMC Plant Methods), the Executive Committee agreed to adopt the open access model for its new journal in the belief that the underlying ideals would have wide appeal but especially for younger scientists.The decision by the Company to adopt an open access model for their new journal was influenced by a growing desire more generally for scientific results to be freely available without charge to readers and for something to be done about the lucrative hold on scientific publishing by profit-hungry international companies. Many of their subscription journals appeared to give poor value for money, with the actual cost of individual journals to university and institutional libraries bearing little relation to the number of pages published or to journal Impact Factors . Open ac[see]. This came not from the Executive Committee but from the Chief Editor of Annals of Botany. This was appropriate since his editorial policies were responsible for Annals of Botany’s high rejection rate. The concept note gave the new journal the interim title of eBotany and sought to establish aims and scope that overcame concerns about how it would distinguish itself from Annals of Botany and also avoid being seen simply as a cascade journal for the parent title. The concept note established the notions of a journal encompassing the breadth of botany and emphasizing excellence of execution of the science as the main selection criterion rather than considerations of broad interest, degree of originality and similar subjective issues. The note was submitted to the Executive Committee in June 2007 and to a senior journals editor at Oxford University Press (OUP) the publisher of Annals of Botany. OUP showed interest and this encouraged the Executive Committee to convene a special meeting at the Langham Court Hotel, London on 17 July 2007 to discuss the concept note. The outcome was cautious approval, with the Chairman and Vice-chairman of the Company retaking responsibility for the project and for putting it to the Company as a whole at its Annual General Meeting (AGM) 2 days later. Opinions at the AGM were mixed. Nevertheless, the meeting gave it guarded support and recommended further consultations with OUP by the Vice-chairman (J. S. Heslop-Harrison) followed by a planning meeting of the whole Company in November/December 2007. But doubts persisted, no detailed plans emerged from the Chairman or Vice-chairman and the intended November/December planning meeting did not take place. The hiatus may have reflected uncertainty about the wisdom of founding a new journal. Certainly, there were worries about the possible costs for the Company and whether botanical science generally was sufficiently well-funded for authors to pay to publish; the former concern being tempered by the Company’s strong financial position  and Executive Committee a month later at Cambridge. Although the name PLANTOME was not much liked, it was agreed that the Company’s Secretary (R. Hunt) would draft a preliminary business plan and that the Chief Editor would reopen discussions with OUP and expand the concept note for consideration by the Executive Committee on 30 June 2008. This more positive stance by the Executive Committee may have been influenced by the recent entry of the open access journal BMC Plant Biology into the top 20 plant science journals based on Institute for Scientific Information (ISI) Impact Factor. For whatever reason, a greater urgency now characterized proceedings. Within a month, OUP had e-mailed a market research questionnaire to ~6000 plant scientists drawn from the Annals of Botany user database with a participant’s prize draw worth £300 helping to achieve a 19 % return. By this time, the name AoB PLANTS had been agreed and it came to be understood at the Cambridge meeting that Annals of Botany’s Chief Editor would relinquish his position in the coming months to become AoB PLANTS’ first Chief Editor.The idea of starting a new journal was resurrected in April 2008, when Annals of Botany’s Chief Editor submitted a second more detailed concept note that included proposing AoB PLANTS until a full analysis was complete. It was left that OUP would clarify its stance once the Questionnaire was assessed fully and that the Chief Editor would prepare a presentation for a special meeting of the full Company in October or November 2008.At the scheduled June meeting, the Executive Committee made minor adjustments to the Chief Editor’s concept note and put it to a full meeting of the Annals of Botany Company at its AGM in London on 2 July 2008. At that meeting, OUP reported that replies to the Questionnaire were broadly favourable but chose to defer a decision on publishing AoB PLANTS and that the title would remain the exclusive property of the Company. OUP had also accepted most of the technical and promotional requirements set-out by the Chief Editor and also agreed to share overt production costs such as editorial office work, website creation and maintenance, archiving and promotional work as well as any income. Actual financial splits were to be finalized later. A special meeting of the Executive Committee and the Chief Editor was then held at the Cotswold Lodge Hotel, Oxford, on 6 October 2008. Preliminary layouts for the home page, ancillary pages and scientific content were approved as were details concerning the Journal’s remit, scope and ambition. The intention to create a top-tier Advisory Board and a separate international Editorial Board was supported and OUP’s initial financial projections were well-received and incorporated into the Company’s business plan. To encourage submissions in the early years, it was resolved that author charges would be low or annulled with the Company and OUP between them standing the losses. An Extraordinary General Meeting (EGM) of the whole Company was set for 2 December 2008 when full details would be up for approval. At the time, it was intended that day-to-day editorial administration, manuscript handling etc. for the new journal would run alongside those of Annals of Botany at its new home at the University of Leicester, UK. But these did not materialize. Instead, an OUP Virtual Office setup was adopted. This would be run from Scotland by Dr Lulu Stader  who had a strong biological background. The new journal would thus be a stand-alone operation managed and operated online by its Chief Editor and Editorial Assistant backed-up by OUP.By the end of September 2008, both a favourable analysis of the questionnaire by OUP and a draft business plan prepared by the Company Secretary (R. Hunt) were in the hands of the Executive Committee. The Chief Editor had also secured verbal assurances from OUP that it was indeed prepared to publish AoB PLANTS. However, at a second meeting in Bristol on 12 January 2009, Wiley-Blackwell were unable to accept a low-cost article processing charge (APC) for authors; their policy being to charge full market price from the start. The impasse proved unsurmountable and, on 16 January 2009, Wiley-Blackwell backed-away. The Company Secretary and Chief Editor were unwilling to lose further momentum by turning to a third publisher since good progress was being made with OUP who, spurred-on by potential competition from Wiley-Blackwell, quickly accepting the principle of a cost-free period for authors. The Company Executive therefore decided to seek to contract with OUP. Two and a half years after the Journal was first conceived an agreement with OUP was finally signed on Thursday 17 April 2009. By then, final designs for the website, and its scientific content . To encourage speedy evaluation of submissions, editorial board members would be paid £25 ($32.3) per manuscript while referees would be offered £30 ($39) in Amazon vouchers for each paper they assessed. An initial plan to graduate referee rewards according to the speed with which their assessments were returned was abandoned. Review articles were encouraged by offering invited authors £250 ($322) for their article when published; an editor with specific responsibility for recruiting review authors being appointed. To speed-up time taken for initial rejections and lighten the load on Editorial Board members, submissions were first assessed by the Editorial Assistant and the Chief Editor against a set of published preliminary criteria of acceptability. Approved papers only would then be passed to a member of the Editorial Board to arrange peer review. These second evaluations were made against a further set of acceptance criteria that emphasized the soundness of the science. By 2016, the Journal was hoping to publish 190 papers a year with an estimated rejection rate of 55 %. Other features included double-blind refereeing, i.e. referees’ identities being concealed from authors and authors’ identities from referees. This policy was rare amongst plant science journals but offered some protection to those worried about conscious or unconscious bias linked to their identity or place of origin. Overall, the intentions were to be fast, fair, inclusive and as transparent as possible with subjective elements of decision-making being minimized. Internal communications were encouraged by the creation of a Listserv bulletin board (‘The Plants-Pub’) with Advisory Board, Editorial Board, editorial staff, Company members and relevant OUP staff as subscribers. A monthly newsletter was distributed in this way by the Chief Editor.Arabidopsis thaliana rosette for the faux-cover image  on 12 October 2009, the first day of a week-long conference entitled Plant Conservation for the Next Decade. An explanatory talk was given by the Chief Editor prior to a reception which, along with champagne and canapes, offered promotional leaflets  where student poster prizes were sponsored. Leaflets and a poster were displayed at the Third International Symposium on Plant Growth Modeling, Simulation, Visualization and Applications at Beijing, China. AoB PLANTS also sponsored a lecture generating a review article on crop trait ontology at a conference in Montpellier, France entitled Biodiversity and Agriculture: Today’s Challenges, Tomorrow’s Research for More Sustainable Farming. In these ways, the Journal had, by the end of 2009, comprehensively announced itself. The following year a promotional stand was arranged at the American Botanical Society’s annual meeting at Providence, Rhode Island, where an Editorial Board member (Dennis Whigham) held a ‘Meet an Editor’ session. The Journal was also promoted at the 10th Conference of the International Society of Plant Anaerobiosis, Volterra, Italy and at the Congress of the Federation of European Societies of Plant Physiology, Valencia, Spain. Arrangements were made to sponsor three sessions at the 2011 International Botanical Congress, Melbourne, Australia and to give financial support to the 2012 International Conference on the Plant Hormone Ethylene, Auckland, New Zealand from which Special Issues would emerge. The botanical coverage of the Journal was extended to include phytoplankton, an editor was appointed to cover this field and promotional material was e-mailed to the phytoplankton community in collaboration with the Chief Editor of Journal of Phytoplankton Research, an OUP journal. In December each year, a seasonal electronic greetings card was sent to authors reminding them of the bibliographic details for their recent papers . This was based on a session at the tropical ecology conference Impacts of Global Change on Tropical Ecosystems sponsored by the Journal and held in Marburg, Germany in 2009.The first two published papers appeared towards the end of 2009. They introduced the new journal and outlined the strengths of open access publishing . Six earThe success of early publicity efforts and conference promotions may be judged by comparing the Journal’s performance with expectations set out in the initial business plan. The comparison reveals a creditable rather than stellar performance. The most recent business plan anticipated four hundred submissions in the first full year (2010) with 90 actually being published. The reality was 114 manuscripts received, with 22 papers published, approximately a quarter of the number planned-for. The managerial response to this sluggish start was to postpone the introduction of author charges and to revise downwards the number of submissions anticipated. In the following year (2012) 169 submissions were received (200 were expected) but a high rejection rate meant that only 34 papers were published rather than 80 although the time taken to publish them following first submission was commendably short (15 weeks).2011 Wetland Biogeochemistry Symposium, Prague, Czech Republic where financial support was given for a session on Phragmites ecology. Promotional material was sent to the Fourth International Orchid Conservation Congress, Hluboka, Czech Republic and a stand was taken with OUP at the 2011 Botanical Congress, Melbourne, Australia where three sessions sponsored by the Journal generated papers on ‘Trace Gas Analysis’, ‘Pollen-pistil Interactions’ and ‘Flooding Stress’. AoB PLANTS also sponsored the opening plenary lecture on computational modelling of development at the 2011 Conference of the Polish Society of Experimental Plant Biology, University of Wrocław. Financial support was given to the 2011 International Symposium on Plant Biotechnology, Birla Institute of Technology, India, the 2011 conference Molecular basis of plant stress, University of Plovdiv, Bulgaria 21–23, the 2012 Latin American Congress of Algal Biotechnology, Universidad de Concepción, Chile, and the 2012 International Conference on Polyploidy, Hybridization and Biodiversity, Zámek, Czech Republic where four student poster prizes were awarded. Finance was also given to the IX International Conference on the Plant Hormone Ethylene in New Zealand (2012) and to a session at the 2012 Society for Experimental Biology Meeting at Salzburg, Austria on ‘Chloroplast biogenesis’. Plans were also laid to support the 2013 Conference of the International Society for Plant Anaerobiosis at the International Rice Research Institute, Philippines from which a sizeable special issue was anticipated. E-mail publicity by OUP and the Editorial Office continued, targeting China in particular. These measures introduced the Journal to numerous countries with sizeable communities of plant biologists thereby enhancing prospects for high-quality papers in the years ahead.In mid-2011, the Chief Editor signalled his wish to stand-down the following summer. This gave the Company the opportunity to recruit internationally for a replacement and, by June 2012, Professor J. Hall Cushman a plant ecologist from Sonoma State University, California, USA had been appointed. In the months leading-up to the change in editorship, promotional activities continued undiminished. Leaflets and pens etc. were distributed at the Annals of Botany previously declined on subjective grounds were improved and this formed the basis of a productive collaboration between the two journals. AoB PLANTS’ overall capability was increased by appointing a Managing Editor (Gail Rice) and a replacement Editorial Assistant (later upgraded to Deputy Managing Editor) to operate the Virtual Editorial Office (Dr Joanne Ferrier). Other changes included an overhaul of the Journal’s website with recent papers and their thumbnail images no longer fronting the landing page. An ‘Editor’s Choice’ feature was introduced to highlight the most promising papers and a simplified, compact PDF format for published papers was adopted. Requirements for a structured abstract and author’s ‘Conclusions and Forward Look’ were also relaxed. The existing Creative Commons Attribution Licence was liberalized to allow unrestricted use, distribution and reproduction of articles by anyone as soon as accepted, provided that authors, citation details and publisher are identified. To encourage invited review articles, payment was raised to £396 ($500) and later to £774 ($1000). These comprehensive changes were partnered with a marketing surge, spearheaded by increased conference attendances, OUP mailshots and by opening a Facebook page, a Twitter account and creating a page on Wikipedia. In addition, numerous promotional messages were sent to various society Listserv bulletin boards. Posting AoB PLANTS content on the AoB Blog (now Botany One) was streamlined while the Journal’s own Listserv bulletin board was replaced by an e-mailed newsletter.Hall Cushman commenced his work as Chief Editor in July 2012 and, within a short time, overhauled most aspects of the Journal. A new image was commissioned from a Californian graphic design firm  and moreAoB PLANTS logo were commissioned for use at these conferences along with a range of display material  with the remit to maintain and develop content for Facebook and Twitter, the AoB Blog and OUP sites whilst also developing new features for the AoB PLANTS website.In addition to the six special issues arranged up to and including 2012, a further seven with ecological themes were published in the years to mid-2018. Review articles continued to be invited as a means of attracting leading authors to the Journal and encouraging citations (20 invited reviews were published from 2013 to mid-2018 incl.). Promotional work also continued much as before with editorially staffed stands at conferences such as FESPB/EPSO Congresses and annual meetings of the British Ecological Society, Botanical Society of America and the Ecological Society of America. Flash drives with links to notable papers; pens, bookmarks and water bottles imprinted with the new material . ArticlePLOS One, a general science journal publishing numerous plant biology papers. Full or partial fee waivers would be given to authors from developing countries, and for selected special issues and invited reviews. To help keep fees low, some economies were necessary. These included abandoning the recently created position of Social Media Editor, stopping incentive payments to associate editors and no longer rewarding referees with Amazon gift vouchers. Bringing-in author charges reduced submissions by about a third over the next 2 years; the effect being much smaller than that experienced 3 years before. Prior to the advent of APCs in 2016, the number of papers published annually had risen almost 7-fold compared with the Journal’s first full year . However, the expense paid-off with submissions rising 300 % during 2014 and 2015. In later years, inevitable cost-cutting brought expenses down a little although they still exceeded those of 2010 almost two and a half times. Despite strong arguments against reintroducing author charges voiced by the Chief Editor, the Company Executive and OUP decided to recommence APCs in January 2016. The starting fee was £781 ($1000) and this was expected to grow annually to £1523 ($1950) by January 2020. These fees were below those charged by most other open access plant science journals but slightly above that charged by . HoweverAoB PLANTS was expanded considerably from 2015, especially electronic publicity for China. This resulted in an increasing proportion of published papers from that country (11 % in 2016 and 14 % in 2017), exceeded only by the USA . OUP’s promotional efforts also included numerous e-mail shots such as an ‘Author and Reviewer Thank you’ campaign, a ‘Ten Reasons to Publish in AoB PLANTS’ campaign and a ‘Highly Cited’ promotion. The Journal was thus enjoying unprecedented support from OUP’s publicity team, an outcome of much lobbying by the Company Chairman and Secretary and the Chief Editor. This heightened support may also have reflected a recognition by OUP that bringing-in author charges required them to initiate compensating support measures.After some initial reluctance, support from OUP to publicize AoB PLANTS 72nd out of 196 plant science journals. In the following year, the Impact Factor rose to 2.73 and, by 2017, it had reached 2.821 placing AoB PLANTS amongst the top 25 % of plant science journals and above traditional competitors such as American Journal of Botany and International Journal of Plant Science. A listing was also secured in the Environment/Ecology category with AoB PLANTS positioned 51st amongst 158 journals. Thus, in its first 10 years and on the basis of Impact Factor, the Journal had overtaken numerous long-established plant science titles and outperformed most of the more recent open access upstarts. The trend in online journal usage, measured in terms of full text downloads, was also healthy. Based on 2010, the first full year of the journal’s operations, downloads had grown 4.5-fold by 2017, while interest in the Journal in terms of requests for e-mailed tables of content rose more than 6-fold. This buoyant picture was complimented by some exemplary service to authors in terms of the time taken to process submissions. Time to provisional acceptance of submitted papers in the first full year of operations (2010) was 52 days but, by 2017, had been shortened to 46 days. By 2018, the average time taken to decide the fate of all submissions was steadily decreased to about 30 days. The time for accepted papers to appear online was maintained throughout at 7–11 days while the time need to convert these to fully formatted HTML and PDF versions was consistent over the years at 5–7 weeks with some recent papers taking only 2–3 weeks. Overall, authors had seen the time taken to publish their papers from first submission slow slightly from just under 4 months in 2010 to 4.5 months in 2017 as a result of authors’ revised papers being scrutinized with increased rigour. A strength of the Journal throughout has been its broad geographical coverage. In 2010, first-author countries of origin numbered 13 but this had grown to 38 by 2015 with submissions that year being drawn from 52 countries. Currently, papers from the USA (24 %), Germany (14 %) and China (10 %) lead the way.In 2014, recognition of the Journal’s academic standing arrived in the form of its first Impact Factor from Thomson-Reuters . The initial rating, covering citations to papers published in 2011 and 2012, was 1.743. This placed In October 2017, the Chief Editor announced his wish to step down. Hall Cushman’s legacy would be a smooth-running journal with fair and transparent decision-making procedures for authors, a large and highly international editorial board, a strong and rising Impact Factor and, since 2016, one now generating income from author charges. The challenge for the future would be to build on the reputation for quality science and excellent service to authors and readers while scaling-up the operation sufficiently to recompense the Company and OUP for their original and ongoing investments. By the end of 2017, the total costs incurred had exceeded £600 000.Agroecology & Environment; Evolution & Diversity; Plants, Ecosystems & Climate, Form and Function; Populations & Communities using acceptance criteria clearly set-out on the Journal’s website. Additional areas would be added later. An overarching consideration was that ‘all good data and sound arguments deserve a voice’. To encourage acceptances and journal loyalty, Buckley also reinstated financial rewards to Associate Editors and increased payments to £40 ($52) per manuscript. The post of Social Media Editor was also restored and, to signal regime change and the accompanying organizational and attitudinal shifts, the Journal’s image was remodelled once more . The Journal has recently revamped its operations under the guidance of a new Chief Editor who believes strongly in the ideals of open access publishing. It is too early to assess the full impact of these recent developments on the performance of AoB PLANTS but at the time of writing, the latest changes had started to prove their worth. After reaching a low point in June 2018, submissions rose steadily and, by January 2019, had grown by 75 % compared to the previous year’s nadir.troversy  whilst aAoB PLANTS will rest on the inherent appeal of a well-run, not-for-profit journal that is owned and managed by plant scientists for plant scientists and has no fiscal demands from shareholders or investors to add to authors’ costs. These features and its timely and transparent evaluation procedures are backed-up by OUP, the world’s largest and oldest university publishing house that also espouses a not-for-profit ethos. This raft of attributes makes AoB PLANTS an increasingly persuasive and appealing prospect for authors and readers alike.Future success for The following additional information is available in the online version of this article—File S1. Initial concept note for the new on-line open access Journal.plz025_suppl_Supporting_InformationClick here for additional data file.


A binary mixture of starch–starch or starch with other biopolymers such as protein and non-starch polysaccharides could provide a new approach in producing starch-based food products. In the context of food processing, a specific adjustment in the rheological properties plays an important role in regulating production processing and optimizing the applicability, stability, and sensory of the final food products. This review examines various biopolymer mixtures based on starch and the influence of their interaction on physicochemical and rheological properties of the starch-based foods. It is evident that the physicochemical and rheological characteristics of the biopolymers mixture are highly dependent on the type of starch and other biopolymers that make them up mixing ratios, mixing procedure and presence of other food ingredients in the mixture. Understanding these properties will lead to improve the formulation of starch–based foods and minimize the need to resort to chemically modified starch.  It has long been recognized that starches derived from various botanical sources differ in the composition, structure and molecular order of their polysaccharide constituents (amylose and amylopectin), which could account for differences in their functionality. Starch owes much of its functionality to the fine structure and relative proportion of these polysaccharides, as well as to the physical organization of starch polymers into larger structural domains in the solid state .Biopolymer mixtures are very important because of their great influences on food properties and formulation . The varet al. [The blending of different types of flours and/or starches is an age-old technique used in the manufacture of various traditional food products. Simple binary mixtures of starch–starch or starch with other biopolymers (such as protein and hydrocolloids) are continuously being studied because such mixtures provide a new approach in producing tailor-made starch-based products. The possibility of formulating starch blends that behave like chemically modified starches; particularly with regard to pasting behavior was reported by Obani et al. . AnotherMany researchers have reported that when two or more biopolymers are mixed together, the mixtures behaved differently from when they are present individually in a single phase ,6,7,8. TThe comprehensive review paper on mixed biopolymer system entitled “Starch–Biopolymer Interactions—A Review” was published in 1997 . There hThe utilization of starch mixtures in food products is not new, but can been traced to the dawn of history. Since ancient times, many types of traditional foods have been made from mixtures of different starches. For example, in the production of rice vermicelli, two different types of starches, usually corn and sago, are used together with rice flour to reduce the raw materials costs. Likewise, fish crackers are usually made from a mixture of tapioca and sago starches. The rationale underlying the use of starch mixtures most likely is due to desirable textural and sensory attributes conferred by the specific mixtures that cannot be obtained from any of the individual starches alone.et al. [Several studies have been conducted based on different properties of starch-starch mixtures . Differeet al. , due to et al. , for exaet al. . The stuThe functional properties of mixed starch system are governed by both the extent of gelatinization and retrogradation. It has been shown that the rate of retrogradation of starch gels on aging generally increases with increasing amylose content of the starch ,20. For Using pulsed low-resolution NMR to monitor retrogradation of mixed rice starch with other starches  in 1: 2 dry starch/water systems, it was found that in any particular mixture, the rate of retrogradation fell between those of the individual component starches . In the et al. [A synergistic interaction was noticed when rice and sago starches are mixed  Figure b. Similaet al. . In bothet al. .mix value will not be calculated as the average value of S’1 and S’2 due to the proportion of two starch components, which might result in the non–additive nature of S’. This idea merits consideration and further investigation. Some researchers reported that at low starch concentration (20%), each individual component in the mixture independently gelatinized, whereas at higher starch concentration (50%) they did not [To explain the sysnergistic interactive effect of starch mixtures, an ideal gelatinized starch mixture system was defined as a fresh mixture of two separately gelatinized starch systems with the same moisture content . This de did not .et al. [P < 0.05) increase in the peak viscosities was notice with increasing starch content in the mixtures. On the other hand, the control wheat showed too low peak of viscosity which also was out of ranges. Thus, it was found that combining starches with wheat flour for optimizing the viscosity was important processing practice. Similar finding of peak viscosity characteristics was reported in the blend of sago starch and wheat flour from 10 to 50% sago in the mixtures [et al. [The pasting properties of mixtures of wheat flour mixed with tuber starches namely sweet potato starch (SPS), potato starch (PS), yam starch (YS), and cassava starch (CS), at 10 to 50% starch in the mixtures was reported by Zaidul et al.   of starch–WPI mixtures during the cooling were different from that of starch gel alone. At a low WPI content (1:0.15), G′ decreased during cooling. When they increased the content of WPI to 1:0.2, G′ still decreased, but the reduction was not as much as that at the 1:0.15 levels. When more WPI (1:0.3) was added, G′ was more than that of pure starch gel at 85 °C. When temperature decreased, G′ initially decreased and at 65 °C, G′ started to increase. It was found that unlike hydrogen bonds, hydrophobic interactions are enhanced in warm temperature (40–60 °C) [G′ and increase in tan δ could be observed. However, there was a continued increase of gel strength due to hydrophobic interactions at warm temperature and hydrogen bond interactions at cold temperature, during the cooling. They concluded that WPI replacement for egg white protein might be beneficial in increasing elastic modulus and the rigid structure of bakery products during the later stage of baking and during cooling.Mixing ratios of 0.15:1, 0.2:1, and 0.3:1 whey protein isolate (WPI)–wheat starch (dry weight basis) and combination of WPI and egg white protein were used by Yang  systems . They fo0–60 °C) . Therefoet al. [Bertolini et al.  reportedG′ of flaxseed protein concentrate, which is similar to that of the addition of potato starch to acid milk [et al. [A recent study was carried out to investigate the effects of starch/protein mass ratio (0.2–1.0) and different cooling rate  on rheological behavior of the composites . The addcid milk  and addicid milk . Starch  [et al. . The stu [et al. .Various starches have been included in meat products such as surimi and low fat sausage for reducing cost and improving texture. Starch added in meat emulsions favors the formation of a more compact and stronger heat induced protein network . Moreoveet al. [G′ decreased slightly due to the temperature effect. At temperatures lower than 55 °C, the starch–meat complex exhibited a similar G′ to pork ham batter. The initial protein denaturation resulted in an increase in G′. Associated with the gelatinization of starch, there existed a dramatic increase in G′ above 60 °C.The effect of adding different starches  to starch–meat complexes was investigated by Li et al. . The autet al. [The thermal processing of fish protein–starch systems was studied by Wu et al. . The reset al. .Polysaccharides or hydrocolloids have been studied by many researchers . It was P ≤ 0.05) increases in peak, breakdown, setback and final viscosities, pasting temperatures and peak times (time to reach the peak viscosity), except for guar addition, in which the peak time was unaffected and xanthan addition, in which case setback viscosity decreased significantly (P ≤ 0.05). A similar tendency was also found for the anionic tapioca starch–guar gum mixture as compared with its control  [Starch–gum mixtures (6% w/w starch and 0.35% w/w gum) were studied using RVA in order to investigate the effects of guar and xanthan gums on pasting and rheological properties of native and anionic tapioca starches . The resh alone) . et al. [−1 was greater for both mixtures, the increase being the greatest after 20 h storage at 5 °C of high amylose and waxy corn starch solutions at a concentration of 25 g·L−1.The rheological description of galactoxyloglucan hydrocolloid (HXG) which contained galactose, xylose, and glucose in a 1:3:4 molar ratio was studied by Freitas et al. . HXG wasn) as low as 0.14–0.24. The n values decreased with increase in xanthan gum concentration. From these results, the authors found that the rheological behaviors of rice starch–xanthan gum mixtures were apparently dependent on the concentration of xanthan gum [Rheological properties of rice starch–xanthan gum mixtures (5% w/w) at different xanthan gum concentrations  were evaluated in steady and dynamic shear . All thethan gum .Increasing xanthan gum concentration from 0.2% to 0.8% caused an increase in the magnitudes of consistency index. This may be attributed to an increase in the viscoelasticity of the continuous phase in starch–gum composite systems due to the thickening properties of xanthan gum . From thThe increasing number of published papers on this subject signifies the potential of fabricating starch-based products by exploiting the complex interactions of starch–starch and starch–other biopolymers that give rise to diverse textural attributes. The rationale underlying the use of mixed biopolymer systems based on starch most likely is due to desirable textural and sensory attributes conferred by the specific mixtures that cannot be obtained from any of the individual starches alone. In addition, the future may see an emphasis on new methods of using starch rather than the proliferation of chemical derivatives. This means that research should be focused on physical processes or investigating possible synergistic/antagonistic relationships that alter starch characteristics, rather than using chemical modification.Many of the studies involving mixed starch–based systems are empirical in nature and did not attempt to elucidate the underlying mechanism involved to explain the manifested changes in some functional properties of the system. Present knowledge appears inadequate to formulate a scientific basis for predicting the effects on retrogradation resulting from the blending of different types of native or modified starch. Systematic study using intensive numerical simulations  could be useful in order to build up various models of mixed polymer–starch system and assist the technologists to predict the behavior of such systems.The synergistic interaction displayed by some starches in mixed systems merits further scrutiny as to the nature of interaction at the molecular level. In cases where the mixture exhibits an additive effect, it is presumed that the starch molecules (amylopectin) from both starches behave independently in the mixture. The rate of retrogradation of the mixtures can, therefore, be predicted by a linear relationship. For synergistic interaction, however, there could be some kind of physical  factors that play a role in accentuating retrogradation rate of the mixture, over and above the predicted additive value. The questions arising under this situation is: Just how exactly the granules from two different populations, containing amylose and amylopectin (from both starches) mix and interact within the composite system? There are apparently many factors  that can influence the course of such interactions.Due to the diverse and complex nature of starch and other biopolymers, a broad range and variation in functional properties of the mixed biopolymers would be expected. From the data obtained so far, it is obvious that interactions (if any) between different starches and between starch and other biopolymers when blended are still not well understood. This is particularly so where the phenomenon of starch retrogradation is concerned. While the retrogradation tendencies of individual starches have been studied extensively, those of mixtures of starches have received little attention. Manipulation of the rate or extent of starch retrogradation by the simple expedience of blending different flours or starches is of technological importance to the food industry.The proper combination of starch–based mixed biopolymer systems provides a convenient approach in achieving desirable functional properties such as pasting, viscosity, viscoelasticity, thermal and textural functionality in the preparation of innovative food products. However, the actual mechanisms of interaction between the polymer constituents are unknown yet and warrant further investigation. An understanding of interactions of various components, especially amylopectin, may be critical to understand the retrogradation behavior of starch mixtures, hence manipulation of the property in formulating starch–based food products. Nevertheless, appropriate blending of different native starches may provide a simple practical avenue for manipulating, at least to some extent, the functional properties and final quality of a starch-based end-product. It is not impossible that starch–based mixture would find wider usage in view of increasing consumer demand for more natural product.


International Journal of Nephrology has retracted the article titled “Management Practice, and Adherence and Its Contributing Factors among Patients with Chronic Kidney Disease at Tikur Anbessa Specialized Hospital: A Hospital Based Cross-Sectional Study” [https://doi.org/10.1371/journal.pone.0200415 [l Study” . The art


NOTCH2NLC. Expanded GGC repeats as the cause of NIID was further confirmed in an additional three NIID-affected families as well as five sporadic NIID-affected case subjects. Moreover, given the clinical heterogeneity of NIID, we examined the size of the GGC repeat among 456 families with a variety of neurological conditions with the known pathogenic genes excluded. Surprisingly, GGC repeat expansion was observed in two Alzheimer disease (AD)-affected families and three parkinsonism-affected families, implicating that the GGC repeat expansions in NOTCH2NLC could also contribute to the pathogenesis of both AD and PD. Therefore, we suggest defining a term NIID-related disorders (NIIDRD), which will include NIID and other related neurodegenerative diseases caused by the expanded GGC repeat within human-specific NOTCH2NLC.Neuronal intranuclear inclusion disease (NIID) is a slowly progressing neurodegenerative disease characterized by eosinophilic intranuclear inclusions in the nervous system and multiple visceral organs. The clinical manifestation of NIID varies widely, and both familial and sporadic cases have been reported. Here we have performed genetic linkage analysis and mapped the disease locus to 1p13.3-q23.1; however, whole-exome sequencing revealed no potential disease-causing mutations. We then performed long-read genome sequencing and identified a large GGC repeat expansion within human-specific  Immunohistochemically, the intranuclear inclusions are positive for ubiquitin and ubiquitin related proteins, including p62, SUMO1, FUS, MYO6, and OPTN-C proteins,NOTCH2NLC (MIM: 618025) via long-read genome sequencing (LRS) in a five-generation Chinese Han NIID family, and the repeat expansions were validated by repeat-primed PCR (RP-PCR) and GC-rich PCR (GC-PCR) in three additional NIID-affected families as well as five sporadic NIID-affected case subjects. In addition, we found two Alzheimer disease (AD)-affected families (1.43%) and three parkinsonism-affected families (1.46%), with GGC expansions in NOTCH2NLC from a cohort that included 140 AD-affected families, 205 parkinsonism-affected families, 51 spinocerebellar ataxia (SCA)-affected families, 16 peripheral neuropathy-affected families, and 44 motor neuron disease (MND)-affected families. Further, skin biopsies confirmed the typical NIID pathology in these two AD-affected families and three parkinsonism-affected families, indicating that GGC repeat expansions could also contribute to AD and PD phenotypes. Thus, we suggest defining a term of NIID-related disorders (NIIDRD), which is a spectrum of diseases including NIID and other diseases caused by the GGC repeat expansions of NOTCH2NLC. The prevalence of NIIDRD is not rare, and screening for expanded GGC repeat within NOTCH2NLC should be done not only in dementia-affected and peripheral neuropathy-affected individuals, but also in parkinsonism-affected individuals.Here we report the identification of an expanded GGC repeat in Twenty affected individuals from four NIID-affected families, five sporadic NIID-affected case subjects, and 211 healthy control subjects from mainland China were recruited for this study. All NIID-affected individuals were recruited from the National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, and subjected to a thorough neurological examination by at least two experienced neurologists. We defined familial NIID-affected case subjects as having at least one NIID-affected case subject diagnosed by skin biopsy, and the additional family member(s) had to have either high signal intensity in the corticomedullary junction using DWI or a clinical examination by a neurologist that confirmed the concordance of phenotype with the pathologically diagnosed NIID individual from the same family. All sporadic NIID-affected case subjects presented with typical high intensity in corticomedullary junction in DWI and received a skin biopsy to confirm the diagnosis. All healthy control subjects were recruited from the medical examination center of Xiangya hospital.Besides NIID cohorts, a cohort of individuals with other neurodegenerative disorders were also recruited, including 140 AD-affected families, 205 parkinsonism-affected families, 51 SCA-affected families, 16 peripheral neuropathy-affected families, and 44 MND-affected families, of which the known pathologic genes had been excluded. All individuals were recruited from the Department of Neurology and National Clinical Research Center for Geriatric Disorders, Xiangya Hospital. All participants were subjected to a thorough neurological examination by at least two experienced neurologists.This study was approved by the Ethics Committee of Xiangya Hospital of the Central South University in China. Written informed consent was obtained from all individuals.Skin biopsies were performed after local anesthesia. A 5-mm-diameter biopsy specimen was obtained at 10 cm above the lateral malleolus of the affected individual. For immunohistochemistry, all samples were fixed in 10% formalin and then were embedded in paraffin and sectioned at 6 mm thickness. Sections of all samples were stained by hematoxylin & eosin (H&E) and immunohistochemical analysis was performed. Anti-ubiquitin  and anti-p62  antibodies were used for ubiquitin and p62 staining. Images were acquired by deconvolution digital microscope .3 size and fixed in 2.5% glutaraldehyde solution with Millonig’s phosphate buffer (pH 7.3). In the following preparations, the samples were incubated in 1% osmium tetroxide, then dehydrated with graded acetone, and embedded with resin. 50–100 nm ultrathin sections were prepared with an ultramicrotome (Leica Microsystems) and a diamond knife. After 3% uranyl acetate and lead nitrate double staining, prepared sections were examined and photographed on a Hitachi HT-7700 electron microscope.Samples for electron microscopy were sliced into 1×1×3 mmWhole-genome SNP scanning on selected members in family 1 A was perLong-read genome sequencing (LRS) on seven affected individuals  and three healthy control subjects was performed on the Oxford Nanopore platform as describe previously.5-3′) were utilized for identifying the CGG repeat expansion. For the GC-rich PCR (GC-PCR) assay, the fluorescein (FAM)-labeled forward primer (5′-AGCGCCAGGGCCTGAGCCTTTGAAGCAG-3′) and reverse primer (5′-TCGCCCCAGGTGGCAGCCCCGGGCGCCGCGGAC-3′) were utilized for repeat size determination. Seven-deaza-2-deoxy guanosine triphosphate (deaza-dGTP) was used in place of dGTP. PCRs were performed using 50 ng of genomic DNA in a 25 μL reaction mix including 10X Expand Long Template Buffer 1, 2.5 mM MgCl2 , 2% 2,4-dimethylsulfolane , 0.2 mM each of deaza-dGTP, dATP, dCTP, and dTTP, 0.2 μM primers  and 1 U of DNA ploymerase from Expand Long Template PCR System , using the following thermal conditions: 98°C for 4 min, followed by 30 cycles of 98°C for 45 s, 60°C for 45 s, 72°C for 4 min, and a final extension at 72°C for 7 min. The PCR products were subjected to capillary electrophoresis using the 3500xL Genetic Analyzer for Human Identification (Applied Biosystems). Allele sizes were determined using GeneScan 1000 ROX Size Standard (Applied Biosystems). GGC repeats less than 200 were determined directly by the size of PCR product as determined by ABI 3500xL and compared with the PCR products of known FMR1 (MIM: 309550) premutation CGG repeat alleles of various sizes. For GGC repeats over 200, the size was determined using an Agilent 2100.For the repeat-primed PCR (RP-PCR) assay, a fluorescein (FAM)-labeled gene-specific primer (5′-CCTCAGCCCGATACTCACCAT-3′) and repeat-containing primers (5′-TACCAATACGCATCCCGCGATTTGTCTTA(CGG)Human postmortem dorsolateral prefrontal cortex (DLPFC) tissue from the subjects of different ages  were obtained from the NIH NeuroBioBank. Total RNA was isolated using Trizol (ThermoFisher). Before reverse transcription, total RNA was treated with ezDNase (Millipore) for 5 min at 37°C to remove contaminated genomic DNA, and then incubated at 55°C for 5 min to inactivate the ezDNase. After ezDNase treatment, total RNA was extracted with phenol: chloroform = 25:24 (pH 5.2) and precipitated with glycogen and ethanol. The reverse transcription was conducted using SuperScript (II)  to generate first strand cDNA. The first strand cDNA served as the template for real-time PCR using FAST-7500 real time PCR equipment (Thermo Fisher). GAPDH TaqMan Gene Expression Assay (Hs02786624_g1) was used as an internal control. NOTCH2NLB primers: Forward (5′-GGGAGATATGAAGGGACGCA-3′); Reverse (5′-GGCACACACCTTCCATTCTC-3′). NOTCH2NLC primers: Forward (5′-CTGACCTTTCAAGATCCTGCTTTCATCCCAGCT-3′); Reverse (5′-AAGTGCCTTACTTTGCGTAGCTGTGTGCTTGGCAGT-3′).NOTCH2NLC in the blood of both NIID-affected individuals and control subjects, statistical analysis was performed using Student’s t test. Differences with p < 0.05 were considered as being statistically significant.For methylation analysis, the dispersion parameters were estimated through a shrinkage estimator based on a Bayesian hierarchical model, then a Wald test was performed as describe previouslyNOTCH2NLC had an expanded GGC repeat in both affected individuals (We recruited a five-generation Chinese NIID-affected family (family 1) with muscle weakness-dominant type A and perividuals C and S1,NOTCH2NLC. NOTCH2NLC is one of the four NOTCH2 paralogs that are present only in the human genome; the other paralogs are NOTCH2NLA (MIM: 618023), NOTCH2NLB (MIM: 618024), and NOTCH2NLR (MIM: 618026). Previous studies have shown that all four NOTCH2NL paralogs contain the first four exons and introns of NOTCH2 as the result of a partial segmental duplication of the NOTCH2 ancestral gene.NOTCH2NLA, B, and C are highly similar to each other, with more than 98% sequence identity over NOTCH2NL exons 1–5.NOTCH2NLC contains the GGC repeat in the reference human genome (hg38), (GGC)9(GGA)2(GGC)2, which is expanded in the NIID-affected family that we have examined here  and GC-rich PCR (GC-PCR) assays to detect both normal and expanded GGC repeats. Among the 211 healthy control subjects that we examined, the GGC repeat sizes range from 5 to 38, with modes at 11 and 16 repeats E. Our RPHowever, we did note that the expanded GGC repeat was also observed in a few individuals that were initially thought to be phenotypically normal, such as F1-III:5 and F1-III:8 A and S2BNOTCH2NLC  was similar to what has been reported for classic NIID where the main and initial clinical manifestation is muscle weakness. The average onset age was around 36  and S1. The parkinsonism-dominant type (families 5–7) presented with parkinsonism as the main clinical manifestation. The onset age was typically around 60 years old  and S2. In the dementia-dominant type families 3, 4, 8, 9), individual characteristics were similar to the previously described dementia-dominant phenotype in NIID., 4, 8, 9FMR1 locus can lead to hypermethylation of the CGG repeat and adjacent CpG islands, resulting in transcriptional silencing.NOTCH2NLC locus in NIID-affected case subjects were greater than 200, we examined the methylation status of GGC repeat and adjacent regions by re-analyzing the signals from Nanopore sequencing. The methylation level around the GGC repeats was very low and no significant methylation difference between NIID and control was detected  in 1q21.1, highly expressed in the brain and thought to be involved in the evolutionary expansion of the human brain.Neuronal intranuclear inclusion disease (NIID) is a slowly progressing neurodegenerative disease characterized by eosinophilic intranuclear inclusions in the nervous system and multiple visceral organs. The clinical manifestation of NIID varies widely, and both familial and sporadic case subjects have been reported.C9ORF72 (MIM: 614260) as a frequent cause of frontotemporal dementia (FTD)/amyotrophic lateral sclerosis (ALS)Almost 30 years ago, expansion of unstable nucleotide (microsatellite) repeats, notably trinucleotide repeats, was identified as a previously unknown mutational mechanism underlying certain human diseases.In addition to GGC repeats, we did observe different repeat units, such as GGT, through our LRS analyses . This isFMR1 locus have shown that there is a threshold for the methylation status of the CGG repeat.FMR1 and fragile X syndrome.XYLT1 (MIM: 608124).in vivo.The key features associated with the disorders caused by unstable nucleotide (microsatellite) repeats are genetic instability and anticipation.NOTCH2NLC could also contribute to the pathogenesis of both AD and PD. Therefore, we suggest defining a term NIID-related disorders (NIIDRD), which include NIID and other related neurodegenerative diseases caused by the expanded GGC repeat. It is likely that the prevalence of NIIDRD could be higher than initially thought. Given that only about 37% of affected individuals showed typical MRI features of NIID, genetic testing for the expanded GGC repeat could potentially be a more sensitive and accurate screening tool for NIIDRD.Our analyses of both classic NIID and other individuals with neurological conditions revealed a broadened clinical spectrum of adult-onset NIID. Besides the suggested dementia-dominant type and muscle weakness-dominant type,300623]), which is also caused by the expanded GGC/CGG repeats.FMR1 mRNA and FMRpolyG peptide have been found in the human postmortem brain inclusions that are characteristic of FXTAS pathology, suggesting that both mechanisms may contribute to the disease.NOTCH2NLC is a human-specific gene.Identification of the GGC repeat expansion as the genetic cause of NIID will help elucidate the molecular pathogenesis in NIID. Our results presented here suggest that the GGC repeat expansion-induced pathogenesis is likely dominant, which suggests that the expression of the expanded GGC repeat could cause the neuronal toxicity associated with NIID and its related disorders. Indeed, a dominant-negative gain-of-function model has been proposed for another neurodegenerative disorder, fragile X-associated tremor/ataxia syndrome (FXTAS MIM: 3003), whichNOTCH2NLC as the genetic cause of NIIDRD, which could include dementia, parkinsonism, and peripheral neuropathy. Our finding will facilitate more accurate clinical diagnosis in the future and help us better understand the molecular pathogenesis of NIIDRD in general.In summary, we have identified an expanded GGC repeat within human-specific The authors declare no competing interests.


The rheological study shows that compared with starch (containing mostly amylose), cellulose contributed more to the solution’s viscosity and has a stronger shear-thinning behavior. A comparison between the experimental and calculated zero-shear-rate viscosities indicates that compact complexes  formed between cellulose and starch with ≤50 wt % cellulose content, whereas a loose structure (phase separation) existed with ≥70 wt % cellulose content. For starch-rich hybrid films prepared by compression molding, less than 7 wt % of cellulose was found to improve the mechanical properties despite the reduced crystallinity of the starch; for cellulose-rich hybrid films, a higher content of starch reduced the material properties, although the chemical interactions were not apparently influenced. It is concluded that the mechanical properties of biopolymer films were mainly affected by the structural conformation, as indicated by the rheological results.Starch and cellulose are two typical natural polymers from plants that have similar chemical structures. The blending of these two biopolymers for materials development is an interesting topic, although how their molecular interactions could influence the conformation and properties of the resultant materials has not been studied extensively. Herein, the rheological properties of cellulose/starch/ZnCl In recent years, sustainable polymers from renewable resources, which can be used for both high-value areas such as biomedical materials and in basic applications such as packaging, have attracted huge interest . Along wd-glucose unit, referred to as homoglucan or glucopyranose, but linked through different glycosidic bonds. Starch contains two types of biomacromolecules, namely amylose and amylopectin. Amylose is a sparsely branched carbohydrate mainly based on α-1,4 linkages with a molecular weight of 105–106 and has a degree of polymerization (DP) as high as 600 [OAc] solutions, as reported previously [aE for our starch/cellulose/ZnCl2 solution was much lower than that for an amylopectin/cellulose/[Emim][OAc] mixture. In Equation (2), n kJ/mol ,91,92. Be solute . This reeviously , althoug2 solution of 65 wt % concentration. However, when the cellulose content was ≤15 wt %, solution casting became unsuitable for preparing the associated films. In this case, the high acidity of the 65-wt % ZnCl2 solution (pH = 0.67) might cause serious acid hydrolysis of the starch macromolecules. Instead, the films could be prepared by compression molding using a 25-wt % ZnCl2 solution [Cl] had a DP of 480 and σt of 138 MPa [2 solution. For cellulose-rich hybrid films, the increased starch content led to higher εb but lower σt. The reason could be that starch may have partly destroyed the intermolecular interactions of cellulose, while the latter was mainly responsible for the mechanical properties. Moreover, as starch and cellulose were phase-separated in the blends , a higher content of starch could have destroyed the continuous phase of cellulose, which may have also led to reduced mechanical properties.  138 MPa . Our lowσt of the pure-starch film C00 was close to the values reported in our previous study [σt and εb of the resultant hybrid films increased initially and then decreased. Both the highest σt (126.7 ± 7.8 MPa) and εb (33.5 ± 4.4%) was achieved at a 7-wt % cellulose content. This suggests that a small amount (7 wt %) of cellulose in starch could provide a reinforcement effect on the hybrid films, although C07 had a lower degree of crystallinity (as shown by XRD results). This may be ascribed to the hydrogen bonding interactions between starch and cellulose due to their similar chemical structures and the inherently better mechanical properties of cellulose. Unfortunately, pure-cellulose films and pure-starch films were unable to be prepared using the same method, so a comparison of the mechanical properties between the two types of biopolymer film was not possible. Moreover, the rheological study shows that for starch-rich materials, interactions between starch (mostly amylose) and cellulose resulted in new compact complexes, which might reinforce the blend films. However, too much cellulose in a starch matrix may lead to phase separation and, thus, deteriorated mechanical properties. us study . With thσt and εb of the starch-rich hybrid films prepared by compression molding , it was likely to cause acid hydrolysis of both the cellulose and starch during the solution casting, which could weaken the mechanical properties of the films. It can be seen that both  molding b were mu molding a. Becaus2) and ethanol of analytical grade were purchased from Guangzhou Chemical Reagent Factory . All the solutions were prepared with distilled water.Gelose 80 maize starch  was supplied by National Starch Pty Ltd. . α-Microcrystalline cellulose  was supplied by Shanghai Macklin Biochemical Co., Ltd. . Anhydrous zinc chloride (ZnCl2 aqueous solutions of two concentrations (65 and 25 wt %) were prepared by mixing ZnCl2 and H2O at ratios of 65/35 and 25/75 (wt./wt.).2 aqueous solution of 65 wt % concentration in a sealed reaction vessel followed by constant stirring at 50 °C for 1.5 h to allow for complete dissolution. Then, about 30 g of the solution was poured onto a petri dish and spread evenly. The biopolymer was then coagulated in two ways: (1) using absolute ethanol for 24 h; and (2) using ethanol solutions with concentrations of 30 wt %, 50 wt %, and 70 wt %, with each concentration kept for 1 h, followed by treatment with absolute ethanol at room temperature for another 21 h. The bath volume was 10 times higher than the solution volume, and the obtained wet films were dried in a blast-drying oven at 50 °C for 1 h. The thickness of the materials was about 0.4–0.5 mm. Our preliminary work showed that ZnCl2 solutions of lower concentrations than 65 wt % cannot dissolve MCC completely.Cellulose-starch hybrid materials with a ratio of cellulose of higher than 50 wt % were prepared via a dissolution-casting-coagulation route . First, 2 solution of 25 wt % concentration. Our previous study showed that this concentration of ZnCl2 can provide a reinforcement effect on starch-based materials [2) with poly(tetrafluoroethylene) (PTFE) glass fabrics located between the starch and the mold. For the subsequent compression molding, a flat sulfuration machine  was used, which has two press areas with the upper one for hot press and the lower one maintained at room temperature for cooling. Each sample in the mold was first hot-pressed in the upper area at a temperature of 120 °C and a pressure of 10 MPa for 6 min and then immediately moved to the lower area and pressed under 10 MPa for cooling for 2 min. Subsequently, the mold was opened and the sample was retrieved (thickness about 1.2 mm). Our preliminary work showed that for compression molding, ZnCl2 solutions of higher concentrations than 25 wt % are not effective for proper plasticization of the biopolymer materials.Cellulose-starch hybrid materials with a ratio of cellulose of less than 15 wt % were prepared by a compression molding method ,61,62,66aterials . Then, tAll the materials were conditioned at 75% relative humidity (with oversaturated sodium chloride solution) for 7 days before characterization of the materials. 2 solutions were carried out using an Anton Paar MCR 92 rheometer  with a 60-mm-diameter plate geometry and a Peltier temperature control system. Silicone oil  was placed around the edge of the measuring cell to prevent the absorption of water from the environment. Silicone oil would hardly affect the experimental results, as it is immiscible with polysaccharide solutions and has a relatively low viscosity (9.5 mPa·s at 20 °C) [2 solutions were recorded at shear rates from 10 to 1000 s−1 at certain temperatures of 25 °C, 40 °C, 55 °C, and 70 °C. The concentration of ZnCl2 solution was fixed at 65%, which can dissolve cellulose and starch totally, and the concentration of starch and cellulose was set at 5% (dry weight). The different starch/cellulose ratios tested were 0 wt % (pure cellulose), 10 wt %, 30 wt %, 50 wt %, 70 wt %, 90 wt %, and 100 wt % (pure starch). The rheological properties of a 65-wt % ZnCl2 solution were also detected. All the rheological tests were carried out at least twice to ensure the consistency of the results. Rheological measurements of cellulose/starch/ZnClt 20 °C) ,91. SteaThe morphologies of the normal and fractured surfaces of the cellulose-starch films were examined using a scanning electron microscope  with an accelerating voltage of 10 kV and a spot size of 6 nm. The blend films were cryo-ground in liquid nitrogen to obtain fractured surfaces, and the pieces were then fixed onto circular metal stubs previously covered with double-sided adhesive, followed by platinum coating for a 5-nm thickness using an Eiko sputter coater under a vacuum.θ) from 5° to 50° with a scanning speed of 10 °/min and a scanning step of 0.033°.X-ray diffraction (XRD) analysis was performed with an Xpert PRO diffractometer  operated at 40 mA and 40 kV using Cu Kα radiation with a wavelength of 0.1542 nm as the X-ray source. The scanning was undertaken with the diffraction angle  accessory, which contained a ZnSe crystal at a nominal incident angle of 45°, yielding about 12 internal reflections at the sample surface. All the resolutions were 4 cm−1, and all the spectra were recorded at room temperature (25 °C).Fourier-transform infrared (FTIR) spectra for the native MCC powder, pure-cellulose films and hybrid films were obtained in the range of 1900–800 cmσt) and elongation at the break (εb) were determined by Instron Merlin software version 2.3 (Instron (Shanghai) Limited, Shanghai, China) from at least 7 specimens for each sample [The mechanical properties of the blend films were determined using an Instron 5566 Universal Testing Machine with a 500 N load cell (Instron (Shanghai) Limited, Shanghai, China). The films were cut into dumbbell-shaped strips, according to ASTM D882-10. The testing was performed with a constant deformation rate of 10 mm/min at room temperature. The tensile strength  and the cellulose resulted in compact complexes , whereas with ≥70-wt % cellulose content, the interactions led to a loose structure (phase separation). These different ways of interactions and structural conformation could also account for the observed differences in the mechanical properties. Thus, our results allow for a further understanding of the molecular interactions between the starch and the cellulose as two polysaccharides with very similar chemical structures. The knowledge obtained from this work could provide insights into the future development of biodegradable materials based on these natural polymers with tailored structures and properties for food packaging and various biomedical  and environmental  applications.In this work, we studied the molecular interactions between starch and cellulose and the properties of such hybrid films based on these two polysaccharides. For starch-rich hybrid films prepared by compression molding, a small amount (≤7 wt %) of cellulose was found to improve the mechanical properties despite the reduced crystallinity of starch; and for cellulose-rich hybrid films, a higher content of starch reduced the material properties although chemical interactions were not apparently influenced. One possible reason could be the inherently better mechanical properties of cellulose, notwithstanding that this could not be verified in this current work. Moreover, the rheological study of the cellulose/starch/ZnCl