Arterial stiffness, glycemic control and blood pressure are risk factors of macrovascular complications in type 2 diabetes. This study aimed to investigate the combined association of arterial stiffness, glycemic control and hypertension status with the occurrence of diabetic macrovascular complication.A total of 1870 patients of diabetes were enrolled from Beijing Health Management Cohort between 2008 and 2018 as baseline, and then followed for macrovascular complication onset. We proposed a composite risk score (0–4) by arterial stiffness severity, pool glycemic control and hypertension status. Cox model was used to estimate the hazard ratio (HR) and 95% confidence interval (CI).The mean age (SD) of this population was 59.90 (12.29) years. During a median follow-up of 4.0 years, 359 (19.2%) patients developed macrovascular complication. Compared to the normal arterial stiffness and good glycemic control group, patients with severe arterial stiffness and pool glycemic control had the highest risk of macrovascular complications . Similarly, those of severe arterial stiffness and hypertension had the highest risk . Patients of the composite score > 2 had a significantly increased risk of macrovascular complication.This study suggested the clinical importance of combined evaluation of arterial stiffness, glycemic control and hypertension status for the risk stratification and management of macrovascular complication of type 2 diabetes.The online version contains supplementary material available at 10.1186/s12933-022-01696-1. Type 2 diabetes is a serious public health issue worldwide with high morbidity and mortality . DiabetePatients with diabetes had increased arterial stiffness level  and highTherefore, this study aimed to evaluate the combined association of arterial stiffness, glycemic control and hypertension status with the occurrence of diabetic macrovascular complication. We hypothesized that the combined assessment of arterial stiffness, glycemic control and hypertension status could improve the risk stratification and prediction of macrovascular complications in type 2 diabetes.We recruited participants from the Beijing Health Management Cohort (BHMC), which is an open, dynamic and population-based cohort conducted in Beijing, China. BHMC aims to collect and investigate the biomarkers of cardiometabolic diseases including diabetes and its complications. Details of the cohort design have been described in a previous study .The original cohort included 10,632 participants with physical and arterial examinations between 2008 and 2018. Then, participants without type 2 diabetes or with type 1 diabetes (n = 7495) or lacking eligible arterial stiffness data (n = 1038) were excluded at baseline. We further excluded participants with a history of macrovascular complication (n = 126), diabetic nephropathy or retinopathy (n = 71) or missing fasting glucose data (n = 32) at baseline. Finally, a total of 1 870 participants was included and followed until the development of macrovascular complication or December 31 of 2019, which came first , and health-related data (diagnosis history of diseases and medication use) were collected using a standardized questionnaire. Smoking status and drinking status were categorized into "current" and "not current". Physical activity was defined as participating in moderate or intense exercise "≥ 20 min per time and ≥ 4 times per week". Self-reported health conditions included a physician-diagnosed history of cardiovascular diseases, diabetes, hypertension, and dyslipidemia. The use of antidiabetic, antihypertensive, and lipid-lowering medications were collected.2 (m)2. Obesity was defined as BMI ≥ 28 kg/m2 In Chinese adults . Differences were compared using Student's t-test or ANOVA for variables with normal distributions, and Mann–Whitney U test or Kruskal–Wallis test for skewed variables. Categorical variables were described as numbers (proportions) and compared using χWe used two approaches to investigate the combined effect of arterial stiffness, glycemic control and hypertension status. Approach 1: the combined effect of arterial stiffness and glycemic control (or hypertension status) was evaluated separately; approach 2: we created two cumulative or weighted scores combining arterial stiffness, glycemic control and hypertension status. The detailed information was shown in Additional file Survival curves were used to present the cumulative hazard of diabetic macrovascular complications and compared by log-rank tests. Cox frailty models corrected for individual random intercept were used to compare the risk of macrovascular complication between groups. Hazard ratio (HR) with 95% confidence interval (CI) were calculated. We adjusted potential confounders in regression analyses: model 1 was adjusted for age and sex; model 2 was further adjusted for obesity, eGFR, LDL cholesterol, hypertension (if not stratified), glycemic control (if not stratified), current smoking, and physical activity. To visualize the dose–response relationship between baseline baPWV and incident macrovascular complication stratified by glycemic control and hypertension status, we carried out restricted cubic spline analysis using three knots at the 10th, 50th, and 90th percentiles.Multiple sensitivity analyses were performed to test the robustness of the results. First, 62 of 1870 participants had missing data of BMI, and then the main analysis was repeated after completing the multiple imputation using Markov Chain Monte Carlo. Second, poor glycemic control was alternatively defined as fasting glucose ≥ 5.6 mmol/L and hypertension was alternatively defined as SBP ≥ 130 mmHg or DBP ≥ 80 mmHg or use of any antihypertensive medication or self-reported diagnosis history of hypertension. All statistical analyses were performed using R software (version 4.1.1), and a two-sided significance level of P value < 0.05 was considered statistically significant.A total of 1 870 individuals with diabetes were included in this study, and 1227 (81.2%) were men. The mean age (SD) of the population was 59.90 (12.29) years. During a median follow-up of 4.0 years, 359 (19.2%) patients developed macrovascular complication. Table Figure Compared with the good glycemic control/normal arterial stiffness group, participants of poor glycemic control/severe arterial stiffness had the highest risk of macrovascular complication . The adjusted HR for good glycemic control/severe arterial stiffness group was 2.24 (95% CI: 1.17–4.30); and 1.98 (95% CI: 1.08–3.61) for poor glycemic control/moderate arterial stiffness group. No significant association was found for the poor glycemic control/normal arterial stiffness and good glycemic control/moderate arterial stiffness groups  of the population had measurements of HbA1c. Fasting blood glucose was used as a measure of glycemic control. Second, we used baPWV as the index of arterial stiffness instead of carotid-femoral pulse wave velocity (cfPWV). We can not compare the difference between these two indices. Nevertheless, studies have confirmed that baPWV correlates closely with cfPWV and has been recommended for assessing arterial stiffness by American Heart Association (AHA). Of note, there are novel techniques to estimate the arterial stiffness in addition to tonometry-derived measures, such as Doppler-derived aortic arch PWV (aa-PWV), which need investigation in further research among diabetic population . Third, In summary, this longitudinal cohort study indicated that the combined evaluation of arterial stiffness, glycemic control and hypertension status could improve the risk stratification of macrovascular complication among diabetic population, which provided novel insights into the preventive strategies against macrovascular complication from the perspective of combined intervention of arterial function and glucose metabolism.Additional file 1. Additional Methods. Table S1. Baseline characteristics of participants according to different arterial stiffness and glycemic control status. Table S2. Baseline characteristics of participants according to different arterial stiffness and hypertension status. Table S3. Association of separate arterial stiffness, glycemic control and hypertension status with the development of macrovascular complication. Table S4. Association of arterial stiffness and glycemic control/hypertension status with macrovascular complications adjusting blood pressure and fasting glucose as continuous variables. Table S5. Association of arterial stiffness and alternatively defined glycemic control status with macrovascular complications. Table S6. Association of arterial stiffness and alternatively defined hypertension status with macrovascular complications. Table S7. Association of per-SD increase of baPWV with diabetic macrovascular complication stratified by glycemic control and hypertension. Table S8. The composition of arterial stiffness, glycemic control and hypertension in each scored population. Table S9. Association of weighted score with diabetic macrovascular complications. Figure S1. Flowchart of this current study. Figure S2. Association of arterial stiffness, glycemic control and hypertension status with development of macrovascular complication in imputed data.


When we decided to write the final editorial for this year's BJORL, we recalled the editorials of 2013 and reviewed what our friends wrote celebrating the journal's 80th birthday. Dr. Sady's editorialWhere did we come from, what has been done, where are we going? Conceived and founded in 1933 by Drs. Otoni de Rezende and Homero Cordeiro, the Oto-Laryngological Journal of São Paulo began as a printed vehicle of the academic discussions of the Medical Society of São Paulo and later, bearing the name of Brazilian Journal of Otorhinolaryngology, became the vehicle for the publication of specific articles for otorhinolaryngologists all over Brazil. In the 1960s, with the institutionalization of postgraduation courses by the Brazilian Ministry of Education and Culture (MEC), the government agencies that managed these postgraduation courses, such as CAPES and CNPq, started to demand that the resulting studies and researches be published in journals of international scope, that were indexed and had a certain impact factor (IF), so that they were awarded grades in a national ranking and thus could secure public funding for research in the courses and grants for the researchers. This was an essential condition for the promotion of research and, at the same time was a call for a action for the scientific journals that wished to survive in Brazil.6SciELO, Lilacs and Experta Medica. In 2005 we were in PubMed and Medline, when we started using an English title next to the title in Portuguese. In 2011 we were indexed by the Institute for Scientific Information (ISI). We are currently in the databases for bibliographic consultation of these international indexers.Even though these demands were debatable and criticized, their adoption became a milestone for the journal's trajectory. After their implementation there was an improvement in the quality of the journal and the published articles. A result of the outstanding efforts of the Boards that came after that, the journal was gradually indexed by in natura to the main universities abroad. Thus, in 2009 the Brazilian Journal of Otorhinolaryngology – BJORL (registered in ISSN) was born. Currently we are the most important journal in our specialty in Latin America. As for our presence abroad, it has been well established, and we reached a privileged position that was unthinkable only decades ago. Today our Impact Factor (IF) is 1.412, and only those who know about the difficulties in reaching this small index assigned by ISI (Institute for Scientific Information) are able to measure its importance. We occupy, according to the Journal Citation Reports, the 25th position among the journals of the specialty in the world. We have an ascending history, and that is undeniable.In parallel to the difficult indexing process, the editors’ perseverance was further impelled by necessary structural changes throughout the entire journal creation process. The main one was the creation of an original publishing platform, that is also currently used by other journals in the country. All issues prior to the journal's indexing have been digitized and are now available for search on the journal's homepage. Among other effects, the advent of informatics and the “wonders” of the internet resulted in an increased number of submissions and in a faster evaluation and approval of articles increasing the journal's exposure abroad. In turn, this last effect brought with it the need for the journal to be published in English, so that it could be distributed However, striving to reach the same levels of excellence as the American and European journals is not compatible with complacence. Our responsibility increases as we face the challenges over the next few years, from the changes and transience of government policies to the training/qualification of our specialists in several courses in this continental country. As Dr. SadyFinally, although we now have other paths and other concerns, we also maintain the same goal as 85 years ago: to make BJORL a reference vehicle, both national and international. History is made, with long strides. It guides us about the starting point, not the point of arrival. The latter will be clarified by the temporal imperatives of the future. Perhaps we are out of our teen years and are now at the age of maturity. Other decades will come. Definitely.The authors declare no conflicts of interest.


The purpose of this study was to determine if the intracecal injection of alkaline solution 2 + Al(OH)3) could stabilize fecal microbiota and clinical changes in horses submitted to starch overload. Clinical signs, gross analysis of the feces, and fecal microbiota were evaluated for 72 h  from ten crossbred horses. Horses were allocated to group I  and group II . Starch overload reduced the richness and diversity of the fecal microbiota. However, the starch–buffer treatment led to a greater increase in amylolytic bacteria and decrease in fibrolytic bacteria than did the starch–saline treatment. This study showed that cecal infusion of buffer did not prevent the intestinal disturbances and their consequences.2 + Al(OH)3) could stabilize these microbiota and clinical changes in horses submitted to corn starch overload. Ten crossbred horses  were allocated to group I  and group II . Clinical signs, gross analysis of the feces, and fecal microbiota were evaluated through 72 h . Corn starch or water were administrated by nasogastric tube at T0, and the buffer injected into the cecum at T8 in starch–buffer and water–buffer treatments. Starch overload reduced the richness (p < 0.001) and diversity (p = 0.001) of the fecal microbiota. However, the starch–buffer treatment showed greater increase in amylolytic bacteria  and decrease in fibrolytic bacteria  than starch–saline treatment. Additionally, animals that received starch–buffer treatment showed more signs of abdominal discomfort and lameness associated with dysbiosis , showing that cecal infusion of buffer did not prevent, but intensified intestinal disturbances and the risk of laminitis.Starch overload in horses causes gastrointestinal and metabolic disorders that are associated with microbiota changes. Therefore, we identified the fecal microbiota and hypothesized that intracecal injection of alkaline solution  The large intestine of horses is an anaerobic fermentation chamber filled with diverse microorganisms  that plaDiet can negatively modulate the intestinal microbiome ,12 since2 + Al(OH)3) have been reported to have therapeutic value in humans and milk-fed calves as antiacid substances to increase the gastrointestinal pH [2 + Al(OH)3 increases pH and water influx into the lumen, pushing the impacted compound through the hindgut until it is eliminated [A low pH in the gut lumen alters the microbiota due to lactate accumulation, which can induce subclinical acidosis . The deatinal pH ,22,23. Itinal pH  and largtinal pH . As an aiminated .2 + Al(OH)3 solution in animals have been conducted to evaluate its effects in gastrointestinal pH alterations [2 + Al(OH)3 on intestinal dysbiosis following starch overload in horses has not been evaluated yet. Therefore, we identified the fecal microbiota and hypothesized that it, and consequently, clinical changes induced by the corn starch overload, could be stabilized by the intracecal injection of this alkaline solution.Most studies related to the Mg(OH)erations ,24,27. M®—Agromix—Jaboticabal, São Paulo, Brazil), 4 kg/day of coast cross (Cynodon dactylon) hay, and free access to fresh water and mineral salt. Seven days after the catheterization, the animals were restricted to the stalls, and the volume of hay was increased to 8 kg/day, with a period of adaptation of two weeks to start the study.This study was approved and supervised by the committee of animal use and care of the São Paulo State University (23391/15). We used 10 crossbred horses (geldings and mares), aged 13 ± 5.6 years and weighing 353 ± 28 kg. Horses were considered healthy based on physical examination and hematology. All horses were submitted to typhlopexy in the right flank [The animals were randomly distributed into two groups (I and II) of five horses. Each group was submitted to two different treatments , evaluation of signs of lameness and abdominal pain, water and food intake, and fecal production were evaluated at T0 to T72. In addition, hydration status was estimated by evaluating the oral mucosa characteristics and skin tenting. Feces production was subjectively evaluated for consistency as firm , pasty (cow pie consistency), or liquid (diarrhea) . We alsoFollowing clinical evaluation, fecal samples were collected via rectal ampulla with gloved hands at each time point, placed into sterile tubes and stored at −18 °C until processing. Bacterial DNA was extracted as previously described .® PowerSoil® DNA Isolation Kit , according to the manufacturer’s protocols. DNA integrity was checked in 1% agarose gels, with 5 µL of metagenomic DNA and 3 µL of carrying buffer  submitted to horizontal electrophoresis with 1X TBE buffer , and stained by sybr gold  at 260 nm and 280 nm, with the 260/280 ratio being used to estimate DNA purity. DNA concentration was determined using a Qubit® 2.0 fluorometer  and a Qubit™ DNA BR Assay Kit , following the manufacturer’s recommendations.Microbial DNA was extracted using the PowerLyzer®  and quantitative PCR, as instructed by the manufacturer. Purified and quantified libraries were pooled in equimolar quantities and sequenced (2 × 250 bp) using the MiSeq® reagent kit v2 (500 cycles), according to the manufacturer’s protocols using the Illumina platform.After extraction, the V4–V5 regions of the bacterial 16S rRNA were amplified using polymerase chain reaction . The primers 515F (5′-GTGCCAGCMGCCGGCCTAA-3′) and 926R (5′-GGACTACHVGGGTWTCTAAT-3′)  were usep < 0.05 for all analyses.Data were submitted to bioinformatic analysis using the Quantitative Insights into Microbial Ecology (QIIME) package, version 1.9.1 . SequencAnimals that received WS  and WB (water–buffer) treatments did not show behavioral and systemic changes. On the other hand, those that received SB (starch–buffer) and SS  treatments showed increased heart rate starting at T8 and T12 up to T72, respectively. In addition, rectal temperature spiked after starch overload, between T8 and T48 .Signs of gastrointestinal discomfort, characterized by diarrhea, were observed in all animals that received SB treatment. Also, 12 to 48 h following starch overload, fecal aspect drastically changed from normal consistency, smell, and color to a soft (pasty) consistency, fetid smell, and white color . Horses Forty-eight hours after starch overload, two animals in the SS treatment group presented Obel grades 2 and 3 lameness, characterized by reluctance to move, as the number of steps increased (from 8 to 10 steps). Horses in the SB treatment group increased the number of steps (from 10 to 14 steps) from T24 onwards. One animal refused to move, while the other three showed slowness and discomfort during the steps, characteristic of Obel grades 2, 3, and 4 .p = 0.001) and richness (p < 0.001) of fecal microbiota were observed in animals exposed to starch. These changes occurred from T12–T72, and between T24 and T72 in animals from the SB and SS treatment groups, respectively  and Actinobacteria (p = 0.011), starting at T48; and the Firmicutes (p < 0.001) phylum increased between T12 and T24. However, the abundance of Spirochaetes (p < 0.001) and Euryarcheota (p = 0.006) phyla was decreased at T12–T72 and T48–T72, respectively. The samples from SS showed prevalence of Proteobacteria (p = 0.05) between 24 and 48 h, and Firmicutes (p < 0.001) at 48 h, after starch overload (Firmicutes (42.0%), Bacteroidetes (3.7%), Euryarchaeota (3.2%), Actinobacteria (2.6%), Spirochaetes (2.2%), and Proteobacteria (1.1%) phyla were prevalent for all treatments. The SB treatment data presented increased relative abundance of Bacteroides (overload .Streptococcus genus (12.2%). The most prevalent subcluster of the Lachnospiraceae family (9.1%) was Clostridium XIVa (4.9%), followed by a non-identified genus (2.1%). Another non-identified and more prevalent genus (4.5%) of the Ruminococcaceae family (9.7%) was also detected. Clostridium III (1.8%) and Ruminococcus (1.3%) comprised the other two more abundant genera. Just one genus predominated in the families Lactobacillaceae (Lactobacillus—3.8%), Prevotellaceae (Prevotella—2.9%), Methanobacteriacae (Methanobrevibacter—2.9%), Spirochaetaceae (Treponema—2.2%), Eubacteriaceae (Eubacterium—1.2%), Bifidobacteriaceae (Bifidobacterium—1.9%), and Erysipelotrichaceae (non-identified genus—1.6%). The families’ relative abundance variations were also observed following starch overload and/or buffer administration relative to their respective genera , specifically, the e genera .Bifidobacterium (p = 0.021), Prevotella (p = 0.003) and a non-identified genus from the Erysipelotrichaceae (p < 0.001) family. The Lactobacillus (p = 0.001) genus increased only at T24, while Streptococcus (p = 0.001) increased at T12–T24. On the other hand, Clostridium XVIa (p < 0.001), Eubacterium (p < 0.001), Ruminococcus (p = 0.05), Clostridium III (p < 0.001), and a non-identified genus of the Ruminococcaceae (p = 0.001) and Lachnospiraciaea (p = 0.002) families showed decreased relative abundance between T12 and T72, which was also observed for Treponema (p < 0.001) and Methanobrevibacter (p = 0.003) genera at T24–T72 and T48–T72, respectively. Samples from SS animals showed an increase in the Lactobacillus (p = 0.001) genus at T24, and a decrease in abundance of Ruminococcus , Clostridium III (p < 0.001), and Eubacterium (p < 0.001) genera  and Proteobacteria  phyla and the clinical signs of diarrhea, inappetence, and dehydration  and positive correlation between the prevalence of the Lactobacillaceae family, its genus (Lactobacillus), and clinical signs of diarrhea and inappetence was revealed. The Eubacteriaceae family and its genus (Eubacterium) correlated negatively with diarrhea and dehydration , as well as did the Clostridium III genera. This one also had a negative  correlation with number of steps  correlation between the Obel grade (2 to 4) and an increase in the abundance of the Actinobacteria phylum. This abundance also had a moderate and positive  correlation with heart rate, decubitus and dehydration. There was also a positive  correlation between diarrhea, dehydration, and the increase in Firmicutes abundance. A negative  correlation occurred between the prevalence of Bacteroidetes phylum and dehydration; and between the decrease of Spirochaetes phylum and heart rate . For these and the Lachnospiraceae family, as well as their respective genera , a negative correlation was observed between their prevalence and most of the clinical alterations. Moreover, the decrease in the Spirochaetaceae family and its genus (Treponema) had a negative correlation  with heart rate. On the other hand, Lactobacillus and Streptococcus, the most representative genera of the Lactobacillaceae and Streptococcaceae families, respectively, correlated moderately and positively  with diarrhea. In addition, the Bifidobacteriaceae family, its genus (Bifidobacterium), and a non-identified genus of the Erysipelotrichaceae family, in addition to a moderate correlation with heart rate , showed a strong  and positive correlation with the increase in Obel grade and the number of steps 3) solution could have prevented or minimized the microbiota changes and clinical signs of starch overload in horses from the SB treatment group, which did not happen. This buffer has been reported to treat large bowel impaction in horses by oral administration [2 + Al(OH)3 solution is administered by nasogastric intubation, 20% of the magnesium is absorbed in the small intestine [2 + Al(OH)3 intracecally, indicating the need for complementary research involving the measurement of magnesium ions to confirm these hypotheses and to describe all the effects of the activity of magnesium salts in the horses’ intestinal tracts.We hypothesized that buffer (Mg(OH)stration ,59. Its stration . On the stration ,60. Althstration  and buffstration . It is wstration ,61, therntestine ,63, whicMost changes in fecal microbiota lasted 48h following starch overload, similar to the signs of diarrhea, mainly in SS treatment. The constant supply of fiber in the diet (hay) throughout the experiment undoubtedly helped, since dietary fiber increases intestinal and fecal pH by buffering fermentation products in the large intestine ,64. In sThe individual vulnerabilities to intestinal and systemic diseases of horses submitted to different managements  vary widely ,17. We n2 + Al(OH)3 solution. On the contrary, the association between this buffer and the corn starch overload intensified microbiota changes and systemic effects, likely due to the cathartic effect of the buffer and the drop in intestinal pH caused by starch overload. These actions lead to deep reduction of some members of the Ruminococcaceae and Lachnospiraceae families, which are essential for gut health, and an increase in amylolytic bacteria, mainly Lactobacillus, Streptococcus, and Bifidobacterium genera. Moreover, dysbiosis was related to gastrointestinal disorders, such as diarrhea, and the onset of systemic effects that can trigger laminitis syndrome, such as hyperthermia, inappetence, high heart rate, and lameness.We conclude that the changes in the fecal microbiota and, consequently, the clinical signs were not stabilized or minimized by the intracecal Mg(OH)


To investigate the effects of repeated dry heat treatment (RDH) and continuous dry heat treatment (CDH) on the structure and physicochemical and digestive properties of potato starch, potato starch was treated continuously and repeatedly at 130 °C for 3–18 h. The results showed that the crystalline form of starch was consistent with the original type B. Still, its physicochemical properties, such as swelling power, transparency, peak viscosity (PV), final viscosity (FV), breakdown (BD) and thermal properties , tended to decrease. At the same time, solubility and RS increased after dry heat treatment. Moreover, RDH-treated starches were higher than CDH-treated ones in terms of molecular weight, crystallinity, swelling power, transparency and final viscosity for the same treatment time. Still, there was no significant difference between the thermal properties of the two. Meanwhile, the resistant starch (RS) content showed a downward trend after the peak value of 9 h of CDH treatment and five cycles of RDH treatment with increasing treatment time and the number of cycles, indicating a decrease in the overall digestibility of the starch. Overall, RDH had a more significant effect on potato starch’s structure and physicochemical properties than CDH. Solanum tuberosum L.) is the fourth largest food crop in the world after wheat, rice and corn [Starch is an abundant natural carbohydrate in our living diet and is present in various food sources, such as grains, tubers, and beans . Starch and corn , where sand corn . As a reand corn . Howeverand corn . Consequand corn . Hence, o, Tp and Tc) and enthalpy (ΔH) value decreased, indicating that the amylose was destroyed. Additionally, the binding force of the crystal was impaired during DHT, thereby improving the starch gelatinization properties.Dry heat treatment (DHT) is a physical modification approach where simplicity, safety and environmental friendliness are the main features . Starch o, Tp, Tc, ΔT) of RDH starch samples were less compared to CDH starch. Zhang et al. [DHT includes continuous dry heat (CDH) treatment and repeated dry heat (RDH) treatment. Most researchers have mainly focused on the study of CDH of starch. Nevertheless, the interaction between starch molecules easily reaches the equilibrium point in the CDH treatment, limiting to a certain extent the modification of starch properties . Howeverg et al.  found thPotato starch was obtained from the Aladdin reagent company. It contains 27.38% amylose, 0.13% crude protein, 0.38% fat and 0.23% ash. 8-Hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (ABTS), Glycosylase (2500 U/mL) and Porcine Pancreatic Alpha-Amylase (USP grade) were provided by Aladdin Bio-chem Technology Co., LTD., Shanghai, China. All other reagents were of analytical grade, and purchased from Sanli Chemical Reagent, Yangling, China.The starch was dried in a 45 °C oven for 13 h until the starch moisture content was lower than 10% and then passed through a 100 mesh sieve. For RDH treatment, the starch was placed in the oven at 130 °C for 3 h, cooled naturally to room temperature 25 °C, making the cooling rate 3.5 °C/min and passed the 100 mesh sieve again; that was, the starch with RDH cycle was obtained (RDH-1). The starch RDH-2, RDH-3, RDH-4, RDH-5 and RDH-6 were labeled after repeated treatment 2 to 6 times. For the CDH treatment, the samples were placed in an oven at 130 °C for 3, 6, 9, 12, 15, and 18 h, cooled naturally to room temperature 25 °C, making the cooling rate 3.5 °C/min and passed the 100 mesh sieve again. The starch samples were labeled CDH-3, CDH-6, CDH-9, CDH-12, CDH-15, and CDH-18. Among them, CDH-3 and RDH-1 are treated similarly, so RDH-1 is used to represent them uniformly.Starch samples were uniformly glued to a conductive adhesive covered with thin gold and carbon under vacuum conditions. The morphology and structure of starch samples were observed by a scanning electron microscope under a 5 kV acceleration voltage.g for 5 min. The residue was washed 5 times with 1 mL of distilled water, then suspended in a 20 μL solution of glycerol-water  and observed by CLSM.The starch samples (2 mg) were spread in a mixture of 4 μL 8-Aminopyrene-1,3,6-trisulfonic acid, trisodium salt (APTS) and 4 μL 1 M cyanoborohydride. The supernatant was removed after a constant temperature water bath at 30 °C for 15 h and centrifuged at 1500× 3 (1 mL) solution were mixed evenly and heated in a boiling water bath for 5 min to dissolve completely. After cooling, the starch was filtered through a 0.22 μm aqueous phase filter. In addition, 20 μL of the liquid was purified through a 0.22 μm aqueous filter for determination by high-performance liquid chromatography.The molecular weight distribution of starch samples was determined by High-Performance Liquid Chromatography. The starch sample (20 mg) and 0.1 mol/L NaNO3 solution and the flow rate was 0.60 mL/min. Dextran standard products with different molecular weights  were determined. The standard curve was:According to Wang et al. , the molThe diffraction pattern of starch crystals was measured by an X-ray diffractometer using CuKα as the radiation source. The scanning range of 2θ from 4° to 60°, the scanning speed of 6°/min and the step size of 0.02 were the operating conditions for X-ray diffraction analysis.g for 15 min, the supernatant was dried at 105 °C to a constant weight. The formula is as follows:The starch sample (0.6 g) was spiked into distilled water to prepare a solution with a mass fraction of 2%, and the solution was bathed at 50, 60, 70, 80, and 90 °C for 30 min, respectively. After cooling and centrifuging at 1500× A starch suspension (1%) was made by adding 0.6 g of starch sample to distilled water and heating it in a boiling water bath until it was wholly pasted. Then, the starch paste was heated for 30 min and cooled to room temperature, and the transmittance was measured at 0, 24, 48, 72, 96 and 120 h using a UV-Vis spectrophotometer at 620 nm.A Rapid Viscosity Analyzer was used to measure the pasting properties of starch. Then, according to the moisture content by the sample calculator, we calculated the amount of starch and water required. A specific mass of starch and 25 mL of distilled water were dispersed in an RVA aluminum canister. After shaking well, starch samples were put into the instrument and pressed on the column lid to start the measurement cycle program. The starch solution was balanced at 50 °C, held for 1 min, and the descending starch was heated to 95 °C to keep it at a rate of 12 °C/min. The starch samples were kept at 95 °C for 2.5 min before cooling to 50 °C and kept at 50 °C for 2 min at the same rate. The speed was 160 rpm during the measurement.The thermodynamic properties of starch were determined by differential scanning calorimetry. The starch samples weighed in the aluminum tray were mixed in 9 μL deionized water for sealing and equilibrating at 4 °C for 12 h. Then, the starch samples were placed in a DSC analysis that was heated from 20 °C to 120 °C at a rate of 10 °C/min.20 and G120 are the contents of glucose released within 20 and 120 min of hydrolysis, respectively.In vitro starch digestibility was determined according to Englyst et al. . The metp < 0.05). Origin Lab (Origin Pro 8.5) software was used to plot our results.All statistical analyses were performed using SPSS  software. Data were analyzed with analysis of variance (ANOVA), which allows for testing relationships between variables. All measurements were repeated 3 times, and differences between the mean values of samples were checked for significance , attaining a minimum value at RDH-4 and CDH-12, respectively, and then inclined, but still smaller than that of native starch. The decline in the molecular weight of potato starch may be related to the cleavage of straight and straight chains in starch by DHT, which shortened the molecular chain and led to a smaller molecular weight. The subsequent increase in Mw may be caused by the rearrangement of amylose and amylopectin, increasing the degree of starch ordering.According to Overall, the Mw of peak I was significantly greater for starch with RDH than for CDH-treated, a result that followed the same trend as for mung bean starch with CDH, probably owing to the more significant disruption of amylopectin by RDH, which reorganized the damaged starch . In addiThe X-ray diffraction pattern indicates an ordered phase mainly determined by the arrangement of amylose . Potato Compared with native starch, RDH and CDH treatments increased the crystallinity of starch in an overall trend, leading to a better microcrystalline structure and a more orderly rearrangement of starch molecules . Liang eThe solubility and swelling power (SP) of potato starch and DHT starch at different temperatures can be seen in Potato native starch has an excellent expansion force. The main reason is that the granule and crystalline area have ample space, and the phosphate group charges are constantly repelling each other, making the starch granules expand continuously. Therefore, under similar processing, the SP of native starch and DHT-treated starch went up with the increase in temperature. At 50 °C, the starch samples’ SP was small and did not begin to gelatinize; therefore, there is not much difference between native and DHT starch. Still, at 60–90 °C, the starch was gelatinized entirely, and the SP increased. Furthermore, the SP of CDH- and RDH-treated starches reduced with increasing treatment time and cycles at the same temperature (except 50 °C). This behavior is ascribed to the realignment and recombination of starch chains, reducing hydrotropic capacity and limiting swelling forces . MeanwhiLight transmittance indicates the transparency of starch paste, reflecting the retrogradation process of starch . It can The light transmittance of starch treated with RDH or CDH decreased simultaneously and increased slightly after reaching the minimum value when treated with CDH-12 and RDH-5. The decrease in light transmittance may be due to the leaching of amylose by the breakdown of starch granules under heating . ConversThe pasting features of potato native starch and DHT starch were measured by RVA. PV stands for peak viscosity, TV for trough viscosity, FV for final viscosity, BD for breakdown, SB for the setback, PT for peak time, and GT for pasting temperature.For the changes in PV, FV and BD, DHT altered potato starch consistently and less than native starch, and these values dropped off with increasing treatment time and cycles . When LiThe PV, TV, FV, BD, and SB of RDH-treated starch were more than those of CDH for the same treatment time. The study reported that PT, TV and TV of sweet potato starch were the same as potato starch under RDH and CDH treatment. This indicates that CDH treatment increases the intramolecular bonding force more strongly than RDH, making starch paste less viscous . CompareDSC is a thermal analysis method that refers to the thermal energy transformations that happen when the hydrogen bonds in the gelatinized starch molecules are broken under the heating of excess water . The theo) and peak temperature (Tp) than native starch after DHT. Similar results were also reported for high-amylose rice starch [o, Tp and Tc demonstrate the integrity of the starch double helix sequence [o is the temperature at which the lowest crystalline structure melts. Tc is the highest-temperature crystalline structure that melts [o, Tp, Tc, and ΔH may be because the molecular chain of starch was broken, and the amorphous region was destroyed during DHT, while moisture reached the interior of starch more conveniently [o, Tp, Tc) of potato starch showed an increasing trend. The increase of To, Tp and Tc values indicated that DHT could strengthen the order of the crystal structure and the thermal stability of starch was, to some extent, improved. Gou et al. [As seen in e starch , tapiocae starch  and glute starch . To, Tp sequence , where Tat melts . The redeniently . As treau et al.  had simiu et al. ,44. SimuFurthermore, the content of RS was slightly higher than potato native starch, among which RS exhibited an increasing trend before CDH-9 (79.73%) and RDH-5 (83.21%). The higher RS is due to the enhanced interactivity between the microcrystalline structure of starch and starch chains, inhibiting the entry of enzymes into the interior to hydrolyze it. In addition, the SDS content increased slightly after decreasing to a minimum of 0.3% and 0.64% with the number of treatment times and cycling (except CDH-12). The reorganization of starch molecular chains to improve the crystal structure may lead to a decrease in SDS content , while tThe significant effects of RDH and CDH treatments on potato starch structure and physicochemical and digestive properties were investigated. The structural properties of starch, such as morphology and crystalline shape, remain the same. The crystal form was still the original B type during the processing. However, DHT changed other properties of the starch. It not only reduced both swelling power and light transmittance of the starch lower than the native starch, but also increased solubility and descended with temperature. PV, FV, BD and thermal properties  decreased during the treatment, indicating that the DHT could improve the thermal stability of the starch. Besides, RDH-treated starch exhibited higher transparency, RS, and peak viscosity than CDH-treated starch under the same treatment time. In a word, RDH is a dry heat treatment performed again after starch reaches equilibrium with the external environmental conditions, which significantly impacts the starch. These results are expected to contribute to the widespread use of dry heat-modified starches in food processing.


Lifetime survival of the kidney graft and normal life expectancy of the recipient have always been the ultimate goals of kidney transplantation. To survive for several decades, the kidney graft must overcome multiple potential threats, including immune-mediated cell damage, ischemia/reperfusion injury-induced cell senescence and fibrosis, suboptimal nephron mass of kidneys obtained from marginal donors, primary kidney disease recurrence; chronic or recurrent graft infections, and drug nephrotoxicity. The antirejection treatment, which protects against alloimmune-mediated injury, may do so at the expense of the patient’s life expectancy. By inhibiting the immune response, anti-rejection drugs increase the risk and severity of infections and cancer. Moreover, some anti-rejection drugs can cause hypertension and severe metabolic complications such as diabetes mellitus, obesity, and hyperlipemia that augment the cardiovascular risk. Cardiovascular abnormalities developed during long exposure to dialysis , patient frailty at time of transplantation, and the effect of long-standing previous immunosuppressive regimens used for the treatment of the primary kidney disease, may also impair long term patient survival, despite a successful transplant procedure.Regardless of these threats, there exists a subset of kidney transplant recipients whose grafts survived for numerous years, formerly represented by young patients who recieved an azathioprine-based immunesuppressive regimen alongside kidneys from donors who died as a result of traffic accidents. Over time this population has been enriched by older patients who received standard calcineurin-based immunosuppressive regimes alongside kidneys from marginal and after-circulatory-death donors (DCD). Understanding the unique characteristics of this population may provide critical information for the management of this population.The determinants and complications that may adversely affect ultra-long term clinical outcomes are highly heterogeneous. Some of those patients might have survived for decades without developing any relevant risk factor or complication. In contrast, others have developed relevant risk factors or complications , but did so too recently with respect to the start of follow-up, therefore the effect on transplant loss could not be yet detected. Some of the risk factors and complications may be still amenable to intervention even decades after transplantation. The benefit to risk ratio of therapeutic intervention at these later stages, such as immunosuppressive treatment for chronic antibody mediated rejection, should consider that decades of exposure to immunosuppression increase the susceptibility to infection and cancer, and that substantial nephron loss unavoidably takes place over decades after transplantation, irrespective of the ongoing alloimmune mediate injury, which leaves little margin for graft function recovery./year) and who were then compared with the remaining patients. As expected, superior graft function was associated with less exposure to pre-transplant dialysis, better graft quality , lower rates of T cell mediated rejection, and primary kidney disease not encompassing glomerulonephritis, which included diseases that can recur very late post-transplantation, such as IgA nephropathy (In this issue of the journal, Reimann et al.  report thropathy . After 1hropathy . Surprishropathy , did nothropathy ,10. LikeGiven the growth of the ultra-long survivor population and its heterogeneous profile, the study from Reimann et al.  provides


Although EMS and ERS had degraded molecular structures, their particle morphology changed from looser polyhedral to more compact with less enzymolysis channels due to the rearrangement of side chain clusters of amylopectin, leading to enzyme resistance. The starch characteristics of IEMS-treated samples significantly differed. EPS had the highest amylose content, medium chains, long chains, and molecular weight but lowest GI, relative crystallinity, and k. ERS showed the opposite results. Thus, IEMS may affect starches with different GIs to varying degrees. In this investigation, we provide a basis for wider applications of conventional crop starch in the food industry corresponding to different nutrition audience.Nowadays, the highly effective modified technology to starch with various digestibility is gaining interest in food science. Here, the interactions between glycemic release characteristics and fine supramolecular structure of cassava (ECS), potato (EPS), jackfruit seed (EJFSS), maize (EMS), wheat (EWS), and rice starches (ERS) prepared with improved extrusion modification technology (IEMS) were investigated. The crystalline structures of all extruded cooking starches changed from the A-type to V-type. IEMS-treated cassava, potato, and rice starches had broken α-1.6-glycosidic amylopectin (long chains). The others sheared α-1.4-glycosidic amylopectin. The molecular weight, medium and long chain counts, and relative crystallinity decreased, whereas the number of amylopectin short chains increased. The glycemic index (GI) and digestive speed rate constant ( Furthermore, native starches have a high weight-average molar mass (Mw), high amylopectin long-chain distribution, and high relative crystallinity (Rc), leading to a slow glycemic release. And it is difficult to maintain the necessary nutrition of the human body  and cassava, rice, wheat, potato, and maize starches were extracted and used to measure the molecular weight distribution, branching chain distribution of amylopectin, crystallization characteristics, amylose content, digestive kinetics, and estimated glycemic index after IEMS treatment. The possibly distinct Fruits of the Malaysia 1 jackfruit cultivar were collected in 2019 from the Xinglong Tropical Botanical plantation  and assigned voucher number 202009. Cassava, maize, potato, wheat, and rice were purchased from a local market .g, 5 min) to remove residual brown impurities. After repeated washing with distilled water, the sediment was collected. The centrifuged sediments were repeatedly cleaned and collected. The resulting wet starch was dried under vacuum (50°C) until the moisture content was lower than 13 g/100 g. After passing through a 200-mesh sieve, the dry starch was stored in a vacuum dryer until use  for 2 min in a colloid grinder. Then pH of starch slurry was adjusted to 7.0, mixed with neutral protease solution (0.015% w/w) , and transferred into a constant-temperature shaking bed . The mixed solution was filtered through a filter cloth (200 mesh) and centrifuged , and the supernatant was collected to determine the glucose content using a glucose oxidase-peroxidase method (Megazyme) and a spectrophotometer  at 510 nm. The RDS, SDS and RS contents in extrusion-cooked starches were calculated as follows:The proportions of resistant starch (RS), slowly digestible starch (SDS), and rapidly digestible starch (RDS) were determined as described by Englyst et al.  with sli20 (%) is the released content of glucose within 20 min, G120 (%) is the released content of glucose at 120 min, GF (%) is the free glucose content, and TS (%) is the total starch content.where Gin vitro digestibility kinetics of starch was determined as described by Goñi et al. , 25 is the dilution factor, and glucose concentration within t min was defined as GC∞) and speed rate constant (k) (h−1) were obtained from the enzyme hydrolysis curves, and the first-order formulas were as follows:The equilibrium concentration .where AUC is the area under the fitted curve, tin vitro digestibility kinetics curve was calculated as the hydrolysis index (HI) and estimated glycemic index (GI) corresponding to white bread as a reference using the following equation  operated at 40 kV, 4 to 40°, 200 mA, and 0.154 nm CuKα radiation was used. Relative crystallinity (Rc) was calculated using MDI Jade v6.5 (The degree of gelatinization (DG) was defined as the glucose content of per gram of gelatinized starch sample after enzymolysis. Therefore, DG could be carried out based on method of enzymatic detection. 100 mg starch sample were passed 200 mesh sieve and hydrolyzed by amyloglucosidase . The glucose oxidase-peroxidase method used to detect glucose content. The DG was measured by the ratio of starch samples to fully gelatinized starch standard.2, and 0.0065 M KI) was used to react with that diluent, then stewing 20 min for Color reaction. The absorbance was measured in an ultraviolet spectrophotometer at 620 nm . The standard curve of potato amylose  was used to calculate the amylose and amylopectin contents.According to Chen et al. , a mixedp < 0.05) was determined using Duncan's multiple.The means, standard deviations, and principal component analysis (PCA) performed using SPSS  were used to determine the interaction between the glycemic release rate characteristics and fine supramolecular structure. The data was analyzed by one-way ANOVA at 5% level of significance. The significance of differences between parameters  of EJFSS, ECS, EPS, ERS, EWS, and EMS were shown in p < 0.05). EPS showed the maximum proportion of RS but minimum RDS values. ERS showed the maximum SDS and lowest RS. The highest RDS content was observed in EWS and lowest SDS content was observed in ECS. Therefore, EPS showed the strongest enzyme resistance, followed by EJFSS, ECS, EMS, and EWS. ERS was also susceptible to enzymolysis. Moreover, according to Zhang et al.  (P < 0.05). These results indicate that most RS and SDS in ECS, ERS, EWS, and EMS were converted into RDS, whereas the RS in EJFSS and EPS was converted to both RDS and SDS. According to Zhang et al.  . After Ig et al. , extrusig et al. , the difto 6.84% .in vitro nutrition fractions contents suggested by Englyst et al.  (As shown in the primary and fitted digestive curves in  < 0.05) , S2. Thi < 0.05) , 20. Zha < 0.05)  reported∞ values were in the following order: EWS > ERS ≈ EMS > ECS > EJFSS > EPS , whereas ERS and EMS showed the opposite trend. The C∞ values of ERS (90.78%) and EMS (90.42%) were considerably lower than those of raw rice starch (91.06%) and maize starch (92.05%), respectively; these results differed from those of enzymatic hydrolysis curve analysis. C∞ is significantly correlated with the enzymatic hydrolysis time intervals  exceeded that of raw starches  was higher than that of raw starch (0.20 h−1).ERS and EWS had higher e higher . Based oe higher , 23, thestarches . This mastarches . AlRabadstarches  reportedp < 0.05), except for ERS and EMS , 0.26–1.13 × 107 g/mol (Mn), 53.9–125.4 nm (Rg), and 1.06–2.24 (PI), respectively, and all absolute molecular weight parameters of EPS, EJSS, and ECS were higher than those of EMS, EWS, and ERS . EWS, with the longest A chain lengths, had the shortest medium (B1 chains), medium-long (B2 chains), and long chains (B3+ chains) compared with that of the other extrudates. EPS showed the opposite trend, resulting in EWS having the highest GI and EPS having the lowest GI as described by Bi et al.  were notably decreased compared to those of the corresponding native samples 15.91–29.39%) , indicat% , indic8–16.22% In addition, the DG values of extrudates followed the order of EPS (98.84%) > EJFSS (98.05%) > ECS (97.63%) > EMS (97.11%) > ERS (96.57%) > EWS (96.08%). The different DG values of extrusion cooking samples might be the diversities of rigidity degree of native starch molecular crosslinking network formed by ordered helices and amorphous single chains. Based on Ren et al. , it was The proportions of amylose and its proportional relationships with amylopectin of starch followed the order EPS (31.34 and 45.65%) > EJFSS (26.98 and 36.98%) > ECS (24.08 and 31.72%) > EWS (22.50 and 29.05%) > EMS (20.56 and 25.87%) > ERS (9.63 and 10.67%) . The amyThe amylose contents and amylose/amylopectin ratios of ECS, EPS, and ERS were notably increased compared to those of the corresponding native samples ; the amyHowever, another degradation mechanism was observed in EJFSS, EMS, and EWS during IEMS. As shown in Previous studies , 27, 43,∞, HI, GI, k, middle-short chain, RDS, and Rc was displayed in PC2 part. A highly remarkably positive relationship was shown among the short chain, middle-long chain, long chain, RS, Mw, and proportion of amylose in PC1 (p < 0.05). C∞, HI, GI, k, middle-short chain, and RDS also showed a significant positive relationship (p < 0.05). Zabidi et al. . SDS had a significantly negative relationship with long chain, RS, Mw, proportion of amylose, and Rc (p < 0.05). Lee and Moon (p < 0.05), which agreed with a published report . The original crystallization structure of all starch samples was altered to V-type from A-type crystallization. The amylose contents in ECS, EPS, and ERS were remarkably higher than those in corresponding native starches (p < 0.05), whereas changes in the amylose content of EMS, EJFSS, and EWS were minimal (p > 0.05). In summary, it was demonstrated that, during IEMT treatment, the α-1,4-glycosidic bonds of maize, jackfruit seed, and wheat starch amylopectin were disrupted because of the high amylose content. The lower amylose content observed for cassava, potato, and rice starches was degraded through α-1,6-glycosidic bonds. Consequently, the van der Waals forces between the branched chains were weakened, and abundant unlinear amylose and amylopectin fragments with side chains were generated. After degradation, the various reassembly patterns of amylose and amylopectin molecules resulted in improved digestibility for ECS, EJFSS, EPS, and EWS and stronger enzyme-resistance capacity for EMS and ERS. PCA further revealed the association between the supramolecular structure and in vitro glycemic release characteristics. Moreover, ECS, EJFSS, and EPS had a more ordered molecular structure and compact granule morphology compared to those of EWS, EMS, and ERS, resulting in lower digestibility because of the higher Mw, proportion of amylose, and long chains of amylopectin in the EWS, EMS, and ERS granules. These results may improve the utilization of starches with various GIs of different food fields for people who require different nutritional adaptations.The glycemic release characteristics and fine supramolecular structure for jackfruit seed, cassava, rice, wheat, potato, and maize starches modified by IEMS were evaluated. ECS, EJFSS, EPS, and EWS showed higher The original contributions presented in the study are included in the article/Conceptualization, software, and validation: CH. Formal analysis, investigation, resources, data curation, and writing-original draft preparation: BL. Writing-original draft preparation, writing-review and editing, and methodology: YZ. Visualization and supervision: WL. Project administration and funding acquisition: JL. All authors have read and agreed to the published version of the manuscript.This work was supported by the College of Light Industry and Food Engineering, Guangxi University, and Spice and Beverage Research Institute, Chinese Academy of Tropical Agricultural Sciences. This study was financially supported by National Key Research and Development Program (2020YFD1001204), Key Research and Development Program of Hainan Province , Natural Science Foundation of Guangxi Province (2019JJD120012), and Key Research and Development Plan of Guangxi (AB18221126).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Echis ocellatus, responsible for the highest casualties in the area, would be covered by 12 different epitopes. Remarkably, this pipeline is versatile and customizable for the analysis of snake venom sequences from any other region of the world.Snakebite envenomation is a neglected tropical disease that causes over 100,000 deaths each year. The only effective treatment consists of antivenoms derived from animal sera, but these have been deemed with highly variable potency and are usually inaccessible and too costly for victims. The production of antivenoms by venom-independent techniques, such as the immunization with multi-epitope constructs, could circumvent those drawbacks. Herein, we present a knowledge-based pipeline to prioritize potential epitopes of therapeutic relevance from toxins of medically important snakes in West Sub-Saharan Africa. It is mainly based on sequence conservation and protein structural features. The ultimately selected 41 epitopes originate from 11 out of 16 snake species considered of highest medical importance in the region and 3 out of 10 of those considered as secondary medical importance.  Snakebite envenomation is a deadly condition categorized as a neglected tropical disease by the WHO since 2017 . At presAs a result of predator–prey coevolution dynamics, snake venoms are cocktails of peptides and proteins from different protein families, with each snake species having different proportions of each protein family . Even wiRemarkably, multi-epitope genetic constructs may contain dozens of epitopes from multiple origins, which upon delivery would be able to elicit a targeted response against them. Thanks to the availability of several bioinformatics tools, the selection of those epitopes can be undertaken computationally at a low cost, for instance, guided by sequence conservation criteria and protein toxicity features. Indeed, a multi-epitope construct that takes into account these requirements could, in theory, neutralize the venom from any given species, independently of their geographical origin. Not only that, considering that cross-neutralization has been observed in previous experiments with pDNA immunization ,15,17, iIn this study, we present a flexible and versatile computational pipeline that relies on publicly available snake venom protein sequences and bioinformatics resources for the analysis and selection of a series of peptides of interest to be carried on by a pDNA construct. Such selection relies on a knowledge-based strategy encompassing evolutionary conservation, protein annotation and lack of sequence identity with susceptible preclinical evaluation models or antivenom-producing hosts. Furthermore, for the prediction of B-cell epitopes, we concede major relevance to the availability of 3D structures or high-quality models to follow a structure-based approach . We use Bitis arietans, Naja nigricollis and Echis ocellatus) providing more than 50% of the sequences , we also found epitopes that would cover proteins from different genera, such as metalloproteinases from Bitis arietans and Echis ocellatus. This would allow covering several toxins using a low number of sequences. Additionally, with this methodology, it would be possible to generate antibodies against protein families absent in current antivenoms, such as in the case of sarafotoxins in Atractaspis spp., either due to difficulties milking the venom or because the toxins show low immunogenicity. While experimental validation is pending, the peptide list presented here, the result of a bioinformatics analytical pipeline, is the first step in the development of innovative epitope-based polyvalent snake antivenom for West Sub-Saharan Africa.From this perspective, we scored and selected the best potential epitopes of as many as possible snakes of medical interest from West Sub-Saharan Africa, taking into consideration the most relevant toxin protein families. Interestingly, our clustering and conservation approach shows that some of the predicted epitopes are conserved in proteins from the same family inside a given species and even in proteins from different species. Even though this is somewhat expected at the genus level , the snakes with a higher number of sequences in the databases were n Africa ,35.E. ocellatus and MAHDGNQCNCG from B. gabonica). A closer inspection of other predicted epitopes revealed that some short motifs within them are indeed part of other already validated epitopes. For instance, it is interesting to find that motif ATCP, present in the predicted epitopes ATCPKPTNVRETI and AATCPKVK that cover Dendroaspis species and which have been predicted through 3D features, has been found in validated 3FTXs epitopes from the Asian Bungarus multicinctus and the American Micrurus corallinus [+ T cell epitopes), our pipeline involves multiple parameters and a scoring system to promote the best possible sequences. In this respect, some of the most important aspects of B-cell epitopes are their accessibility to the protein surface and their flexibility [E. ocellatus, have a higher proportion of lower quality (tier 7) epitopes. In future studies, recent advances in 3D protein modeling such as AlphaFold2 might aid in the structure-based prediction of epitopes from those large proteins [Remarkably, the substring IEDB legacy-experimentation approach found three matches with already validated B-cell epitopes  [H was calculated only by taking into account residues that did not correspond to terminal gaps. While any real gap was taken into account in the entropy calculation, unknown residues (marked with “X”s) were not considered  ,52. Eachnsidered . The entA BLASTP  web searProtein models were generated for the clusters with a 3D structure identity that fell between 90% and 50% with the homology modeling software Modeller ,54. The BZ is the new standardized B-value, B is the raw B-value, and Bμ and Bσ are the mean and standard deviation of all the B-values of the structure, respectively. These were used as a measure of flexibility [BepiPred2  was run xibility ,39. SincH = 0 as a cutoff. Upon defining totally conserved peptide sequences of all reference sequences, these were split into conserved fragments and considered separately. A minimum length of eight residues was established for any given epitope, so shorter conserved fragments were automatically discarded. Then, all possible 8-mers from a given fragment were calculated for all conserved fragments.In this study, all peptide sequences selected were based on absolute conservation within MSA, so we used To identify potential cross-reactive peptides, this 8-mer database was locally blasted against human (taxid 9606), chicken (taxid 9031), rabbit (taxid 9986), horse (taxid 9796), donkey (taxid 9793), dromedary (taxid 9838), llama (taxid 9844), mouse (taxid 10090) and sheep (taxid 9940) non-redundant sequences protein databases, in separate blast searches. The blastp-short task was used because of the short length of the sequences. In addition, these were blasted using the PAM30 matrix, a word size of 2, no composition-based statistics, and gap penalties of open 9 and extend 1. An expect value  threshold of 10,000 was used. The identity of each queried 8-mer to a given hit was calculated by dividing by eight (the queried length) the number of identical positions, minus any gaps that appeared. The hit with the lowest e-value was selected as the representative for each 8-mer. This meant that each 8-mer had seven hits associated, one for each blasted organism.Epitopes were predicted by analyzing each conserved fragment 8-mer by 8-mer, calculating the mean value for each feature from the eight positions. For any given 8-mer, a tier value was selected according to the established thresholds of flexibility , RSA (≥50), hydrophobicity , and BepiPred2 score (≥0.5) . A new eWe queried the Immune Epitope DataBase  to downlAtractaspis irregularis (Lamprophiidae), the same proportions were assigned to each protein family.On the other hand, we classified each cluster, with its corresponding reference sequence, into one of ten venom protein families: three-finger toxins (3FTX), Cysteine-rich secretory proteins (CRISP), disintegrins (DIS), Kunitz peptides (KUN), L-amino acid oxidases (LAAO), natriuretic peptides (NP), phospholipase A2s (PLA2), snake C-type lectins (SNACLEC), snake venom metalloproteinases (SVMP) and snake venom serine proteases (SVSP). For this, the protein description and features data of each reference sequence were consulted on the NCBI database, parsed, and compared to lists of keywords corresponding to each family . If therFor predicted epitopes in which their best tier value  was betwIf a predicted epitope covered two species, one extra point was given. If it covered three or more, two extra points were given.If the cluster depth (number of peptide sequences within a given cluster) was higher than 10, two extra points were given. If it was between 5 and 10, one extra point was given.If any predicted epitope was found inside an IEDB epitope or the other way around, three extra points were given.For the comparison and selection of the best epitopes, these were scored as follows:Dendroaspis spp. and sarafotoxins from Atractaspis spp. were also kept due to their relevance in these particular species [This baseline score, which ranged from 1 to 10, was multiplied by the corresponding proportion of each venom family in a particular snake species. If multiple species were covered, the proportion was calculated as the mean between the covered species. Then, the resulting epitope scores were normalized from 0 to 100 and rounded to the nearest integer. Finally, any epitope found inside a bigger epitope was merged, keeping the best score of the merged pair. To compose a final list of epitopes, the highest scored epitope for each venom cluster was selected. Any predicted epitope that had a match with validated epitopes from IEDB was automatically selected. Since the most toxic families are 3FTXs, SVMPs, SVSPs and PLA2s , only ep species . NoteworPlasmodium falciparum antigen [We designed a genetic construct encoding the 41 selected B-cell epitopes shaped like a “string-of-beads” . AAY lin antigen .https://www.genscript.com/tools/gensmart-codon-optimization (accessed on 6 June 2022)) for codon optimization using Mus musculus as the host organism. The resulting nucleic acid sequence was then inserted into a eukaryotic expression plasmid based on the InVitrogen pVAX2 backbone. This plasmid backbone is commonly used as an in vivo mammalian expression vector and is used in the first and only DNA vaccine approved for human use against the SARS-Cov-2 coronavirus, ZyCov-D [Thinking of the next experimental proof-of-concept, the full peptide sequence of this ensemble was then subjected to the Genscript GenSmart™ Codon Optimization Tool ( ZyCov-D . Briefly


Temp), solar radiation (Rad), precipitation (Prec) and CO2) on crop phenology, yield and water consumption for the rice (Oryza sativa L.) -wheat (Triticum aestivum L.) rotation. Due to the increase in Temp, the key phenological periods (flowering and maturity) of wheat in the future mainly tend to advance, while the phenological changes of rice show different trends at different sites. Both rice and wheat yields were negatively correlated with Temp, but positively correlated with Rad, Prec, and CO2 concentration ([CO2]). However, crop ET was positively correlated with Rad, but negatively correlated with [CO2], as elevated [CO2] decreased stomatal conductance. Moreover, the water use efficiency (WUE) of rice and wheat was negatively correlated with Temp, but positively correlated with [CO2]. Overall, climate change will have a significant impact on the crop growth process, yield and water consumption.Quantitatively exploring the impact of climate change on crop production and water consumption  is very important to achieve sustainable regional agricultural development. In this study, based on daily downscaled climate data from 22 Global Climate Models (GCMs), we applied the Agricultural Production Systems sIMulator (APSIM) to investigate the possible impact of climate change  and solar radiation (Rad), and annual total precipitation (Prec) at four investigated stations, except Rad under SSP370. Climate change mainly leads to a consistent advance in wheat phenology, but inconsistent trends in rice phenology across four stations. Moreover, the reproductive growth period (RGP) of wheat was prolonged while that of rice was shorted at three of four stations. Both rice and wheat yields were negatively correlated with Temp, but positively correlated with Rad, Prec, and CO2 concentration ([CO2]). However, crop ET was positively correlated with Rad, but negatively correlated with [CO2], as elevated [CO2] decreased stomatal conductance. Moreover, the water use efficiency (WUE) of rice and wheat was negatively correlated with Temp, but positively correlated with [CO2]. Overall, our study indicated that the change in Temp, Rad, Prec, and [CO2] have different impacts on different crops and at different stations. Therefore, in the impact assessment for climate change, it is necessary to explore and analyze different crops in different regions. Additionally, our study helps to improve understanding of the impacts of climate change on crop production and water consumption and provides data support for the sustainable development of agriculture.Global climate change has had a significant impact on crop production and agricultural water use. Investigating different future climate scenarios and their possible impacts on crop production and water consumption is critical for proposing effective responses to climate change. In this study, based on daily downscaled climate data from 22 Global Climate Models (GCMs) provided by Coupled Model Intercomparison Project Phase 6 (CMIP6), we applied the well-validated Agricultural Production Systems sIMulator (APSIM) to simulate crop phenology, yield, and water use of the rice–wheat rotation at four representative stations (including Hefei and Shouxian stations in Anhui province and Kunshan and Xuzhou stations in Jiangsu province) across the Huang-Huai-Hai Plain, China during the 2041–2070 period (2050s) under four Shared Socioeconomic Pathways . The results showed a significant increase in annual mean temperature ( With elevated atmosphere [CO2] under future scenarios, crop growth process can be affected significantly. We fitted the yearly atmosphere [CO2] under four SSPs in the future for integration into APSIM model using the empirical equation obtained by nonlinear least-squares regression .As shown in −1, 1.7–2.4 kg mm−1, 2.3–3.0 kg mm−1 and 2.4–4.0 kg mm−1, respectively  at the four selected stations . The ricectively d. For toectively e. The WU limited f.Tmean and Rad but negatively correlated with Pre and [CO2]. Conversely, wheat ET was positively correlated with Rad but negatively correlated with [CO2]. Further, the WUE for wheat, rice and total WUE of two crops were negatively correlated with Tmean but positively correlated with [CO2].In the meantime, we analyzed the impact of climatic variable change on the ET and WUE . The ET Generally, climate warming can accelerate crop growth process and has a significant impact on crop phenology, yield accumulation, and water consumption ,54. In oThe change in crop growth period can exert a significant influence on crop yield formation ,54,56. I4, SO2, and black carbon (BC) . In addition, climate change had a considerable influence on crop water consumption. Under the comprehensive influence of future climate factors, we predict that the ET and WUE of crops would change remarkably. There was a significant positive correlation between solar radiation and ET, while the ET of crops was negatively correlated with [CO2]. Moreover, the WUE of crops was negatively correlated with temperature but positively correlated with [CO2]. It is beneficial to develop adaptation strategies to alleviate or mitigate the negative effects of climate change on crop productivity and water resource use.


Bioengineering & Translational Medicine is completing its seventh year of publication in 2022. The journal was launched with the mission of providing a platform for featuring cutting‐edge research and technologies at the interface of engineering and medicine, with a particular focus on translation. Many transformative medical technologies find their roots in engineering, and the journal aims to provide a forum to celebrate the role of technological innovation in medicine.Bioengineering & Translational Medicine was published in March 2016 which included seven articles focused on nanomedicine. The journal quickly established itself in the expansive landscape of bioengineering journals. Over the last 5 years, the journal has witnessed tremendous growth; the number of submitted manuscripts has grown by more than 10‐fold and the manuscript download rate has increased by 5‐fold. Bioengineering & Translational Medicine articles have been downloaded in more than 200 countries, thus attesting to the global reach of the journal. The journal received its first impact factor in 2020 and the impact factor now stands at 10.68. These metrics speak to the solid foundation that the journal has built in its nucleation phase.The first issue of Bioengineering & Translational Medicine. I am deeply thankful to our advisory board who provided strong support and guidance in the journal's formative days. I am especially grateful to our Associate Editors, Profs. Elizabeth Nance, Kaushal Rege, Zongmin Zhao, Hadley Sikes, and Amir Sheikhi who have been instrumental in operating the journal during its rapid growth on multiple fronts, especially in handling the ever‐increasing manuscript submission volume. Sincere thanks also go to Dr. Aaron Anselmo who served as an Associate Editor in the early days of the journal. I express my sincere appreciation for the AIChE and Wiley teams for their strong support to Bioengineering & Translational Medicine. I also express my deep gratitude to our authors who trusted the journal in its formative days and submitted their manuscripts. Without their trust, the journal would not have gotten off to such a strong start.As the journal enters its next growth phase, I have decided to step down as the Editor in Chief. It has been a pleasure and privilege to serve as the founding Editor in Chief of Bioengineering & Translational Medicine reflects the needs and views of the broad translational bioengineering community. As such, a leadership transition will provide additional perspectives for the journal's future growth. Now is a natural time for a transition in leadership. I am thankful to AIChE for appointing me as the Editor in Chief to launch Bioengineering & Translational Medicine and after a successful launch, it is my pleasure to hand off this responsibility to the next Editor in Chief to take the journal to new heights. I am delighted that Prof. Elizabeth Nance has agreed to serve as the interim Editor in Chief of the journal. Prof. Nance has already made tremendous contributions to the journal as Associate Editor and has launched multiple initiatives of high importance to the journal. I am looking forward to her leadership as the interim Editor in Chief.Bioengineering & Translational Medicine over the past 7 years, and I hope that you continue to support the journal in years ahead.To the readers, authors, and reviewers of the journal, I would like to reiterate my deep appreciation for your staunch support to Samir Mitragotri: Conceptualization (lead); writing – original draft (lead); writing – review and editing (lead).https://publons.com/publon/10.1002/btm2.10422.The peer review history for this article is available at


N-acetylcysteine (NAC) uptake. The feasibility of detecting NAC-induced glutathione (GSH) biosynthesis using CEST MRI was also investigated. The detectability of the GSH amide and NAC thiol CEST effect at B0 = 7 T was determined in phantom experiments and simulations. C57BL/6 mice were injected intravenously (IV) with 50 g L−1 NAC in PBS (pH 7) during MRI acquisition. The dynamic magnetisation transfer ratio (MTR) and partial Z-spectral data were generated from the acquisition of measurements of the upfield NAC thiol and downfield GSH amide CEST effects in the liver. The 1H-NMR spectroscopy on aqueous mouse liver extracts, post-NAC-injection, was performed to verify hepatic NAC uptake. The dynamic MTR and partial Z-spectral data revealed a significant attenuation of the mouse liver MR signal when a saturation pulse was applied at −2.7 ppm  after the IV injection of the NAC solution. The 1H-NMR data revealed the presence of hepatic NAC, which coincided strongly with the increased upfield MTR in the dynamic CEST data, providing strong evidence that hepatic NAC uptake was detected. However, this MTR enhancement was attributed to a combination of NAC thiol CEST and some other upfield MT-generating mechanism(s) to be identified in future studies. The detection of hepatic GSH via its amide CEST MRI contrast was inconclusive based on the current results.This proof-of-concept study looked at the feasibility of using a thiol–water proton exchange  MRI contrast to detect in vivo hepatic  N-acetylcysteine (NAC) is a pharmaceutical drug with widespread applications, including the treatment of acetaminophen (paracetamol) poisoning . A. AT2-wein FL1 in C, and onn FL1 in C. For FLwitching  [24]. FoB0-shift maps. The acquisition parameters were the same as in the IV injection scans, but with the saturation txω being swept ±5 ppm with a frequency resolution of 0.2 ppm.Pre- and post-dynamic scan (FL1) Z-spectra were acquired with the CEST-UTE pulse sequence to generate n = 6) and a control group (n = 6) were used. The preparation of the extracts was performed according to the procedure reported by Beckonert et al. , bu, bu14C-lAvg curves  [p-values > 0.05 in the corresponding cells in B0 drift from the recurring RF pulse and pulsed field gradient switching in the CEST-UTE pulse sequence, which would cause the shifting and broadening of the water resonance [Avg values at −1.1 ppm. This large data variance was evident when considering that the hypo-intense and hyper-intense MTRAvg values at −1.1 ppm were not statistically significant  , this waesonance ,28. The nificant B. In addas WASSR , could nAvg spike with a subsequent MTR/MTRAvg increase when saturating at 3.6 ppm  in Avg profiles also showed a similar MTRAvg increase at 3.3 ppm. This suggested that the increased CEST effects from other endogenous amides/amines, which also had their proton resonances in the same region [Avg increases, since only a small number (n = 3) of experimental repetitions were performed due to time constraints. The MTRAvg increase at 3.6 ppm suggested a possible increase in the GSH amide MTR. However, previous studies have shown that significant hepatic GSH replenishment occurs only after a NAC dosage of almost four times that used in this study [The CEST MRI detection of GSH was also investigated , since Ge region ,52,53,54is study ,56. The is study , so thatAvg spike when saturating the NAC thiol proton resonance was indeed due to the presence of NAC in the mouse liver, a 1H-NMR spectral analysis was performed on the aqueous liver extracts from the test and control mice  via the distinct NAC methyl proton singlet peak at ~2.06 ppm. Whilst the small hepatic NAC concentration derived from these results may not have provided strong evidence that the post-injection upfield MTR/MTRAvg spikes observed in the test group’s dynamic MTR/MTRAvg curves between 24 and 39 min . The hepatic NAC uptake was verified by the presence of the NAC methyl singlet peak at ~2.06 ppm in the 1H-NMR spectra of aqueous mouse liver extracts obtained ~8 min after the injection of 50 g L−1 NAC in PBS (pH 7); however, the estimated in vivo hepatic NAC concentration, 0.12 ± 0.03 mM, from the spectral deconvolution was too low to generate a detectable CEST effect. These results suggested that the MTR/MTRAvg spike was due to an apparent combination of an NAC thiol CEST effect and other MT-generating mechanism(s), possibly as a result of NAC metabolism—this should be investigated in a future study. The MTR/MTRAvg increases observed after 17 min post-injection could not be verified as being due to an NAC accumulation in the test mouse liver tissues based on the current results. The downfield MTR/MTRAvg increase in the dynamic MTR/MTRAvg curves obtained from the test mice could not be verified as being due to the GSH replenishment from the hepatic NAC uptake because changes in the 1H-NMR signal intensities of the GSH non-labile protons could not be detected.In conclusion, the detection of hepatic NAC uptake in mice using MRI was shown to be feasible. The upfield dynamic MTR/MTR


Background and objectiveGraft performance is the most important postoperative parameter for patients undergoing kidney transplantation (KTx). The renal function of the donor is reported to be correlated with graft function after KTx. The body mass index (BMI) is also one of the important parameters involved in the prediction of graft function. The aim of this retrospective study was to examine the relationship between early postoperative graft function in patients undergoing KTx and donor cystatin C and estimated glomerular filtration rate (e-GFR) levels, graft weight/recipient BMI (G/B) ratio, and human leukocyte antigen (HLA) tissue compatibility.Materials and methodsA total of 215 cases  who underwent KTx at our center between January 2018 and December 2022 were included in the study. Patients' age, sex, BMI, preoperative donor serum cystatin C and e-GFR levels, HLA tissue compatibility, graft weights, and recipient creatinine levels were recorded one week postoperatively. The Kolmogorov-Smirnov test and histogram plots were used to analyze the conformity of the variables to the normal distribution and Spearman's correlation test was used to analyze the relationship between variables.ResultsA negative correlation was identified between recipient creatinine level and G/B ratio and donor e-GFR . Donor cystatin C level showed a positive correlation with recipient creatinine level (r = 0.242). No significant correlation was noted in terms of tissue matching rates (p = 0.616).ConclusionAlthough these three parameters are correlated with early graft functions, the graft weight/recipient BMI ratio has the strongest correlation. Kidney transplantation (KTx) is an effective treatment strategy for patients with end-stage renal disease (ESRD) . Graft pThe renal function of the donor is reported to be correlated with graft function after transplantation . The expThe aim of this retrospective study was to examine the relationship between early postoperative graft function in patients undergoing KTx and donor cystatin C and estimated glomerular filtration rate (e-GFR) levels, graft weight/recipient BMI ratio, and human leukocyte antigen (HLA) tissue compatibility.This study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the local ethics committee .The data of patients who underwent KTx at our center between January 2018 and December 2022 were retrospectively reviewed. After excluding patients whose archival records could not be accessed, patients who developed acute rejection in the early period, and patients receiving cadaveric transplants, a total of 215 KTx cases  were included in the study. Cadaveric kidney transplants were excluded because all pertinent parameters were not evaluated in those cases. Furthermore, the delayed graft function rate - observed at a higher probability in cadaveric transplants compared to living transplants - could have caused interpretation errors in the statistical analyses.2) (G/B) ratio. HLA tissue compatibility was statistically analyzed by examining the number of matches in the three HLA pairs .Patients' age, sex, BMI, preoperative donor serum cystatin C and e-GFR levels, HLA tissue compatibility, graft weights, and recipient creatinine levels were recorded one week postoperatively. Graft weights were acquired from data recorded by weighing the graft kidney after removing perinephric adipose tissue following bench surgery. The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula was used to calculate e-GFR. The effect of graft weight on postoperative graft function was evaluated by calculating the graft weight (g)/recipient BMI , tacrolimus , mycophenolate mofetil (2 × 1000 mg), and prednisolone. The doses were adjusted for a target blood level of 10 mg/ml for FK506 and a target CD3 count of 20-50 for ATG.Statistical analyses were performed using the IBM SPSS Statistics version 25.0 . The Kolmogorov-Smirnov test and histogram plots were used to analyze the conformity of the variables to normal distribution. Mean, standard deviation (SD), and median (min-max) values were used when presenting descriptive analyses. Spearman's correlation test was used to analyze the relationship between variables. P-values below 0.05 indicate statistical significance.Table The correlation between recipient creatinine level and donor cystatin C level, donor e-GFR level, G/B ratio, and HLA compatibility at postoperative week one was examined. A negative correlation was identified between recipient creatinine level and G/B ratio and donor e-GFR . Donor cystatin C level showed a positive correlation with recipient creatinine level r = 0.242). No significant correlation was noted in terms of tissue matching rates (p = 0.616)  on early recipient serum creatinine level revealed that the relationship between cystatin C level and graft function was relatively stronger (r = 0.242 vs. −0.137) than that between e-GFR and graft function. Studies evaluating cystatin C have reported that it is not affected by muscle mass, sex, and body surface area ,18. The Various studies have reported that recipient BMI has a significant effect on graft function, especially in the early period -23. Xin HLA tissue compatibility, which was evaluated for the prediction of early graft function, did not show a statistically significant correlation with the recipient’s creatinine level at one week post-KTx (p = 0.616). It is known that HLA tissue compatibility is an important factor affecting long-term graft function . High-doAmong the parameters examined in the present study, the G/B ratio showed the strongest correlation with early graft function, followed by donor cystatin C level and e-GFR, respectively. The main limitations of the study are its single-center and retrospective design and the exclusion of some immunological factors, comorbidities, and certain parameters  that may have had an impact on graft function in the statistical analysis. The main reason for this exclusion was to compare the predictive ability of the parameters we focused on, rather than determining the parameters that affect the graft functions.Donor serum cystatin C and e-GFR levels are strongly correlated with early postoperative serum creatinine levels in patients who have undergone kidney transplantation. Graft weight and recipient BMI are also parameters that can be used for the estimation of graft functions, and it is possible to use the “graft weight/recipient BMI” ratio in order to use both parameters effectively. Although these three parameters are correlated with early graft functions, the graft weight/recipient BMI ratio has the strongest correlation. Multicenter studies with a prospective design are needed to validate the findings obtained in the present study.


Neuronal intranuclear inclusion disease (NIID) is a rare and slowly progressive neurodegenerative disease characterized by the presence of eosinophilic neuronal intranuclear inclusions. The clinical manifestations of NIID are diverse, and the most common initial feature in cases of sporadic NIID is dementia. Herein, we report an adult female with keratitis as the initial presentation with subsequent bilateral limb tremor, gait disturbances, overemotional behavior, sweating and constipation. Diffusion-weighted imaging (DWI) showed hyperintensity in the bilateral fronto-parieto-occipital corticomedullary junction. Skin biopsy specimens revealed eosinophilic hyaline intranuclear inclusions in fibroblast cells, sweat gland cells and adipose cells. In vivo confocal microscopy of the cornea indicated the absence of corneal nerves in both affected eyes. The patient’s diagnosis of NIID was based on the presence of intranuclear inclusions in biopsied skin and the characteristic high-intensity signal in the corticomedullary junction obtained with DWI. This case report emphasizes that the clinical heterogeneity of NIID and an examination of the corneal nerves may offer valuable clues to its early diagnosis in some patients. Neuronal intranuclear inclusion disease (NIID) is a rare and slowly progressive neurodegenerative disease characterized by the presence of eosinophilic and ubiquitin-positive neuronal intranuclear inclusions in the central and peripheral nervous systems, as well as in visceral organs ,2. NIID The ocular manifestations of NIID have recently attracted attention and have expanded the clinical spectrum of NIID in neuro-ophthalmology. The novel ophthalmological manifestations of the disease involve miosis, night blindness, oculogyric crisis, reduced eye movements, nystagmus, blepharospasm, ptosis and rod–cone dysfunction with progressive outer retinal degeneration . In a prIn the present article, we report for the first time a case of sporadic adult-onset NIID with neurotrophic keratitis as the initial manifestation and fewer corneal nerve fibers, as detected with IVCM. The purpose of this paper is two-fold: (1) to raise awareness that neurotrophic keratitis is an initial manifestation of NIID and (2) to describe the corneal morphological presentations that can contribute to the diagnosis of NIID-related ophthalmopathy.In April of 2016, a 57-year-old female was admitted with progressively decreasing vision in the right eye over the previous 8 years. Eye pain and redness, photophobia and lacrimation were also reported. The patient was diagnosed with viral keratitis at a local hospital on admission. Despite receiving antiviral therapy and an amniotic membrane transplant in the right eye, the patient’s vision continued to worsen. The patient denied a history of diabetes mellitus, trauma and stroke. She also did not report any other ocular manifestations, such as rod–cone dysfunction, retinal degeneration, night blindness and miosis, during the 8 years prior to being admitted to the hospital.Upon examination, the best-corrected visual acuity was light perception in the right eye and 10/20 based on the Snellen chart in the left eye. Slit-lamp microscopy revealed conjunctival hyperemia, nubecula, keratoleukoma and neovascularization of the cornea in the right eye A. A cottIn May of 2018, the patient was re-admitted to the ophthalmic inpatient department because she noticed reduced visual acuity in the left eye over the previous 4 months. The sensitivity of the left cornea was reduced, as demonstrated with corneal reflex testing. Slit-lamp microscopy revealed a distinct corneal scar in the right corneal transplant with nubecula and vascularization C. Additi2. The patient was oriented and had fluent speech. Her cognitive function was intact. The left eye had a normal pupillary reaction to light and fundoscopy, whereas the right pupil could not be visualized because of decreased corneal transparency. The patient’s strength and sensation of the trunk and the limbs were normal. The patient had mild hypermyotonia and a resting tremor in the jaw, hands and feet. The patient had dysdiadochokinesia and deficits in the bilateral finger-to-nose test  and the heel–knee–tibia slide test. The Romberg test was positive, but there was no Babinski or Chaddock sign. The remainder of the examinations were unremarkable.During her second hospitalization, the patient consulted with a neurologist because of some neurological symptoms, including bilateral limb tremor, gait disturbances, overemotional behavior, sweating and constipation, which may have existed for the previous 2 years. There was no bradykinesia, rigidity, limb weakness or numbness. The patient’s body mass index was 22.9 kg/mSimilar to the assessment of neurological signs and symptoms, neuropsychiatric and autonomic functions were also evaluated. The Hamilton Anxiety Scale score was 15 (definite anxiety), and the Hamilton Depression Scale score was 26 (definite depression). The Mini-Mental State Examination score was 30 , and the Montreal Cognitive Assessment score was 26 . The Neuropsychiatric Inventory (NPI) score was 24 . Autonomic (SCOPA-AUT) score was 11 . There were no obvious abnormalities in the laboratory tests, including complete blood count, urine and stool analyses, liver and kidney function, and lipid profile, and blood glucose concentration was unremarkable. The following indicators were negative: infectious indicators, including HIV, treponema pallidum and hepatitis virus; purified protein derivative test; galactomannan test; glucan test; thyroid hormone test; immunological index, including anti-nuclear antibody (ANA), anti-neutrophil cytoplasmic antibody (ANCA), immunoglobulin (Ig) A, IgG, IgM, IgE, complement-3/4, and λ and κ light chain immunoglobulins; and tumor markers, including carbohydrate antigen 724 (CA724), carcinoembryonic antigen (CEA), alphafetoprotein (AFP), neuron-specific enolase (NSE), CA19-9, CA125, CA153, β-human chorionic gonadotrophin (hCG), cytokeratin-19-fragment (CYFRA21-1), CA50 and human epididymis protein 4 (HE4); the risk of ovarian malignancy algorithm (ROMA); and squamous cell carcinoma antigen (SCCAg). The axial planes of T2-weighted images of the brain taken using magnetic resonance imaging (MRI) revealed symmetrically diffuse and high signal leukodystrophy. Such areas presented isointense or slight hypointensities in T1-weighted imaging. The diffusion-weighted imaging (DWI) sequence showed a high-signal-intensity zigzag edging sign in the bilateral fronto-parieto-occipital corticomedullary junction, also known as the “cortical line sign” . The corThe typical “cortical line sign” in DWI of the brain combined with eosinophilic hyaline intranuclear inclusions in the skin biopsy supported the diagnosis of NIID. No family member had similar symptoms or the FMR1 gene premutation. The patient was finally diagnosed with sporadic NIID. With regard to the corneal impairment, we reviewed the pathogenic factors of the secondary neurotrophic keratitis along the pathways of the corneal sensory nerve, including the following causes: (1) injuries of corneal sensory nerves induced by viral infection, refractive surgery, chemical or thermal burns, ocular medication and wearing contact lenses; (2) damage of the ophthalmic branch of the trigeminal nerve, as well as ciliary nerves, due to tumor formation and surgeries ; (3) trigeminal ganglion lesions due to herpetic viral infections, surgery for trigeminal neuralgia, preganglionic trigeminal nerve root injury  and some systemic diseases (such as diabetes and leprosy) that can decrease sensory nerve function or damage sensory fibers; and (4) damage to the lowest part of the spinal trigeminal nuclei due to dorsolateral medullary infarction and tumor formation ,10. HoweNIID exhibits highly variable clinical manifestations, and the initial manifestation in adult-onset NIID is usually cognitive impairment, often accompanied by pyramidal signs and peripheral neuropathy with or without autonomic nervous system dysfunction ,2. The dPrevious studies reported several ophthalmological manifestations in cases of familial NIID, including miosis, night blindness, oculogyric crisis, eye movement disorders, nystagmus, blepharospasm, ptosis and retinopathy ,7,11,12.To date, there is no evidence for the onset of NIID being marked with impaired vision caused by keratitis. In the current case, the intractable corneal epithelial defect was initially thought to be caused by a corneal infection. However, the affected cornea was negative for the presence of microbes, and IVCM revealed its structural abnormality but not signs of infection. Furthermore, the corneal exfoliation and opacification in the epithelium and the reduction of corneal nerves were not consistent with corneal degeneration and congenital dystrophy but were consistent with corneal neurotrophic dystrophy. The deteriorating keratopathy evolved during the progression of the disease, and the eyes were threatened despite the fact that the patient received several amniotic membrane transplants and underwent keratoplasties. We therefore attributed the lingering keratitis to neurotrophic keratitis of autonomic nervous dysfunction of NIID. In fact, autonomic innervation (mostly sympathetic nerves and a paucity of parasympathetic nerves) of the cornea played a vital role in maintaining the quality and quantity of tears and established homeostasis of the ocular environment ,14,15. TBiopsy is recommended as an effective and less invasive tool to diagnose NIID ,16,17. UIn brain imaging studies, a high-signal-intensity zigzag edging sign in the bilateral corticomedullary junction on a DWI sequence is considered as the hallmark of NIID. It reveals a neuropathological result of spongiotic changes proximal to the U-fibers or diffuse myelin pallor in subcortical white matter. This type of leukoencephalopathy may always be an anticipatory clue for clinicians to explore whether causative intranuclear inclusions are seen upon biopsy .We herein report a case of sporadic adult-onset NIID with autonomic impairment manifesting as neurotrophic keratitis. Neurotrophic keratitis may serve as the initial presentation and the most prominent ophthalmological characteristic in NIID. The absence of NIIs in the affected cornea may indicate that the corneal impairment is due to the dysfunction of autonomic nerves rather than the cytotoxicity of NIIs in corneal neurodegeneration. This paper strengthens the awareness that quantitative NIID-related changes in the corneal nerves detected using IVCM may precede the systemic neurological manifestations and, thus, may assist in the early treatment and staging of the disease. Future studies are warranted to reveal the underlying mechanism of keratitis in patients with NIID.