The application of steatotic liver graft has been increased significantly due to the severe donor shortage and prevalence of non-alcoholic fatty liver disease. However, steatotic donor livers are vulnerable to acute phase inflammatory injury, which may result in cancer recurrence. Alternative splicing events (ASEs) are critical for diverse transcriptional variants in hepatocellular carcinoma (HCC). Here, we aimed to depict the landscape of ASEs, as well as to identify the differential ASEs in steatotic liver graft and their association with tumor recurrence after transplantation. The overall portrait of intragraft transcripts and ASEs were elucidated through RNA sequencing with the liver graft biopsies from patients and rat transplant models. Various differential ASEs were identified in steatotic liver grafts. CYP2E1, ADH1A, CYP2C8, ADH1C, and HGD, as corresponding genes to the common pathways involved differential ASEs in human and rats, were significantly associated with HCC patients’ survival. The differential ASEs related RNA-binding proteins (RBPs) were enriched in metabolic pathways. The altered immune cell distribution, particularly macrophages and neutrophils, were perturbated by differential ASEs. The cancer hallmarks were enriched in steatotic liver grafts and closely associated with differential ASEs. Our work identified the differential ASE network with metabolic RBPs, immune cell distribution, and cancer hallmarks in steatotic liver grafts. We verified the link between steatotic liver graft injury and tumor recurrence at post-transcriptional level, offered new evidence to explore metabolism and immune responses, and provided the potential prognostic and therapeutic markers for tumor recurrence. Liver transplantation provides an effective treatment for selected patients with hepatocellular carcinoma (HCC), which is the second cause of cancer-mortality worldwide . The extAlternative splicing is a process that removes introns and concatenate specific exons, playing important roles in post-transcriptional regulation of gene expression ,9,10. VaThe dysfunction of metabolism in liver is closely associated with the development of HCC, such as NAFLD, which has become a leading aetiology underlying many cases of HCC . Our recThe inflammatory responses resulting from liver graft injury reshape the immune microenvironment. Our previous findings showed that the release of inflammatory cytokines due to liver graft injury recruited immune cells, such as regulatory T cells and myeloid derived suppressor cells (MDSCs), which subsequently promoted the tumor recurrence post liver transplantation ,21. FurtIn this study, we aimed to comprehensively analyze the ASEs in steatotic and normal liver grafts both from our clinical cohort and rat model post transplantation through RNA-seq. Our data illustrated the landscape of transcription and ASEs in liver grafts at early phase after transplantation. We also identified the differential ASEs of steatotic liver graft, together with the network of metabolic-related RBPs and the distribution of immune cells and hallmarks of cancer. The findings may not only elucidate the mechanism bridging steatotic graft injury and tumor recurrence at post-transcriptional level, but also provided potential therapeutic targets to reduce tumor recurrence. Moreover, our work about transcript and ASE based on RNA-seq in steatotic and normal liver grafts may fill in the blank of the transplant data pool and provide resources for further exploration.All the raw sequencing data were qualified for the analysis. A total of 188,424 transcripts were detected to express in the liver graft transcriptome, which were categorized into five types of genes A, left. For further exploration, the rat orthotopic liver transplantation model with normal or steatotic donors was established. The transcriptome of liver grafts was subsequently analyzed. Protein coding genes occupied almost three quarters among the 30,195 transcripts D, left. Alternative splicing is a major contributor to the transcriptional diversities . Next, wIn rat liver grafts, 8146 high confidence ASEs were detected with seven types. Consistent with the clinical findings, AF and SE were the most frequently detected ASEs, particularly in protein coding genes D. More tTo explore the aberrant ASEs, the differentially spliced events between steatotic and normal liver grafts were identified. A total of seventy ASEs were detected as differentially spliced in steatotic liver grafts. All the differential ASEs were found in more than 70% patients, and most of the differential ASEs were detected in 100% of patients A. FurtheIn the rat liver transplantation model, the differential ASEs in steatotic grafts were detected in contrast with normal grafts. In total, seventy-four differential ASEs were identified in steatotic liver grafts. The majority of differential ASEs were found in more than 85% rats with few ones in over 70% rats A. SE andTo further explore the role of differential ASEs in steatotic liver graft injury and their association with tumor recurrence, enrichment analysis was implemented on genes that harbour differential ASEs through KEGG. The genes with differential ASEs in humans were enriched in metabolism-related pathways . Different types of ASEs were over-represented in distinctive pathways. For example, only differential SE and AL ASEs were over-represented in complement coagulation cascades A. In a r4502 (CYP2)C8, CYP2D6, CYP2E1, and homogentisate 1,2-dioxygenase (HGD) were the genes corresponding to the common pathways. Gstz1, Adh6, and RGD were corresponding genes in rats 1A, ADH1B, ADH1C, cytochrome P in rats C. Throug in rats D. In add in rats . These rThe association of splicing factors with differential ASEs were further analysed. Each type of ASEs could have positive and negative correlation with one splicing related gene set. One splicing related gene set could be associated with multiple types of ASEs. The differential ASEs were strongly correlated with Post mRNA release spliceosomal complex and prespliceosome . These sA substantial portion of abnormal ASEs might be modulated by RBPs. Firstly, we paired the differential ASEs with corresponding RBPs to build the RBP dysregulation network. A total of 95 RBP genes were differentially expressed in human steatotic liver grafts, including 72 up-regulated and 23 down-regulated RBP genes. These RBPs were involved in many biological pathways. Importantly, the related RBPs were mainly enriched in metabolic pathways, which was consistent with our previous KEGG analysis A. SpeciaThe inflammation signature was analyzed to explore the liver graft injury in steatotic donor. The inflammation was obviously increased in steatotic grafts compared with normal ones through the enrichment analysis . These rThe immuno-suppressive environment due to inflammation has contributed to the tumor recurrence as reported in our previous studies ,21. ThroTo further investigate the differential ASEs, their association with cancer hallmarks was analysed. The majority of the pathways in cancer hallmarks were enriched in steatotic liver grafts, for example, glycolysis, IL-2-STAT5, inflammatory response, complement, and reactive oxygen species, which not only indicated the strong link between steatotic liver graft injury and cancer development, but also suggested the importance of both metabolism and immune response in steatotic liver graft injury A. FurtheThe overall portrait of transcription and ASEs in steatotic liver graft both of human and rat post-transplantation were first demonstrated based on our comprehensive analysis of high-through-put RNA-seq data. In addition, we analysed the differential ASE network with metabolic RBPs, inflammation signature, immune cell distribution, and cancer hallmarks in depth. It not only provided the link between steatotic liver graft injury and tumor recurrence, but also suggested the research direction between metabolism and immune cell responses at the post-transcriptional level. The steatosis was associated with increased histological damage, hepatic function derangement, and reduced survival post liver transplantation . The priThe rat transplant model, using a steatotic donor, mimicked the clinical scenario, but it avoided patient-related confounding factors. The transcriptome analysis both in the patient and rat showed the diversity of transcripts. It was noteworthy that the substantial part of protein coding genes with transcripts were unannotated ones, which might be new players during liver graft injury. The accounts of ASEs with A3, AF, and SE type were top three in patients and consistent in the rat model. In particular, ASEs of A3 and A5 type constituted the highest fraction both in humans and rats, although the distribution of PSI value and length of ASEs were different. It will be worthwhile to further explore the specific characteristics of ASE types in liver grafts. Regarding the differential ASEs between steatotic and normal grafts, SE and AF were the most frequently observed types in human steatotic liver grafts, which was consistent with the differential ASEs in HCC patients . The oveThe genes with differential ASEs were significantly enriched in metabolism-related pathways in HCC . ConsistThe alteration of inflammation and immune cell distribution reshaped the liver graft immune environment . ImportaTaken together, our study not only offered the portrait of transcription and ASEs in liver grafts both from human and rat, but also identified the differential ASE network with metabolic RBPs, immune cell distribution, and cancer hallmarks in steatotic liver grafts. Our analysis verified the link between steatotic liver graft injury and tumor recurrence at the post-transcriptional level, provided new resources to explore the metabolism and immune cell responses, and offered the potential prognostic markers and therapeutics for tumor recurrence after liver transplantation.Twenty-eight patients that underwent liver transplantation in Queen Mary Hospital, The University of Hong Kong, were included in this study. Eighteen patients received steatotic donor livers , while the other 10 patients received normal donors. The clinical characteristics of patients were listed in Guide for the Care and Use of Laboratory Animals . Experimental procedures were approved by the Committee on the Use of Live Animals in Teaching and Research, The University of Hong Kong.Male Sprague Dawley (SD) rats (six to eight weeks old) were obtained from the Laboratory Animal Unit, The University of Hong Kong. All animals were housed in a standard animal facility at 22 ± 2 °C under controlled 12-h light/dark cycles and had free access to chow and autoclaved water. Rats received humane care following the criteria outlined in n = 3). The detailed protocols were implemented according to the previous studies [The steatotic and normal donor rats were fed with 45% high-fat diet  or regular diet for two weeks, respectively. Carbon tetrachloride  was injected into the recipient rats subcutaneously for four weeks to induce liver cirrhosis before the operation. The orthotopic liver transplantation model was established with small-for-size graft (ratio of graft weight to recipient liver weight was about 50%). The surgical procedure was briefly three steps: donor operation, recipient operation, and liver implantation. Generally, the survival rate was over 80%. Liver tissues were harvested at 6 h post transplantation .Clean reads were obtained by fastp (version 0.21.0) . Then, ap value < 0.05 were considered as differential ASEs. The parent genes of these differential ASEs were applied to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. The survival analysis of corresponding genes to common enriched pathways with differential ASEs in liver grafts of human and rats were analyzed by Gene Expression Profiling Interactive Analysis ).The splicing events were quantified by the percent spliced in (PSI) value using SUPPA (version 2.3) based on the assembled transcriptome and transcript levels . PSI ranp value < 0.05) were selected.A catalogue of 1756 RBPs was retrieved from two previous reports ,43. Diffp value less than 0.05 were considered significantly correlated. In addition, the differential RBPs correlated with differential ASEs were analyzed to explore the related KEGG pathways. Then, a dysregulation network was built, wherein RBP-ASE pairs containing both differentially expressed RBP genes and differential ASEs were extracted.Expression of RBP genes was expected to be correlated with the PSI level of target ASEs. Therefore, we calculated the Spearman correlation for each RBP-ASE pair. RBP-ASE pairs with Spearman correlation coefficients greater than 0.5 (or less than −0.5) and a corresponding http://bioinfo.life.hust.edu.cn/ImmuCellAI#!/resource (accessed on 6 March 2022)) [The immune cell infiltration levels were downloaded from Immune Cell Abundance Identifier ) ,45. The http://geneontology.org/ (accessed on 27 December 2020)), the inflammation markers provided by He et al. [http://www.gseamsigdb.org/gsea/msigdb/collections.jsp (accessed on 27 March 2022)). The PSI values of differential ASEs were performed with Spearman correlation with GSVA scores of each gene set in liver graft biopsies.Gene set variation analysis (GSVA) was used to perform the enrichment analysis through several gene sets for splicing factor , inflammation signature, and cancer hallmark . The gene et al. , and canStatistical analysis and data visualization statistical analyses were performed using the R software (version 4.0.1), Python (version 3.7) and Cytoscape (version 3.9.0). Data analysis and visualization tools in R software included the ggplot2, complexheatmap, GSVA, and clusterProfiler packages.


Opioid maintenance treatment (OMT) has the potential to reduce mortality rates substantially. We aimed to compare all-cause and overdose mortality among OMT patients while in or out of OMT in two different countries with different approaches to OMT.Two nation-wide, registry-based cohorts were linked by using similar analytical strategies. These included 3,637 male and 1,580 female patients enrolled in OMT in Czechia (years 2000–2019), and 6,387 male and 2,078 female patients enrolled in OMT in Denmark (years 2007–2018). The direct standardization method using the European (EU-27 plus EFTA 2011–2030) Standard was employed to calculate age-standardized rate to weight for age. All-cause and overdose crude mortality rates (CMR) as number of deaths per 1,000 person years (PY) in and out of OMT were calculated for all patients. CMRs were stratified by sex and OMT medication modality .Age-standardized rate for OMT patients in Czechia and Denmark was 9.7/1,000 PY and 29.8/1,000 PY, respectively. In Czechia, the all-cause CMR was 4.3/1,000 PY in treatment and 10.8/1,000 PY out of treatment. The overdose CMR was 0.5/1,000 PY in treatment and 1.2/1,000 PY out of treatment. In Denmark, the all-cause CMR was 26.6/1,000 PY in treatment and 28.2/1,000 PY out of treatment and the overdose CMR was 7.3/1,000 PY in treatment and 7.0/1,000 PY out of treatment.Country-specific differences in mortality while in and out of OMT in Czechia and Denmark may be partly explained by different patient characteristics and treatment systems in the two countries. The findings contribute to the public health debate about OMT management and may be of interest to practitioners, policy and decision makers when balancing the safety and accessibility of OMT. Opioid use disorders (OUD) are linked to elevated mortality rates among users of opioids across regions , 2. MortOMT is the first-line treatment for OUD. Generally, OMT is a well-established treatment approach following international guidelines . DespiteOver the past few decades, an increasing number of countries, such as Denmark and Norway, have embraced a more inclusive approach to OMT. This approach is characterized by extensive treatment coverage and less stringent criteria for remaining in treatment, which has resulted in higher retention rates . A recenThere is evidence from both experimental and observational studies that OMT contributes to the reduction in mortality rates among patients in OMT compared to patients who discontinue or terminate OMT , 12. NevMethadone and buprenorphine are currently the most widely used medications in OMT in Europe and elsewhere . CurrentNation-wide OMT registers with detailed information on an individual level about time of enrolment in treatment, discontinuation, re-entry, and termination of OMT medication exist in Czechia and Denmark , and mayThe aim of this study was to investigate mortality rates among OMT patients during periods of both being in and out of OMT, within two countries that represent distinct treatment approaches. Specifically, within each of the two national OMT cohorts, we aimed to investigate the crude mortality rates (CMR) related to: (i) treatment status (episodes in and out of OMT); (ii) sex; (iii) cause of death (overdose or non-overdose); and (iv) OMT medication modality .We used a prospective cohort approach linking the data sources by using the unique civil registration number assigned to all residents in both countries .OMT has been available as standard treatment in Czechia since 2000. The procedures for OMT are defined in the Standards for Substitution Treatment .In 2017, the OMT patient rate in Czechia was 0.6 per 1,000 population . As oppoDenmark was one of the first countries in Europe that introduced OMT in 1970), and one of the first countries that focused on achieving high treatment coverage and retention through a more inclusive approach. In 2017, Denmark had an OMT patient rate of 1.7 per 1,000 population 0, and on, and morIn Czechia, we included data from 1,580 females and 3,637 males who had been prescribed OMT medication at any time during the study period from January 1st 2000 to December 31st 2019.In Denmark, we included 2,078 females and 6,387 males who had been prescribed OMT medication during the study period at any time from January 2nd 2007 to December 31st 2018. A total of 22 OMT patients who died during the study period were excluded from the analysis because of missing data on either cause or date of death.In Czechia and Denmark, both data on exposure (OMT treatment) and outcome (death) were retrieved from national registers (20). Reporting to these registers is mandatory for the relevant professionals in both countries. A personal identification number is registered for each record, which enables linking data from various health registers at the individual level.Czechia :The national registry of addiction treatmentThe national registry of addiction treatment (NRAT) contains information on patients in OMT, including diagnoses, OMT treatment details, and basic socio-demographic data of all patients.The information system on deathsThe information system on deaths (ISZEM) is a general mortality register, which includes information on deaths of individuals with permanent or long-term residence in Czechia. The data is based on the information provided in the death certificate. The ISZEM contains the underlying and contributing causes of death based on ICD-10 codes. The data have been available in electronic form since 1994.Denmark :The Danish registry of drug abusers undergoing treatmentThe registry contains information on individuals enrolled in treatment of drug use disorders due to illicit drug use since 1996. The register contains brief socio-demographic information, information on past year drug use, and dates of admission and discharge, and provides information to the european monitoring centre for drugs and drug addiction (EMCDDA) treatment demand indicator.Cause of death registryDates and causes of death were identified using the general mortality register  that incin treatment or out of treatment. In treatment refers to the period that the individual patient was enrolled in OMT and was eligible to receive OMT medication. Out of treatment refers to the period that the individual patient was in between OMT treatment episodes or between the end of OMT episode and the end of follow-up if no new treatment episode was initiated  and 95% confidence intervals (CI) were calculated as the number of deaths per 1,000 person years (PY) in treatment and out of treatment  see Fig. We calcBased on EMCDDA selection B, overdose was defined as deaths, where the underlying cause was mental and behavioural disorders caused by illicit drug use , or accidental, intentional, or undetermined illicit drug poisoning, i.e., a combination of the mechanism of death listed under X or Y with the diagnoses for substance poisoning (dg. T40 and T43.6) .The direct standardization method using EU-27 + EFTA standard population 2011–2030 was employed to standardize for age .The study period was 2000–2019 in Czechia and 2007–2018 in Denmark. Therefore, we performed sensitivity analysis on in treatment and out of treatment CMR in Czechia for the same period as in Denmark (2007–2018).Statistical analyses were performed using SPSS (version 24)  and StatApproval from the Ethics committee in Czechia (no. 36/19GrantAZVVES20201.LFUK) has been obtained. In Denmark, ethical evaluation of studies utilizing quality assurance data is not required by law but was approved by the data authority . No human studies are presented in this manuscript.In Czechia, 1,580 (30.3%) out of 5,219 OMT patients were females, while 2,078 (24.5%) out of 8,465 OMT patients were females in Denmark . The aveIn Czechia, 8.1% (421) of OMT patients  died during the study period . In DenmIn Czechia, a quarter of all deaths occurred while in treatment, the CMR was 4.3/1,000 PY , the all-cause CMR out of treatment was higher  . The proIn Denmark, 65.2% of deaths occurred while in treatment. The all-cause CMR in treatment was 26.6/1,000 PY, and out of treatment 28.2/1,000 PY. The proportion of overdose deaths in treatment was 27.4% while overdose-specific CMR was 7.3/1,000 PY. Poisoning by methadone (T40.3) in overdose deaths was found in 63.9% of female and 62.0% of male OMT patients while in treatment as opposed to 40.0% of females and 35.5% of males out of treatment.In Czechia, all-cause CMR in treatment was quite similar across the types of OMT medication; methadone 3.8/1,000 PY (CI 95% 2.5–5.0), buprenorphine 4.9/1,000 PY (3.5–6.2), buprenorphine with naloxone 4.3/1,000 PY (2.5–6.0).In Denmark, all-cause CMR in treatment specific to types of OMT medication was the highest for methadone 29.6/1,000 PY (27.8–31.5) and comparable for buprenorphine 8.2/1,000 PY (5.7–11.9) and buprenorphine with naloxone 10.8/1,000 PY (7.2–16.4).Age-standardized mortality rate for OMT patients in Czechia and Denmark was 9.7/1,000 PY and 29.8/1,000 PY, respectively. Age-standardized overdose mortality rate for OMT patients in Czechia was 0.6/1,000 PY and 5.2/1,000 PY for Denmark.Sensitivity analysis on CMR in Czechia for the same period as in Denmark (2007–2018) showed similar results as for the longer observation period (2000–2019). In treatment, the CMR was 4.6/1,000 PY , while the all-cause CMR out of treatment was  .In this large cohort study involving over 13,000 patients ever enrolled in OMT, we found higher mortality rates both in and out of OMT among patients in Denmark than in Czechia. The proportion of overdose deaths while in OMT was nearly 15 times higher among Danish patients than their Czech counterparts. In contrast to previous studies  and while out of treatment (nearly 40%) in Denmark. The inclusivity of the OMT system in Denmark, along with its minimum requirements for supervised intake of OMT medication, urine testing, or other safety measures , 26 combOur findings indicate that there is a potential for improvement in clinical practices by applying a systematic evaluation and monitoring of mortality risk, increased involvement of psychosocial services, and enhanced safety measures to manage overdoses and other health risks. The differences between the Czech and Danish clinical approaches are substantial; one approach focuses more on safety and restrictions while the other focuses more on inclusion and reduced control measures , 22, 23.We should not think of the three main features in OMT - access, safety, and quality - as mutually exclusive, but rather as important dimensions of treatment that must be addressed and optimized. This would indeed be in accordance with the Dole, Nyswander, and Kreek recommendations for OMT from the 1960s .This study was unique because we were able to apply similar analytic approaches to study differences in mortality of OMT patients based on a comparison of nation-wide patient cohorts from two countries with different OMT settings. Further, we were able to study mortality rates among those in and out of OMT and while using different OMT medications  by causes of death , age, and sex. Information about OMT patients was drawn from the nation-wide health and population-based registers in Czechia and Denmark, minimizing selection and information bias. Finally, yet importantly, the information from the registers includes the specific dates for entering, terminating, and re-entering OMT on an individual patient level. Thus, the individual patient follow-up can be precisely calculated with respect to CMR. Additionally, this study provided new information on mortality rates linked to buprenorphine with naloxone.Our study also had limitations. The main limitation is that some important clinical information was not included in this study , 42, incWe observed significant differences in all-cause and overdose mortality rates in patients while in or out of OMT in Czechia and Denmark. Higher mortality rates were observed across the different types of OMT medication as well as in both sexes in the Danish cohort compared to the Czech cohort. These differences may be attributed to the clinical characteristics in both patient populations and to differences in clinical practice in OMT in the two countries. The findings are indicative and call for more studies on risk factors related to premature mortality as well as risk management.In Denmark, provision of liberal access to OMT did not necessarily lead to expected reduction in overdose deaths while in treatment. In Czechia, more inclusive clinical practice might lead to higher OMT coverage. Perhaps, a better balance of patient safety and access to OMT would lead to a reduced risk of overdose deaths.Our study shows how important yet challenging it is to extrapolate mortality estimates from one setting to another and to generalize findings across settings and populations. The findings contribute to the discussion about OMT management and may be of interest to practitioners as well as policy and decision makers when balancing the safety and accessibility of OMT.roman.gabrhelik@lf1.cuni.cz. Denmark: Requests for data sharing/case pooling may be directed to mh.crf@psy.au.dk. Requests to access these datasets should be directed to roman.gabrhelik@lf1.cuni.cz; mh.crf@psy.au.dk.The data analyzed in this study is subject to the following licenses/restrictions: this project uses third-party data derived from State government registries and databases, which are ultimately governed by their ethics committees and data custodians. Thus, any requests to share these data will be subject to formal approval from each data source used in this project. Czechia: Requests for data sharing/case pooling may be directed to the corresponding author RG on email: The studies involving humans were approved by the study has been reviewed and approved by the responsible ethics committee (the respective reference numbers are: 36/19GrantAZVVES20201.LFUK). For Denmark, the project is not under the legislation for ethical evaluation, since it is based entirely on administrative data, but the data has been approved by the data authority . The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements.SS, RG, and MoH designed the study. SS and BN analyzed the Czech data. MoH and AS analyzed the Danish data. All authors contributed to the interpretation of data, refinement of the paper, read, and approved the final version of the manuscript.


The pasting and rheological properties of starch gels from different botanical origins have been widely used to evaluate the application of these starches in pharmaceutical and food products. However, the ways in which these properties are modified by starch concentration and their dependence on amylose content and thermal and hydration properties have not been adequately established so far. An exhaustive study of the pasting and rheological properties of starch gels , wheat, potato, and tapioca) at concentrations of 6.4, 7.8, 9.2, 10.6, and 11.9 g/100 g was performed. The results were evaluated in terms of a potential equation fit between each parameter and each gel concentration. The parameters determined for the gels at the studied concentrations were correlated with the hydration properties and thermal properties by applying principal component analysis (PCA). Wheat starch, followed by normal maize and normal rice starches, presented a greater capacity to modulate their gels’ pasting and viscoelastic properties via their concentration in water. On the contrary, the characteristics of waxy rice and maize, potato, and tapioca starches were barely modified by concentration in pasting assays, but the gels of potato and tapioca showed noticeable changes in their viscoelastic properties as functions of concentration. In the PCA plot, the non-waxy cereal samples  were located close to each other. Wheat starch gels were the most dispersed on the graph, which is consistent with the high dependence on the concentration of the gel shown in most of the studied parameters. The waxy starches had close positions not too distant from those of the tapioca and potato samples and with little influence from amylose concentration. The potato and tapioca samples were close to the vectors of the crossover point in rheology and peak viscosity in their pasting properties. The knowledge gained from this work allows a better understanding of the effects of starch concentration on food formulations. The use of native starches is one of the most popular strategies for the development of gluten-free products. They are used in a wide variety of food products as thickeners, gelling agents, stabilizers, bulking agents, and water absorbers .Rice and maize starches are the most commonly used starches in the gluten-free bakery industry ,3, whereSwelling power, amylose content, and paste viscosity provide important information to infer their potential in the development of products such as biscuits , whereasStarch dispersions are subjected to combined heating and shearing effects during processing, which affect their rheology and the final product’s characteristics . HoweverIn the present work, the pasting and rheological properties of starch gels , wheat, potato, and tapioca) at concentrations ranging from 6.4 to 11.9 g/100 g were studied. The results were evaluated in terms of the fit to a potential equation between the studied parameters and the concentrations of the gels. The parameters determined for the gels at the concentrations studied were correlated with their hydration properties (Water Absorption Capacity (WAC), Water Absorption Index (WAI), Water Solubility Index (WSI), and Swelling Power (SP)) and thermal properties by applying principal component analysis (PCA). Normal maize (C*Gel 03401), waxy maize (C*Gel 04201), wheat (C*Gel 20006), potato (C*Gel 30002), and tapioca (C*CreamGel 70001) starches were manufactured by Cargill  and kindly donated by Brenntag Química S. A. U. . Waxy rice (Remyline XS) and normal rice (Remy DR) starches were obtained from BENEO  and kindly provided by Ferrer Alimentación . The starches analyzed had a purity greater than 99% on a dry basis, according to the commercial information available.Moisture determination was carried out by applying the AACC method 44-19.01 [The amylose concentration was determined using the Amylose/Amylopectin Determination Kit (K-AMYL 06/18) from Megazyme , applying the procedure of Gibson et al. (1997) . As descg for 30 min. The supernatant was discarded, and the weight of the precipitate (hydrated starch weight) was recorded, from which the mass of incorporated water was calculated. The WAC value is expressed as g water/g starch. The water absorption capacity (WAC) of the starches was determined by using the centrifugation method described by several authors ,22,23,24g for 10 min. The supernatant was collected in capsules and dried at 110 °C for 24 h. The weight of the drained supernatant was recorded. WAI, WSI, and SP values were expressed in terms of g gel/g starch, g soluble solids/100 g starch, and g gel/g insoluble matter in starch, respectively. Water absorption index (WAI), water solubility index (WSI), and swelling power (SP) tests were performed as described by Abebe et al. (2015)  with sliAll values obtained were corrected for the moisture content of each sample and are expressed on a dry basis. Samples were measured in duplicate.w/w. Each capsule was hermetically sealed and placed in the equipment for scanning from 0 to 120 °C at a heating rate of 5 °C/min, using an empty pan as reference. Indium and zinc were used for calorimeter calibration. The enthalpy change  and onset (To), peak (Tp), and ending (Te) temperatures were recorded for the gelatinization (G), retrogradation (R), and amylose–lipid dissociation (A-L) peaks. Endothermic transitions of retrograded starches were evaluated with a second scan conducted after storing the gelatinized samples in DSC pans for 7 days at 4 °C, following the same procedure used to evaluate gelatinization. This low storage temperature allowed faster amylopectin recrystallization, resulting in higher retrogradation enthalpies and more accurate results. At this temperature, the melting enthalpy of the recrystallized amylopectin reached a levelling-off value in about 7 days [A differential scanning calorimeter (DSC)  was used to evaluate the thermal transitions, including gelatinization, retrogradation, and amylose–lipid complex dissociation in starch samples using a protocol previously applied to other samples ,25. About 7 days . The degt 7 days . Each saThe pasting tests were carried out using a Rapid Visco Analyzer (RVA), a Perten RVA 4500 instrument , following the STD1 test detailed in the official AACC method 76–21.02 . Five coOnce the pasting profiles were obtained, the viscosity parameters were identified as follows ,30,31: pF): (1) G′ = G′1·Fa, (2) G″ = G″1·Fb, and (3) tan (δ) = tan (δ)1·Fc. This model proved to be valid and robust, as all fits achieved correlation coefficients R2 > 99.9%, confirming that these viscoelastic properties increase with concentration following a potential evolution. The coefficients of the potential equation, which represent the elastic and viscous moduli as well as the loss tangent, respectively, at a frequency of 1 Hz were named G′1, G″1, and tan(δ)1. The exponents of the potential equation, named a, b, and c, quantify the dependence of the elastic and viscous moduli and the loss tangent, respectively, to the oscillation frequency. Strain sweeps were performed at 1 Hz in the range of 0.1 to 1000% except for tapioca and potato starches, where the range was extended to 2500%. The maximum stress in the LVR and the stress at the cross point (G′ = G″) were determined from strain sweeps. The end of the LVR (τmax) was identified as the sharp decrease in the G′ modulus, coinciding with the sudden increase in tan(δ). A dynamic oscillatory test was conducted to analyze the gels using a Kinexus Pro+ rheometer  equipped with 40 mm parallel plates of serrated surface, working at a 1 mm gap and a constant temperature of 25 °C. Gel samples were prepared using the method described in Shapiro–Wilk normality tests, statistical analyses of mean, standard deviation, ANOVA with Tukey’s test of comparisons, and principal component analysis (PCA) were performed using the free software R version 4.2.2 (The R Foundation) under the IDE RStudio 2022.12.0 + 353 . The fitting of the studied parameters as a function of starch concentration to the potential equation was performed with the statistical package Statgraphics Centurion 19—Version 19.4.01 . The amylose content and hydration properties  are presented in The WAC values allowed grouping the samples into two main groups: one containing wheat, waxy rice, normal rice, and tapioca that showed the lowest values (0.82–0.89 g/g), and one containing normal maize, waxy maize, and potato that showed the higher values (1.01–1.08 g/g). The variation in WAC could be due to variations in starch structure, resulting in variations in the formation of covalent and hydrogen bonds between the starch chains and, thereby, in the availability of water binding sites ,40. WAI The results from the DSC analyses are presented in Wheat starch showed the lowest onset temperature of gelatinization (To), followed by potato and waxy rice, whereas normal rice had the highest value. In rice starch samples, a higher amylose content has been correlated with a higher gelatinization temperature ; howeverIn general, peak (Tp) and ending (Te) temperatures of gelatinization varied among starches in the same order as To, with some exceptions due to the different shapes and widths (ΔT = Te − To) of the gelatinization peaks. Potato recorded the narrowest gelatinization peak (ΔT = 9.2 °C), whereas waxy rice showed the widest (19 °C), denoting a more complex and heterogeneous crystalline structure.The enthalpy of the melting of amylopectin recrystallized after 7 days of storage at 2 ± 2 °C; the DR and the temperatures of this transition are also shown in The amylose–lipid complex dissociation peak, which appeared in the range of 85 and 105 °C, was only recorded in normal maize, normal rice, and wheat starches. Normal rice showed the highest amylose–lipid dissociation enthalpy. As expected, it was not detected in waxy starches due to their low amylose content; it was not observed in tuber starches such as potato and tapioca either, despite their significant amounts of amylose. The occurrence of amylose–lipid complex dissociation peaks is common in cereals, which may contain about 1% lipid content, whereas starches from tubers, legumes, and waxy starches contain practically no lipids ; thus, ve, are summarized in 2 coefficients ranging 94–99) except for potato starch, which was always far from this potential evolution, and tapioca starch, which did not follow it in the cases of TV and SV. This procedure facilitates the comparison between starches regarding the effect that concentration has on their pasting properties by using only two values: the fitting coefficient k  and the exponent e (which informs the rate of increase of the P parameter with concentration).The pasting properties of the starches at all concentrations studied are presented in k coefficient for PV and the very low e exponent, indicating little dependence of this variable on concentration. Normal rice, normal maize, and wheat starches showed the lowest PV values at low concentrations and the highest rate of increase with concentration, as evidenced by their low k coefficient values and high e exponents , which presented significantly equal values at concentrations ≥9.2 g/100 g. This can also be evaluated from its very high xponents . On the e  values (4801–9129 mPa·s) denoting its lowest stability against heating and stirring B. Breakde . A signi2 values >98%) except potato starch. As shown in FV increased potentially with concentration in all studied starches , a value that then decreased at 11.9% w/w, indicating that tapioca starch, like potato starch, experienced saturation behavior at high gel concentrations.The potato starch gels followed an inverted U-shaped trend in SV values with increasing concentration, showing a maximum value (2176 mPa-s) at 9.2% w/w D, which w/w . This bee exponents and values <1 (Increasing the concentration of the gels tends to maintain or decrease the peak time (Pt), with negative alues <1 . Among aalues <1 E. Cereale) and on high Pt values at each concentration, which is explained by the high coefficient k. Samples with lower amylose content registered variations of Pt ~1 °C between the lowest and highest gel concentration values . A similar behavior was observed for tuber starches , showing once again the low dependence of the measured parameters on the amylose content of these two samples. On the other hand, non-waxy cereal samples with higher amylose content showed a marked decrease in Pt with increasing starch concentration in the gels. Thus, wheat starch recorded the highest difference in Pt (25.71 °C) between the 6.4 and 11.9% w/w concentration gels, followed by normal maize (14.39 °C) and normal rice (12.80 °C). This marked change in the cereal samples shows more clearly that as gel concentration increases, amylose loses the ability to delay granule swelling, requiring a lower temperature to initiate the pasting process. The PT provides important information about the quality of gluten-free bakery products. A significant positive correlation between the specific volume of bread and PT has been reported [Pt tended to decrease with increasing gel concentration in all cases . The nonreported . Given t1, G″1, and tan δ1 coefficients and a, b, and c exponents (obtained from the frequency sweeps tests) versus gel concentration is illustrated in 2 < 84).The evolution of the G′1) were higher than those of the viscous modulus (G″1), resulting in a loss tangent of (tan (δ)) < 1, indicating an elastic-like behavior in the studied gels. The value of G′1 in each material increased with concentration, where its evolution was influenced by the nature of each starch. In all cases, the values determined for the elastic modulus  and by potato starch gel at 11.9% 1 values, denoting their more elastic behavior. The normal and waxy rice samples had close values at low concentration, but though the tan(δ)1 of waxy rice gels increased at higher concentrations, with an e exponent similar to that of waxy maize, the normal rice gels tended to decrease with concentration similarly to the other two non-waxy cereal starches .At all concentration levels, waxy maize starch gels showed the highest tan(δ)ntration C. Tapioca, b, and c exponents  with frequency) with gel concentration is illustrated in 1 and G″1 moduli, waxy maize and tapioca, showed the highest a and b exponents at almost all concentration levels, whereas wheat and normal maize starch gels, with the highest viscoelastic moduli, had the lowest values. This indicates that gels of softer consistency are, in general, more dependent on frequency than those of stronger structure. The evolution of the a, b, and c exponents with concentration did not follow a clear trend. At high concentrations, potato starch gels showed a significant drop in their frequency dependence (concomitant with the increase in their viscoelastic moduli), whereas for wheat starch gels, the drop of these exponents was determined at lower concentrations. The values of coefficient c did not follow a particular pattern in wheat and normal maize starch gels ; thus, a low value of c indicates a similar dependence of both viscoelastic moduli on frequency.The evolution of the rch gels F. Gels o1 = G″1), where the structure of the gel passes from a predominantly elastic behavior to one with a more pronounced viscous character, and the corresponding deformation of the sample at this point in function of the gel concentration, are shown in k and e obtained from fitting the evolution of these values with concentration to a potential equation are shown in The stress required to reach the crossover point  for crossing point stress and crossing point strain, which is possibly because of the low final viscosity values of the sample. Normal rice showed a lack of fit in both crossing point stress and maximum strain. Crossing point strain also showed non-adjustment in the potato gels, which is a consequence of the erratic behavior shown in The fits to the potential equation in the gels of wheat and normal maize starch were good in all cases, with R than 80 . The fite of wheat (3.2) and normal maize (2.5). However, the strain reached at the crossover point by both wheat and normal maize gels did not show much variation with respect to concentration (e exponents in crossing point stress (2.5 and 1.7 respectively) but lower k coefficient values, which can be seen in k coefficient of tapioca starch gels in crossing point strain was much higher than those of the other gels, although its low e exponent  and maximum strain before their structures are destroyed. The variations of the maximum stress that marks the end of the LVR and the corresponding maximum strain with the gel concentration are shown in The conducted principal component analysis (PCA) is illustrated in The model explains 56.8% of the total variation between samples. It consists of two dimensions, where Dim1 is presented in the horizontal axis and Dim2 in the vertical axis. The central point of the graph, which defines four quadrants, represents the zero-contribution value from which the studied parameters emerge as vectors, depending on whether they are positively or negatively correlated with each of the two dimensions.1, G″1 and crossing point stress), which is in agreement with the high e coefficients shown by this starch in these parameters. The normal maize starch gels have a very similar distribution on the graph as wheat starch, but they are less dispersed, whereas normal rice starch has a much narrower distribution. With respect to the range of positive Dim1 values, waxy rice and waxy maize samples were placed in the positive quadrant of Dim2, whereas potato starch gels are in the negative quadrant of Dim2, and tapioca starch gels are in an intermediate position. Gels of waxy samples, potato, and tapioca were found to be opposite to the effect of amylose concentration and are also shown to be less affected by parameters such as pasting temperature, crossing point stress, and maximum stress, which did not vary greatly with increasing concentrations in these four materials.Cereal samples with higher amounts of amylose  are located in the zone of negative Dim1 values, where the greatest effect of amylose concentration and calorimetric parameters of dissociation of the amylose–lipid complex are found. The wheat starch gels at different concentrations show an enormous dispersion along Dim1, which is related to the pronounced separation of pasting temperature with increasing concentration as well as a stronger relationship of the more concentrated gels with the vectors of FV, SV and some rheological parameters  together with normal maize and tapioca gels. 1 values in the waxy rice and tapioca gels coincide with the position of these samples in the vicinity of the vector of this variable. Potato starch gels showed lower tan(δ)1 values for samples of higher concentration, which is illustrated by the greater distance of the higher concentration gels to the vector of this variable. The waxy starch gels, together with potato and tapioca, had the highest ΔH(G) values, justifying the closeness of these samples to the vector of this variable. The high tan(δ)The concentration of starch gels influences their viscoelastic behavior in different ways. The gels that underwent the greatest changes were those from non-waxy cereals, with wheat starch gels showing the greatest changes, followed by gels from normal maize. The amylose content influenced the behavior of maize gels when comparing the waxy and normal samples, whereas waxy and normal rice gels did not show such a great distance between the measured parameters, indicating that the molecular structure of the granule in rice samples have more weight in the explanation of the observed phenomena. Potato and tapioca gels showed different behaviors than cereal gels, being one of the most important differences in the little effect of amylose concentration on pasting properties, with similar results to waxy samples but with higher viscosities. The structure of the amylopectin chains probably also exerts an important effect on these parameters. The calorimetric parameters and the hydration properties allowed for a better clustering of the parameters in the PCA, helping to determine the characteristics of each starch and contributing to the inference of the behavior that each material would have in the elaboration of foods and, in particular, gluten-free products.


In living-donor liver transplantation (LDLT), portal Y-graft interposition using the recipient’s portal vein (PV) bifurcation has been used for right lobe grafts with double PV orifices. We herein report the use of thrombectomized autologous portal Y-graft interposition for a recipient with preoperative portal vein thrombosis (PVT) in a right lobe LDLT with double PV orifices.The recipient was a 54-year-old male with end-stage liver disease due to alcoholic liver cirrhosis. There was PV thrombus in the recipient’s PV. The living liver donor was his 53-year-old spouse, and a right lobe graft was planned for the transplantation. Since the donor's liver had a type III PV anomaly, autologous portal Y-graft interposition after thrombectomy was planned for PV reconstruction in the LDLT. The portal Y-graft was resected from the recipient and a thrombus extending from the main PV to the right PV branch was removed on the back table. The portal Y-graft was anastomosed to the anterior and posterior portal branches of the right lobe graft. Followed by venous reconstruction, the Y-graft was anastomosed to the recipient’s main PV. The operation time was 545 min and the intraoperative blood loss was 1355 ml. The recipient was discharged on postoperative day 13 without any complications. The recipient remains well with the patency of the portal Y-graft one year after the liver transplantation.We herein report the successful use of autologous portal Y-graft interposition after thrombectomy on the back table for a recipient with PVT in a right lobe LDLT. Portal vein (PV) reconstruction using a graft has been required for living-donor liver transplantation (LDLT) in the case of the right lobe graft with double PV orifices. Several methods have been proposed for the vascular reconstruction of the double PVs. Among them, portal Y-shaped graft interposition using the recipient’s PV bifurcation has been used worldwide due to its technical feasibility and good long-term patency , 2. AlthThe recipient was a 54-year-old male patient with end-stage liver disease due to alcoholic liver cirrhosis. He had undergone radiofrequency ablation for hepatocellular carcinoma in segment 8, endoscopic variceal ligation for esophageal varices, and thrombolytic therapy for a PV thrombus extending from the superior mesenteric vein (SMV) to the right PV branch Fig. A–C. BecaThe living liver donor was his 53-year-old spouse with no pre-existing disease, and a right lobe graft was planned for the transplantation. The graft-to-recipient weight ratio (GRWR) calculated from computed tomography liver volumetry was 0.96. The estimated GWRW of the left lobe graft was 0.55, which did not meet our criteria of GRWR > 0.80 in our institution. The recipient was ABO blood type-incompatible, and thus preoperative rituximab and mycophenolate mofetil were administered according to the institutional protocol . Since tIn the donor surgery, in order to prevent iatrogenic injury to the donor's remnant PV, two right liver PV branches were cut separately. To use the thrombosed PV as autologous Y-graft interposition, after ligation of all the small branches from the hilar PV, the portal Y-graft was resected from the recipient. On the back table, a thrombus extending from the main PV to the right PV branch was easily removed without damaging the PV wall Fig. A, B. SubThe operation time was 545 min and the intraoperative blood loss was 1355 ml. No anticoagulant was administered after PV reconstruction according to the protocol for the cases in which PV thrombectomy was performed [In right lobe LDLT, some of the right lobe grafts with a type II PV anomaly and all of those with a type III PV anomaly have double PV orifices . When thIt has been reported that 13% of recipients have PVT in LDLT . TherefoWe successfully performed autologous portal Y-graft interposition after thrombectomy on the back table for a recipient with PVT in a right lobe LDLT.


Background Burr hole covering in brain surgical procedures can avoid complications and unaesthetic results. The aim of this registry was to assess the safety and performance of a new polymeric burr hole covering device .Methods A multicenter, prospective, clinical registry design was used for the study. All the patients who fulfilled the inclusion criteria were included in the study and followed up for 6 months. Baseline clinical parameters, surgical variables , postoperative variables , and adverse events were evaluated.Results Forty-three Cranial COVER devices were implanted in 30 patients. Most of them were implanted in frontal locations (53.5%). After implantation, 97.7% of the devices completely covered the burr hole, and 100% perfectly adapted to the skull surface. All surgeons ranked their satisfaction with the implantation procedure as very high or high. No artifacts were detected in any of the neuroimaging studies performed and no adverse events related with the device or its implantation were reported during the follow-up. There were significantly more scalp depressions associated with uncovered than with Cranial COVER–covered burr holes (p = 0.040). Patient satisfaction with covered burr holes located in the frontal and parietal areas was 9.0 ± 1.4 over 10.Conclusion Cranial COVER is a safe and reliable burr hole covering system that offers excellent cosmetic results and high satisfaction rates for both surgeons and patients. Cranial COVER is highly adaptable to the skull surface, and it was predominantly used in frontal locations due to their cosmetic importance. Burr holes are the most common way to access the brain, either as stand-alone holes or as part of a craniotomy. Multiple systems have been used for the re-fixation of the bone flap after a craniotomy, but the covering of burr holes has been less considered throughout history. Considering their small size , frequently the burr holes are not specifically covered once the surgery is completed. This can lead to complications The first obvious solution to cover a bone defect, particularly if it is small , is to use bone itself. Some have proposed the possibility of using buttons made of bone autografts, obtained from the inner table of the bone flap, to guarantee that there is no reaction to foreign materials and make the end of the surgery easier.The use of metal plates and screws, particularly made of titanium, in cranial surgery is also frequent.In this study, we assess a conceptionally new product for covering burr holes: a polymeric clamplike device . This clamplike device can be used both in isolated and craniotomy burr holes. This registry had the main aim of evaluating the clinical safety and performance of Cranial COVER as a burr hole cover in neurosurgical procedures in a “real-world” cohort of patients.This is an observational, prospective, single-arm, multicenter cohort study. Recruitment and follow-up took place between March 2019 and August 2020, at four tertiary centers in Spain. The study protocol was approved by the Ethics Committee of each hospital (References: HSCSP 18/325 (PS); GHQSB 53/2018; PSM 2019/8693/I; HUB PR389/18) and the investigation was conducted according to the principles and rules laid down in the Declaration of Helsinki and its subsequent amendments. This report follows the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement: guidelines for reporting observational studies.Patients were eligible for inclusion in the registry if they were >3 years of age and had to undergo a cranial surgery, where at least one burr hole was planned to be performed. Key exclusion criteria were fever or leukocytosis, allergy to implant material, degenerative bone disease, and use of artificial cranial bone. The eligibility of subjects was also assessed intraoperatively: the burr hole cover was implanted in patients with burr holes whose diameter was between 10 and 14 mm, and that did not have infection, inflammation, or bone tumors in the operated area. Patients with burr holes located in the sinus or in the inferior occipital cranial area, or without a suitable tissue cover in the operative field, were intraoperatively excluded from the study.Patients included in this registry were followed up according to standard clinical practice of each study center for 6 months after surgery, until they were clinically discharged, lost to follow-up, or dead.The device evaluated in this clinical investigation was Cranial COVER (NEOS Surgery S.L), a CE-marked device. Cranial COVER is intended to cover burr holes, either stand-alone holes or holes being part of a craniotomy. Cranial COVER is totally made of the polymer polyether ether ketone (PEEK) and is based on the principle of a clamp: it consists of two platforms linked by two adjustable cable ties, and it is kept tightened to the skull with a double locking system in the upper platform. This is based on two ratchets that engage with the lower platform's cable ties' teeth that allow its movement toward the lower platform and, at the same time, impede its backward movement. The device is available in two sizes: the large size, for use in the most common burr holes, made with standard perforators of diameters 14/11 and 13/9 mm; and the small size, for use in burr holes with a diameter in the range of 10 to 12 mm made with high-speed spherical drills.The implantation of Cranial COVER does not require any specific surgical instrument. It can be implanted following two different approaches:For stand-alone burr holes or for craniotomy burr holes where the bone flap has been previously repositioned in its original location: First, the Cranial COVER's elongated lower platform is introduced through the burr hole, into the subcranial area, between the bone and the dura mater and centered in the hole axis. Then, the device is tightened by pulling the handle and adjusting the upper platform on the burr hole surface by sliding it down with the help of the applier, until the upper platform is properly adapted to the skull surface. The upper platform then remains in its position thanks to its double locking system. Afterward, the cable ties are cut using standard instruments  to remove the handle and the applier. Finally, the protruding excesses of the cable ties are cut and removed taking advantage of their self-cutting feature, by bending them repeatedly up and down.For craniotomy burr holes where the bone flap has not been previously repositioned in its original location only: First, the lower platform is prepositioned in the burr hole area, between the bone and the dura mater, before positioning of the bone flap. Then, the bone flap is placed back and the device is initially adjusted to prevent its movement while the bone flap is fixed with specific products . Finally, the Cranial COVER is completely adjusted to the skull surface, and the handle and applier are removed as described in the first approach and2.The primary endpoint of the study was to assess the prevalence of patients who have a complete covering of the cranial burr hole at the end of the surgery. Burr hole covering and adaptation was evaluated according to the following classification with four levels: <25% of the upper platform contacts the cranial surface; between 25 and 50% of the upper platform contacts the cranial surface; between 50 and 75% of the upper platform contacts the cranial surface; and >75% of the upper platform contacts the cranial surface.Secondary outcomes comprised both safety endpoints  and performance endpoints. The latter included the surgeon's overall satisfaction with the procedure of covering the burr hole, assessed by a Likert scale ; the surgeon's evaluation of the presence of scalp depression, by direct visual control of the surgical area during follow-up visits, measured as a dichotomic variable (yes/no); the satisfaction of patients with the aesthetic result of covered and uncovered burr holes, assessed by a Visual Analog Scale ; the prevalence of patients who presented discomfort or functional handicaps associated with the covered and uncovered burr holes; and the presence of artifacts generated by the implanted products on computed tomography (CT) or magnetic resonance (MR) imaging.Utest when appropriate. Ap-value <0.05 was considered statistically significant. GraphPad Prism 6.01  was used to perform the analysis.Descriptive statistics were used to characterize the population studied and the different variables evaluated in all time points: in the preoperative period, in the surgery visit and at different follow-up times. For continuous variables , arithmetic mean, standard deviation, median, minimum, maximum, and interquartile ranges (IQR) are presented. Categorical variables  are presented in relative and absolute frequencies. Comparison of covered and uncovered burr holes was performed by means of Fisher's exact test or Mann–Whitneyn = 30 patients is the sample size needed to detect an adverse event that occurs in the following conditions: 95% of confidence interval of the probability of occurrence of the event (α = 0.05) and 10% of probability of occurrence of the adverse event. To account for possible losses (10%) at follow-up , this number was increased to reachn = 33 patients.For sample size calculation, the primary endpoint of the study was considered: the aim was to confirm that a complete and solid covering of the burr hole was achieved in 90% of the patients. Bearing in mind that this threshold can be considered both in terms of performance and of safety, it is reasonable that, for the sake of calculating the sample size, the value for adequate burr hole covering (90%) was transformed into the frequency of an adverse event. This corresponds, in this case, to considering that the frequency of incomplete and/or nonsolid covering of the burr hole was 10%. According to Hanley's formula,Thirty-three patients were screened for inclusion in the study. All of them fulfilled the inclusion/exclusion criteria. However, three patients were finally not included as written informed consent was not obtained. Of the 30 patients included, 19 patients completed the 6-month follow-up, 9 patients performed at least one follow-up visit, but the follow-up time was less than 6 months, and 2 patients were discontinued because of an adverse event (not related to the study product or procedures). Results from the analysis of these 30 patients are presented in the following sections. The main sociodemographic and baseline clinical characteristics of the population studied are presented inA total of 82 burr holes were performed in the 30 patients, 43 of which were covered with Cranial COVER (52.4%), 5 were covered with titanium burr hole plates (6.2%), and 34 were left uncovered (41.5%). Twenty patients were implanted with one Cranial COVER, 7 patients were implanted with two, and 3 patients were implanted with three devices. Details about the surgical procedure, the number and the location of burr holes performed, and the covering used in each of them are provided inThe primary endpoint of the study was to assess the prevalence of patients who present a complete covering of the cranial burr hole at the end of the surgery.After the surgery, 42 of 43 (97.7%) Cranial COVER devices completely covered the burr hole, and all devices (43) had their upper platform completely adapted to the skull surface.The surgeon overall satisfaction with the procedure of covering the burr hole with Cranial COVER was evaluated after surgery. Surgeons rated it as “satisfied” or “very satisfied” in all cases.p = 0.04; Fisher's exact test) scalp depressions in those burr holes that were left uncovered  than in those burr holes that were covered with Cranial COVER . A subanalysis of frontal and parietal burr holes was then performed to assess those burr holes with a higher aesthetic impact. Patients reported a higher satisfaction score for parietal and temporal burr holes covered with Cranial COVER (9.0 ± 1.4) than for those left uncovered . Differences were less pronounced in burr holes with a lower aesthetic impact : patient satisfaction score with covered burr holes was 8.9 ± 0.6 in these locations, while it was 8.5 ± 1.4 with uncovered holes in these areas (p = 0.85).During follow-up visits, satisfaction with the aesthetic results of Cranial COVER was evaluated by 25 patients, while satisfaction with the aesthetic results of uncovered burr holes was evaluated by 14 of these patients . Patient satisfaction score with covered burr holes was 8.9 ± 1.1, while it was 8.1 ± 1.7 with uncovered burr holes .In follow-up visits, patients were also asked if they presented discomfort associated with the covering system and if they presented any functional handicaps during activities of daily living, such as hairdressing, combing, scratching the scalp, or washing their hair. Only three patients reported some kind of discomfort associated with the device implanted and one patient reported functional handicaps. Patients also reported a similar degree of discomfort and functional handicaps associated with uncovered burr holes  were reported during the study (two brain tumor recurrences). Neither of them was related to the product, but to the baseline pathology progression.This study demonstrates that Cranial COVER is a safe and reliable burr hole covering system. A total of 82 burr holes were performed in the 30 patients included in the study in the four participating sites, 43 of whom were covered with Cranial COVER devices.Cranial COVER was mainly implanted to cover burr holes performed in craniotomy or cranioplasty procedures (28 patients), but in 2 patients, it was used in single burr holes performed for hematoma drainages (three devices implanted in single burr holes). The fact that most of the devices were implanted in craniotomy burr holes reflects the daily clinical practice of most neurosurgery departments, where craniotomy procedures are much more frequent than those where single burr holes are performed. Moreover, two additional circumstances that limit the implantation of Cranial COVER in single burr holes should be considered: the first one is that Cranial COVER cannot be implanted in single burr holes where a drainage catheter is used and the second one is that, in some hospitals, small burr holes  are performed to treat subdural hematomas.The implantation technique used to implant Cranial COVER in craniotomy procedures depended on surgeon preference, and both implantation techniques (before or after positioning the bone flap) were similarly used.Regarding location, it is worth noting that Cranial COVER was mainly implanted in frontal and parietal locations, while temporal and occipital burr holes were predominantly left uncovered. Both situations point in the same direction: Cranial COVER devices are preferentially used in areas that have a higher aesthetic impact, as frontal and parietal areas are more prone to the presence of visible scalp defects due to, for example, the smaller presence of hair. Indeed, the fact that patients can potentially develop cosmetic complexes caused by the scalp depressions associated with burr holes is a frequent reason to cover these with a specific device, particularly in young patients and/or with good prognosis. The presence of thin skin in the area  is another aspect that may lead to the use of burr hole covering devices. In fact, in these patients, to avoid the ulcerations that have been associated with plates before,The primary study goal was to assess if Cranial COVER completely covers the burr hole: this was achieved with almost all of the Cranial COVER devices implanted, which proves the product's adequate performance. In addition, all 43 devices were completely adapted to the skull surface, thus further confirming the appropriate performance of the device. In fact, surgeons ended all surgical interventions satisfied or very satisfied with the implantation procedure of Cranial COVER.Secondarily, the study also aimed to assess aesthetic results of the implant and self-perception patient evaluations.Based on the surgeons' evaluation, there were significantly more skin concavities present over uncovered holes than over holes covered with Cranial COVER. However, patients ranked their satisfaction with covered and uncovered burr holes similarly. A potential explanation for this is that, as previously discussed, Cranial COVER was predominantly implanted in areas with a higher aesthetic impact , while uncovered burr holes were predominantly located in temporal and occipital regions, which have a smaller cosmetic importance; thus, their negative aesthetic impact may be considered less relevant. In fact, when analyzing only those burr holes performed in frontal and parietal locations, patients did give higher satisfaction scores when burr holes were covered with Cranial COVER than when burr holes were left uncovered.We also confirmed that Cranial COVER does not generate any artifacts in the neuroimages (neither CT scan nor MRI).From a safety point of view, it is relevant to note that neither intraoperative nor follow-up complications were registered in the study.This is the first study that has prospectively and systematically evaluated the use of a burr hole covering system after a neurosurgical procedure, adding an important level of clinical evidence to the procedure of covering burr holes after cranial surgery, which has been seldom investigated so far. A previous pilot retrospective study evaluated a total of 14 burr hole covers (titanium plates) placed in 11 patients and compared them to 50 burr holes that were not covered.Although in our study the incidence of scalp depressions over uncovered burr holes was smaller, this can be explained by at least two factors: first, because the evaluation was performed visually by a neurosurgeon ; it does not require any particular instrument for implantation . Based on this, Cranial COVER is plausibly a cost-effective solution to cover burr holes.It is worth noting that in a craniotomy fixed with the Cranial LOOP system, the XL size could be used for both covering and fixing the bone flapOther not so large devices for covering burr holes are the so-called buttons. These usually consist of a small piece, in the shape of a mushroom and with a diameter that fits with the hole diameter, which is plugged into the hole at the end of the surgical procedure. Multiple materials have been proposed for these devices, including both absorbable (polycaprolactone)Covering the burr hole with bone dust or polymethyl methacrylate (PMMA) is a less costly solution. However, after a variable period of time, cranial defects treated only with bone dust demonstrate local skin depressions, probably caused by a high level of bone resorption, which leads to unfavorable cosmetic resultsThis clinical investigation has some limitations. First, the lack of a control group does not allow for a direct comparison of covered versus uncovered burr holes in similar conditions, thus limiting the interpretation of results. However, this study aimed at obtaining “real-world” clinical data, and it sought not to interfere with the usual clinical practice, or the neurosurgeons' decision to cover burr holes with an implant, or in the scheduled follow-up visits.Also, the study aimed at collecting 6 months of follow-up data, but this follow-up time was not reached in 11 patients, due to several reasons . Again, the design of the study to collect real-world clinical data and to minimize the interference with daily clinical practice explains this situation: a fixed scheduled timing for follow-up visits was not established. Future studies including longer follow-up times and a larger cohort could also be of interest to confirm the safety and efficacy of the device in the long term.In summary, the results of this clinical registry prove that Cranial COVER is a safe device that completely covers both stand-alone and craniotomy burr holes, perfectly adapting to the skull surface. Furthermore, the use of Cranial COVER could have a positive aesthetic impact on patients, particularly in frontal and parietal burr hole locations, where cosmetic defects are more disturbing. Finally, it has been confirmed that Cranial COVER does not interfere with follow-up imaging, as it does not generate any artifact in CT and MRI.


Amiota and six representative species in the genus Phortica. We performed a series of comparative and phylogenetic analyses for these 12 Steganinae mitogenomes, paying special attention to the commonalities and differences in the D-loop sequences. Primarily determined by the lengths of the D-loop regions, the sizes of the Amiota and Phortica mitogenomes ranged from 16,143–16,803 bp and 15,933–16,290 bp, respectively. Our results indicated that the sizes of genes and intergenic nucleotides (IGNs), codon usage and amino acid usage, compositional skewness levels, evolutionary rates of protein-coding genes (PCGs), and D-loop sequence variability all showed unambiguous genus-specific characteristics and provided novel insights into the evolutionary implications between and within Amiota and Phortica. Most of the consensus motifs were found downstream of the D-loop regions, and some of them showed distinct genus-specific patterns. In addition, the D-loop sequences were phylogenetically informative as the data sets of PCGs and/or rRNAs, especially within the genus Phortica.To address the limited number of mitochondrial genomes (mitogenomes) in the subfamily Steganinae (Diptera: Drosophilidae), we assembled 12 complete mitogenomes for six representative species in the genus  As the secondary genome in eukaryotes, the mitochondrial genome (mitogenome) is far from being independent of the nuclear genome and the rest of the cell . The mitIn general, a typical mitogenome is a circular, double-stranded DNA molecule that is 14–20 kb long and contains 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and an A + T-rich, noncoding displacement loop (D-loop) region  ,13 relatAmiota Loew, 1862  as outgroup taxa. We performed phylogenetic analyses on the following three data sets of mitogenomes: (1) nucleotides of concatenated 13 PCGs and two rRNAs; (2) amino acids of concatenated 13 PCGs; and (3) nucleotides of the D-loop regions. The three data sets were aligned using MAFFT v7.490 [To rigorously test the monophyly of Steganinae, a total of 14 complete drosophilid mitogenomes were included in subsequent phylogenetic analyses, including the 12 assembled Steganinae mitogenomes and two Drosophilinae mitogenomes [T v7.490  with theT v7.490  to searcT v7.490 , which rT v7.490 . The conT v7.490 ,45. UBPsT v7.490 . All resT v7.490  with adjAmiota dentata Okada, Amiota femorata Chen & Takamori, Amiota nagatai Okada, Amiota setosa Zhang & Chen, Amiota spinifemorata Li & Chen, Amiota yifengi Zhang & Chen, Phortica (Ashima) longipenis Chen & Gao, Phortica (Ashima) tanabei Chen & Toda, Phortica (Phortica) huazhii Cheng & Chen, Phortica (Phortica) pseudogigas Zhang & Gan, P. (P.) variegata, and Phortica (Shangrila) hani Zhang & Shi (Amiota (A. femorata) and Portica species (P. (P.) variegata), all 12 Steganinae mitogenomes contained the typical set of 37 genes  and had an expected D-loop region located between srRNA and trnI (Drosophila (Sophophora) yakuba Burla  tanabei to 16,290 bp in P. (A.) longipenis (Drosophila (Hawaiian Drosophila) grimshawi Oldenberg (BK006341) to 19,951 bp in Drosophila (Sophophora) melanogaster Meigen (CM010568). In the principal component analysis (PCA) based on the sizes of genes, PC1 and PC2 explained 45.94% of the total variance  variegata mitogenomes contained a total of 121 bp gene overlaps or IGNs ranging from −1 to 18 bp, whereas they were located in 21 and 19 pairs of adjacent genes, respectively (A. femorata and P. (P.) variegata mitogenomes were located between trnE and trnF. However, we found another 18 bp IGNs between trnS1 and ND1 in the latter  longipenis had 100 bp IGNs between trnI and trnQ. The generation of these long IGNs between tRNAs might be related to the evolutionary divergence of these species.The variation of gene intergenic nucleotides (IGNs) is another important characteristic of the mitogenome. For instance, both the ectively . The lone latter . Accordie latter B. The AmAmiota mitogenomes generally had a higher A + T content, whereas the Phortica mitogenomes generally had a higher G + C content  (Amiota and Phortica were relatively less overlapped in the 2nd (Amiota and the Phortica (reflected by less overlap). Altogether, our results showed unambiguous genus-specific characteristics between and within the Amiota and Phortica from mitochondrial base composition to skewness.The base compositions of the 12 Steganinae mitogenomes were A = 39.1–40.7%, T = 38.1–40.3%, G = 8.1–9.5%, and C = 11.2–13.6% . Among t content . The J-sGC-skews . By estaogenomes , we foun5–0.106) A; (2) th the 2nd D and 3rd the 2nd E, tRNAs  the 2nd G, and D- the 2nd H; and  variegata mitogenomes were putative start codons ATD, except for COX1, which used TCG  variegata mitogenome  variegata mitogenome shared identical termination codons excluding COX2, which also used an incomplete stop codon T  longipenis and P. (A.) tanabei, were both missing three degenerate codons. Only the codon family of Leu1 had codons per thousand codons (CDspT) values over 100 in all of the Steganinae mitogenomes (Amiota mitogenomes (100.51–106.17) than in the Phortica mitogenomes (93.97–99.89)  values of the PCGs, the analysis A. The usanalysis . Overallanalysis . Includianalysis A. The loogenomes . As a hy7–99.89) . PC1 and7–99.89) B and CDs7–99.89) C, respec7–99.89) A, the Phus level B,C. Furtus level B,C. All ω) values of the 13 PCGs were computed for the Amiota and Phortica mitogenomes  of the ATPase subunits (ATP6 and ATP8) was inferior to those of other mitochondrial PCGs (p < 0.001)  indicated that the PCGs evolved under purifying selection [COX1, CYTB, and NAD1 , synonymous substitutions per synonymous site (Ks), and Ka/Ks (ogenomes . In thisogenomes . The genogenomes . In addi< 0.001) . All ω velection . Our resand NAD1 , are bettrnC) to 72 bp (trnV)  stem but held the DHU loop. Although trnSer1 in some other dipteran mitogenomes also did not form complete cloverleaf secondary structures, they generally had neither the DHU stem nor the DHU loop [The typical set of 22 tRNA genes was scattered throughout each circular, double-stranded DNA molecule ranging from 62–64 bp (p (trnV) . The sizectively . All theDHU loop ,14,56.trnL2 and trnV (lrRNA) and between trnV and the D-loop region (srRNA), respectively  of the 12 Steganinae mitogenomes were all located between srRNA and ectively . Furtherogenomes A.trnI in the D-loop sequences. The upstream regions 200–600 bp away from srRNA had only two or three consensus motifs . The phylogenetic relationships within the Phortica were also attached with high confidence  with the exception between P. (P.) huazhii and P. (P.) variegata in the ML analyses   was primarily separated from the clade of the remaining five Amiota species   and A. femorata + A. spinifemorata (the taurusata species group), and between A. yifengi (the basdeni species group) and A. dentata (ungrouped species)  A. The suPhortica . A. nagao acids) A. The tospecies) A.A. nagatai and A. setosa, the topology of all other nodes was the same as in the above analyses  (P. (P.) huazhii and P. (P.) variegata was highly supported by the D-loop sequences   B. In addP = 100) B. The coogenomes .Amiota were significantly greater than those within the Phortica. Therefore, we suggest further studies to investigate the necessity, possibility, and feasibility of establishing subgenera within the Amiota.The D-loop region is inferred to play important roles in mtDNA gene transcription and mitochondrial replication ,14. FurtAmiota, the genus Phortica, the subgenera Phortica, and the Shangrila as a monophyletic group, respectively. The downstream D-loop sequences were also found to be phylogenetically informative and provided a similar topology to the Steganinae as the non-D-loop mitochondrial regions. Overall, these results not only broaden our knowledge of the mitochondrial characteristics of the Steganinae but also improve our understanding of the phylogenetic relationships within the subfamily. In addition, the D-loop sequence variability within these mitogenomes showed unambiguous genus-specific characteristics and provided novel insights into the evolutionary implications between and within the Amiota and Phortica. These newly available mitogenomes will contribute to further species identification, evolutionary biology, and conservation biology, and help to reveal the phylogenetic relationships and evolutionary history of the Drosophilidae.A total of 12 novel mitogenomes belonging to two large genera in the Steganinae were sequenced, assembled, annotated, compared, and analyzed in the present study. Our mitogenomic results strongly supported the Steganinae, the


Nitrogen application contributed to the hardness and stickiness of cooked rice, reducing the texture quality. The amylose content and pasting properties decreased significantly, while the relative crystallinity increased with the increasing nitrogen rates, and the starch granules became smaller with an increase in uneven and pitted surfaces. The proportion of short-chain amylopectin rose, and long-chain amylopectin declined, which increased the external short-range order by 1045/1022 cm−1. These changes in hierarchical structure and grain size, regulated by nitrogen rates, synergistically increased the setback viscosity, gelatinization enthalpy and temperature and reduced the overall viscosity and breakdown viscosity, indicating that gelatinization and pasting properties were the result of the joint action of several factors. All results showed that increasing nitrogen altered the structure and properties of starch, eventually resulting in a deterioration in eating quality and starch functional properties. A moderate reduction in nitrogen application could improve the texture and starch quality of rice while not impacting on the grain yield.The quality and starch properties of rice are significantly affected by nitrogen. The effect of the nitrogen application rate (0, 180, and 230 kg ha Oryza sativa L.) is the only staple food that is consumed mostly in the form of whole grains after cooking [Rice  the composition of the grain and textural properties, (ii) the fine structure of amylopectin, and (iii) the thermal and pasting properties of rice starch. The results will enhance the understanding of rice starch, and how to improve the starch quality and rice quality to conform to the concept of sustainable agriculture by reducing nitrogen application.japonica rice cultivars, Bengal and Shendao 505 (SD505), were cultivated in the field at Xi’an farm in Panjin, Liaoning province, China  during the rice cultivation season in 2020 and 2021. The Liaohe River Delta plain is a major rice growing region where the rice planted takes up 15% of the land in Liaoning. With the advantages of river irrigation and climate, rice produced in Panjin has a better quality and palatability than rice from other places. The soil type is littoral saline paddy soil with 26.4 g kg−1 of organic matter, 82.57 mg kg−1 of available nitrogen, 13.28 mg kg−1 of available phosphorus, 215.83 mg kg−1 of available potassium and a pH of 8.10. The daily temperature and precipitation from transplanting to harvesting are shown in Three replicates of two −1 was applied as basal, striking root, and tillering fertilizers with proportions of 65%, 15%, and 20%, respectively. Phosphorus (90 kg ha−1 as superphosphate) and 50% potassium (110 kg ha−1 as K2SO4) were applied and incorporated as a basal fertilizer before transplanting. The other potassium was applied during the tillering period. Other practices were carried out according to local recommendations. The total nitrogen application of 230 kg ha−1 was the application practice of local farmers.The experiment setup employed a split plot design, with nitrogen application level as the main plot and cultivar as the split plot. Seeds were sown on 18 April 2020 and 20 April 2021 and sequentially transplanted on 25 May 2020 and 26 May 2021, respectively. Three seedlings per hill with a spacing of 30 cm × 18 cm were transplanted. The plot dimensions were 17 m × 3 m. A total nitrogen amount of 0, 180, and 230 kg haThe rice grains were processed using a rice huller, followed by three rounds of grinding into fine flour using a DFT-small-sized flour milling machine . The rice flour was sieved (100-mesh) and stored for further use. −1 enzyme activity Macklin/Macklin Shanghai, Shanghai, China) at a temperature of 42 °C for 24 h. The starch slurry obtained was then sieved through a 200-mesh sieve, and the residue collected on the mesh was mixed with 30 mL of deionized water. After stirring for 2 min, further sieving was carried out to minimize the presence of residue. The filtrates were combined with the initial filtrate, discarding any residue remaining on the mesh. The combined starch slurry filtrates were centrifuged at 4000 g for 20 min. After discarding the supernatant, the faint yellow upper layer was removed, and the remaining precipitate was re-suspended in 20 mL of deionized water. Centrifugation at 4000 g for 20 min was repeated, followed by the removal of the supernatant. To ensure the thorough removal of impurities, the centrifugation process was repeated five times. Subsequently, the starch was dried at 35 °C under ambient pressure and filtered through a 200-mesh sieve. The flour and starch samples were placed into plastic zip-lock bags and stored at 4 °C until further use.Starch isolation was conducted based on the method described by Zhu et al. (2016) with minor modifications. To eliminate protein in the rice flour, 5 g of the sample was steeped in a pH 9–11 aqueous solution (40 mL) of sodium hydroxide containing 0.05% of alkaline protease (200 u g2 site (except border plants) in each plot. The recorded yield values were adjusted to 14.5% moisture content uniformly before statistical analysis. The grain yield was assessed by harvesting all plants within a 3 mThe total starch content (TSC) was measured using a commercial kit  and the AOAC Method 996.11 (AACC Method 76-13.01). The apparent amylose content (AAC) was measured using the iodine colorimetric method . The AACThe polished rice (5 g) was placed into an aluminum cylinder with 7.5 g of water, rinsed after 0.5 min, and then soaked for 30 min. The rice was cooked with an electric cooker by heating it to 100 °C for 30 min followed by 10 min of steaming. The rice was gently stirred to loosen it, and then it was transferred to a cooling device for 30 min with a filter paper cover and kept at room temperature for 90 min. A texture profile analyzer  was employed to measure the texture attributes of the cooked rice using a two-cycle compression, force versus distance program , accordiThe morphology of starch granules was studied by scanning electron microscopy . Rice starch was affixed to specimen stubs and coated with a layer of gold. Microscopic images were captured at an accelerating voltage of 5.0 kV and a magnification of ×2000.The granule size was established using a laser diffraction particle size analyzer  according to a method described previously . The sta−1. The degree of relative crystallinity was calculated by using MDI Jade 6.5 .XRD patterns were scanned using a D8 Advance X-ray diffractometer . The starch samples were analyzed under the following conditions: a current of 40 mA, a voltage of 40 kV, and a diffraction angle (2θ) range of 3–40° with a scanning speed of 4° min−1) by OMNIC software 8.2  over a range from 1200 to 800 cm−1. Intensity measurements at 1045, 1022, and 995 cm−1 were performed on the deconvoluted spectra.The starch was analyzed with a Fourier transform infrared spectrometer  following the method of a previous study . To invew/v) at 37 °C after it had been dissolved in a boiling water bath for 60 min. The branch chain-length distribution of amylopectin was assessed with an ICS-5000 high-performance anion-exchange chromatograph  using a DionexTM CarboPacTM PA100 anion-exchange column  according to the method described by Zhou et al. (2015).The experimental conditions included a flow rate of 0.4 mL min−1, an injection volume of 5 μL, and a solvent system consisting of 0.2 M NaOH and 0.2 M NaAc.Acetate buffer  and isoamylase  were used to debranch the starch  was mixed at a constant stir speed of 160 g using the standard profile. The temperature profile employed was as follows: the start temperature was 50 °C held for 1 min, followed by heating to 95 °C at 11.84 °C/min, held at 95 °C for 2.5 min, and then cooling at the same speed to 50 °C and held for 1 min.The starch pasting properties were tested using a rapid viscosity analyzer (RVA)  according to the method of Lu and Lu . BrieflyThe thermal properties were investigated by a differential scanning calorimetry (DSC) with a Model 200 F3 Maia . Using an empty aluminum pan as a reference, the ratio of the weight of starch to water was 1:3 for each sample, which was sealed in a hermetic aluminum pan and scanned from 30 to 95 °C at a heating rate of 10 °C/min after overnight equilibration at room temperature . The thew/v) then heated in a water bath at 95 °C for 30 min. The sample was cooled, centrifuged at 8000 g for 10 min, the supernatant was discarded, the colloid was weighed (W1), and the sediments were dried to a consistent weight (W2). The swelling power = W1/W2 (g g−1) and the solubility (%) = (1 − W2/W0) × 100%.Swelling power and water solubility were measured according to the method of Zhu et al. . The stap < 0.05) using the data processing system DPS 9.50 . Graphs were produced using Origin 2021 .All the data reported are presented as the average of three replicate observations unless otherwise specified. Duncan’s new multiple range test was used to analyze the variance of the experimental group means  at different nitrogen application levels were significantly different, as shown in −1 from the deconvoluted FTIR spectra were three typical characteristic bands of native starch, which were sensitive to the crystalline regions, amorphous regions, and the bonding in the helical structures in the carbohydrate, respectively [−1 and 995/1022 cm−1 can serve as quantitative indices to determine the degree of short-range molecular order in the external region of the granules [FTIR was used to evaluate the short-range ordered structure in the external region of the starch granules . The absectively ,25 , followed by the nitrogen fertilizer treatment groups N1 (0.72) and N2 (0.73–0.78). The absorbance ratios of 1045/1022 cm−1 and 995/1022 cm−1 increased along with the nitrogen application levels , B1 chains (DP 12–24), B2 chains (DP 24–36), and B3 chains (DP > 36) ,67. The Nitrogen fertilizer significantly decreased the average chain length, by increasing the proportion of short A and B1 chains while decreasing the B2 and B3 chain proportion slightly. This further elevated the (A + B1)/(B2 + B3) ratio, which was found at a high ratio in cereal crop starch . The differences between samples treated with N fertilizer and their controls N1–N0 and N2–N0 show the same trend, which is an increase in the B1 chain (DP 10–20) and a decrease in the proportion of mid to long chains. The change was observed more in N2–N0 than N1–N0. We can speculate that it might be due to the extension of the grain-filling period by the nitrogen treatment; most of the inferior grains developed late with poor plumpness, when the short chain branches had not developed into longer chain branched amylopectin. Short chains (A + B1 chains) form double helices that are involved in crystal formation in the starch granules  in accorThe RVA response curve and characteristic values correctly reflect the gelatinization properties of rice flour under hydrothermal conditions by visually depicting the molecular changes of starch granules during gelatinization and swelling . The pasThe gelatinization temperature (GT) is the minimum temperature for pasting starch, which is related to the stability of starch granules. The higher GT in the N2 treatment may be ascribed to the higher crystallinity structure, external short-range order molecular structure, and double helix structure of starch.Eating and cooking quality was significantly positively correlated with breakdown and negatively correlated with setback viscosity values . High brThe thermal characteristics of starch in different nitrogen fertilizer treatment measured by DSC are illustrated in Swelling power reflects the ability of starch granules to bind to water, and solubility signifies the dissolution of starch molecules in water at a certain temperature. These two parameters define the interaction between starch and water molecules and are responsible for several factors. The solubility of starch in the different treatments of the two varieties ranged from 16.5% to 21.7%, with a swelling range of 15–18.1%, both with an increasing trend with the application of nitrogen. Compared to the N0 treatment, the solubility and swelling of starch in the N2 treatment increased 1.58 and 2.93 percentage points on average, respectively. The highest levels of starch solubility were observed in the N2 treatments with lower amylose content. The granules’ swelling may have been caused by prior water uptake into the amorphous region, which contains a large proportion of amylose. Amylose’s double helix can inhibit the further access of solvents and swelling of granules and, therefore, hinder the hydration of amorphous regions and the entire starch granule ,80,81. IThe Pearson correlation coefficient for starch structure and properties is shown in a plot . As the The gelatinization of starch has been found to be influenced by interactive factors, including granule morphology, amylose/amylopectin ratio, the microstructure of starch and the trace composition of the flour . It has Starch granules swell during gelatinization and pasting. Amylose suppresses the granules’ expansion, and it has been considered as an acknowledged explanation for differences in eating quality. However, it is not that simple, and amylose content alone is insufficient to explain pasting properties. These may be attributed to super long debranched amylopectin, which, like amylose, leaches out and forms a network around the granules, inhibiting further swelling and maintaining the swollen starch granules . The smaIn fact, the structure, size, and fine distribution of starch granules were not separate from each other, but closely related to each other . They cojaponica rice were employed to investigate grain yield and eating quality and their associations with starch structure and properties at three nitrogen application rates. The nitrogen application level significantly affects the composition and structure of starch, regulating the starch properties and texture quality of cooked rice. With nitrogen treatments (N1 and N2) the yield was increased by 49~87%; starch granules’ size decreased, and the surface of the starch granules became uneven and porous. With a greater relative crystallinity and a higher external short-range order, the structure of long branch-chain amylopectin was lower, and the proportion of short branch amylopectin was higher. Gelatinization enthalpy and temperatures rose, and viscosity declined. All of these changes in structure and proportion led to a harder texture and deteriorated the eating and cooking quality. The change in functional properties contributed to multiple factors synergistically and antagonistically in terms of the structure and contents, such as the granule size, fine structure of amylopectin, compositions, and hierarchical structure. Overall, a moderate reduction in nitrogen application improves rice texture and starch quality without affecting grain yield.Two cultivars of