The mean ± standard deviation (SD) BFSs for DC and ML were the highest in the control groups. Surface air abrasion with 50 µm Al2O3 particles and 2 bar pressure is recommended for 3Y-TZP translucent zirconia, while no abrasion of 5Y-ZP translucent zirconia ceramic.In this study, the biaxial flexural strength (BFS) and fractography of high/ultra-translucent monolithic zirconia ceramics subjected to different mechanical surface pretreatments were evaluated. A total of 108 disc-shaped samples  of three zirconia materials (5Y-ZP KATANA Zirconia UTML (ML), 3Y-TZP DD Bio ZX2 (DB), and 5Y-ZP DD cube X2 (DC)) were used. The BFS was investigated after subjecting the samples to surface treatment using air abrasion particles of two types . The data were analyzed using two-way analysis of variance, followed by Scheffe’s post hoc test for multiple comparisons. The mean ± standard deviation BFS for DB was highest after treatment with 50 µm Al All-ceramic-based dental restorations are of particular importance due to their superior biocompatibility and aesthetics compared to porcelain fused to metal restorations . More sp2O3) [To improve the material translucency and overcome this shortcoming, newer generations of zirconia with higher translucency parameters have been developed. More specifically, first-generation materials consisted of 3Y-TZP and contained 0.25 wt% alumina (Al2O3) . Althoug2O3) . This ty2O3) . Such im2O3) .Subsequently, third generation materials were developed using an yttria content of 5 mol% (5Y-ZP), which is referred to by manufacturers as ultra-translucent (or super-high-translucent) zirconia, while fourth generation materials were based on zirconia with an yttria content of 4 mol% (4Y-ZP), which exhibited enhanced mechanical properties . Both thTo alter the internal surface of zirconia and increase both its mechanical retention and its bonding to the resin cement, air abrasion is commonly employed  using aiHowever, the effect of air particle abrasion on the flexural strength of 3Y-TZP zirconia is controversial, since it depends on the size of the abrading particles, their type, and the air pressure employed . For exaCurrently, limited evidence is available regarding the use of low abrasive particles and low air pressures when treating high-translucent zirconia . Air abrTo date, the effects of different air particle abrasion protocols on the flexural strength of highly translucent zirconia have yet to be examined in detail. The aim of this study is therefore to evaluate the biaxial flexural strength (BFS) values and carry out a qualitative fractographic analysis of high-translucent and ultra-translucent monolithic zirconia ceramics subjected to different mechanical surface treatment protocols. Two null hypotheses are tested: (1) the biaxial flexural strengths of high-translucent and ultra-translucent zirconia ceramics are not affected by air abrasion surface treatment with 50 µm aluminum oxide particles, and (2) the BFSs of high-translucent zirconia and ultra-translucent zirconia ceramics are not affected by air abrasion surface treatment with 50 µm glass microbead particles.n = 12 per group) according to the surface treatment employed  were prepared from pre-sintered zirconia blocks  using computer-aided design and computer-aided-manufacture techniques  . The sinemployed . More spEach air abrasion procedure was performed under a standardized pressure of 2 bar with a nozzle placed at a 90° incidence angle from the center of the sample. The nozzle was placed at a distance of 10 mm and air abrasion was carried out for 20 s using a sandblaster  ,29,30.1 is the radius of the support circle (5 mm), r2 is the radius of the loaded area (0.6 mm), and r3 is the radius of the specimen (6 mm).A total of 108 samples were ultrasonically cleaned with 99% isopropanol for 180 s, air-dried, and subjected to biaxial flexural strength testing according to ISO 6872 . The samThe fractured surfaces were ultrasonically cleaned for 10 min using distilled water, then air-dried and examined using a digital microscope  for determination of the fracture origin in relation to the fractography principles of ceramics . The selp > 0.05). Two-way analysis of variance (ANOVA) was conducted to evaluate the null hypothesis, followed by Scheffe’s post hoc tests for multiple comparisons (p < 0.05). The level of significance was set at p ≤ 0.05.Data were collected and grouped for statistical analysis using a statistical software package (SPSS version 23). Statistical analysis was performed using the Shapiro–Wilk test of normal distribution (p < 0.05), and the interaction effect between the ceramic material group and the surface pre-treatment group was also found to be statistically significant (p < 0.05). In addition, one-way ANOVA showed a statistically significant difference between the BFS values of the surface pre-treatment groups for all ceramic materials (p < 0.05)  .2O3, with a lower value being obtained following treatment with 50 µm glass microbeads, and the lowest value being observed for the control. In contrast, the mean ± SD BFSs for the DC and ML samples reached their highest values in the control and became lower after treatment with the 50 µm glass microbeads and the 50 µm Al2O3 particles (The mean ± SD BFS for the DD Bio ZX2 (DB) sample reached its highest value after treatment with 50 µm Alarticles .p < 0.05). More specifically, for the DB samples, the mean ± SD BFS after treatment with 50 µm Al2O3 was significantly higher than the control sample and after treatment with the 50 µm glass microbeads (p < 0.05). In contrast, both the DC and ML samples showed mean ± SD BFS values that were statistically significantly higher in the control group than after treatment with either the 50 µm Al2O3 beads or the 50 µm glass microbeads (p < 0.05)  .2O3 particles resulted in the generation of rougher surfaces, while for the groups treated with 50 µm glass microbeads, pitting of the surface was observed in all cases. The various fractured discs obtained following surface treatment of the samples are shown in Significant differences in the topography of the abraded ceramic surfaces in comparison with control can be seen . The useSEM examinations revealed numerous characteristic features for the fractured zirconia ceramic surfaces, wherein the fracture origins (*), direction of crack propagation (dotted arrows), secondary cracks, hackles, river delta, arrest lines, and compression curls were clearly evident . Twist hIn the present study, we evaluated the BFSs of three highly translucent monolithic zirconia ceramics after subjecting them to different mechanical surface treatment protocols. Previous studies have demonstrated that sandblasting with aluminum oxide particles at 0.2 MPa improved the BFSs of both conventional and high-translucent 3Y-TZP zirconia ceramics ,35,36. IThe flexural strength of dental ceramics can be determined using 4-point and 3-point bending tests or a BFS test . HoweverAir abrasion by alumina particles is known to promote a transformation toughening mechanism within the zirconia, in which the tetragonal to monoclinic phase transformation is accompanied by a volumetric expansion that limits the propagation of the initiated cracks ,37,43. TFractography allows for the accurate examination of fractured surfaces that contain microscopic features in which the direction of crack propagation is pointing toward the fracture origin site . The quaWe found that the DB sample exhibited multiple secondary cracks that likely led to shattering of the ceramic disc into multiple fractured pieces of varying sizes and the loss of smaller fragments of ceramics. For the DB sample, the resulting surface and subsurface porosities promoted crack initiation in these areas, and progress in multiple planes was frequently observed wherein secondary cracks were also present. Furthermore, in the case of the ML sample, a smooth fracture surface with clear fracture lines was generally observed.Thus, based on the results of this study, the use of 50 µm aluminum oxide particles for air abrasion at 0.2 MPa pressure could be recommended for the surface treatment of high-translucent 3Y-TZP zirconia ceramics. In the case of 5Y-ZP ceramics, the use of 50 µm glass microbeads for surface treatment negatively affected the BFS values, and so a smaller particle size should be considered in further studies.The limitations of the current study include the fact that we concentrated on the type of air abrasion particles, while all other parameters were maintained constant. A limitation of the methodology used in this research is that the samples were machined, leaving micro-grooves that could affect the transformation at the surface, and this could impact durability and strength of zirconia. Moreover, the effect of aging was not examined. Future studies should therefore investigate additional parameters, such as different air abrasion pressures and particle sizes.2O3 particles at a pressure of 2 bar resulted in the highest BFS values. In addition, the BFS of the tested 5Y-ZP ultra-translucent zirconia ceramic was negatively affected when air abrasion was carried out using either 50 µm Al2O3 particles or 50 µm glass microbeads. Smaller particle size should be considered in future research.Within the limitations of this study, it could be concluded that the air abrasion of a 3 mol% yttria-stabilized tetragonal zirconia polycrystalline (3Y-TZP) translucent zirconia surface with 50 µm Al


Acceptable or poor  organ quality were independent predictors of EAD. Our data do not support the notion that aprotinin prevents nor attenuates PRS, EAD or mortality.The number of patients awaiting liver transplantation still widely exceeds the number of donated organs available. Patients receiving extended criteria donor (ECD) organs are especially prone to an aggravated ischemia reperfusion syndrome during liver transplantation leading to massive hemodynamic stress and possible impairment in organ function. Previous studies have demonstrated aprotinin to ameliorate reperfusion injury and early graft survival. In this single center retrospective analysis of 84 propensity score matched patients out of 274 liver transplantation patients between 2010 and 2014 (OLT), we describe the association of aprotinin with postreperfusion syndrome (PRS), early allograft dysfunction  and recipient survival. The incidence of PRS (52.4% vs. 47.6%) and 30-day mortality did not differ (4.8 vs. 0%;  Increasing organ shortage for liver transplantation is a major challenge for transplant hepatology. To address this situation and to overcome the discrepancy between organ demand and supply, marginal donors or extended criteria donors (ECD) are more often accepted to increase the number of available donor organs ,2.www.eurotransplant.org/organs/liver/, accessed on 22 September 2021). In the Eurotransplant region, available donor organs are primarily offered to patients matching the ABO blood group with the highest model of end stage liver disease (MELD) score nearest to the explantation site ) + 6.4 ×  ) + 11.2 × logherwise)  were recherwise) , Bilirub−1; (3) increase of continuous norepinephrine (NE) infusion of ≥0.1 μg kg BW−1 within 5 to 30 min after reperfusion; or (4) initiation of continuous vasopressin infusion after reperfusion. According to our department’s SOP, PRS was treated as follows: (i) 0.5 mg atropine before reperfusion if heart rate < 80; (ii) NE boli and NE infusion to maintain MAP; (iii) epinephrine boli and infusion in the case of significant bradycardia with hypotension and the decrease of SVO2 during reperfusion; (iv) infusion of vasopressin if high doses of NA are necessary or NA therapy is ineffective.Postreperfusion syndrome was defined as the occurrence of one of the following criteria: (1) decrease in mean arterial pressure (MAP) of at least 30% at time of reperfusion; (2) administration of an intravenous bolus of norepinephrine >2 μg kg body weight (BW)p-value ≤0.05 was considered statistically significant.Using organ quality, donor age and CIT, we identified a propensity score matched control group with the nearest neighbor method . Matching variables were selected a priori, as these were the criteria used to apply aprotinin at our center at that time. Differences between groups were analyzed using the t-test for continuous, Chi-Square test for categorical and variance analysis for repeated measurements for continuous variables over time. Kaplan–Meyers curves were generated to display the effect of aprotinin on patient and graft survival. A logistic regression analysis was calculated to estimate the effect of aprotinin on early allograft dysfunction and a linear regression to estimate the effect on peak AST. Covariates  were introduced because they were deemed relevant by the literature or by clinical judgement. Parameters were included in the multivariate approach if the univariate analysis was significant (SPSS 24.0). Results were displayed as mean and standard deviation or absolute and relative number of cases. Figures were created using Prism 6.0 . A two-sided 2 balance test showed no significant imbalance  in the matched cohort (n = 84).A total of 274 patients received a liver transplantation graft during the five-year study period and 42 of these patients were treated with aprotinin. Whether the patient was treated with aprotinin to attenuate PRS and EAD had been a shared decision of the attending anesthesiologist and the transplant surgeon based on the following criteria: (i) organ quality ; (ii) cold ischemia time; and (iii) age of the donor. In a propensity score analysis using these three criteria, 42 statistically similar patients were identified who were not treated with aprotinin and were assigned to the control group. Matching reduced the relative multivariate imbalance L1 (0.634 vs. 0.782) and the χp = 0.061).Demographic and clinical characteristics of the study population are described in detail in p = 0.018) and a higher ALT .Demographic and clinical characteristics of the liver graft donors for the matched study population of liver graft recipients are described in p = 0.414) and hyperfibrinolysis . Similarly, the number of transfusion units of packed red blood cells, fresh frozen plasma and platelets, as well as the amount of fibrinogen and four-factor prothrombin complex concentrate, did not differ between liver graft recipients who were treated with aprotinin compared to liver graft recipients who were not.Differences in outcome between liver graft recipients who were treated with aprotinin compared to recipients who were not treated with aprotinin are displayed in p = 0.029), suffered more often from acute kidney injury  and more often required renal replacement therapy  than controls. Multivariable regression analysis confirmed this finding: Intraoperative treatment with aprotinin was associated with a 4-fold risk , acceptable donor organ quality with a 5-fold risk  and poor donor organ quality with a 12-fold risk  of developing early allograft dysfunction. The multivariable regression analysis adjusted for recipient age, sex, BMI and MELD, donor age, BMI and AST, cold ischemic time and organ quality .Liver graft recipients who were treated with aprotinin had more postoperative complications than recipients who were not treated with aprotinin—they suffered more often from early allograft dysfunction  and ALT  after transplantation of the graft. A multivariable linear regression model confirmed this by demonstrating that having been treated intraoperatively with aprotinin was associated with a 1.324 U/L (95%CI 354–2.295) higher maximum AST after adjusting for recipient age, sex, BMI and MELD, donor age, BMI and AST, cold ischemic time and organ quality  and had higher postoperative peak transaminases compared to controls. In fact, multivariable regression analyses determined intraoperative treatment with aprotinin to be independently associated with a four-fold risk of EAD and an on average 1.300 U/L higher peak AST after liver graft transplantation in patients receiving organs from extended criteria donors.All of the 84 liver graft recipients in this analysis were treated at the same center with the same operative technique   and withIn our sample, the incidence of EAD was 52%. While the incidence of EAD has been reported in the literature between 23%  and 39% Aprotinin is a protease inhibitor derived from bovine or porcine lungs. As such, aprotinin inhibits human proteases such as, for example, trypsin or kallikrein but moreover plasmin, thus decelerating hyperfibrinolysis. In 1989, aprotinin was first demonstrated to reduce blood loss, transfusion requirements and duration of surgery in liver graft recipients , a findiAprotinin is a serine protease inhibitor. Of these proteases, interaction with plasmin and kallikrein are likely the most important, for hemostasis and the reduction of inflammation. Additionally, aprotinin also inhibits matrix metalloproteinases (MMP). MMPs are zinc-binding proteolytic enzymes, which are responsible for the degradation of extracellular matrix proteins and basement membranes. Detriments in transplant livers following cold storage and ischemia reperfusion are essentially the liver sinusoidal endothelial cells (LSEC) ; for exaAprotinin is transiently stored in the tubular cells for 5–6 days . This maThe idea of pharmacologic preconditioning by aprotinin of the OLT recipient in the case of liver transplantation of ECD organs did not improve the short nor the long-term outcomes in this study. Regarding our results presented in this study, we changed our standard protocol for liver transplantations and do not further applicate aprotinin regularly.


Prostate-specific antigen (PSA) is considered neither sensitive nor specific for prostate cancer (PCa). We aimed to compare total PSA (tPSA), percentage of free PSA (%fPSA), the PSA density (PSAD), Prostate Health Index (PHI), and the PHI density (PHID) to see which one could best predict clinically significant prostate cancer (csPCa): a potentially lethal disease.A total of 412 men with PSA of 2–20 ng/mL were prospectively included. Serum biomarkers for PCa was collected before transrectal ultrasound guided prostate biopsy. PHI was calculated by the formula: (p2PSA/fPSA) x √tPSA. PHID was calculated as PHI divided by prostate volume measured by transrectal ultrasound.p=0.004). At 90% sensitivity, PHID had the highest specificity (54.1%) for csPCa and could reduce the most unnecessary biopsies (43.7%) and miss the fewest csPCa (8.5%) when PHID ≥ 0.67. In addition to AUC, DCA re-confirmed the clinical benefit of PHID over all PSA-related parameters and PHI in csPCa diagnosis. The PHID cut-off value was positively correlated with the csPCa ratio in the PHID risk table, which is useful for evaluating csPCa risk in a clinical setting.Of the 412 men, 134 (32.5%) and 94(22.8%) were diagnosed with PCa and csPCa, respectively. We used the area under the receiver operating characteristic curve (AUC) and decision curve analyses (DCA) to compare the performance of PSA related parameters, PHI and PHID in diagnosing csPCa. AUC for tPSA, %fPSA, %p2PSA, PSAD, PHI and PHID were 0.56、0.63、0.76、0.74、0.77 and 0.82 respectively for csPCa detection. In the univariate analysis, the prostate volume, tPSA, %fPSA, %p2PSA, PHI, PSAD, and PHID were all significantly associated with csPCa, and PHID was the most important predictor . Besides, The AUC of PHID was significantly larger than PHI in csPCa diagnosis (The PHID is an excellent predictor of csPCa. The PHID risk table may be used in standard clinical practice to pre-select men at the highest risk of harboring csPCa. Prostate cancer (PCa) is one of the most common malignancies in both Western and Asian countries. The introduction of the prostate-specific antigen (PSA) test in 1987 is one of the reasons for the growing incidence of PCa. Produced by prostate epithelial cells, PSA is regarded as an organ-specific rather than a disease-specific marker. The correlation between PSA and benign prostate hyperplasia, prostate inflammation, and PCa makes it a marker with broad clinical utility; however, it is a complex tool in terms of confirming the cancer diagnosis, with a 60%–70% false positive rate .About 2% of patients have post-biopsy complications, such as infection, bleeding, or voiding difficulty . MoreoveProstate Health Index (PHI), a novel PCa screening alternative, is calculated with total PSA (tPSA), free PSA (fPSA), and [-2]pro-PSA (p2PSA) using the following formula: (p2PSA/fPSA) x √tPSA. PHI is proved to be better at predicting the presence of PCa and its aggressiveness than tPSA, fPSA, and PSA density (PSAD) in multiple studies in both Western and Asian countries –9. CurreIn recent years, PHI density (PHID) has been a focus of research for its clinical utility. One prospective study of 118 men in Western society receiving prostate biopsy showed PHID is associated with clinically significant prostate cancer (csPCa) and outperformed PHI in the area under the receiver operating characteristic curve (AUC) analysis . CsPCa  x 100. Prostate volume was estimated with transrectal ultrasound with the standard ellipsoid formula: width x height x length x 0.52. PSA density (PSAD) was calculated with (tPSA/prostate volume), and PHI density (PHID) was calculated with (PHI/prostate volume).Biopsy specimens were graded according to the updated Gleason grading system of the International Society of Urological Pathology . The speThe primary end point was to evaluate the sensitivity, specificity, diagnostic accuracy, and clinical benefit of %fPSA, PSAD, %p2PSA, PHI and PHID (index tests) in determining the presence of PCa and csPCa at prostate biopsy in comparison to tPSA (standard tests).The primary outcome was csPCa found on biopsy. Continuous variables were reported as median and interquartile range (IQR). Statistical differences were assessed with Mann–Whitney U test for continuous variables and Chi-square test for categorical variables. Univariable logistic regression was used to determine the association between measured covariates and prostate cancer and clinically significant prostate cancer. The area under the receiver operating characteristic curve (AUC) was used to examine the diagnostic ability of each PSA derivative. Difference between AUCs were evaluated with DeLong test. Decision curve analysis (DCA) was applied to compare different diagnostic strategies with regards to maximizing clinical net benefit at different threshold probability . StatistOf the 412 men included, 134 (32.5%) were diagnosed with PCa and 57 (42.5%) had a GS of 6 PCa. 94 of 412 men (22.8%) were diagnosed with csPCa, of which 77 (81.9%) were GS ≥7 and the rest 17 men (18.1%) had a GS of 6 fulfilling the Epstein criteria. Table 1.The univariable logistic regression Table 2 p=0.007 and 0.004, respectively). Among the tested biomarkers, PHID showed the highest discriminative ability for PCa and csPCa.AUC was used to examine the ability of each diagnostic marker to indicate PCa  under different PHID values Table 4.In our prospective cohort, we compared the performance of tPSA, %fPSA, %p2PSA, PSAD, PHI, and PHID in terms of predicting csPCa without missing the diagnosis. We found PHID was the best predictor of csPCa and could greatly reduce the number of unnecessary biopsies. We also found that the PHID cut-off value was positively correlated with the ratio of csPCa. We could further evaluate the patient’s csPCa risks based on this PHID risk table to decide whether to arrange a prostate biopsy. In our understanding, this is the first time a PHID risk table to evaluate the csPCa risk has been established.PSA is neither sensitive nor specific in csPCa prediction, leading to many unnecessary biopsies and indolent cancer detected –3. ThereThere is no denying that multiparametric magnetic resonance imaging (mpMRI) is the best tool for predicting csPCa –23, but To make up for the shortcomings of the low specificity and low sensitivity of PSA, PHI was developed. The first prospective PCa screening study in 2010 found PHI and %p2PSA (AUC= 0.77 and 0.76) could distinguish PCa from benign diseases more accurately than tPSA AUC= 0.50) . Afterwa0 . AfterPHI shows an excellent ability to accurately diagnose csPCa in different races. Our previous study  shows thLarger prostate volume is associated with increased PSA levels . Benson p=0.004, Prostate volume is an important factor for csPCa and should be added to PSA-related factors to improve PCa detection. Filella et al. found PHI to be associated with prostate volume. The AUCs of PHI in patients with small, medium, and large prostate volumes were 0.818, 0.716, and 0.654, respectively, suggesting that a larger prostate size would decrease PHI diagnostic ability . RecentlThe optimal PHID cut-off value was still not determined in as a result of the scarcity of the related studies. Tosoian et al. proposed a cut-off value 0.43 to detect Epstein significant disease with a 97.9% sensitivity and a 38.0% specificity . BarisieAssociation of different PCa biomarkers with mpMRI findings is another interesting topic worth researching. The combination of biomarkers and mpMRI may result in more clinical benefit than PSA plus mpMRI, especially in those who had equivocal PI-RADS scores , 57, 58.Our study had several limitations. First, the study cohort was heterogenous with biopsy-naïve subjects and those with biopsy histories. This may have confounded the results, as less PCa was detected in those who had prior biopsies. Secondly, the sample size of our study was relatively small. The PHID results and PHID risk table for csPCa need further external verification by other studies focused on different ethnicities. Third, mpMRI was not routinely performed in our entire cohort. We are incorporating PHI and mpMRI data from our cohort database. Further study results will be analyzed when more subjects are enrolled in the coming future.In our prospective cohort, we found that PHID had the best performance, could reduce the most unnecessary biopsies, and missed the fewest csPCa cases. The PHID cut-off value is positively correlated with the csPCa ratio in the PHID risk table. In conclusion, the PHID has excellent ability to predict csPCa before biopsy. The PHID risk table may be used in standard clinical practice to pre-select men at a higher risk of harboring csPCa.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.The studies involving human participants were reviewed and approved by National Taiwan University Hospital . The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.Study design and conceptualization: C-YH, C-HC, Y-TC. Data collection: Y-TC and S-TC. Formal analysis: Y-CL, J-HH, and S-DC. Writing—original draft preparation: Y-TC and S-TC. Writing—review and editing: Y-SP, C-YH, and C-HC. 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Living cells orchestrate enzyme activities to produce myriads of biopolymers but cell-biological understanding of such processes is scarce. Starch, a plant biopolymer forming discrete, semi-crystalline granules within plastids, plays a central role in glucose storage, which is fundamental to life. Combining complementary imaging techniques and Arabidopsis genetics we reveal that, in chloroplasts, multiple starch granules initiate in stromal pockets between thylakoid membranes. These initials coalesce, then grow anisotropically to form lenticular granules. The major starch polymer, amylopectin, is synthesized at the granule surface, while the minor amylose component is deposited internally. The non-enzymatic domain of STARCH SYNTHASE 4, which controls the protein’s localization, is required for anisotropic growth. These results present us with a conceptual framework for understanding the biosynthesis of this key nutrient. Starch is the major form of energy storage in plant cells and forms discrete, semi-crystalline granules within plastids. Here the authors use electron tomography and nanoSIMS to show that Arabidopsis starch granules initiate in stromal pockets between thylakoid membranes that coalesce before growing anisotropically. Starch forms as discrete, semi-crystalline granules within sub-cellular plastid compartments, i.e., the chloroplasts of green plants and algae and the amyloplasts of non-green heterotrophic tissues, such as seeds, roots, and tubers. The main molecular constituents of starch are the glucans amylopectin (70–90%) and amylose (10–30%). Amylopectin is a branched polymer, synthesized by a suite of interdependent enzymes, and underpins the semi-crystalline nature of starch. Amylopectin fine-structure varies between plant species as a function of the relative amounts and complex interplay between the starch biosynthesis enzymes2. Amylose is an essentially linear polymer made by a single enzyme, Granule-Bound Starch Synthase (GBSS)3. Observed variations in the number, size, and morphology of starch granules are highly species specific5 and the morphological characteristics of starch in archeological remains are used as identifiers of crop species used by early civilizations7. However, despite the global importance of starch for human civilizations, the genetic and cell-biological basis for the structural and morphological diversity of starch are largely unknown.The major source of calories for society is plant-derived carbohydrates. The most important of these carbohydrates is starch—a remarkable, insoluble biopolymer2 and degraded to support metabolism at night, when photosynthesis cannot occur. Arabidopsis chloroplasts were reported to produce 5–7 lenticular starch granules on average8. The starch biosynthetic enzymes are well known9: α-1,4-linked glucan chains are elongated by a set of five starch synthases (SS: E.C. 2.4.1.21). The α-1,6-branches are introduced by two branching enzymes (BE: E.C. 2.4.1.18) and the structure is finally tailored by isoamylase-type debranching enzymes (ISA: E.C. 3.2.1.68) to promote its crystallization into a lamellar structure with a 9–10-nm periodicity. The capacity to form insoluble, semi-crystalline starch-like granules was recently engineered into the non-starch synthesizing yeast Saccharomyces cerevisiae through the introduction of the Arabidopsis enzymes. This synthetic biology approach demonstrated that the core components of starch biosynthetic apparatus are identified10.In leaves, starch is formed in chloroplasts during the day from photo-assimilated COss4 mutant chloroplasts exhibiting a strongly diminished number of granules13. Furthermore, ss4 mutant granules are aberrant in morphology, i.e., more spherical than lenticular. The abilities of SS4 to both promote granule initiation and control their growth depend on its C-terminal glucosyltransferase domain and on its non-enzymatic N-terminus, respectively14. However, the molecular mechanisms underpinning SS4 function are not understood. Recent genetic studies have identified additional proteins involved in granule initiation and growth16. These include PROTEIN TARGETING TO STARCH (PTST) family proteins, which are proposed to deliver glucan substrates to SS417, SS5, a non-enzymatic homolog of SS418, and two coiled-coil domain proteins MAR-BINDING FILAMENT-LIKE PROTEIN 1 (MFP1) and MYOSIN-RELATED CHLOROPLASTIC PROTEIN (MRC) that bind to and help localize PTST2 and/or SS420. The precise molecular functions of these proteins are also unclear due to the lack of knowledge about the actual process of starch granule initiation.The starch synthase isoform STARCH SYNTHASE 4 (SS4) is known to play a key role in starch granule initiation, with To discover how starch granule initiation occurs and to track subsequent granule development, we employed a suite of complementary microscopic methods. Serial block face scanning electron microscopy (SBF-SEM) allowed us to visualize the initiation processes in 3D and quantify changes in granule numbers. The additional use of carbon isotope labeling and quantitative nanoscale secondary ion mass spectrometry (NanoSIMS), in combination with molecular genetic approaches, enabled us to understand the role of granule initials and follow granule expansion patterns during the day. These findings provide a new level of mechanistic insight into the cell biology of starch biosynthesis.−1 fresh weight (FW) starch  and their chloroplasts contained 12.8 ± 2.3  lenticular granules . Leaves sampled at the end of the night had 0.24 ± 0.03 mg g−1 FW  starch and 4.4 ± 1.0  granules per chloroplasts, which appeared as small discs, thinned at the poles  and decreased by ~50% after 30 min light without a change in the number of starch-containing pockets suggests that either some starch granule initials are degraded again, or granule initials coalesced as starch synthesis proceeded. The irregular appearance of granules after 30 min is suggestive of the latter, implying that granules can have multiple initiations. Furthermore, the fact that after 8 h light, the numbers of both starch granules and starch-containing pockets increased suggests that new granules continued to be initiated during the day.13CO2 stable isotope labeling of illuminated plants, followed by transmission electron microscopy (TEM) and subsequent high resolution, quantitative isotope mapping of the imaged samples using NanoSIMS21. After de-starching chloroplasts by extending the night to 16 h, a pulse of 13CO2 air was given at the start of the day to label newly formed starch during the first 15 min of photosynthesis. Leaf samples harvested immediately after the pulse, or after a 15-min, 45-min, or 4-h chase in normal air, were chemically fixed and embedded in resin for TEM analysis. During fixation and dehydration, most soluble 13C-labeled compounds are lost from the sample, while 13C-labeled cellular structures, including starch, are preserved. All of the starch initials clustering in pockets were labeled after the pulse of 13CO2, showing that they were newly synthesized . As for the first experiment, most label was incorporated non-uniformly onto the granule surface, with stronger enrichment along the margins than on the faces  were allowed to photosynthesize for 45 min in the light, then labeled for 15 min with les Fig. . This inces Fig. . Interesces Fig. . Howeverss4 mutant, which is defective in the initiation and growth of granules11. The chloroplasts of ss4 have few large, rounded starch granules . SBF-SEM revealed that, after a 4-h night extension, some ss4 chloroplasts still contained a granule, unlike the wild type , followed by immediate sampling. The almost uniform, isotropic labeling of the granule surface in ss4 suggests that SS4 is required for normal anisotropic granule growth  displayed a similar surface-labeling pattern as in the wild type , granules from the amylose-free ype Fig. . Howeverype Fig. . In contype Fig. . These d25. By combining high-resolution spatio-temporal imaging techniques with genetics, we bridge these disciplines to address previously intractable questions about biopolymer formation in vivo. Collectively, the parallel initiation, followed by fusion and localized starch accumulation, challenge the classical view of granule development and illustrates its dependence on the emerging network of proteins that includes both enzymes and scaffolding factors.Understanding the in vivo biosynthesis of biomacromolecules requires genetic, biochemical, and cell-biological insight. We have brought together a new combination of experimental methods and analytical techniques to bridge these disciplines in the study of starch—arguably the most important storage biopolymer produced in plants and a vital agricultural product. As with many biopolymers, starch granule formation is brought about by the concerted actions of numerous enzymes, the coordination of which is evidently genetically controlled, but poorly understood at the cell-biological level. The study of granule initiation in Arabidopsis provides us with a tractable genetic system with which to identify the genes and proteins controlling these processes19 that starch granule initiation in chloroplasts occurs at defined sites in-between the thylakoid membranes, i.e., the stromal pockets we describe here. Some proteins implicated in initiation are themselves partly  or wholly (MFP1) thylakoid-associated and localize to chloroplast subdomains25. Further, our SBF-SEM analysis of de-starched plants, i.e., after 16-h nights, revealed thylakoid-bounded regions differing in appearance to the rest of the stroma. We propose that these are the subdomains where the granule initiation proteins accumulate, thereby defining stromal pockets where granule initials will form. This is a hypothesis we are testing with other correlative microscopy methods. Previous models of starch granule biosynthesis generally imply a single point of initiation—the hilum—and suggest that when multiple initiations occur, compound starch granules are formed, with each with its own hilum . Our study unambiguously demonstrates that single granules can arise from multiple parallel initiations that subsequently coalesce. In chloroplasts, this pattern of multiple initiations may be functionally important in allowing the rapid establishment of sufficient granule surface area, which is key to the high rate of starch deposition that accompanies photosynthesis. It will be important to determine next whether similar granule initiation patterns occur in the plastids of other plant storage organs, such as in amyloplasts of seeds and tubers, and whether this process influences starch yields.There is strong evidence from this work Fig.  and prev28. It is unclear if these semi-crystalline structures are already formed in the observed granule initials. If semi-crystalline lamellae are present, they are unlikely to be organized in a concentric way considering that, at ~50 nm in diameter, the initials are not much larger than the lamellae themselves. Rather, each initial may be composed of a stack of a few, similarly oriented lamellae. We speculate that, for adjacent granule initials to coalesce, the orientations of the constituent polymers may need to be similar , and that initials with opposing polymer orientations  might not fuse. Indeed, not all adjacent granules in the same pockets fuse, despite the fact that our NanoSIMS imaging revealed deposition of new material onto their flat, abutting surfaces  must be concentrated at the equatorial regions of the granule. In other starch synthesizing tissues, starch granules also take on very specific morphologies. By analogy with the observations made here, we suggest this also happens via the non-random patterns of deposition of new material. Remarkably, our data show that SS4 is critically required for growth anisotropy in Arabidopsis; when missing, newly synthesized starch is deposited almost uniformly onto the granule surface, explaining why the starch granules in 34, which we demonstrate to be correct through isotope labeling and NanoSIMS imaging.It is possible that the sites of granule synthesis in stromal pockets may be examples of liquid–liquid phase-separation brought about by many low-affinity protein–protein and protein-glucan interactions. A number of the aforementioned proteins have amino-acid regions predicted to be intrinsically disordered—a common feature in proteins capable of condensing into phase-separated organelles not enveloped by membranes. Starch biosynthetic enzyme activities may also be significantly increased by high local concentrations of glucan substrates within such condensates (while the actions of degradative enzymes might be simultaneously excluded). This type of enzyme stimulation has been demonstrated for GBSS, the activity of which shows biphasic kinetics as soluble glucan substrate concentrations breached a threshold, above which viscosity and turbidity increased, signifying the onset of phase transition. This, together with other observations, prompted the hypothesis that GBSS is active within the granule matrixIn conclusion, the power of combining genetics with innovative imaging techniques to capture temporal and spatial information with high resolution has allowed us to answer previously intractable questions about starch biosynthesis. This approach, extended to the study of other important biopolymers in vivo, could enable similar breakthroughs in those fields.Arabidopsis thaliana L., ecotype Col-0, grown on individual pots of fertilized bedding compost (Klasmann-Deilmann Substrate 2) for 35 days in controlled environment cabinets  with a 12-h light (150 µmoles photons m–2 s–1)/12-h dark regime, with regular watering. The relative humidity was 60% and the temperature was 20 °C. Plants defective in SS4 (At4g18240) and their transformed lines expressing Agrobacterium tumefaciens glycogen synthase, or glycogen synthase linked to the N-terminus of SS4, are those that were described previously14. Plants defective in GBSS (At1g32900) were also described previously35.All experiments were carried out with 13CO2 labeling , plants were transferred to an airtight chamber within the growth cabinet. Air was passed through the chamber in which CO2 was substituted with 380 ppm 13CO2 . Labeling to investigate granule initiation was started 5 min before dawn and continued for the first 15 min of the light period. Labeling to investigate granule growth was done for 60 min at midday. Plants were harvested immediately after the 13CO2 pulse or transferred to the growth cabinet for a defined chase period.For 36. The blocks were trimmed, glued on pins, and sectioned/scanned ca. In all, 500 times using a scanning electron microscope  at 1.8–2 kV with an integrated 3View stage and a back-scattered electron detector . The structural features were marked out manually with Amira .Samples were taken from the upper quadrant of the lamina of unshaded leaf #6, between the major vein and the leaf margin, for all imaging experiments. Each fixation and staining step was followed by three washing steps with the corresponding buffer. Leaf samples were fixed for 6 h at 20 °C, 200 mbar in fixation solution . Samples were then stained in a medium containing 1.5% (w/v) potassium ferrocyanide, 2% (w/v) osmium tetroxide, 4 mM calcium chloride for 1 h on ice, followed by an incubation in pre-filtered 1% (w/v) thiocarbohydrazide solution  then in 2% (w/v) osmium tetroxide . Samples were then stained with 1% (w/v) uranyl acetate for 12 h at 4 °C and then with Walton’s lead aspartate for 30 min at 60 °C . Samples were dehydrated with a six-step ethanol series , followed by 100% acetone, each for 20 min at 4 °C. Samples were infiltrated in epoxy resin  with increasing concentrations  in acetone. The infiltrated samples were cured in molds for 48 h at 60 °CTmax = 22 °C settings for embedding18. Ultrathin sections (70 nm) were transferred onto formvar-coated copper grids , post-stained with uranyl acetate (10 min) and with lead citrate (10 min). Images were acquired with a transmission electron microscope  at 120 kV.When TEM was used for section imaging, leaf samples were fixed with fixation solution , stained with 1% (w/v) osmium tetroxide, dehydrated with a six-step ethanol series, infiltrated with a four-step  epoxy resin series in ethanol  and cured for 48 h at 60 °C. All steps except the last were microwave-assisted using a BioWave Pro+ , with 40 s/250 W settings for dehydration steps and 5 min/200 W/37, adapted as follows. Fixed samples were stained with 3% (w/v) potassium ferrocyanide (II) in 0.1 M sodium cacodylate, pH 7.4, with an equal volume of 4% (w/v) osmium tetroxide solution. Next, samples were incubated with pre-filtered 1% (w/v) thiocarbohydrazide solution and finally stained with 2% (w/v) osmium tetroxide solution. After embedding, 200-nm-thick sections were cut, collected on hydrophilised wafers (PELCO easiGlowTM Glow Discharge Cleaning System), and imaged with a GeminiSEM 500  scanning electron microscope (3 kV) using the back-scattered electron detector.When SEM was used for section imaging, samples were processed as for TEM, but osmium staining prior to dehydration and embedding was based on the OTO method20, pre-sputtered regions of interest (ROI) of 20 × 20 µm2 or 10 × 10 µm2 (256 × 256 pixels) were scanned with a 16 keV primary Cs+ beam, focused to a spot size  of ~100 nm with a pixel dwell time of 5 ms. For each image, the counts for the secondary ions 12C2−, 13C12C−, and 14N12C− were recorded over five rasters (planes) in electron multipliers at a mass resolving power of around 9000 (Cameca definition), enough to resolve the ions of interest from potential interferences in the mass spectrum. For each session, three analyses were done on similarly prepared, unlabeled plant material serving as isotopic standard against which 13C-enrichments were reported in the δ-notation, according to the equation:12C13C−/12C2− ratio of the sample and C_nat is the average 12C13C−/12C2− ratio measured in unlabeled samples (isotopic controls).Grids previously imaged with TEM or by SEM (semi-thin samples deposited on silicon wafers) were coated with ca. 15 nm gold. Using a NanoSIMS 50 L instrument14N12C– map, which shared the highest similarity with the corresponding electron micrograph. All further image processing tasks were carried out in Python the ImageJ plugin OpenMIMS. The aligned and accumulated planes of 12C12C– and 13C12C– were used to compute the 13C-enrichment maps (in δ13C) and electron micrographs were then overlaid onto these maps using the 14N12C– map as a reference. Differences observed in the degree of enrichment could result from variation in (i) assimilation rates between plants, (ii) in the incident light and/or CO2 reaching a given chloroplast, and (iii) the amount of glucan present, i.e., the number stromal pockets in which granules can form or existing granule surfaces available for elaboration. To compare the 13C-enrichment in granule cores between the wild type and the gbss mutant, the ion maps were manually segmented into three regions: background, granule surface, and granule core. For each region, the average value of the enrichment along with the standard deviation was calculated.Using the ImageJ plugin OpenMIMS, ion map files (*.im) were corrected for dead time and aligned using the 38 to draw posterior samples from the probability density of generalized linear models ). The parameters were estimated using the Hamiltonian Monte-Carlo NUTS sampler using four chains, each with 16,000 iterations. The last 8000 iterations were used for analysis. The plots were drawn using the Python plot library Matplotlib.For each inferential procedure, we used StanFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Reporting Summary


There is a general concern about the rationalization of resources and the management of waste. Plant residues can contribute to the development of new non-polluting construction materials. The objective of this study was to valorize a plant residue such as the giant reed and obtain a particleboard with cement using potato starch as a plasticizer in a manufacturing process involving compression and heat. The influence of cement and starch in different proportions and its stability over time were analyzed. Finally, their physical and mechanical properties were evaluated and compared to European Standards. High-quality sustainable particleboards  were obtained and can be classified as P6 according to European Standards. Mechanical properties were improved by increasing the starch content and pressing time, whereas greater resistance to water was obtained by increasing the cement content. Giant reed particles seem to tolerate the alkalinity of the cement since there was no sign of degradation of its fibers. The use of these residues in the manufacture of construction materials offers a very attractive alternative in terms of price, technology and sustainability. This indicates that there was a water shortage that prevented a correct absorption of water by the cement. It is possible that water contained in the mixture before hot pressing was attracted by the reed particles and then evaporated when pressed. Boards made with starch could have retained this water and later released it to the cement to hydrate it properly, thereby improving the mechanical performance of the board.Particleboards are classified based on their mechanical properties and dimensional stability by European Standards , which eAccording to The boards with the best performance were types A4 and C4, which met European Standards  and coulHigher proportions of cement produced lower TH, whereas higher proportions of starch resulted in better mechanical performances. Starch could have retained part of the water previously added onto the mixture to later transfer it to the cement, favoring its correct hydration. This confirmed a study that suggested that in order to improve the properties of cementitious compounds, an additive of cellulose and starch was added because it behaves as a good water-retention agent .The two manufacturing variables considered in this study were the type of binder and the time in the hot plate press. The type of binder is the most influential parameter since all physical and mechanical properties varied according to it . The pressing time affected half of the properties tested .Several authors noticed that during the setting time of cement composites with plant fibers, there was a delay in the formation of hydration products ,28,79. TSEM micrographs showed calcium compounds at eight days of setting and therefore the hydration process had not been delayed. In addition, micrographs showed how the gelled starch enveloped the cement grains, which would make difficult its dilution and the interaction of the reed components with the cement.The hydration of the cement in the experimental boards is in accordance with the study carried out with cement without plant fibers at seven days . Some reOther researchers obtained composites made of cement and wood or plant fibers. Their results are shown in Industrial boards are normally manufactured with a wood/cement ratio of 1/5.5. The boards in this work have been made with a small amount of cement, with the ratio in weight of giant reed/cement being 1/0.2 maximum. Pressing time was a maximum of 4 h at 100 °C, while industrial boards need 8 h at 75 °C and drying times of 8 h at 90 °C. The experimental boards described in this study have been made with less time and with a much lower proportion of cement than in the production of industrial wood–cement boards. This represents significant energy savings in their manufacture and makes them potentially a more ecological product.Arundo donax L.) particles were investigated.The effects of the binder (starch–cement) on the physical and mechanical properties of boards made of giant reed  were obtained using giant reed and can be classified as P6 according to European Standards. Mechanical properties were improved by increasing the starch content, whereas greater resistance to water was obtained by increasing the cement content.The pressing time of the boards in the hot plate press at 100 °C had a great influence in MOR, MOE and IB, which increased performance. Further tests are needed to determine the optimum pressing temperature and time for the manufacturing process.Cement is mineralized in the giant reed–cement–starch particleboards following the same hydration process that occurred without fibers or additives. Giant reed particles seem to tolerate the alkalinity of the cement since there was no sign of degradation of its fibers, with MOR and MOE even increasing at 365 days of setting.Using giant reed particles, cementitious composites can be made which could be used in the manufacture of various construction components. Therefore, the use of these residues in the manufacture of construction materials offers a very attractive alternative in terms of price, technology and sustainability.


The study on non-host tomato environment correlated the dose × time aspects of xenobiosis by examining the correlation between bacterial survival in terms of log-reduction and defense responses at the level of gene expression. In E. coli, low (1 μg O3/g of fruit) and moderate (2 μg O3/g of fruit) doses caused insignificant reduction in survival, while high dose (3 μg/g of fruit) caused significant reduction in survival in a time-dependent manner. In L. monocytogenes, moderate dose caused significant reduction even with short-duration exposure. Distinct responses to O3 xenobiosis between E. coli and L. monocytogenes are likely related to differences in membrane and cytoplasmic structure and components. Transcriptome profiling by RNA-Seq showed that primary defenses in E. coli were attenuated after exposure to a low dose, while the responses at moderate dose were characterized by massive upregulation of pathogenesis and stress-related genes, which implied the activation of defense responses. More genes were downregulated during the first hour at high dose, with a large number of such genes getting significantly upregulated after 2 hr and 3 hr. This trend suggests that prolonged exposure led to potential adaptation. In contrast, massive downregulation of genes was observed in L. monocytogenes regardless of dose and exposure duration, implying a mechanism of defense distinct from that of E. coli. The nature of bacterial responses revealed by this study should guide the selection of xenobiotic agents for eliminating bacterial contamination on fresh produce without overlooking the potential risks of adaptation.Because of the continuous rise of foodborne illnesses caused by the consumption of raw fruits and vegetables, effective post-harvest anti-microbial strategies are necessary. The aim of this study was to evaluate the anti-microbial efficacy of ozone (O Label numbers from 0 to 19 denote genes from most lowly expressed to most highly expressed. Only those falls into either label 0 or 19 at least in one treatment were selected. Genes differentially expressed based on PCC were shown in columns L-N. (Sheet 3) Propensity of E. coli genes selected for 3 μg O3 network analysis. The 250 most highly expressed and 250 most lowly expressed genes from each treatment . Label numbers from 0 to 19 denote genes from most lowly expressed to most highly expressed. Only those fall into either label 0 or 19 at least in one treatment were selected. Genes differentially expressed based on Pearson Correlation Coefficient were shown in columns L-N. (Sheet 4) Co-expression network of E. coli genes for 1 μg O3 at 1 hr. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 5) Co-expression network of E. coli genes for 1 μg O3 at 2 hr. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 6) Co-expression network of E. coli genes for 1 μg O3 at 3 hr. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 7) Co-expression network of E. coli genes for 1 μg O3 control. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 8) Co-expression network of E. coli genes for 2 μg O3 at 1 hr. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 9) Co-expression network of E. coli genes for 2 μg O3 at 2 hr. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 10) Co-expression network of E. coli genes for 2 μg O3 at 3 hr. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 11) Co-expression network of E. coli genes for 2 μg O3 control. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 12) Co-expression network of E. coli genes for 3 μg O3 at 1 hr. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 13) Co-expression network of E. coli genes for 3 μg O3 at 2 hr. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 14) Co-expression network of E. coli genes for 3 μg O3 at 3 hr. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 15) Co-expression network of E. coli genes for 3 μg O3 control. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC.The 250 most highly expressed and 250 most lowly expressed genes from each treatment . Label numbers from 0 to 19 denote genes from most lowly expressed to most highly expressed. Only those falls into either label 0 or 19 at least in one treatment were selected. Genes differentially expressed based on Pearson Correlation Coefficient (PCC) were shown in columns L-N. ((XLSX)Click here for additional data file.S3 Table(Sheet 2) Propensity of L. monocytogenes genes selected for 2 μg O3 network analysis. The 250 most highly expressed and 250 most lowly expressed genes from each treatment . Label numbers from 0 to 19 denote genes from most lowly expressed to most highly expressed. Only those falls into either label 0 or 19 at least in one treatment were selected. Genes differentially expressed based on PCC were shown in columns L-N. (Sheet 3) Propensity of L. monocytogenes genes selected for 3 μg O3 network analysis. The 250 most highly expressed and 250 most lowly expressed genes from each treatment . Label numbers from 0 to 19 denote genes from most lowly expressed to most highly expressed. Only those falls into either label 0 or 19 at least in one treatment were selected. Genes differentially expressed based on Pearson Correlation Coefficient were shown in columns L-N. (Sheet 4) Co-expression network of L. monocytogenes genes for 1 μg O3 at 1 hr. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 5) Co-expression network of L. monocytogenes genes for 1 μg O3 at 2 hr. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 6) Co-expression network of L. monocytogenes genes for 1 μg O3 at 3 hr. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 7) Co-expression network of L. monocytogenes genes for 1 μg O3 control. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 8) Co-expression network of L. monocytogenes genes for 2 μg O3 at 1 hr. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 9) Co-expression network of L. monocytogenes genes for 2 μg O3 at 2 hr. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 10) Co-expression network of L. monocytogenes genes for 2 μg O3 at 3 hr. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 11) Co-expression network of L. monocytogenes genes for 2 μg O3 control. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 12) Co-expression network of L. monocytogenes genes for 3 μg O3 at 1 hr. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 13) Co-expression network of L. monocytogenes genes for 3 μg O3 at 2 hr. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 14) Co-expression network of L. monocytogenes genes for 3 μg O3 at 3 hr. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC. (Sheet 15) Co-expression network of L. monocytogenes genes for 3 μg O3 control. ’color’: ’brown’ demotes positively correlated by PCC; ’color’: ’green’ demotes negatively correlated by PCC.The 250 most highly expressed and 250 most lowly expressed genes from each treatment . Label numbers from 0 to 19 denote genes from most lowly expressed to most highly-expressed. Only those falls into either label 0 or 19 at least in one treatment were selected. Genes differentially expressed based on Pearson Correlation Coefficient (PCC) were shown in columns L-N. (XLSX)Click here for additional data file.


Southern African Journal of Infectious Diseases (SAJID) had undergone numerous changes over the years, largely because of evolutionary changes in journal publication practices. Amongst these changes were the following:The Modernisation of the look of the journal.Placement of the journal on an open access platform.Southern African Journal of Epidemiology and Infection to the Southern African Journal of Infectious Diseases.Changing the name of the journal from the The journal was placed on a sound financial footing, with the introduction of ‘page fees’.Despite the introduction of costs for publishing in the journal, the journal still continues to elicit numerous manuscript submissions, and pleasingly we have also seen an increase in submissions from sub-Saharan Africa, and beyond that including some submissions from the Middle East and even Europe.The journal remains the ‘mouthpiece’ of the Federation of Infectious Diseases Societies of Southern Africa (FIDSSA), an umbrella group of most infectious diseases societies in South Africa, and has been Department of Higher Education and Training (DHET) accredited for many years, both by way of being on the DHET SA list, and since 2021, by being on the Directory of Open Access Journals (DOAJ). What this means, amongst other considerations, is that articles published in the journal accrue governmental subsidy for the universities of the authors.Remarkably, all articles published in the journal are included in the African Index Medicus, DOAJ, Ebsco Host, Gale, Cengage Learning, Google Scholar and Hinari.The major next step that all potential authors hoped for was the indexing of the journal in PubMed Central. We are thrilled to announce, officially, as many of you may already know that the journal is now indexed in PubMed Central and also included in the Web of Science Other Coverage, Emerging Sources Citation Index (ESCI). As the Pubmed Central website indicates, for journals in the medical and life sciences, being indexed in PubMed is a high priority goal. Clearly the benefits are that it significantly increases the trust in the journals processes and its commitment to science, so that it becomes easier to attract high quality submissions. It also increases the journal’s visibility, leading to an increased reach and exposure of the journal with the hope of an increase in citations and eventually achieving an impact factor. With regard to indexing, the journal is highly appreciative of the role of all our loyal reviewers, the editorial board members and our publishers, AOSIS (Pty) Ltd, for their great professionalism.However, we cannot stop here and rest on our laurels. Recent activity that has aimed to achieve even greater heights for the journal include the following:Revision of the editorial board, in line with the common recommendations.The introduction of an improved process of facilitating the award of continuing professional development (CPD) points for both authors and reviewers for the journal.We need to strive further to increase our citations and therefore be granted an impact factor.Furthermore, it is time for the journal to continue succession planning for the next editor-in-chief (EIC). I had hoped that during my tenure as EIC I would at least be able to get the journal to the point where we are now. Having got there, I think it is therefore time for the appointment of a new leader, who as the saying goes, could be the new broom that sweeps clean, and who could lead the journal to even greater heights.I thank you for your confidence in me over all these years.


Properties of modified starch and its interaction with functional raw materials are of great interest to the food industry. Thus, this study aimed to evaluate the rheological and technological characterization of starches modified by the action of the enzymes α-amylase and amyloglucosidase and their mixtures with jaboticaba peel powder. The parameters of firmness, gumminess, and final viscosity of starches paste increased, and the tendency to setback was reduced with the addition of jaboticaba peel powder. Starches and mixtures presented shear-thinning behavior. The addition of jaboticaba peel powder to starches increased water, oil, and milk absorption capacity, while syneresis remained stable over the storage period. The addition of jaboticaba peel powder had a positive effect on native and modified starches' rheological and technological properties, qualifying it as an alternative for developing new functional food products. However, native starches have technological limitations3. Thus, there is substantial interest in modifying their structures to improve properties and increase starch application4. Enzymatic hydrolysis is considered the most promising approach offering more environmentally friendly processing since no solvents or chemical reagents are used5. Partial enzyme treatment modulates the granule starch's external macromolecular structure, thereby producing a slow-digesting starch6. Additionally, alterations of the starch molecular structure must obtain the desired viscoelastic properties and develop a more palatable food and increase shelf-life stability7.A wide range of native starches with profoundly different functionalities is required to meet market needs8. Rice (Oryza sativa L.) is part of the essential human diet and is consumed worldwide. Rice is composed mainly of carbohydrates as starch, comprising 90% of its total dry weight10. According to the study by4, native red rice starch comprises 18% amylose and 64% amylopectin. However, starches from different sources exhibit specific physicochemical, textural, rheological, and digestive properties of each particular plant12. Starch processing into food products is often performed at relatively high temperatures. These thermal treatments alter the starch rheological properties that influence several textural properties, nutritional quality, and ultimately, starch products' acceptability14.Besides, new starch sources specifically targeted to have a specific structure and functionality are required to meet market needs. This challenge may require new ingredients to help meet those market needs15. However, the low shear and thermal resistance while cooking and the syneresis of native starch preclude its use in specific food industry applications17. The peel of the jaboticaba (Myrciaria cauliflora) fruit is a food byproduct with nutritional and properties that can be explored. It has recently attracted researchers' attention who found high soluble and insoluble fibers, mineral salts, and phenolic compounds. Among the phenolic compounds, it is highlighted the high contents of anthocyanins, flavonoids, ellagitannins, and condensed tannins. These bioactive molecules have a powerful antioxidant effect that plays an essential role in the body's defense system inhibiting free radical scavenging19.Starch is the main ingredient in many foods and significantly contributes to the textural properties of the food. Moreover, starch can be used as a thickener, stabilizer, gelling agent, and water-retaining agent for many industrial applications20. The ingestion of insoluble fibers can cause a satiety sensation since it absorbs water and thus increases its size during the GI tract, which can cause pronounced effects on the intestinal transit and feces volume21. Some products, available in the Brazilian market, use fruit byproducts claiming health benefits to human health because of their high fiber content20. Dietetic fibers present in jabuticaba peel can increase the nutritional value of different foods, including breakfast cereals, bakery products, dairy, pet food, and can be used to formulate farmaceutical products using slow delivery of antioxidants18.Dietetic fibers, either soluble or insoluble, are edible polysaccharides that cannot be hydrolyzed by the enzymes present in the human gastrointestinal (GI) tract23. Besides the nutritional improvement, the inclusion of fruit byproducts can improve the starch's technological properties. The present work will provide helpful information regarding the rheological and technological characterization of a mixture between a red rice starch modified by hydrolysis with two enzymes (α-amylase and amyloglucosidase) and powdered jaboticaba peels at 10% wt.Therefore, the inclusion of such fruit byproducts can have a positive and synergic effect on starchy foods since they can increase the dietary and bioactive contentsOryza Sativa) were purchased in the local market. Starch was extracted by the method previously described elsewhere with adaptations24. Briefly, the red rice was firstly immersed in a sodium metabisulfite solution (0.2%)  in a ratio of 1:2 (w/v) for 48 h and then rinsed in running water. The treated red rice was then homogenized along with distilled water at a ratio of 1:2 (w/v) using an industrial blender  for 5 min and then filtered to obtain the starch suspension. The filtration residue was again homogenized to increase the process yield. The starch suspension was decanted for 24 h in a refrigerated environment at 7 °C to avoid enzymatic or fermentative action during the settling process. The decanted starch was submitted to convective drying at 50 °C until constant weight. Finally, the native starch was vacuum packed and stored at room temperature (25 ± 3 °C) and kept away from light exposure to avoid bioactive compounds' losses.The red rice grains (−1), mass: volume ratio (2 RZ), pH 8, time 18 h, starch mass 4 g, 60 °C, and stirring at 150 rpm.The commercial enzymes α-amylase and amyloglucosidase were kindly supplied by . Both products had their enzymatic activity determined. First, the native starch (SN) was hydrolyzed with the α-amylase enzyme producing the formulation (MS1). Another hydrolysis was performed with the amyloglucosidase enzyme, thus acting on the α-amylase modified starch mass in the first hydrolysis and producing the formulation (MS2). For the hydrolysis of native starch by α-amylase, the process conditions were: enzymatic charge (3UE g−1), mass: volume ratio (2 RZ), pH 6, time 3 h, starch mass 4 g, temperature 60 °C, and stirring at 150 rpm. Both processes were performed following the optimal hydrolysis conditions for this material, according to the study by4. The percentage of hydrolysis characterized the hydrolysis process. The absorbance measurements were taken at the wavelength of 540 nm25.For the starch hydrolysis by the amyloglucosidase, the process conditions were: enzymatic load (3UE g−1) of free chlorine for 15 min and then rinsed with running water. The fractions were separated  by manual pulping and submitted to the convective drying process, carried out in an oven with air circulation (Marconi—MA048) temperature 50 °C and an airspeed of 1.0 m s−1 for 24 h. The jaboticaba peels were crushed in an industrial blender (Kohlbach—KM42A) within 3 min. The sample was packaged in a polyethylene package with a zip lock under the light shelter.The jaboticaba fruits were purchased in the local market and selected, washed, and sanitized with sodium hypochlorite in a solution , in neutral and acid detergent27, total carbohydrate content was calculated by difference. Water activity (aw) was determined using the Decagon Aqualab CX − 2 T device at 25 °C. Anthocyanins and flavonoids28, total carotenoids (lycopene)29, total tannins30 methodology with adaptations, total phenolic compounds31. The antioxidant activity was determined by the ABTS•+ method using the method proposed by32, with modifications made by33. The antioxidant activity by DPPH• was performed using the methodology described by34, with adaptations. For both analyzes, distilled water was used as the extractive solvent.The methodologies used were: moisture (method 934.06), ash (method 940.26), lipid (method 948.22), protein (method 920.152)The powdered jabuticaba peel (JPP) was added to all starch samples  at the same concentration of 10%. Therefore three new formulations were composed of JPP at 10% and 90% (w/w) of one of the starch samples . The batch mixing process used the diffusion mechanism, using a non-segregating Y-mixer  with a stainless steel chamber and a residence time of 10 min. The mixture was considered complete when both materials were randomly mixed against each other.35. Standard profile STD1 supplied with the instrument was used with 3.0 g of one of the mixtures (corrected to 14% moisture content) with 25 mL of deionized water. The total cycle time was 13 min. Peak viscosity (PV), viscosity at trough , and final viscosity (FV) were recorded. Breakdown  and setback  were calculated using the software Thermocline for Windows, version 3 provided with the instrument.Pasting properties were determined with a Rapid Visco Analyzer 36. The experiments were performed in triplicate, and the results were analyzed with the aid of Origin software version 8.037. Results were modeled using the Ostwald-de-Waele (Power Law), Eq. (n), γ is the shear rate (s−1), and n the flow behavior index38.The six starch: water suspensions 1:10 w/v) were kept at 80 °C for 30 min for complete gelatinization and then cooled to temperatures of 70 °C and then 25 °C. The temperature was maintained using a thermostatically controlled water bath connected to a jacketed vessel. Viscosity and torque measurements were performed using a Brookfield viscometer  with spindles RV06 and RV07 at nine spindle speeds . Measurements were performed at 70 ± 2 °C and then at room temperature (25 ± 1 °C). Shear stress and shear rates were calculated by the method of:10 w/v wSix suspensions were produced using a ratio of powder to the water of (1:10 w/v). After complete mixing, each suspension was heated at 80 ± 1 °C for 30 min. The obtained gels were poured into a cylindrical container  and stored at 6 °C. After 24 h, the samples were taken from refrigeration and placed at room temperature.−1; post-test speed, 1.0 mm s−1; test speed, 1.0 mm s−1; compression height, 40 mm; strain, 40%; interval between two compressions, 5 s; compression times, 2 s. Each sample was measured twice at room temperature (25 ± 1 °C), and the final result was the average of both tests. The firmness, cohesiveness, gumminess, and adhesiveness values were calculated from the force–time plots obtained39.The texture profile was determined using the TA-XT PLUS texture analyzer . Instrument settings were compression mode, trigger type, auto 5 g; pretest speed, 2.0 mm s40.−1); MWet is the mass of the sample moist after centrifugation (g); Mdry is the mass of dry starch (g).One gram of each of the six starch + JPP samples was combined with 10 mL of water or sunflower oil in centrifuge tubes. Slurries were kept at 24 °C and occasionally stirred with a glass rod over a 30 min period and then centrifuged at 15000 g for 15 min. The volume of decanted supernatant fluid was measured, and the amount of water or oil retained per gram of sample was calculated according to Eq. ; MS = sample weight (g), dry basis; MCR = weight of centrifugation residue (g).The milk absorption index was measured using a technique developed for cereals43, with modifications performed for all gel formulations  and followed for 28 days at 7-day intervals for each reading, as follows: .Syneresis index capacity was determined according to the methodology proposed byg for 10 min under refrigeration at 6 °C, in duplicate. The water mass separated from the gel network during centrifugation, divided by the initial gel weight and multiplied by 100, according to Eq. (w is the water's weight separated from the gel after centrifugation (g), and Mi is the initial gel weight (g).Three days after the gel preparation and storage at 6 °C, the samples (15 g) were centrifuged at 3000 g to Eq. , calcula44.All experiments were executed in triplicate, and the results were submitted to analysis of variance (ANOVA) at 5% of probability. The significant qualitative responses were submitted to the Tukey test adopting the same level of 5% of significance. All statistical analyses were performed using Statistica software version 7.04, the ash content ranged from 0.45 to 0.60 g 100 g−1, the lipids content ranged from 0.06 to 0.09 g 100 g−1 proteín content ranged from 0.60 to 0.74 g 100 g−1 and did not significantly change after the enzymatic treatment.The result of enzymatic hydrolysis with α-amylase (MS1) expressed in the percentage of hydrolysis was 5.64%. The value obtained for reducing sugar at the second hydrolysis with amyloglucosidase (MS2), after the initial hydrolysis with α-amylase, corresponding to a percentage of hydrolysis 8.63%, totaling 14.27% of hydrolysis with two enzymes. According to Almeida et al., 201947.Partial or total hydrolysis of starch in its subunits (glucose) is a standard process for preparing starch products and controlling the starch content in different plant matrices. This hydrolytic process generally involves enzymes capable of disrupting the polysaccharide's glycosidic bonds under mild conditions. This process is based on two enzymes' complementary action: alpha-amylase followed by amyloglucosidase, responsible for the cleavage of glycosidic bonds , respectively, which generates more fragments and, consequently, more non-reducing ends4, since the purpose of the present work is the rheological and technological analysis of the modified starch produced previously.In the present work, the red rice starch did not undergo a heat pre-treatment, gelatinization, and paste formation before or during the enzymatic process; the substrate was raw starch in the powder form. Therefore, only partial hydrolysis was obtained, which can be confirmed with the data presented in the study carried out byTable −1, which is in accordance with the Brazilian Legislation requirement for the dust/flour identity and quality standard, which recommends values in the range of 5 to 15 g 100 g−148. In49 while characterizing jaboticaba flour found a moisture content of 14.45 g 100 g−1. In50 found a value of 15.33 g 100 g−1 for the jaboticaba peel flour.The moisture value found for jaboticaba peel powder was 9.46 g 100 g−1, consistent with those determined elsewhere51: found higher values, 4.23 g 100 g−1 for forced-air-dried jaboticaba peel, and 3.52 g 100 g−1 for freeze-dried peel, respectively. In52 found ash content values of 3.89 g 100 g−1 for JPP obtained after convection drying at a temperature of 60 °C.Ash content was 3.06 g 100 g−1, lower than the value found by51 of 1.72 g 100 g−1 for the lyophilized peel. According to53, the low lipid content of jaboticaba peel may bring benefits to human health since a large part of the population consumes hyperlipidemic diets, which contributes to the emergence of obesity to many chronic diseases.The value found in the present work for the lipid content of jaboticaba peel powder (JPP) was 1.22 g 100 g−1 and is similar to the value found by51 of 5.23 g 100 g−1. In53 evaluated the influence of drying temperature and obtained a crude protein content of 6.58, 6.06, and 5.95 g 100 g−1 at temperatures of 30, 45, and 60 °C, respectively.The protein content found was 6.13 g 100 g−1. In54 found for the fresh peel the value of 1.84 g 100 g−1 and for the lyophilized peel, 3.89 g 100 g−1, which is lower than the value found in the present work. Carbohydrates were the macronutrient with the highest content fraction on JPP (51.77 g 100 g−1). In50 found a lower value of 58.70 g 100 g−1. Since fiber content is included in total carbohydrates, the method used for carbohydrates estimation may be overestimating its value.The crude fiber content of jaboticaba peel powder was 4.36 g 100 g−1, higher than the 5 g 100 g−1 found by49. In55, while characterizing the jaboticaba fruit and its byproducts, found insoluble fiber content of 27.03 g 100 g−1 and 26.43 g 100 g−1, for the Paulista and Sabará variety, respectively. The acid detergent fiber content represents the soluble fraction of total fibers. The acid detergent fiber content found was 12.32 g 100 g−1, whereas50 obtained a value of 20 g 100 g−1 of soluble fibers.The neutral detergent fiber content found was 12.11 g 100 g56. The jaboticaba peel powder produced in the present work presented relevant anthocyanin contents of 83.59 mg 100 g−1. In57 found average values of total anthocyanins ranging from 4.9 to 147 mg 100 g−1 at five maturation stages. The authors also found that anthocyanin concentration increased with the maturation stage. Jaboticaba peel powder presented a flavonoid content of 108.91 mg 100 g−1, a lower value than the one found by34, which was 147.0 mg 100 g−1 for jaboticaba (peel + pulp) in natura.Anthocyanins are extremely unstable to environmental conditions such as light, pH, oxygen, temperature, and the presence of metal ions that can affect their stability−1 lycopene or 1.867 mg 100 g−1. In34, in their study with 18 non-traditional Brazilian fruits, including jaboticaba. The authors obtained for the fresh fruit (peel and pulp) a carotenoid content of 0.32 mg 100 g−1. Thus, the present work's value was higher, which can be justified by a higher presence of carotenoids on the peel than the pulp. Alternatively, the dehydration concentrated the carotenoids, and thus the value is higher.The total carotenoid content found in jaboticaba peel powder (JPP) was 18.67 μg g−1 after extraction for aqueous extract. In58 found for lyophilized jaboticaba peel 3612.00mgGAE 100 g−1 and considered it a good source of bioactive compounds. In59 found for jaboticaba powder a value of 2149.58mgGAE 100 g−1. In50 characterizing both the fresh jaboticaba whole fruit and peel, presented a total phenolic content of 11.400 and 3.215mgGAE 100 g−1, respectively.The value of total phenolic compounds found for (JPP) in the present study was in the range of 2549.65mgGAE 100 g•+ method in the present work was 403.51 µmol Trolox g−1 for jaboticaba peel powder (JPP). In34 presented an antioxidant activity of 37.5 and 317 µmol Trolox g−1, for fresh and dried jaboticaba, values lower than those found for (JPP). The value found for the antioxidant activity for jaboticaba peel powder (JPP) by the DPPH• method expressed by the EC50, after aqueous extraction, was 180.58 g g−1. A similar was found by34 with 138 g g−1 DPPH• in dry matter using three consecutive extractions with organic solvents  to obtain a final aqueous extract. Climatic and soil conditions affect grapes' phenolic composition since these factors influence their biochemical synthesis and, consequently, their biological properties61.The value obtained for antioxidant activity by the ABTSTable 61, the highest values of peak viscosity or maximum viscosity were associated with a high proportion of ungelatinized starch, while the lowest values of viscosity were related to more considerable degradation gelatinization of starch. This degradation is generally attributed to the depolymerization and molecular entanglement resulting from the starch modification. The highest peak viscosity value was found for enzymatically modified starch 1 (MS1) with 5164.61 mPa.s, while the formulations SN and SN + JPP presented no significant differences. When comparing the mixtures' other values with the samples without jaboticaba peel powder, it was observed that for MS1 + JPP and MS2 + JPP, the values were lower. In62 found an increase in peak viscosity due to pre-gelatinized starch to rice flour for dumpling production, ranging from 4716 to 5036 mPa.s.According to63 observed no significant difference for minimum viscosity during the production of starchy rice balls. In64 found differences between wheat starch incorporated with different proportions of barley starch citrate, obtaining values ranging from 610 to 1075 mPa.s.The minimum viscosity value was determined by the final viscosity minus the tendency to retrograde. No significant difference between the formulations SN and MS2 was found for minimum viscosity, revealing that the two enzymatic hydrolyzes were not enough to change this parameter if compared to native starch. The same trend was found for their respective mixes SN + JPP and MS2 + JPP, which shows that jaboticaba peel powder also did not change this pasting property. However, results reveal that the minimum viscosity for (MS1) presented a higher value with or without the addition of jaboticaba peel powder (JPP). In65. The addition of jaboticaba peel powder in the SN and MS2 tests did not influence this parameter but reduced the breakdown in the MS1 + JPP. The higher the breakdown viscosity, the stronger the starch gel. The viscosity drop value is the peak viscosity value minus the minimum viscosity value66. The highest values for the viscosity drop were obtained for the formulations MS1 and SN + JPP, followed by native starch (SN).Breakdown viscosity is caused by the disruption of gelatinized starch granules when a high shear rate combined with temperature is applied, causing a sudden viscosity lossp > 0.05). The addition of jaboticaba peel powder (JPP) decreased the retrogradation tendency primarily for MS1.Table 62 obtained 2960 to 2969 mPa.s for glutinous rice flour with pre-gelatinized starch.SN and MS1 presented the highest values for the final viscosity with significant differences comparing to the other formulations. Final viscosity can be related to the textural firmness parameter. The firmness values for the enzymatically modified starch 1 (MS1) and native starch (SN) formulations are similar. Alternatively, a different characteristic is observed when the texture profile is correlated to the rheological profile of jaboticaba peel powder (JPP) mixes. In63 also found no changes in the pasting temperature. In67 studying rice and tapioca starch obtained pasting temperatures of 91.43 °C and 71.67 °C, respectively. In68 found variations in pasting temperature from 72.5 to 79.3 °C for quinoa flour.Table 67 for rice flour, 6.78 min.Furthermore, only MS1 presented a significant difference for peak viscosity time than the other formulations, with values ranging from 5.16 to 6.15 min. This time is shorter than that found by68 in their studies with quinoa.Observing the rheological behavior of red rice starch paste and jaboticaba peel powder through Table 70. Dynamic viscoelastic properties can be used to describe three-dimensional starch and hydrocolloid network structures71. Generally, the contribution to intermolecular interaction in the final gel formation of the mixture depends on two main factors: (1) the appropriate concentration and (2) the intermolecular synergistic effect, which is affected by molecular conformation, molecular weight, and starch structure73.The pseudoplastic behavior can be explained by the progressive orientation of soluble starch molecules in the flow direction and the rupture of the intra-and intermolecular bonding system in the starch network that increases shear force sensitivity2) and the mean square deviation (MSD).Table 62 for the mixture between wheat starch with barley starch citrate and69 for cornstarch. The consistency index (K) presented lower values at 70 °C, except for (MS2), indicating temperature dependence. The native starch formulations presented the most considerable difference between the temperatures of 25 °C and 70 °C.The fluid behavior index in the Ostwald-de-Waele model was found to be less than 1 for all samples. The same behavior was found by73 obtained firmness results similar to (MS2) since the starches modified by acid hydrolysis presented lower firmness when compared to the native starch. In the present work, only after the addition of jaboticaba peel powder , this desired characteristic was obtained.Table 74 obtained cake with softer texture when native cornstarch was replaced by hydrolyzed and acetylated starch. This finding shows that modification of starch forms a smoother paste, and smoothness increases as the number of modifications applied to a native starch increases. However, this did not happen in the formulation (MS1) because the enzymatic hydrolysis process using α-amylase selectively attacks the amylose present in the starch. Subsequently, the amylopectin, mainly responsible for the texture characteristics, was not affected during the first hydrolysis. Moreover, in the hydrolysis second step, now with amyloglucosidase, the hydrolysis percentage was higher, and thus differences in firmness were found.In76 reported that the addition of mango peel powder on bread production prevents the formation of a three-dimensional network structure, increasing the product firmness78. Firmness increase is generally a phenomenon observed when the fiber is added to products79.According to80 define gumminess as the energy required to disintegrate a semi-solid food. The native red rice starch presented a gumminess value of 0.28 N, which is higher than formulation MS2 (0.23 N) and similar to MS1 (0.30 N). All starch + jaboticaba mixtures presented higher firmness and gumminess than the formulations . This result confirms that the jaboticaba peel is rich in fibers, which gives the paste more support when gelatinized.In81 obtained corn starch cohesion values of up to 0.8, close to that obtained in the present study. In67 obtained values of 0.23 for rice flour and 0.90 for tapioca starch. In76 observed that the bread cohesiveness significantly decreased with increasing mango peel powder. Cohesiveness is a parameter that reflects the damage tolerance and sample integrity and is defined as the sample internal contraction force23.The cohesiveness of the native starch paste was higher than the modified starch paste (MS1 and MS2). This result suggests that the higher the number of hydrolysis steps, the lower the starch paste value's cohesiveness. However, the only significant difference noted for this parameter was between native starch (SN) and its respective mixture (SN + JPP), in which an increase in cohesiveness was noticeable. InAll formulations presented a significant difference between them for the adhesiveness parameter. The sample SN presented higher adhesiveness than all the other starches without JPP but lower adhesiveness than the mixture with JPP. Inversely, the enzymatic modified starch 1 (MS1) presented lower adhesiveness than its respective mix (MS1 + JPP), which obtained the lowest adhesiveness of the study.75 observed that mango peel powder's addition did not influence adhesiveness, unlike what was found for jaboticaba peel powder in the present study.This result suggests that after starch pastes were heated, the intermolecular bonds were broken, and new bonds with the water molecules were formed, thereby increasing its adhesiveness. InWater, oil, and milk absorption profiles for all formulations are found in Table 82. On average undamaged starch absorbs 33% of its weight in water82. The difference between the absorption of different components was more significant for the formulation (MS2) in water absorption than the other formulations, while for the formulation (MS1), no significant differences were observed between oil and milk absorption.The ability of modified starches to absorb water, oil, and milk is higher when compared to native starch. This result suggests that water absorption is related to the paste's cold viscosity since only damaged starch granules absorb water at room temperature. The absorption mechanism is explained by binding water molecules to hydrophilic groups (–OH), resulting in gel formation with a higher viscosity83 obtained as a result  for water absorption and  for oil absorption before a freeze–thaw cycle, respectively for corn, wheat, and tapioca starch. In64 found water absorption values of 98% for wheat starch and values ranging from 73 to 89% for wheat starch mixed with different proportions of barley starch citrate. In84 found that oil absorption was higher than water absorption for native and gamma-irradiated wheat starch. In83 found that the addition of pre-gelatinized starch on rice balls decreased, from 98.34 to 93.73%, the percentage of immobilized water, indicating that pre-gelatinized starch has a stronger water absorption and retention capacity.In85 found a range of 70.09 to 85.66% in oil absorption capacity for jackfruit seed starches. In86 obtained values for water-binding capacity ranging from 0.93 to 2.01 g water/g solid for the mix consisting of starchy rice flour and protein to produce gluten-free cookies, whereas, for oil absorption, the values ranged from 1.82 to 1.96 g oil/g solid.The addition of jaboticaba peel powder to the starches contributed to increased water, oil, and milk absorption capacity. The highest absorption for water and oil was found for the MS2 + JPP formulation. However, no significant differences were found between MS1 + JPP and MS2 + JPP for milk absorption. InThe high water, oil, and milk absorption capacity of enzymatically modified red rice starch with the addition of jaboticaba peel powder demonstrates a desirable property for employing as an ingredient in food products requiring high water, fat, and milk retention. The obtained results suggest its best use as a thickener and stabilizer in fluids and emulsions.Table Analyzing the formulations on the same day of storage, a significant difference between SN and enzymatically modified starches (MS1 and MS2), this behavior is confirmed for all storage days.The addition of jaboticaba peel powder in the mixture's preparation helps decrease the syneresis index of the formulations MS1 + JPP and MS2 + JPP, which showed lower retrogradation on days D0 and D7. However, in the following days, the mixture MS2 + JPP obtained a significant difference and became the formulation with a lower value for this parameter. For MS2 and its respective mixture, the differences started at D7. These two formulations presented the lowest value for all storage days. MS2 + JPP was able to trap the water molecules avoiding further material retrogradation, and consequently, a lower syneresis rate was found for this sample at D28.87.Syneresis results for formulations SN and MS1 obtained a significant difference only after D14, whereas their respective mixtures with JPP, only after D21 such significant difference was found. The amount of syneresis reflects the stability of starch in the freeze–thaw cycle. Additionally, the freeze–thaw stability is a critical feature in determining starch resilience to undesirable physical changes during freeze–thaw processes, which is also an indicator of starch retrogradation70 obtained for corn starch with the addition of different proportions of konjac-glucomannan, syneresis values ranging from 24.78 to 60.45%. In84, his study with native and irradiated wheat starch, the authors obtained syneresis values ranging from 0.77 to 46.66%, being the highest value found for native starch after 120 h of analysis, a value considered intermediate to that found in the present study. The increased percentage of syneresis may be attributed to macromolecules' association, particularly amylose, due to their linear structure and reinforced by amylopectin chains. During the freezing of a starch gel, the transformation of liquids into ice crystals increases the local concentration of starch molecules. This phenomenon accelerates intermolecular associations classified mainly as hydrogen bonding. When thawed, the ice turns to water and is likely to be released from the gel, producing a spongy texture. This phenomenon is known as syneresis88.InEnzymatic hydrolysis of red rice starch using a two-step treatment with α-amylase and then amyloglucosidase caused rheological and textural alterations in paste formation. Fiber-rich jaboticaba peel powder inclusion on starch revealed to be a texture modifier. The firmness, gumminess, and final viscosity of starches increased with the inclusion of JPP, while the tendency to setback was reduced. Besides texture, JPP proved to be a source of anthocyanins and phenolic compounds with antioxidant power. A shear-thinning behavior was found for all samples, with JPP causing a further deviation from the Newtonian behavior. The addition of jaboticaba peel powder to starches increased water, oil, and milk absorption capacity, while syneresis remained stable over a 28-day storage period. According to the rheological and technological properties presented in each formulation, some applications can be suggested. For example, the modified starch can be used as a thickener or as an emulsifier or can even be applied as a humectant to increase water retention while lowering water activity. Simultaneously, the produced mixtures are appropriate in the food industry to be formulated in sponge cakes, bread, cookies, yogurts, and dairy beverages. Finally, considering all results, it is possible to conclude that the addition of jaboticaba peel powder had a positive effect on native and modified starches' technological properties and that its inclusion is a positive alternative for the development of new functional food products.