To introduce a new method to map the mechanical stiffness of healthy and keratoconic corneas.Numerical modeling based on the finite element method was used to carry out inverse analysis of simulated healthy and keratoconic corneas to determine the regional variation of mechanical stiffness across the corneal surface based on established trends in collagen fibril distribution. The Stress–Strain Index (SSI), developed and validated in an earlier study and presented as a parameter that can estimate the overall stress–strain behavior of corneal tissue, was adopted in this research as a measure of corneal stiffness. The regional variation of SSI across the corneal surface was estimated using inverse analysis while referring to the common features of collagen fibrils’ distribution obtained from earlier x-ray scattering studies. Additionally, for keratoconic corneas, a method relating keratoconic cone features and cornea’s refractive power to the reduction in collagen fibril density inside the cone was implemented in the development of SSI maps. In addition to the simulated cases, the study also included two keratoconus cases, for which SSI maps were developed.SSI values varied slightly across corneal surface in the simulated healthy eyes. In contrast, both simulated and clinical keratoconic corneas demonstrated substantial reductions in SSI values inside the cone. These SSI reductions depended on the extent of the disease and increased with more considerable simulated losses in fibril density in the cone area. SSI values and their regional variation showed little change with changes in IOP, corneal thickness, and curvature.SSI maps provide an estimation of the regional variation of biomechanical stiffness across the corneal surface. The maps could be particularly useful in keratoconic corneas, demonstrating the dependence of corneal biomechanical behavior on the tissue’s microstructure and offering a tool to fundamentally understand the mechanics of keratoconus progression in individual patients. With thAn earlier study has shown evidence of SSI’s independence of corneal thickness and IOP, and demonstrated its increase with age . This stThe SSI maps rely on the microstructure of corneal tissue, and in particular the distribution of collagen fibrils . Assuminex vivo microstructure study, the areas outside the cone in keratoconic corneas were compared with corresponding areas in healthy corneas in terms of the collagen fibril distribution , the minimum corneal thickness (T in mm), and the central refractive power (P in diopters)  :The process of developing the SSI maps is described below in detail and the implementation was demonstrated using both simulated and clinical topography data to facilitate consideration of keratoconic cases with wide ranges of geometry, stiffness, IOP, and disease severity.Seven healthy corneas were scanned in an earlier study using x-ray scattering to determine their collagen fibril distribution . The fibAn inverse analysis process was then established to convert the single SSI reading obtainable with the CorVis ST into an SSI map providing the value of SSI at any point across corneal surface. This process was carried out numerically and was based on an objective function that minimized the difference between the apical displacement under IOP of a cornea model Model 1,  adoptingIn this equation, δ is the apical displacement of the two models: Model 1 with a homogeneous material model and Model 2 with a stiffness distribution that matched the collagen fibril distribution; i refers to different IOP application steps and n is the total number of IOP steps.In the analysis, a wide range of the percentage change (used with the stiffness levels in Model 2) was applied and the RMS associated with each change was determined. The percentage change values and the resulting RMS were then fitted to a third-order polynomial, and the percentage change in stiffness that corresponded to the minimum value of RMS was used to generate the SSI map.3 to simulate the aqueous and the vitreous. IOP was applied through controlling the pressure in this internal fluid. This technique enabled the internal eye pressure to vary from the initial IOP with the deformation experienced under the CorVis air pressure  models representing the corneas and matched their assumed geometry . The models included 800 fifteen-noded continuum elements organized in one layer, 10 cornea element rings, and 10 sclera element rings . The modpressure . To prevpressure . This coThe process to develop SSI maps for KC corneas followed a similar route with two distinctive differences . First, In order to demonstrate the construction and illustrate the benefits of the SSI maps, a parametric study was carried out to cover cases with a KC cone of radius of 0.5, 1.0, 1.5, and 2.0 mm; a distance between cone center and corneal apex of 0.0, 1.0, 2.0, and 3.0 mm; and an angle of 0°, 45°, and 90° between the horizontal center line of cornea and line from apex to cone center. In all cases, the fibril density within the cone area was reduced by a maximum of 60% in steps of 10%. This maximum reduction was compatible with cases of severe KC as was found in our earlier study . AdditioIn all cases, IOP was assumed to be 15 mmHg, with an anterior central radius of 7.8 mm, an anterior shape factor of 0.82 (shape factor = 1 - asphericity), a central corneal thickness of 545 microns, and a limbal diameter (or white-to-white distance) of 11.85 mm. These values represented the means of clinical measurements reported in the literature . For simFurther, in order to recognize the fact that KC leads to not only a reduction in fibril density within the cone but also a change in fibril arrangement (which has not been quantified), part of the study was repeated to consider the effect of assuming either total isotropy or random distribution at each measurement point within the KC cone.The methods described above to generate SSI maps were implemented in two clinical cases with mild and advanced keratoconus, respectively. Tomography and biomechanical data on these two cases were obtained from the Vincieye Clinic in Milan, Italy. Institutional review board (IRB) ruled that approval was not obligatory for this record review study. However, the ethical standards set in the Declaration of Helsinki were observed. Both patients provided informed consent before using their data in research. They had a complete ophthalmic examination, including CorVis ST and Pentacam  exams. Both patients had bilateral keratoconus without any previous ocular surgeries, such as collagen CXL or intracorneal rings.The first parameter to consider is the fibril density reduction within the cone area, which varied from 0  to 60% (in severe KC cases). The results presented in In these analyses, the SSI of the healthy cornea was assumed at 0.7, while for KC corneas, an optimization process was carried out to determine the value of the overall SSI (the CorVis ST single SSI reading), for which the SSI values in the parts outside the cones of the KC corneas would remain at the same level as in the corresponding areas in the healthy eye. This exercise was carried out for fibril density reduction factors of 20, 40, and 60%.Comparisons were also held to show the effect of cone fibril arrangement on the results. The comparisons were between three KC models, all with the same fibril density reduction (60%) in the cone area. While one model maintained the same fibril arrangement as in healthy tissue, another assumed uniform and isotropic fibril distribution. A third model was also included in which the variation of fibril density from point to point within the cone, as well as the anisotropy at each point, was made random . However, in all three models, it was ensured that the total fibril content within the cones remained the same.All three models had a 2.0-mm-radius cone, whose center was 1.0 mm away from apex and located on the horizontal central meridian. The results presented in The next step was to consider the effect of cone size, distance from cone to apex, and location of cone center on the SSI maps. Here, the focus was on SSI values along the horizontal central meridian of the cornea – normalized against the corresponding values obtained for healthy corneas . In all 2. Also, using Equation 1, the collagen fibril reduction factor (within the cone area) was 19%. On the other hand, the advanced keratoconic case had an age of 27 years, a min thickness of 377 microns, a refractive power of 65 D, and an SSI value of 0.43. Further, cone mean radius was estimated at 1.57 mm, cone height was 0.258 mm, cone area was 6.562 mm2, and fibril reduction factor was 41%. The two cases were analyzed to generate the SSI maps following the process explained above  that satisfies the mechanical equilibrium between the applied pressure (IOP) and corneal stiffness; this corneal stiffness has two components: the material stiffness and the geometric stiffness. Therefore, knowing the IOP and both the material stiffness and the geometric stiffness would allow estimating corneal topography. However, for a 3D object like the cornea with a significant thickness variation across the surface, it would be difficult to estimate the material stiffness distribution from the topography maps, as the topography is not solely dependent on the material stiffness. For this reason, we sought to determine the material stiffness maps from first principles – based on reliance on the tissue microstructure. Once these maps are developed, weak areas can be identified to show the scale of stiffness deterioration associated with KC progression.in vivo mapping of the microstructure is developed. However, this assumption meant that the small variations that exist between individual eyes were not considered. Another limitation of the study is the reliance on a method developed earlier to relate cone geometric features to the fibril density reductions within the cone  have the same microstructure was necessary for our study and will perhaps remain necessary until a technique for the the cone . That stThe goal of this study in producing stiffness maps of the cornea is shared with other technologies, including most notably BM. BM relies on an inelastic scattering process, which is associated with a medium’s acoustic phonons or density fluctuations. Measurement of spectral changes provides information on the phonons’ properties and is highly correlated with viscoelastic properties . While iin vivo and in both healthy and keratoconic eyes. The resulting maps can potentially offer a tool to improve fundamental understanding of the mechanics of keratoconus progression in individual patients.This study provided a method to map the distribution of mechanical stiffness of the cornea, which can be used ashkan@eliasy.com.The data analyzed in this study is subject to the following licenses/restrictions: There is a material transfer agreement in place. Requests to access these datasets should be directed to AsE, The studies involving human participants were reviewed and approved by the Vincieye Clinic in Milan, Italy and University of Liverpool in Liverpool, United Kingdom. The patients/participants provided their written informed consent to participate in this study.HZ and AsE drafted the manuscript, carried out the study, performed the analysis, and acquired the results. AsE developed the software used in the work and supervised the conduct of the study. BL and AA interpretated the data and supported the analysis. RV, PV, RA, and CR critically reviewed the manuscript, interpreted the data, and contributed in the design of the work. AhE designed and conceptualized the study, supervised the entire project, drafted the manuscript, and interpreted the data. All authors have reviewed and approved the submitted version, and agreed both to be personally accountable for the author’s own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, even the ones in which the author was not personally involved, are appropriately investigated and resolved, and the resolution is documented in the literature.RV, PV, RA, CR, and AhE are consultants for Oculus. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sulfation is an expedient strategy to increase the solubility, bioavailability, and bioactivity of nutraceuticals and clinically important drugs. However, chemical or biological synthesis of sulfoconjugates is challenging. Fusarium graminearum PH-1. We expressed FgSULT1 in a Saccharomyces cerevisiae chassis to modify a broad range of benzenediol lactones and their nonmacrocyclic congeners, together with an anthraquinone, with the resulting unnatural natural product (uNP) sulfates displaying increased solubility. FgSULT1 shares low similarity with known animal and plant sulfotransferases. Instead, it forms a sulfotransferase family with putative bacterial and fungal enzymes for phase II detoxification of xenobiotics and allelochemicals. Among fungi, putative FgSULT1 homologues are encoded in the genomes of Fusarium spp. and a few other genera in nonsyntenic regions, some of which may be related to catabolic sulfur recycling. Computational structure modeling combined with site-directed mutagenesis revealed that FgSULT1 retains the key catalytic residues and the typical fold of characterized animal and plant sulfotransferases. Our work opens the way for the discovery of hitherto unknown fungal sulfotransferases and provides a synthetic biological and enzymatic platform that can be adapted to produce bioactive sulfates, together with sulfate ester standards and probes for masked mycotoxins, precarcinogenic toxins, and xenobiotics.Total biosynthesis or whole-cell biocatalytic production of sulfated small molecules relies on the discovery and implementation of appropriate sulfotransferase enzymes. Although fungi are prominent biocatalysts and have been used to sulfate drug-like phenolics, no gene encoding a sulfotransferase enzyme has been functionally characterized from these organisms. Here, we identify a phenolic sulfotransferase, FgSULT1, by genome mining from the plant-pathogenic fungus IMPORTANCE Sulfation is an expedient strategy to increase the solubility, bioavailability, and bioactivity of nutraceuticals and clinically important drugs. However, chemical or biological synthesis of sulfoconjugates is challenging. Genome mining, heterologous expression, homology structural modeling, and site-directed mutagenesis identified FgSULT1 of Fusarium graminearum PH-1 as a cytosolic sulfotransferase with the typical fold and active site architecture of characterized animal and plant sulfotransferases, despite low sequence similarity. FgSULT1 homologues are sparse in fungi but form a distinct clade with bacterial sulfotransferases. This study extends the functionally characterized sulfotransferase superfamily to the kingdom Fungi and demonstrates total biosynthetic and biocatalytic synthetic biological platforms to produce unnatural natural product (uNP) sulfoconjugates. Such uNP sulfates may be utilized for drug discovery in human and veterinary medicine and crop protection. Our synthetic biological methods may also be adapted to generate masked mycotoxin standards for food safety and environmental monitoring applications and to expose precarcinogenic xenobiotics. Sulfotransferases (SULTs) have been extensively studied as important enzymes for the metabolism of xenobiotics and drugs and for the modulation of endobiotics  in humans and other organisms. However, much less attention was paid to their application in synthetic biology despite the importance of sulfate esters in drug development. Compared to the parent molecules, sulfated derivatives show better water solubility –3 and ma–8–in vitro, although the required sulfo group donor cosubstrate 3′-phosphoadenosine 5′-phosphosulfate (PAPS) is expensive, and its in situ regeneration is challenging  displays cancer cell antiproliferative and heat shock response modulatory activities, while the RAL12 lasiodiplodin displays mineralocorticoid receptor antagonist and prostaglandin biosynthesis inhibitory activities . Thus, we established polyketide synthase (PKS) domain and subunit shuffling as practical methods to obtain uNP BDL scaffolds  was shown to be sulfated during the course of phase II (conjugative) detoxification by plants and fungi (Benzenediol lactones (BDLs) are drug-like polyketide natural products from fungi with wide-ranging bioactivities –27. BDLs–tivities . The DALegregase , 30. We caffolds , 24, 31.ormation , 33. Hownd fungi , 22, butFusarium graminearum PH-1 as a candidate cytosolic PAPS-dependent SULT for the biotransformation of phenolic natural products  is able to biotransform the model BDL substrate lasilarin 1 to the more polar products 1a and 1b  of the [M-H]– ions of products 1a and 1b were both 80 atomic mass units (amu) higher than that of lasilarin, indicating that these products are sulfated derivatives . To validate these presumed sulfoconjugates, we used high-performance liquid chromatography (HPLC)-HRMS/MS to verify that the parent ions of products 1a and 1b give rise to daughter ions of 319 amu (the m/z of the pseudomolecular ion of lasilarin 1 in the negative mode [M-H]–) and 275 amu  was available in the data banks, we used the amino acid sequence of the human phenolic sulfotransferase 1A1  as a bait to query the predicted proteome of F. graminearum PH-1  that had been annotated to feature a sulfotransferase conserved domain each . This enzyme, designated FgSULT1, shares 26% identity over 81% coverage with human SULT1A1 and is expressed under culture conditions conducive to lasilarin 1 sulfation, as shown by reverse transcription-PCR of the mRNA derived from gene FGSG_02887 (fgsult1)  RIL. The His-tagged recombinant FgSULT1 protein was purified to homogeneity using Ni2+-nitrilotriacetic acid (NTA) affinity chromatography, and the reaction was reconstituted in vitro with lasilarin as the substrate and PAPS as the sulfo group donor cosubstrate. As shown in in vivo by other enzymes of the host during biotransformation.The intron-free gene encoding FgSULT1 was cloned into the pACYCDuet-1 vector and expressed in in vitro reactions. Compound 1a was obtained in a sufficient amount  for structure elucidation using nuclear magnetic resonance spectroscopy (NMR). The 1H NMR spectrum of compound 1a closely resembled that of lasilarin   was too low for NMR characterization despite our best efforts. Nevertheless, the structure of compound 1b could still be elucidated as lasilarin 5-O-sulfate, considering that (i) The high resolution electrospray ionization mass spectrometry (HRESIMS)/MS profile of compound 1b is perfectly consistent with that of compound 1a, indicating that compounds 1a and 1b are lasilarin-O-sulfate regioisomers, and (ii) lasilarin has only two positions that can be sulfated: 7-OH, which is modified in compound 1a, and 5-OH, which consequently has to be the sulfation site in compound 1b. Lasilarin sulfates 1a and 1b are both new to nature.The two apparent lasilarin sulfates 1a and 1b were isolated from large-scale asilarin , but con14, DAL12, RAL16, RAL14, and RAL12 compounds, and nonmacrocyclic BDL congeners such as isocoumarins, acyl-resorcylic acid (ARA) ethyl esters, acyl-dihydroxyphenylacetic acid (ADA) ethyl esters, and an acyl benzaldehyde . A modest number of more distant fungal homologues were also found to be members of the same SULT family as FgSULT1 . Most of these putative enzymes are encoded in the genomes of Microdochium, Astrocystis, and Xylaria spp. that belong to a different order  of the Sordariomycetes class than the Fusaria. One additional homologous family member was also found in Stanjemonium grisellum and Acremonium strictum  and in Microascus trigonosporus . Several putative enzymes belonging to the FgSULT1 family were also detected in two genera (Hortaea and Aureobasidium) that belong to the Dothideomycetes, a different class of the Ascomycota  showed that homologues of this enzyme have a very sporadic phylogenetic distribution in the kingdom Fungi. Thus, homologues that belong to the same SULT subfamily  exist only in Fusarium, Xylaria, Michrodochium, Hortea and Aureobasidium spp., together with bacterial homologues. This clade is basal to characterized SULTs from animals and plants, including those that conjugate xenobiotics and phenolic small molecules . The four sulfotransferase domain-containing hypothetical fungal proteins that display low similarities (23 to 27% identities) to FgSULT1 form a different clade, which is nestled among the animal SULT clades.Instead of additional fungal homologues, the SULT family that FgSULT1 belongs to is typified by a large variety of enzymes from Proteobacteria and Cyanobacteria that feature the Sulfotransfer_1 domain  clusters with a gene (D0864_01955) encoding a major facilitator superfamily transporter similar to the hydroxamate and catechol-type siderophore uptake transporters MirB in Fusaria , the S. toyocaensis glycopeptide antibiotic SULT StaL , and the A. thaliana AtSOT18 . This large cavity in FgSULT1 is sufficient to allow the binding of a variety of phenolic xenobiotics and natural products, such as the various BDL congeners  as the scaffold. This was the human cytosolic sulfotransferase SULT1B1 , complexed with PAP or PAPS and the corresponding substrate, resveratrol . The hom-strands , 49–51 . Ac. Ac55). O-sulfate 1a against untransformed Vero cells (African green monkey kidney epithelium) and human cancer cell lines HeLa , HepG2 (hepatocellular carcinoma), MCF-7 (breast cancer), and A549 (lung cancer) as the targets. However, no cytotoxicity was observed with either compound, even at the highest dose (50 μM).BDLs such as desmethyl-lasiodiplodin 5 show potent cytotoxicity against various cancer cells , 58. ThuFusarium genus. Additional family members are present in selected species from the Hypocreales, Xylariales, and Microascales among the Sordariomycetes and in only two genera among the Dothideomycetes. Intriguingly, the family to which FgSULT1 belongs contains many predicted bacterial SULTs, mainly from Proteobacteria. Together with the patchy distribution of these enzymes in fungi and the lack of synteny in the fungal genomic loci harboring the genes for the FgSULT1 homologues, this may indicate horizontal gene transfer events from bacteria followed by limited retention of the transferred gene among selected fungal lineages. However, the presence of an intron in fgsult1 and the absence of codon usage divergence in this gene compared to other F. graminearum genes suggests that any transfer event may have been ancient. While more extensive phylogenetic analyses may help to explain the complex evolutionary paths of these interesting genes in more detail, sequence comparison, structure modeling, and site-directed mutagenesis of selected residues reveals that FgSULT1 shares the classical fold of cytosolic SULTs, including the conserved catalytic residues, the PAPS-binding regions, and the flexible loops surrounding the substrate binding site (Research on sulfotransferases (SULTs) has focused on the twin roles that these enzymes play in phase II detoxification of various xenobiotics and the modulation of the activities of various endobiotics such as hormones, peptides, lipids, and carbohydrates. Naturally, most attention was paid to human SULTs, although an increasing number of enzymes from other mammals , insects, plants, and prokaryotes are also being analyzed 13, 50, 51ing site . Just liF. graminearum PH-1 is proficient in conjugating BDL congeners and other phenolic polyketides to yield the corresponding sulfate esters. Using 49 potential substrates with varied carbon skeletons, we detected sulfation with 14 scaffolds and isolated and elucidated the structure of one representative sulfated derivative, the novel compound lasilarin 7-O-sulfate 1a. Except for a 2,4-dihydroxybenzaldehyde motif that must be part of a more complex scaffold, no other, obvious structural constraint was evident for substrate acceptance by FgSULT1. Even with this motif present, the enzyme still shows idiosyncratic preferences for accepting or refusing closely related BDL analogues as potential substrates . Considering the numerous homologues of FgSULT1 in fusaria that belong to the same subfamily of SULTs, suitable enzymes to sulfate other BDLs or related polyketides not recognized by FgSULT1 are likely to be discovered later.We established that FgSULT1 from S. cerevisiae biocatalyst expressing FgSULT1, or in a one-pot total biosynthetic format  encountered by these fungi in their native environments.We demonstrated that the generation of sulfated polyketides is feasible in a one-step format without tedious and environmentally problematic protection/deprotection chemistries by feeding preformed substrates to an c format , 60 by ce medium . Considechoderma ; neverthin vivo) or to expose precarcinogenic xenobiotics that are accidentally activated and rendered toxic by sulfation during human and animal metabolism or upon catabolism/detoxification by the microbiomes resident in animals or the environment.Importantly, BDL sulfate congeners and other sulfated uNP polyketides detected in this study display improved solubility and achieve more drug-like ClogP values (Table S3 in reference Escherichia coli DH10B and plasmid pJET1.2 (Thermo Fisher) were used for routine cloning and sequencing. Saccharomyces cerevisiae BJ5464-NpgA (MATα ura3-52 his3-Δ200 leu2-Δ1 trp1 pep4::HIS3 prb1 Δ1.6R can1 GAL) was used as the host for expression vectors based on plasmids YEpADH2p-URA, YEpADH2p-TRP, and YEpADH2p-LEU , and product isolation were conducted as described in references 1H and 13C NMR as described in reference DH2p-LEU , 32, 41.The FgSULT1 homology protein structure model was built with I-TASSER , and the2+-NTA column affinity chromatography. Native PAGE and MALDI-TOF were used to measure the molecular mass of FgSULT1. Enzyme activity assays were performed at 30°C in 100-μl reaction mixtures containing 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 0.2% Tween 20, 1 mM lasilarin 1 as the substrate, 1 mM PAPS as the sulfate donor cosubstrate, and 10 μg FgSULT1. Reactions were stopped by extraction with ethyl acetate.The His-tagged FgSULT1 protein was purified to homogeneity (as judged by SDS-PAGE) using Ni


The COVID-19 outbreak is a global crisis that has placed small and medium enterprises (SMEs) under huge pressure to survive, requiring them to respond effectively to the crisis. SMEs have adopted various digital technologies to cope with this crisis. Using a data set from a survey with 518 Chinese SMEs, the study examines the relationship between SMEs’ digitalization and their public crisis responses. The empirical results show that digitalization has enabled SMEs to respond effectively to the public crisis by making use of their dynamic capabilities. In addition, digitalization can help improve SMEs’ performance. We propose a theoretical framework of digitalization and crisis responses for SMEs and present three avenues for future research. At the end of 2019, a novel coronavirus disease (COVID-19) broke out suddenly and spread rapidly to become a global pandemic. By late June 2020, COVID-19 had infected more than 8 million people worldwide, including more than 80,000 people in China. This public health crisis has posed great challenges for the survival and development of firms, with small- and medium-sized enterprises (SMEs) suffering in particular. The COVID-19 pandemic has been economically destructive in many ways. First, as more and more governments block cities in order to control the pandemic, the global supply chain has been significantly disrupted as both imports and exports are blocked. Second, delays in the resumption of work have greatly reduced firms’ production capacity while fixed costs such as salary and rent remain unchanged, leading to serious cash flow issues. Third, the reduction in demand due to the outbreak has posed serious threats to service sectors such as catering, hospitality, and cultural tourism. Worse, the damage caused by the COVID-19 outbreak is expected to be long-lasting and have a chilling effect on global economic growth.SMEs play a vital role in promoting technological innovation, improving employment, and maintaining social stability  students from key universities in China.By February 23, 935 valid samples had been collected from 518 SMEs (with less than 500 employees), accounting for 55.4% of the full sample. As Table overall digitalization degree of a firm, which is reflected through five items. (1) Digital artifacts refers to applications or media content with specific functions and values embedded in digital products or services, such as positioning applications in mobile phones to track travel trajectories during the pandemic  A dic Ekbia . (3) Theic Ekbia . (4) Theadoption of digital technologies, which is reflected through seven items. These are (1) social, (2) mobile, (3) big data, (4) cloud computing, (5) IoT, (6) platform development, and (7) AI  and employee protection strategies (0.71), while the adoption rate of the donation strategy is relatively low (0.32). These results suggest that the most important task for SMEs is to survive the crisis. For long-term crisis responses, SMEs prefer to implement digital transformation strategies (0.71) instead of strategic changes in products, markets, and external relations (0.54). These results imply that SMEs have realized the unique value of digitalization in the COVID-19 outbreak.Regarding crisis response performance, the results of the survey show that SMEs have been adversely affected by the COVID-19 pandemic in various aspects. Costs (0.70) have increased less than 10%, but revenue in the first quarter (0.56) has decreased by 10%–50% on average, and cash flow (0.59) can only be maintained for about half a year, on average. Overall, under the COVID-19 outbreak, there is an inevitable decline in predicted performance (0.59) compared with firms’ performance in 2019.Province (the province in which the firm is located), industry, property, and year (the year in which the firm was established) are set as control variables. The results show that the digitalization of SMEs is positively associated with the implementation of crisis response strategies and performance, and that crisis response strategies are also positively associated with performance. Therefore, it is necessary to continue to explore the functional relationship between variables. We calculated the variance inflation factor (VIF) and the result shows that the maximum value is 1.10, far below the cutoff value of 10. Our results show no evidence of multicollinearity among all variables.Table As this is a survey study, a more intuitive and concise approach should be adopted to clearly present the results in a simple manner. To this end, we divided the value of each variable into three degrees, namely low, medium, and high. For example, the value of the degree of digitalization was sorted from the largest to the smallest, and then divided into three equal groups according to the number of samples, namely the high group, the middle group, and the low group. We then calculated the average value of the variable at each degree. This grouping method has the advantages of simplicity, intuition, and clarity in the presentation of data, which enhances the readability of the paper. The values were reserved to two decimal places, except for special notes.p < 0.05) and long-term strategic responses (p < 0.001). In the face of the COVID-19 outbreak, highly digitalized SMEs can more effectively use short-term responses. For SMEs with a high degree of digitalization or digital technology adoption, the average scores of short-term crisis responses are 0.62 and 0.64, respectively, both of which are higher than those for SMEs with a medium or low degree of digitalization. Specifically, in terms of digitalization, the scores for production recovery strategy, employee protection strategy, and donation strategy are 0.70, 0.74, and 0.34, respectively. In terms of digital technology adoption, the three scores are 0.71, 0.75, and 0.34, respectively. The policy-based shutdown under the pandemic has left many firms facing production stagnation. Highly digitalized firms with dynamic capabilities are more likely to integrate their internal and external resources quickly to resume production and operation activities through methods such as adopting online telecommuting or divesting less-profitable units. In addition, digital firms can make donations to the pandemic area through existing digital channels. Internet firms, which have attracted plenty of well-deserved attention during this pandemic, have higher philanthropic efficiency than traditional firms and charities.As shown in Table Similarly, highly digitalized SMEs are more determined to make long-term responses in order to deal with the crisis. For SMEs with a high degree of digitalization, the average scores of digital transformation intention and strategic change intention are 0.76 and 0.58, respectively, all higher than those for SMEs with a medium or low degree of digitalization. Similarly, for SMEs with a high degree of digital technology adoption, the average scores of digital transformation intention and strategic change intention are 0.78 and 0.60, respectively, all higher than those for SMEs with a medium or low degree of digital technology adoption. The agility and openness of digital technologies greatly improves the accuracy of business analysis for the firms that employ such technologies. This explains why highly digitalized firms are more likely to find potential opportunities in disruptive environments and integrate resources for strategic transformation and changes.p < 0.1), which is mainly reflected in their long-term crisis responses . Specifically, regarding other crisis response strategies, the performance of SMEs that conduct their business online is no worse than that of those that conduct their business offline, and the difference is not highly significant.In addition, SMEs, with the primary business conducted online instead of offline, will perform better in responding to the COVID-19 outbreak , but do not necessarily help SMEs improve their current performance. Excessive production recovery and employee protection will consume a large amount of a firm’s resources, putting even greater pressure on already thinly stretched SMEs during the outbreak. Therefore, a moderate degree of production recovery and employee protection may be a better option. Donation strategies have an overall positive impact on SME performance (0.58) and can help SMEs obtain more revenue (0.49), leading to stronger cash flow (0.63) and better predicted performance (0.61). SMEs and stakeholders are a community of interests in the public crisis. By donating to relevant causes, SMEs can obtain support from the government and the community to mitigate the harm of the crisis in an atmosphere of mutual assistance.On the one hand, short-term crisis response strategies can improve the performance of SMEs, though this does not occur in a uniform manner. Production recovery strategies (0.60) and employee protection strategies (0.60) can lead to better predicted performance . However, strategic changes are associated with lower SME performance (0.55), indicating that an SME’s intention to change its strategy seems to merely be a reflection of poor performance. Strategic changes will consume a large amount of human, material, and financial resources, placing huge pressure on SMEs that are already struggling in the outbreak. However, this does not mean that strategic changes are not a good choice because the benefits of successful changes may be long-term and sustainable even if the temporal performance may decline.As shown in Table p < 0.05). Specifically, digitalization could help SMEs maintain cash flow , generate more revenue , and lead to perceived best predicted performance . As identified above, highly digitalized firms are more likely to resume production, effectively curbing revenue and profit declines. However, the advantage of digitalization for cost control is not obvious, leading to a lack of significant advantages for firms’ overall performance.First, a high degree of digitalization will lead to higher SME performance . Specifically, the adoption of digital technologies is associated with a higher degree of cash flow maintenance , all higher than the rest. However, this cannot help SMEs control costs or obtain revenue most effectively.Second, similarly, a high degree of digital technology adoption is associated with optimal SME performance , cash flow maintenance (0.58), revenue (0.49), and predicated performance (0.59). However, these results require further and more rigorous verification.β = 0.100, p < 0.05). Technologies such like 4G, instant messaging, and GPS together with a variety of mobile applications could support SMEs in refined management and improve their organizational efficiency. For example, trajectory tracking applications can help SMEs efficiently conduct human resource management. Second, big data technologies are regarded as most useful for maintaining cash flow  and generating revenue . In the digital economy, big data not only helps firms improve the efficiency of their existing businesses, it also enables the creation of new businesses. During the COVID-19 pandemic, big data technology may not only help SMEs evaluate the spread of the pandemic, guiding them to reactivate work and production, it may also help them identify and take advantage of new business opportunities in the crisis and thus guide the direction of future developments. Finally, cloud computing technology seems to be most important for SMEs’ overall predicted performance . This indicates that a new era of cloud computing is being welcomed.Table province, industry, property, and year, we conducted a simple whole regression test among the main variables as a robustness test.Descriptive analyses and simple correlation analyses are often conducted in survey research. However, entirely abandoning the regression may cause problems hazarding research rigor such as neglecting the partial correlation variables. Therefore, controlling the four variables of β = 0.161, p < 0.001) and long-term responses . Second, digital technology adoption also has a significant positive impact on the implementation of short-term responses  and long-term crisis responses . Third, SMEs that conduct their primary business online instead of offline will perform better in crisis responses, especially in long-term responses . In addition, overall digitalization degree  and digital technology adoption  both have a significant positive impact on SMEs’ performance. The implementation of crisis response strategies is further positively associated with SMEs’ performance, which is mainly reflected in their short-term strategies . These empirical results support our arguments.As shown in Table We have constructed a theoretical framework based on the above survey results Fig. . The surDigital technologies are at the center of digitalization. Sebastian et al.  further However, the connotations of dynamic capabilities are changing under the context of digital transformation. On the one hand, the widespread use of digital technologies is reshaping the nature and purpose of dynamic capabilities  reshape the business environment? Second, what is the nature of firms’ strategic decision-making in crisis environments characterized by sudden and violent environmental changes? Third, what are the similarities and differences between short-term emergency response strategies and long-term strategic response strategies? Finally, what is the underlying mechanism through which digitalization impacts crisis response strategies?The results of the survey indicate that digitalization has a positive effect on SMEs’ performance. Although this paper only focuses on SMEs’ present performance, future research should consider both present and future performance, as digitalization strategies have long-lasting impacts on firms. Digitalization has greatly altered the ways in which businesses are run (Ofek and Wathieu As digitalization often occurs at the firm level, firms must make holistic strategic deployments (Fitzgerald It is thus a promising research direction to explore digital transformation strategies as well as their impact on firms’ competitive advantages. We suggest the following research questions. First, is digitalization a new source of competitive advantages for firms? Second, how should firms’ digital business strategies be designed? Third, how does a digital business strategy guide a firm’s adoption of digital technologies? Finally, how does digital technology adoption change a firm’s value creation paths?The COVID-19 outbreak is a public health crisis that has posed great challenges for the survival and growth of SMEs. The crisis has also highlighted the important role of digital technologies in the response to the COVID-19 outbreak. Data from a questionnaire survey was used in this study to investigate the relationships among digitalization, public crisis responses, and SME performance in the context of the COVID-19 outbreak. The results of our survey show that an SME’s efforts towards digitalization, manifested by their degree of digitalization, adoption of digital technologies, and business mode can help them better respond to public crises. Further, digitalization contributes to improvements in SMEs’ performance through the implementation of public crisis response strategies. To conclude the study, we construct a theoretical framework that links digitalization with public crisis responses from the dynamic capabilities perspective and propose three avenues for future research.


Inadequate liver volume and weight is a major source of morbidity and mortality after adult living donor liver transplantation (LDLT). The purpose of our study was to investigate HCC recurrence, graft failure, and patient survival according to change in right liver graft weight after histidine-tryptophan-ketoglutarate (HTK) solution perfusion in LDLT.Two hundred twenty-eight patients underwent LDLT between 2013 and 2017. We calculated the change in graft weight by subtracting pre-perfusion graft weight from post-perfusion graft weight. Patients with increased graft weight were defined as the positive group, and patients with decreased graft weight were defined as the negative group.After excluding patients who did not meet study criteria, 148 patients underwent right or extended right hepatectomy. The negative group included 89 patients (60.1%), and the positive group included 59 patients (39.9%). Median graft weight change was -28 g  in the negative group and 21 g  in the positive group (P<0.001). Median hospitalization time was longer for the positive group than the negative group . There were no statistical differences in tumor characteristics, postoperative complications, early allograft dysfunction, or acute rejection between the two groups. Disease-free survival, death-censored graft survival, and patient survival were lower in the positive group than the negative group. Additionally, the positive group showed strong association with HCC recurrence, death-censored graft survival, and patient survival in multivariate analysis.This study suggests that positive graft weight change during HTK solution perfusion indicates poor prognosis in LDLT. In Asia, where there is a shortage of deceased donors, living donor liver transplantation (LDLT) is frequently performed in hepatocellular carcinoma (HCC) patients using right or left hemi-liver graft from living donors. Grafts from living donors are higher quality than livers from deceased donors. Numerous factors have been identified as predictors of short-term outcomes of LDLT such as age, model for end-stage liver disease (MELD) score, cold ischemic time, graft-to-recipient weight ratio (GRWR), transfusion demands, and laboratory test findings . Among tHistidine-Tryptophan-Ketoglutarate (HTK) solution is the only organ preservation solution used in Korea . HTK solPartial liver grafts are flushed with a chilled HTK solution through the portal vein in the back-table procedure. After this process, the liver graft weight decreases after perfusion compared to before perfusion. However, some patients have increased graft weight after perfusion. No previous study has investigated the effect of right liver graft weight change during organ preservation solution perfusion on recipient outcomes in LDLT patients. Herein, we investigated HCC recurrence, graft failure, and patient survival according to right liver graft weight change in adult LDLT patients.Two hundred twenty-eight patients underwent LDLT between January 2013 and March 2017 at Samsung Medical Center, Korea. There were 155 HCC patients who received LDLT with HCC. The present study was approved by the Institutional Review Board (IRB) of Samsung Medical Center (SMC-2020-07-027). The need for patient consent was waived by the IRB because this was a retrospective observational study of data that used patient medical records. We reviewed all patients’ medical records and excluded those who did not have records of graft weight (n=4), patients without HCC (n=48), multiple organ transplantation (n=2), re-transplantation (n=3), received a left-side graft (n=10), pediatric liver transplantation patients (n=8), or were lost during follow-up or had incomplete medical records (n=5). Finally, 148 HCC patients were identified in the study and underwent right or extended right hepatectomy.Our immunosuppressive regimen has been described in previous studies . Basilixvia the portal vein.At the beginning living liver donor surgery, liver wedge resection is performed in segment 4 to check for hepatic steatosis. The portal vein of the right liver graft is clamped using a vascular clamp, but the hepatic artery and hepatic vein are not clamped during liver graft extraction. The right liver graft comes out of the body immediately after right hepatic vein division and the vascular clamp is released. We manually and gently press the right liver graft to remove blood within the graft. The graft was then perfused with 3L of HTK solution We routinely measured the right liver graft weight twice before and after HTK preservation solution perfusion in the back-table procedure: (1) immediately after procurement after the blood was drained (blood-free graft weight) and (2) after perfusion with HTK solution (graft weight after perfusion). We calculated the change in liver graft weight by subtracting pre-perfusion graft weight from post-perfusion graft weight.Patients with increased graft weight were defined as the positive group and patients with decreased graft weight as the negative group. GRWR was calculated by dividing graft weight by recipient body weight before and after perfusion. EAD was defined on the basis of abnormal increases in total bilirubin, international normalized ratio (INR), and aminotransferase within 7 days after LDLT , death-censored graft survival, and patient survival (PS) were calculated as the duration from the starting date of LDLT to the date when a new event was first detected or, if the entire follow-up period was event-free, to the date of the last follow-up visit. DFS rates, death-censored graft survival rates, and PS rates between the positive and negative groups were estimated using the Kaplan-Meier method, and survival curves for those rates were compared with the log-rank test. Significant variables in univariate analyses (P < 0.05) were entered into a Cox multivariate proportional hazards model to determine which factors independently predicted DFS, death-censored graft survival, and patient survival.To compare the differences between the positive and negative groups, Fisher’s exact test for categorical variables and the Mann-Whitney All statistical analyses were performed using SPSS 24.0 for Windows . P < 0.05 was considered statistically significant and all statistical tests were evaluated as two-sided.There were 89 people (60.1%) in the negative group and 59 people (39.9%) in the positive group. Baseline characteristics are summarized in Perioperative and pathologic characteristics are outlined in Median hospitalization time was longer in the positive group than the negative group . Total bilirubin, AST, ALT, and INR, tested repeatedly up to 30 days after LDLT, did not differ between the two groups  in the negative group and 10.2% (n=6) in the positive group. Only one patient in each group had antibody-mediated rejection.The incidence of HCC recurrence was 14.6% (n=13) in the negative group and 25.4% (n=15) in the positive group. The 1-, 2-, and 3-year cumulative disease-free survival rates were 92.0%, 86.2%, and 86.2%, respectively, in the negative group and 82.0%, 76.3%, and 71.5% in the positive group  and alcoholic patients in the positive group were slightly more prevalent in the negative group, and median hospitalization in the positive group was longer than that in the negative group. There were no statistically significant differences in tumor characteristics, postoperative complications, EAD, or acute rejection between the two groups. Interestingly, disease-free survival, death-censored graft survival, and patient survival were lower in the positive group than the negative group. Additionally, the positive group was strongly associated with HCC recurrence, death-censored graft survival, and patient survival in multivariate analysis.We suspect that the increase in liver graft weight after HTK solution perfusion was due to deficiency in liver graft elasticity, which is thought to have adverse effects the patient after graft implantation. The increase in kidney graft weight during perfusion showed vascular disruption by endothelial disruption and inflammatory infiltration . The volAfter graft implantation, hepatocyte injury after reperfusion is mediated by release of reactive oxygen species with subsequent oxidative stress . In addiHCC recurrence after LDLT increases the likelihood of death in liver transplant patients. The most common risk factors for HCC recurrence after liver transplantation were tumor size and tumors, tumor differentiation, tumor markers included AFP or PIVKA-II, vascular invasion, and neutrophil-lymphocyte ratio . The AFPHCC patients may have a higher probability of HCC recurrence after LDLT than DDLT. When partial liver graft is implanted, growth factor and vascular inflow increase for regeneration of liver graft . This prThis study has several imitations. First, we do not know the degree of liver stiffness of the liver donor, so it is difficult to measure liver elasticity precisely. Thus, we do not know the relationship between liver graft weight change and liver elasticity. Second, we were not able to control for confounding factors because of the retrospective nature of this observational study. In addition, it is difficult to completely remove blood and HTK solution in the graft and exactly measure the liver graft weight. The study population was small and enrolled from a single center, making it difficult to obtain high statistical power. Third, biological factors before and after perfusion were unknown in present study. Prospective research on peripheral blood, perfusate solutions, and liver biopsy before and after perfusion is required. Fourth, we are not sure how positive liver graft weights change contribute to HCC recurrence, graft survival, or patient death. Randomized controlled prospective studies are required to validate our findings.This study showed that positive graft weight change during HTK solution perfusion strongly contributed to HCC recurrence, graft failure, and patient death. However, this change in graft weight change has no effect initially after LDLT. Our study revealed that positive graft weight change during HTK solution perfusion is an indicator of poor prognosis in LDLT patients. However, we do not know the exact mechanism between graft weight change and poor prognosis. More research is needed to clarify this mechanism or relationship.The datasets generated for this study are available on request to the corresponding author.This study was approved by the Samsung Medical Center Institutional Review Board (IRB) (SMC-2020-07-027). Patient consent was waived by the IRB. Written informed consent was not obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.JMK designed the study, conducted the literature search, acquired, analyzed, and interpreted the data and wrote the manuscript. J-WJ and YJC designed the study and interpreted the data. SK analyzed the data. JR and G-SC acquired and analyzed the data. All authors read and approved the final manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


This study analyzed the characteristics of a small reciprocating pump with a cantilever valve driven by a piezo actuator. Three types of valves were fabricated to investigate the effect of the valve stiffness on the pump performance and to measure the variation in the flow rate according to the frequency. The flow rate increased with the driving frequency until a certain frequency was reached, and then it started to decrease. The rise in the pressure of the pump was found to increase as the stiffness decreased. The pump performance could be clearly distinguished according to the stiffness of the valve. The observation of the valve movements revealed that the valve opening time did not change regardless of the operating frequency, but it changed with the valve stiffness. The delay in time for the outlet valve increased significantly with an increase in the frequency. It seems that the overlap of the opening time of the inlet valve and the outlet valve plays an important role in pump performance. Therefore, it is advisable to use different designs for the inlet and outlet valves, where the shape and stiffness of the valve are adjusted. Hydraulic pumps are used in various applications, including for robots, precision machinery, and medical applications. There is a large variation in their specifications given their widespread use ,2,3,4, aTypical driving mechanisms of micropumps are reciprocating, rotary, and peristaltic. Rotary pumps require external motors, making it difficult to implement miniaturization and are not used much, and reciprocating pumps and peristaltic pumps are often used . AlthougIn this study, an experimental study was conducted on the effect of valve stiffness on the performance of micropump. The basic working principle of the pump was analyzed and the pump was built to conduct experimental research. A cantilever valve was used for the check valve, and the valve stiffness was changed by adjusting the neck width of the valve, and three different stiffness designs were adopted. Basic performance measurements were made on the pump and the movement of the valve was observed using visualization techniques with a high-speed camera. The effect of the stiffness of the valve was investigated in view of the valve behavior.P means the total power, This study used piezoelectric elements that move in one direction, and it repeatedly contracts and expands depending on the applied voltage. This operation is periodically repeated to generate the flow and pressure of the pump. When the piezoelectric element expands, the fluid in the chamber is discharged to the outlet as the chamber volume contracts, and conversely, when the piezoelectric element contracts, fluid is sucked into the chamber. At this time, the fluid dynamic performance of the pump can be expressed as shown in Equation (1) (1)P=14QAt this time, the flow rate A pump operated by the frequency can be described as a vibration of the spring system. Equation (3) is a spring system that vibrates a pump piston using a piezoelectric element .(3) considering the light transmission and very similar refractive index with the working fluid. The valve and the membrane were made of PET  with thicknesses of 0.2 and 0.1 mm, respectively. Since the performance of the pump is greatly affected by the stiffness of the valve, three valves were designed according to the stiffness shown in The fluid used in a pump is one of the most important aspects of the pump. In the previous study, the results of the analysis showed that the flow rate produced by the pump was inversely proportional to the dynamic viscosity ,29,32,33The entire experimental setup was constructed as shown in In this study, the shadowgraphic image technique was used to analyze the movement of the valves. This technique observes the valve inside the pump by using the shadow generated by an obstruction of the light source. Although both the inlet and outlet valves were designed to be visible, both valve images could not be taken simultaneously due to the limited view of the high magnitude lens. Therefore, the motion of the two valves was taken separately including a piston and the valve movement was synchronized according to the piston movement. The actual imaging area for the valve movement is shown in Since the performance of the pump varies depending on the driving frequency, the pressure and flow rate were measured by varying the operating frequency of the piezoelectric element. The driving frequency is 10 to 150 Hz, and the driving waveform is a sinusoidal wave.The flow rate measurement results are depicted in The backpressure for the pump with the flow rate for each valve model was measured with a driving frequency of 60 Hz, and the results are shown in The previous analysis of the performance confirmed that the stiffness of the valve influenced the rise in pressure of the pump. The inside of the pump was visualized to investigate the mechanism of this phenomenon occurring inside the pump.The normalized valve movement according to the frequency is presented in The stiffness is important to improve the pump performance, and the behavior of the inlet and outlet valves is different, so it is recommended to produce the corresponding design. Both valves can be designed with a reduced stiffness to allow for operation, but at the same time, an improper stiffness can induce unstable operation. Therefore, when designing a valve, both the stiffness and the valve operation must be considered.This study investigated the behavior of the valves with various stiffnesses and frequencies to assess the performance in a reciprocating pump driven by a piezoelectric element. The flow rate and pressure that were measured to increase as the driving frequency increased, as expected. In addition, the backpressure of the pump was found to increase as the stiffness of the valve decreased. The effect of the resonance in the specific frequency region could be observed. The behavior of the valve according to the driving frequency of the pump and valve stiffness was analyzed using visualization techniques, and the valve opening time did not change regardless of the operating frequency, but it changed with the valve stiffness. The delay in time for the outlet valve increased significantly with an increase in the frequency. As a concluding remark, the inlet and outlet valves could have different designs to improve performance. Improvements in performance and operational stability can be expected through optimization studies on inlet and outlet valves with different stiffness.


To evaluate marginal fit and internal adaptation of three-unit Zr frameworks fabricated from four Zr CAD/CAM milling systems.Fixed partial denture models were replicated (40 stone models) using Polyvinyl Siloxane impression material (PVS) and type IV stone for Zr framework fabrication. FPDs were milled with four CAD/CAM systems, Group-II: LAVAL Zirconia milled by LAVA , Group-2: Vita In-Ceram YZ milled by Cerec®, Group-3: Zirconia milled by GM1000 and Group-4: Zirconia milled by DWX-50N. Twelve marginal gap measurements per framework were performed at pre-established points, with a metallurgical microscope  at 500X magnification. Eight measurements of cement space per section were performed for adaptation. Data was analyzed using ANOVA and Tukey post hoc test.Zirconia FPD frameworks exhibited gaps ranging from 16 to 50.1 µm for marginal fit and 26.8 to 102.5 µm for internal adaptation. Group-3 [20.8 (8.3) µm & 50.3 (11.4) µm] and Group-4 [16.0 (4.0) µm & 40.2 (8.8) µm] specimens showed significantly lower marginal fit and internal adaptation gaps compared to Group-I [50.1 (13.4) µm & 100.5 (16.7) µm] and Group-2 [38.9 (8.2) µm & 102.5 (13.4) µm] specimens respectively.Different CAD-CAM systems for fabrication of Zr FPD frameworks displayed a significant influence on marginal fit and internal adaptation of restorations. There has been a paradigm shift in the field of dentistry, especially with the growing awareness for high-quality cosmetic dentistry and biocompatibility.CAD-CAM systems employ contemporary technologies for milling restorations from densely sintered or partially sintered ceramic blocks.5One critical criterion for the success of CAD-CAM manufacturing systems is the marginal integrity and internal fit accuracy of the restorations. Accurate fit of restorations is extremely essential to achieve acceptable longevity.8It is reported that parameters specific to the milling and machining process including number and dimensions of burs, axis of milling and machining type (soft or hard) may effect the fit and adaptation of CAD-CAM restorations.st 2019)This experimental study was performed over a period of twelve months and reported inline with the checklist for reporting in-vitro studies (CRIS). The project was approved by the ethics and research committee at specialist practice and research center. (Ref. FR-031 June 21Acrylic resin teeth (second premolar and second molar) were mounted in clear EpoxiCure® Epoxy Resin  with space for a missing first molar tooth to form the master model . The mouUsing polyvinyl siloxane impression material  in sectioned stock tray, the “master model” was impressed with a light body material at the finish line and heavy body material in the sectioned tray. After 5-6 minutes of setting, each impression was examined for defects or air bubbles. A total of 40 impressions were made.All impressions were carefully boxed with boxing wax (Kerr). Thirty impressions were poured with type IV stone (Whip mix), generating thirty stone models for fixed partial denture (FPDs) frameworks fabrication. The other ten impressions were poured with scan able stone  according to manufacture’s instructions . Powder was added gradually to water and vacuum mixed for 20 to 40 seconds. A total of 40 models were fabricated.Partially stabilized Zirconia fixed partial denture (FPD’s) frameworks were milled for all models (forty) using four CAD-CAM systems. This resulted in four study groups based on the type of manufacturing techniques and systems (n=10). A total of forty zirconia frameworks were fabricated using the recommended materials and following the manufacturers instructions . The fouGroup-II: LAVAG Zirconia  frameworks milled by LAVA CNC 240.Group-2: Vita In-Ceram YZ  frameworks, scanned with InEos Red Scan , milled with Cerec® inLab .Group-3: AadvaG Zirconia (Zr) (GC Advanced technologies Inc.) frameworks milled by GM1000 (GC Advanced technologies Inc.).Group-4: Katana Zirconia  frameworks milled by DWX-50N .Each framework was secured on the respective stone model with a special clamp with uniform force at the pontic area. A metallurgical microscope  connected to a high precision digital video-micrometer  was used as for marginal gap measurements at 500x magnification. A total of twelve marginal gap measurements per framework were performed at pre-established points, six (6) measurements per tooth “mesio-buccal, mid-buccal, disto-buccal, mesio-lingual, mid-lingual, disto-lingual” for both prepared abutment teeth . The imaAll frameworks were cemented on respective stone die using dual cure resin cement RelyX™ Unicem Aplicap™ 3M ESPE). Cementation was done individually and one RelyX™ Unicem Aplicap™ capsule was used per framework. The RelyX™ Unicem Aplicap™ was activated and triturated for 15 seconds  utilizing a 3M ESPE CapMix™ Triturator according to manufacturer’s instructions. The cement was dispensed into the framework and then placed on the die. A weight device axially loaded frameworks on the dies at 50N for 10 minutes with the metal ball centrally at the occlusal part of the pontic. Excess cement was cleaned and allowed to auto cure (eight minutes). All cemented frameworks were carefully boxed (boxing wax-Kerr) and embedded using clear epoxy resin . Specimens were sectioned  under 200 grams load at a speed of 500 rpm under copious irrigation, mesio-distally and bucco-lingually through the center of abutment teeth, dividing each specimen six parts. . The finM ESPE. CThe maximum and minimum marginal fit gap was observed in Group-II (50.1 ± 13.4 µm) (LAVAm Zirconia- LAVA CNC 240) and group-4 (16.0 ± 4.0 µm) (Katana Zirconia-DWX-50N) specimens respectively . HoweverThe means and standard deviations for internal adaptation of frameworks in each group are presented in The present study was based on the hypothesis, partially stabilized zirconia restorations when fabricated using different CAD-CAM milling systems will show a significant difference in their marginal fit and adaptation. It was observed that FPD fabricated with Katana Zirconia-DWX-50N (group-4) and AadvaD Zirconia-GM1000 (group-3) showed better marginal fit and internal adaptation compared to LAVA Zirconia- LAVA CNC 240 (Group-II) and Vita In-Ceram YZ- Cerec® inLab (group-2) specimens. Therefore the hypothesis was accepted. A myriad of reasons are implicated for the findings including material properties, milling parameters  and cement space and viscosity.Multiple factors involved in the methodology of the study can influence the marginal gap and internal adaptation, including, cement type, cement thickness, cement mixing, seating pressure and preparation margin type.18In the present study, AadvaA Zirconia (Zr) frameworks milled by GM1000 and Katana Zirconia frameworks milled by DWX-50N were compared to LAVA Zirconia frameworks milled by LAVAs CNC 240 and Vita In-Ceram YZ frameworks milled by Cerec® inLab. There is limited data reporting the use of Katana zirconia and AadvaA Zirconia in the literature.The present study revealed, that the internal adaptation of fixed partial denture specimens was significantly different based on the type of CAD-CAM system used. Specimens milled in group-3 (Aadva Zirconia GM1000) and group-4 (Katana Zirconia-DWX-50N) showed significantly lower internal adaptation gaps than Group-II (LAVAL CNC 240) specimens. A possible explanation of these findings can be derived from the fact that LAVA Zr milling undergoes post milling sintering for milling (soft machining) efficiency of Zr substructure, resulting in a linear shrinkage of nearly 20%.24Outcomes of the present study should be interpreted in light of the limitations. The Zr materials used in the study groups were manufactured by different companies, therefore may differ in composition. In addition, FPD frameworks were seated on artificial teeth abutments with a static force of 50N, however intraoral forces are dynamic and can be upto 400N in some cases. The cemented specimens were sectioned using a destructive method, which may have possibly influenced the fit and adaptation measurement outcomes. Hard machining with a 5-axis milling although showed better fit and adaptation outcomes for Zr frameworks in the present study, however there is a continuous need for further improvements in the technology of hard machining with regards to cutting efficiency, time consumption and surface properties of milled materials. In addition, the introduction of additive technology (3D Printing) for the manufacture of metal and Zr frameworks of dental restorations provide improvement potential in restorative fit and adaptation. Therefore, further investigations on the properties of multiple axis, laser milled and printed, hard machined Zr framework including surface topography, flexural strength, fracture toughness and color stability are recommended.The Zr CAD-CAM systems assessed exhibited marginal fit and internal adaptations measurements of 16.0 to 50.1 µm and 40.2 to 102.5 µm respectively. For both fit and adaptation, specimens in Group-3 (Aadvam Zirconia (Zr)- GM1000) and Group-4 (Katana Zirconia- DWX-50N) showed better outcomes then specimens in Group-II (LAVA Zirconia/ LAVA/ CNC 240) and group-2 (Vita In-Ceram YZ/ Cerec® inLab). Marginal fit and internal adaptation values of all materials and systems tested were within clinically acceptable standards.KA: Experimentation, Data collection, study design, manuscript writing, final manuscript approval.AM: Data collection, study design, manuscript drafting, data analysis, funding and equipment, manuscript approval.RA: Specimen design and preparation, Data collection, manuscript approval and data interpretation.FV & TA: Data collection, sample preparation, writing, revision, editing, final manuscript approval, table and figure designing.As per the ICMJE authorship requirement, all authors are responsible and accountable for the accuracy or integrity of the work.


Allograft tissue stiffness (both cortex and medulla) was significantly negatively correlated with body mass index , and positively correlated with Proteinuria/Creatinuria ratio  but remained statistically significant only in cases with stable function. The cortical tissue stiffness proved significantly higher values for patients with allograft dysfunction as compared to patients with stable function, but to evolve as an additional tool for the evaluation of patients with a kidney transplant and to change the clinical practice, more extensive studies are needed.Shear-wave elastography (SWE) showed the absence or presence of significant differences among stable kidney allograft function and allograft dysfunction. We evaluated the variability of kidney allograft stiffness in relation to allograft dysfunction, respectively, in terms of a correlation of stiffness with patients’ characteristics. A single-center prospective study on patients who had undergone renal transplantation was conducted between October 2017 and November 2018. Patients were clinically classified as having a stable allograft function or allograft dysfunction. SWE examinations performed by the same radiologist with a LOGIQ E9 were evaluated. Ten measurements were done for Young’s modulus (kPa) at the level of allograft cortex and another ten at the level of medulla. Eighty-three SWE examinations from 63 patients, 69 stable allografts, and 14 allografts with dysfunction were included in the analysis. The intra-examinations stiffness showed high variability, with the quantile covariation coefficient ranging from 2.21% to 45.04%. The inter-examinations stiffness showed heterogeneity (from 28.66% to 42.38%). The kidney allograft cortex stiffness showed significantly higher values in cases with dysfunction  = (25.68–31.98) kPa) as compared to those with stable function  kPa;  The kidney is one of the most frequently transplanted organs with 58.9% kidney transplants in the United States of America (USA) from 1988 to 2019 /(CPPt0/CPPt-1), were CPP = cortex-pelvis proportion, PI = pulsatility index, t0 = values from the day of biopsy, t-1 = values from three to seven days before biopsy) and investigated how its value varies on normal kidney graft function, acute tubular necrosis, acute cellular rejection and acute vascular rejection [The presence of similar morphological and functional characteristics of the kidney allografts among those with dysfunction as compared to those with stable function indicate no feature characteristics among these two groups . Howeverejection . Howeverejection , pulse pejection  affect tejection ,42,43,44The CQV is a measure of spread that depends on the estimation of location (first and third quartile in our study) and is used in laboratory medicine to assess the precision of measurements. An inter- and intra-assay CQV under 15% is considered acceptable ,46, and p-value < 0.02). The values of the tissue stiffness at cortex in our study are similar to those reported by Ma et al. [In our study, the SWE stiffness showed significantly higher YM cortex values as compared to the medulla, regardless of the presence or absence of allograft dysfunction . Furthermore, the patients with allograft dysfunction had a significantly higher stiffness at the cortex as compared to those with stable allograft function  [p-values < 0.03) positive correlations of cortical (0.26) and medullary stiffness (0.59) with interstitial fibrosis [p-value = 0.006), a similar result being also found in our cohort.The association analysis for tissue stiffness showed a significant association with BMI (negative association) for both cortex and medulla values and Proteinuria/Creatinuria ratio (positive association) . Conside < 0.05) ,50,51. T < 0.05) . Ma et afibrosis . Järv etfibrosis  have alsSeveral limitations of our study need to be considered. First, this is a single-center study conducted over one-year, and therefore the investigated sample size is limited, and the reported results do not support generalizability or the change of clinical practice in the evaluation of kidney allograft function. Furthermore, the measurement of kidney allograft stiffness is dependent on a series of biological factors such as blood and urinary pressure, the recipient and donor atherosclerosis and age, as well as mechanical factors such as the pressure on the probe, the subjective evaluation of anisotropy in post-processing, etc. The experimental design reduces the possible influence of some above-listed factors , but the small sample size did not support the correction of such factors during the statistical analysis. The development of a SWE investigation guideline for kidney allografts evaluation and inclusion of more centers over a more extended period will assure a reliable assessment of the stiffness variability as well as validation of the identified results. Second, the function of patients was clinically classified as kidney allograft dysfunction or stable function, and only patients with clinical dysfunction underwent kidney biopsy. Establishing the true thresholds for tissue stiffness requires a gold standard diagnosis, namely allograft biopsy. An experimental design based on the gold standard diagnostic (biopsy) will help establish reliable thresholds of tissue stiffness that allow the identification of allograft dysfunction and classification on different dysfunction grades. Third, the SWE examination was performed by one radiologist during a single sitting, and therefore the interobserver variations could not be determined. Fourth, the investigated cohort was relatively small, most of the transplants were from deceased donors, and the patients were predominately male; thus, generalizability is not recommended. The evaluation of kidney allografts from living and deceased donors, as well as the evaluation of donor features, could also be beneficial in the SWE assessment of kidney allografts.The cortical tissue stiffness proved significantly higher values on patients with allograft dysfunction as compared to patients with stable function but to evolve as an additional tool for evaluation of patients with a kidney transplant and change the clinical practice more extensive studies are needed. Kidney tissue stiffness showed high intra- and inter-examination variability, and this variability need to be explained. Extensive studies are needed to identify the kidney allograft stiffness in order to assess the capacity of SWE to discriminate between patients with and without allograft dysfunction.


Prunus dulcis, Pistacia vera and Juglans regia. Presented is the initial technical analysis of the first 3 years. With no exception, levels of reserves changed continuously throughout the year suggesting that even during dormancy, the average concentration of NSC, starch and sugars varies seasonally. In general, carbohydrate reserves are highest entering dormancy. During winter, NSCs slowly decrease to depletion during bloom time and remain low during summer until recovery near harvest. Starch is the major reserve compound in the wood of P. dulcis and P. vera while soluble sugars are the major reserves in J. regia. NSC content fluctuates throughout a season and significantly varies between years suggesting intrinsic and climatic effects on trees’ energy reserves.Perennial plants in temperate climates evolved short and long-term strategies to store and manage reserves in the form of non-structural carbohydrates  and starch (St)). NSC storage allows plants to survive seasonal periods of photosynthetic inactivity (dormancy). To study year-to-year seasonal patterns of trees’ NSC dynamics that control phenology and yields, we established a large scale, multi-year study called the “Carbohydrate Observatory” using a citizen science approach with ~ 590 sites throughout the Central Valley of California. Monthly sampling tracked seasonal trends of starch and sugar levels in both xylem and phloem of twigs in  These two pools of carbohydrates are under biological control that evolved to meet demands of species-specific internal (physiology) and external  properties4. Allocation of NSC to the aforementioned functions must be balanced with storage in order to buffer asynchronous supply and demand on different temporal and spatial scales5. All plants evolved the capacity to overcome a daily cycle of discontinuous photosynthate availability by accumulating carbohydrate reserves to survive nights13. It is also known that perennial plants had to evolve long term strategies to acquire energy reserves as they cycle through periods of growth and reproduction (activity) that are associated with photosynthetic activity and periods of inactivity (dormancy)14.Most basic plant functions, like growth, energy metabolism, maintenance respiration, osmosis and transport, are dependent on non-structural carbohydrates (NSC). Generally, NSC exist as two pools: chemically active soluble carbohydrates (SC) present as mono and disaccharide sugars like glucose, fructose, and sucrose and sometimes sugar alcohols like sorbitol, and inactive, insoluble starch (St)16. During bud break, fast and efficient mobilization of stored carbohydrates are needed to sustain bud growth18. Thus, the amount of NSC reserves ahead of the winter should be enough to sustain winter respiration and spring demands for bloom and leafing. Species vary in their seasonal pattern of NSC reserve pools based upon vegetative habit, reproduction, growth, and duration of dormancy14. These evolved NSC management traits are being challenged now by fast climatic changes such as the rise of global temperatures, the increase in the frequency of droughts, and the length and severity of winter with significant, often detrimental, effects on tree phenology21 endangering the future of orchards and natural ecosystems. Therefore, it is important to study inter-seasonality of NSC patterns, that occur during adverse conditions like low temperature or prolonged drought23.These long-term strategies require that during the times of activity, NSC reserves are accumulated and allocated to processes that require energy or structural input such as growth, reproduction, and defense. To survive the continuous metabolic requirements during dormancy and later allow for the resumption of post dormancy growth (spring flowering and leafing), deciduous perennial plants must acquire and store an ample supply of carbohydrates in their woody tissue24. As orchard trees were presumably selected for high productivity in expense of energy reserves security, even small changes to NSC management induced by climate change or management practices can disproportionally affect productivity and orchard health.Interestingly, during the active growing period, the pools of NSC are relatively low and constant while the highest variability of the NSC pools are observed during the time of dormancy and spring bloom14 conducted a detailed within-year NSC study at the organ and whole tree level of five dominant temperate tree species in the Northeast to quantify total NSC storage over the course of a year and found that in general, whole-tree total NSC pools built up over the growing season and declined over the dormant season. However, there are no long-term observational studies over multiple years that look at interannual variability. Thus, the questions of how active and variable the pool of carbohydrate reserves in trees are and when reserves are being used and replenished in respect to trees’ phenology and climatic conditions still remains open. Therefore, to begin to understand intra and inter-seasonal dynamics and patterns of tree NSC, a large scale, multi-year, multi-species study called the “Carbohydrate Observatory” was established in 2016, Using a citizen science approach, samples were obtained from ~ 590 orchard sites throughout the Central Valley of California , P. vera L. and J. regia L. trees. This analysis spans a time period from January, 2017 to December 2019. We analysed 590 orchard sites, with a total of 5330 individual observations, split between P. dulcis (2502) P. vera (1781) and J. regia (1047). We encouraged growers to collect samples once a month, although frequency and participation level varied over time.Using a Citizen Science approach, growers spanning the entire Central Valley of California sent samples of current-season wood and bark from twigs of A specific unified protocol for sample collection required that one current season twig from three trees per orchard were cut at the base of the twig where the current season’s wood met last year’s wood. The bark from the lower 10 cm of the twig was removed using a razor blade. Both the bark and the wood of the three twigs were put in a paper envelope and mailed to the laboratory for NSC analysis. In total, we performed 31,980 soluble sugars and 31,980 starch analyses for bark and wood. It is important to note that prior to the project we tested the integrity of the NSC content over shipping time and found that sugar and starch do not break down within 4 days . Analysis of the NSC integrity was tested for each species on five separate twigs. Each twig was collected using the standard protocol and separated to four sections. One piece was immediately place in the oven while the remaining samples were left in the shipping envelopes. On day 2 another piece was removed from the envelope, similarly another sample was taken on day 3 and 4 . We used a linear mixed model with twig as random factor to determine presence of changes in NSC, SC and St content in wood and bark. There was no missing data. Results of the analysis are presented in Table .26; with modifications described in Tixier et al.24. Specifically, 25 mg of powder per sample was placed in 1.5 ml tubes. Tubes were then treated with 1 ml of sodium acetate buffer , vortexed, and incubated in a 70 °C water bath for 15 min and centrifuged . 50 µl of supernatant was extracted and diluted in ultra-pure (UP) water (1:20 v:v) and vortexed. SC content was quantified from diluted supernatant tubes using an anthrone/sulfuric acid colorizing reagent (0.1% (m:v) in 98% sulfuric acid) and reading absorbance at 620 nm in a spectrophotometer. The remaining centrifuged tubes with the pellet and buffer were used for starch quantification. To extract starch, the tubes were vortexed to suspend the pellet, boiled at 100 °C for 10 min to allow starch gelatinization and let sit for 20 min at room temperature (22 °C). Once cooled, 100 µl of amyglucosidase  and 100 µl amylase  were added to the tubes and incubated for 4 h at 37 °C in a rotating incubator. Samples were then centrifuged . 50 µl of supernatant was extracted and diluted in 1 ml UP water (1:20 v:v) and vortexed. Total soluble sugar content was analysed using the same method described above. Starch content was determined by subtracting original SC content from post-digestion SC content. All samples were plated on 96-well plates. To account for any procedural variability , each plate contained a glucose standard curve and wood/bark standard tissue samples with known SC and St content (four per plate) that were concurrently undergoing all steps in the same chemical analysis. The wood/bark standard tissue is a sample from a homogenous mix of several thousand ground samples leftover from a 2016 preliminary part of the study. We measured out 25 mg of 96 samples at of the mix and analyzed them for NSC (starch and sugar) using our protocol.Upon arrival, samples were put in the dryer for 48 h at 75 °C. The bark and wood were chopped into small < 1 mm pieces separately and ~ 100 mg of each were ground into a fine powder (~ 1 µm) using a ball grinder . To analyse for sugar and starch we used the previously described protocol from Leyva et alFor statistical analysis we used R. For Kruskal Wallis using ‘kw’ function from R package ‘seastests v0.14.2’ with 12 months lag time. For the augmented Dickey Fuller (ADF) test we used ‘adf.test’ function from R package ‘tseries v0.10-47’. For ANOVA analysis to compare carbohydrate content between months in consecutive years, we used the ‘aov’ function from R package ‘stats v3.6.2’.J. regia, all NSC, St, and SC in bark and wood in all three species showed significant seasonal trends with a 12-month frequency   suggests that, with the only exception of soluble sugar content in the wood of e) Table . To detee) Table , thus sue) Table . Generalch Table .Table 3KP. vera and lowest in P. dulcis. The average maximum NSC content was observed in fall months (September to October) preceding the typical leaf senescence and ranged from 113 to 148 mg/g DW in P. dulcis, 156–189 mg/g DW for P. vera, and 154–165 mg/g DW in J. regia. These high levels were followed by the dormancy period during which, NSC content in branches is depleted prior to spring bud break. Throughout the bud break period, NSC depletion was continuous in P. dulcis but in both P. vera and J. regia we observed a characteristic increase in NSC content in April/May that coincides with leafing. Lowest levels of NSC were observed in April and May in P. dulcis (45–71 mg/g DW), May in P. vera (45–52 mg/g DW) and July in J. regia (58–84 mg/g DW), which typically coincides with the most active period of vegetative growth (diameter and height) and initial nut filling. From June to July we observed a recovery of the NSC content to maximum in the fall . In P. dulcis wood NSC content was 1.5 times higher (~ 77 to 49 mg/g DW) in the fall than in bark and in J. regia NSC content in the wood was only 1.3 times higher than in bark (~ 89 to 68 mg/g DW). Those differences were very consistent in consecutive years. Absolute content of NSC in wood was relatively constant for each month between years in all three species. However, content in the bark varied significantly among the years in P. vera and P. dulcis but was invariable in J. regia , and xylem are tissues that have the capacity to store NSC. For all three species, the general pattern of NSC content is similar to each other with maximum in the fall and minimum in the early summer Fig. . NSC conP. dulcis SC content varied from 23 to 53 mg/g DW and St content varied from 1.7 to 13 mg/g DW, in P. vera SC content varied from 14 to 29 while St content varied from 2 to 23 mg/g DW, and in J. regia SC content varied from 26 to 57 with St varying from 4 to 37 mg/g DW. SC content was less variable than in the wood tissue with variation being only ~ 2 times different between low content in spring and high in winter. St content was much more variable. During summer, levels of St reserves in the bark were near complete exhaustion (near < 5 mg/g DW) but recovered by fall (September–October) contributing significantly to the NSC reserves. Surprisingly, the bark St reserves were depleted before the winter (by December) and remain low through the rest of the dormancy period. This pattern was highly consistent across the 3 years of observations and similar in all three species. Year-to-year SC content variation in bark was significant in all three species while starch content only varied in J. regia  fluctuation suggests year-round activity, supporting the notion that there is no true dormancy. There is no time to rest for tree carbohydrate metabolism.The seasonal patterns of NSC, starch, and sugar show global similarities and some species-specific differences. One major result of this study is that NSC content in twigs is highly variable throughout the season with fall content being two-to-three times higher than during the active time of spring growth. The highest level of NSC reserves occurs between September and October, typically preceding leaf senescence by 1 or 2 months. This peak is followed by a continuous slow decrease of NSC content throughout the winter. These findings are consistent with other seasonal NSC studies. Matinez-Vilalta27 seemingly during the highest level of overall demand. This suggests that starch reserve accumulation is not a passive activity occurring in response to an excess of soluble sugars but is instead a competitive process to other carbon demanding processes like growth and fruit development29. Starch content reaches a maximum in bark in late summer (August–September) and in wood (September–October) and is followed by slow degradation while soluble sugars still accumulate in both tissues over winter. This suggests that starch degradation (as starch accumulation) is not a passive response to a low soluble sugar content level, but rather an active process performing biological functions potentially related to chill response and bloom timing28. The large swings of SC concentration in summer and winter and the counterintuitive response of St content might also suggest that SC levels are controlled at different concentrations in trees during the active period and dormancy. Interestingly, we did not observe any spike in NSC accumulation postharvest when one would expect an excess of photosynthates due to removal of fruits and significant slowdown in growth while maintaining leaf photosynthetic capacity. However, we only analyzed the twigs, thus it is possible that, at the end of the tree growth cycle during fall, most of the photosynthetic output is exported to basal parts of the tree–trunk and roots27.In all three species in all 3 years, accumulation of NSC reserves in twigs starts in mid-summer, during the nut filling period and active girth growth30 and possibly whole tree redistribution of sugars24. However, the large swings between NSC forms needs a deeper look. Starch degradation over winter seemed to be associated with an increase of SC that lasts till bloom/leafing. The increase in SC during winter is often explained as part of the tree’s response to frost that requires input of osmotica and energy to counter frost, but in the Central Valley in California, frost is a highly infrequent event and cannot be used to explain the observed trend. It is more likely that accumulation of SC and degradation of starch reflects underlying biological activity where SC-starch balance during winter can be associated with time/temperature memory of dormancy28 and could be linked to the concept of chilling in future studies.During winter, a slow loss of total NSC observed in all three species is accompanied by highly dynamic changes in soluble sugar and starch content. The slow loss could be explained by respiration activity that is affected by winter temperaturesP. vera has the most yearly variation of ~ 300%, P. dulcis ~ 200%, while J. regia only ~ 150%. It seems that this yearly variation is not related to maximum pre-winter reserves , but rather minimum amounts tolerated by each species in the summer. Thus, pre-winter accumulation of NSC that could reflect climatic constraints related to dormancy length seemed of lesser importance in seasonal patterns of NSC. However, tolerance to summer risks and yield capacity can be more important in shaping seasonal variation of NSC content. P. vera and P. dulcis seemed to tolerate very low reserve levels (risk taking) in late spring but rebuilt the reserves in competition to fruit and vegetative growth enforcing the notion of reserves being a strong competitive sink during summer in preparation for dormancy. J. regia reached a minimum of NSC in mid-summer before restoration of reserves is initiated allowing only a short period of time for reserves to build up. Overall though, J. regia seemed to maintain higher NSC levels in summer, potentially representing a lower risk strategy to summer stress.The three species differed in variation between minimum and maximum NSC content: 29, the year-to-year variation might be related to some stochastic parameters, potentially weather variation, tree breeding patterns (masting) and/or horticultural practices. However, this study spans only spans 3 years. The determination of the non-stationarity (year-to-year variation) using climatic , management (changes in applied management strategies) or tree species-specific intrinsic responses  is premature, but possible in the near future.In general, NSC, SC, and St content in wood and bark show highly significant seasonality in addition to significant interannual variation that suggests that while a general NSC pattern is most likely driven by seasonal/phenological eventsP. dulcis and P. vera which show significant variation between months in the year-to-year analysis. The variations were especially pronounced in SC content. Interestingly, starch content was less variable suggesting that low summer values are persistent in the current climate under applied management practices, while high winter values potentially reflect physical space limitation for starch storage in twigs. In addition, year-to-year levels of variation of SC might be a result of not only climatic variability, but also yield history especially in alternate bearing species. Nevertheless, assuming those levels of NSC and their composition is physiologically important; the year-to-year variation can provide useful information to assess orchard performance, potential yield, and orchard health.While the annual pattern of NSC content in twigs, their distribution between bark and wood, and the dynamics of a starch and soluble sugar fraction showed high levels of seasonality and a similar pattern in three consecutive years, the absolute content shows some variability between specific months in a year-to-year analysis (patterns were non-stationary). This was especially true for P. dulcis and P. vera are running early summer growth and fruit set with almost zero reserves illustrates that this is the most vulnerable period, as even a small level of stress could result in carbon deficit and affect carbon demanding processes causing fruit drop, reduced vegetative growth or slowdown in NSC reserves buildup. The fact that there is no time to rest for carbohydrate management in trees underlines the importance of winter conditions on carbohydrate activity—their redistribution and twig preparation for bloom. Knowledge on minimum requirements to survive winter to assure synchronous bloom can offer new options to mitigate changing climatic conditions when evolved traits might be inadequate for new challenges like winter drought and extreme temperature swings. In addition, observed year-to-year variation in total NSC content and minimum/maximum values might have an impact on species productivity and explain yield variability. This unique large-scale multiyear study on both bark and wood dynamics and both intra and inter-seasonality variation, provides a solid foundation for future studies analyzing effects of climate and phenology.The presented study provides a temporal analysis of NSC reserves in perennial cropping systems and opens a door for designing NSC-based novel management practices. Specifically, a better understanding of the post-harvest recovery of NSC content and its redistribution is vital as the tree prepares for dormancy. Perhaps the timing of irrigation, fertilization and other management practices should be carefully considered for this period in order to most efficiently recharge carbohydrate storage for the following season. Current practices like chemical defoliation might result in unexpected side effects related to their effect on fall NSC management. For example, reduced fall photosynthates supply may not affect NSC content in twigs but significantly reduce their export to trunk and roots affecting spring growth. Further, knowledge that