The conditions of adhesive connections testing can significantly affect the recorded findings. The standards, under which the investigations are conducted, do not take into account numerous factors that can greatly influence the outcome of the tests. Moreover, the research parameters in the standards are not specified. It is not defined in what manner their change, or any failure to comply with the standards, will affect test results. This article presents the results of experimental research, completed with numerical simulations, designed to test to what extent the recorded impact strength of adhesive connections is affected by the stiffness of the test stand, and the speed and energy of the impactor. In the experimental research, the authors used block samples whose substrates were made of an aluminum alloy. The elements of the samples were bonded by means of three different epoxy adhesives. The models used in numerical calculations were built on the basis of the real dimensions of the specimens used in this experimental research. As a result of the performed tests and conducted calculations, it was found that the use of test stands of lesser stiffness resulted in an increased registered impact strength due to an increased energy value of elastic deformations. Examinations of impact strength of adhesive connections are becoming increasingly popular and necessary due to the constantly rising overall use of this type of connection in contemporary design, and due to the fact that glued structures may undergo an impact load. Dynamic, single, or multiple loads often occur during the operation of means of transport ,2,3,4 orOne of the most popular methods of impact strength testing of glued connections is the standard method ,12,13. TIn order to test the impact strength of the samples, the authors prepared batches of adhesively bonded block samples , whose aThe adhesive materials were Epidian 57 with Z1 hardener, as well as construction adhesives Loctite 3421 and 3430, selected due to their significant difference in the Young’s modulus . The thicknesses of the joints were the same and measured 0.1 mm.Prior to bonding, the metal parts were cleaned and their surface was given proper structure and roughness through abrasive blasting ,15,16, wAfter curing the joints, the authors performed a visual inspection of the quality of the obtained connections and removed excess glue and threads outside the bonded surfaces. Removing the threads soaked in glue was particularly relevant, since they may be caught up during an impact strength test by a torn upper element and consequently slow down the dropping tool, which would translate into a higher value of impact strength of the connection. The excess glue may result in raising the strength of the joint  , in which it is possible to use two pendulums of different maximum energy of 10 J or 25 J. The pendulum of 10 J has a smaller flexural stiffness (arm diameter of 12 mm), whereas the pendulum of 25 J has higher bending stiffness (arm diameter of 20 mm). Moreover, the testing device allows for the modification of the initial energy of the impactor through moving the angle of the pendulum, which is also associated with changing the speed of the impact. The maximum shifting of the pendulum loads the examined sample at a speed of 3.81 m/s. In order to check whether the eleven-piece batch of samples is sufficiently large to conduct impact strength tests, for all the examined adhesives the authors conducted testing of two eleven-piece batches in the same conditions and then compared their results . The invIn In order to evaluate the influence of the pendulum stiffness upon the impact strength of block connections, batches of identical specimens were loaded by a pendulum of 10 J and 25 J, at a speed of 3.81 m/s. The findings of the tests are presented in The registered lesser impact strength of samples loaded with the pendulum of 25 J may be explained by a smaller value of the deformation energy of the stiffer pendulum arm “25 J” (Φ = 20 mm) than of the “10 J” (Φ = 12 mm). This might indicate a considerable impact of the test stand stiffness upon the recorded findings. Additionally, investigations were conducted in which identical samples were loaded with the energy of 10 J using different pendulums (“10 J” and “J 25”). The pendulum “10 J” struck the sample at a speed of 3.81 m/s, whereas the pendulum “25 J” struck at a speed of 2.42 m/s. The findings of these tests are presented in The obtained results suggest that reducing the impact speed increases the recorded impact strength of adhesive connections. However, taking into consideration greater stiffness of the pendulum “25 J”, the result requires a more in-depth analysis.The calculations were conducted in the software ANSYS 16.2, using the Explicit Dynamics module. A numerical model of the tested samples was built .The impactor was modelled with two elements, in which the striking piece that directly struck the upper element of the sample had a stiffness equal to steel (E = 200 GPa); the stiffness of the other element was higher by one row in value (E = 2000 GPa) in order to ensure adequate stiffness of the impactor . The mat−4, and at the speed of 3.81 m/s and the time of 8 × 10−5. For these times, it was possible to estimate the force of the impactor striking the spacer during the time of the occurrence of the stresses in the joint, being close to the destructive ones, based on stresses σx The recorded energy of impact damage of adhesively bonded connections is a sum of the energy of the joint’s destruction and the elastic deformations of the test stand.(2)The use of research stands of lesser stiffness resulted in increasing the registered impact strength due to a higher energy value of elastic deformations.(3)Reducing the speed of impact loading in research investigations led to an extended time of the research stand, and thus an increase in the elastic strain energy. Therefore, it caused an increase in the recorded impact strength.(4)The comparative study of the impact strength of adhesive connections should be executed on test devices, whose stiffness and speed of load application has been explicitly specified.


Background: In patients with allogenic hematopoietic stem cell transplantation , immune-checkpoint inhibitors (ICI) are used to treat malignancy recurrence. However, ICI are also associated with graft vs. host disease (GVHD). In this pharmacovigilance analysis, we aimed to characterize cases of GVHD associated with ICI, drawn from the World Health Organization pharmacovigilance database, VigiBase®, and from literature.Methods: We performed VigiBase® query of cases of GVHD associated with ICI. These cases were combined with those of literature, not reported in VigiBase®. The Bayesian estimate of disproportionality analysis, the information component, was considered significant if its 95% credibility interval lower bound was positive; denoting a significant association between GVHD and the suspected ICI. Time to onset between ICI and GVHD onset and subsequent mortality were assessed.Results: Disproportionality analysis yielded 93 cases of GVHD associated with ICI . Cases were mostly associated with nivolumab , pembrolizumab  and ipilimumab  monotherapies. GVHD events occurred after 1  injection of ICI, with a time to onset of 35  days. Immediate subsequent mortality after GVHD was 24/93, 25.8%. There was no significant difference in mortality depending on the molecule (p = 0.41) or the combination regimen . Previous history of GVHD was present in 11/18, 61.1% in cases reported in literature.Conclusion: In this worldwide pharmacovigilance study, disproportionality yielded significant association between GVHD and ICI, with subsequent mortality of 25.8%. Previous history of GVHD was reported in more than half of cases.Clinicaltrials.gov identifier:NCT03492242 Immune checkpoint inhibitors (ICI) may be used to treat aggressive hematologic malignancies, either in refractory or relapsed lymphoma frequently before being treated by allogeneic hematopoietic stem cell transplantation  or in relapse after allo-HSCT. Indeed, in both situations, relapse mechanisms include immune escape by the tumor, T-cell anergy, down-regulation of regulatory T cells and activation of immune checkpoints . They inLike other therapies used in these indications , the aim of ICI is to enhance the immune system so that it may be effective against malignancy, by restoring T-cell function, activating lymphocytes and inducing a sustainable graft-versus-tumor (GVT) effect.However, ICI are associated with immune-related adverse events, secondary to the over-activation of the immune system, which may, in turn, cause auto-immune-like complications, including graft-versus-host disease (GVHD). GVHD is serious adverse event in patients with allo-HSCT, initially described as an aggravated manifestation of regular inflammation, in which, donor lymphocytes interact with recipient antigens which may cause multi-organ dysfunction .Previously, cohort studies showed that ICI used before allo-HSCT were associated with an incidence of GVHD varying from 41 to 56% . When usTo comfort these results yielded from cohort studies; in the present work, we used disproportionality methodology to present characteristics of GVHD following ICI administration. This method is based on a large pharmacovigilance database, VigiBase®, which collects worldwide reports of drug-related adverse events in the World Health Organization network . In addihttp://www.vigiaccess.org/). ICSRs include administrative information , patient data , date of onset of reaction(s) and nature of the outcome using the latest version of MedDRA  terms (currently v22.1). Drug(s) involved  are also indicated.This work combines a worldwide pharmacovigilance observational case-control cross-sectional study focusing on GVHD related to the usage of ICI, and a systematic literature case report analysis. The pharmacovigilance part relies on VigiBase®, a database encompassing 22 million individual case safety reports (ICSR) received worldwide . It is fWe searched for cases flagged with MedDRA preferred terms (PT) reflecting GVHD (see below), and associated with ICI molecules  from VigiBase® creation through January 05, 2020.clinicaltrials.gov registry number NCT03492242).This work is ancillary to the Immune CHeckpoint Inhibitors Monitoring of Adverse Drug ReAction (CHIMeRA) registry  methodology. Briefly, the estimate of disproportionality analysis can be calculated by the information component (IC) for which ICnificant . The IC nificant .Calculation of the IC using a Bayesian confidence propagation neural network was developed and validated by the Uppsala Monitoring Center as a flexible, automated indicator value for disproportionate reporting that compares observed and expected drug–ADR associations to find new drug–ADR signals with identification of probability difference from the background data (full database) . ProbabiThe statistical formula is as follows:Nexpected is the number of case reports expected for the drug–ADR combination.Nobserved is the actual number of case reports for the drug–ADR combination.Ndrug is the number of case reports for the drug, regardless of ADR.Neffect is the number of case reports for the ADR, regardless of drug.Ntotal is the total number of case reports in the database.025 is the lower end of a 95% credibility interval for the IC.IC025 value (>0) is the traditional threshold deemed statistically significant. IC025 values have only been validated for comparison of drug-specific ADR vs. the full database and cannot be used to compare disproportionate reporting among different ICI regimens. All patients were included in these analyses.A positive ICList of all MedDRA preferred terms (PT) related to GVHD, used in VigiBase® query.Acute graft vs. host disease (PT), Acute graft vs. host disease in intestine (PT), Acute graft vs. host disease in liver (PT), Acute graft vs. host disease in skin (PT), Chronic graft vs. host disease (PT), Chronic graft vs. host disease in intestine (PT), Chronic graft vs. host disease in liver (PT), Chronic graft vs. host disease in skin (PT), Graft vs. host disease (PT), Graft vs. host disease in eye (PT), Graft vs. host disease in gastrointestinal tract (PT), Graft vs. host disease in liver (PT), Graft vs. host disease in lung (PT), Graft vs. host disease in skin (PT).A systematic literature review of all published cases of GVHD associated with ICI was performed, spanning from January 1st, 2010 to February 1st, 2020, on MEDLINE using PubMed® search engine. The methodological search strategy included keywords associated with Medical Subject Headings (MeSH) terms related to ICI and GVHD: “immune checkpoints inhibitors” OR (“immune” AND “checkpoints”) OR (“checkpoint” AND “inhibitor”) OR “nivolumab” OR “pembrolizumab” OR “ipilimumab” OR “cemiplimab” OR “avelumab” OR “durvalumab” OR “atezolizumab” OR “PD-1 blockade”) AND (“graft vs. host disease” OR (“graft vs. host” AND “disease”) AND . From this search, 715 articles were found which contained any of these terms, but the combination of these terms in the format of case reports or case series after adequate filtering yielded 22 cases.Continuous variables are presented as median [interquartile-range] and categorical variables as number (percentage), with relevant comparison tests, accounting for data distribution. GraphPad Prism v6.0  was used for statistics and figures, except 025 = 1.7 (see 025 = 1.9). The most common ICI indications reported were Hodgkin disease in 47/81, 58.0% and non-Hodgkin lymphoma in 14/81, 17.3%. ICI were prescribed in monotherapy, in 88/93, 94.6% . There was no relevant information regarding GVHD grade in VigiBase®.Overall, we included 93 deduplicated cases . In VigiBase®, disproportionality analysis yielded significant association between GVHD events and ICI molecules, with an IC = 2.0 and IC 1.7 see . The firIn the aggregated dataset of 93 cases, men represented 42/68, 61.8% of cases . Median p = 0.60). Mortality was not affected by the type of molecule (p = 0.41). Immune-related adverse events were co-reported in 23/93, 24.7%, mostly with hepatitis in 6/23, 26.1%.Overall mortality associated with ICI-associated GVHD was 24/93, 25.8%. This mortality was that reported in VigiBase® and implies a very short follow-up after the episode (less than a month). Therapy regimen (combination vs. monotherapy) did not affect subsequent mortality  . As of yInterestingly, we found similar results as those found in the largest dataset to date, of GVHD due to ICI, aggregating 283 cases from several studies albeit with a reported overall mortality after GVHD of 11%, lower than that observed in our study. There were 107 cases occurring prior to allo-HSCT, and 176 after, akin to the results we observed in our dataset .Our findings support the fact that ICI use in patients with allo-HSCT needs to be carefully monitored, as these patients are at high risk of developing GVHD. Regarding event prediction, risk factors of developing immune-related adverse events are currently under investigation , and bio025) have been demonstrated to accurately to identify signals of cardiovascular adverse events associated with anti-cancer drugs in various settings (We acknowledge several limitations to the present work. First, retrospective pharmacovigilance analyses present inherent intrinsic limitations; while disproportionality reporting used here (ICsettings , risk ofThis worldwide pharmacovigilance database analysis confirmed a significant association between GVHD and ICI.http://www.vigiaccess.org.Publicly available datasets were analyzed in this study. This data can be found here: http://www.vigiaccess.org/.The supplied data from VigiBase come from various sources. The likelihood of a causal relationship is not the same in all reports. The information does not represent the opinion of WHO. The authors have nothing to disclose related to this study. The data, analytic methods, and study materials are available to other researchers for purposes of reproducing the results or replicating the procedure at LN and LR equally contributed to the manuscript. They wrote the draft, performed analyses, reviewed literature. BL-V provided VigiBase data and provided critical review to the manuscript. J-ES supervised the project, performed analyses and provided critical review to the manuscript.J-ES has participated to BMS advisory boards.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Several methods are currently employed in the modification of starch obtained from different botanical sources. Starch in its native form is limited in application due to retrogradation, syneresis, inability to withstand shear stress as well as its unstable nature at varying temperatures and pH environment. Modification of starch is therefore needed to enhance its food and industrial application. A primary and safe means of modifying starch for food and industrial use is through hydrothermal methods which involves heat-moisture treatment and annealing. Heat-moisture treatment (HMT) is a physical modification technique that improves the functional and physicochemical properties of starch without changing its molecular composition. Upon modification through HMT, starches from cereals, legumes and tuber crops serve as important ingredients in diverse food, pharmaceutical and industrial processes. Although changes in starch initiated by HMT have been studied in starches of different plant origin, this work further provides insight on the composition, thermal and functional properties of heat-moisture treated starch obtained from cereals, legumes and tuber crops. D-glucose units with an α- linkage while AP comprise branches of D-glucose from an α- linkage at the branches, to α- linkage at the branch points , the minor component and amylopectin (AP) which is the major component in most starches  which account for approximately 98–99% (dry weight) Sulaiman . StarcheStarch granules comprises alternating amorphous and semi-crystalline rings. Granule morphology, size and surface properties, however, plays a great role in food and industrial utilization of starches. The functional properties of starch depend on the composition of the molecular structure of AM and AP as well as their arrangement in starch granules and this confers vital effects in food formulations , but lower than the onset temperature (To) of gelatinization for a specified time. Kweon et al.  or at intermediate water content . During the annealing process, starch granules are kept at temperatures above their Annealing modification technique is further associated with increase in granule structure stability, formation of perfect crystalline, starch chain connections in amorphous and crystalline domains of granule, as well as in the emergence of double helices in starch granule Fig. . It alsoAdebowale et al.  discoverRecent studies on the effect of HMT on starches include studies on amylomaize, maize, dull waxy maize, waxy maize . Literatures on the effect of HMT on RS content of starches from various sources, have presented increases in the RS concentration of these starch. Li et al. , noted tThe potential of increasing the functional properties of flour from tuber crops such as sweet potato using physical modification has been noted and this could assist in generating new applications of the flour  and concluding temperature (Tc) of pea and lentil starches with increase in temperature  has been observed to significantly affect modified starches by influencing its gelatinization and enthalpy change. Cooke and Gidley  reportedts Table . Lim et ts Table , showed Starch retrogradation is an unstable thermo changeable re-crystallization process which occurs through three successive stages of propagation, nucleation and maturation  as well as the functional properties of the modified starches. Modification of starch through HMT therefore, results in improved quality starch readily available for use and application by food processors and industries. However, further studies are needed on the optimization of the HMT temperature times as well as its effect on other nutritional properties of the starch.


Based on tension–tension fatigue tests, this paper investigated the mechanical property degradation of carbon fiber reinforced polymer (CFRP) tendons from a macroscopic perspective. According to the degradation regularity, this paper proposed a normalized phenomenological fatigue model based on the residual stiffness/strength of CFRP tendons during the fatigue loading process. In this paper, the residual stiffness of CFRP tendons were tested at five stress ranges, while the residual strength was tested at four stress ranges. In order to validate the reliability and applicability of proposed fatigue damage model, the predictions of proposed model and cited models from the literature are discussed and compared. Furthermore, experimental results from literatures were adopted to verify the accuracy of the proposed model. The results showed that the proposed model is applicable to predict both residual stiffness and residual strength throughout fatigue life cycle and has a better accuracy than models from the literature. Moreover, the three-stage degradation can be observed from the degradation processes of stiffness and strength at each stress level.  Fiber reinforced polymer (FRP) composite is a high-performance material achieved by mixing fibers with resin matrix through a proprietary re-engineering process. Due to its several unique characteristics, such as lightweight, high strength, high corrosion resistance, etc., the vigorous development of FRP composites has been promoted. FRP composites have been vastly utilized in various bridge structures, such as external prestressed tendons in Hisho Bridge , internaFatigue research on the FRP composite increased in the 1970s. Due to the anisotropy, brittleness, and heterogeneity of composite materials, the failure mechanism and failure characteristics of the FRP composite subjected to fatigue loading are significantly different from those of metallic materials. The fatigue damage of the FRP composite is manifested as the initiation and development of micro-cracks, the degradation of mechanical properties, etc. Degrieck and Van Paepegem  classifiResidual stiffness is a macroscopic parameter to characterize the fatigue performance of the FRP composites. Whitworth  conducteResidual strength is another macroscopic parameter to characterize the fatigue performance of the FRP composites. Broutman and Sahu  assumed The above mentioned residual stiffness models and residual strength models demonstrated the property degradation regularity under cyclic loading from a macroscopic perspective, and accordingly provided theoretical bases for research works on the fatigue property of FRP composites. However, the fatigue models proposed in above literature cannot be applicable to not only predict residual strength but also predict residual stiffness of FRP composites under cyclic loading. This paper investigated the stiffness and strength degradation of CFRP tendons based on tension–tension fatigue tests. It can be observed from the aforementioned literature that the residual stiffness and residual strength of FRP composites have a potential relationship with stress range and maximum stress. Therefore, in order to investigate the influence of stress range on mechanical property degradation, the maximum stress was kept consistent. The residual stiffness of CFRP tendons were measured at five stress ranges, while the residual strength was measured at four stress ranges. Based on the degradation regularity in the experiment, this paper proposed a normalized phenomenological model that can be used to predict both the residual stiffness and residual strength of CFRP tendons subjected to cyclic loading. In order to investigate the reliability of this proposed model, comparisons between the predictions of residual stiffness/strength models reported in the literature were performed and of this model were performed as well. Furthermore, this paper investigated the influence of stress range on stiffness/strength degradation regularity at a given maximum cyclic stress.CFRP tendons tested in this paper are manufactured by Hengshen Co., Ltd. , which is pultruded from T700-12K carbon fiber and epoxy resin. The fiber volume ratio of this CFRP tendon is 65%. The CFRP tendon has a smooth round surface with a diameter of 8mm and is held by a wedge-type anchorage system. A servo-hydraulic fatigue testing machine, Instron 8800, is adopted to carry out the experiment. In the fatigue tests, CFRP tendons were subjected to constant amplitude axial cyclic loadings. Tension–tension fatigue tests of CFRP tendons were conducted following ASTM D3479/D3479M , which a9/D3039M . In the When the cyclic loading achieves a given cycle, Based on the above obtained fatigue life, There are various damage mechanisms in the FRP composite, such as matrix cracking, fiber breakage, matrix/fiber debonding, and delamination. During the process of cyclic loading, single or multiple damage mechanisms appear in the FRP composite. The mechanical properties degrade during this process, macroscopically shown as stiffness degradation and strength degradation. Because the damage mechanism of the FRP composite is complicated during the fatigue loading process, it is impractical to establish a fatigue damage model from a microscopic perspective. Based on experimental data, Reifsnider  pointed This paper proposed a normalized phenomenological fatigue model based on the experimental residual stiffness/strength of CFRP tendons during the fatigue loading process. In order to illustrate the reliability and applicability of proposed fatigue model, the predicted results of the proposed model are compared with the predicted results of reported residual stiffness models and residual strength models from the literature, respectively. As previously mentioned, several recommended residual stiffness and residual strength fatigue model are listed below.H. A. Whitworth (1987) :(1)S(n)J. N. Yang et al. (1990) :(2)S(n)A. T. Echtermeyer et al. (1995) :(3)S(n)ST. P. Philippidis and V. A. Passipoularidis (2000) :(4)S(n)SW. X. Yao et al. (2012) :(5)S(n)SWhitworth  assumed L. J. Brountman and S. Sahu (1972) :(6)S(n)SJ. R. Scaff and B. D. Davidson (1997) :(7)S(n)SW. X. Yao and N. Himmel (2000) :(8)S(n)ST. P. Philippidis and V. A. Passipoularidis (2007) :(9)S(n)SN. Stojković et al. (2017) :(10)S(n)where Brountman and Sahu  assumed The experimental results from fatigue tests were analyzed in this paper and were compared with predicted results of above proposed residual stiffness models from the literature. The above mentioned residual stiffness models from the literature were adopted to fit experimental results, which use the least squares method. The fitting curves of the residual stiffness models from the literature are plotted in It can be observed from (a)Under cyclic loading with various stress ranges, both the stiffness and strength of CFRP tendon have three-stage degradations throughout the fatigue life cycle.(b)Stiffness/strength degradation rate of the CFRP tendon increases with the growth of stress range. This means that if the stress range gets larger, stiffness/strength degradation of the CFRP tendon become more obvious. (c)When the stress range gets smaller, the stiffness/strength degradation rates of initial stage and final stage become closer to that of the second stage.It can be observed from In order to verify the accuracy of proposed fatigue model, this paper adopted the experimental results from Xu  and Post(a)Both the stiffness and strength of CFRP tendon degrade during the fatigue loading process. Also, it can be observed that as the stress range increases, stiffness and strength of CFRP tendons degrade more obviously.(b)The three-stage regularity can be observed from degradation processes of stiffness and strength when CFRP tendons and other FRP composites subjected to fatigue loading. In the first stage, transverse cracks become more saturated when CFRP tendons subjected to lower stress range. Therefore, in the first stage, mechanical property degrades more obviously at lower range. The damage mechanism in the first stage is matrix cracking and is matrix/fiber debonding in the second stage at lower stress range. However, the damage mechanism in the first and second stage becomes the mixing of matrix cracking, matrix/fiber debonding and fiber breakage at higher stress range. Therefore, the boundaries between adjacent stages become more obvious when stress range decreases.(c)The proposed fatigue damage model is applicable to predict both residual stiffness and residual strength throughout fatigue life cycle. This new proposed model has a better accuracy than the models from the literature based on the experimental results of CFRP tendons and results from the literature.In order to investigate the mechanical property degradation of composite material subjected to fatigue loading, tension–tension fatigue tests of CFRP tendons were conducted. In this paper, the residual stiffness and residual strength of CFRP tendons were tested at different stress ranges, and a new fatigue damage model was proposed according to the experimental results. The following conclusions can be drawn from the investigations presented here:


Compared with boiled and steamed noodles, the microwaved noodles showed a more compact morphology without porous holes on the surface, whereas fried noodles showed irregular morphology. The results indicated that the digestive properties of noodles made with the same ingredients can be greatly altered by using different cooking methods, and the digestive properties of different cooked noodles are worthy of confirmation using in vivo analysis.Noodles are widely consumed in China, which can be cooked in different ways. The effects of different cooking methods  on the resistance starch (RS) content and digestive properties  value) of noodles were investigated. The RS content was greatly affected by the cooking time, and it was varied when the noodles were optimally cooked using different cooking methods. The RS contents of the microwaved and stir-fried noodles were relatively high (0.59%–0.99%), but it was lower (0.43%–0.44%) in the boiled and steamed noodles. Microwaved noodles showed the slowest digestion rate and the lowest eGI. Due to the limited water within fried noodles, none RS was found in the fried noodles, whereas stir-fried noodles showed RS Noodles are staple foods for many Asian countries . In Chin1 is the starch that is physically inaccessible to digestion by entrapment in a non-digestible matrix. RS2 is composed of native starch granules of selected botanical sources. RS3 is retrograded starch and is created when gelatinization is followed by a retrogradation process. RS4, which is formed by chemical modification. RS5 is the amylose complexed with lipid, where the helical structure is hardly digested by the enzyme. However, RS, is not a ‘property’ of starch, but is a kinetic parameter largely determined by access of enzyme to the substrate (binding) and subsequent enzyme action  as discussed elsewhere [Starch is the major ingredient of noodles, which can be classified as rapidly digestive starch (RDS), slowly digested starch (SDS) and resistant starch (RS)  based onlsewhere .Compared with raw starch, the RS content of cooked starchy food is significantly reduced, and further influenced by different cooking methods. For example, the RS content of potato chips has been reported to increase after roasting, baking and shallow frying (frying in limited oil), whereas it decreases after steaming and frying . After cNoticeably, noodles are considered as a high glycemic index (GI) food  as the sTherefore, the objective of this study is to investigate the effects of different cooking methods  on the RS content and digestive properties  value of noodles. The thermal properties, crystalline structure, and morphology of cooked noodles were explored using differential scanning calorimetry (DSC), X-Ray diffraction (XRD) and scanning electron microscope (SEM), respectively, to understand how the micro-structure and macro-structure affect the digestive properties of noodles using different cooking methods.Wheat flour of Ningchun 4, a gift of Northwest Agricultural and Forestry University, was prepared using a laboratory Buhler mill  and passed through an 80-mesh sieve. Soybean oil  was purchased from a local grocery store.2 and 0.49 M of MgCl2) were used for the in vitro digestion experiments. All of the chemicals used were of analytical grade.Resistant starch (RS) assay kit  was used to measure the RS content of noodles prepared using different cooking methods and storage conditions. Pancreatin , Amyloglucosidase (AMG) , and Acetate buffer  and the time before and after OCT were prepared. This will capture the eating habit of consumer that prefer the firm  or softer noodles. OCT was obtained through taking out a noodle string every 30 s and squeezed by a pair of transparent glass plates to observe the presence of the white core. The time when the white core disappeared was considered as the OCT for the noodles cooked with different methods . Boiling: Noodles (20 g) were placed in 200 mL boiling water and boiled for 120, 150, 180, 210, and 240 s, with 210 s the OCT.Steaming: Noodles (20g) were spread out evenly in trays with a thickness of about 2mm and steamed at 100 °C for 600, 900, 1200, and 1800 s, with 900 s the OCT.Stir-frying: Raw noodles tend to stick together when stir-fried directly, thus noodles were partially gelatinized before stir-frying. Noodles (20 g) were boiled in 200 mL boiling water for 120 s and then cooled in running water for 30s. The boiled noodles were stir-fried in a pan (180 °C) with 5, 10, and 15 mL soybean oil for 30 and 120 s. Among them, 10 mL of oil stir-fried for 30 s is the optimum cooking condition.Frying: Noodles (20 g) were fried in soybean oil (400 mL) in an electric frying pan  at 140, 160, 180, and 200 °C for 30 s and 120 s. Among them, fried at 180 °C for 30 s is the optimum cooking condition.Microwave-cooking: Noodles (20 g) were put in a glass bowl with 200 mL boiling water and microwaved for 60, 120, 150, 180, and 240 s in a microwave oven  (on high) with 150 s the OCT.The noodles cooked at different conditions were frozen immediately in liquid nitrogen. The samples were lyophilized, ground  and passed through a 100-mesh sieve for further analysis.Moisture contents (MC) of noodles cooked using different methods were determined according to AACC method 44-15A. The analysis was performed in triplicate. Freshly cooked noodles  at room temperature for 12 h. The diffractogram scan was run between 2° and 40° (2θ) with a step size of 0.02° and at a rate of 5°/min. The relative crystallinity was calculated from the ratio of the crystalline area to the total diffractogram area using the methods of Li et al. [A D8 Advance X-ray diffractometer  was used with Cu Ki et al.  using a Tp) and retrogradation enthalpy change (∆HR) or gelatinization enthalpy change (∆HG) were determined from each endotherm using a built-in software . The thermal properties of freshly cooked noodles (cooking under OCT) were analyzed in duplicate using a DSC  based on the method described by Teng et al. . For theNoodles cooked using different methods were freeze-dried by ALPHA 1-2 LD plus freeze dryer . Noodle strands with a length of about 5 mm were fixed on the sample stage, which was sprayed with gold using JFC-1600 ion sputter . The images were taken under a JSM-6510 LV scanning electron microscope  at a magnification of 1500 times.The RS content of cooked noodles was determined using a K-RSTAR 06/18 kit from Megazyme , according to the AOAC Method 2002.02 . The samv/v), followed by centrifuging at 4000× g for 5 min. The hydrolyzed glucose content was measured using GOPOD reagent.In vitro starch digestibility was analyzed following the method of Wang  with slit is the concentration of product at a given time (t), Ct may be expressed as the amount of starch digested as a percentage of the total starch content of the sample. A Logarithm of Slope (LOS) plot was obtained by expressing the first derivative of the first-order equation in logarithmic form (Equation (3)).k and The digestograms were fitted to first-order kinetics ,25 as shexpAUC) were calculated with Equation (4):To calculate the estimated GIs of the samples, the areas under the digestograms (The hydrolysis index (HI) of each sample was calculated by dividing the area under its digestogram by the area under the digestogram of fresh white bread , as showFrom the hydrolysis index obtained, the estimated glycemic index (eGI) (bread = 100) was calculated using the equation established by Goni et al. : eGI = 3The RS content of raw noodles was 13.83 g/100 g, but it decreased significantly after cooking. After boiling, steaming and microwave cooking, the RS content decreased significantly with the cooking time, whereas the RS content did not change further after the noodles were cooked for the optimal cooking time . The decThe temperature, the amount of oil used and the cooking time varied during the stir-frying and frying process. The RS content of stir-fried noodles was significantly changed with the amount of oil used . When thAfter frying, only a small amount of RS (0.47 g/100 g) remained in the noodles when the oil temperature was 140 °C; it decreased significantly (0.25 g/100 g) as the frying time increased. There was no RS be detected by using the assay kit when the frying temperature was above 160 °C .Comparing the optimal cooking conditions of the five different cooking methods, noodles cooked using microwave showed the highest RS content (0.99 g/100 g), followed by the stir-fried noodles (0.59 g/100 g). The RS contents of the steamed and boiled noodles were similar, 0.44 and 0.43 g/100 g, respectively. With the same cooking time and temperature (180 °C for 30 s), the RS content of stir-fired noodles was ranged between 0.52–0.61 g/100 g, while no RS was detected in the fried noodles .k) and the extent of starch digested (∞C) during starch amylolysis [−1. The two digestion phases were similar to the results of Yao [k1 value is more physiologically relevant, as the food digestion in the first 120 min can reflect the release rate of glucose after a meal and is given more importance compared to k2. Besides, there were no significant differences in the second stage digestion rates (k2) among the noodles cooked using different methods. Thus, we mainly focused on the changes in the k1, which showed the main differences in the digestive rates among different noodles.The digestive properties of optimally cooked noodles were investigated. The LOS plot was used in this study to quantify the differences in the digestion rate  than noodles prepared using other cooking methods, which was followed by steamed noodles (0.0354 min−1). The digestion rates of noodles after microwave cooking and stir-frying were slower, 0.0202 and 0.0290 min−1, respectively. The boiled and fried noodles were digested quicker  and 96.22% (boiling). Among the five cooking methods, the steamed and boiled noodles showed the largest digestibility . The digestibility of starch in stir-fried noodles was slightly lower (90.46%), which is similar to the study of Reed [The maximum hydrolysis extent,  of Reed . The digk and ∞C. The eGI value of noodles followed the order, boiled noodle (98.33) > steamed noodle (95.55) > stir-fried noodle (90.46) > microwave cooked noodle (86.93) > fried noodle (86.18). Although there were some differences in the eGI of the noodles obtained by the five cooking methods, they were still among the high GI food (GI ≥ 75).The values of estimated glycemic index (eGI) of noodles cooked using different methods are shown in The peak gelatinization temperature of raw wheat starch was 69.61 °C . This isThe crystalline polymorph of raw wheat starch in the uncooked noodles was of the type-A crystal B, with t5 (amylose-lipid complex) was ranged from 98.6 to 99.8 °C. The ΔH value of stir-fried noodles (0.84 J/g) was similar to the result of Yang [H of the starch-lipid complexes of the steamed-fried noodles ranged from 1.01 to 1.40 J/g. However, this peak was not found in the fried noodles. There was a pre-boiling step in the preparation of stir-fried noodles, which enabled water  inside the noodles. On the other hand, from the results of XRD  easily [5 was formed in the stir-fried noodles and its digestion rate was lower.For stir-fried and fried noodles, starch can form amylose-lipid compounds (RS) easily , as the ) easily . The RS ) easily  also fou∞C was not consistent. For example, there was no RS within the fried noodles but the extent of starch digested was 77.48%, which was the lowest among cooked noodles. The reason for the difference between the RS content and the final digestibility may be due to the methods for measuring the RS content of in vitro digestion, which is mainly different in the duration of digestion. The test method for RS content using kit lasted for 16 hours, while it only lasted 5 hours for the in vitro digestion method. Similar difference in the RS content and ∞C was also observed by Hasjim [The RS content of noodles had a significant negative correlation with its digestion rate , that isy Hasjim , but it According to the K-RSTAR 06/18 Megazyme kit, higher errors can be obtained for samples with RS contents < 2%. The repeatability to measure the RS content was justified using some of the cooked noodles, where the standard deviation was between 0.01 and 0.12 (g/100 g). Thus, although the RS content was low in some cooked noodles, the value can still be compared in this investigation.5 formation during noodle cooking, since more RS5 was formed in stir-fried noodles. The eGI value of the noodles obtained using different cooking methods was also different, which indicates that it is not accurate to estimate the GI value of the type of food using single value, as the eGI can be varied by the way of cooking. Besides the cooking methods, food storage conditions may also impact the structure and digestive properties of starch-based foods differently, which merits further investigation. In addition, the digestive properties of different cooked noodles in this study were analyzed through in vitro experiments, whether similar results can be obtained through in vivo analysis requires further experiments.Cooking conditions affect the amount of RS in noodles, and the RS content of the noodles was not changed when further extended the cooking time when the optimal cooking time is reached. The RS content in noodles after microwave cooking and stir-frying was highest, while there was no RS in the fried noodles. Moisture content may influence the RS


Castanea henryi. Seed germination is the beginning of the plant’s life cycle, and starch metabolism is important for seed germination. As a complex metabolic pathway, the regulation of starch metabolism in C. henryi is still poorly understood. To explore the mechanism of starch metabolism during the germination of C. henryi, we conducted a comparative gene expression analysis at the transcriptional level using RNA-seq across four different germination stages, and analyzed the changes in the starch and soluble sugar contents. The results showed that the starch content increased in 0–10 days and decreased in 10–35 days, while the soluble sugar content continuously decreased in 0–30 days and increased in 30–35 days. We identified 49 candidate genes that may be associated with starch and sucrose metabolism. Three ADP-glucose pyrophosphorylase (AGPase) genes, two nucleotide pyrophosphatase/phosphodiesterases (NPPS) genes and three starch synthases (SS) genes may be related to starch accumulation. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the expression levels of these genes. Our study combined transcriptome data with physiological and biochemical data, revealing potential candidate genes that affect starch metabolism during seed germination, and provides important data about starch metabolism and seed germination in seed plants.Starch is the most important form of carbohydrate storage and is the major energy reserve in some seeds, especially  It is related to the subsequent growth and final yield of plants. Seeds are a vital component of diets worldwide, and seed biology is one of the most extensively researched areas in plant physiology. During the period of germination, the metabolism of fats, proteins and carbohydrates provide energy for seedling growth [Seed germination is the basis of plant formation and is the period in the life of a plant with the highest g growth ,2,3. Carg growth . Starch g growth ,6. Amylog growth ,8,9.Seed starch is a primary source of carbohydrate for both human and animal diets. Seed starch is the major storage compound that accumulates in the cereal endosperm, and more than half of the world’s population uses rice as a source of carbon intake every day . Starch Sucrose is hydrolyzed by sucrose synthases (SuSy) and invertases (INVs) into monomer sugars, glucose/UDP-glucose (UGP2) and fructose, which are then converted through a series of reactions into glucose-1-phosphate, which is considered to be the most efficient starch synthesis precursor . Starch Fagaceae are the main tree species of tropical, subtropical and temperate forests, and they are also the most significant dominant species of angiosperms. Many of the Fagaceae plants are important economic plants and dual-purpose plants [Castanea henryi is a member of Castanea genus of the Fagaceaethe  family. Castanea trees are extensively distributed between eastern Asia and eastern North America and are used to produce wood, tanning agents and food [Candidate genes associated with starch biosynthesis and metabolism have been widely studied in many plants . Howevere plants ,24. Castand food ,26.Fagaceae plants play an important role in the forest ecosystem; however, previous studies have not focused on the germination of Fagaceae seeds [C. henryi seeds at four germination stages to identify the candidate genes involved in starch and sucrose metabolism. The expression profiles of a few genes were further validated by quantitative real-time polymerase chain reaction (qRT-PCR). The results presented herein may be useful for characterizing the molecular mechanism underlying starch and sucrose metabolism in seed germination.Chestnut fruits have considerable economic value. Chestnuts are rich in nutrients and contain starch, soluble sugar, protein, lipids and 18 types of amino acids, including eight that are essential to the human body. The nutritional value of chestnut fruits is higher than flour, rice and potato ,28,29. Tae seeds ,32. In tC. henryi seeds were obvious (Seeds were sampled from 0 days after sowing (DAS) to 35 DAS, approximately every 5 days, throughout the germination of the seeds. The morphological changes in the  obvious . The whidry matter were measured. The starch content increased from 50.60% to 60.00% from day 0 to 10 DAS, then slowly decreased from 60.00% to 53.07% from 10 DAS to 30 DAS, followed by rapidly decreasing to 38.83% , were selected for comparative transcriptome analysis to better explore the molecular and metabolic regulatory mechanisms of starch and sucrose metabolism during the germination of C. henryi genome, and 52.94% of the mapped reads were unique to the C. henryi genome. An overview of the sequencing statistics is shown in C. henryi genes is shown in In total, twelve cDNA libraries with three repetitions for each stage were constructed and sequenced across the stages of seed germination: 70.19, 67.68 and 70.19 million raw reads were generated for T01 stage; 67.68, 70.19 and 70.19 million raw reads were generated for T02 stage; 70.19, 67.69 and 70.19 million raw reads were generated for T03 stage; and 70.19, 65.7 and 67.68 million raw reads were generated for T04 stage. After removing the adaptor sequences, low-quality and high content of unknown base N reads, the clean rates were all higher than 92.28%, and the Q20 values were all over 92%. Thus, a total of 76.903 Gb-cleaned reads (81.25%) were mapped to the Among the 36,734 genes, 32,766 (89.20%) could be annotated, and 100 genes could be matched with all of the databases . In partTo assign the genes capable of encoding transcription factors (TF), 1391 genes were annotated to 59 TF families . Among tA total of 15,275 (41.58%) genes had significant hits in the GO database, which further classified them into 47 terms: biological process (20 terms), cellular component (15 terms), and molecular function (12 terms) . In partTo identify the active biological pathways in seed germination, the assembled genes were mapped to the KEGG protein database. Genes were classified into 21 terms: metabolism (11 terms), genetic information processing (four terms), environmental information processing (two terms), organismal systems (one term), cellular processes (one term) and human diseases (two terms) . In partC. henryi seed germination were explored with the DEGseq Package [p-value ≤ 0.001), 12,943, 11,999, 13,103, 5641, 5952 and 6206 were searched through the transcriptome analysis and comparisons were made between T01 and T02 stage, T01 and T03 stage, T01 and T04 stage, T02 and T03 stage, T02 and T04 stage, and T03 and T04 stage, respectively. The T02/T03 comparison produced the fewest DEGs (2222 genes upregulated and 3491 genes downregulated), while the T01/T04 comparison produced the most DEGs (6748 genes upregulated and 6355 genes downregulated)  . Moreovegulated) .The starch and sucrose metabolism pathways are well characterized, and many critical genes have been identified ,35,36,37C. henryi transcriptome gene dataset, two of which were highly expressed with FPKM values > 100 (Che007374 and Che013145) and two of which were significantly decreased in T01 to T03 (Che003775 and Che013145).In the first step of starch biosynthesis, AGPase (EC 2.7.7.27) catalyzes the conversion of glucose-1-phosphate into ADP-glucose . We idenNucleotide pyrophosphatase/phosphodiesterases  are widely distributed N-glycosylated enzymes that catalyze the hydrolytic breakdown of the pyrophosphate and phosphodiester bonds of numerous nucleotides and nucleotide sugars, and previous studies have suggested that NPP1 exhibits the highest hydrolyzing activity towards ADP-glucose, ADP-ribose, ATP and UDP-glucose ,39,40,41Additionally, two GBSS genes were predicted in our dataset; one gene was highly expressed with an FPKM value > 3000 (Che029279). Eight SS genes and five SBE genes were also predicted, and four of the eight SS genes were highly expressed  in T01 and similarly expressed in T02 to T04. One SBE gene was highly expressed and continued to increase from T01 to T04. Starch synthases  utilize ADPG to elongate linear chains by catalysing the formation of new α-(1→4) linkages. One particular SS, granule-bound starch synthase , is responsible for the production of the long linear chains in the amylose component of starch. Starch branching enzymes  introduce α-(1→6) branch linkages by cleaving an internal α-(1→4) bond within a linear chain and transferring the released reducing end to a C6 hydroxyl .Starch is degraded by alpha-amylase , beta-amylase  or via the phosphorylation pathway. Starch phosphorylase  catalyzes starch degradation via the reversible phosphorylation of α-glucan . In thisSucrose synthase  mainly decomposes sucrose into UDP-glucose and fructose to provide substrates for starch synthesis and catalyzes the reversible conversion of UDP-glucose and fructose into sucrose ,47. Ten To verify the reliability of the RNA-seq data, seven candidate genes with differentially expressed transcripts involved in starch and sucrose metabolism were selected for real-time quantitative polymerase chain reaction (qRT-PCR) analysis . The qRTCastanea trees are found all over the world, and their chestnut fruits are rich in nutritional elements and have considerable economic value [C. henryi during germination is not fully understood, and full elucidation of the expression levels of genes involved in starch metabolism during germination has not been reported yet.ic value . The cheic value ,51, and Pinus tabulaeformis [Oryza sativa, Oryza sativa, Sorghum bicolor, Linum usitatissimum, Medicago truncatula, and Arabidopsis thaliana [During the germination, the quiescent dry seed rapidly resumes metabolic activity upon imbibition, and the first change is the resumption of respiratory activity. After a steep initial increase in oxygen consumption, the rate declines until the radicle penetrates the surrounding structures, when another burst of respiratory activity occurs ,53. The aeformis , the sugthaliana .C. henryi are different to those in most plants. The starch content increased from the T01 to T02 stages, and then began slowly decreasing, while the soluble sugar continued decreasing throughout the germination. The observed changes in the starch and soluble sugar content were consistent with the results of previous studies involving other plant species. For example, in Hildebrand’s study, the changes in starch and soluble sugar content for four different genotypes were similar in that the starch content increased in the early stage and continuously decreased in the later stage, while the soluble sugar content decreased throughout the germination [Interestingly, in this study, we measured the contents of starch and soluble sugar throughout germination, and the results showed that the changes in the starch and soluble sugar contents in mination . In Guzmmination . Thus, tIn the present study, we used RNA sequencing technology to completely analyze the chestnut seed transcriptome at four germination stages. A total of 15 categories and 49 functional genes in our transcriptome dataset were identified based on the known starch and sucrose metabolism pathway ,63,64,65Starch biosynthesis is a complex system composed of multiple subunits or isoforms of four classes of enzymes: ADP-glucose pyrophosphorylase (AGPase), starch synthase/granule-bound starch synthase (SS/GBSS), and starch branching enzyme (SBE). Each enzyme plays a distinct role, but presumably functions as part of a network ,65,66. TSucrose is mainly metabolized in two ways: via invertase (INV) and via sucrose synthase (SUSY). INV catalyzes sucrose conversion into glucose and fructose in the apoplast and intracellularly, and sucrose synthase (SUSY) catalyzes the reversible conversion of UDP-glucose and fructose into sucrose. In addition, sucrose metabolism includes sucrose–phosphate synthase (SPS), sucrose–phosphatase (SPP) and UDP-glucose pyrophosphorylase (UGPase) ,47,48,50The seeds which were genetically related were germinated in a greenhouse at Fujian Agriculture and Forestry University in Fuzhou, China. A total of 30 seeds sown in six different flowerpots were sampled from 0 days after sowing (DAS) to 35 DAS, approximately every 5 days, between 11 and 12 am. Samples were flash frozen in liquid nitrogen and stored at −80 °C until use. In this study, four periods of seed sampling  were selected for transcriptome sequencing.Sample preparation: first, we dried the seeds at 50 °C for 72 h to a constant weight and ground them, including radicles, etc. Next, we placed 50 mg into a 10 mL test tube, added 4 mL of 80% ethanol, stirred and heated it in a water bath for 40 min, and, after cooling, collected the supernatant by centrifugation. We added 2 mL of 80% ethanol to the residue, extracted it twice, combined all of the supernatants and then added 10 mg of active carbon. We decolorized this mixture at 80 °C for 30 min, and then diluted it to 10 mL with 80% ethanol for soluble sugar determination. After drying the residue, we transferred it into a 50 mL volumetric flask, added 20 mL of hot distilled water, heated it in a boiling water bath for 15 min, added 2 mL of 9.2 mol/L perchloric acid to extract it for 15 min, kept it at a constant volume after room temperature cooling, mixed it evenly, filtered it, and measured the starch content of the filtrate. The content of soluble sugar and starch was determined referencing the anthrone colorimetry method [C. henryi tissue according to the manual instructions: grind tissue samples about 80 mg with liquid nitrogen into powder and transfer the powder samples in 1.5 mL of preheated 65 °C CTAB-pBIOZOL reagents. The samples were incubated by Thermo mixer for 15 min at 65 °C to permit the complete dissociation of nucleoprotein complexes. After centrifuge at 12,000× g for 5 min at 4 °C, the supernatant was added—400 μL of chloroform per 1.5 mL of CTAB-pBIOZOL reagent—and was centrifuged at 12,000× g for 10 min at 4 °C. The supernatant was transferred to a new 2.0 mL tube, then 700 μL acidic phenol and 200 μL chloroform were added, followed by centrifuging 12,000× g for 10 min at 4 °C. An equal volume of aqueous phase of chloroform was added and centrifuged at 12,000× g for 10 min at 4 °C. An equal volume of a supernatant of isopropyl alcohol was added and placed at −20 °C for 2 h for precipitation. After that, the mix was centrifuged at 12,000× g for 20 min at 4 °C and then the supernatant was removed. After being washed with 1 mL of 75% ethanol, the RNA pellet was air-dried in the biosafety cabinet and was dissolved by adding 50 µL of DEPC-treated water.Based on the previous results of the physical indicators of and changes in soluble sugar and starch content, the samples from four periods  were selected as materials for comparative transcriptome analysis, and three biological replicates were used in this work. An ethanol precipitation protocol and CTAB-PBIOZOL reagent were used for the purification of total RNA from the Subsequently, the total RNA was qualified and quantified using a Nano Drop and Agilent 2100 Bioanalyzer . Oligo(dT)-attached magnetic beads were used to purified the mRNA. The purified mRNA was fragmented into small pieces with the fragment buffer at the appropriate temperature. Then, first-strand cDNA was generated using random hexamer-primed reverse transcription, followed by second-strand cDNA synthesis. Then, A-Tailing Mix and RNA Index Adapters were added by incubation for end repair. The cDNA fragments obtained from the previous step were amplified by PCR, and the products were purified with Ampure XP Beads then dissolved in the EB solution. The product was validated with the Agilent Technologies 2100 Bioanalyzer for quality control. The double-stranded PCR products from the previous step were heat-denatured and circularized by the splint oligo sequence to produce the final library. The single-strand circular DNA (ssCir DNA) was formatted as the final library. The final library was amplified with phi29 to make DNA nanoballs (DNBs), which had more than 300 copies of one molecule. The DNBs were loaded into the patterned nanoarray and pair-end 100 base reads were generated on the BGIseq500 platform . The measurements were conducted in triplicate.parameters: -l 5 -q 0.5 -n 0.1) and filtered by using Trimmomatic . The filtered “clean reads” were saved in fastq format. Then, we compared the clean reads to the reference genome sequence by HISAT2 (v2.0.4) [Clean reads were obtained by removing the adaptor sequences, and reads containing poly-N and low-quality reads were counted by using Soapnuke   and to t(v2.0.4) , and we (v2.0.4) ,78,79,80p-value ≤ 0.001). p-values were corrected to Q-values by methods [The FPKM method was used to eliminate the effect of different gene lengths and sequencing levels on the calculation of gene expression. Therefore, the FPKM values were used to compare gene expression differences between different samples. The DEGseq method is based on the Poisson distribution   . The qRT-PCR, performed by TransStart® Tip Green qPCR SuperMix  with the total system was 20 μL, including 2 μL cDNA. The qRT-PCR conditions followed the manufacturer’s instructions. The primers were designed using Primer 6.0 software. The thermal cycling protocol was 30 s at 94 °C, then 40 cycles of 94 °C for 5 s and 60 °C for 30 s for annealing and extension. The specificity was assessed by the melt curve and size estimation of the amplified product. The expression levels of the DEGs were determined using the 2−∆∆t method. The reference large subunit ribosomal protein gene used for normalization of the assayed genes was Che034253 because its expression is stable throughout seed germination. The results shown are the average of three independent biological replicates, repeated three times.Total RNA from the seeds was isolated using the RNAprep Pure Plant Kit  according to the manufacturer’s instructions. Three duplicates of total RNA extracts were reverse transcribed for first-strand cDNA synthesis for qRT-PCR using the TransScriptC. henryi seeds during germination. Most of the seeds of Fagaceae plants contain high starch content, and improving the understanding of starch metabolism during seed germination is helpful to understand the significance of starch to Fagaceae plants. Consequently, the genes associated with starch metabolism during germination will need to be more comprehensively characterized in future studies.In this study, the physiological data showed that starch accumulated briefly during germination, while soluble sugar continuously decreased before the leaves began to grow. Transcriptome analysis identified 15 categories and 49 functional genes were associated with starch and sucrose metabolism pathways, and the expression levels of the GBSS gene (Che029279) revealed that GBSS may be the key regulatory factor of starch synthesis. In addition, the transcriptome data suggested that β-amylase may be an important starch degrading enzyme and that SUSY mainly catalyzes the decomposition of sucrose during germination. By integrating RNA-Seq data with physiological and biochemical data, we have provided insights into the molecular mechanisms that regulate starch and sucrose metabolism in


Ceramic inlay-retained fixed partial denture (IRFPD) is a conservative prosthetic option but the mechanical durability of new high strength zirconia reinforced glass ceramic FPDs is not investigated. The purpose of this study was to compare fracture load of 3-unit ceramic FPDs.N = 64) were used to create three test groups (IRFPDs) and one control group (full coverage FPD) (n = 8). The teeth were embedded in PMMA resin with a mesiodistal distance of 6 mm. Premolars had a distal and molars had a mesial inlay preparation  in the test groups. IRFPDs were made from a zirconia reinforced lithium silicate (VS) or a monolithic zirconia. Zirconia IRFPDs received 2 types of surface treatments: sandblasting (Zr-IRFPD) or internal coating with feldspathic porcelain (ZrC-IRFPD). Control group was made from monolithic zirconia with the same connector size and zirconia surfaces were sandblasted (Zr-FPD). All restorations were cemented using a resin luting cement. After 5000 thermo-cycles, fracture load values (N) were determined with a universal testing machine at a crosshead speed of 0.75 mm/min. Data were analyzed using 1-way ANOVA and Tukey`s post hoc test (p ˂ .05). Extracted premolars and molars (p > .05). VS-IRFPD exhibited statically lower values (391 ± 136). The predominant mode of failure was fracture at the connector area in all groups. Fracture load (mean ± SD) of Zr-FPDs, Zr-IRFPDs and ZrC-IRFPDs were 672 ± 183, 672 ± 123 and 638 ± 59, respectively, being not statistically different ( The fracture load of 3-unit IRFPD was significantly affected by types of ceramics but the retainer design and surface treatment in Zr groups did not show a significant effect. Replacement of missing teeth could be achieved with numerous artificial materials. Conventional full-coverage tooth-supported  or impla2O3 [Dental ceramics have evolved significantly during the several decades one of which is zirconia, a high-strength dental ceramic . Zirconi2O3 . 3 mol% 2O3 ,9. Howev2O3 ,9. Never2O3 .2O3) is the most commonly used mechanical treatment [One of the methods of improving the translucency is to increase the yttria content to 5% or more. However, the resultant microstructure consists more of cubic phase which has lower mechanical properties ,11. Therreatment ,15. Amonreatment . Anotherreatment ,13.0.5 compared with lithium disilicate which presented lower values for the same properties 348 ± 29 MPa, 60.6 ± 1.6 MPa and 2 MPa m0.5, respectively [Lithium-disilicate glass-ceramic is another high strength material with impressive esthetic quality . Howeverectively . This ceectively .This study was designed to evaluate the load at fracture and failure types of 3 units all ceramic FPDs with two different retainer designs, namely full-coverage versus inlay retained FPDs. The ceramic types included either monolithic zirconia that received different types of surface conditioning or a zirconia reinforced lithium silicate glass-ceramic when adopted with a protocol concerning preparation. 3-Unit full-coverage monolithic zirconia FPDs were considered as the control group. The null hypothesis were that neither retainer design, full coverage versus inlay, nor type of material, monolithic zirconia or zirconia reinforced lithium silicate, would affect the fracture load of 3-unit FPDs.The materials used in this study along with their batch numbers are summarized in The experimental models simulating two maxillary abutments (first premolar and first molar) for the replacement of maxillary second premolar (having a span of 6 mm) were used to fabricate full-coverage FPDs or IRFPDs. Sixty-four intact non-caries human maxillary premolars and molars of similar size which were extracted for periodontal or orthodontic reasons were selected and cleaned by scaling and stored in 0.5% chloramine solution . All teeIn order to homogenize the groups, teeth were selected for the study if the variation in length and width was within 1 mm of the mean values  = 5 mm, buccolingual dimension of premolars at the cementoenamel junction (CEJ) = 8 mm, mesiodistal dimension of molars at the CEJ= 8 mm and buccolingual dimension of molars at the CEJ = 9 mm.n = 8 per group):Control group (full-coverage monolithic zirconia FPD) (Zr-FPD).IRFPD made of monolithic zirconia (Zr-IRFPD).IRFPD made of monolithic zircona internally coated by a feldspathic porcelain layer (ZrC-IRFPD).IRFPD made of zirconia reinforced lithium silicate, Vita Suprinity (VS-IRFPD).Pairs of premolars and molars were randomly assigned into one of the following groups  in order to simulate periodontal ligament membrane. In order to make it comparable to the biological width, the impression material was removed 2 mm short of the CEJ, using a scalpel. Each pair of premolar and molar was embedded in a metal box filled with an auto-polymerizing acrylic resin (Acropars. Marlic Co.) at a distance of 6 mm to simulate a missing second premolar. The abutments were aligned both vertically and horizontally and the occlusal tables of the abutments were set parallel to the horizontal plane.For IRFPD groups, the previously described inlay preparation design  was follAll preparations were made with a high-speed hand piece utilizing water spray coolant by one operator. A new diamond was used after every four preparations.All preparations were scanned  and a virtual spacer layer of 40 µm were chosen. Eight full-coverage zirconia bridges and 16 inlay-retained zirconia bridges were manufactured by a CAD/CAM system  using partially sintered monolithic zirconia . After the milling procedures, the enlarged restorations were removed from the CAD machine and final sintering was performed in a special furnace at 1450 °C for two hours. For the ZrC-IRFPD group of the study, the same procedure was followed but 30 µm additional space layer was chosen in CAD system for internal coating of zirconia IRFPDs by a silica-based ceramic  with a matching coefficient of thermal expansion. A layer of a separating medium  was applied on the die before the porcelain mixture was added into the intaglio surfaces of the retainers of the fabricated zirconia IRFPDs. The separating medium facilitated removal of zirconia IRFPDs without any distortion of the applied porcelain prior to firing. ZrC-IRFPD was then seated on the abutments with gentle finger pressure. Extra amount of porcelain extruded from the cavity margins were removed by a brush. The porcelain was then fired at 810 °C for 1 min under vacuum.The VS-IRFPDs with the required dimensions were cut out from the respective blocks (Vita Superinity PC) using a milling machine  which was followed by an additional thermal cycle in the furnace  according to the manufacturer`s instruction.The prepared prostheses were finally cleaned with steam and then seated on the abutment teeth. While constant finger pressure was applied onto the FPDs, they were evaluated on the abutment teeth by visual inspection under a microscope  at a magnification of 8× for marginal discrepancy. The inspection was performed on the buccal, lingual and mesial aspects of premolar abutments and the buccal, lingual and distal aspects of the molar abutments for the Zr-FPDs. For IRFPDs, the gaps were measured at the facial and lingual interproximal margins and along the occlusal margins. The restorations were rejected if the marginal discrepancy was greater than 60 µm according to the literature . New FPDA dual-polymerized resin cement (Panavia F 2.0) was used for cementation of all groups according to the manufacturer`s instruction.2O3) for 20 s at 2 bar pressure from a distance of 10 mm. In order to ensure that the air-abraded surfaces were free from loose alumina particles, the FPDs were cleaned with steam. The internal surfaces of ZrC-IRFPD and VS-IRFPD were etched with 9.5% hydrofluoric acid gel  for 1 min and 20 s, respectively. They were then washed thoroughly and dried.For groups Zr-FPD and Zr-IRFPD, the surfaces were air-abraded  and gently air-dried.All abutment teeth were etched with 35% phosphoric acid  for 15 s, rinsed and blot-dried prior to cementation procedure. Afterwards, equal amounts of ED PRIMER A and B were mixed and applied to the abutment surfaces and dispersed with an air syringe. Then, equal amount of paste A and B of Panavia F 2.0 cement were dispensed and mixed and applied on the inner surface of all retainers. The FPDs were placed on the abutments and held in place with finger pressure for 40 s. Any excess cement was removed with a micro-brush and glycerin gel was applied in the marginal areas . The marAll specimens in each group were then subjected to thermo-cycling (5000 × 5–55 °C) with 30 s dwell time before the testing procedures. The FPDs were then centrally loaded in a universal testing machine  at a cross-head speed of 0.75 mm/min with a stainless steel ball (diameter: 5 mm) placed on the center of the pontic. To achieve an even force distribution, a 0.5 mm tin foil was placed between the pontic and the loading ball. The fracture loads were determined when a sudden decrease in the applied load occurred. Values of fracture loads (N) were recorded and modes of failure were determined with a stereomicroscope . The nature of the fracture patterns was classified as following: Cohesive fracture in ceramic, pontic or connector area and adhesive failure at the ceramic-resin interface.p Values less than .05 were considered significant in all tests.Normal distribution of data was tested using one sample Kolmogorov-Smirnov test . The data were then analyzed using one-way analysis of variance (ANOVA) and multiple comparisons were performed with the post-hoc test (Tukey-HSD). p ˂ .001). The values for VS-IRFPD (391 ± 136 N) were significantly lower than those of all the other groups  (p ≤ .025). The mean failure load for Zr-FPD group (672 ± 183 N) was not statistically different from those of Zr-IRFPD (672 ± 123 N) and ZrC-IRFPD (638 ± 59 N) (p ≥ .998).Mean and standard deviations of the fracture load values are presented in All groups failed cohesively and no adhesive failures were observed. The failure sites are illustrated on In this study, the fracture load of 3-unit inlay-retained fixed partial dentures made from either monolithic zirconia or zirconia reinforced lithium silicate were tested and compared with that of 3-unit full-coverage fixed partial dentures made from monolithic zirconia. The inlay-retained monolithic zirconia FPDs received two types of surface treatments. All FPDs were designed to restore maxillary second premolar. Inlay-retained monolithic zirconia FPDs showed fracture load which were not statistically different from 3-unit full-coverage monolithic zirconia FPDs, regardless of type of surface treatment. Inlay-retained FPDs made from zirconia reinforced lithium silicate failed at a significantly lower load than the other types of FPDs. Thus, the first null hypothesis about the non-significant effect of retainer design on fracture load was accepted. However, the second null hypothesis that ceramic type is inconsequential on fracture load was rejected.In agreement with these results, several previous studies showed that zirconia inlay-retained FPDs exhibited higher resistance to fracture when compared to lithium disilicate inlay-retained FPDs ,24. The The average load bearing capacity of 3-unit full-coverage and inlay-retained FPDs recorded in this study exhibited mean values ranging between 391 and 672 N. Numerous authors have investigated the maximum bite forces during mastication in different region  and haveIn late 1990s, Kelly recommended four factors for in vitro examination of load to failure test of all ceramic restorations to simulate clinical situation: (1) die material with elasticity similar to dentin (2) avoiding point contact but having contact dimensions of 0.5 to 3 mm, (3) cyclic loading, and (4) testing in wet conditions. At the current study, 2 out of 4 the requirements were fulfilled. Another point fulfilled in the current study is the minimum span length required in uniaxial flexural strength test. Minimum length of 20 mm has been allowed in ASTM C 1161, ISO 17565 and ENV 843-1 for ceramics . In the The inlay-retained FPDs were subjected to a thermo-cycling process at the present study. It has been demonstrated that aging accelerate degradation of adhesive interface . HoweverFailure modes at full-coverage and inlay retained FPDs were fractures in ceramic either in connector or pontic, being predominantly at connector area. No de-bonding was observed in any of the specimens. It might indicate a favorable surface conditioning of monolithic zirconia through feldspathic layering and/or air-abrasion. In addition, use of an MDP-containing resin cement might have resulted in a stable adhesive interface during 5000 thermocycling and static loading. Furthermore, two methods of surface conditioning of monolithic zirconia inlay-retained FPDs were comparable regarding load bearing capacity. The findings could indicate the high importance of bonding for the success of inlay-retained ceramic FPDs considering the reduced surface area for bonding.Several studies have demonstrated that the connector areas are highly influential in failure and failure rate is relatively high in 3-unit all-ceramic FPDs associated with the connector area ,24,29. B2 to 16 mm2 [The ideal preparation design for ceramic inlay-retained FPDs described by Thompson et al.  was follo 16 mm2 ,28. Occlo 16 mm2 ,28. Thomo 16 mm2 . Assessmo 16 mm2 . Howevero 16 mm2 . High coA systematic review on all-ceramic inlay-retained FPDs for replacing posterior missing teeth was conducted in 2018 . The inlIn the present study, fracture load of two types of ceramics in 3-unit FPDs were tested to determine the more suitable material for replacement of single posterior missing tooth. Among the materials tested, monolithic zirconia-based inlay-retained FPDs showed promising results, yielding fracture loads comparable to the fracture loads of full-coverage monolithic zirconia FPDs. The similarity in the fracture load of ceramic FPDs could be attributed to the material properties and connector dimensions ,40. In a2 was provided. In contrast, inlay-retained zirconia reinforced lithium silicate fixed partial dentures showed significantly lower load bearing capacity. All failures were cohesive in nature in ceramic materials and the connectors were the weakest parts of the reconstructions.Based on the findings of this study, for replacement of second premolar tooth, there was no significant difference in fracture load of inlay-retained and full-coverage fixed partial dentures made of monolithic zirconia under static loading when a connector size of 12 mm


Canine Genetics and Epidemiology as Canine Medicine and Genetics. This is to reflect the expansion in journal scope from an emphasis on genetic and epidemiological research into all aspects of canine health and medicine.We are very pleased to re-launch Canine Medicine and Genetics will continue to be uniquely focussed on research relating to canine health. Thus, as well as considering genetic and epidemiological studies, we will now also welcome medical research including the areas of internal medicine, emergency medicine and critical care, cardiology, oncology, neurology, dermatology, ophthalmology, clinical and anatomic pathology, and diagnostic imaging, in addition to clinical genetics.As the only journal to date that exclusively publishes canine science, This expansion in scope was driven by the desire to publish a wider range of research relating to canine biology, in order to better serve the journal’s mission of disseminating information for the improvement of dog welfare. We are honoured to welcome our new co-Editor in Chief, Prof. Frédéric Gaschen, a small animal internist and expert in small animal gastroenterology, to help drive growth into new canine medicine topics. The founding co-Editor in Chief, Prof. Bill Ollier, and Managing Editor, Dr. Lorna Kennedy, continue to provide invaluable expertise and support in maintaining our strength in genetic and epidemiology papers.editorial from the journal launch, it was important to us that this open access journal disseminates findings not only between scientists, but also veterinarians, dog breeders and owners through lay summaries for non-scientific readers.Reflecting on the story of the journal so far, the inception of the journal was inspired by the increasing public awareness over the health of dog breeds, with growing concerns over breeds being particularly predisposed to certain diseases. It was founded with the backing of the UK Kennel Club, the UK’s largest organisation dedicated to promoting the welfare of all dogs. The UK Kennel Club greatly contributed to the establishment of the journal but will not be a part of the new direction of the journal. The overall aim was to create a forum to advance our understanding of dog breeds and to make these scientific findings as accessible as possible. As outlined in the Labrador retrievers under primary veterinary care in the UK: demography, mortality and disorders” receiving coverage in over 555 news articles around the globe. We featured in the top 100 most-discussed journal articles of 2016, for our paper “A genetic assessment of the English bulldog”. Our most read blog, which gives up-to-date knowledge on the vulnerability of the French Bulldog to a number of health conditions, has amassed over 10,000 views. The blog educates the public on the negative consequences that can occur to the health of a breed when there are dramatic rises in demand and popularity. Thus we are clearly embracing public engagement with science, and for the academic community, we are evidently publishing findings of high quality and interest to an international audience. According to 2019 Google Analytic data, the journal had website visits from 156 different countries with USA, UK, Australia, Canada, India and New Zealand appearing in the top 10 countries.We feel that we have achieved this goal and that the journal has now been firmly established as a valuable scientific resource. Our content has been accessed over half a million times since the launch of the journal in 2014, and we have accrued over 500 citations to date. This is noteworthy considering that this is a specialist journal with fewer than 100 publications thus far. The journal has had significant attention in the press with our paper “3rd and 4th International Dog Health Workshops and the Companion Animal Genetic Health conference.To aid academic collaboration for canine related research, we provide a platform for the publication of meeting reports from relevant conferences, including the Moving forward, we intend to unite the various sub-disciplines related to canine health and continue our endeavour to provide an efficient open access platform for scientists and clinicians to share their research results and their expertise with their peers and with the public at large.Canine Medicine and Genetics for your future submissions on canine science.We are excited by the new phase of the journal and we would like to encourage you to consider


Vascular composite allotransplantation (VCA) is a field under research and has emerged as an alternative option for the repair of severe disfiguring defects that result from infections or traumatic amputation in a selected group of patients. VCA is performed in centers with appropriate expertise, experience and adequate resources to effectively manage the complexity and complications of this treatment. Lifelong immunosuppressive therapy, immunosuppression associated complications, and the effects of the host immune response in the graft are major concerns in VCA. VCA is considered a quality of life transplant and the risk-benefit ratio is dissimilar to life saving transplants. Belatacept seems a promising drug that prolongs patient and graft survival in kidney transplantation and it could also be an alternative approach to VCA immunosuppression. In this review, we are summarizing current literature about the role of costimulation blockade, with a focus on belatacept in VCA. Vascular composite allotransplantation (VCA) is a field under research and has emerged as an alternative option for the repair of severe disfiguring defects that result from infections or traumatic amputation in a selected group of patients . VCA conThe first attempt for a human hand transplantation was done in 1964 in Ecuador, but acute rejection (AR) led to graft amputation at 3 weeks postoperatively, despite the use of combined immunosuppression therapy with azathioprine and prednisone  Table 1Table 1. In 2005, the first face transplantation was reported by the Amiens transplantation team in a woman suffering from traumatic amputation of the lower face, including distal nose, lips, chin, and lateral face parts . Since tAdvances in the field of VCA led to the first time uterine transplantation (UTx). It was first attempted by Fageeh et al. in Saudi Arabia in 2000 on a 26 year-old female in the context of previously performed hysterectomy due to post-partum hemorrhage. The uterus graft underwent acute thrombosis 3 months post-transplantation and eventually hysterectomy was performed . The firThe main limitation of currently used immunosuppression regimens are side effects. Specifically, tacrolimus, which is a calcineurin inhibitor (CNI) is a core component of maintenance regimens in VCA worldwide and has been reported as one of the main causes of complications in VCA. Barth et al. reviewed the VCA experience and identified 4 cases (3 limb and 1 face) with renal failure or progressive renal dysfunction up to 8 years post-transplantation. One patient has been transplanted and 3 are listed for renal transplantation . AlternaThe increasing numbers of VCA along with advances in immunomodulation schemes mandate the need of a universally accepted histological classification. The Banff 2007 classification was a milestone in the characterization and appropriate reporting of VCA rejection. The skin as a visible component of the transplant provides an easily and accessible monitored graft area facilitating AR recognition. According to Banff 2007 the skin rejection severity is classified in five grades ranging between 0 and IV . AR manide novo tumorigenesis, associated with immunosuppression per se or viral reactivation (EBV related lymphomas), or tumor recurrence  have been tested as promising agents in VCA immunosuppressive regimens.Alemtuzumab, a humanized mAb targeting CD52 (GPI-linked surface protein of mature lymphocytes) that causes B and T lymphocyte depletion, has been used in the prevention of SOT AR as well as in the treatment of VCA AR episodes , 79, 80.The development of belatacept along with its prototype molecule CTLA4-Ig introduced a new class of immunosuppressive agents called costimulation blockade agents . T-cellsp < 0.01) and all CTLA4-Ig treated histologic specimens remained unaffected at 7 days post-transplantation  after belatacept conversion in the context of tacrolimus and rapamycin adverse effects . InitialRecently, belatacept was tested as maintenance treatment in 4 male hand-transplanted patients with beneficial results and limitations as well. In two DSA-negative bilateral hand recipients, belatacept treatment was not associated with rejection in spite of decreases in tacrolimus dosage (patient 1) or cessation of everolimus administration (patient 2) . Patientde-novo belatacept in VCA  . The CTLA4-Ig regimen resulted in a prolonged survival in animals  while the tacrolimus alone group resulted in rejection 2 days after tacrolimus cessation . Our sturegimen) . Based o healing  followed healing .A main clinical concern with belatacept treatment is the risk of post-transplantation lymphoproliferative disorder (PTLD) in EBV seronegative transplant recipients , 116. InCostimulation blockade alone is ineffective in inducing long-term graft allograft survival or tolerance in animal transplant models , 118. Thde novo belatacept to avoid chronic CNI exposure.VCA is a field under development and is performed in centers with appropriate expertise, experience and adequate resources to effectively manage the complexity and complications of this treatment option. Similar to other solid organ transplants, lifelong immunosuppressive therapy, their complications, and the effects of the alloimmune response in the graft are major concerns in VCA. VCA is a quality of life transplant and the risk-benefit ratio is dissimilar to life saving transplants. Belatacept seems a promising drug that prolongs patient and graft survival in solid organ transplantation and it could also be an alternative approach to VCA immunosuppression. A regimen that deserves ongoing consideration is DM and LC: conception or design of the work. DG and DM: data collection. DG, DM, and LC: data analysis and interpretation, drafting the article, critical revision of the article, and final approval of the version to be published. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.