Background: To analyze the fracture resistance of zirconia implants within the anterior region and evaluate whether zirconia–zirconia implants can be a viable alternative to titanium implants. Methods: Four online databases  were searched for the period of January 2011 to July 2021. All studies that analyzed the in vivo clinical outcome of two-piece implants in the anterior region in English language were included. Results: The search strategy identified 242 studies. Of these studies, three studies were included for qualitative synthesis based on the pre-determined eligibility criteria. The results showed that there is significant difference in biological results, fractal behavior and other complications between one-piece and two-piece zirconia implants. Two-piece zirconia implants demonstrated favorable longevity and success rates within anterior maxillary in short-term trials. Conclusions: Although factors involved in fractures have been identified—sandblasting, implant diameter, occlusal load, age and implant coating—there is limited quantitative assessment to gauge the fracture resistance of two-piece zirconia implants. Hence, further research with long-term clinical evidence is required. Dental implants are increasingly being considered and established as a reliable option for the replacement of missing teeth, and titanium is widely accepted as the gold-standard material used in implant-borne reconstructions ,2,3. TitSurvival rates of titanium implants have been extensively researched over the past few decades with reports suggesting 67–100% survival specifically in patients with head and neck cancer after surgical treatment ,6,7,8. ICeramic materials are commonly used in dentistry for various applications, such as the fabrication of crowns and bridges, orthodontic brackets and implant abutments, due to its tooth colored, excellent esthetics and high biocompatibility ,10. In fZirconia implant systems have been available commercially as either one-piece or two-piece implant systems for more than a decade. From a mechanical perspective, one-piece designs may be superior to the two-piece systems, due to increased fracture resistance and reduced susceptibility for low-grade temperature degradation . In addiSeveral two-piece zirconia implants are being commercially marketed and clinically utilized wherein implant-abutment ”attachment” is usually achieved through cementing the abutment and the crown to the endosseous implant, or by mechanical means (screw). Although cement can seal the microgaps and initial adjustment of implant angulation is possible, the processes are mostly irreversible, and therefore, limited options for replacement of restorations over the implant exist. In contrast, ceramic implants restored by screw retention are still utilizing titanium screws, and hence are not truly ”metal-free”.Previous in vitro studies have noted that the biomechanical stability of both one-piece and two-piece zirconia implants could withstand a loading duration of 10 million cycles at 95 N force ,13. AlthNonetheless, there are still concerns regarding the fracture resistance of zirconia, as standardized testing protocols addressing the aging behavior of zirconia implants have mostly focused on zirconia abutments, rather than the implant itself. Despite the fracture resistance of zirconia implants in in vitro studies being widely reported, there is a distinct lack of data derived from in vivo specifically human studies. Furthermore, Garcia-Hammaker et al. highlighted that the variability of in vitro study designs makes it difficult to have a consensus on the fracture strength and to correlate it to clinical circumstances . As suchThis systematic review aimed to address the following focused research question in PICOS format “What are the mechanical factors influencing fracture resistance (outcome) in two-piece (Intervention) versus one-piece zirconia implants (comparison) in humans (Population) within the anterior region?” and secondary questions included “What is the fractal behavior of anterior zirconia implants?” and “How does the longevity of two-piece zirconia implants compare to one-piece designs?”https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42021271790 (accessed on 25 January 2022).This systematic review used the Preferred Reporting Items for Systematic Review and Meta Analyses (PRISMA) guidelines . The proStudies were included if they fulfilled these criteria: all original research articles and case series, and all study designs including prospective, retrospective and randomized controlled clinical trials on patients treated with one- or two-piece zirconia implants within the anterior region of the oral cavity that qualitatively addressed the incidence and factors implicated in the fracture of zirconia implants within the anterior region. Furthermore, two-piece implants, anterior/aesthetics/aesthetic zone, zirconia and coated zirconia fracture were considered as inclusion criteria. Exclusion criteria were: implants placed in animals, in vitro studies, posterior implants, implants not fabricated with pure zirconia titanium screw/abutment/implant, lithium disilicate, Emax, porcelain-fused-to-metal, studies that were not peer reviewed, grey literature, non-English literature, case reports and reviews of any type, including systematic reviews.A comprehensive literature search was performed using the following electronic databases: Scopus, PubMed, Ovid, and Cochrane Library in July 2021. Articles published from 1 January 2011 up to and including 16 July 2021 were considered. No other restrictions were placed during each initial search of the databases. The articles were retrieved using different combinations of MeSH terms .Independently, four reviewers  commenced the identification of relevant articles employing the predetermined search strategy, and citations of relevant articles were exported into reference management software (EndNote X8). After removal of duplicates, the screening phase was completed by the reviewers, wherein the titles and abstracts of all remaining studies were screened for studies that potentially met the inclusion criteria. In the eligibility phase, the full-text versions of all remaining studies were independently assessed by the same reviewers for eligibility for the included phase. Any disagreements over the eligibility of studies were resolved through discussion between the reviewers and arbitration by the lead of the review team (DS), to avoid inter-reviewer variability.The PRISMA flow chart  demonstrRelevant data were extracted and collated into the JBI SUMARI online tool by six authors independently , using the data-extraction template provided ,18. ExtrFull-text articles were collated and data were extracted using the JBI SUMARI Tool . Six revThe eligible articles were all in vivo or ex vivo studies, with one study categorized as a Randomized Control Trial   whilst tThe quality and risk of bias assessments for the three articles included in the review were carried out using the appraisal tool created by JBI SUMARI ,23, and The relevant data collected for qualitative synthesis are summarized in two custom-made characteristic tables: Randomized Clinical Trial  and CohoComplications with dental implants leading to structural failure whilst functional within the oral cavity for a period of time is well documented ,25,26,27Analyzing the two cohort studies identified a range of factors that have the greatest impact on fractal behavior and longevity in one- and two-piece zirconia implants in the anterior region: diameter, sandblasting, occlusal load, age, coating, bruxism and location. Roehling et al. reported that zirconia implants with a reduced diameter of 3.25 mm had a survival rate of 58.5% in comparison to an implant with a diameter of 4.0 mm, which had a survival rate of 88.9% . In addiSurface characteristics, both macro and micro features incorporated into the implant surface, have been known to influence the osseointegration and thereby the survival of dental implants. Two studies discussed the impact of using alumina large-grit sandblasting on one- and two-piece zirconia implants ,22. TheyThe magnitude, frequency, duration and direction of the occlusal load endured on the implant-supported tooth has been well recognized as a factor in the overall success of implant therapy. When an implant undergoes occlusal loading under eccentric force, the force is dissipated to the supporting bone tissue. As such, the implant body must be designed in such a way as to reduce micro-movements and correct force dissipation. Scherrer et al. noted that the fractures of the first-generation implants developed under the cyclic load of masticatory forces . FurtherThe relation between patient age and implant survival was reported in one of three included studies. Roehling et al. noted that the highest seven-year survival rate was in patients between 40–59 years, compared to patient groups aged between 19–39 years or 60–85 years . This hiFractographic analysis of the implant microstructure has been used to confirm the presence of a transgranular fracture within the bulk of the implant, suggesting that this occurs when cracks or defects are inherent within the coating and arise during processing prior to sintering of the implants. These cracks present within the coating were reported to reach the implant bulk and influenced the location of crack propagation . FurtherAll included studies acknowledged the limitations within their study design. Due to the inherent limitations of a retrospective cohort study, the authors were unable to perform follow-up or comparative analysis of their results, thus making it difficult to relate to clinical-based scenarios due to limited information on the original scenario for implant fracture. Specifically, the Roehling et al. study on zirconia implant fracture, although insightful, came with its limitations . AssessoAlthough the study by Paolantani et al. is a randomized control trial, the risk of selection bias is high, as the assignment of participants to treatment groups were chosen with coin-toss randomization . This crEvolution in ceramic manufacturing technology has led to significant and predictable improvements in the characteristic of dental materials. Recently, the focus has shifted towards additive manufacturing techniques for fabrication of customized root-form zirconia implants utilizing a combination of stereolithography and digital light processing ,30. HoweThere were some limitations within this systematic review. Only three studies satisfied all the inclusion criteria limiting the extent of data available for consideration. Furthermore, this paper only considered implants placed in anterior dentition, and the factors that were implicated in this review may not be applicable to posterior dentition due to a significant difference in the magnitude and direction of forces applied on the implant.This systematic review discusses the application of zirconia implant designs within the anterior region and their associated factors influencing fracture and incidence. Despite short term trials, literature confirms the benefits of anterior zirconia implants having good technical and biological results, and there is currently limited clinical evidence on the long-term results of two-piece zirconia implants. Although various factors implicated in zirconia implant fractures have been identified as sandblasting, implant diameter, occlusal load, age and implant coating, there is limited quantitative assessment to gauge the fracture resistance of two-piece zirconia implants clinically. Hence, further research into long-term survival rates to provide evidence on the fracture resistance of two-piece zirconia implants is required, to support evidence-based designs and guidelines for routine clinical use, specifically in the anterior region of the oral cavity.


The aim of the study was to evaluate the impact of selected fatty acids: palmitic, stearic or oleic on functional properties of cassava and wheat starches. Effectiveness of complexation procedure was evaluated by determination of complexing index, lipids content and fatty acid composition. Native starches and their complexes with fatty acids were analyzed for water binding capacity and solubility in water, in vitro digestibility, pasting characteristic and rheological properties. On the basis of the obtained results it was found that values of complexing index values for both starches complexed with oleic acid were remarkably higher than those determined in palmitic or stearic counterparts. Starches complexation with fatty acids resulted in a significant increase in the percentage share of used fatty acid in total fatty acid composition. Presence of oleic acid in cassava starch significantly limited its water binding capacity and solubility in water. Complexation with all fatty acids used in the study resulted in an increase in final viscosity and rheological stability of cassava starch pastes. Wheat starch complexes with fatty acids were characterised by higher pasting temperatures as compared to non-complexed counterpart, with the greatest effect observed for starch–oleic acid complex. In most cases complexation of starches with fatty acids contribute to an increase in contents of resistant starch fraction. Analysis of rheological model parameters revealed that procedure of oleic acid complexation increased by twice the values of consistency coefficient, and significantly decreased flow index of both wheat and cassava starches.The online version contains supplementary material available at 10.1007/s13197-021-05153-x. Fatty acids as a part of triacyloglycerols are the principal contributors of dietary fat in human diet. A major methabolic role of fatty acids is as a source of energy, as a substrates for cell membrane biogenesis or as a precursors of many intracellular signaling molecules  with a hydrophobic molecules including fatty acids  were obtained from Sigma-Aldrich Chemical Company . All other chemicals used in the study were of analytical grade.Cassava(C*CreamGel 70001) and wheat (C*Gel 20006) starches provided by Cargill BV  were used in this study. Amylose contents in cassava and wheat starches were 26.60 ± 1.89 and 35.05 ± 0.78, respectively. The contents of protein and ash were 0.37% ± 0.01 and 0.16% ± 0.01 in cassava starch, or 0.35% ± 0.02 and0.19% ± 0.01 in wheat starch. Oleic (OA), stearic (SA), and palmitic (PA) acids were purchased from Chempur . α-amylase from porcine pancreas (type VI-B) and amyloglucosidase from The complexation process of starch with fatty acids  was carried out following a procedure described by Wang et al. . Fatty a2 in distilled water). The maximum absorbance of analyzed starch derivatives with iodine complexes was measured at 690 nm. Native cassava and wheat starches were used as reference sample. CI values were calculated as follows (1):Acontrol is the absorbance of the solution of starch without added fatty acids, and Asample is the absorbance of the solution of starch complexed with fatty acids.Starch–fatty acid complexes were examined for the complexing index (CI) according to Wang et al. . InitialIn order to determination of total lipids content the lipids were Soxhlet extracted from tested starch preparations in a Büchi B-811 extractor  with petroleum ether at 80 °C. The amount of total lipids was expressed as a sum of the extracted lipids after evaporation of organic solvent from the recipients and its drying to a constant weight.2SO4) and dried using nitrogen at 45 °C.Prior to analysis of fatty acid composition, lipids were extracted from tested samples using chloroform–methanol mixture (2:1 v/v) according to slightly modified Folch et al.  procedurf 0.25 μm, Sigma Aldrich Chemical Company, St. Louis, MO, USA). The temperature of the column was programmed from 160 °C to 210 °C at 3 °C/min, then kept constant at 210 °C for 25 min. A helium was used as a carrier gas and a flow rate was set to 1 mL/min.The injector and detector temperatures were 220 °C and 250 °C, respectively.Afterwards, fatty acids present in extracted lipids were esterified according to standard analytical method 991.39 AOAC . The comg for 30 min, the supernatant was poured out in a glass pan and precipitate was weighed (Wp). The supernatant was dried in an air oven at 100 °C to constant weight (Ws). The water binding capacity of starch (2) and its solubility in water (3) were calculated as follows:Water binding capacity and solubility in water of tested starch–fatty acid complexes at temperatures of 60 °C, 70 °C and 80 °C were determined by the method described by Li et al. . Starch t) were obtained from viscograms.Effect of cassava or whet starches complexation with fatty acid on their pasting characteristic were determined by a Rapid Visco Analyzer (RVA) . The pasting parameters of 8% (w/w) starch suspensions were measured using the following temperature program. The slurries were first held at 50 °C for 1 min, then heated to 95 °C within 5.5 min, and held at 95 °C for 5 min. The hot samples were subsequently cooled to 50 °C within 5 min, and maintained at 50 °C for another 5 min. The agitation speed of paddle during the run was 160 rpm. Pasting parameters including: peak viscosity at heating (PV), hot paste viscosity at 95 °C (HPV), final viscosity at 50 °C (FV), breakdown value (BD = PV–HPV), setback value (SB = FV–HPV), and pasting temperature :n;−1; and n – flow behaviour index.The rheological properties of 5% (w/w) starch pastes were evaluated by shear flow curves measurement using a rotational rheometer Rheolab QC  with system of coaxial cylinders . Tests were controlled by Rheo Compass software . Starch pastes were obtained by heating the 5% (m/m) starch suspension in boiling water bath for 30 min at constant stirring. Prepared pastes were placed in measuring system, relaxed and thermostated at 50 °C. Afterwards, samples were measured at a shear rates ranging between 1 sr) were calculated to describe the association between selected starch properties.All determinations were performed at least in triplicate. The obtained data were evaluate by one-factor analysis of variance, and significant differences were determined by Fisher’s test at significance level of 0.05. Pearson correlation coefficients . Fatty acids used in the study were probably partially complexed with amylose and partially cover the starch surface with a film . In native cassava and wheat starches the dominant fatty acid was palmitic acid .The pasting characteristic parameters of native and complexed with fatty acids starches are shown in Table The starch–fatty acids complexes were analyzed for contents of different starch fractions depending on in vitro digestibility Table . ComplexK) values corresponds to the apparent viscosity changes of the tested starch pastes. Introduction of fatty acids into native starches in most cases resulted in higher values of consistency coefficient. An exceptions were the pastes of wheat starch complexed with palmitic or stearic acids which did not differ statistically in values of this parameter from uncomplexed starch. It was concluded that pastes formed by starches complexed with oleic acid were more viscous compared to native counterpart. Their K values were about two times greater than those determined for native starches. Obtained results are in good agreement with final viscosity values determined using RVA (Table n) of all tested samples was found to be less than one  indicating more resistance to shearing forces compared to wheat starch pastes.Pastes formed by cassava starch complexed with fatty acids exhibited lower values of shear stresses in the examined shear rate range compared to the system prepared from native starch . Additionally, introduction of oleic acid into cassava starch resulted in the greatest decrease in values of shear stress. When comparing the flow curves of wheat starch systems it is evident that paste formed by complex of starch with stearic acid showed the lowest values of shear stress. But still, systems prepared with native wheat starch and its complexes with stearic or palmitic acids did not differ between each other. Values of Power-Law Model used to describe the experimental flow curves are collected in Table ne Table , which dne Table . After wPerformed wheat and cassava starch complexation with palmitic, stearic or oleic acids was effective as was corroborated by determinations of complexing index, lipids content and fatty acid composition. In both cassava and wheat starches the presence of oleic acid resulted in higher complexing index compared to starch complexes with other fatty acids. Procedure of cassava starch complexation with oleic acid decreased in the highest extent its water binding capacity and solubility in water. An increase in final viscosity and rheological stability of cassava starch pastes was observed as a result as a result of cassava starch complexation with all fatty acids used in the study. Pasting temperatures of wheat starch complexes with fatty acids were higher compared to non-complexed counterpart with the greatest effect observed for starch with oleic acid complex. In most cases complexation of starches with fatty acids contribute to an increase in contents of resistant starch fraction. Procedure of oleic acid complexation increased by twice the values of consistency coefficient and significantly decreased flow index of both native wheat and cassava starches.Supplementary file1 (DOCX 135 kb)Below is the link to the electronic supplementary material.


Ginkgo biloba L. is an important economic tree species in China, and its kernels have been used as a popular food in Asian countries. Herein, the morphology, basic chemical components, starch granule structures, and physicochemical properties of kernel flours from seven ginkgo cultivars were investigated, and their relationships were analyzed. The kernels were oval or spherical in shape, with variable sizes. The starch granules exhibited both regular and irregular Maltese cross patterns. Amylose was mainly distributed in amorphous growth rings. A spatial variation in the 865/942 cm−1 ratio was observed within individual starch granules. Variations in total starch content, apparent amylose content (AAC), crude protein content (CPC), total amino acid content (TAAC), starch fine structure, and thermal and pasting properties were observed among the seven kernel flours. Pearson correlation coefficients and principle component analyses showed that the thermal properties were affected by kernel CPC, TAAC, AAC, and starch fine structure, while the pasting properties were affected by AAC and starch fine structure. Furthermore, experiments showed that the seed protein structure and α-amylase activity affected the pasting properties of ginkgo kernel flours. Ginkgo biloba L. is among the most primitive extant gymnosperms and a “living fossil” plant [G. biloba L. is among the most important economic tree species in China.l” plant . In Chinl” plant ,3, wherel” plant ,5. Ginkgl” plant . Owing tl” plant . Furtherl” plant ,9,10. ToA-type crystallinities, with a degree of crystallinity of 27–41%. These granules have an oval or spherical shape with a central hilum, containing high levels of A and B1 chains of amylopectin [When eating ginkgo kernels as an intact grain, consumers prefer cooked ginkgo kernels, which have a soft and sticky texture. Ginkgo kernels are the main edible part of the seed, containing 60–70% starch, 10–20% protein, 2–4% lipids, 0.8–1.2% pectin, and about 6% sucrose, on a dry weight basis ,12. Furtlopectin ,18,19. Wlopectin . The endlopectin . Furtherlopectin . These sThe grain quality of ginkgo largely depends on the physicochemical properties of kernel flours. Seven ginkgo cultivars commonly found in South China were used in this study. Their seed kernel morphology and components, together with starch granule fine structure, thermal and pasting properties of the corresponding kernel flours, and their interrelationships were investigated. Furthermore, in vitro experiments were conducted to analyze the effects of protein and starch structures on their physicochemical properties. This paper not only provides a comparative profiling of kernel starch and protein in terms of their structure and components, and their functional impact on the physicochemical properties of kernel flours, but also provides vital information for the potential improvement of ginkgo kernel quality for food and industrial applications.G. biloba L. cv. ‘Qixingguo’ (QXG), G. biloba L. cv. ‘Dafozhi’ (DFZ), G. biloba L. cv. ‘Dongtinghuang’ (DTH), G. biloba L. cv. ‘Fozhi’ (FZ), G. biloba L. cv. ‘Maling’ (ML), G. biloba L. cv. ‘Dalongyan’ (DLY), and G. biloba L. cv. ‘Longyan’ (LY). Mature ginkgo seeds were collected from ten plants of the same variety at the Ginkgo experimental farm in Yangzhou University, in early October 2019.Seven Ginkgo cultivars were selected in this study, namely The mature ginkgo seeds were harvested by a stripping machine (Xing Yuan-Xin/BGTP), and the nuts were dried in the shade for about a week. The ginkgo kernels were then shelled manually and dried in an oven, at 37 °C, for one week, prior to milling into flour, using a sample crusher .The seed, nut, and kernel of ginkgo were photographed, using an EOS-60D Canon digital camera, as previously described . The cel2 and 2% KI, w/v). This mixture was diluted to 50 mL with distilled water, and the absorbance was measured at 620 nm. The amylose content was determined from a standard curve developed by using amylose and amylopectin blends. The crude protein content (CPC) was measured by using a nitrogen analyzer  according to AOAC standard method 990.03 [g for 10 min. Following removal of the supernatant, the pellet was dried by blowing with a nitrogen stream. The dried samples were then dissolved in Na-S buffer (1 mL) for 30 min in a mixer and extracted for 10 min by ultrasonication. After centrifugation , the supernatant was collected and filtered through a 0.45 μm nylon membrane syringe filter  before instrumental analysis. The relative amino acid content was calculated and normalized with the level of L(+)-norleucine. For each measurement, two technical replicates were performed.The moisture content (MC) was measured by using a halogen moisture analyzer  . The totd 990.03 . The totd 990.03 . Brieflyg for 10 min. The sediment was then washed with distilled water and centrifuged for 10 min, which was repeated five times. For lipid removal, the starch sediment was further washed with ethanol (100%) twice prior to adding CHCl3/methanol . Following incubation at 45 °C in a shaker with speed of 200 rpm for 2 h, the resulting starch slurry was centrifuged at 3600× g for 10 min. The starch precipitate was washed four times with 85% ethanol prior to drying in a convection oven at 40 °C for 48 h.Ginkgo starch was isolated by using the protease method, as described by Lu with some minor modifications . Brieflyw/v) was mounted on a microscope slide in 50% glycerol aqueous solution. The morphology and Maltese cross pattern of starch were observed by confocal laser scanning microscopy  under bright field and polarizing light models, respectively. Starch granules were stained with 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt (APTS) (Beckman Coulter) prior to suspension in 50% glycerol (0.5 mL) and analyzed by using CLSM, as described by Naguleswaran [A diluted sample of the starch slurry obtained above  with a step size of 1.0 μm and an exposure time of 0.01 s per pixel, performing 10 scans per image, ranging from 200 to 2000 cm−1. Several sections of starch from seven ginkgo cultivars were examined, and images were recorded from different areas in each section. Raman images were analyzed by using Thermo Scientific OMNIC xi Raman imaging software .Raman images were acquired on a Thermo Scientific DXR xi Raman imaging microscope . Raman spectra were collected over a 30 × 30 μmTo), peak temperature (Tp), conclusion temperature (Tc), and enthalpy of gelatinization (ΔH), which were set as default. Enthalpies were calculated on the kernel flour dry-weight basis. The pasting properties of kernel flours were analyzed by using a Rapid Visco Analyzer (RVA)  [3; 0.5 mmol/L), or dithiothreitol solution . The viscosity was determined by using a standard procedure, as described by Zhang et al. [Thermal properties of both the native and gelatinized kernel flours were investigated by using differential scanning calorimetry (DSC)  [stralia) . Brieflyg et al. .p < 0.05), and the bivariate Pearson correlation. Principle component analysis (PCA) was performed by using the R programming language to further analyze the effects of starch and protein structure on the physicochemical properties of ginkgo kernel flour.All data are presented as means ± standard deviation of three replicates. SPSS 25.0 statistical software was used to compare the data from one-way analysis of variance (ANOVA), Tukey’s test  within the individual starch granules [−1 ratio, in both the vertical and horizontal direction regions, varied among the ginkgo starches derived from different cultivars, ranging from 0.69 ± 0.05 to 0.96 ± 0.07, and from 0.33 ± 0.02 to 0.53 ± 0.03, respectively (−1 ratio was used to determine the ratio of linear residues to branched residues (branching) within starch granules [−1 ratio corresponded to a higher linear-to-branched ratio. The ratio in the map was homogeneous for ginkgo starches, both within individual and between starch granules within a cultivar  that were reported in our previous study [−1, which were lower than the corresponding values of ginkgo starch (9.34–12.86 J·G−1) in our previous report [The gelatinization properties of the seven ginkgo kernel flours were determined by DSC. The seven ginkgo kernel flours showed similarly broad endothermic peaks . The gell flours . The To,us study . The ΔH s report . It suffs report . It is pTo’, Tp’, and Tc’ retrogradation temperatures and retrogradation enthalpy (ΔretH) varied among the seven ginkgo kernel flours, giving ranges of 44.00–46.70 °C, 54.80–58.80 °C, 66.90–68.45 °C, and 3.11–4.24 J·G−1, respectively . The AAC positively affected Tp’  and Tc’ . The CPC was significantly negatively correlated with ΔgelH . The 865/942 ratio was significantly positively correlated with Tc’, but negatively correlated with ΔgelH and To’. The 952/942 ratio was negatively correlated with Tc’, but positively correlated with ΔgelH and To’. PCA was performed to further analyze the relationships between the basic components and thermal properties of kernel flours from the seven ginkgo cultivars  and the 865/942 cm−1 ratio , while the PV , HV , FV , and SV  were positively correlated with the 480/942 cm−1 ratio, respectively. A negative correlation was observed between the 952/942 cm−1 ratio and PTemp . PCA was also performed, with PC1 and PC2 describing 50% and 19% of the variance, respectively , which is an α-amylase inhibitor, to the kernel flours, in comparison with adding water as a control. When AgNO3 solution was added to the ginkgo kernel flours, significant increases in the PV, HV, BV, and FV were observed, accompanied by large decreases in the SV, pasting time, and pasting temperature  were, to a large extent, determined by α-amylase activity. Furthermore, with the aid of chemical analysis, it was concluded that the gelatinization properties and retrogradation properties of kernel flours were affected by CPC, TAC, AAC, starch crystallinity, starch chain orientation, and starch branching. This study may help elucidate the relationships between the starch structure and physicochemical properties of ginkgo kernel flours, and provide vital information for ginkgo kernel applications in both food and non-food industries.


Background. Interfacial failures at the cement‒restoration interface highlights the importance of effective surface treatment with no adverse effect on the zirconia’s mechanical properties. This study aimed to determine the effect of different surface treatments on dental graded zirconia’s surface roughness and certain mechanical properties.Methods. Forty sintered zirconia specimens were randomly divided into four groups (n=10): control (no surface treatment), sandblasting (SA), grinding with diamond bur (GB), and Er,Cr:YSGG laser (LS). Following surface treatment, the surface roughness and surface topography of the specimens were examined. X-ray diffraction (XRD) was conducted. In addition, the biaxial flexural strengths of specimens were evaluated. The data were analyzed using one-way analysis of variance (ANOVA) and post hoc Tukey tests; the Pearson correlation coefficient was calculated between either volumetric percentage of monoclinic phase or roughness and flexural strength of specimens (α=0.05).Results. The GB group exhibited significantly greater surface roughness compared to the other groups (P < 0.005). The LS and control groups exhibited a significantly lower volumetric percentage of the monoclinic phase (P < 0.001) than the GB and SA treatments. The SA group exhibited significantly higher flexural strength than the control (P = 0.02) and GB groups (P < 0.01). Furthermore, the Weibull analysis for the LS showed higher reliability for the flexural strength than other treatments.Conclusion. Er,Cr:YSGG laser treatment, with the lowest extent of phase transformation and reliable flexural strength, can be a promising choice for surface treatment of zirconia. In this regard, the stabilized tetragonal zirconia (Y-TZP) exhibits some superiority over other ceramic types with improved mechanical properties, biocompatibility, and esthetic characteristics.7 They are commonly caused by the weak interfacial strength between the cement and ceramic crowns that cannot tolerate the mechanical stresses in the oral environment.8Despite its robust mechanical properties, zirconia crowns clinically suffer from interfacial failures.9 In restorative dentistry, this can be achieved by combining multiple methods, including some well-established treatments, such as grinding/abrasion with a diamond bur, sandblasting, acid etching, and silanization.12 More recently, laser irradiation, including Er,Cr:YSGG laser, has been proposed in this regard.14 Lasers can gather and concentrate high magnitudes of energy on target areas. Lasers, in some cases, induce chemical reactions that alter the shape and, in other situations, only cause physical changes. Er,Cr:YSGG laser has the potential to remove particles through a mechanism called ablation, including micro-explosions and vaporization.15 During vaporization, the internal pressure builds up within the tissue until the inorganic material is explosively destroyed before the melting point is reached.13 However, laser treatment needs special care for any use in dental crown surface treatment.There are different methods to improve the interfacial adhesion, and the purpose is to increase the surface area and decrease the stress levels at the interfaces.16 Kurtulmus-Yilmaz et al,17 Liu et al,18 and Kosmac et al19 have determined the effects of different surface treatments on the zirconia and have reported contradictory results.Martins et al,. This study aimed to evaluate the effect of different surface treatments, including grinding by a bur, sandblasting, and laser irradiation, on the properties of dental graded zirconia. The null hypothesis was that these surface treatments do not affect the surface roughness, surface topography, and flexural strength of zirconia specimens.20A pre-sintered 98-mm zirconia block  was milled using a computer-aided design/manufacturing system to produce forty disk-shaped specimens with a diameter of 12 mm and a thickness of 1.2 (±0.2) mm. The disks were sintered at 1450ºC according to the manufacturer’s instructions in the furnace  and then polished with 600-, 800-, and 1200-grit silicon carbide papers (Struers A/S) for a minimum of 30 minutes under a 10-N load using a grinding/polishing machine  at 300 rpm.The finished specimens were randomly divided into a nominally flaw-free control group evaluated directly (n = 10) and three other groups with different surface treatments as follows:Grinding with bur (GB): The specimens in this group were processed using a high-speed hand tool with a diamond bur  (Germany)  at 200-kSandblasting with alumina (SA): The specimens were subjected to sandblasting by 110-µm aluminum oxide particles with 2.5-bar pressure at a 30-mm distance for 30 seconds.Laser treatment (LS): The specimens were subjected to Er,Cr:YSGG laser irradiation  with 2940-nm wavelength fiber-optic system (1 mm in diameter) and 400-µm diameter head handpiece for 10 seconds under 80% water (32 mL/min) and 80% air settings. The laser was irradiated at a 1-mm distance from the surface in non-contact mode. The laser pulse in this study was 74 µs at 1.5-W power output and the frequency of 15 Hz. The proper power output for the laser was selected through a pilot study on four extra disc-shaped samples irradiated at 1.5, 2.25, 3, and 3.75 W . Then, the surface topography of the specimens was examined under a scanning electron microscope (SEM)  at ×600 magnification. There was less surface damage in the specimens treated with 1.5-W power intensity  and peak-to-valley height (Rz) of the specimens were assessed under an optical microscope  equipped with a ×1000 objective lens, following the ISO 25178 .specimen .23:X-ray diffraction  was conducted to evaluate the relative percentage of the monoclinic phase on the treated specimens operated at 150 mA and 50 kV at 2Ø range, 5‒80 degrees, 0.02º step size and 50-second stop at each step. Three specimens were randomly selected from each group for this measurement. The relative percentage of monoclinic phase and phase transformation were determined from the integral intensities of the monoclinic M (-111) and M (111), and the tetragonal T (111) peaks according to the equation belowm is the ratio of monoclinic peak intensity, Vm is the volumetric percentage of the monoclinic phase content, Im (-111) and Im (111) are the severity of monoclinic peak at 2Ø = 28.2°, 2Ø = 31.4°, and 1t (111) is the intensity of tetragonal phase at 2Ø = 31.1°.where X24The biaxial flexural strengths were evaluated using a universal testing frame  with a 1000-N load cell. The specimens were placed on a fixture with three equidistance stainless steel spherical balls, distributed on the periphery of a 10-mm diameter circle. The load was applied at a rate of 1 mm/min to the opposite side of the treated surface by a cylindrical head piston (1.4 mm in diameter) so that the treated surface was subjected to the flexural tension (ISO6872:2008).The biaxial flexural strength was calculated using the formula below:d is the thickness of the specimen. X and Y were also calculated as follows:where S is the flexural strength, P is the load required for fracture, and 1 and r2 are the radii of supported and loading balls, respectively, and r3 is the radius of the zirconia disk. The value of 0.25 was used for the Poisson’s ratio-ѵ of dental zirconia in the equation above. In addition, the two-parameter Weibull analysis was applied to characterize the flaw size distribution of different groups in this study following the method introduced by Quinn and Quinn.26 Based on the description of the Weibull distribution, the probability of failure can be defined as:where r0 is the characteristic strength, m is the Weibull modulus, and Pf denotes the failure probability. Taking double algorithm of eq. 6 yields:where σ is the failure strength for each test, σ0, and Weibull modulus, m.That would allow simple linear regression to calculate characteristic strength, σP < 0.05. Moreover, Pearson’s correlation coefficient test was carried out to find potential correlations between the variables.One-way ANOVA and post hoc Tukey tests were conducted using SPSS 22 (SPSS Inc.) to compare different groups in this study regarding roughness, XRD, and flexural strength. The level of significance was set at a) and peak-to-valley roughness (Rz) of different groups are summarized in a and Rz values for the control group were 0.19±0.05 μm and 1.36±0.14 μm, respectively. Pairwise comparisons between the groups showed that Ra and Rz roughness values in GB were greater compared with the other three groups (P < 0.005). However, there were no significant differences in surface roughness between the control, LS, and SA groups (P > 0.7).P < 0.001). As evident in P < 0.001) than the laser and control groups.A representative XRD pattern for a dental zirconia disk is shown in P < 0.01). The SA group (1023.0±74.8 MPa) exhibited a significantly higher flexural strength than only the control group (926.3±65.5 MPa) (P = 0.02) and GB (909.8±87.3 MPa) (P < 0.01). However, there were no significant differences between the flexural strength of the LS group (994.5±56.8 MPa) and the control, GB, and SA groups (P > 0.05).The mean biaxial flexural strengths of the control and treated samples are shown in 0), the LS (1020.2 MPa) and SA groups (1023.0 MPa) exhibited similar and the highest values among the tested groups. Those values for the control and GB groups were 947.5 and 955.3 MPa, respectively.Regarding the Weibull analysis, the probability of the flaw distribution and Weibull parameters are presented in a and Rz) and the biaxial flexural strength in control (P = 0.44), LS (P = 0.63), GB (P = 0.33), and SA groups (P = 0.98). Furthermore, there was no significant relationship between the volumetric percentage of the monoclinic phase and the flexural strength of zirconia in control (P = 0.560), LS (P = 0.516), GB (P = 0.632), and SA groups (P = 0.396).Pearson’s correlation coefficient test indicated no significant relationship between the surface roughness  were significantly higher than the corresponding values in other groups (P < 0.005). However, there were no significant differences in the surface roughness between the control, sandblasted (SA), and laser-treated (LS) specimens (P > 0.05).Concerning the surface roughness and topography, the results showed that the mean R27 Roughening the internal surface of ceramic restorations increases the surface area for penetration and polymerization of resin cement, leading to better adhesion.29 Both grinding and sandblasting could result in contamination removal, increased surface area, and enhanced wettability.31 Surface roughening by Er,Cr:YSGG laser is caused by the ablation of surface particles that can improve adhesion.32 In some few cases, discoloration and microcracks have been reported following laser irradiation.17 Extreme roughening and micro-cracks were also observed in our pilot study at higher power. However, lower power laser treatments resulted in more moderate roughness with no microcracks or discoloration on the surface . However, Weibull analysis showed that the laser-treated specimens exhibited more reliable flexural strength , implying that the distribution of flaw generated in laser treatment was more controlled and uniform.25Furthermore, phase transformation in the zirconia is a crucial tool to consider to evaluate surface treatment. The stresses induced during surface roughening processes could result in phase transformation in zirconia.P < 0.001) with comparable flexural strengths. These results are consistent with those of previous studies,42 which reported that zirconia’s structural integrity after laser treatment had promising durability. In this study, there were no significant differences in the flexural strength between the control, GB, and LS groups (P = 0.05). However, the Weibull characteristic strength and Weibull modulus showed that the GB group had lower reliability among all the other treatment methods. Furthermore, the monoclinic phase on the surfaces of the GB group increased significantly (P < 0.001). This finding contrasts with Kurtulmus‐Yilmaz et al,43 who reported superior reliability from the highest to lowest in the post-sintered grinding, post-sintered laser irradiation, and post-sintered sandblasting, respectively. On the other hand, Kosmač et al19 reported that the highest Weibull modulus was obtained in the control group, followed by the sandblasted and grinding treatment groups.This study showed that laser could roughen the surface, while the percentage of monoclinic phase in the laser-treated specimens was significantly lower than that of sandblasted ones  was significantly higher than the control and LS groups, the mean flexural strength value and Weibull modulus for the SA group were significantly higher than the control group and also comparable with that of LS group. This finding contrasts with a study by Hallmann et al,45 reported that the flexural strengths of the sandblasted and laser-treated zirconia samples were higher than the control group. However, no significant difference in the flexural strength was observed between the sandblasted and laser groups, consistent with the present study. In general, discrepancies in the flexural results can also be attributed to numerous differences in the treatment protocols, testing configuration, zirconia types, and sample size.On the other hand, Çağlar and YanıkoğluP > 0.05). This finding is consistent with another study.46 The authors stated that despite phase transformation in the samples as a result of treatment, the mechanical performance of Yttrium-Stabilized Tetragonal Zirconia (YTZP) did not deteriorate. Perhaps, volumetric phase transformation on the surface caused by different treatments is not large enough to adversely affect zirconia’s biaxial flexural strength. Other studies have also reported that grinding, laser, and sandblasting treatments on the post-sintered samples positively affected zirconia’s flexural strength.47Pearson’s correlation coefficient demonstrated no significant relationship between either the surface roughness or the volumetric percentage of the monoclinic phase and the biaxial flexural strength (Despite some promising results in some of the surface treatment methods for zirconia, the results of this study clearly showed the lack of common knowledge in the form of a standardized surface treatment method for zirconia. Finally, the effect of other variables, such as different surface treatments, different sandblasting particles, different grinding burs, different laser types and energies, aging and environmental durability, and fatigue responses, on the mechanical properties of zirconia should be evaluated further.Under the limitations of this study, the grinding of zirconia surfaces with a diamond bur resulted in high surface roughness, phase transformation, and deterioration of the flexural strength of zirconia. Sandblasting of zirconia surfaces by alumina with a great extent of phase transformation exhibited the highest flexural strength. However, a more reliable mechanical property concerning the flexural strength was obtained by the Er,Cr:YSGG laser treatment with less surface roughness and phase transformation in the zirconia. In addition, there was no significant relationship between the surface roughness or the extent of phase transformation and the biaxial flexural strength of zirconia.NY, MY, and TH were responsible for investigation and writing the original draft. SSS contributed to the concept, supervision, writing, reviewing, and editing. MAZ was responsible for formal analysis, data collection, writing, reviewing and, editing. SMRH contributed to the methodology, supervision, writing, reviewing, and editing. William M. Johnston for his expert advice throughout this project.The authors would like to acknowledge Dr.None.None.Not applicable.


There exists a dearth of supplementary programs to educate physician–scientist trainees on anti‐racism and topics surrounding social justice in medicine and science. Education on these topics is critical to prevent the perpetuation of systemic racism within the institutions of academia and medicine. Students in the Washington University School of Medicine Medical Scientist Training Program and the Tri‐Institutional MD‐PhD Program developed journal clubs with curricula focused on social justice and anti‐racism for the summer of 2020. In this article, we describe the impact of the Washington University journal club on the education of first year MD‐PhD students and summarize the progress to date. The role of the journal club in the midst of the “double pandemic” of COVID‐19 and generational systemic racism is discussed, highlighting the need for such supplemental curricula in MD‐PhD programs nation‐wide. As we embark on our physician‐scientist training in the community of St. Louis, and in the United States, where science and medicine have histories steeped in injustices towards communities of color, we sought to create a platform to educate ourselves in order to uplift these communities in our careers and personal lives. Recently, societal inequalities have been exacerbated by the COVID‐19 pandemic, with marginalized communities being disproportionately affected.As physician‐scientists in training, we have a fundamental responsibility to society to understand and respond appropriately to this “double pandemic.” Inspired by like‐minded students at the Weill Cornell Medical College, The Rockefeller University, and Memorial Sloan Kettering Tri‐Institutional (Tri‐I) MD‐PhD Program, we decided that creating a journal club to educate each other and discuss important issues regarding social justice and anti‐racism was a first step in our life‐long learning of these topics. In creating this journal club, we aimed to garner the knowledge necessary to understand historical traumas suffered by communities of color in the institutions of science and medicine. We felt the need to gain a deeper understanding of the inherent mistrust of the medical community that exists and how to dismantle it. These topics were addressed in the context of the social and structural determinants of health that already plague the health outcomes of populations of color. At the same time, this journal club served as a space for us to challenge each other to think about how to use our multifaceted roles as medical students and scientists, to address the issues that have led to poorer health outcomes for communities of color.Currently, there is an urgency to put an end to the COVID‐19 pandemic, but the pandemic of systemic racism has plagued this nation since its conception and has taken countless lives and oppressed countless others in the process. Therefore, it is just as urgent for all of us to work together to end systemic racism. As future physician‐scientists, this journal club provides a critical component of our training to best advocate for our patients and continue to push the medical and scientific establishments to fight towards racial equity, instead of being complicit in a system that has taken so many lives.2In July of 2020, the onslaught of institutional violence against marginalized communities in the United States prompted two of the largest MD‐PhD programs in the country, the Tri‐I MD‐PhD Program and the Washington University School of Medicine (WUSM) Medical Scientist Training Program (MSTP), to take action. The entering Tri‐I MD‐PhD class was concerned that the formal education on social determinants of health through the medical school was not extensive, rigorous, or frequent enough to explicitly focus on how science and medicine contribute to structural racism. With the support of the Tri‐I leadership, they created an intersectional anti‐racist journal club to learn how to recognize and begin to address their own internalized racism and biases, identify how modern‐day institutions continue to enforce racial oppression, and discuss concrete solutions and actionable items to implement. The works featured in the syllabus were based on resources that academics, historians, activists, artists, scientists, and authors  have put together over the years to learn, reflect, and shape behaviors that actively oppose racism and injustice.3The incoming MSTP students at WUSM in St. Louis became aware of the journal club started by the entering Tri‐I MD‐PhD class. Not only were we, as the incoming MSTP class, inspired to start our own journal club focused on these themes, but we were cognizant of the need to establish educational opportunities like this for MD‐PhD programs across the country. Since we had not officially started our formal MD‐PhD training, this journal club represented a timely opportunity to positively shape our class culture. The journal club was designed to serve as a safe environment to discuss social injustices in science and medicine within our MD‐PhD community. By sculpting the shared goals and values of our class around addressing bias, discrimination, and racial injustice, we hoped to go beyond enhancing our individual knowledge and influence positive change at a structural level.4The two main educational goals of the journal club were to increase our group awareness of the systemic racism that exists in the fields of medicine and academia, and to learn how to take specific and longitudinal actions to combat oppression and discrimination in our communities. Because of WUSM’s location in St. Louis, MO, specific consideration was given to the history of St. Louis to educate incoming students on the community they would be engaging with. Both scientific research and medical practice have long histories of exploiting marginalized communities in the United States, especially Black and Indigenous populations.For decades, medical school students and administrators have advocated for a specific curriculum centered around social justice.Unfortunately, patients, trainees, scientists, and physicians from marginalized groups continue to face injustice.As future physician‐scientists, this journal club provides a critical component of our training to learn how to best advocate for our patients and continue to push the medical and scientific establishment to fight towards racial equity. As MD‐PhD students, we function at the intersection of the medical field and scientific academia. In our unique and privileged position, we not only have an increased obligation to educate ourselves on the historical and current forms of institutional racism that persist in this country, but are dually empowered to effect change throughout our training and our future careers.5To design the structure of the journal club, all interested students communicated over email and attended two preliminary Zoom meetings. The purpose of these meetings was to brainstorm a syllabus, journal club structure, and community guidelines to be followed in each session. In designing our journal club leadership format, we decided to use an empowered leadership approach.In the preliminary meetings, we brainstormed the topics that we felt were critical to address in a curriculum focused on the injustices present in both science and medicine. A survey was completed by the participants and used to assess which topics were perceived as most important to cover during the five journal club discussions to be held over the summer. The top five most voted topics were: the history of race in St. Louis, environmental racism, race in science, health disparities and cost, and microaggressions in science and medicine Figure . The othOne week prior to each presentation, student presenters finalized a list of literature and media resources and sent out required readings to all journal club participants. The readings were chosen from academic journals, local and national news sources, as well as educational videos. There were no strict expectations for presentation content and resource lists, allowing each group to have creative freedom in choosing what and how to present so long as it followed our community guidelines to support respectful and thoughtful discussion. The freedom to individually design each presentation was believed to increase the level of presenter engagement and commitment to presentation topics. During the presentations, each group was responsible for teaching topic content, moderating group discussions, and fielding questions.One of the key components of the journal club was creating discussion questions related to the required readings and key learning objectives for each topic. These questions were proposed to the group during the presentation and were discussed at length. Some examples of discussion questions included: “How might social and biological interpretations of race be used in basic research?”, “How does environmental racism factor in health disparities?”, “What surprised you about your Implicit Association Test results?”, and “What is different about the current conversations surrounding race, policing, and protests, compared to those at the time of Ferguson?” Discussion questions were chosen by student presenters and they were formed to be open‐ended and evoke open conversation to dive deeper into the topics. They also served to connect the topics to our future profession as physician‐scientists.Following each presentation, resources and presentation slides were uploaded to a centralized folder on Dropbox so that the information could be accessed at a later time. All journal club meetings and presentations were held over Zoom to remain socially distanced. At the final journal club meeting, a post‐journal club survey was sent out to the group to determine overall satisfaction with the journal club and to assess comfort level and awareness of topics discussed throughout the summer. The survey was also used to guide further journal club programming to be continued throughout the school year.66.1Twenty‐one students were invited to take part in the journal club. Participation in the journal club was optional. Out of the class of 21 students, 4 chose not to attend. The remaining 17 students attended at least one meeting over the course of the summer. Weekly attendance varied between 11 and 15 students per session, although a formal attendance was never taken. The demographic composition of the attendees consisted of 44.4% White students, 38.9% Asian students, and 22.2% Hispanic or Latino students based on a self‐reported survey completed by the participating students. There were no participants from the following demographics: Black or African American, Native Hawaiian or Pacific Islander, American Indian or Alaska Native. Since 38.9% of our participants were temporary visa holders, and would not be considered as an underrepresented minority (URM) by the National Institutes of Health guidelines, we analyzed the demographic composition based only on race and ethnicity and not based on citizenship status. Overall, 22.2% of our participants came from underrepresented backgrounds as defined by the National Institutes of Health racial and ethnic categories.Surveys were administered through Google Forms both before and after our 5‐week curriculum. Questions were aimed at understanding the personal perspective of cultural competency, perceived understanding of racism, comfort discussing race, perceived understanding of systemic racism, perceived knowledge of the history of St. Louis with regards to racism, perceived understanding of microaggressions, degree of feeling empowered to act on eliminating racism, familiarity with Black pioneers in science and medicine, and perceived knowledge of how to deal with microaggressions. There was no formal assessment to test true knowledge of each subject, so all measures are indicative of each students’ perception of their mastery of the material. Fifteen students responded to both surveys. According to the pre‐survey results, 11 incoming students (73.3%) had previously learned about racism in an academic setting and 4 had not.One of our main goals in hosting a journal club was to discuss difficult topics, such as racism in medicine, in a safe, open environment, with the hope that this would facilitate collaborative learning. Based on the survey data Table , there w6.2During the period in which we worked to design the curriculum and identify topics of interest, it became abundantly clear that participants had a diverse array of perspectives on which issues were important for us to learn about as a cohort of physician‐scientists in training. Two approaches that contributed to the efficacy of the curriculum's execution were allowing students’ self‐identification of topics of interest and disseminating teaching responsibility uniformly throughout our class. Both of these decisions in parallel gave students a voice in the direction and nature of the teaching materials presented, and additionally set expectations to produce high‐quality lecture and discussion materials for our classmates. This utilized students’ varying familiarities with preparation materials across a wide range of topics  and sources  and produced a curriculum that was more interdisciplinary than could have been conceived otherwise. Doing so also capitalized on the respect fostered by our leadership structure by giving students the shared experience of facilitating a session. This ultimately increased engagement with the material and produced consistent attendance and participation across the cohort.The use of defined discussion questions presented to the group during each session allowed students to interact and get to know one another, and actively engage with the week's material. Using discussion questions interspersed throughout the presentation, we were able to stimulate conversation regarding the material at hand; the questions not only gave students a prompt to which they could respond, but also a starting point to share their own thoughts and solicit the perspective of others in the group. Questions aimed towards assessing pre‐existing knowledge of the concepts presented also allowed facilitators and other students to understand the prior perspective of their peers and catalyzed the collective building of definitions. The community guidelines helped ensure that students remained respectful and mindful of others, creating a safe space that made students more comfortable and willing to share thoughts and experiences, especially when discussion questions solicited personal experiences. While it was understood that students need only answer if they felt comfortable sharing, many students were willing to share their personal experiences with concepts such as microaggressions.6.3Washington University School of Medicine underwent a curriculum renewal this academic year. The new curriculum places an increased emphasis on longitudinal community engagement, education of students on social and structural determinants of health, and improved understanding of the ways that inequities manifest in healthcare systems. The Gateway Curriculum topics strongly aligned with the self‐identified topics chosen by the MSTP journal club participants. Consequently, our journal club discussions prepared us with the background and familiarity to engage with these concepts throughout our medical education.The Gateway Curriculum covered some of the topics previously discussed in our summer journal club, such as racism, ethics, and social justice in medicine, facilitating additional reinforcement and depth of knowledge. However, with our MSTP journal club, we were able to have intimate and open conversations amongst a smaller group of students with fewer time constraints as compared to the large lecture settings and substantially shorter discussions amongst students within the Gateway Curriculum. A second exposure to concepts such as microaggressions and bias, shared between the journal club's syllabus and the WUSM Gateway Curriculum, has allowed us to think about these topics not only through a medical lens, but also through a scientific one. Further, our journal club curriculum had a more in‐depth coverage of topics surrounding race in science. A deeper understanding of racial inequities in academia and science is especially useful to our MSTP class given our trajectory as physician‐scientists. We placed a large emphasis on learning how to conduct scientific research in an equitable, inclusive, and just way. We feel that longitudinal exposure to these concepts throughout our MSTP training will make our anti‐racism and social justice work more effective, impactful, and central to our careers one day as physician‐scientists.6.4At a time when health, safety, and social connection were dramatically impacted by the global pandemic, our journal club created a unique opportunity to socially connect with future classmates over topics that are critical to effectively addressing health and safety in our medical and scientific communities. After three planning meetings and five journal club sessions, participants felt comfortable contributing to a diverse array of discussions within our MSTP cohort. It was evident that the journal club dramatically enhanced class bonding, despite all encounters being virtual. The ability to establish class bonds through Zoom encounters was something both we as students and our administration were pleasantly surprised by. Not only did students get to know one another better through rich, and oftentimes personal, discussion, but presenter groups also bonded over creating a portion of the collective syllabus and coordinating a presentation of the material.The development of the journal club also coincided with the unexpected, and later retracted, Immigration and Customs Enforcement directive to deport F1 and M1 visa students participating in 100% online curricula in the coming semester.7There were challenges associated with executing an entirely student‐directed curriculum as the entering MSTP class during a global pandemic. One of these challenges is related to the empowered leadership structure that we adopted. While it was a strength that all participants had the opportunity to educate the class on a specific topic, none of us had expertise in the topics covered. This lack of expertise made it difficult to bring in opposing viewpoints, where different academic branches had different stances on a topic. For example, some academic sociology circles oppose the idea that there is any shared biological basis within a racial category, while there are trends in academic medicine which rely on race‐specific pharmacological or physiological differences to explain racial differences in outcomes and metrics.Another challenge we faced was taking specific actions based on and inspired by our curriculum. While COVID‐19 has posed an unprecedented obstacle in utilizing our knowledge, the desire to funnel our efforts to supporting community‐initiated programs with an emphasis on responding to the needs of the community has impelled us to pause and thoroughly research how we can help in a positive way. An avenue through which we may take action in the near future is through community engagement opportunities introduced by the facilitators of the WUSM Gateway Curriculum. These community engagement opportunities have been vetted by the facilitators as ways to collaborate with the community to bring about change and improve well‐being and health, while deferring to the expertise of organizations and activists local to St. Louis. Further, these opportunities are in response to conversations with community leaders to ensure that Washington University's actions are specifically addressing the needs that the community has voiced and intervening to the degree the community desires.8t‐test approach. In addition, the sample size was low because this journal club was proposed to our 21 person MSTP class, which resulted in low power in our analysis. The creation of the survey to measure the learning of self‐perceived growth in understanding health disparities and racism was difficult to quantify and does not necessarily correlate with objective learning. Eleven students already had a self‐perceived understanding of racism before attending the journal club, which made it harder to determine the impact of some of our sessions. An improved way to measure learning across this small cohort will be to create more data points as our journal club progresses and continue to implement surveys to understand the benefits of this type of engagement. For example, we could implement more objective measures of learning based on material taught and discussed during each journal club meeting over longitudinal timepoints, similar to previous studies focusing on assessing the effects of an indigenous health curriculum through knowledge recall or measures of ethnocultural empathy.While the journal club was designed to be voluntary, this limited the strength of our data analysis. A variable number of students joined our online discussion every week and the survey respondents from the beginning and end surveys were not paired. This variability in participants meant that statistics had to be analyzed using an unpaired student Another important limitation of our journal club was the lack of specific underrepresented groups, including Black and Indigenous students. In total, 22.2% of the participants of our journal club fit the NIH criteria of URM, contributing to the varied perspectives in our presentations and discussions. However, since anti‐racism was a major value behind the implementation of the journal club, the lack of Black students in our group was very apparent, which we discussed during our sessions. Presenters made sure to reference Black authors and experts, including them in mandatory and optional resources. However, this does not replace the voices of Black students. We have expressed this dearth of representation to our MSTP directors and we have been in communication with upper year Black students in the WUSM MSTP. We have voiced our support of their efforts for increased diversity and inclusion in the program, we have solicited input on topics and resources for our future journal clubs, and we have opened the opportunity for them to join our journal club to share their experiences and perspectives. The MSTP office has responded to the concerns of our classmates by hiring a Diversity, Equity, and Inclusion administrator and focusing on undergraduate diversity recruitment programs. Further, the MSTP students and administration gathered at a virtual Town Hall meeting to discuss and begin the creation of a student‐run “Diversity Sub‐Committee" within the WUSM MSTP. This sub‐committee will work on recruitment programs, education, and improvement of MSTP culture around racism.Our exact journal club structure may be difficult to replicate across other MSTP programs since the topics of discussion were chosen to be specific and connected to the St. Louis community. We believe that this approach of planning, implementing, and reflecting is still very valuable for other cohorts that are interested in addressing health disparities in science and medicine. This curriculum was created in the time of a global pandemic and was completely virtual, so this model may look different for future years where a shift back to in‐person learning may occur. In addition, there was no centralized leadership for this journal club. This made the presentations, content, and engagement completely voluntary. The main incentive to participate was the willingness to invest in learning about racism and health disparities in healthcare and research. Students that participated in this journal club felt empowered to participate in these discussions because there was a safe space and environment, which our MSTP directors contributed to. If other program directors wish to encourage students to start something similar, it is important to create a space where discussions about addressing racism are brought to the fore as a commitment of the program.9As MSTP students, we are training to play integral leadership roles in science, medicine, and the translational interface between the two fields. Given our future leadership roles, we will have the platform and authority to advocate for change and move our fields in directions that actively oppose racism and injustice. This is why it is especially important for us to be aware of different issues surrounding social injustice and to be comfortable discussing these issues and the different points of view around them.Additionally, as MSTP students, we will be residing in St. Louis for at least 8 years, which provides us the opportunity to be involved with the community in a longitudinal and significant manner. Long‐term community engagement would allow us to build trust and deeper connections with community leaders, making community members more comfortable and more receptive to our involvement in addressing their needs. To be able to truly make the most out of our time spent engaging with the community, we must be aware of social and structural issues that individuals face. The MSTP journal club provides the framework for us to learn, discuss, and repeatedly think about these issues. Repeated exposure and application of these frameworks will allow us to practice trauma‐informed care, community engagement, and ethical scientific research.As a cohort, we plan to continue this journal club throughout our tenure as MSTP students at WUSM. So far, we have had several organizational meetings and integrated feedback from the summer surveys in order to continue meeting as a journal club on a monthly basis. We plan to revisit the community guidelines each semester to ensure it still aligns with our goals. During the academic calendar year, we have continued to hold journal club meetings once per month for two hours. Discussions continue to be student‐lead and focus on different topics each month. Students are able to sign up to present articles, media, books, or other materials relevant to the topic for that month. We also plan on holding an action item meeting once per month, where we will discuss ways in which we can actively contribute to the St. Louis community. With the leadership skills we have and will continue to develop, we hope to make a long‐lasting impact in the St. Louis community.The inaugural MSTP journal club participants have two long‐term goals: to officially incorporate the “Social Justice in Medicine and Science Journal Club” into the WUSM MSTP curriculum and to facilitate the implementation of curricula like this in other MD‐PhD programs across the country. The long‐term effects of this journal club will positively benefit physician‐scientist communities at all levels of training. The Tri‐I MD‐PhD class also plans to continue their journal club in future years and has been working with Tri‐I program leaders to incorporate the journal club into the formal summer curriculum for future Tri‐I classes. Similar initiatives of social justice and anti‐racism journal clubs are currently implemented at only a few other institutions like the University of Cincinnati, University of California San Diego, and University of California Irvine, all of which appear to be mostly faculty‐led.10The development and implementation of a social justice and anti‐racism journal club for MSTP students at Washington University School of Medicine was motivated by an urgency for action during an extraordinary global pandemic which has reinvigorated the ongoing movement for racial justice, along with the police killings of Ahmaud Arbery, Breonna Taylor, George Floyd, and countless others. For us first year MD‐PhD students, the journal club provided us with not only an opportunity to learn about the ever‐present structural inequalities built into medicine and academia, but also a safe space where we could reflect on our current and future roles within these pre‐existing frameworks. In particular, the flexible “shared leadership” structure of the journal club was critical to our success in developing a well‐rounded curriculum and promoting respectful dialogue among students. Looking forward, we recognize that this journal club ultimately represents a small step in a lifelong journey to learn about and ultimately combat systemic racism in our future careers as physician‐scientists. However, we are committed to the continued evolution of this journal club as a vehicle for positive impact in our local and national communities during our longitudinal training as MD‐PhD students. We hope that our experiences will serve as a much‐needed prototype for student‐led social justice initiatives in medical education.


The use of heterografts is widely applied for the production of several important commercial crops, but the molecular mechanism of graft union formation remains poorly understood. Here, cucumber grafted onto pumpkin was used to study graft union development, and genome-wide tempo-spatial gene expression at the graft interface was comprehensively investigated. Histological analysis suggested that resumption of the rootstock growth occurred after both phloem and xylem reconnection, and the scion showed evident callus production compared with the rootstock 3 days after grafting. Consistently, transcriptome data revealed specific responses between the scion and rootstock in the expression of genes related to cambium development, the cell cycle, and sugar metabolism during both vascular reconnection and healing, indicating distinct mechanisms. Additionally, lower levels of sugars and significantly changed sugar enzyme activities at the graft junction were observed during vascular reconnection. Next, we found that the healing process of grafted etiolated seedlings was significantly delayed, and graft success, xylem reconnection, and the growth of grafted plants were enhanced by exogenous glucose. This demonstrates that graft union formation requires the correct sugar content. Furthermore, we also found that graft union formation was delayed with a lower energy charge by the target of rapamycin (TOR) inhibitor AZD-8055, and xylem reconnection and the growth of grafted plants were enhanced under AZD-8055 with exogenous glucose treatment. Taken together, our results reveal that sugars play a positive role in graft union formation by promoting the growth of cucumber/pumpkin and provide useful information for understanding graft union healing and the application of heterografting in the future. In production, the achievement of a well-developed graft union determines compatible or incompatible grafting. The reconnections of functional vasculature tissues that differentiate from calli between scions and stocks are essential for compatible grafts4. In China, approximately 30% of cucumber cultivars are grafted onto pumpkin to overcome soil-borne diseases and increase resistance against abiotic stress, and farmers utilize heterografting methods5. There is intricate communication between the two different species at the graft junction6. Many research studies have examined the mechanisms of graft union formation based on morphological and metabolic changes8. However, the molecular development mechanisms of heterografting are less well understood.Grafting is a widely used technique to improve horticultural crop product quality, quantity, and disease resistance, with effects on controlling tree vigor and fruit size9. Calluses, with cells that are close to the cut and attached to the opposing tissue, give rise to cambium, phloem, and xylem10. Calluses also fill the gaps between adhering tissues to strengthen the adhesion of opposing tissues12. However, the process of callus formation at the graft interface may be species specific. In grafted Arabidopsis hypocotyls, little callus is produced, although in many plants, callus formation is critical for effective grafting10. For plant samples that are cut and not grafted, significant amounts of wound-induced calli are formed, and transcriptional analysis indicates that wound healing proceeds via different mechanisms depending on the presence or absence of adjoining tissues13. Cell division is rapidly activated during cutting and grafting. Removal of the cotyledons results in the inhibition of cell division and wound healing in cucumber14. Sufficient connection of functional vascular bundles between the scion and rootstock is essential for the graft union of woody trees16. The establishment of phloem connections generally precedes xylem connection at the graft junction in Arabidopsis4. However, Arabidopsis scions that are grafted onto tomato rootstocks can flower and produce seeds in the absence of the scion/stock vascular connection17. In addition, plasmodesmata provide an important channel for cell–cell communication at the graft junction, with higher plasmodesmal coupling observed between callus cells than between cortex cells in stem unions of Prunus spp.18. Despite these important findings, a more detailed study of the anatomy of graft union development is needed for many crops, especially in the field of horticulture.Previous work describes a time course for graft formation at the graft junction, including cell adhesion, the formation of a necrotic layer of dead cells, callus formation through cell division, and finally the reconstruction of vascular bundles across the graft junction19. The absolute auxin level is not a key indicator of graft success, but how auxin is perceived may activate auxin responses involving ABERRANT LATERAL ROOT FORMATION 4 (ALF4) and may promote vascular connection at the graft junction4. The potential effects of other phytohormones  in graft formation are poorly understood, and other molecules  may have little effect19. Sugar accumulation in the grafted top and sugar depletion in the grafted bottom can cause asymmetric expression levels of sugar-induced genes  and sugar-repressed genes  in the scion and rootstock. This asymmetric gene expression is a critical feature of grafting and tissue reunion and may play an important role in vascular connections at the graft junction13. Adding 0.5% sugar to the grafting medium resulted in faster recovery after grafting and a higher graft success rate compared to 0% sugar plants20. Sucrose affects the quantity of calli deposited on sieve plates and further promotes the formation of discontinuous vascular bundles21. Sugar transporters SWEET15 and SWEET19 activate cell–cell communication between scion and rootstock cells through the CBL interacting protein kinase 14 (CIPK14) during the healing process9. Recent research demonstrates that β−1,4-glucanase, encoded by the member of the glycosyl hydrolase 9B (GH9B) family (GH9B3), is secreted into the extracellular region and functions in cell wall digestion and enables the success of Nicotiana interfamily grafting with a diverse range of angiosperms22.Plant hormones and sugars accumulate above the cut site and are depleted below due to the severity of vascular tissues24. Only one TOR gene was identified in Arabidopsis, and its complex1 (TORC1) consists of TOR kinase, the regulatory-associated protein of TOR (RAPTOR) and lethal with SEC13 protein 8 (LST8). In plants, TOR kinase activity is chemically inhibited by rapamycin, AZD-8055, TORIN 1, and KU-6379425. The repression of TOR always results in less biomass, smaller leaves and roots, whereas overexpression of TOR or kinase domains also causes abnormalities in Arabidopsis, such as severe shoot and inflorescence meristem defects26. Therefore, only a moderate increase in the TOR expression level (less than 2-fold) can promote root and shoot growth, cell size, and seed yield without visibly affecting plant morphology27. In the Arabidopsis meristem, glucose and sucrose can activate the S-phase in a CYC-CDK-RBR-independent fashion by activating TOR kinase, which directly phosphorylates E2F (a/b) transcription factors25. However, sugar acts independently of TOR activity in promoting maize germination28. Additionally, TOR protein kinase is also an energy sensor that regulates cellular homeostasis in balance with sucrose nonfermenting-related kinase 1 (SnRK1), which triggers adaptive responses during carbon/energy deficiency24. However, the function of TOR in graft union formation has not yet been studied.Sugars are upstream regulators of the target of rapamycin (TOR), a highly conserved master regulator that plays pivotal roles in controlling cell proliferation, cell size, transcription, photosynthesis, carbon and nitrogen metabolism, and autophagyIn this study, we performed an anatomical analysis and in-depth RNA-seq analysis to observe the cellular and transcriptional changes that occur during the development of cucumber–pumpkin heterografts. Furthermore, etiolated seedling grafting and the application of exogenous glucose or the TOR inhibitor AZD-8055 demonstrated that sugar and TOR play important roles in cucumber/pumpkin graft union formation. This study provides new insights into the understanding of the mechanism of graft union formation and the application of heterografting in horticultural plants.29  enrichment analysis of the DEGs for the comparisons of 3 d vs 0 d, 6 d vs 0 d, and 9 d vs 0 d in the scion and rootstock. Genes that were only differentially expressed at 3 DAG or 6 DAG in the cucumber scion were enriched in the “chloroplast”, “plastid”, “thylakoid”, “cell wall organization or biogenesis”, and “cell wall macromolecule metabolic process” pathways Figs. , S6. In The KEGG analysis also showed a significantly distinct response between the scion and rootstock Figs. , S8, S9.ApL3 and the sugar-repressed genes STP1, DIN6. We found decreased expression of CsApL3 and increased expression of CsSTP1 in both the scion and rootstock at 3 DAG. In addition, the expression of CsDIN6 was significantly increased only in the rootstock, i.e., not in the scion  activity showed similar development patterns in the scion and rootstock, with marked decreases from 1 DAG to 6 DAG, which potentially caused decreased raffinose and stachyose levels during graft union development Fig. . The actTo investigate the role of sugar levels in this process, we sprayed a range of concentrations of exogenous glucose solution  on the scion after grafting. The results showed that phloem reconnection was not significantly different under exogenous glucose treatments, and xylem reconnection occurred at 4.5 days after grafting with 0.5% exogenous glucose. However, the graft efficiency was lowered by higher levels of exogenous glucose Fig. S. Next, wTOR, small ribosome protein S6 kinase (S6K2), and E2F transcription factors (E2Fa), and the energy response factors SnRK1α and SnRK2. As shown in Fig. CsTOR, CsS6K2, and CsE2Fa was significantly downregulated in the scion of normal heterografts (WT/P) during graft union formation, whereas the expression levels of these genes were significantly increased with the application of exogenous glucose  signaling pathways possibly regulated graft union formation by balancing growth conditions and energy metabolism.To investigate whether TOR signaling pathways are involved in graft union formation, we also checked the expression of ess Fig. . Interes9. Previous studies have shown that this process is species specific, and there are significantly different responses between homografts and heterografts in Arabidopsis and grapes30. In this study, we performed an anatomical study and in-depth RNA-seq analysis using cucumber–pumpkin heterografts to observe the cellular and transcriptional changes that occur during the development of heterografts. We conclude the results in Fig. Graft union formation is an interesting and complex process31. In this study, the functional phloem connection occurred two days earlier than the xylem connection  and sugar levels exhibited a sugar-starvation response at the graft junction at 3 DAG. Sugar starvation affects several central aspects of development by regulating photosynthetic activities and carbon remobilization  signaling was first used to explain the normal TOR activity by glucose through glycolysis and mitochondrial bioenergetics to reprogram the transcriptome and activate meristems25. Depletion of endogenous sugars led to inhibition of TOR activity, whereas exogenously applied glucose reactivated TOR activity25. In this study, TOR expression levels were downregulated compared with those at 0 DAG and were upregulated after phloem connection during graft union formation. Exogenous glucose had a positive effect, whereas lower endogenous sugar had a negative effect on TOR expression  is the upstream signal for TOR activation, and the effect of TOR inhibitors could not be recovered by the feeding of exogenous sugars42. In our study, exogenous sugar still promoted xylem reconnection and growth of grafted plants under AZD-8055 treatment. We speculated that sugars might promote plant growth independently of the Glc-TOR pathways. Consistent with research on maize germination, Glc stimulation of growth requires TOR kinase during the first 48 h, and Glc could also have a positive effect on germination with the TOR inhibitor AZD-805528. Strikingly, TOR and S6K expression was significantly upregulated by AZD-8055 in the scion, especially at 3 DAG. On the one hand, the degree of TOR transcript repression does not always fully correlate with the amount of active TOR protein, which is affected by posttranscriptional regulation44. In addition, only the appropriate increases in the expression of TOR levels (less than 2-fold) positively stimulate root and shoot growth, and strong overexpression of full-length TOR results in developmental abnormalities27. Therefore, the precise regulation of both temporal and spatial TOR expression is critical and complicated for the growth of plants. However, E2Fa and SnRK1α were significantly downregulated and upregulated, respectively, by the application of AZD8055. These results indicated that the Glc-TOR pathway might play a role in promoting vascular reconnection and the growth of grafted plants during graft union formation. How TOR regulates graft union formation requires further study.AZD-8055 is a specific TOR kinase activity inhibitor and is always used to discover TOR functions in plantsts Fig. S. Graft eIn conclusion, we present a model Fig.  with graCucumis sativus L.) and pumpkin (Cucurbita moschata Dutch.) were planted in an artificial chamber at the Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences, Beijing, China. Plants were cultivated with a day/night (12/12 h) cycle at 25 °C/18 °C and 60–70% relative humidity. For etiolated cucumber, cucumber seeds were germinated on moisture filter paper in the dark at 28 °C for 2 days, and germinated seedlings were transferred to a growth chamber filled with vermiculite and grown in light incubators maintained at 28 °C:15 °C (day:night) under dark conditions with 50–60% humidity. The etiolated cucumber was readied for grafting when cotyledons had fully opened (6 d after sowing). The cucumber was grafted onto the pumpkin by splice grafting  acid fuchsin, and the cucumber hypocotyls were examined after 40 min17.Cut and separated shoot, cut and separated root, and grafted plant samples that were separated into scion and rootstock 1-10 DAG were cleaned with deionized water and weighed. The samples were placed in an oven at 105 °C for 15 min and then dried at 75 °C to constant weight. Eight to fifteen plants were weighed for each experiment, and three replicates were performed for each time point. Phloem and xylem connectivity were measured by CFDA and acid fuchsin movement across the graft union, respectively Fig. S. To assa45. Samples were sectioned to 10 μm vertically using a rotary microtome , dewaxed, rehydrated, cleaned, stained with Fast Green, counterstained with safranin, and then fixed with neutral balata. Sections were examined using a light microscope , and representative sections were photographed.The samples were collected at 0, 1, 3, 6, and 9 DAG. The collected samples were placed in FAA for 2 days and then dehydrated through an ethanol/xylene series followed by a xylene/paraplast series. The samples were then embedded in paraplast and sectioned as described previously® UltraTM RNA Library Prep Kit for Illumina®  following the manufacturer’s instructions. Briefly, the steps included mRNA enrichment, mRNA fragmentation, second-strand cDNA synthesis, size selection, and PCR amplification as previously described46. The prepared libraries were then sequenced on an Illumina HiSeq 2000 platform .Total RNA was isolated from different samples using RNeasy plant mini kits  according to the manufacturer’s instructions and treated with RNase-free DNase I  to degrade genomic DNA. The quality and quantity of RNA were checked by a Nanodrop 1000 Spectrophotometer  and Agilent 2100 Bioanalyzer . A total of 3 μg RNA per sample was used for cDNA library preparation. Sequencing libraries were constructed by the NEBNext48. The reference genome and gene database were downloaded from an open genomic resource platform (http://cucurbitgenomics.org)49. The reads were then assembled through StingTie48. Based on protein database annotations (NCBI nonredundant (NR), Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes), sequences with the highest similarities were classified by KEGG and Gene Ontology (GO) using Blast2GO. The sequenced raw data in our study were deposited in the NGDC GSA database .To ensure the accuracy of the data, low-quality reads were filtered by FastQC software. Qualified reads were mapped to the reference sequences using HISAT2 with the default parametersArabidopsis database (http://www.arabidopsis.org/index.jsp). DEGs significantly enriched in functional classifications or pathways at 3 d vs 0 d, 6 d vs 0 d, and 9 d vs 0 d were identified through GO and KEGG enrichment analyses.The gene expression level was calculated based on TPM (transcripts per million). The DEGs of the scion/rootstock combination at 3 d vs 0 d, 6 d vs 0 d, and 9 d vs 0 d after grafting were identified as genes with absolute fold change >2 and FDR < 0.01. Homology gene function prediction was performed using the 51 with minor modifications. Samples (2 g FW) were harvested from the scion part 3 cm above the graft junction and rootstock part 3 cm below the graft junction at 0, 1, 3, 6, and 9 d after grafting. Samples were extracted three times in 80% (v/v) ethanol for 45 min at 80 °C. The extracts were combined, decolorized with activated carbon, and evaporated to dryness at 40 °C with applied vacuum. The dried extracts were redissolved in 500 μL distilled water and filtered through a 0.45 μm filter. Next, 20 μL samples were analyzed for the carbohydrate content using high-performance liquid chromatography (HPLC) (Agilent 2100 system) and a Sugar-Pak I column . Separation was performed at 75 °C with water as the eluent at 0.5 mL min−1 and a refractive index detector (G1362A RID Agilent USA). Eluted sugars were identified and quantified based on retention time and peak heights of sugar standards (Sigma). Our study specifically focused on the contents of sucrose, raffinose, galactinol, and stachyose. Starch content was measured using a Starch Content Assay Kit  following the manufacturer’s protocols.Soluble carbohydrates were extracted and assayed according to previous reports50 with minor modifications. Samples (2 g FW) were finely ground in a chilled mortar with four volumes of chilled extraction buffer containing 50 mM HEPES–NaOH (pH 7.0) and 20 mM 2-mercaptoethanol. The extract was then centrifuged at 28,000 × g for 20 min. The resulting supernatant was used for the STS activity assay after being dialyzed with 20 mM HEPES-NaOH buffer (pH 7.0) containing 20 mM 2-mercaptoethanol for 16 h at 4 °C. Dialyzed extract (100 μL) was added to 100 μL reaction buffer containing 50 mM HEPES-NaOH (pH 7.0), 20 mM 2-mercaptoethanol, 10 mM galactinol, and 40 mM raffinose. Normals were similar but lacked galactinol. Reaction mixtures were typically incubated at 25 °C for 90 min and then terminated by the addition of 100 μL of 0.1 M NaOH. The mixtures were heated for 30 s in a boiling water bath and cooled to 25 °C for 40 min in the presence of 2 mM NAD, 0.1 U myo-inositol dehydrogenase, and 50 mM Na2CO3 (pH 9.5) in a total reaction volume of 1.0 mL. The reduction of NAD was measured spectrophotometrically at 340 nm.STS activity was determined according to a previous reportOther enzyme activities were assayed by corresponding assay kits from Solarbio Life Sciences  according to the manufacturer’s protocols. Briefly, enzyme activities involved in sugar metabolism  were assayed by a sucrose synthase (decomposition direction SS-I) activity detection kit , S-AI detection kit , NI activity detection kit , SS activity detection kit , sucrose phosphate synthase (SPS) activity detection kit , α-galactosidase (GAL) activity detection kit , SSS activity detection kit , and SBE activity detection kit , with spectrometric analyses.To test the effect of sugars on grafting, the grafted cucumber/pumpkin was sprayed with different concentrations of glucose  (W/V) from 0 to 7 days after grafting. Fifty plant replicates were performed for every treatment. To test the effect of TOR inhibitors (rapamycin and AZD-8055) on cucumber, we chose two treatment modes, spraying or adding them to the nutrient solution. When the cotyledon of cucumber had just opened, one part was sprayed with a range of concentrations of rapamycin  and AZD-8055  for 5 days. The other parts were transferred to a 5 L (33 cm 33 5 cm 33 5 cm) plastic tank filled with ½-strength Hoagland’s nutrient solution, and a range of concentrations of rapamycin  were added after 3 days. The phenotype was observed after 5 days. Fifteen plant replicates were performed for every treatment. For the grafted cucumber, we sprayed with 10 μM AZD-8055 with/without 0.5% glucose 0–7 days after grafting, and normal cucumber was sprayed with water. Rapamycin and AZD-8055 were stored and dissolved in DMSO according to the manufacturer’s instructions.52. ATP, ADP, and AMP measurements were performed on a Waters 2695 high-performance liquid chromatograph  using a Diamonsil ll-C18 column  and an ultraviolet detector at 254 nm. Sample aliquots of 10 µl were injected into the HPLC. The concentrations were calculated according to the external standard program. The energy charge (EC) was calculated as [ATP + 1/2ADP]/[(ATP + ADP + AMP)].Samples (0.2 g FW) were harvested from the scion part 3 cm above the graft junction and rootstock part 3 cm below the graft junction at 0, 1, 3, 6, and 9 days after grafting. ATP, ADP, and AMP levels were extracted and assayed according to previous reports−ΔΔCt method and were normalized to the level of the reference gene LEA2653.RNA preparations were extracted as described above in triplicate with three biological replications using the RNAprep Pure Plant Plus Kit . First-strand cDNA synthesis was performed using a FastQuant cDNA Synthesis kit  according to the manufacturer’s instructions. All gene-specific primers were designed using Primer 5.0  Table , and qRTP < 0.05) using SPSS software.All the data are the average values of at least three replicates and their standard deviations. The statistical evaluations were performed via one-way analysis of variance, followed by individual comparisons with Duncan’s multiple range tests (Sugars promote graft union development in the heterograft cucumber onto pumpkinTable S1 . The list of DEGs in RNA-seq analysis during graft union formation