Ramaria species. The assembled mitogenomes of Ramaria cfr. rubripermanens and R. rubella were circularized, with sizes of 126,497 bp and 143,271 bp, respectively. Comparative mitogenome analysis showed that intron region contributed the most  to the size variations of Ramaria mitogenomes. The genetic contents, gene length, tRNAs, and codon usages of the two Ramaria mitogenomes varied greatly. In addition, the evolutionary rates of different core protein coding genes (PCGs) in Phallomycetidae mitogenomes varied. We detected large-scale gene rearrangements between Phallomycetidae mitogenomes, including gene displacement and tRNA doubling. A total of 4499 bp and 7746 bp aligned fragments were detected between the mitochondrial and nuclear genomes of R. cfr. rubripermanens and R. rubella, respectively, indicating possible gene transferring events. We further found frequent intron loss/gain and potential intron transfer events in Phallomycetidae mitogenomes during the evolution, and the mitogenomes of R. rubella contained a novel intron P44. Phylogenetic analyses using both Bayesian inference (BI) and Maximum Likelihood (ML) methods based on a combined mitochondrial gene dataset obtained an identical and well-supported phylogenetic tree for Basidiomycota, wherein R. cfr. rubripermanens and Turbinellus floccosus are sister species. This study served as the first report on mitogenomes from the genus Ramaria, which provides a basis for understanding the evolution, genetics, and taxonomy of this important fungal group.In the present study, we assembled and analyzed the mitogenomes of two The online version contains supplementary material available at 10.1186/s43008-022-00100-7. Ramaria are usually known as coral fungi because of their colorful and finely branched sporocarps. Ramaria species are conspicuous ectomycorrhizal fungi associated with conifers, which are widely distributed in the northern hemisphere  have been used to infer inter- and intra-specific relationships among Ramaria and closely related genera, which reveal that Gomphales species are closely related to taxa in the Geastrales, Phallales, and Hysterangiales , while hundreds of thousands of basidiomycetes have been described in nature. The limited number of available mitogenomes limits our understanding of the genetics and evolution of this diverse fungal group (Basidiomycota).Mitochondrial genome is called the ‘second genome’ of eukaryotes, which is considered to be obtained from bacteria through endosymbiosis  to reveal the characterizations of the two Ramaria mitogenomes; 2) to reveal the variations or conservativeness between Ramaria and other Phallomycetidae mitogenomes by comparative mitogenomic analysis; 3) to reveal the intron dynamics of Phallomycetidae mitogenomes; 4) to understand the phylogenetic status of Ramaria in the phylum Basidiomycota based on the combined mitochondrial gene set. This study served as the first report on the mitogenome from the genus Ramaria, which will promote our understanding of the origin, evolution, and genetics of Ramaria species and closely related genera.In the present study, the mitogenomes of two R. cfr. rubripermanens and R. rubella using the raw sequencing data from the Sequence Read Archive (SRA) database under the accession numbers of SRR5801920 and SRR3747475, respectively, which are publically available  for the 1000 Fungal Genome project, and is stored in the USDA Forest Products Laboratory collection, Madison, WI (number UT-36052-T). Quality control steps were conducted to obtained clean reads from the raw sequencing data, which included removing adapter reads using AdapterRemoval v2 . Strand asymmetries of the tested Phallomycetidae mitogenomes were calculated according to the following formulas: AT skew = [A − T]/[A + T], and GC skew = [G − C]/[G + C]. We determined codon usages of the two Ramaria mitogenomes based on Sequence Manipulation Suite (Stothard Ka) and synonymous (Ks) substitution rates for the 15 core PCGs  in the 6 Phallomycetidae mitogenomes were calculated using the DnaSP v6.10.01 . Tandem repeats in the two Ramaria mitogenomes were determined by using Tandem Repeats Finder  according to previously described methods , which included Bayesian inference (BI) method and Maximum Likelihood (ML) methods. The phylogenetic status of Ramaria species in the phylum Basidiomycota was investigated according to the phylogenetic tree. The phylogenetic tree was constructed based on previously described methods  was greater than 100, and the potential scale reduction factor (PSRF) approached 1.0. The first 50% samples were discarded as burn-in, and the remaining trees were used to calculate Bayesian posterior probabilities (BPP) in a 50% majority-rule consensus tree  and the large subunit ribosomal RNA (rnl)  and trnL(tag), contained larger extra arms than other tRNAs, indicating the size variations of tRNAs were mainly due to size variations of extra arms in the Ramaria mitogenomes. Of the 25 tRNA genes shared by the two Ramaria mitogenomes, 23 contained mutational sites that varied between the two mitogenomes. A total of 83 mutational sites were detected in the 25 tRNA genes between the two Ramaria mitogenomes, of which 24.10% variable sites were located on the extra arm, followed by the D arm.We detected two rRNA genes in both the R. cfr. rubripermanens and R. rubella both contained one overlapping nucleotide . The length of intergenic sequences in the two Ramaria mitogenomes ranged from 1 to 4480 bp, and the longest intergenic sequence was located between cox3 and atp8 in the R. cfr. rubripermanens mitogenome.The mitogenomes of R. cfr. rubripermanens and R. rubella mitogenomes, which accounted for 47.26% and 46.85% of their mitogenomes, respectively . The R. rubella mitogenome was 16,774 bp larger than that of R. cfr. rubripermanens. Intronic regions contributed the most to the size expansion of the R. rubella mitogenome, with the contributing rate of 43.74%. Intergenic regions contributed to 38.17% of the size variation, and protein coding regions contributed to 17.23% of the R. rubella mitogenome expansion.The intronic regions accounted for the largest proportions of the ely Fig. . IntergePhallomycetidae mitogenomes used ATG as start codons, except that the cox2 gene of Sphaerobolus stellatus used GTG, the nad3 gene of R. cfr. rubripermanens used TTG, and the nad6 gene of S. stellatus used TTG as start codons , TTT , TTA , AAT , ATT , and TAT  .Codon usage analysis indicated that the most frequently used codons in the two yr) Fig. ). The frRamaria mitogenomes against themselves, and identified 142 and 74 repeat elements in the mitogenomes of R. cfr. rubripermanens and R. rubella, respectively  was found located in the first exon of nad2. A total of 4499 bp and 7746 bp aligned fragment were detected in the mitogenomes of R. cfr. rubripermanens and R. rubella, respectively.We further conducted BLAST searches of the two atp9, cox3 and nad4L, which had identical gene length among the 6 Phallomycetidae species  genetic distance between the 6 Phallomycetidae species among the 15 core PCGs detected, followed by the nad3 gene, which showed that these genes differentiated greatly in evolution  among the 15 core PCGs, followed by the nad2 and nad3 genes. While the nad4L gene had the smallest Ka value. The average synonymous substitution rate (Ks) of nad3 gene was the largest, while that of nad2 gene was the smallest among the 15 core PCGs. The Ka/Ks values for all the 15 core PCGs were < 1, indicating that these genes were subjected to purifying selection.The Phallomycetidae mitogenomes, which were distributed in atp6, cob, cox1, cox2, cox3, nad1, nad2, nad4, nad4L, nad5, rns, and rnl genes  using the cox1 gene of medical fungus Ganoderma calidophilum  in Phallomycetidae, which were distributed in 4 of the 6 Phallomycetidae species. Intron P209, P273, P717, P731, and P1057 were the second common introns (belonging to group I), which could be detected in 3 of the 6 Phallomycetidae mitogenomes. However, some rare Pcls could only be detected in one of the 6 Phallomycetidae species, such as P237, P369, P807, and P971 (belonging to group I). These rare introns in Phallomycetidae could be detected in distantly related species, such as Pleurotus eryngii  from the order Geastrales. The GC content of the 6 Phallomycetidae mitogenomes ranged from 24.3 to 31.69%, with an average GC content of 27.46%. The two Ramaria mitogenomes had a higher GC content than the average value. In addition, there is a large gap (2.77%) in GC content between the two Ramaria mitogenomes. Four mitogenomes from Gomphales and Geastrales had negative AT skews, while the other two mitogenomes from Phallales had positive AT skews. Four of the six mitogenomes had positive GC skews, including R. cfr. rubripermanens, R. rubella, Dictyophora indusiata, and Phallus echinovolvatus. Each mitogenomes contained 16–58 PCGs, and the two Ramaria mitogenomes contained the greatest number of PCGs. There were 4–38 introns harboured in the mitogenomes of the 6 Phallomycetidae species, and the two Ramaria mitogenomes contained the greatest number of introns. All the 6 Phallomycetidae mitogenomes contained two rRNA genes. In addition, 24–26 tRNA genes were detected in the 6 Phallomycetidae mitogenomes.The size of the 6 Basidiomycota species could be divided into 18 major clades, corresponding to the orders Agaricales, Boletales, Cantharellales, Filobasidiales, Geastrales, Gomphales, Hymenochaetales, Microbotryales, Microstromatales, Phallales, Polyporales, Pucciniales, Russulales, Sporidiobolales, Tilletiales, Tremellales, Trichosporonales, and Ustilaginales  and Maximum Likelihood (ML) methods Fig. . All majPhallomycetidae, the largest mitogenome  . We further detected large variations in genetic contents, gene length, tRNAs, and codon usages of the two Ramaria mitogenomes. Comparative genomic analysis detected large-scale gene rearrangements between Phallomycetidae mitogenomes, including gene displacement and tRNA doubling. We first found possible gene transferring events between the mitochondrial and nuclear genomes of the two Ramaria species. The Phallomycetidae mitogenomes have been experienced frequent intron loss/gain and potential intron transfer events during the evolution, and a novel intron P44 was found in the R. rubella mitogenome. Phylogenetic analyses showed that phylogenetic relationships of R. cfr. rubripermanens and T. floccosus species is closer than that between the two Ramaria species. The present study provides the first record of mitogenomes from this genus Ramaria, thus providing a foundation for studying the evolution, genetics, and taxonomy of this important genus and related fungal group.In this study, we obtained two novel mitogenomes from the Additional file 1. Table S1. Comparison on mitogenomes among six Phallomycetidae species. Table S2. Characterization and annotation of the two Ramaria mitogenomes. Table S3. Start and stop codons analysis of 6 species from Phallomycetidae. Table S4. Local BLAST analysis of the two Ramaria mitogenomes against themselves. Table S5 Tandem repeats detected in the two Ramaria mitogenomes. Table S6. Aligned fragments between mitogenomes and nuclear genomes of the two Ramaria species. Table S7. Species information used for phylogenetic analysis in this study.


The 20-year survival rate in pediatric patients after liver transplantation (LT) was no more than 70%. Hepatic fibrosis is one of the principal factors affecting the long-term prognosis. Imaging evaluation was the first-line examination for pediatric liver graft assessment. However, the sensitivity and specificity were insufficient. Thus, two-dimensional shear wave elastography (2D-SWE) was performed to evaluate liver graft stiffness and complication in post-transplant pediatric receipt.In this retrospective cohort, 343 pediatric recipients who underwent liver graft biopsy in our tertiary LT center were recruited between June 2018 and December 2020. The 2D-SWE evaluation, laboratory examination, routine post-transplant biopsy, and hepatic pathological assessment were performed.p < 0.0001). The LSM value of patients with post-transplant biliary complications (p < 0.0001) and biopsy-proven rejection  also rose compared to regular recovery patients. Concerning the sensitivity and specificity of 2D-SWE in diagnosing liver graft fibrosis, the area under the ROC curve (AUC) was 88%, and the optimal cutoff value was 10.3 kPa.Ninety-eight of the 343 pediatric patients were included according to the protocol. The Liver Stiffness Measurements (LSM) value of 2D-SWE was significantly elevated in post-transplant fibrosis (Pediatric LSM by 2D-SWE was efficient. Routine 2D-SWE evaluation could be optimal to predict significant liver graft fibrosis. However, the 5 and 10-year survival rates in pediatric patients after LT were 83 and 80%, respectively, and the 20-year survival rate was no more than 70% –7. ThougImaging evaluation, including ultrasound, was the first-line examination for liver graft assessment, which was considered non-invasive and non-radiation, especially for children. However, the sensitivity and specificity were insufficient . By contIn this study, 2D-SWE was performed to evaluate liver graft stiffness in post-transplant pediatric patients. The results were compared with pathologic findings of liver graft biopsy to clarify the value of 2D-SWE for pediatric patients.n = 12) or serum liver function (n = 13), or unsuccessful 2D SWE examination (n = 4) were excluded from the study. Additionally, in a normal situation, measurement of LSM in the left lobe is inappropriate because it is affected by cardiac pulsation. However, in pediatric recipients, as the majority of the pediatric recipients have a body weight of <25 kg, donation of the left lateral lobe (segments II + III) is sufficient in most cases. Considering the potential differences in the shape and texture of different liver segments, for comparability of data, the pediatric patients with whole liver or right lobe transplantation (n = 13) were also excluded from the study  was used with a curvilinear transducer (1–6 MHz) and a linear array transducer (4–15 MHz). Ultrasound examination was performed by one radiologist (G.L.H. with 10 years of experience in liver transplantation ultrasound and 4 years of experience in 2D SWE measurement). The time interval between 2D SWE measurement and liver biopsy was less than 2 days. The pediatric patients were fed with milk during the examination or sedated with oral chloral hydrate (0.5–1 mg/kg body weight) before the test to keep quiet if necessary. A high-frequency linear transducer SL15-4 (4–15 MHz) was used. The pediatric patient was maintained in the supine position, and the left lateral (or left) lobe of the graft liver was scanned. The SWE sampling frame was 2 cm × 2 cm and was put at 0.5 cm below the liver capsule to avoid the influence. A 10-mm-diameter region of interest was positioned in the center of the sample frame with a complete and homogeneous filling color map. The examination was considered successful when most of the area of the interest box (>90%) could be filled with homogeneous color and maintained at least one calm breath. When taking the measurement, major blood vessels and biliary ducts in the liver graft should be avoided. Five qualified data were obtained from each patient, and the median values (with their range) were calculated . The 2D SWE value was 3.5–4 kPa for healthy children and 7.5–8.5 kPa for post-transplant pediatric patients with normal liver function.Ultrasound-guided liver biopsy was performed with an 18-gauge needle automatic biopsy gun, and the pediatric patient was in the supine position. The length of the liver specimen was at least 15 mm with six portal spaces. The liver tissue strips were routinely fixed in formalin and embedded in paraffin. Hematoxylin-eosin (HE) staining was used to find the histologic change. Two hepato-pathologists blinded to clinical data and 2D SWE results made the pathological diagnosis. Masson staining was used to stage liver graft fibrosis based on the Scheuer score system. Substantial was defined as S2 or more significant.P-value of < 0.05 was considered statistically significant.All data were first analyzed for normality of distribution using the Kolmogorov-Smirnov test of normality. Continuous normal distribution variables were expressed as mean ± standard deviation (SD). Non-normally distributed variables are represented by median  and range. Categorical variables are represented by counts and percentages. A receiver operating characteristic (ROC) curve was performed to determine the liver fibrosis stage. A frequency distribution was obtained for choosing the optimal cutoff values of 2D SWE to maximize the sum of sensitivity and specificity for different fibrosis thresholds. Optimal cutoff values to predict fibrosis stages were identified. Sensitivity, specificity, positive predictive values, negative predictive values, and accuracy were calculated. Statistical analysis was performed using software . The graph was performed by GraphPad Prism . A Ninety-eight pediatric patients underwent liver graft biopsy 2 years after LT . The medp < 0.0001, p < 0.0001) and biopsy-proven rejection  also rose compared to regular recovery patients, with the difference between means at 9.025 ± 1.530 kPa , the optimal cutoff value was 10.3 kPa, and the optimal cutoff value was 18.3 and 21.6 kPa for Grades 2-3 and Grade 4, respectively . HoweverAlthough LSM also increased with elevated serum ALT levels , the sensitivity and specificity of 2D SWE were still reassuring. For instance, in 44 pediatric patients with ALT ≦ 1ULN who had routine liver graft biopsy, Masson-staining was performed on 29 subjects. Scheuer’s score was graded as S0 in 10 subjects, S1 in nine recipients, S2 in seven recipients, and S3 in three recipients . SWE couTwo-dimensional shear wave elastography is a newer elastography method using several lines of acoustic radiation force impulse to generate shear waves over a large region of interest within the liver in real-time to obtain quantitative images of the elasticity. 2D SWE can detect the propagation of the shear wave, obtain elastic images based on conventional ultrasonic images, and quantitatively measure liver stiffness . The sucLiver stiffness measurements increased in pediatric patients with liver graft cirrhosis, liver graft rejection, drug-induced liver injury, EBV/CMV infection, bile duct complication, hepatic venous-occlusive disease, and hepatitis. Due to the liver capsule around the liver, the size of the liver is relatively fixed; when inflammation, edema, congestion, and cholestasis make the liver volume increase, the intrahepatic pressure rises. In addition, literature reported that patients with liver cholestasis, acute inflammation, bile duct complication, and hepatic vein obstruction lead to an elevated LSM, which is consistent with our findings , 22.Recently, the pilot study reported by Vo et al.  has indiIn our study, the pathologic findings in thirteen patients were liver graft rejection, LSM was 7.8, 10.7, and 12.6 kPa in ALT ≦ 1 ULN, 1 ULN < ALT≦2 ULN, and ALT > 2 ULN groups, respectively. LSM increased with the elevated serum ALT level. In the patients with liver graft rejection but with normal serum ALT levels, LSM was not significantly increased. Yoon et al.  performeScheenstra et al.  found thIn this study, we analyzed the pediatric recipients who received liver graft biopsy with normal ALT levels after LT; the pathological results showed liver fibrosis stages ≥S1, ≥S2, and ≥S3 were accounted for 65.5% (19/29), 34.5% (10/29), and 10.3% (3/29), respectively. LSM was satisfactory in diagnosing significant liver graft fibrosis with a sensitivity and specificity of 100%. However, the sensitivity and specificity for diagnosing mild liver graft fibrosis and significant liver graft fibrosis were 73.7 and 60%, 70 and 84.2%, respectively. Zhuang et al.  performeFirstly, the enrolled children may not widely represent the pediatric recipients. In recent years, a routine liver biopsy was performed in our center, and the parents were not preferred for the invasive and possible complications after liver graft biopsy. Secondly, the pediatric recipient was not strictly fasting when taking the SWE examination, and the measuring was started while feeding , 33. ThiThe LSM by 2D-SWE in the left liver graft was efficient. Besides liver graft fibrosis, elevated post-transplant pediatric LSM could also be present by liver graft rejection and biliary duct complications. Routine 2D-SWE evaluation could be optimal to predict significant liver graft fibrosis.The original contributions presented in this 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 Ethical Committee of Renji Hospital. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.HFe, L-HG, and Z-FX: substantial contributions to conception and design, acquisition of data, analysis and interpretation of the data, and drafting of the manuscript. Y-CH, Z-CL, R-LG, HFa, and L-XJ: revising for critically important intellectual content. HFe and QX: final approval of the versions to be published. 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.


Coccinia abyssinica (Lam.) Cogn) is a potentially important source of starch and an underutilized root and tuber crop indigenous to Ethiopia. In this study, the physicochemical, morphological, thermal, and rheological properties of native starches isolated from four cultivars of anchote tubers were studied and compared to potato and cassava starches, which were considered as references. The amylose content of anchote starches varied from 15.8–22.3%. The anchote cultivars showed different granule sizes, but all revealed a B-type crystalline structure, identical to potato starch. The phosphorus content of anchote starches ranged from 82–93 mg/100 g and was much higher than that of potato and cassava . This characteristic could govern several functional properties of anchote starches, making them suitable for applications in different types of noodles, glucose syrups, and viscous products. The gelatinization temperature and enthalpy of anchote starches, which ranged from 60.97 °C to 69.33 °C and 16.87 to 18.38 J/g, respectively, were considerably different compared to potato and cassava starches. Significant variations were also observed among the pasting properties of starches from anchote cultivars. They showed a higher stability to heating and shearing, having higher TV (2046 to 2280 mPa·s) and lower BV (248 to 487 mPa·s) values, and a higher final viscosity (3409 to 3686 mPa·s) than potato and cassava, which are important characteristics in food processing and when high gel viscosity is required after cooling. Anchote starch gels exhibited rheological characteristics of true gels, showing much lower (tan δ)1 values and significantly higher viscoelastic moduli than those found in cassava and potato gels. The present study revealed significant differences among the physicochemical properties of anchote starches, depending on the cultivar, and demonstrated their promising potential in food product development and other industrial applications.Anchote ( Starch constitutes a significant component of our diet and has a variety of industrial applications . DespiteCoccinia abyssinica (Lam.) Cogn) is an indigenous tuber crop grown in Ethiopia for economic, nutritional, medical, and socio-cultural benefits [Cucurbitales and the family Cucurbitaceae. Anchote cultivation and utilization have been a common practice in western parts of Ethiopia, mostly in the Wollega, Illu-Abba Bora, and Jimma areas, but recently, its use has expanded to the southern and southwestern areas of the country [Anchote  of all starch samples varied in the following order: D01S (11.58 μm) < REDS (13.31 μm) < CS (13.45 μm) < D24S (14.41 μm) < WHTS (14.48 μm) < PS (36.21 μm) /D50) of anchote starch (15.45–22.47) were considerably higher than those of PS (1.27) and CS (1.0) starches. The surface-  and volume-weighted mean diameter  of anchote starches varied from 12 μm (D01S) to 14.7 μm (WHTS) and 63.9 μm (D01S) to 106.9 μm (WHTS), respectively. Significant differences (p < 0.05) in all parameters were observed among the four cultivars of anchote, potato, and cassava starches. The differences in the particle size of the starches are likely attributed to cultivar differences, growing conditions, and plant physiology [The particle size of starch samples was measured as surface- and volume-weighted mean diameter. The mean diameter D6.21 μm) . The D501 μm . The starch .45–22.47The four cultivars of anchote, potato, and cassava starches showed significantly different morphologies, as observed from scanning electron micrographs . AnchoteThe X-ray diffraction pattern and relative crystallinity of the studied starches are shown in p < 0.05), and D01S has higher moisture content compared to other anchote cultivars. The ash contents of anchote starches varied between 0.38% and 0.49% and were significantly higher than those recorded in PS and CS starches. The D24S had the highest fat content (0.14%), while the rest of the anchote starches had comparable fat content to PS (0.09–0.11%), and CS showed significantly lower fat content compared to all studied starch samples.The proximate compositions of anchote, potato, and cassava starches are shown in p < 0.05) than that of either the PS or CS starches. The protein content in tuber starch relies on the species and variety of starch [The protein content of the anchote starches varied between 0.51% (WHTS) and 0.65% (D01S)  and was f starch . The higf starch . The resp < 0.05), between 30.4 mg/100 g (WHTS) to 55.7 mg/100 g (D24S)  . The PS S . The Pp < 0.05) variations, ranging from 15.8% (D01S) to 22.3% (D24S)  to 80.8% (D01S)  and was % D24S) . High am4S . Highp < 0.05) than that of PS (380 mPa·s) and CS (426 mPa·s). SB is related to the retrogradation tendency, or gelling ability, of amylose [p < 0.05) than that of the PS (2322 mPa·s) and CS (1222 mPa·s) starches. Thus, the FV of anchote starch suggested the higher stability of the gel after cooling and the suitability for applications which require higher final viscosities. The PT of the starches from the anchote cultivars varied significantly (p < 0.05) among themselves, with the highest value obtained from the D24S (72.65 °C), followed by D01S and WHTS, while REDS scored the lowest value (69.50 °C). The PT of the anchote starches fell between those of the reference starches, PS (68.8 °C) and CS (75.65 °C). The highest Ptime was obtained in the REDS (5.15 min), while the lowest was recorded in D01S (4.89 min). In all anchote starches, the Ptime was higher than in PS (3.27 min) and CS (4.47 min). The high content of phosphate groups in the anchote starches can influence their pasting properties and contribute to the high peak viscosities obtained, regardless of the cultivar [The pasting properties play a crucial role in defining the quality and utilization of starch for various applications in the industry ,12,14. T amylose . Thus, t amylose . In thiscultivar .max) and the stress at the cross point (G′ = G′′ and tan δ = 1) obtained from the strain sweep tests were also recorded. The strain sweeps were carried out to establish the linear viscoelastic region (LVR), and two different regions were observed in the four cultivars of anchote starch gels (1 began to increase with increasing strain until the curves of G′ and G′′ intersected (G′ = G′′ and tan δ = 1). The cross point is a singular and interesting point that can be considered as a yield stress. It represents the stress where the rheological behavior of the gels changes from a viscoelastic solid to that of an elasto-viscous liquid [max values within the LVR: it was not possible to establish them for the PS and CS gels due to the small drops observed in the G’ and G’’ moduli during their strain sweeps over the entire studied range. This finding was in agreement with the finding in previous studies [The rheological properties of the starch gels formed from four cultivars of anchote were investigated and compared to the starch gels made from PS and CS starches . The parrch gels : the LVRs liquid . It is a studies .max) resisted by the gels before the disruption of their structure, which occurred at the end of the LVR. The highest τmax (181 Pa) was obtained in D01S anchote starch, while the lowest τmax (131 Pa) was obtained in WHTS anchote starch. The cross point (G′ = G′′ and tan δ = 1) of anchote starch varied from 242 Pa (REDS) to 285 Pa (D01S). Therefore, the stability of the gels made with starches from the four anchote cultivars was lower than that of the PS and CS gels. According to the values of τmax and the stress at the cross point , denoting their lower elastic-like behavior than that of the anchote starch gels. PS and CS gels can be classified rheologically as typical weak gels [The viscoelastic behavior of starch was studied by measuring the evolution with the frequency of G′ (storage modulus) and G″ (loss modulus), which reveal elasticity and viscosity respectively, and tan δ (loss tangent), which quantifies the ratio between them (G’’/G’). Frequency sweeps  showed t1 values , rangingeak gels ,34.p < 0.05) on the cultivar. The G1′ and G1″ ranged from 168–202 Pa and 28–39 Pa, respectively, with the most consistent gels, with the highest viscoelastic moduli, being those made from D24S and REDS. The anchote starch gels, regardless of the cultivar, had G1′ values that were six and ten times higher than those corresponding to the PS and CS gels, respectively. This means that anchote starches are appropriate ingredients for preparing gel-like products. The dependency of the elastic and viscous moduli with frequency (a and b exponents) was also significantly lower in anchote starch gels than in PS and CS gels, denoting a more stable consistency of anchote gels versus frequency. It was reported that the rheological properties of starch gels were influenced by amylose content, branch chain length distribution of amylopectin, and starch variety [1′ and G1″ viscoelastic moduli can be concluded, in agreement with that observed by Tangsrianugul et al. [The viscoelastic properties of anchote starch gels depended significantly , peak (Tp), and conclusion (Tc) temperature of the four cultivars of anchote starches ranged from 60.97 °C (WHTS) to 61.74 °C (D24S), from 63.86 °C (WHTS) to 65.07 °C (D24S), and from 67.21 °C (WHTS) to 69.33 °C (D24S), respectively. These results show significant (p < 0.05) variation among the cultivars of anchote starches. Abera et al. [o), 70.18 °C (Tp), and 73.98 °C (Tc), which were higher than those in our results, while the gelatinization temperatures of PS and CS starches were 59.42 °C and 64.7 °C (for To), 63.45 °C and 70.76 °C (for Tp), 69.16 °C and 81.17 °C (for Tc), respectively. The gelatinization temperatures of anchote starches showed comparable results with those for PS starches. However, the CS starch showed significantly (p < 0.05) higher values than the anchote and PS starches; this could be due to the crystalline structure of CS starch (A-type), since it was reported that A-type starch has a higher gelatinization temperature than B-type starch, due to a closely packed double-helices and low water content [p < 0.05) higher than those for anchote starches. The range between the onset and conclusion temperatures of the gelatinization peak represents the homogeneity degree of crystallites [p < 0.05) with the cultivar in the range of 16.87 J/g to 18.38 J/g. The highest value was obtained in WHTS, while the lowest value was found in REDS. The ΔH of anchote starches from the four cultivars was always above that of CS and similar to PS. This denotes a more stable crystalline structure in anchote starch granules, requiring higher energy to destroy it. The thermal properties of starches are altered for several reasons, including amylose content, granule size, and crystalline structure [The thermal properties and gelatinization parameters of the four anchote cultivars, along with PS and CS starches, are summarized in  content . The geltallites . Therefotallites . The ΔH tructure ,22. The tructure ,22.−1 indicates the O–H bond stretching of the starches. The maximum of this band was detected at different wavenumbers, depending on the anchote cultivar: 3284.49, 3281.12, 3277.61, and 3288.71 cm−1, for D01S, D24S, WHTS, and REDS, respectively. The weak peak observed at ~2900 cm−1 is related to C–H stretching. The characteristic peaks that represent the H–O–H bending vibrations of tightly bound water molecules in starch samples were observed between 1634.96 cm−1 , and 1644.83 cm−1 .FTIR spectra of four anchote cultivars, potato, and cassava starch are presented in −1 (D24S) and 1338.25 cm−1 (REDS); the value was in accordance with a previous report [−1, specifically for anchote cultivars (996.27 to 996.78 cm−1), potato (993.12 cm−1), and cassava (995.12 cm−1) starches. This peak was believed to be a characteristic of C–O bond stretching [−1 are related to C−O, C−C, and C−O−H stretching and C−O−H bending [The peaks associated with O–C–H, C–C–H, and C–O–H vibrations were observed for all studied starch samples between 1337.40 cmIn this study, the physicochemical, morphological, thermal, and rheological properties of native starches isolated from four cultivars of anchote tubers were studied and compared to potato (PS) and cassava (CS) starches, which were considered as references. A significant impact of the cultivar on the properties of the studied anchote starches was concluded. The phosphorus content of the studied anchote starch was considerably higher than that of the reference starches, favoring the functional characteristics of the starch due to its enhanced paste viscosity, clarity, and lightness, making it a possible alternative to potato starch. The result shows the amylose contents of starches from anchote cultivars were within the range reported for most root and tuber starches, and these were categorized in the range of normal/regular starch. Similar morphologies were observed among the studied cultivars of anchote starch granules; however, they had different particle sizes. The four cultivars of anchote and PS starches had a B-type crystalline structure, while CS starch had an A-type crystalline structure. The results of pasting properties showed that anchote starch had a higher peak viscosity than CS starch, but a lower peak viscosity than PS starch. The starches of anchote cultivars showed higher stability to heating and shearing, exhibiting a higher trough and final viscosity than PS and CS starches, which are important characteristics for products that require a higher stability of the gel after cooling, making it suitable for applications that require higher viscosities. Anchote starch gels showed rheological characteristics of true gels, with significantly higher elastic behavior (lower tan δ) and stronger consistency (much higher viscoelastic moduli) than those found in CS and PS gels. Therefore, the results revealed that anchote starches are suitable ingredients for preparing gel-like products. Therefore, the present study reveals the promising potential utilizations of anchote starch in as a functional ingredient in food processing, product development, novel food product formulations, and other industrial applications, particularly in the formulation of gel-like products.Two anchote tubers Desta 01 (D01) and Desta 24 (D24) were collected from Debre Zeit Agricultural Research Center (DZARC), Bishoftu, Ethiopia. The Desta 01 is the only released variety of anchote, while Desta 24 is a promising accession to be released as a second variety. The other two types of local anchote cultivar tubers,  and cassava (CS) starch used in this study as references were supplied by Ferrer Alimentación S.A. , and Cargill S.L. , respectively.v/v) for 4 min using a laboratory-grade blender . The slurry was then filtered using a fine muslin cloth. The filtrate was allowed to stand for 14 h for sedimentation, the supernatant was drained, and the residue was washed repeatedly with distilled water until the color became white. The obtained clear sediment was dried at 45 °C for 24 h in a drying oven. The dried starch was ground, packed in a zip-locked polythene bag, and kept in a cool, dry place for further analysis. Anchote starch samples were named: D01S = Desta 01 starch, D24S = Desta 24 starch, WHTS = white starch, and REDS = red starch.The starch was isolated from anchote tubers according to the methods of Falade and Okafor , with sl50) and the dispersion (D90 − D10)/D50, as described in [The particle size of anchote starches was analyzed by a laser diffraction particle size analyzer . The results were obtained as the volume distribution and mean diameter of starch granules, which were expressed by the median diameter  of the starch samples were studied using a Bruker-D8-Discover-A25 diffractometer  following the method previously described by Vela et al. . The XRDThe micromorphology of the starch samples was examined using a Quanta 200FEG scanning electron microscope , equipped with an X-ray detector, which allowed for the study of the starch samples without metallization . The samThe moisture, ash, and fat content of anchote, potato, and cassava starches were determined using the standard methods of the Association of Official Analytical Chemists (AOAC) . The nitThe amylose and total starch content of isolated anchote starches, as well as potato and cassava starches, were determined using the Megazyme amylose/amylopectin assay kit , according to the manufacturer’s instructions.Pasting properties were determined using the Rapid Visco Analyser  following the RVA method for potato starch (RVA Method 07.04). Briefly, two grams of starch (at 14% moisture basis) were weighed in the RVA canister and mixed with 25 mL of distilled water. The prepared starch-water slurry was subjected to RVA analysis, where it was analyzed under programmed heating and cooling conditions. The starch-water slurry was mixed at 960 rpm for the first 10 s, and then at 160 rpm throughout the analysis. The programmed temperature-time variation was: holding for 1 min at 50 °C, heating gradually to 95 °C at 12 °C/min, holding for 2.5 min at 95 °C, cooling down at 12 °C/min up to 50 °C, and finally holding at 50 °C for 2 min. The pasting property parameters recorded were peak viscosity (PV), trough viscosity (TV), breakdown viscosity (BV), setback viscosity (SV), final viscosity (FV), pasting temperature (PT), and peak time (P time). All samples were analyzed in triplicate.1′, G1″, and (tan δ)1, represent the elastic and viscous moduli and the loss tangent, respectively, at a frequency of 1 Hz. Then, a, b, and c, are the exponents of the corresponding potential equations (power-law model) and quantify the dependence of the dynamic moduli on the oscillation frequency. All tests were carried out in triplicate.The fresh gels of starch samples obtained from the pasting property analysis described in e System, Mettler Toledo, Switzerland) according to the method utilized in Vela et al. [o), peak (Tp), and conclusion (Tc) temperatures (°C), and the enthalpy of gelatinization (ΔH) (J/g dry starch). All samples were measured in duplicate.The thermal properties of the starch samples were measured by differential scanning calorimetry .The functional group analysis of anchote starch and control starches (potato and cassava) was performed using an FTIR Nicolet iS50 spectrophotometer  coupled with an attenuated total reflectance (ATR) device equipped with a diamond crystal. The starch samples were equilibrated at a humidity of 15% following the method previously used by Vela et al. . The powp < 0.05 using Statgraphics Centurion v.18 . All the results were reported as means and corresponding standard deviations.The results were examined using a one-way analysis of variance (ANOVA), and Tukey’s honest significant difference (HSD) test was used to assess significant differences at


Objective Different post-sintering processes are expected to be a reason for alteration in the strength of zirconia. This study evaluated the effect of post-sintering processes on the flexural strength of different types of monolithic zirconia.Materials and Methods A total of 120 classical- (Cz) and high-translucent (Hz) monolithic zirconia discs (1.2 mm thickness and 14 mm in Ø) were prepared, sintered, and randomly divided into four groups to be surface-treated with (1) as-glazed (AG); (2) finished and polished (FP); (3) finished, polished, and overglazed (FPOG); and (4) finished, polished, and heat-treated (FPHT) technique (n = 15). Biaxial flexural strength (σ) was determined on a piston-on-three ball in a universal testing machine at a speed of 0.5 mm/min.Statistical Analysis Analysis of variance, and post hoc Bonferroni multiple comparisons were determined for significant differences (α = 0.05). Weibull analysis was applied for survival probability, Weibull modulus (m), and characteristic strength (σ0). The microstructures were examined with a scanning electron microscope and X-ray diffraction.Results The mean ± standard deviation value of σ (MPa), m, and σ0were 1,626.43 ± 184.38, 9.51, and 1,709.79 for CzAG; 1,734.98 ± 136.15, 12.83, and 1,799.17 for CzFP; 1,636.92 ± 130.11, 14.66, and 1,697.63 for CzFPOG; and 1,590.78 ± 161.74, 10.13, and 1,663.82 for CzFPHT; 643.30 ± 118.59, 5.59, and 695.55 for HzAG; 671.52 ± 96.77, 3.28, and 782.61 for HzFP; 556.33 ± 122.85, 4.76, and 607.01 for HzFPOG; and 598.36 ± 57.96, 11.22, and 624.89 for HzFPHT. The σ was significantly affected by the post-sintering process and type of zirconia (p < 0.05), but not by their interactions (p > 0.05). The Cz indicated a significantly higher σ than Hz. The FP process significantly enhanced σ more than other treatment procedures.Conclusion Post-sintering processes enabled an alteration in σ of zirconia. FP enhanced σ, while FPOG and FPHT resulted in a reduction of σ. Glazing tends to induce defects at the glazing interface, while heat treatment induces a phase change to tetragonal, both resulted in reducing σ. Finishing and polishing for both Cz and Hz monolithic zirconia is recommended, while overglazed or heat-treated is not suggested. When the material is subjected to surface stress and subsequent cracks, high compressive stress can be developed at the crack tips, leading to t- → m-phases transformation with 4 to 4.5% volumetric expansion, rendering crack inhibition phenomenon, known as transformation toughening.2O3. Adding 5% mol. of Y2O3yields a high amount of cubic (c) phase with a smaller grain size of 5-yttrium partially stabilized zirconia (5Y-PSZ). It shows the best enhancement of translucency and aging resistance over the classical 3Y-TZP.Nowadays, all-ceramic restoration has become popular and plays an important role in contemporary restorative dentistry, which is capable of providing a natural esthetic restoration. The ceramic materials must possess high esthetics and be fracture-resistant, especially in the load-bearing area.Post-sintering processes are clinical procedures that clinicians need to perform on the zirconia restorations before delivery to the patients. The restorations need to be ground, adjusted, finished, polished, glazed, or heat-treated.The zirconia blanks were milled into a cylindrical shape with an 18 mm diameter (Ø) from precolored classical translucent monolithic zirconia , and high translucent monolithic zirconia  and sectioned into a disc shape of 1.6 mm in thickness by using a sectioning machine . The dimension of zirconia discs was compensated for sintering shrinkage with the enlargement factor of 1.2302 for Cz and 1.2334 for Hz. The discs were ground flat on both surfaces by silicon carbide abrasive paper with grit 500, 800, and 1,200, respectively, with water coolant on a polishing machine  at a speed of 300 rounds per minute (rpm). Then, the specimens were sintered in a furnace , following the manufacturer's recommendation. A total of 120 zirconia discs of thickness (1.2 ± 0.2 mm) and Ø (14 ± 0.2 mm) were derived. The zirconia discs were sandblasted with 50 µm of alumina oxide powder with a pressure of 30 psi. The specimens were cleaned and allowed to dry at room temperature. The mixing of glazing paste and liquid  was applied over the blasted surface and fired in a ceramic furnace  to produce a glazed surface.All specimens were randomly divided into four groups according to post-sintering surface treatment: AG (as-glazed), FP (finished and polished), FPOG , and FPHT  groups. The specimens in the AG did not receive any surface treatment. The specimens in the FP were ground by cylinder fine diamond finishing bur  by an air-rotor with a speed of 400,000 rpm and water coolant. The contact pressure was exactly 50 g and 30 seconds finishing time for each step in a continuous stroke. The horizontal movement was conducted in one direction with the custom-made load and direction-controlled machine with a fixture for holding the grinding handpiece . The fiThe specimens were tested on the piston-on-three-ball apparatus . The te1is the radius of support circle (mm), r2is the radius of loaded area (mm), and r3is the radius of the specimen (mm).Where P is a load at fracture (N), and b is the specimen thickness (mm), υ is Poisson's ratio = 0.35, ro) as well as the Weibull modulus (m) by using Weibull++statistics  according toThe mean and standard deviation (SD) of σ for each group were compared and analyzed by using ANOVA and post hoc Bonferroni multiple comparisons using statistical software  to determine significant differences in the flexural strength with different post-sintering processes. The result was considered statistically significant at the 95% confidence interval (CI). Weibull analysis was used to determine the reliability of flexural strength and to estimate characteristic strength (σf(σ) is fracture probability, σ is fracture strength, σ0is characteristic strength, and m is Weibull modulus.Where PThe surface topography and fracture surface of the specimens were evaluated with a scanning electron microscope . The crystalline phases of zirconia were determined by their relative proportion of microstructures using an X-ray diffractometer . The crystal structures were examined at a diffraction angle (2θ degree) of 20 to 90 degrees with a 0.02 degrees step size per second intervals by using copper k-alpha radiation. The crystalline phase was analyzed by cross-reference with the standards database of powder diffraction and measured the intensity of the peaks using X'Pert Plus software .o, and m for each group are shown inp < 0.05), but no interaction effect (p > 0.05) was found . The mean ± SD values of flexural strength upon post-sintering surface treatment with AG, FP, FPOG, and FPHT were 1,134.87 ± 523.19; 1,241.23 ± 552.70; 1,116.64 ± 564.06; and 1,111.68 ± 518.99 MPa, respectively (The mean, SD, 95% confidence interval of σ, σs found . The rectively . Post hctively . The poctively .The XRD analysis of the crystalline contents of the Cz and Hz was illustrated inThe SEM photomicrographs revealed the irregularities of the surfaces of the CzAG, CzFPOG, HzAG, and HzFPOG due to small particles of glazing material, and some areas which possibly indicated the incomplete adhesion of the glazing materials as well as several voids inside the glazed layer . The toThis study indicated that post-sintering processes significantly affected the biaxial flexural strength of different types of monolithic zirconia. Therefore, the null hypothesis was rejected for the post-sintering processes and types of zirconia. The grinding, finishing, and polishing procedures were the stepwise method, which was needed to proceed from the coarsest to the finest grit size. The purpose of these procedures was to achieve a smooth, mirror-like surface that provided less susceptibility to bacterial plaque accumulation.Grinding with a coarse diamond bur produced a higher degree of surface roughness than that for the polished surface. The ground zirconia showed significant deterioration in its long-term mechanical properties, which are negatively affected by aging.The multistep zirconia polishing kit can reduce the zirconia surface by a depth of approximately 3 to 4 µm, which is higher than the coarse-diamond ground-induced transformation layer with a thickness of 0.3 µm.The gloss finish was also produced by applying glaze material. Flexural strength results in this study were significantly lowered, possibly because of moisture in the glazing mixture and heat from the glaze firing.Heat treatment can reverse the t- → m-phase transformation when heat is applied at 910°C for 1 minute.o, and survival probability. The m in ceramic was used to determine the reliability of the material and the distribution of flaws. A higher m had higher reliability or homogenous distribution of flaws and greater reliability.owas different in each post-sintering process. The FP had the highest σo, which means that finished and polished zirconia can tolerate more force before it fails and is more durable for long-term use. Moreover, when comparing the σoof the overglazed and heat-treated group to the as-glazed group in both Cz and Hz, the former was found to be lower than the latter.The Weibull analysis provided the m, σobecause of the incompatibility between the glazed layer and the zirconia itself.This study showed that the group subjected to grinding and then glazing exhibited lower σBased on the results of the study, the biaxial flexural strength of the Cz was stronger than that of the Hz. Post-sintering processes affected the flexural strength of monolithic zirconia. The flexural strength of monolithic zirconia can be enhanced through a proper finishing and polishing procedure. Overglazed- and heat-treated processes after finishing and polishing are not necessary because they can reduce the flexural strength of the zirconia. Heat treatment can reverse the phase back to tetragonal but result in a reduction in the flexural strength. However, overglazing shall be done in the esthetic region of restoration to achieve a natural-looking restoration as a gloss surface is also needed for the translucent monolithic zirconia.


There is a need to improve personalized immunosuppression in organ transplantation to reduce premature graft loss. Biomarkers are needed to better detect rejection, asymptomatic graft injury, and under-immunosuppression. Assessment of minimal necessary exposure to guide tapering and prevent immune activation is also important. There is robust clinical evidence from a large number of published studies supporting the role of dd-cfDNA for monitoring graft integrity and detection or exclusion of rejection. Dd-cfDNA indicates graft cell death without being rejection specific. It can be determined in plasma through droplet digital PCR using preselected SNPs or next generation sequencing. Changes in recipient cfDNA  can affect the results of dd-cfDNA fractional determination. This limitation can be overcome using absolute dd-cfDNA quantification. The combination of fractional and absolute determination including total cfDNA is recommended for meaningful interpretation of the results. The value proposition for the patient includes earlier transplant injury detection and intervention, less full blown rejection risk, an alternative to invasive biopsies, and personalized immunosuppression with potential for improved long-term outcome. Transplant physicians benefit from better immunosuppressive guidance and having an alternative when biopsies are refused or contraindicated. Further advantages are improved biopsy interpretation, less trial and error changes in immunosuppression, and less time dealing with complications. The laboratory medicine specialist can provide more effective services. Hospital management and insurance companies could benefit from more cost-effective surveillance of transplant recipients. Potential cost savings would result from fewer biopsies as a result of the tests’ high negative predictive value, fewer re-transplantations, and less organ failure with return to dialysis. A pathway to implementation and metrics is suggested to measure the effectiveness of dd-cfDNA testing. Improvement of personalized immunosuppression is necessary in kidney transplantation to reduce premature graft loss . TherefoCurrent approaches used for kidney recipient surveillance, including plasma creatinine, immunosuppressive drug levels, proteinuria, hematuria, donor-specific antibodies (DSA) and duplex ultrasonography, have diagnostic limitations . Plasma Immunosuppressive drug monitoring indicates the risk of toxicity, but is a poor predictor of graft damage . BiopsieAgainst this background noninvasive donor-derived cell-free DNA (dd-cfDNA) determination has great potential to early detect graft injury and/or rejection. The rationale for using dd-cfDNA as a quantitative biomarker (“liquid biopsy”) in transplantation is based on the fact that organ transplants are also genome transplants which opens up the possibility to use them to directly monitor allograft health . In the HLA-DRB1 locus using optimized droplet-digital PCR  that allow discrimination between heterologous alleles in the recipient and donor . Currentital PCR . Fractioital PCR . Most ofital PCR . Studiesital PCR , 0.04%  from 0.8% to 2.1% (90th percentile) was observed as a result of a systematic concurrent decrease of total cfDNA, presumably due to a decrease of calcineurin inhibitor exposure over time . The resIn patients with either T-cell mediated rejection (TCMR) or antibody-mediated rejection (ABMR) elevations of dd-cfDNA were observed in the vast majority of studies . ABMR isIn DSA + patients early detection of ABMR is important and dd-cfDNA may also be helpful to identify patients with “indolent” DSA from those with graft injury due to DSA . A cliniIn the multicenter DART study  acute anIt has been demonstrated using INDEL q PCR that dd-cfDNA quantification can be substantially improved using smaller amplicon sizes . In a fuA problem with fractional dd-cfDNA determination is that it is influenced by variations in total cf-DNA levels due to leukopenia, leukocytosis and inflammatory illness; leading to falsely elevated or decreased results . Of partThe overall diagnostic performance of dd-cfDNA for detection of rejection after KTx seems to be adequate based on data reported to date . Based oDd-cfDNA detects rejection also in liver , heart , lung R and simuMonitoring of dd-cfDNA may also be helpful to guide immunosuppression minimization. Increased dd-cfDNA values were associated with subtherapeutic tacrolimus concentrations after kidney transplantation in patients with variable tacrolimus levels . TherefoIn a recent study, fractional dd-cfDNA was tested during subclinical graft injury after liver transplantation confirmed by surveillance biopsies . The lowA majority of studies have found that dd-cfDNA was clearly superior compared with eGFR in the discrimination of patients with or without acute rejection . Dd-cfDNA previously published value proposition concept  can be uVarious stakeholders are involved in delivering and receiving care in transplantation. There are the transplant patients whose care could be altered by the less invasive graft injury detection, clinicians who manage solid organ transplant patients, laboratory medicine specialists who analyze and interpret test results and hospital management, insurance companies, public payers and policy makers who are involved in providing value-based health care. Value in this context is defined as outcomes relative to costs. For patients the following benefits are expected relative to outcome: detection or exclusion of graft injury or rejection, earlier transplant injury detection and subsequent intervention, biopsy alternative, early diagnosis of subclinical antibody-mediated rejection, detection of under-immunosuppression, evaluation of infectious complications of kidney allografts and personalized immunosuppression. Benefits for transplant physicians include better personalized immunosuppression guidance; for example during tapering, enhanced biopsy interpretation, less trial and error changing of immunosuppression, less time dealing with complications, and indication of response to rejection treatment. Laboratory medicine specialists would have increased involvement in molecular diagnostics regarding the use and interpretation of tests. Hospital management, insurance companies, public payers and policy makers can expect cost savings due to a decreased burden for care-givers; for example due to fewer re-transplantations or less returns to dialysis in KTx.Dd-cfDNA has great potential for cost savings. Acute rejection is associated with a significant increase in the cost of care  . TCMR anFor implementation of dd-cfDNA in clinical practice the following points are relevant. Depending on the required turnaround time, transplant centers can either set up the test themselves or utilize a qualified external central laboratory. Using ddPCR results can be obtained in about 1 working day and by the use of NGS in 2-3 working days . ImplemeOverall, evidence regarding clinical validity suggests that dd-cfDNA monitoring can facilitate personalized immunosuppression and thereby potentially decrease premature graft loss and health care costs . TherefoIntegration of all available data on relevant clinical and diagnostic findings with dd-cfDNA results should enable individualized transplant patient therapy .


A study was conducted on selected physicochemical properties of blackberry jelly-like desserts (kissel) prepared from physically modified starches (with various degrees of inhibition) and chemically modified starches (with various degrees of cross-linking). The desserts were conventionally sweetened with saccharose (S) or, as a dietary alternative, xylitol (X). The characteristics of changes in the viscosity of the kissels as a function of temperature and time were determined. It was noted that regardless of the sweetener used, the viscosity of the kissels increased with the decreasing degree of inhibition (high < medium < low). Regardless of the kind of modification of the starch used for the preparation of the kissels and of the kind of sweetener, thixotropy was observed. Desserts prepared from inhibited starch with xylitol (CL + X) were characterised by the biggest range of their hysteresis loop. Progressing retrogradation was noted with the decrease in the temperature of the experiment (+20 °C and +4 °C). After 7 days of storage, kissels sweetened with saccharose were characterised by a low transparency, which may indicate retarded retrogradation; however, on day 28, the transparency significantly increased, exceeding the values of transmittance for samples sweetened with xylitol. The tendency towards syneresis was tested at +4 °C and −22 °C. The substitution of saccharose with xylitol only caused a slight modification of viscosity. Regardless of the sweetener used and of the level of starch inhibition, lower ranges of the hysteresis loop were noted (apart from CL + X) than in the case of kissels obtained from chemically modified starches. Distinctly lower values of kissel “aging” indices were noted in the case of samples obtained from inhibited starches, and their colour did not significantly differ in relation to the dessert prepared from native starch. Starch is a fundamental component of carbohydrate food products. It is used for the thickening of sauces, soups, sweet puddings, jelly-like desserts (kissel) and many other processed foods. The use of native starches in the food industry is rather limited, which has led to ongoing research aimed at the obtainment of starches with new, universal functional properties that will satisfy food producers, and products with their admixture will meet the expectations of consumers. Modified starches display considerably higher temperature stability during heating, cooling, freezing and thawing relative to their native counterparts. This allows for the creation of products with constant quality determinants and high taste values during product turnover, i.e., in storage (under refrigeration or freezing) and transport .Producers of food additives more and more frequently offer physically modified starches, conforming with the “clean label” idea, as substitutes for the chemically modified starches that enjoyed extreme popularity for decades. Starches obtained through physical modification more and more often attain the same level of application universality as that of their chemically modified counterparts ,3.The innovative aspect of this study is the analysis of the interactions and use of “inhibited starch” in food systems. The conditions of the modification involve the effect of high temperature on starch granules in a dehydrated environment. However, these are not the classic conditions of hydrothermal modification (in the solid phase or in the form of paste), as the modification in this case is conducted in alcohol solutions (>80%) under atmospheric pressure or elevated pressure. The conditions of the modification and the possibility of their modelling  do not cause any degradation of starch granules or their gelatinization, so the starch retains its granular and “hardened” structure, which is more resistant to culinary thermal treatment than native starch. Such starches are obtained through physical modification under the effect of high temperature at a reduced water availability. This allows for the retention of the granular structure, and the products of such a modification are inhibited starches . These sThe available literature of the subject only provides reports concerning the properties of inhibited starches . There hWhen designing a new product, it worth considering the substitution of popular sweeteners (saccharose and glucose-fructose syrups) with natural ones such as beech sugar (xylitol). Temperature sensitivity, specific taste, colour, and allergising properties often disqualify honey as a sweetener. The physicochemical, sensory and health-promoting properties of xylitol mean that it can be a good substitute for saccharose, as its sweetness is identical, its calorific value is 40% lower, and its glycaemic index is 10-fold lower ,7,8,9.This study was focused on the analysis of selected physicochemical properties of blackberry jelly-like desserts prepared from chemically or physically modified corn starches in which saccharose was substituted with xylitol without any significant quality changes. The realization of this research objective involved the analysis of the rheological properties of the desserts (flow curves and viscosity curves in the gradient of temperature and time), changes taking place during long-term storage in drastic temperature conditions (gel syneresis and clarity), and changes in the index that determines the acceptability of blackberry jelly-like desserts for consumers (colour in the L*a*b* system).The viscosity of starch gels with the addition of sweetened blackberry fruits was measured as a function of temperature and time (viscosity vs. temperature and viscosity vs. time). The shapes of the viscosity change curves fundamentally differed for the kissels obtained from physically and chemically modified corn starches . The shaRegardless of the sweetener used, gels obtained from inhibited starches were characterised by a lower initial viscosity compared with their final viscosity. This was attributed to the aggregation of starch chains with the possibility of the recreation of their three-dimensional network .It was also demonstrated that in the case of samples with low degrees of cross-linking  and low inhibition , the highest level of viscosity was attained over the entire range of measurement, regardless of the sweetener used. A probable cause of this is the reduction in the water-plastifying effect and the increased ease of the formation of sugar bridges between starch chains in amorphous areas of starch, which inhibits the penetration of water molecules and stabilises newly formed structures ,14,15,16The phenomenon of thixotropy is related to shear thinning (mixing) and can be attributed to the aggregation of particles in the unstressed state, which causes the formation of an internal suspension structure and the partial or total disintegration of the formed structure of a given non-Newtonian fluid subsequently subjected to shear . The oppSikora et al.  describeBasically, thixotropy is a measure of the instability of systems, and it is defined as an unfavourable effect, hence the continued research on physical, chemical or biochemical modifications of starch that will contribute to a reduction in the area of the hysteresis loop .Retrogradation is an undesirable phenomenon that causes a change in the texture and stability of food products. The factors that determine this phenomenon include the botanical variety of starch, temperature and storage time. The determination of the degree of clouding through the measurement of transmittance provides information on the intensity of starch retrogradation. In the course of retrogradation, the spaces between starch chains (amylose and amylopectin) decrease. Starch transforms from a partially amorphous state to an ordered form, with semi-crystalline networks, that is observed as separation of the product into layers. In the case of liquid products, one can observe a “sediment” forming on the bottom of the vessel, above which the clarity of the liquid increases ,25,26.Here, the intensity of retrogradation was determined by measuring the transmittance of the blackberry jelly-like desserts at +4 °C a,c and +The shapes of the curves illustrating the progress of retrogradation differed in relation to the kind of sweetener used. Blackberry jelly-like desserts sweetened with saccharose were characterised by fairly low values of transmittance (%) after 7 days compared with samples sweetened with xylitol. In spite of the initially (after the 7th day) intensive retrogradation of the samples with xylitol, a significant slow-down of the process was noted over subsequent days . Regardless of the kind of sweetener used, the blackberry jelly-like desserts obtained from inhibited starch had lower values of transmittance (%) compared with those sweetened with saccharose.The process of starch retrogradation under the effect of added sugars has been extensively researched, but the obtained results are not coherent. Marsh et al.  argued tMeasurements of the degree of syneresis of blackberry kissels can be an indicator of starch (amylopectin) tendency towards the liberation of water as a consequence of retrogradation. Here, the susceptibility of blackberry kissels to syneresis was analysed under refrigeration conditions at +4 °C a,c and aIn the context of the current research, available reports have primarily considered the analysis of the botanical origins of starch and its susceptibility to syneresis. Gel-like, spongy, layered structures of de-frozen maize and potato sauces with a flaking tendency were mentioned by authors such as ,34, whilThe perception of the colour of food products often has a bearing on their acceptance or rejection. Among the analysed samples, the kind of sweeter used had a strong differentiating effect. An approximately 11% increase in L* and an approximately 50% increase in a* were noted for samples with the addition of xylitol .The analysis of ΔE revealed that the gels obtained from inhibited starches only slightly differed from the reference samples (native starch), as the range of ΔE values was from 0.88 to 1.88, due to which the colour differences could be described as undetectable or slight. Notably greater differences were noted for samples obtained from chemically modified starches, for which the values ranged as 1.26 < ΔE < 4.45—which indicates colour differences that are distinct and observable by consumers relative to the control.A distinct change in the colour of maize gels (−a*) compared with wheat gels (+a*) was mentioned by Martínez et al. . Those aDesserts prepared on the basis of physically modified starches with varying degrees of inhibition show similar or better stability compared to desserts obtained from chemically modified starches. Such satisfactory results suggest that physically modified starches can also be used to stabilise, thicken, and add texture to other food products, such as sauces, ketchups and creams. However, due to the differences in the qualitative composition of these products, the use of inhibited starches requires further research.The thickening material used in this study consisted of physically and chemically modified commercial corn starches acquired from the company TATE & LYLE. The method of preparation and the properties of the starches were described by Włodarczyk-Stasiak ,27,36. TThe quantitative and qualitative composition of the desserts is presented in Blackberry fruits with the sweetener and water (40 mL) were brought to the boiling point and then kept at a temperature of 100 °C for 5 min with constant stirring. The obtained extract was separated and mixed with a prepared starch gel obtained by heating (90–100 °C for 6 min) modified starches (3.2 g) with the remaining volume of water (20 mL). The dessert prepared in this manner was cooled down to 24 °C and then subjected to analysis. The flow curves and the measurements of viscosity in the gradient of temperature and time were determined using a Rheotest 2 tester .Measurements of the viscosity of the modified starch gels were performed according to the Winkler method after certain necessary modifications . The visThe flow curves of the blackberry kissels were determined using the Rheotest 2 tester. The determinations were conducted using the S/S-1 set of measuring cylinders with sequence “a” (12 measurement points). The results were taken at 1 min intervals at increasing and decreasing shear rates. The shearing stress τ (Pa) was calculated from the product of the value of α and the constant “Z” (Pa) for the given set of cylinders.The obtained results were used to plot the graphs of shear stress versus shear rate.The blackberry jelly-like desserts were stored at +20 °C or +4 °C for 4 weeks. Measurements of transmittance (T) were taken at 7-day intervals at λ = 650 nm and a temperature of 20 °C, referenced to distilled water. Samples stored at +4 °C were pre-measured and then heated for about 90 min to +20 °C .The blackberry jelly-like desserts were stored for 4 weeks at −22 °C or +4 °C. The degree of syneresis was determined from the volume of liberated water  after centrifugation . Measurements were taken at 7-day intervals at 20 °C .The analysis of the colour of the obtained blackberry kissels was conducted with an NH310 colour tester and the use of the CIE LAB (L*a*b*) method.The measurements were taken at room temperature (20 °C), changing the position of the cuvette with the samples ten times. Averaged colour parameters were used to calculate the total colour difference according to Formula (1).The results were processed based on the criterion according to which the absolute colour differences ΔE form the following ranges:ΔE 0–1—undetectable differences.ΔE 1–2—small differences.ΔE 2–3.5—medium, detectable differences.ΔE 3.5–5—distinct differences.ΔE above 5 means big differences in the hue of the colour.x) and the standard deviation (σ) were calculated from the range ; data out of this range were rejected. An ANOVA was used to calculate significant differences in treatment means and LSD (p < 0.05).The reported data are expressed as the average of triplicate observations. In the study, the mean values  obtained from inhibited and chemically modified (cross-linked) starches that were sweetened with saccharose or xylitol. The results of the analyses did not provide a clear-cut indication of which variables (method of starch modification or kind of sweetener used) produced a stronger differentiation of the physicochemical properties of the analysed blackberry kissels. The use of inhibited starches  resulted in the obtainment of products with similar viscosity change characteristics to their chemically modified counterparts. The substitution of saccharose with xylitol only caused a slight modification of viscosity. Regardless of the sweetener used and the level of starch inhibition, lower ranges of the hysteresis loop were noted (apart from CL + X) than in the case of kissels obtained from chemically modified starches. Distinctly lower values of kissel “ageing” indices were noted in the case of samples obtained from inhibited starches, and their colour did not significantly differ in relation to the dessert prepared from native starch.In view of the above results, one can state with complete conviction that the use of physically modified (inhibited) starch is fully acceptable in the preparation of desserts with diverse composition (in terms of the sweetener used) destined for long-term storage within a broad range of storage temperatures. The use of xylitol as a substitute for saccharose  allows for such a product to be defined as dietary, thus conforming to the clean label idea.


Some static patterns evoke the perception of an illusory expanding central region or “hole.” We asked observers to rate the magnitudes of illusory motion or expansion of black holes, and these predicted the degree of dilation of the pupil, measured with an eye tracker. In contrast, when the “holes” were colored (including white), i.e., emitted light, these patterns constricted the pupils, but the subjective expansions were also weaker compared with the black holes. The change rates of pupil diameters were significantly related to the illusory motion phenomenology only with the black holes. These findings can be accounted for within a perceiving-the-present account of visual illusions, where both the illusory motion and the pupillary adjustments represent compensatory mechanisms to the perception of the next moment, based on shared experiences with the ecological regularities of light. A large class of optical patterns evoke conscious dynamic sensations of illusory movement, despite being static. These illusory motions can be described as a variety of changes in shape or space, like drifting, rotating, oscillating, waving, fluttering, contracting, or expanding. An example of this type of illusion, which we call “expanding hole” is illustrated in via an observer’s phenomenology, consisting of conscious verbal reports, but also by examining a concurrent, involuntary, physiological index: the diameter of the eye pupil.Given that the above type of illusion also implies an illusory change in luminance, since it implies expanding darkness, it is possible to probe the impact of the illusion not only Such a two-pronged approach, uniting phenomenology with psychophysiology, has been used previously with other illusions of brightness, showing spontaneous pupil adjustments to illusions of luminosity from gradient patterns e.g., .1 we surmise that the visual system is prepared from individual experience (or that of the species) with the natural statistics of the visual world  light. In fact, glare can temporarily incapacitate sight and lead to life-threatening situations . Although, as in any illusion, this virtual expanding darkness is experienced at the cost of veridicality, since the observer is neither moving forward nor entering any dark space, such a cost is likely to be less severe than if there were no corrections when an observer really moved forward into a dark space . Hence, In this study, we showed several “hole” patterns for a few seconds on a computer screen while, using an infrared eye tracker, we recorded the concurrent adjustments of the eye pupil to the perceived illusory expansion. After each pattern, we also collected from each participant a phenomenological report about the magnitude of the experienced illusory motion by obtaining a rating  of the amount of expansion of each stimulus. Specifically, we aimed to show that the pupil response is related to the magnitude of the illusory effect, that is, when the central region is black, the pupil will show a dilation response, from an equiluminant baseline, that is proportional to the subjective degree of expansion. The reason for expecting a pupillary dilation is that the black hole will evoke a sensation of a gradual loss of light, like entering a dark space. In contrast, central light patterns of different colors  will suggest an expansion of light, as when moving toward a differently illuminated space. Hence, we expected to observe constrictions of the pupil, from an equiluminant baseline, to the colored central regions and that these pupillary changes will also be proportional to the subjective degree of expansion.However, considerations about the ecology of light lead us also to expect greater subjective expansions when the central region appears black than any other color. That is, when an observer moves into a dark place, this is invariably associated to the experience of an absence of light and necessarily of color. Any other scenario involving shifts in the spectrum between colored holes and surrounding colored or dark fields cannot be linked to the same type of ecological regularity or statistical learning. Hence, we predicted that both the magnitude of perceived expansion and the concomitant pupil response should be stronger when viewing black holes than in the other instances.SD = 4.6) with normal vision were recruited. The participants were treated according to the Declaration of At the University of Oslo, fifty healthy participants , while gaze and pupil diameters were monitored by an infrared SMI R.E.D. 500 , and a minimum gaze point accuracy was 0.5.Participants were seated 68 cm from the screen and followed, at the start of the session, a standard 4-point calibration procedure. The head was stabilized with a chinrest aimed to reduce artifacts in pupil measurement with gaze and head movement . All eyeWe extracted expansion ratings and gaze and pupil data in each trial and for each participant using the BeGaze software (SMI). For some analyses, data were averaged across images to compare the black expanding holes to the others with no black central region, hereafter labeled “colored” .Pupil data were based on pupil diameters (in mm) during fixations only, as computed by BeGaze, effectively excluding possible artifacts due to blinks or saccades, which can be processed and analyzed separately. No trial was excluded since the tracking ratio was above 90% in all trials. For each image, we averaged pupil diameters across fixations during each 8 s period. We subtracted the averaged pupil diameter when looking at each baseline array from that to each corresponding expanding hole stimulus. This corrected measure expresses diameter as the mean change (in mm) from baseline, with positive values indicating a dilation and negative values indicating a constriction. In addition, we also obtained standardized pupil z-scores for each expanding hole and for each participant by subtracting mean pupillary change from an individual’s mean pupillary change and then dividing by the standard deviation for each expanding hole.We found that participants varied considerably in their perceptions of subjective expansion. As visible in the plots in t-test of mean subjective expansions to black vs. colored expanding holes revealed a significant difference (t(49) = 3.05, p = 0.004, Cohen’s d = 0.432.A paired fference ; t(49) =F = 5.5, p < 0.001]; white: . Post hoc comparisons with Bonferroni correction showed that only two background colors were significantly associated with changes in expansion of black/white holes .Because both the black and the white holes were tested against a background with each of the other colors, we run two separate ANOVAs on the black or white hole images with expansion  as the dependent variable and background  as a factor. Both analyses revealed significant effect of background; black: [r = 0.009, or the “dispersion” of eye position during fixations (indexing microsaccades), r = 0.003. Moreover, the observers who showed no susceptibility (ratings < 1) did not show significantly fewer saccades than those who were susceptible, t(48) = 0.48, P = 0.63.Since a common report from observers is that the illusory motion is enhanced by freely moving the eyes, we assessed the possibility that rates of gaze shifts may be proportionally related to the magnitude of the illusion and that observers who were not susceptible moved their eyes less than those who experienced subjective expansion. We found no support for these ideas since there was no relation between saccade frequency (counts per second) and ratings of expansion, SD = 0.69; colored holes’ baselines: mean = 3.98 mm, SD = 0.74).Pupil diameters during fixations were averaged within each half second period, yielding sixteen epochs. t-test to the grouped means when looking at the black holes vs. the colored holes. As shown in t(49) = 69.2, p < 0.001 (Cohen’s d = 9.8).To analyze pupillary changes, we computed baseline-corrected and z-scored pupillary changes for each pattern and participant and applied a paired z-scores as the dependent variable and with brightness of the central region or “hole”  and contrast  as the independent variables. This analysis revealed that the hole brightness significantly explained about 64% of the pupil responses, standardized coefficient = 0.81, t = 6.16, p < 0.0001. However, contrast had no significant relation to the pupil change, standardized coefficient = 0.01, t = 0.14, p = 0.89. Interestingly, an analogous multiple regression analysis with the subjective expansion ratings as the dependent variable shows no significant relation with both hole’s brightness, standardized coefficient = 0.39, t = 1.94, p = 0.065, or its contrast’s, standardized coefficient = 0.04, t = 0.21, p = 0.84. Since a sizable portion of pupillary changes remains unexplained by either brightness or contrast, it is possible that the pupillary response also relates to the conscious perception of expansion, which is what we examined in the following section.Given that the central regions of the black and colored holes are necessarily dark or bright, respectively, in relation to the baselines, it is likely that a good portion of the pupil responses simply reflect differences in luminance and contrast of this region centered on the screen in relation to the background. To assess this possibility, we performed a multiple regression analysis on the pupil z-scored) pupillary changes of each participant. Kendall correlations are the (non-parametric) alternative to Pearson’s correlation and the best alternative to Spearman correlation with a small sample size and when a variable is ordinal  for each hole pattern  and the subjective expansions, in this case, averaged over all participants.To capture the relation between the growth of the pupil and subjective expansions for each pattern, we additionally computed simple regression analyses of each rating of subjective expansion for each hole pattern as the regressor and the corresponding p = 0.02. On the other hand, the same analysis with the colored holes showed no significant relationship; Tau = 0.076, p = 0.65. This was also the case when considering only the patterns with white holes (N = 7); Tau = 0.048, p = 0.88. The analysis on black holes revealed a moderately strong relationship between expansion and the rate of dilation of the pupil; Kendall’s Tau coefficient = 0.71, We collected subjective reports of illusory expansion to the pupillary adjustments while viewing the patterns shown in We based this expectation on the idea that the illusory pattern contains optical cues like a circular smear or shadow gradient of the central black hole that evokes an impression of optic flow, as if the observer were heading forward into place where light is absent or a dark hole. The illusion of a virtual expanding darkness is experienced at the cost of veridicality, since the observer is not moving forward into a real space, but both the illusory motion and the preparatory pupil adjustments are useful according to the “perceiving-the-present” account of illusions , since tAs expected, we also found that the pupil constricted to the colored patterns in relation to an equiluminant baseline . HoweverOur analyses also confirmed that, although differences in luminance across patterns did account for strength of the pupillary adjustments to different hole patterns, a portion of pupillary changes remained unexplained by either pixel brightness of the holes or their contrast with the background. We suggested that the measured pupillary responses were also related to the conscious perception of expansion and reflected oculomotor adjustments to the corresponding neural representations of the patterns, as previously suggested for brightness illusions .We also found, based on the phenomenological reports when rating expansion, that a minority of observers appeared to be not susceptible to the present illusions . It remaWe note that this account is based on the account by Finally, we found that color combinations of hole and background yielded a similar phenomenology, and most patterns did not differ in their effectiveness in evoking illusory motion . HoweverThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Departmental IRB (ref. no: 2088238) at, Department of Psychology, University of Oslo. The patients/participants provided their written informed consent to participate in this study.BL, SN, and AK conceived and designed the study. AK created the visual stimuli. BL performed the statistical analyses and wrote the first draft of the manuscript. SN and AK edited the parts of the manuscript. All authors contributed to the manuscript revision, read, and approved the submitted manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.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.


The Bosnian Journal of Basic Medical Sciences (BJBMS) was founded in 1998 in Sarajevo, Bosnia and Herzegovina, by Professor Muzafer Mujić, Professor of Physiology at the Faculty of Medicine, University of Sarajevo. Most of the scientific communication at that time, particularly in developing countries, took place through printed media with little access to a broader audience, and editorial communication took place via ground mail. Consequently, communication was much slower than today, and the journal’s readership was primarily composed of members of the local or, at best, national scientific community.Nevertheless, the early editors of the BJBMS set high scientific standards, and this, combined with their diligence, resulted in the BJBMS being listed in PubMed/MEDLINE and Web of Science. These were the crucial initial steps in the journal’s internationalization. The journal was well received by the global scientific community, and submissions from other countries soon began to arrive.A new era began with the widespread use of the Internet. The Internet changed almost every aspect of our lives, including our work. Scientific publishing underwent a revolution due to the vastly improved speed, efficiency, and number of communication channels. Following the trend, the BJBMS launched its first online edition, the website, in 2009. The journal’s online edition was well received by readers and increased the journal’s visibility and reputation, which was reflected in an increase in submissions from the global community. Since then, the BJBMS has experienced continuous growth in terms of quality, readership, visibility, reputation, and scientific metrics.Today, the BJBMS is included in the most relevant scientific databases and is well-known worldwide. The journal has outgrown its national and regional boundaries given that more than 90% of the articles published in BJBMS come from the international community.Because of aforesaid, the national designation “Bosnian” in the title of the journal does not reflect the essence and global significance of the journal. Therefore, the publisher and editors decided to change the title of the journal to more accurately reflect its international character and the content of the articles published.From February 2023 , the title of the Bosnian Journal of Basic Medical Sciences will be changed to Biomolecules and Biomedicine. The new title reflects the increasing number of published research done on subcellular/molecular level as well as translational and clinical research contained in the term Biomedicine. Biomolecules and Biomedicine will continue to be published by the Association of Basic Medical Sciences of the Federation of Bosnia and Herzegovina.