Regardless of the kind of biomaterial used for the graft, it is clear that, over time, the graft undergoes dimensional changes that could influence the final bone volume obtained, which could alter the stability of the installed implants. The aim of the present study was to compared and correlated the graft behavior with the amount (in grams) of xenogeneic and alloplastic biomaterials used in grafts for maxillary sinus lift. This retrospective cohort study used 148 CBCT images of 74 grafts from 68 maxillary sinuses lift patients in a university, post-graduate clinic. The weights of biomaterials, categorized in intervals according to amount used, were correlated with the graft volumes at V1 (10 days) and V2 (180 days). Kruskal-Wallis test was used to evaluate the possible bias effect of weight on graft maintenance. p>0.05). Were found for the influence of these mean amounts in graft maintenance: Bio-Oss Small® (18); Bio-Oss Large® (10); Endobon® (17); BoneCeramic®+Emdogaim® (10); Cerasorb® (11); and Osteogen® (08) at V1 and V2. However, when biomaterials were categorized by intervals, all Cerasorb® interval groups showed statistically significant differences (p<0.001) in graft volume at V2. Mean weights of biomaterials used were: Bio-Oss Small® (1.58g); Bio-Oss Large® (1.35g); Endobon® (0.72g); BoneCeramic®+Emdogaim® (0.96g); Cerasorb® (1.13g) and Osteogen® (2.70g). No significant differences (The amounts of the biomaterials used could influence the final volume; depending on the biomaterial characteristics. Implant installation was possible with all studied grafts, although graft volume shrinkage should be considered when selecting biomaterial for sinus lift. Key words:Biocompatible materials; cone beam computed tomography; maxillary sinus; hydroxyapatites. Biomaterials with osteoconductive capacity were evaluated in maxillary sinus lift, obtaining results equivalent to those of autogenous grafts . The xenRegardless of the kind of biomaterial used for the graft, it is clear that, over time, the graft undergoes dimensional changes that could influence the final bone volume obtained, which could alter the stability of the installed implants -6.The use of computed tomography (CT) to analyze the bone volume achieved after maxillary sinus is reported in the literature as an accurate method, which produces reliable images with little distortion -14.In this context, there are studies ,6,11-22 There being several alternatives regarding the choice of graft biomaterials for maxillary sinus lift, the present study aims to evaluate the possible bias effect of the amount of biomaterials on graft volume maintenance, comparing and correlating graft volumes with the amount (in grams) of different xenogeneic and alloplastic biomaterials, used for maxillary sinus lift grafts.The present study was approved by the PUCMINAS - Catholic University, Post-graduate Research Implant Clinic, Belo Horizonte, Brazil, - ethics committee, number CAAE 02663212.9.0000.5137.This retrospective cohort observational study analyzed 80 maxillary sinuses lift patients records from PUCMINAS between 2014 and 2018. After inclusion and exclusion patients’ criteria, the present study used 68 patients records, whose was planning maxillary implant overdenture prosthesis.-Patient selectionThe inclusion criteria patients’ data selection was determined by the complete maxillary tomographic exams obtained between the 5th and 7th day and 180-day post-surgery. It’s important to stress that the tomographic exam in the first period of the study at 7 to 10 days was done as part of the University Surgical Clinical protocol -13, to cAs exclusion criteria patients had autoimmune diseases, diabetes mellitus, alcoholism, smoker, stress (required sedation), active periodontal diseases, maxillary sinus diseases or surgeries, or that had some intercurrences like membrane perforations, biomaterials sinus incursions, sinus infections, during or after the sinus lift graft procedure, were removed from the data study.-Surgical procedureAll surgeries were performed by clinical specialists according PUCMINAS Implant Dentistry Department protocol ,12. AfteThe sinus cavity obtained was filled with a selected biomaterial according to the previously choice of surgeons and patients. The biomaterials used were: anorganic bovine bone ; anorganic bovine ; biphasic phosphate Bone Ceramic® + protein enamel matrix Emdogain® , tricalcium phosphate ; and hydroxyapatite .The amount of biomaterial used during the sinus lift surgery is determined according to the number of implants and their spatial distribution based on the prosthetic planning for overdentures using a total of four implants. All the biomaterials were carefully applied dry in the surgical sinus areas, according to the necessary amount. Regarding the intended degree of the height sinus filling, all the grafts must permit the installation of 9mm to 11mm long implants, after a healing period of 180 days. A sample of each biomaterial was weighed using a precision balance to check that it complies with the manufacturer’s information, to a precision of 0.01g. The bone window was covered with a collagen membrane  and the flap was repositioned without tension and sutured appropriately by first intention using 0.5 Nylon  suture. Medications were prescribed, including: 0.12% chlorhexidine gel twice daily for 15 days in the operated area, 500mg of Amoxicillin every 8 hours for 10 days, Paracetamol 750mg every 6 hours for three days and Benzalkonium Chloride (0.1mg + 9.0 mg Sodium Chloride - Sorine®) nasal solution four drops in each nostril six times per day for 10 days. The sutures were removed 10 days after the surgery and the patients were monitored throughout the entire period.The biomaterials used to graft the 74 sinuses analyzed were: Bio-Oss® Small=18, Bio-Oss® Large=10, Osteogen®=08, Bone Ceramic® + Emdogain®=10, Cerasorb®=11, Endobon®=17.-CBCT evaluationDuring the study period, at V1 (7 days) and V2 (180 days), a total of 148 CBCT images, were obtained. The tomography images were analyzed by a single, expert, trained and blinded operator using Osirix® MD Imaging 6.5 software , with a Kappa concordance index of 0.79. The variables maxillary sinus size , with the mesiodistal, buccal-palatal distances and height, were measured, and correlated with the volumetric changes of the grafts during the two healing periods. The images were delimited manually by the evaluator, and the program semi-automatically defined all sagittal, axial and coronal reconstructions of the images as described for Favato ,  images of 74 grafts from 68 posterior maxillary edentulous patients, 23 males and 45 females, mean age of 56 years, referred for sinus lift.The amounts of biomaterials used were categorized in the following intervals, show in p=0.415.The mean amounts of biomaterials used were: 1.58g of Bio-Oss® Small, 1.35g of Bio-Oss® Large, 0.72g of Endobon®, 0.96g of BoneCeramic® + Emdogaim®, 1.13g of Cerasorb® and 2.70g of Osteogen®. The means, medians and standard deviations of the graft volumes obtained by all biomaterials in the two periods are described in The purpose of regression analysis is determining the dependence of a variable on the independent or predictive variable, assuming a linear model. In this way, the degree of linear relationship between the variables is strong or weak, depends on how the group of the points are located around an imaginary line that passes through the set of points.Figure Figure Comparisons of the initial volume (V1) and final volume (V2) according to the biomaterials groups were conducted.p=0.00003) can be found only in comparisons of four groups: Cerasorb® G8 x Bio-Oss® Small G2, Cerasorb® G8 x Endobon® G5, Cerasorb® G8 x Endobon® G6 and Cerasorb® G8 x Bio-Oss® Large G3  was found, showing a strong correlation between the variables V1 versus V2. Thus, all biomaterials showed significant contraction between V1 and V2.The present retrospective cross-sectional cohort observational study has established a correlation of the amount in weight of the xenogeneic hydroxyapatite and alloplastic hydroxyapatite biomaterials with the initial and final volumes of the grafts obtained using these biomaterials in sinus lift. It has also determined the final volume changes of grafts obtained, using CBCT images. The relationships and variables assessed in the present study have still not been identified in the literature.et al. , Bio-Oss® Large (1.35g), Endobon® (0.72g), BoneCeramic® + Emdogaim® (0.96), Cerasorb® (1.13g) and Osteogen® (2.70g) did not present statistically significant differences. These biomaterials mean amounts were used to establish the correlation between initial and final graft volumes obtained. Therefore, the influence of this variable could be evaluated, and this proposition has not been reported in the literature.et al. , present an average 18% to 23% volumetric reduction. Consistent with the literature, ,11,25. tet al. (Individually analyzed, the mean contractions found for the grafts/biomaterials were: Bio-Oss® Small= 14.98%, Bio-Oss Large= 11.93%, Endobon®= 8.04%, Bone-Ceramic®= 21.83%, Cerasorb®= 43.36% and Osteogen®= 20.43%. A high-volume reduction was observed for Cerasorb® graft, similar to the autogenous graft contraction of up to 40% reduction of the final graft volume. This graft behavior data is consistent with that reported by Browaeys To determine the influence of the amount of biomaterial on the volumetric changes of the grafts, the present study categorized groups by gram intervals and established multiple comparisons. It was found, at final volume V2, that all Cerasorb® groups presented a significant difference in the contractions. These data are very important for the biomaterial choice in sinus lift graft planning.et al. (et al. (et al. (Concerning the amounts of biomaterials used and the final contractions found with xenogeneic hydroxyapatites in maxillary sinus lift, the similarity of the results to Chackartchi et al. , Cosso e (et al. , Testori (et al.  can be i (et al.  the pres (et al. . Endobon (et al. ,28 was tAs reported in the literature ,10,25-30Further controlled and longitudinal investigations are needed to verify the impact of the biomaterial amount on the graft volumetric changes using different biomaterials over longer control periods. In this context, studies like the present one take on increasing importance with regard the predictability of osseointegrated implants installed in grafted sinus lift areas.Tricalcium phosphate Cerasorb® presented statistically significant contraction were compared to the other biomaterial groups.The amount of biomaterial used could influence the final graft volume depending on the biomaterial characteristics.


The carbon-fiber-reinforced polymer (CFRP) is a mainstream material for lightweight products from the end of the 20th century to the present day. Its compression molding process has obvious advantages in mass production. This paper attempts to establish the constitutive models of compression molding of the CFRP materials and study their mechanism. Based on anisotropic linear elastic mechanics, viscoelastic mechanics, and thermodynamics, as well as the Maxwell viscoelastic constitutive model, we first establish the constitutive model of thermorheologically simple CFRP materials (TSMs). Then, considering the influence of temperature on the initial stiffness and equilibrium stiffness, the concept of temperature stiffness coefficient is introduced, and the Cartier coordinate system is converted into a cylindrical coordinate system, thereby establishing the constitutive model of thermorheologically complex materials (TCMs) using the tensor form. Finally, by comparing to the structure of the Zocher model, the two constitutive models established in this study are verified. The research findings have important theoretical research significance for studying the compression molding mechanism of carbon fiber and further improving the quality of product molding. Carbon fiber is one of the most relevant materials from the end of the 20th century to the present day. Due to its characteristics such as high specific strength, high specific modulus, fatigue resistance, good molding process, good breakage safety, and strong performance designability, the carbon-fiber-reinforced polymer (CFRP) has been increasingly applied in aerospace, wind turbine blades, sports equipment, and automotive parts, etc. It is the mainstream material for lightweight products in the 21st century , gradualCommonly used molding methods of the CFRP materials include compression molding, autoclave, winding, and the squeezing method, etc. Among them, the compression molding method has the advantages of low cost, high efficiency, low internal stress, small warpage, good mechanical stability, and high repeatability, and it has a strong competitive advantage in the batch production of product parts, especially in large batch production ,5. HowevScholars worldwide have conducted a lot of research based on the constitutive model of the CFRP materials and achieved rich results. The linear elastic constitutive equation combined with laminate theory is a commonly used method to predict the residual stress of composite materials. Stango et al.  assumed Johnston  stated tr et al.  used ther et al. ,13 assumr et al.  used a dr et al.  combinedr et al.  proposedBased on the previous research, this paper establishes the CFRP anisotropic viscoelastic constitutive models of thermorheologically simple CFRP materials (TSMs) and thermorheologically complex materials (TCMs) by combining with anisotropic linear elastic mechanics, viscoelastic theory, and thermodynamics, and the Maxwell viscoelastic model. Considering the effect of material temperature on the stiffness and equilibrium stiffness of the carbon fiber molding process, the temperature-dependent thermoelastic stiffness coefficient was introduced, and the Cartesian coordinate system was converted into a cylindrical coordinate system, thereby establishing the CFRP anisotropic elastic constitutive equation and increment equations of the TCM materials. The establishment of the CFRP anisotropic viscoelastic constitutive models, especially for TCM materials, will lay a theoretical foundation for revealing the mechanism of carbon-fiber-reinforced polymers , and theGenerally, if the performance of each point in the object is the same, it is a homogeneous material, otherwise, a heterogeneous material. In addition, materials that show the same properties in every direction at every point in the object are called isotropic materials, while those with different properties at every point in the object are anisotropic materials. For a CFRP material, the matrix is an isotropic material, and the reinforcing fiber is a transversely isotropic body, so the CFRP material is an anisotropic material. In the molding process of CFRP materials, the matrix material has undergone a transition process from the viscous fluid state to rubber state to glass state, etc. It is necessary to combine the elastic mechanics of the composite material and the viscoelastic mechanics in the research.To study the mechanical properties of composite materials and solve the stress and strain of a continuous elastomer under external load, we need to balance the equations, geometric equations, constitutive equations, etc.In the space coordinate system x, y, and z, then the geometric equation of any point in the elastic body along the Similarly, in the space coordinate system According to Equations (1) and (2), in the space coordinate system Among them, The matrix of the stiffness coefficient is symmetric, and only 21 stiffness coefficients are independent. Equation (3) establishes the relationship between stress and strain, which is called the generalized Hooke’s law or elastic constitutive equation.For the matrix of a CFRP material, the number of independent stiffness coefficients is 2, and its stress–strain relationship isThe reinforced fiber has 5 independent stiffness coefficients. Assuming that the The mechanical properties of viscoelastic materials such as shear modulus, loss modulus, and loss factor, etc. are usually related to ambient temperature, vibration frequency, strain amplitude, etc. Therefore, the constitutive relationship of viscoelastic materials is very complicated.When describing the viscoelastic behavior of materials, the generalized Maxwell model is generally used, which consists of multiple Maxwell elements in parallel with a spring, as shown in Each Maxwell element contains a spring with elastic modulus E and a viscous strain damper with strain viscosity aterials ,19,20,21The effective strain tensor can be expressed by the total strain tensor If the material exhibits simple thermo-rheological properties when cured, the above equation can be changed to:Among them, The stiffness matrix During the molding process of the composite material, the matrix undergoes a curing reaction to release heat, which is regarded as an internal heat source and controls the entire heat transfer process together with the applied temperature.According to the Fourier heat conduction equation, the heat transfer process can be expressed as:In Equation (13), The boundary conditions of the heat conduction model are:The viscoelastic constitutive modulus can reflect the creep and relaxation characteristics of the material. In this paper, the generalized Maxwell viscoelastic constitutive equation was used to reflect the viscoelastic characteristics of the simple thermal-rheological composite material.For a single Maxwell element, the strain of the element is a combination of spring and viscous element strain, which is expressed asUsing the relaxation modulus to express the relaxation characteristics of the material,For the generalized Maxwell model, the stress relaxation modulus is given asIt can be seen from The stress of the m-th branch in the generalized Maxwell model at time t is expressed asIts difference form isAmong them, Combining the stresses of all branches of the Maxwell model and the balance spring, we obtain the total stress at time t:Based on the above, the stress increment at time t can be expressed asr.The conversion equation can be expressed asAmong them, When the CFRP materials are TSMs, the material’s equilibrium stiffness and initial stiffness are unrelated with the degree of curing. According to Equations (11), (19), and (27), the relaxation stiffness of the CFRPs can be expressed as:For anisotropic materials, the internal stress is expressed as: ,22,23Δσi=To simulate the high-temperature viscoelastic behavior of composite materials, it is generally assumed that the composite material is a simple thermal-rheological material, that is, the equilibrium stiffness and initial stiffness of the material are related to temperature and curing degree. Many experiments have shown that the initial stiffness and equilibrium stiffness are related to the material temperature . In thisIn the curing process of composite materials, the molding temperature and the curing degree of the matrix change with time, and the elastic modulus of the material is related to the temperature and the curing degree. On the one hand, it affects the conversion factor and relaxation time, similar to the performance of the viscous elements; on the other hand, it also affects When constructing the viscoelastic model of the material in this paper, the vertical movement coefficient representing the correlation between d by Kim ,13, the For a better description in the study of the CFRP mechanical properties, it is necessary to establish a local coordinate system for the geometric elements or convert the global coordinate system. In this paper, the Cartesian coordinate axis was converted into a cylindrical coordinate system for reducing the amount of calculation.In the Cartesian coordinate system , the stress value at time t is:According to Equations (44) and (45), the stress The relaxation modulus Referring to (46), the stress In the formula, the reduced time Thus, The stress increment equation Among them, At very small time increments, the temperature T and the curing degree are extremely small and negligible, and the conversion factor is constant, so it can be expressed as:In addition,Then,Use the same method to deal with Finally, the stress increment In addition,To verify the constitutive models of both TSM and TCM CFRP materials, this paper selects the molded flat structure of a CFRP material based on Kim’s research ,13. FourIt can be seen from On the basis of the constitutive model of CFRP molding and viscoelastic theory, viscoelastic mechanics, and thermodynamics, the CFRP viscoelastic constitutive equation and incremental equation of the TSMs were established, which lays a foundation for explaining the solidification and relaxation behavior of the molding process.Considering the effects of temperature on the initial stiffness and equilibrium stiffness, the thermoelastic stiffness coefficient was introduced, and the Cartesian coordinate system was converted to the cylindrical coordinate system. Thus, the viscoelastic constitutive equation and incremental equation of the TCMs were established, and the accuracy and application range of the constitutive model were improved while the calculation scale was reduced.On the basis of the viscoelastic constitutive model proposed by Zocher, the correctness and validity of the constitutive models for TSMs and TCMs were verified by comparing the change in curing degree under the anisotropic viscoelastic constitutive model.This paper studies the constitutive models of the CFRP during the compression molding process. The main content and conclusions are as follows:


This article presents a multi-criteria analysis of the errors that may occur while measuring the geometric deviations of crankshafts that require multi-point support. The analysis included in the paper confirmed that the currently used conventional support method—in which the journals of large crankshafts rest on a set of fixed rigid vee-blocks—significantly limits the detectability of their geometric deviations, especially those of the main journal axes’ positions. Insights for performing practical measurements, which will improve measurement procedures and increase measurement accuracy, are provided. The results are presented both graphically and as discrete amplitude spectra to make a visual, qualitative comparison, which is complemented by a quantitative assessment based on correlation analysis. Methodological errors occur when a model fails to include the factors of the measurement method and related phenomena. This quantity is the discrepancy between the model characteristics of the method and its real characteristics. The accuracy of measurements requires for the possible errors to be analyzed and eliminated as much as possible or treated as correction factors, especially for measurements where analysis will significantly affect the total method error. Examining and analyzing individual error components facilitates the determining of how they influence the measurement accuracy . Taking Specific procedures for measuring large machinery components have been discussed in several studies ,3,4. TheThe error analysis elaborated in ,10,11,12As shown in , the reaTherefore, to accurately assess the crankshaft geometry, measurements must be performed by controlling the reaction forces at the supports, which must be articulated and susceptible, i.e., adaptable to possible mutual displacements of the main journals due to geometric deviations of the item being measured.The aspect related to measurement inaccuracies caused by shaft deflection under its own weight was investigated in our previous study , in whicThe continuation of the study presented in  was 12]12]. In tThe latest study  describeIn this paper, we investigated the effect of elastic deformations on geometrical deviation and shape profile measurements of large crankshafts with uncontrolled supports. We considered the influence of the difference in the height of the supports and the influence of the journal eccentricity on the measurement results of the shaft geometry. The main motivation of this study was to indicate the limitation of the rigid vee-blocks measuring method. The results presented in this paper confirm that the currently used conventional support method—in which the main journals of a shaft are supported by a set of fixed, rigid vee-blocks—significantly limits the detectability of geometric deviations, especially those of the journal and pin axes’ positions. The results of this study also provide insights to be considered during measurements, thereby improving the measurement procedures and increasing measurement accuracy.The structure of the article is as follows: in As part of the methods and techniques currently used, measurements are performed with the shaft axis fixed in the horizontal or vertical plane. Large crankshafts are placed in the horizontal plane because of their large masses and dimensions. The main journals of those shafts are rested on a set of rigid vee-block supports a, but thFor a horizontal shaft axis with equally elevated vee-blocks supporting all the main journals and a perfectly manufactured crankshaft , the shaft does not undergo elastic deformations; however, the actual manufacture of machine parts always deviates from the ideal shape. Therefore, we must assume that a shaft will have geometric deviations, which qualify such shafts as usable for operation if they are within the engineering limits. Even when the geometrical deviations are within permissible limits, they cause elastic deformations of the shaft, which directly affects geometrical quantity measurements.The object subjected to the analysis was the crankshaft of the main propulsion medium-speed Buckau Wolf R8 DV-136 engine , measuring 3630 mm in length and weighing 9280 N, equipped with ten 149 mm main journals and eight 144 mm crankpins. The geometrical model with main journal numeration used in further analysis is shown in It was assumed that the material of which the shaft was made is AISI 1060-2 steel, characterized by Young modulus Case 1: individual main journals are perfectly coaxial, while one of the supports  is offset upwards by 0.03 mm relative to others;Case 2: the main journals of all crankshafts are perfectly coaxial, while one of the supports  is offset downwards by 0.03 mm relative to others;Case 3: the axis of one of the main journals (no. 5 counting from the timing gear end) is offset upwards by 0.03 mm from the others, while the supports are at set at the same height;Case 4: the axis of one of the main journals , is offset downwards by 0.03 mm from the others, while the supports are set at the same height.A study plan was developed in order to prove that the currently used methods of measuring crankshaft geometrical deviations, briefly described in A graphic representation of the cases under consideration is shown in To assess the deflections and reaction forces distribution at the contact between vee-block support heads with the main journals, a finite element model ,24 of thThe gravity load was applied to the model. An analysis consisting of determining shaft deflections and reaction forces acting on a supported shaft was performed using linear static Nastran solver (SOL101). Subsequent cases formulated in Experimental measurements were performed using a constructed system consisting of a MUK 25-600 measuring head and SAJD software, which enabled a complete qualitative evaluation of the roundness profiles a [2]. ThAn important advantage of this system is that measurements can be made directly in the work environment and the measured object does not need to be dismantled. Similar features have different design solutions of the measuring heads equipped with multi-contact self-adjusting vee blocks cooperating with one or more dial sensors. However, with this method it is only possible to evaluate the deviations and shape profiles, which, from the perspective of performing a comprehensive evaluation of the journal geometry, provides only a partial control of the measurement accuracy. Measuring axis deviation remains difficult.The roundness profiles were then superimposed on the displacement profile of the center of the journal moving eccentrically and the displacement profile subject to support limitations. To completely depict the issue, the measured roundness contour was repeatedly superimposed on the displacement profile limited by the support. It was moved angularly to the defined starting position. Detailed description of the experimental system was presented in ,11.To implement the study plan formulated in For this type of support and a shaft rotation angle of 90°, In this case, the shaft is cyclically bent upwards and support no. five carries very high loads, completely relieving supports no. three, four, six, and seven. As support no. five is offset, the shaft is lifted, and the aforementioned supports lose contact with the journals.Lowering the support relative to the others removes the support from under the shaft at its location. It was assumed that support no. five was offset downwards by 0.03 mm relative to the others. The results from exemplary finite element analysis for case two are shown in The shaft bends down under its own weight in locations without support. Deflections at non-supported journals are insignificant (−0.00228 mm to −0.00335 mm), while the reaction forces at journals adjacent to the non-supported ones increase significantly, reaching 1713 N to 1773 N in journal no. four and 1541 N to 1749.9 N in journal no. six.The situation is slightly different when the supports are at the same height and the main journal axes positions deviate . In case three, the supports are located at the same height, while the axis of journal no. five (for the shaft’s reference angular position) is offset eccentrically upwards by +0.03 mm relative to the other journals. The form of deformation of the shaft is analogous to that presented in the Changing the shaft rotation angle by 180° causes the eccentrically located journal axis to move to an extreme location opposite the reference angle used in the previous case. The considered relative positions of the journals correspond to case four and are an alternative version of case three . Using tA detailed analysis of cases three and four shows that for deviations in the position of main journal axes, the detectability of journals’ misalignment was limited by supporting the shaft with a set of fixed rigid vee-blocks located at the same height. The analysis of the graphs of journal axes’ deflections  shows thO1 was offset upwards relative to the others by 0.03 mm in the initial angular shaft position. In case three, when the shaft was rotated in the angular range from 0 to 90°, journal no. five was unsupported, and the axis of this journal moved eccentrically with respect to the main axis O of the shaft (relative to the shaft’s axis of rotation). The displacement sensor, whose probe stylus is located vertically, measures the deflection of the journal resulting from its eccentric movement during the shaft rotation , the reaction force will gradually increase at the point where journal no. five and the support get into contact. Simultaneously, journals adjacent to journal no. five  will be lifted upwards, losing contact with their supports due to bending of the shaft . Journal no. five is permanently in contact with its support, so the sensor will indicate a constant deflection of 0.03 mm, the same as for the angle of 90°. At shaft angles ranging from 180° to 270°, journal no. five will remain in contact with the support, which will exert gradually decreasing reaction forces on the journal. At the same time, the shaft becomes less bent at the locations of journals no. four, three, six, and seven. At 270° rotation, journal no. five loses contact with its support, and journals no. four, three, six, and seven rest on their supports. For shaft angles ranging from 180° to 270°, the displacement sensor still shows a constant deflection of –0.03 mm. For shaft rotation angles from 270° to 360°, journal no. five is gradually lifted upwards, and the displacement sensor indicates a change from –0.03 mm (at 270°) to 0.00 (at 360°). w of eccentricity x of the support  takes the angular position e sensor ,2.ϑ and the arrow of deformations Thus, considering the location of the section to be measured relative to the support points, the location of the center of the section being measured moves with respect to the axis of rotation (determined by the measuring system) by the value of the elastic (ormation . Consequr series ).In the considerations presented so far, it has been assumed that journals have an ideal circular profile, but machining processes involve unavoidable errors that give journals irregular roundness profiles. In general, when the shaft is fixed on vee-blocks, the shape and axial position deviations are measured in individual cross-sections of the main journals of the rotating crankshaft. In the case of a misaligned journal position, the center of the measured journal’s profile may move relative to the axis of rotation determined by the measuring system. In this case, the measurements describe the shape profile of the given cross-section, as well as the eccentricity that represents the profile center position of the section measured relative to the axis of rotation determined by the measuring system. Taking into account that a rigid support limits the detectability of geometric deviations in the main journal axes of a crankshaft (which has been demonstrated), an analysis was conducted to determine how the limited detectability of axis position deviations affects the evaluation of the main journal’s roundness profile. To accomplish this, the deviations and shape profiles of the main journals were measured for the tested crankshaft. e = 0.03 mm, assuming that the support does not limit the shaft displacement. The corresponding discrete amplitude spectrum is shown in The image of the eccentric profile of the measured roundness contour center at an eccentricity The image of the profile corresponding to the eccentric displacement of the center of the measured roundness profile, presented in the Cartesian system for the case when all the supports were situated at the same height (x = 0 mm), with the eccentricity of one of the main pins equal to When the measured round contour was superimposed on the displacement profile of the pin center without being limited by the support, the total profile and the discrete spectrum shown in When the measured round contour was superimposed on the displacement profile of the pin center at the support limit, the total profile was obtained, which is shown in The image of the profile obtained after superimposition and shifting by 60°, followed by 90° (relative to the assumed starting point) of the measured roundness contour of pin no. 5, on the simultaneously displayed profile of the eccentric movement at the measured roundness contour center limited by the support are shown in According to the accepted interpretation of the measured round contour geometrical features of the analysis based on harmonics, the first term in the Fourier series of the function characterizing the course is the deviation of the axis position, namely the eccentricity. Eliminating this harmonic makes it possible to treat the sum of the remaining harmonics as the theoretically measured roundness contour. This interpretation was used to qualitatively and quantitatively compare the measured and theoretical (excluding harmonic no. 1) roundness contours, obtained from superimposing the measured roundness contour of pin no. 5 on the full eccentric profile and the eccentricity profile of the measured round outline center at the support limit. A graphical representation of the compared profiles of journal no. 5  is helpfThe quantitative evaluation was conducted by determining the roundness deviations between the compared profiles and finding the correlation coefficient between the profiles provided by the formula :The adopted procedure involved repeatedly superimposing the measured roundness profile (with angular rotation) onto the displacement profile using the support to create a limitation. This approach allowed the relative angular position of the compared profiles to be determined. It can also be used to determine the maximum and minimum correlation coefficients between the actual standard profile and the theoretical profile obtained by superimposing the measured profile onto the displacement profile, as well as the roundness deviations resulting from this procedure.ρ as a function of the angular shift between the compared profiles. The minimum correlation coefficient was 27.03 μm d. The ma41.39 μm e. e, the support location x, and the location of the measured journal profile reflecting the journal axis displacement. The study presented in this article confirmed that the detectability of geometric deviations is limited when the shaft is supported in an uncontrolled manner (with a set of rigid vee-block supports), which was especially true for the main journal deviations. However, the limited detectability of large crankshafts due to the support conditions was observed for positional deviations of journal axes and also for shape profile deviations in journals. The shape deviation measurements may vary significantly in terms of their values and profiles relative to the actual shape of an object. Referring to the denotations used in the article, when a journal axis moved eccentrically as the shaft rotated, the investigated parameter values were directly influenced by the eccentricity The results of this study show the importance of ensuring appropriate support conditions to eliminate deflections, and thus elastic deformations of the crankshaft under the influence of its own weight, as well as those caused by its geometric deviations. These deflections can only be eliminated if there is constant contact between the supports and the main journals of the shaft. Such conditions cannot be guaranteed by supporting the shaft with a set of rigid vee-block supports maintaining a fixed height. For deviations in the position of main journal axes, unintentional pre-deflections generate elastic deformations when the shaft rotates. This state causes interactions between geometrical deviations and elastic deformations (which are interrelated), and the geometric evaluation of the shaft geometry becomes unreliable.Therefore, to ensure correct measurement conditions, the main journals of the shaft should be supported with a set of supports that compensate for its deflections and elastic deformations under the influence of its own weight, as well as those caused by geometric deviations of the shaft. The reaction forces at the contact between support heads and main journals vary along the shaft, and also depend on the angle of rotation of the shaft being supported, thereby ensuring zero deflections at the journals.1. Nozdrzykowski, K. Device for measuring positional deviation of axis of crankshaft pivot set. Polish Patent Office, PL393829-A1; PL218653-B1.


Increased systolic blood pressure (SBP), pulse pressure and carotid-femoral pulse wave velocity (cfPWV) are strong predictors of cardiovascular events. The aorta and carotids are both elastic arteries susceptible to arterial stiffening and its deleterious complications. The relative importance of ascending aortic and carotid stiffness on central arterial stiffness remains unclear.Our aim was to study the relationship of local arterial stiffness in the carotid and ascending aorta to central arterial stiffness assessed by cfPWV and peripheral BP.We studied 22 healthy subjects . Central arterial stiffness was determined using carotid and femoral tonometry and transit surface distances (cfPWV). Distensibility of the ascending aorta was calculated as the ratio of its transverse area change on MRI to the central pulse pressure from radial tonometry arterial waveforms using a transfer function. Carotid distensibility was calculated as the ratio of transverse area change by sonography to carotid pulse pressure by tonometry. Local stiffness indexes of carotid and aorta were calculated from distensibility by the reverse Moens-Korteweg equation. Peripheral SBP and pulse pressure were averages of 6 brachial measurements. Linear regression was used to study correlations between aortic and carotid stiffness and cfPWVAortic stiffness has a significantly stronger correlation than carotid stiffness with: age , peripheral SBP , pulse pressure , and cfPWV . In multivariate analysis with simultaneous adjustment for age, body mass index, SBP, pulse pressure, aortic stiffness and carotid stiffness, aortic stiffness is the strongest independent determinant of cfPWV (p = 0.013), SBP (p = 0.005) and pulse pressure (p = 0.01).Stiffness of the ascending aorta is a stronger predictor of cfPWV and peripheral SBP and pulse pressure than carotid stiffness among healthy individuals. This is consistent with heterogenous age-related stiffening of large arteries.


There are many vertically deflected building structures in the world that require rectification. Temporary supports installed in the building bearing walls can be used to perform such a rectification. The supports consist of a hydraulic piston jack, a stack of parallelepiped steel elements, and a concrete grout. The structure is unevenly raised and reaches the desired vertical position using such supports. The support in which the piston extension is forced at the given time is an active support. The aim was to determine the stiffness of an active support. The investigations were performed in in situ conditions during experimental building rectification. No such investigations have been performed to date. It has been demonstrated that the stiffness of the investigated support results from the stiffness of the serially connected elements forming the support. In general, the support stiffness depends on the value of the force occurring in the support and is rising linearly along with the load for the investigated range. It was also shown that the force existing in the active support also depends on the stiffness of the building being rectified. The investigations carried out show that it is advantageous to use supports with smaller stiffness for rectification, as forces with smaller values must be induced in them. The application of forces with lower values also allows the avoidance of unfavorable penetration of the unlifted part of the building into the ground. One of the issues emerging in civil engineering is the vertical deflection of building structures. The issue concerns all types of buildings. For example,  shows veThe deflection of adjacent buildings may lead to their interaction. Such a situation took place as a result of simultaneous changes of deformations and slope of the ground surface . The defTilted structures are most often stabilized to prevent the deterioration of deformations. Such method was applied for the Leaning Tower of Pisa  where thSuch experimental removal of tilting by lifting was carried out in Silesia, Poland. In this case, hydraulic piston jacks were installed in the load-bearing walls of the building cellar. The building was detached with their help a. The houext) was forced in the given cycle, was called an active support (Q) in this support. Moreover, in the place where the active support was installed, there was an upward movement of the elevated part of the building (uobj) and a slight downward movement of the part remaining in the ground (ufou). Moreover, as the support force was rising, the support was shortened (Δlsup). The support in which no piston extension was forced, was called a passive support. In the process of uneven lifting, depending on the lifting phase, the given support can be either active or passive.The pistons of the jacks were extended while removing the deflection. The support, in which the piston extension  1.0 m/0.4 m, and under the internal walls, with the dimensions 1.4 m/0.4 m. The subsoil layers below the foundations were determined with geotechnical boreholes drilled to the depth of 6 m. There is a 0.5 m thick layer of sand with a degree of density of ID = 0.7 directly underneath the strip foundations. Below, sandy clay in a hard-plastic state with a plasticity degree of IL = 0.2 is deposited to the depth being recognized.The building where the investigations were planned and carried out is completely cellared and has five above-ground floors. Its height above the ground level is 16.1 m . The proDuring the investigations, the building rested on 36 temporary supports, whose location is shown in uext), forces occurring in the support (Q), displacement of the part of the building remaining in the ground (ufou), displacement of the straightened part of the building in relation to the part of the building remaining in the ground (uo-f), change of the length of the temporary support (Δlsup), change of the length of the jack and the stack of steel parallelepiped elements (Δljack + Δlund). The measurement of displacement ufou was designed in relation to a steel rod driven into the ground at a depth of 1.5 m , it was reduced to the minimum value (Qmin). For technical reasons, force reduction was carried out in two ways. First, the force in the adjacent support no. 31  are distinguished in this system: 100–780 kN, 200–780 kN, 300–780 kN, and 400–780 kN. The load value increased at an average speed of 5.8 kN/s. The measured values were recorded at a frequency of 10 Hz.The following load cycles . When comparing the dependency Q−uobj with the dependency Q−uext, one can see that only part of the piston extension shows itself as an upward movement of the object.In turn, Q−ufou were provided in Q is accompanied by the settlement of the foundation under the jack. In the beginning, when increasing the load, the displacements ufou are small. Then, a further increase in the value Q is accompanied by a more significant increase in the displacements ufou. The remark applies to each load cycle Qmin–Qmax.The dependencies uext and displacements uo-f, uobj, ufou corresponding to the change ΔQ in the range of Qmin–Qmax. The stiffness k of the system is then defined asuext corresponds to the change of ΔQ.k of the system is influenced by the properties of all its constituent elements: the part of the building being lifted, the part remaining in the ground, the ground and the properties of the supports, in particular the investigated active support. The stiffness kobj—stiffness of the elevated part of the buildingThe following was additionally defined: kfou—stiffness of the part of the building remaining in the groundQmax−Qmin and listed in The stiffnesses described by the dependencies (5)–(7) were determined for the four examined load cycles Q = Qmax − Qmin). With the increase of the value Qmin (with constant Qmax = 680 kN), higher values of the parameter were obtained. The smallest values, from 99 MN/m to 154 MN/m, are assumed by k. On the other hand, the highest values, from 895 MN/m to 2223 MN/m, are assumed by kfou. The stiffness kobj assumes the values from 169 MN/m to 250 MN/m and ko-f from 142 MN/m to 225 MN/m. The greatest variability, together with the load, is characteristic for kfou.The stiffnesses are graphically presented in the diagrams in Q on the system parameters was analyzed. For this purpose, the displacement values corresponding to the force Q of 100 kN, 200 kN, 300 kN, 400 kN, 500 kN, and 600 kN were read from a load curve of 100 kN–680 kN. Displacement increments , corresponding to increments ΔQ of the force, were determined on such basis for the ranges of 100–200 kN, 200–300 kN, 300–400 kN, 400–500 kN, and 500–600 kN. For these ranges, the stiffnesses k, kf-o, kobj and kfou were determined according to the dependency (4)–(7). The determined displacement and stiffness values are listed in The influence of the value of the force kfou is most dependent on the load value. Its value decreases significantly as the load increases, falling from 32,452 MN/m (for Q from 100 kN to 200 kN) to 646 MN/m (for Q from 500 kN to 600 kN). Other parameters of the structure do not show such significant changes along with the change Q. It can be concluded that all stiffnesses, except for kfou, slightly grow with an increase in the value Q from 100 kN to 200 kN and then stabilize. The stiffness k rises from 73 MN/m to 115 MN/m, ko-f from 109 kN/m to 153 kN/m and kobj from 110 kN/m to 198 kN/m.It can be seen that the stiffness lsup) caused by a change in the value Q. A load increasing monotonically from the value Qmin to Qmax was distinguished there. The aim of the investigations is to analyze variations in the length of the support and its elements corresponding to changes in force in the range of Qmin–Qmax.lsup + Δljack) caused by a change in the force Q. An increase in the load Q resulted in shortening the length of the elements. It can be noticed that this shortening is not fully proportional to the increase in the value of the force Q. This is because a change in the stack length is caused by three deformations. The first one is the deformations of the material of parallelepiped elements. The second one is the mutual displacement of the profiles, from which a parallelepiped element is created by welding. The third one is the deformations occurring around interfaces of parallelepiped elements which are not perfectly contiguous. The latter are characterized by a non-linear force-deformation dependency.ljack) under the influence of the increasing load Q. The change Δljack, obviously, is smaller than the changes shown in Q in the range of 100 kN to 780 kN, is 0.912 mm. Such a significant change in the jack length derives from the fact that it is the result of both the deformations of the cylinder material as well as the deformations occurring at the contact between the sheet metal fixed in the concrete grout and the jack base. In turn, lgrout) under the influence of the load. The value (Δlgrout) was not the subject of direct measurement, but it was determined from the dependency (3).Qmin–Qmax. The stiffnesses of the support were then definedkjack is the stiffness of the jack, kund stiffness of the stack of parallelepiped elements, kgrout stiffness of the grout.Q = Qmax − Qmin). With the increase of the value Qmin, with constant Qmax equal to 680 kN, higher values of the parameters were obtained. The smallest values, from 307 MN/m to 457 MN/m, are assumed by ksup. On the other hand, the highest values are assumed by the stiffness kgrout and kund; from 1541 MN/m to 1700 MN/m and from 916 MN/m to 1832 MN/m, respectively. The stiffness kjack assumes the value from 658 MN/m to 1020 MN/m.The stiffnesses of the support and its elements determined according to (8) and (9) were compiled in Q on the support parameters was then analyzed. For this purpose, the displacement values corresponding to the force Q of 100 kN, 200 kN, 300 kN, 400 kN, 500 kN, and 600 kN were read from a load curve of 100–600 kN. Displacement increments , corresponding to increments ΔQ of the force, were determined on such basis for the ranges of 100–200 kN, 200–300 kN, 300–400 kN, 400–500 kN, and 500–600 kN. For these ranges, the stiffnesses ksup, kjack, kund and kgrout were determined according to the dependency (8) and (9). The determined displacement and stiffness values are listed in The influence of the value of the force ksup of the support increases with the increase in the force Q. This stiffness increases from 213 MN/m with the load Q of 100–200 kN to 430 MN/m with the load Q of 500–600 kN. The greatest change in stiffness is distinct for the stack of steel elements, whose stiffness increases from 443 MN/m for Q in the range of 100–200 kN to 1391 MN/m for Q in the range of 500–600 MN/m. On the other hand, the force Q has the smallest effect on the jack stiffness, which varies from 503 MN/m for Q for the range of 100–200 kN to 985 MN/m for Q for the range of 500–600 kN.The stiffnesses corresponding to the centers of the analyzed ranges were marked with points. The figures clearly show that the stiffness (k), building structure elements  and supports (ksup) corresponding to the ranges ΔQ. The relationship between these stiffnesses results from Equations (4)–(6) and (8) and from the fact that the displacement uext, corresponding to the stiffness of the whole system, is equal to the sum of displacements of the part remaining in the ground (ufou), the lifted part (uobj) and the change in the length of the support (Δlsup)—Equation (1). Hence, the stiffness k results from the serial combination of the stiffness kfou, kobj, and ksup, thusk can also be expressed by the stiffness ko-fkobj, kfou, ksup, ko-f are dependent on Q. The values k, calculated according to (4), (10) and (11), are compiled in k, determined according to these dependencies, do not differ significantly, which proves the correctness of the adopted model. At the same time, it should be noted that the stiffness k of the model increases from 73 MN/m with the force Q between 100 kN and 200 kN and then stabilizes for the value Q greater than 200 kN, assuming the values between 104 MN/m and 115 MN/m. This is mainly influenced by the stiffness of the lifted part, which at Q of about 200 kN stabilizes with a value of about 200 MN/m. The stiffness of the support (ksup) consists of the stiffness of the jack (kjack), a stack of elements installed under the jack (kund) and grout (ktgrou). The values of these stiffnesses, corresponding to the examined ranges ΔQ, are shown in The stiffness kjack, kund, and kgrout are dependent on the load Q. The model of the system adopted above is shown in The elements forming the support rest on top of each other, hence, according to (3), the total change in the length of a support is the sum of changes in the length of its constituent elements. Thus, the support stiffness can be determined from the binding dependency of the three connected stiffnesses in seriesk) was analyzed based on the model defined in kfou and kobj were studied successively.The influence of the particular parameters on the system stiffness , was provided in black in kund = ∞). In addition, the line to which the values refer  is marked in grey. The dependencies obtained are similar in nature. It is shown by comparing the respective values that the removal of the parallelepiped elements has increased the system stiffness by 13%–55%.The dependency kgrout = ∞ black line). It can be stated, by referring the values obtained in this way to the results obtained for the support with the grout (grey line), that the removal of the grout has increased the stiffness of the system by 5%–10%.The analogous diagram is given in kfou = ∞; , the stiffness of the system increases by 10%–35% depending on the force Q.However, assuming that the supports rest on an undeformable substrate . The case for n = 0 corresponds to the situation where the support consists only of grout and a jack. Within the investigated range, the stiffness of the support may vary from 124 MN/m  to 985 MN/m .The stack in the investigations consisted of four parallelepiped elements, 72.5 mm long each. Hence, the stack length was 290 mm. The results obtained can be generalized into stacks consisting of a different number of elements. One of the issues in civil engineering is the vertical deflection of building structures. For buildings, such deflections can be removed by means of temporary supports installed into its walls. Temporary supports consist of hydraulic piston jacks, stacks of steel parallelepiped elements and a concrete grout. With their help, the building is torn apart in a horizontal plane. Then, the part of the building situated under the tearing area is elevated unevenly until a vertical position is reached. The support in which the piston extension is forced at the given stage is an active support.Piston extension in the support increases the force value in this support. The investigations performed in situ conditions reveal that the force increase in the support is unfavorable, as it results in the deformation of the building part being lifted and in the unfavorable movement of the part remaining in the ground towards the bottom. The force increase in the active support depends on the stiffness of the system consisting of the unevenly elevated part of the building, the part remaining in the ground and temporary supports. The stiffness of the whole system in the place and direction of piston extension of the active support for the range of 100–200 kN was 73 MN/m and for the load range of 500–600 kN it was 115 MN/m.The stiffness of the support itself has the greatest influence on the system stiffness in the place and direction of piston extension of the active support. The stiffness of the support consisting of a jack, a stack of four parallelepiped elements with a total length of 290 mm and 150 mm thick grout depends on the load value. For the load range of 100–200 kN it was 213 MN/m and for the load range 500–600 kN it was 430 MN/m.The support stiffness relies upon the stiffness of the serially connected elements forming the support. Concrete grout has the greatest stiffness. For the load range of 100–200 kN, its stiffness was 2258 MN/m and for the load range 500–600 kN, it was 1692 MN/m. The jack with the stiffness of, respectively, 503 MN/m and 985 MN/m, had lowest stiffness. On the other hand, the stiffness of the stack consisting of four parallelepiped elements was 443 MN/m for the load range of 100–200 kN and 1391 MN/m for the load range of 500–600 kN.The defined model of the support and the stiffnesses of its elements determined during the in situ tests made it possible to calculate the stiffness of the supports which are also characterized by other lengths of the stack of steel elements. The support stiffness is declining along with the growing length of the stack of parallelepiped elements.Apart from the active support, the stiffness of the elevated building part resting on supports has the greatest influence on the stiffness of the entire system. This was 110 MN/m for the load range of 100–200 kN and 194 MN/m for the load range of 500–600 kN. In the case where the stiffness of this part reaches an unlimited value, then the stiffness of the system would increase three times. This would be the case if the stiffness of the adjacent passive supports was unlimited.There have been no studies up to now of buildings rectified by uneven lifting. Therefore, the results provided in this work also allow conclusions to be drawn about the rectification process. When using supports with smaller stiffness, it is necessary to induce forces with smaller values, which is beneficial. However, the use of supports with insufficient stiffness can be dangerous due to the loss of system stability. Moreover, to ensure that the unlifted part of the building does not deepen into the ground, it is necessary to change the values of forces in the supports by as little as possible. This means that small extensions have to be applied to the jack pistons due to the fact that while the forces in the supports are growing, the stiffness of the part of the building remaining in the ground is decreasing.


As the present year comes to an end, the world, for obvious reasons, hopes for a better one to come, with positive developments and promising perspectives. On a much smaller scale, the new year will bring some, as we hope, interesting and welcome changes to this journal. The world as a whole will most certainly be unaware of them, but they will hopefully be registered favorably by the community of physiologists in Germany, Europe and even beyond: Pflügers Archiv - European Journal of Physiology and the German Society of Physiology will team up, and as of January 2021 this publication will be the society’s official journal.At a first glance it may look strange to have the word “European” in the title of a journal that is about to become the figurehead of a German society. But in fact, it is not. Obviously, the ties between physiological research in Germany and Pflügers Archiv have a long history. In fact, it is the longest one possible since, worldwide, it was the first publication dedicated to this field when it was founded by the German physiologist Eduard Pflüger in 1868. Renamed in his honor in 1910, it is published under its present name since 1968 to reflect the widened scope of nationalities of the researchers who published their work in this journal, but perhaps also to acknowledge the growing importance of international collaborations for the field.Looking back on close to 120 years of history, the German Physiological Society (DPG) is a youngster by comparison. Today, though, its name is almost more an indication of its linguistic rather than strictly national focus since its membership also includes scientists from Austria and Switzerland. And while its basis lies in this geographic region, the DPG is also firmly rooted in Europe, interacting with its fellow societies, most notably by organizing joint international meetings. So, joining forces with a reputed journal that unmistakably sees itself as an international publication makes perfect sense.Pflügers Archiv will continue to have a team of Executive Editors and an Editorial Board consisting of distinguished scientists from all over the world, and it will welcome manuscript submissions from any country and on any area and aspect of physiology, including translational research.Needless to say, this new partnership will not affect the management and general scope of the journal. Pflügers Archiv with its editors and the DPG are looking forward to a fruitful collaboration under the roof of the Springer Nature as the publisher, to ensure its success and to shape the future of the journal for, who knows, maybe a further 150 years…Markus HeckerPresident of the German Physiological SocietyArmin KurtzEiC Pflügers Archiv – Eur J Physiol


Castanea mollissima Blume) seed kernels (CCSK) with high quality and quantity of starch has emerged as a potential raw material for food industry, but the molecular regulatory mechanism of starch accumulation in developing CCSK is still unclear. In this study, we firstly analyzed the fruit development, starch accumulation, and microscopic observation of dynamic accumulation of starch granules of developing CCSK from 10 days after flowering (DAF) to 100 DAF, of which six representative CCSK samples (50–100 DAF) were selected for transcriptome sequencing analysis. Approximately 40 million valid reads were obtained, with an average length of 124.95 bp, which were searched against a reference genome, returning 38,146 unigenes (mean size = 1164.19 bp). Using the DESeq method, 1968, 1573, 1187, 1274, and 1494 differentially expressed unigenes were identified at 60:50, 70:60, 80:70, 90:80 and 100:90 DAF, respectively. The relationship between the unigene transcriptional profiles and starch dynamic patterns in developing CCSK was comparatively analyzed, and the specific unigenes encoding for metabolic enzymes  were characterized to be involved potentially in the biosynthesis of G-1-P, ADPG, and starch. Finally, the temporal transcript profiles of genes encoding key enzymes  were validated by quantitative real-time PCR (qRT-PCR). Our findings could help to reveal the molecular regulatory mechanism of starch accumulation in developing CCSK and may also provide potential candidate genes for increasing starch content in Chinese chestnut or other starchy crops.Chinese chestnut ( Castanea mollissima Blume), a deciduous tree in the family Fagaceae and genus Castanea, is a chestnut variety of considerable economic importance that is widely distributed in temperate and subtropical regions of the Northern Hemisphere3. In China, the chestnut has been cultivated for more than 3000 years, currently covering a total area of about 335,904 ha, and Chinese annual production (1.94 million tons) accounts for about 83.34% of global chestnut production (2.33 million tons)4. Most importantly, chestnut has high nutritional value, containing essential fatty acids, minerals , vitamins, dietary fiber, and amino acids5, and is thus considered the king of dried fruits. In addition to direct consumption, chestnut is also used in the food processing industry, such as in marron glace production, chestnut flour for bread-making, and confectionery paste for desserts and jams8. Chestnut is a major arboreal nut crop and its chemical composition reveals that starch is the most abundant component, comprising approximately 47–80% of the dry kernel weight10, differing significantly from other temperate seeds, which are oily11. This starchy property also makes Chinese chestnut a potential substrate for fermentation, which could be developed for biological production in addition to utilization in the food industry.Chinese chestnut  can be applied in the industrial processing of paste foods and frozen food as a thickening agent23. In addition, recent researches on chestnut starch have investigated its characteristics for industrial utilization, chemical composition, isolation methods, functional properties, physicochemical characteristics, modification of its structure and digestibility25, as well as its morphology and starch characteristics with different cooking styles26, but little is known about the synthesis of chestnut starch. Thus, it is essential to investigate the regulatory mechanisms of starch biosynthesis in chestnut to improve its starch content and extend its application in food industry.Starch, as the main storage carbohydrate in vascular plants, is the most important dietary source of energy for humans, accounting for 80% of daily caloric intake, and thus plays an essential role in the food industry30. Although a Chinese chestnut reference genome has been published, its annotation remains incomplete. RNA-Seq analysis of developing CCSK could support a more comprehensive and reliable genetic information database, which could be used for research into chestnut molecular breeding to promote the development of chestnut resources for food, forest biomass energy or industrial material.Recently, Illumina RNA sequencing (RNA-Seq) technology has been used to investigate gene expression in various plant tissues at different developmental stagesHerein, fruit development was surveyed and kinetic changes of starch granules were observed using a microscope during growth period . In addition, dynamic patterns of sucrose, starch and its components (amylopectin and amylose) were measured at different developmental stages . Thus, the major periods of starch biosynthesis, which were optimal for transcriptomic analysis, were determined in CCSK. Transcriptome sequencing was carried out using Illumina-Solexa technology, and the obtained unigenes were functionally annotated. Furthermore, differentially expressed genes for key enzymes involved in starch synthesis in CCSK were screened using the DESeq method, and the results were verified via qRT-PCR. Overall, this study presents a systematic analysis for dynamic pattern of starch accumulation and transcript profiles of metabolic enzymes involved in starch biosynthesis in developing CCSK, which will contribute to clarifying the mechanisms of starch accumulation, thus supporting future work to increase starch biosynthesis and accumulation in CCSK and to extend the application of chestnut starch in industry.C. sativa9. We also noticed that the fresh weights of developing fruits gradually increased with a significant increase (6.1-fold) from 70 DAF (1.54 ± 0.07 g) to 90 DAF (9.33 ± 0.03 g), followed by a slight increase (10.9%) at 100 DAF (9.95 ± 0.03 g) . We found that 2–4 nuts were contained within the spherical spiked involucre, which was up to 11 cm in diameter at maturity. The nuts were pale yellow during the early growth period and gradually darkened to dark brown. After removal of the chestnut shell, no seed kernels were observed during development until 50 DAF, and the kernel color gradually changed from the initial white to yellow during fruit ripening  staining was conducted on chestnut fruits at 10–40 DAF (removing the spiked involucres) and kernels  at 50–100 DAF. Through microscopy, we found that the ovary of the fruit consisted of six to eight ventricles, each with two ovules Fig. a, c, and36 as well as the carbon and energy source from photosynthesis which are supplied to heterotrophic organs to initiate the biosynthesis of starch37. To explain the starch accumulation pattern and to address the relationship between sucrose content and starch accumulation, we analyzed dynamic changes in the sucrose and starch contents in developing CCSK at 50–100 DAF, and found that the starch content was about 60-fold higher at 90 DAF (335.60 ± 7.45 mg/g DW) than at 60 DAF (5.65 ± 0.24 mg/g DW), followed by stabilization at 100 DAF  during CCSK development, of which amylopectin showed more abundant, and its content gradually increased to a peak at 100 DAF, a similar pattern was noted for amylose 42 and C. sativa (73.4%)7, but lower than that reported in C. mollissima (81.7%)43 and higher than another study in C. sativa (67.1%)44. These differences may be attributed to analysis of chestnuts with different geographic origins, or to different methods used for amylopectin determination and starch isolation7. In terms of amylopectin content of starch plants, Chinese chestnut fell in the range of normal level, compared with that low amylopectin starches were reported in corn varieties46, while wheat and potato were reported to be regular starches31. Owing to that amylose and amylopectin possess different properties and uses, the corresponding functions and products can be developed according to the content of starch components in chestnut fruit.Notably, the ratio of amylopectin to amylose is a critical factor affecting starch structure, properties and functionalities of starchose Fig. b. Amylopose Fig. b, compar29, Centella asiatica (474 bp)47, and Yellow Horn (462 bp)48. These results implied that our Illumina sequencing successfully captured most expressed genes, ensuring the reliability of the transcriptome data.To explore molecular regulatory mechanism of starch biosynthesis in developing CCSK, CCSK samples from six crucial periods  were selected for transcriptomic sequencing analysis based on the above results for fruit growth and starch granule accumulation as well as dynamic patterns of starch content during CCSK development Figs. , 3. A to−5. Of 38,146 unigenes, 36,284 (95.12%), 29,424 (77.14%), 21,244 (55.69%), 12,091 (31.70%), and 8345 (21.88%) unigenes in developing CCSK showed high similarity to known proteins in the National Center for Biotechnology Information nonredundant (NCBI Nr), Swiss protein (SwissProt), eukaryotic orthologous groups (KOG), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, respectively  in developing CCSK, the above obtained unigenes were screened by fragments per kilobase per million reads (FPKM) values using the DESeq method  to fructose (Fru) and UDP-glucose (UDPG) in the cytoplasm of the storage organ, whereas INV catalyzes the irreversible hydrolysis of sucrose to glucose (Glu) and Fru54. Thus, to fully understand the allocation of available carbon from sucrose to starch synthesis in the developing CCSK, it was essential to concretely analyze the differential transcription profiles of genes for sucrose-cleaving enzymes in developing CCSK. Through sequence analysis, we identified three cytosolic SUS isoforms (SUS2/3/4) and three INV isoforms  with differential transcription in developing CCSK, of which only SUS2 displayed high transcription levels at 50–100 DAF, whereas INV1 and INV4 showed upregulated transcripts at 50–60 DAF and 50–70 DAF, respectively  or invertase (INV)rms SUS2//4 and th61. Therefore, it was necessary to determine the potential source of G1P for starch biosynthesis in our experiments. G1P, which is mainly derived from cytosolic G6P in developing seeds of plants, is transported by glucose 6-phosphate (G6P) translocator (GPT) to the plastid, where it is converted to G1P by plastidial phosphoglucomutase (PGM)63. Meanwhile, both PGM and glucose-6-phosphate isomerase (PGI) have been shown to have roles in the interconversion of hexose phosphate 64.G1P is known to act as a glucosyl donor or substrate for the biosynthesis of ADPG, a critical precursor for starch biosynthesis66. This finding prompted us to explore interchange of glycolytic intermediates between the cytosol and plastid in developing CCSK. Intriguingly, differentially expressed genes encoding a glucose transporter (GLT1/2/4), G6P transporter (GPT1/2), triose phosphate transporter (TPT), and phosphoenolpyruvate (PEP) transporter (PPT2) were annotated in the developing CCSK  was much higher than that of PFK (downregulation), which catalyzes phosphorylation of F6P to fructose-1,6-bisphosphate , a key regulatory step in glycolytic pathway. Upregulation was observed for cytosolic PGI2 transcripts, an enzyme that converts F1P to G6P , granule-bound starch synthase (GBSS), starch synthase (SS), starch branching enzymes (SBE), isoamylase-type starch-debranching enzyme (ISA), and phosphorylase (PHO)74, which is supported by our finding that AGP2/3 exhibited a highly correlated temporal pattern with active starch accumulation in CCSK at 50–90 DAF , among which GBSS1 and SS1 exhibited markedly upregulated expression at 50–90 DAF and SS3 showed upregulated expression at 50–70 DAF, whereas GBSS2 and SS4 showed low and stable expression, in accordance with the results of qRT-PCR analysis  and ISA (ISA1/2/3), as well as PHO, were identified in developing CCSK, and importantly, transcript abundance of SBE2.1/2.2, ISA1/2/3 and PHO was found to be highly correlated with the accumulation of amylopectin  increased over the active starch synthesis period  of CCSK development  in developing CCSK were validated by qRT-PCR detection. Together, our findings will provide a better insight into regulatory mechanism of high starch production and may also provide both a rich source of data and of considerable interest to those studying starch plants.The starchy Chinese chestnut is a potential substrate for fermentation and holds promise for development as industrial raw materials or biological products. To fully understand the temporal patterns of starch accumulation, ten samples collected throughout the developmental period of CCSK were analyzed. Based on starch and sucrose content measurement and microscopic observation of CCSK at various developmental stages, six experimental samples from crucial periods  were selected for comparative deep transcriptomic analysis. The resulting 38,146 unigenes with a mean length of 1164.19 bp were obtained and deposited in public database, massively enriching the dataset available for Chinese chestnut. By using the DESeq method, a total of 1968, 1573, 1187, 1274, and 1494 differentially expressed unigenes were identified specifically at 60:50, 70:60, 80:70, 90:80, and 100:90 DAF, respectively, of which 239 unigenes were characterized to be potentially involved in starch accumulation in developing CCSK. Notably, the application of an integrated analysis of comparative transcriptome sequencing and temporal pattern of starch accumulation has led to the identification of some crucial regulatory enzymes  responsible for the biosynthesis of G-1-P, ADPG, and starch of developing CCSK. Also, the dynamic transcript patterns of some key related genes  of Beijing, China. The developmental stages of fruits from flowering to seed maturity were observed from June to September. Flowers that were at the anthesis stage simultaneously were marked, and fruits were harvested at 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 DAF. Fifteen fruits from the randomly selected 3 trees (5 fruits each tree) of each developing stage were collected each time, and then immediately frozen in liquid nitrogen and stored at − 80 °C until use.Developing fruits from 85, stained using the PAS method, and finally observed and photographed using a Leica DM 6000 light microscope.The characteristics of chestnut fruits were observed throughout fruit growth (10–100 DAF). Fruit development was expressed using the fruit shape index (length/diameter) and fresh weight at different developmental stages. To investigate the accumulation pattern of starch, we examined dynamic changes of starch granules in the fruits. Fresh fruits were collected from the tree, fixed in formalin-acetic acid-alcohol (FAA) solution and stored in 70% ethanol at 4 °C until use. Then, samples were prepared as 8-μm sections using the conventional paraffin section method86. The contents of starch components  were determined through dual wavelength spectroscopy, with amylopectin measured at 550 nm and 695 nm and amylose measured at 617 nm and 475 nm, using a spectrophotometer (UV-2102C)87. All the determinations were performed in triplicate.After the kernels (50–100 DAF) were dried in a 45 °C oven, each dried sample was ground into fine powder with a mortar and pestle, followed by filtration through a 30-mesh sieve prior to use. The sucrose content (about 50 mg of sample powder) was determined using a previously described method29. Illumina sequencing was conducted on the HiSeq 2500 sequencing system. After removal of reads containing poly-N and low quality reads, the remaining clean reads were mapped to the reference C. mollissima genome using Tophat or bowtie289 (http://tophat.cbcb.umd.edu/), from which unigenes were obtained.Based on microscopic inspection of starch grain accumulation and analysis of starch content in developing CCSK, samples from six crucial stages  were selected for transcriptomic analysis. Two fresh seeds of each fruit (3 samples per developing period) were cut into small pieces and mixed together, and then the equal weights of three biological duplicates from each developmental stage were fully mixed for total RNA extraction using the RNA Isolation Kit following the manufacturer’s protocol. Purified RNA was quality-tested and quantified using a Nanodrop ND-1000 spectrophotometer , and all samples showed 260/280 nm ratios of 2.1–2.2. cDNA library construction and normalization were performed as described previously90, and read counts for each gene were obtained using htseq-count91. The levels of gene expression in various samples were compared using the DESeq method92, with P-value < 0.05 and fold-change > 2 or fold-change < 0.5 used as thresholds indicating significant differences in gene expression. Databases including the NCBI Nr (https://www.ncbi.nlm.nih.gov/), SwissProt93, and KOG94 were used to annotate the unigenes with an E-value cut-off of 10−5. GO95 and KEGG97 pathway enrichment analyses of DEGs were both performed using R, based on a hypergeometric distribution48.Unigene expression levels were calculated as FPKM using the Cufflinks program−ΔΔCt method98.Total RNA was extracted as described for cDNA library preparation and was reverse transcribed using HiScript II QRT SuperMix for qPCR (+ gDNA wiper) . The primers were designed using Roche LCPDS2 software based on mRNA sequences obtained from the NCBI database  reported in the figures are averages of at least three different determination. Microsoft Excel Statistical Software (Microsoft Office Excel 2016) software was used for statistical analysis. The level of significance used for all the statistical tests was 95%.Supplementary Legend.Supplementary Table S1.Supplementary Table S2.Supplementary Table S3.Supplementary Figure S1.Supplementary Figure S2.Supplementary Figure S3.Supplementary Figure S4.Supplementary Table S4.Supplementary Table S5.Supplementary Table S6.


Unlike high amylose maize starch, it had a gelatinization temperature that could be achieved during cooking, and had good gelling properties.Starches and flours used commonly in Asia  were characterized in terms of their chemical composition, morphological, functional, pasting, thermal, gelling and in vitro digestibility properties. It was observed that the differences in their chemical composition and structure influenced their properties. High amylose maize was the most stable, thus it required the highest gelatinization temperature which was observed in both the differential scanning calorimetry (DSC) and pasting profiles. Kithul flour had a significantly lower rate of digestion ( Starch is the main carbohydrate in foods and is an important food ingredient for many food products. Most of the research have focused on the mainstream starches such as maize, potato, rice, cassava and wheat starches, which are the major sources used for the commercial extraction of starch globally . In receIpomoea batatas), sago (Metroxylon sagu), water chestnut (Eleocharis dulcis) starches, red rice (Oryza rufipogon) and kithul flour (Caryota urens), have not been extensively characterized. Starch is extracted from the roots of the sweet potato, and the corm of the water chestnut. Sago and kithul are from the palm family and extraction from the pith of the stem yields the flour. These starches/flours are available commercially in native state but not much research has been done in terms of systematically characterizing them and studying their functional properties. Some characterization work of these starches/ flours have been carried out comparing between species after starch isolation [There is a growing demand for starchy foods with low glycemic index (GI), which are more slowly digested and metabolized, leading to a more gradual and smaller rise in blood glucose. Much work has been done on mainstream starches in a bid to alter the structure to influence the digestibility of the starch. However, many of the unconventional starches and flours commonly used in Asia such as sweet potato  starch is selected as a basis of comparison against these starches to evaluate the functional properties with a mainstream commercial starch commonly used in Asia. High amylose maize starch is selected as it is a commercial food ingredient that is used in low GI food products. All samples were sieved through a 250 µm sieve to remove impurities, and sealed in bags for future analysis. The abbreviations for the samples are in brackets: high amylose maize starch (HAMS), kithul flour (KF), red rice flour (RRF), sago starch (SGS), sweet potato starch (SPS), tapioca starch (TS) and water chestnut starch (WCS). The starch and flour samples used in this study were commercial samples: tapioca starch , sweet potato starch , sago starch , water chestnut starch , high amylose maize starch , red rice flour  and kithul flour (Sri Lanka). Tapioca  using the following methodology: Moisture (AOAC 950.46), protein , fat (AOAC 996.06), ash (AOAC 930.30), dietary fiber (AOAC 985.29), available carbohydrates by difference (FAO 1998) and phosphorus by inductively coupled plasma-optical emission spectrometry (ICP-OES) after microwave digestion [The total starch and amylose content of the starches and flours were determined using the megazyme amylose/amylopectin assay procedure, ultilizing the commercial kit .The granule shapes and “maltese cross” were viewed under a light microscope  equipped with a CCD camera, with and without a polarizing plate, according to the method used by Cai and Wei . Starch The granule size distribution was examined by a laser diffraction particle size analyzer . Starch granules were fully suspended in water and an obscuration of 15% ± 1% was achieved for all measurements. The volume-weighted mean diameter (µm), D ,4, was rThe crystalline patterns of starches were performed using an X-ray diffractometer  equipped with a two-dimensional detector Vantec 500 and micro focus Cu source operated at 50 kV, 800 µA and 40 watt. The diffraction patterns were obtained from a scanning angle range between 3° and 30° (2θ) at a step size of 0.02°. Swelling power of starches were determined using a modified method by Leach et al. . Starch g for 20 min and the released water (%) was measured as freeze thaw stability.Freeze thaw stability was determined by the method of Hoover and Ratnayake . Starch The pasting profile of the starch suspensions  were obtained using the starch analyzer . A total weight of 20 g starch suspension was placed in the starch cell and dispersed at a vane speed of 960 rpm, which reduced to 160 rpm after 1 min. The heating and cooling cycle was programmed according to the following: with the exception of high amylose maize starch, samples were held at 50 °C for 1 min, heated to 95 °C at a rate of 2 °C/min, maintained at 95 °C for 5 min, cooled to 50 °C and held for 2 min. A pressure cell system  is required for high amylose maize starch to heat the starch above 100 °C in order for gelatinization to occur. High amylose maize starch was held at 50 °C for 1 min, heated to 150 °C at a rate of 2 °C/min, maintained at 150 °C for 5 min, cooled to 50 °C and held for 2 min. The parameters including pasting temperature, peak viscosity, trough viscosity (minimum viscosity at 95/150 °C), final viscosity (viscosity at 50 °C), breakdown value (peak viscosity–trough viscosity) and setback value  were recorded. Duplicate pasting curves were measured. o), peak (Tp) and conclusion (Tc) temperatures (°C), and enthalpy change (J/g) were determined. Proteus® software  was used to calculate the enthalpy of the endothermic peak. The onset (To), peak (Tp), and completed (Tc) temperatures of the gelatinization peak were determined from the intersection of tangents fitted to the leading and trailing flanks of the peak with the baseline. The thermal properties of the starches were measured using a differential scanning calorimeter  according to Lan et al. . Three mGel texture properties were analyzed using methods by Wang et al.  and KaurAn in vitro digestion model that mimics human gastrointestional digestion ,15,16,17Oral phase: After stirring for 30 min, a solution of 0.1 Ml of 10% α-amylase (≥10 units/mg solid) dissolved in distilled water was added and stirred for 1 min. A total of 0.8Ml of 1 M HCL was added to adjust the Ph to Ph 2.5 (±0.2), using a Ph meter .Gastric phase: 1 Ml of 10% pepsin (≥250 units/mg solid) dissolved in 0.05 M HCl was added. Gastric digestion was concluded after 30 min of stirring at 37 °C. For neutralizing the gastric phase HCl, 2 Ml of 1 M NaHCO3 and 5 Ml of 0.2 M maleate buffer (Ph 6) were added into the mixture. Distilled water was added to the mixture to a final volume of 55 Ml and stirred continuously at 150 rpm until the temperature of the mixture reached 37 °C.Pancreatic phase: 0.1 Ml of amyloglucosidase  and 1 Ml of 5% pancreatin in 0.2 M maleate buffer was added and stirred continuously.A total of 0.5 Ml aliquots was taken at baseline , at the end of oral and gastric phase, and at 20, 60, 90, 120 and 180 min from when the pancreatic phase started. To stop the enzymatic digestion, the drawn aliquots were added to 2 Ml of ethanol. The ethanolic digesta samples were stored at 4 °C for the analysis of reducing sugar.g for 10 min. 0.5 Ml aliquots of the supernatant were drawn and added to 0.25 Ml of 1% amyloglucosidase  dissolved in 0.1 M acetate buffer (Ph 5.2). The mixture was vortexed and incubated at 37 °C for 10 min. Then, 0.75Ml of DNS mixture was then added. This was prepared by mixing 0.5 mg/Ml of glucose, 4 M NaOH and DNS reagent in a ratio of 1:1:5. After the mixture was heated for 15 min at 95 °C, 4 Ml of distilled water was added to each sample and cooled for 10 min. Absorbance was measured at 530 nm by a UV-VIS spectrophotometer . The blank and standard was distilled water and glucose (10 mg/Ml), respectively. The results of the glucose concentration were presented as mg/g starch.The analysis of reducing sugars in the ethanolic digesta samples was measured by a dinitrosalicylic acid (DNS) colourimetric method ,18. The p < 0.05) using GraphPad Prism version 8.0.0 for Windows, GraphPad Software . The data reported in all the tables were mean values and standard deviations of triplicate observations unless otherwise stated. There were four replicates for both the gel texture analysis and the in vitro digestion. The comparisons between samples for the texture analysis and in vitro digestibility were analyzed by a one-way ANOVA with Tukey’s multiple comparison test  for red rice flour and tapioca starch was 0.22, 0.35, 0.60 µm and 0.31, 0.44, 1.60 µm, respectively. The hardness, springiness, cohesiveness and chewiness values of the 10% w/w starch and flour gels with the exception of high amylose maize starch, are shown in Starches and flours that are non-mainstream but commonly used in Asia were studied. The chemical composition and structure of the starches varied, which resulted in the significant differences in the in vitro digestibility, functional, thermal, pasting and textural properties. From this study, it was discovered that kithul flour had a significantly lower rate of digestion than the other samples (except for high amylose maize starch), had a gelatinization temperature that could be achieved during cooking and had good gelling properties. It would be interesting to have a greater understanding of its chemical composition such as how the amylopectin fine structure may influence its properties. Future research on unconventional sources of starch from the tropical region enable us to secure starches with unusual rheological, sensory, nutritional or organoleptic properties.