P < 0.05) of resistant starch (RS) content. The hydrolysis kinetic analysis suggested a decelerating influence of CH on the hydrolysis content with lower values of equilibrium hydrolysis percentage (C∞) while CS on hydrolysis rate with lower kinetic constant (K). The estimated glycemic index (eGI) of the CH (15-20%) samples were below 55. These results are of great significance in delaying starch digestion and provided a better choice in design of fried puffed snacks for special crowd with chronic diseases such as diabetes, cardiovascular disease, and obesity.Chitin is a polysaccharide and possesses numerous beneficial properties such as nontoxicity, biodegradability and biocompatibility, which draws much attention to its applications in food. Crayfish shell is a source of chitin alongside an antioxidants and a potential source of beneficial dietary fiber. In this study, chitin (CH) and crayfish shell (CS) with different concentrations were used to study their impact on pasting characteristics of flour mixture (wheat flour and glutinous rice flour) and influence on physicochemical and starch digestion property of puffed biscuit. The Rapid Visco-Analyzer results showed that the viscosity of powder mixture was decreased with the ratio of CH and CS increased. CH resulted in lowest peak viscosity and breakdown values of mixed powder. It was indicated that increasing amounts of CH and CS led to significantly reduced moisture content, expansion ratio but raised density of biscuits. CH and CS inhibited starch digestion and promoted a remarkable increase ( Fried puffed foods are popular with consumers globally mainly due to their unique flavor, crispness, color, attractive taste and affordable cost . The maiDietary fiber, consisting of indigestible cellulose, hemicellulose, lignin, chitin, gums etc, are indigestible polysaccharides which is resistant to digestion in the small intestine and fermentation in the large intestine . In receProcambarus clarkii) has become common food source and approximately 100,000 tons of crayfish shells were generated every year, with about 80% of crayfish shells having been discarded as waste -2-acetamido-2-deowy-β-D-glucose, which has antioxidant, antidiabetic, lipid-lowering and antibacterial effects –12. It ias waste . The craas waste . Consumpas waste .Although many efforts have been made to investigate the applications of chitin or chitosan in starch-based products , from thin vitro starch digestibility of biscuits was also investigated. The research are of significance in inhibiting starch digestion and maintaining the health of special crowd with chronic diseases such as diabetes, cardiovascular disease, and obesity.The purpose of our study was to develop an efficient method to control starch digestion in fried biscuit and made low glycemic index (GI) food by incorporating chitin and crayfish shell into flour mixture. The chitin and crayfish shell inhibited the starch digestion by forming barrier to prevent contact between starch and amylase. Chitin-biscuits (CH-biscuits) and crayfish shell-biscuits (CS-biscuits) containing chitin and crayfish shell at different ratios were prepared. The pasting, physicochemical property and morphology of biscuits was studied by scanning electron microscopy (SEM), texture profile analysis (TPA) and rapid visco analyzer (RVA). The The chitin (CH) from crayfish shell was purchased from Shanghai Macklin Biochemical Co., Ltd. . The crayfish shell (CS) was kindly supplied by Wuhan Liangzihu Aquatic Processing Co. Ltd. . The α-amylase from porcine pancreas (4 u/mg) and Glycosylase  were from Shanghai Yuanye Biotechnology Co., Ltd. . The low gluten wheat flour was purchased from Wuhan Suntyh Food Co., Ltd. and the glutinous rice flour was from Henan Enmiao Food Co., Ltd. Other ingredients including soybean oil, baking soda, yeast, salt and eggs were all available at local supermarket. All chemical reagents were of analytical grade purchased from Sigma-Aldrich (Germany).The crayfish shells were washed thoroughly with distilled water to remove impurities, dried in a hot air oven at 105°C for 24 h, and pulverized to powder in a ultrafine pulverizer. After pulverizing, the crayfish shell powder was filtered through a 25 μm sieve to obtain a uniform powder and kept in a desiccator until application. Proximate composition of CS was determined in accordance with the methods in AOAC  and RuthThe procedure for making the biscuits was followed the method of AACC . FirstlyA Rapid Visco Analyzer  was used to measure the pasting characteristics of the mixed powder (wheat flour and glutinous rice flour with different content of CH and CS) according to the method of Feng et al.  with sliThe volume of biscuit was determined using a seed displacement method. The bulk density was calculated by dividing its determined volume by the mass of biscuit . The expThe moisture content of the samples was calculated based on the weight difference measured before and after drying the samples in a hot air oven at 105°C for 24 h . The oilg for 15 min. The supernatant was then poured into a pan of known weight. WSI refers to the weight of dried solids in the supernatant, expressed as a percentage of the original weight of the sample. WAI refers to the weight of gel obtained by removing the supernatant from the original dried solid of unit weight. Such measurement was subject to three samples. The expressions of WAI and WSI were as follows:Both WSI and WAI were defined by Kowalski et al. . 1 g of The textural characteristics of biscuits were measured with the help of a TA-XTPlus Texture analyzer  with a P36/R aluminum cylindrical probe. The parameters were set as: pretest speed 1.0 mm/s, test speed 0.5 mm/s, post-test speed 1.0 mm/s, trigger force 5.0 g, and deformation level 75%. The harness and springiness was obtained from the force-time curve of the texture profile, respectively.The surface morphology of CH-biscuits and CS-biscuits at different magnification (× 100 and × 30) were observed using a scanning electron microscope  with an accelerating voltage of 2 kV. The freeze-dried biscuits were adhered to brass slip under argon environment by a gold sputter module in a high vacuum evaporator to form a gold layer with thickness of 20 nm.Twenty well-trained assessors  evaluated the finished CH-based biscuits and CS-based biscuits for their appearance, color, taste, texture and overall acceptability. The trained sensory panel were all from Institute of Agro-Products Processing and Nuclear-Agricultural Technology . A nine-point preference scale was used to evaluate the overall acceptability for determining sensory attributes of the above samples  . The bisThe sensory evaluation experiment was conducted according to the guidelines of the Declaration of Helsinkiwas. All work with human subjects performed here was reviewed and approved by the Hubei Academy of Agricultural Sciences Institutional Review Board (IRB). The products tested were safe for consumption.in vitro starch digestion of CH and CS-based biscuits samples was determined based on the modified method of Zhang et al. . A factor 0.9 indicated the conversion factor of glucose into starch.Where: GThe rate of starch digestion kinetics were estimated based on the approach described elsewhere . The sta–1) is the starch digestion rate constant. The areas under hydrolysis curves  were calculated as the integral of the kinetic equation and used to obtain the HI (Hydrolysis Index). The HI was calculated by dividing the AUC of the samples by that of white bread (as a reference). The following formula proposed by Goñi et al. (Where: Ct (%) is the percentage of starch digested at t time ; C∞ is the estimated percentage of starch digested at 180 min; K. Experimental data were analyzed using analysis of variance (ANOVA), expressed as the mean value ± SD, and significant differences among means were determined by Duncan’s test. All analyses were performed using IBM SPSS Statistics 25 . The significance level in all cases was set at The effect of CH and CS on the pasting properties of flour mixture was showed in P < 0.05). The addition of CH led to a significant decrease in peak viscosity from 2879 mPa⋅s (control) to 1787 mPa⋅s (CH20%). This could be attributed to the fact that chitin interacted with wheat starch and glutinous rice starch granules through hydrogen bonding, inhibiting the swelling of the granules and leading to lower peak viscosity. The experimental results of Liu et al. . The smaller breakdown viscosity indicated that the structure of starch granules during starch gelatinization was more stable and the damage of starch granules by heating and shear force was reduced (P < 0.05), the addition of CH and CS made the starch granules with more stable structures. System viscosity was mainly affected by three factors: granules swelling degree; granules disruption and the surface interaction especially the system was added with polysaccharide, which would influence the amylose leaching out the swollen granules  and 1,076.5 mPa⋅s (CS20%) with the content of CH (CS) increased from 0 to 20%. Setback viscosity was an index of starch retrogradation, demonstrating the trend of starch paste to retrograde. The decrease in the setback viscosity implied a prevention of short-term retrogradation. It might be explained that –OH groups of CH interacted with the –OH groups of wheat and glutinous starches to form a hydrogen bridge of CH-amylose, thus reducing the association of amylose molecules and inhibiting the amylose rearrangement  Qin et aP < 0.05) (CH and CS both had significant effects on the puffing degree and density of biscuits ( < 0.05) . The den < 0.05) . The mol < 0.05) . The res < 0.05) . The pufAccording to P < 0.05) due to higher chitin quantity.Water absorption index (WAI) is a measure of the water-holding capacity of the starch, cellulose and protein in products after swelling in excessive water . It is tP < 0.05) with a corresponding increase in CH or CS concentration. In 20% CH and 20% CS, the hardness reached 39.08 ± 0.69 kg and 26.51 ± 0.36 kg. The hardness value correlated with density, expansion and thickness of cell walls  of the cross section of the fried biscuits were showed in The incorporation of either CH or CS at different addition ratios  on judging scores of sensory quality characteristics: appearance, color, taste, texture and overall acceptability of produced biscuits were studied and the results were shown in P < 0.05).The digestion property is an important property of starch-based samples. The RDS, SDS, and RS contents of control, CH-biscuits and CS-biscuits were shown in in vitro method based on the same starch content in The digestion of the various biscuits samples  was measured using an ∞), the kinetic constant (K), and the estimated glycemic index (eGI) of each of CH-biscuits and CS-biscuits samples were calculated using a fitting model that assumes first-order kinetics (Eq. 5) to the data . The results suggested that the addition of CH decreased the extent of starch digestibility, while the CS decreased the rate of starch digestibility. The nature of chitin determines its physico-chemical behavior and this may affect the rate of digestion of carbohydrates and absorption of sugars in the small intestine. The estimated glycemic index (eGI) of biscuits decreased gradually with the increasing addition of CH and CS, and significant difference (P < 0.05) was observed between the eGI of the CH and CS groups. It has been reported that eGI above 70 is classified as high blood glucose food, 55 to 69 as middle blood glucose food, and below 55 as low blood glucose food  and slow digestible starch (SDS) by 28 and 20% while increasing the resistant starch (RS). Both CH and CS could slow down starch hydrolysis of biscuits. It was indicated that the structure formed by chitin and crayfish shell around the starch surface, which provided protection to the starch granules, prevented the entry of amylase, and slowed starch digestion. The protecting also restricted the swelling of the starch granules. The addition of CH and CS to hinder starch digestion in fried biscuits had healthy consequences, which indicated that CH-biscuits and CS-biscuits were belong to middle eGI food, even some were low eGI food. The CH and CS may help to lower the risk of high blood glucose levels caused by fried biscuits and maintain sensory quality at same time. The research results are of great significance in delaying starch digestion. However, the oil content of the fried biscuits was also a potential danger for health, and future research should identify methods of simultaneously reduce the oil content and the digestibility of starch.The chitin and crayfish shell had significant effects on the physicochemical properties of biscuits and the digestion properties of starch. The moisture content and expansion ratio of the biscuits decreased with the increasing amount of CH and CS, while the oil content and density showed the opposite. The original contributions presented in this study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.CB: conceptualization, verification, draft writing, and project administration. JZ: review and editing and supervision. GX: data curation, validation, and formal analysis. WW: data collection, data interpretation, validation, and language polish. JW: investigation, validation, and data curation. LQ: methodology and validation. QZ: project administration and conceptualization. TL: editing, methodology, resources, and funding acquisition. All authors contributed to the article and approved the submitted version.


A 58-year-old female who underwent renal transplantation and closure of arteriovenous fistula (AVF) for hemodialysis at age 24 was presented with left forearm pain and cyanosis. Computed tomography revealed an obstructed true brachial aneurysm at the anterior aspect of the elbow joint. Under a diagnosis of true brachial aneurysm associated with AVF, aneurysm resection and brachial to ulnar artery bypass grafting using a reversed great saphenous vein were performed. To prevent graft occlusion due to elbow flexion, it was routed through the ulnar side of the elbow joint. One year after surgery, the patient was asymptomatic with a patent graft. A true brachial aneurysm is a very rare complication of an arteriovenous fistula (AVF) created for hemodialysis access . ReporteA 58-year-old female was presented with pain and cyanosis of the left forearm, and referred to our hospital. At the age of 24 years, she suffered from end-stage renal failure due to IgA glomerulonephritis and underwent AVF creation for hemodialysis. Later that same year, she received living-donor renal transplantation and then the AVF was closed. At the age of 56, pain and cyanosis of the left forearm developed, and ultrasound findings led to a diagnosis of true brachial aneurysm. Antiplatelet therapy with aspirin was started because the symptoms were mild and intermittent. However, symptoms gradually worsened and she was referred to our hospital for surgical treatment.Upon admission, the patient received immunosuppressive treatment with prednisolone (5 mg/day), tacrolimus (1.5 mg/day) and everolimus (0.5 mg/day) for renal transplantation. A physical examination revealed a hard mass in the left antecubital fossa of the volar aspect , and conUnder a diagnosis of symptomatic true brachial aneurysm, a procedure for resection of the aneurysm and bypass grafting to the ulnar artery was scheduled. Intraoperative findings revealed a brachial aneurysm just proximal to the brachial bifurcation. Following incision of the aneurysm, the orifices of the afferent and efferent arteries were closed, then the thrombus was removed for volume reduction. Brachial to ulnar artery bypass grafting using a reversed great saphenous vein was subsequently performed. To prevent graft occlusion due to elbow flexion, the graft was routed through the ulnar side of the elbow joint. Accounting for natural graft routing, a distal anastomosis was made in an end-to-side manner, whereas a proximal anastomosis was also done in an end-to-side manner because of a size mismatch between the dilated brachial artery and saphenous vein graft.Follow-up CT showed patency of the great saphenous vein graft , and vasThe true brachial aneurysm associated with hemodialysis access was reported in a very limited number of patients . A revieet al. [The mechanism underlying aneurysmal development in patients with AVF is not fully understood, though Basile et al.  reportedet al. . The preResection of the aneurysm and revascularization using a reversed great saphenous vein graft is the gold standard treatment for this disease . Only exWe report a surgical case of a true brachial aneurysm associated with AVF for hemodialysis. When a large brachial aneurysm is found at the anterior aspect of the elbow joint, resection followed by revascularization using a reversed great saphenous vein graft routed through the ulnar side of the elbow is considered to be an optimal strategy.None declared.None.The patient consented to the publication of this report.Data for this study are available from the corresponding author upon reasonable request.


A decreasing hazard of graft loss was observed also with 1-year, but not with 30-day graft survival. During 1-year follow-up, 953 (12.1%) patients had an acute rejection, and longer procurement interval was also associated with less acute rejections  in the multivariable model. In conclusion, longer procurement interval is associated with improved long-term outcomes in pancreas transplantation.A brain-death-induced cytokine storm damages organs in an organ donor. However, a longer time period between declaration of brain death and organ procurement  is associated with improved outcomes in kidney, liver, heart, and lung transplantation. The aim of this study was to find the optimal procurement interval for pancreas transplantation. Association of procurement interval with pancreas graft outcomes was analyzed using multivariable models adjusted for variables possibly affecting procurement interval and outcomes. Altogether 10,119 pancreas transplantations were included from the Scientific Registry of Transplant Recipients. The median follow-up was 3.2 (IQR 1.01–6.50) years. During the first year, 832 (9.0%) grafts were lost, including 555 (6.0%) within the first 30 days. Longer procurement interval was associated with increased death-censored graft survival in a multivariable model (HR 0.944 95% CI 0.917–0.972, per 10-h increase,   As practically all pancreas allografts are obtained from deceased donors, and as there continues to be hesitation regarding using pancreata from donors after circulatory death, roughly 97% of transplanted pancreata are affected by brain death and the resulting “cytokine storm” , 2. The To minimize this possible damage, some transplant centers have aimed to minimize time to procurement; although, in recent decades, evidence to support continuous organ injury is sparse, and donor losses from hemodynamic instability are rare –9. InterIn other retrospective studies of kidney, lung, liver, and heart transplantation, waiting before procurement seems beneficial, at least up to 50 h after brain death –18. OrgaThis study aimed to analyze the association of procurement interval (time from declaration of brain death to organ cold perfusion) with pancreas allograft survival and acute rejections in a retrospective cohort from the United States.This study used data from the Scientific Registry of Transplant Recipients (SRTR). The SRTR data system includes data on all donors, wait-listed candidates, and transplant recipients in the US, submitted by the members of the Organ Procurement and Transplantation Network (OPTN). The Health Resources and Services Administration (HRSA), U.S. Department of Health and Human Services provides oversight to the activities of the OPTN and SRTR contractors.Pancreas transplantations from brain-dead donors recorded in the SRTR database in the US between January 2010 and September 2021 were included. Follow-up was recovered for all patients from SRTR Standard Analysis Files. Living and donation after circulatory death (DCD) donors were excluded.This study was approved by the Institutional Review Board of Helsinki University Hospital (HUS/459/2018) and SRTR. The clinical and research activities being reported are consistent with the Principles of the Declaration of Istanbul as outlined in the “Declaration of Istanbul on Organ Trafficking and Transplant Tourism” and the Declaration of Helsinki.in situ cold perfusion. Brain death is generally diagnosed according to strict criteria, which include having a cause of death, testing for the absence of brainstem-, and pain reflexes, and apnea. Additional testing proceeds from uncertainty of any of the above [Procurement interval was defined as the time between the declaration of brain death and the start of he above .The following donor data were gathered: the time of declaration of brain death, start time of cold perfusion in organ procurement surgery, location, donor age, gender, cause of death, body mass index and history of resuscitation, inotrope use, hypertension, and diabetes. The obtained recipient and transplantation data included recipient age, gender, body mass index, history of hypertension, human leukocyte antigen mismatches, calculated panel-reactive antibodies (CPRA), time in dialysis, previous transplants, graft cold ischemia time, organ location, acute rejection episodes before discharge from hospital and before follow-ups, and graft survival during the follow-up.Death-censored graft survival (measured as centers’ reporting to follow-up forms) was chosen as the primary dependent outcome measure. Secondary endpoints were graft survival at 30 days after the operation, graft survival 1 year after the operation and acute rejections during the first year after transplantation. The definition of pancreas graft failure has evolved from center-specific definitions of either a degree of insulin-independence or C-peptide production, to (from 2018 onwards) a uniform measurement of either: 1. Removal of graft, 2. Patient waitlisted for retransplantation or islet transplantation, 3. Patient death, or 4. Recipients total insulin need is ≥0.5 units/kg/day for 90 consecutive days . In thisCharacteristics of data in the tables are reported with median and interquartile range (IQR) for continuous data and frequencies with percentages for categorical data. Number of patients with missing values are stated in Confounder analysis for the multivariable model was constructed as a directed acyclic graph (DAG) . The DAGp-values. All associations were linear in the final results.The associations were modelled as both linear and non-linear. Potentially non-linear associations were checked for by using restricted cubic spline functions between procurement interval and endpoints, as logistic and Cox regression models require the assumption of linearity for continuous data. The associations are modelled in the figures as non-linear for realization of confidence intervals and data visualization. Linear associations were reported using hazard ratio (HR) and odds ratio (OR) with a 95% confidence interval (CI), and significance of non-linearity was reported with Bias was addressed by the DAG, including all transplantations, testing endpoints for non-linearity, confounder adjustment, and sensitivity analyses for including only SPK recipients and for comparing different eras.The significance level was set at 5% and analyses were carried out as two-tailed. Analyses were performed using R software, utilizing survival and rms packages .From 2010 to September 2021, 11,919 pancreas transplantations were performed and recorded to the SRTR database in the United States. The final cohort of complete cases included 8,046 simultaneous pancreas and kidney (SPK) transplantations and 2,073 pancreas transplant alone (PTA) or pancreas after kidney (PAK) transplantations, as 311 DCD, 57 transplantations with unreliable procurement interval (>120 h), 449 pediatric recipient transplantations and 983 transplantations with missing brain death time, survival status or follow-up time were excluded.Median procurement interval was 41.7 h (IQR 32.1–54.3). The distribution is provided in p = 0.88, p < 0.001). The association remained independent in the adjusted model . For graphical purposes in the Kaplan-Meier curve  grafts were lost and 986 (9.7%) patients died. Altogether 593 (5.9%) were lost during the first 30 days and 832 (9.0%) during the first year. In a univariable Cox regression model the association of procurement interval with death-censored graft survival was linear (non-linearity er curve  the cohop < 0.001), but 30-day graft survival was not associated with procurement interval . As procurement intervals grew longer with time in the cohort, transplant year was added to the 1-year adjusted model as a confounder. In this model, transplant year and the interaction between procurement interval and transplant year were significant, and a longer procurement interval remained significantly associated with improved 1-year graft survival .In the adjusted model, longer procurement interval was associated with better 1-year graft survival  and linear  patients had an acute rejection episode before 1-year of follow-up. In a univariable logistic regression model the association of procurement interval with acute rejection within 1 year was significant , but not significantly associated with delayed graft function  or 1-year graft survival . For 30-day graft survival the association was slightly significant . When the association of procurement interval with outcomes was adjusted with inotrope use the association of procurement interval remained significant . The association also remained significant when the interaction between procurement interval and inotrope use was considered . The association of procurement interval with 30-day graft survival became significant when inotrope use and the interaction with procurement interval were analyzed .Donor inotrope use at the start of the procurement operation decreased with longer procurement interval . InotropIn this study, longer procurement interval was associated with improved long-term pancreas graft survival and fewer rejections within 1 year. Most importantly, a longer procurement interval posed no additional risk.Potential donors are seldom lost to cardiovascular collapse, possibly due to improved donor management protocols –9. EarliThis is outlined in recent retrospective studies which point to benefit in outcomes from longer procurement intervals in kidneys, livers, hearts, or lungs –18. ThisDonor inotrope use decreased with procurement interval, which could be expected with cytokine storm cooling and stabilization of the donor. It could also serve as a surrogate marker for instability. Interestingly, in sensitivity analyses the use of inotropes in the management of the organ donor was not significantly associated with pancreas graft outcomes. However, dichotomous inotrope use before procurement operation is probably insufficient as a marker for donor instability. Unfortunately, the use of three or more inotropes was only reported for a few patients.Acute rejections have not been associated with procurement times in other organs in earlier studies. The reason for this discrepancy remains unclear, but may on one hand be related to different granularity of reporting acute rejections to large registries, and on the other hand relate to variable sensitization of different organs during the process of brain death .Procurement intervals grew longer during the period of the study cohort, and possibly better donor treatment practices during the later years are associated with the better outcomes of longer procurement intervals. Therefore, sensitivity analyses by transplantation year were conducted, which showed the association of a longer procurement interval with improved graft survival to be significant only in recent years, and the association of less acute rejections to dissipate.The difference in procurement intervals between Europe and the US is notable, which may arise from scheduling procurement during office hours and more time consuming consent obtainment in the United States (Nijboer). Obviously much less concern about longer procurement intervals exists in the United States.This study is an observational registry analysis and therefore cannot prove causality. Retrospective studies can be susceptible to non-random allocation and confounding, and residual bias, which cannot be completely overcome by multivariable adjustment or sensitivity analyses. However, the use of the multivariable model and non-linear associations, together with the sensitivity analyses, provide greater confidence in the conclusion. The start of the procurement interval was defined by the declaration of brain death, although the exact time or progression of the fatal brain insult cannot be known. Similarly, the urgency of obtaining the diagnosis of brain death and declaration time and thus the start of the interval can vary between different centers and practises. However, this variance should be balanced in a large cohort. Graft failure for pancreas allografts has been uniformly defined in the US only from 2020 onwards, which can also have an influence on our findings. A lack of definition for pancreas graft function could have resulted in variation in reporting complications. Still, a large cohort can alleviate many of these concerns.A possible selection bias follows from longer procurement times distributing to more recent years. We sought to limit this with sensitivity analyses, which do not point to the effect resulting from better care in the later years, but did show significantly better results with a longer procurement interval only for later years in terms of graft survival, and no significance for acute rejections. Also, other short-term complications, such as graft thrombosis and leaks would have been of interest in this study, but are unfortunately outside the scope of the used registry data.A concern in optimizing other organs by lengthening the procurement interval is that more pancreas grafts would become edematous or firm and lead to more discard of the pancreas grafts. Before suggesting delaying procurement, information on discard rates with a longer procurement interval would be insightful.Future studies with randomization by procurement interval would be welcomed but could prove ethically challenging and unwarranted since so many other factors weigh first in organ procurement. Also, studies on how many donors are lost to cardiovascular collapse during the current era of donor management protocols would be of interest. These studies would have clinical implications for transplantation logistics and patient outcomes.In conclusion, based on this study, pancreas procurement from a brain-dead donor can be postponed if needed, and a longer procurement interval may lead to better outcomes.


Balantioides coli is a zoonotic parasite with a global distribution that inhabits the cecum and colon of humans and animals. This pathogen frequently causes diarrhea in weaned piglets undergoing the stress of transitioning from sow milk to pellet feed containing starch, leading to significant economic losses in the swine industry. Starch is necessary for the growth of the ciliated parasite. Here, our study revealed that B. coli is able to effectively consume and digest starch into glucose moieties. The molecular mechanism of the effect of starch on the growth and proliferation of B. coli by promoting the cell cycle and suppressing the autophagy of trophozoites has been elucidated. These findings will provide a novel insight to the pathogenesis of balantidial diarrhea in weaned piglets and an important reference for the control of balantiosis via adjusting the starch level in pellet feed.B. coli, supplying energy for cell growth and development. The research aimed at investigating the mechanism of starch on the growth and replication of B. coli. Single-cell separation was used to isolate single trophozoites of B. coli under a stereomicroscope, transcriptomic profiling was conducted based on the SMART-seq2 single-cell RNA-seq method. Comparative genomic analysis was performed on B. coli and eight other ciliates to obtain specific and expanded gene families of B. coli. GO and KEGG enrichment analysis were used to analyze the key genes of B. coli under the action of starch in the present study. The results of single-cell RNA-seq depicts starch affected the growth and replication of B. coli in two ways: (1) the cell cycle was positively promoted by the activation of the cAMP/PKA signaling pathway via glycolysis; (2) the cell autophagy was suppressed through the PI3K/AKT/mTOR pathway. Genes involved in endocytosis, carbohydrate utilization, and the cAMP/PKA signaling pathway were highly enriched in both specific and expanded gene families of B. coli. Starch can be ingested and hydrolyzed into glucose, in turn affecting various biological processes of B. coli. The molecular mechanism of the effect of starch on the growth and proliferation of B. coli by promoting cell cycle and inhibiting the autophagy of trophozoites has been elucidated in our study.Carbohydrates are the main source of nutrition for  Balantioides coli is an important zoonotic protozoan known to infect various hosts including humans, non-human primates, and pigs [B. coli can invade the colon and cecum of hosts and potentially cause intestinal diseases such as dysentery and even death [and pigs . It is tand pigs . The paren death , which ien death , where tB. coli mainly focus on the diagnosis and treatment of clinical cases and epidemiological investigations, while the interaction of B. coli with its hosts, and its biochemistry and pathogenicity are not well understood. Barbosa et al. added sterile rice starch to the xenic medium Pavlova modified by Jones, and B. coli isolated from pigs and non-human primates were maintained in vitro for more than two years. The trophozoite died at the early stages of culture in media without starch [B. coli was increased, and the survival time was elongated [B. coli and the parasite could use starch as its source of carbohydrates. Some previous investigations combined with our observations indicated that the prevalence and amount of B. coli in weaned piglets were higher than in other age groups [B. coli often cause diarrhea in weaned piglets when there is a sudden change of food from sow milk to pellet feed containing starch. Therefore, it is possible that the proportion of starch in feed will affect proliferation of the parasite in the large intestine of weaned piglets. However, the molecular basis of starch promoting the propagation of B. coli remains unclear.So far, studies of t starch . In our longated . These de groups . More imToxoplasma gondii, a small RNA expressed by this parasite may be related to the response of the host immune cells and the adaptation of T. gondii to its host environment. In addition, some transcriptomic studies have been reported in Cryptosporidium [B. coli. Recently, the single-cell RNA sequencing has gained substantial attention for its powerful ability to avoid cell heterogeneity when revealing gene expression and regulation in single cells. Here, both transcriptomic profiling based on single-cell RNA sequencing and comparative genomic analysis were performed to reveal the molecular mechanism of the effect of starch on the proliferation of B. coli in vitro.Transcriptomic analysis is a powerful tool used to reveal metabolic pathways and relationships among various functional genes in cells or parasites, and to screen key functional genes under specific conditions ,9. In trporidium ,11. To oB. coli strain P011 was originally isolated from a diarrheic fecal sample of the 43-day-old weaned piglet in Hongchang pig farm of Yichuan County, Henan Province used in this study, and the trophozoites were cultured in a modified DMEM medium at 28 °C as described previously. The detailed procedure and the composition of the modified DMEM culture medium were described previously [The eviously . WrittenB. coli under a stereomicroscope [B. The trophozoite concentration (A) was calculated according to the following formula: A = B × 50 × 10 [A mouth pipette that is an aspirator tube assembly for a calibrated microcapillary pipette  was used to isolate a single trophozoite of croscope ,13. The  50 × 10 . Three rFor transcriptomic analysis, ten cells were isolated from trophozoite samples at the peak of growth as the initial sample (labeled as IS subgroup); at 72 h post starch addition, ten cells from T group as the starch treating sample (labeled as TS subgroup), and ten cells from N group as the negative control sample (labeled as NS subgroup). The isolated cells were washed 3–5 times in RNase-free water . Ten celThe released RNAs were reverse-transcribed, template-switched, and amplified, and then cDNA libraries were constructed by modification of the SMART-seq2 single-cell RNA-seq method . The cDNhttps://github.com/jstjohn/SeqPrep, accessed on 5 August 2022) and Sickle v1.0  are as follows: (1) Filter out the adapter sequences in reads, and remove the reads that did not insert the fragments due to the adapter self-connection and other reasons. (2) Trim off the reads with N content more than 10% and the bases with low quality  at the end of the sequence (3’ end). (3) Discard adapters and sequences with less than 50 bp after trimming. After quality control, the statistical analysis and quality evaluation of the data were carried out again, and the clean reads were obtained.After sequencing, the raw data needed to be processed to obtain clean reads that could be used for subsequent analysis. The steps for filtering the raw sequencing data with software SeqPrep v 1.2  using Hisat2 (v 2.2.1) to obtain mapped reads for subsequent analysis. The gene expression values calculated by FeatureCounts software (v 1.6.0) were subsequently standardized by reads per kilobase per million (RPKM). To reduce the effect of genes with low expression levels, the genes with a change of RPKM value below 10 were firstly filtered. Then, the differentially expressed genes (DEGs) within the three comparison groups were determined using the “edgeR” package (v 3.39.6) according to the criteria of fold change ≥ 2 and FDR < 0.05 [The clean reads were aligned with the R < 0.05 . The DEGIn transcriptomic data analysis, clustering analysis is usually performed based on the number of genes to reflect the differences in gene expression between samples. Hence, in order to further explore the expression pattern among the three sets, DEGs of TS vs. IS, NS vs. IS, and TS vs. NS sets were combined for a hierarchical clustering analysis by the ‘pheatmap’ package of R software (v4.2.0).http://wego.genomics.org.cn/, accessed on 30 August 2022) was first used for visualizing, comparing, and plotting Gene Ontology (GO) annotation results for DEGs from three sets, and the BiNGo plug-in of Cytoscape software v3.9.1  was used to perform GO enrichment analysis using the default parameters. The false discovery rate (FDR) was used to correct the statistical significance  of each GO term. The protein sequences of all genes of B. coli were aligned to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database , and the KEGG Orthology (KO) results were obtained. According to the KO annotations corresponding to upregulated and downregulated genes, the metabolic pathways involved in the DEGs were searched by the KEGG Mapper tool .WEGO  utilizing the primers listed in B. coli  was sequenced using the PacBio Sequel sequencing platform. The de novo assembly of B. coli genome was conducted using software and tools including Canu v2.0, WTDBG , Quickmerge v0.3 , Numer and Pilon v1.24 , BUSCO v2.0 and CEGMA v2.5. The prediction and annotation of B. coli genome were completed by main tools and software including Augustus v2.4, GeMoMa v1.3.1, Hisat v2.0, TransDecoder v2.0, PASA v2.0, Infenal 1.1, BLAST v2.2, and Blast2GO. Finally, the B. coli genomic data have been obtained in the Parasitology Laboratory, Henan University of Science and Technology .The genome of Tetrahymena thermophila, Ichthyophthirius multifiliis, Paramecia tetraurelia, Stylonychia lemnae, Pseudocohnilembus persalinus, Oxytricha trifallax, Stentor coeruleus, and Halteria grandinella) were downloaded and used for comparative genomic analysis of B. coli. Gene family clustering of nine ciliate species was performed with Orthofinder v2.4 to identify specific gene families of B. coli [B. coli [B. coli were annotated with the PANTHER v15 database. Both GO and KEGG enrichment analyses were performed for specific and expanded gene families of B. coli using ClusterProfile v3.14.0.The genomic data of eight closely related ciliate species , Molecular Function (MF), and Biological Process (BP) groups were greater than the respective numbers of upregulated genes , while iMeanwhile, the GO functional enrichment analysis of BP was further elucidated. The significantly upregulated genes under starch treatment were involved in processes such as protein post-translational modifications (phosphorylation), and microtubule-based movement in the TS vs. IS set B, while To understand the dynamic changes of DEGs among the three subgroups, 11,451 genes in total were divided into four patterns (clusters 1–4) utilizing the hierarchical clustering method in the heatmap package. In B. coli under the action of starch, the specific DEGs of the TS subgroup were mapped to the KEGG database. KEGG enrichment analysis results showed a significant alteration in the 48 pathways . Meanwhile, the comparative bar plot for corresponding genes expression of the two methods were shown in detail  and 36 expanded gene families (including 510 genes) were detected in the genome of B. coli. The expansion and contraction analysis of gene families among the nine species were estimated across a phylogenetic tree  and other maltase were upregulated for expression in B. coli after starch treatment in this study. These findings imply that B. coli is able to produce a number of amylases and maltase for the hydrolyzation of starch into glucose, and then the generated glucose is transported to the right sites in the cytoplasm through glucose transporters. Our previous data suggest that B. coli may be a facultative anaerobic organism [B. coli.To develop a comprehensive overview of starch promoting the growth of organism . TherefoSaccharomyces cerevisiae, the cAMP/PKA signaling pathway is dually activated by glucose via glycolysis and the G-protein coupled receptor Gpr1 [S. cerevisiae [B. coli, the Gpr1-like receptor may not have occurred on the membranes of trophozoites due to the lack of growth or even death in the DMEM medium containing 4500 mg/L glucose without starch after 48 h post inoculation. Conversely, the trophozoites were able to grow well in the modified DMEM culture medium with 5 mg/mL starch. Consequently, we speculated that trophozoites failed to use extracellular glucose directly, and the intake and hydrolysis of starch may be the only way to acquire glucose by B. coli. In this study, genes related to the glycolysis and cAMP/PKA pathways such as PYG, pfk, Cdc25, and Cyr1 were enriched and upregulated in expression in the trophozoites of B. coli after starch treatment. The data imply that the product of glycolysis in B. coli, F-1,6-BP can activate the cAMP/PKA pathway and in turn regulate downstream biological process including the cell cycle [In tor Gpr1 . Glycolyrevisiae . In B. cll cycle ,20,21.Tetrahymena and other protists, elevated cAMP can directly activate Epac that indirectly affects AKT and PDK1 via PI3K. The phosphorylation of AKT promotes the upregulation of Cyclin and CDK; the former plays an important part in cell mitosis, while the latter is a considerable factor in cell cycle regulation in eukaryotes [B. coli after starch treatment. Our data support the hypothesis that the cAMP promoted by F-1,6-BP from glycolysis can activate the cell cycle of B. coli trophozoites via the related regulatory factors.In the cell cycle of karyotes ,23,24. Akaryotes , and SGKkaryotes . In addikaryotes . Here, gB. coli at 72 h post starch treatment. These regulatory factors were engaged in the suppression of autophagy of the trophozoites.cAMP depends on AKT in regulating autophagy. The PI3K/AKT/mTOR signaling pathway has been demonstrated to be the main pathway for cells to regulate autophagy ,29,30,31B. coli trophozoites after starch treatment combined with comparative genomic analysis, a hypothetical molecular mechanism model for the effect of starch on the growth of B. coli was constructed (B. coli can constantly propagate and grow in the culture medium containing the right starch level.According to the data of transcriptomic analysis based on single-cell RNA sequencing of structed  as folloB. coli. In fact, transcriptomic analysis still has certain limitations, including some of replicates in subgroups failing to be sequenced, and not directly proving whether the phenotype has changed, further multi-omics studies including transcriptomics and metabolomics with a greater sample size are needed to explain the complexity and integrity of biological processes of B. coli.To our knowledge, this is the first report of single-cell transcriptomic study of B. coli was supported through comparative genomic analysis and transcriptomic profiling. The molecular basis of starch promoting the growth and proliferation of B. coli was revealed by activation of the cAMP/PKA signaling pathway via glycolysis, which positively promoted the cell cycle, and suppression of cell autophagy through the PI3K/AKT/mTOR pathway in our study. These findings will be helpful for us to understand the pathogenesis of balantidial diarrhea and provide an important reference for the control of balantiosis in weaned piglets and other young animals by adjusting the starch level in their feed. However, the functions of the key genes regulating starch utilization, glycolysis, and other related metabolic pathways will need to be further confirmed using other tools such as gene knock-out strategy.Taken together, the effective utilization of starch in


Proso millet starch (PMS) as an unconventional and underutilized millet starch is becoming increasingly popular worldwide due to its health-promoting properties. This review summarizes research progress in the isolation, characterization, modification, and applications of PMS. PMS can be isolated from proso millet grains by acidic, alkaline, or enzymatic extraction. PMS exhibits typical A-type polymorphic diffraction patterns and shows polygonal and spherical granular structures with a granule size of 0.3–17 µm. PMS is modified by chemical, physical, and biological methods. The native and modified PMS are analyzed for swelling power, solubility, pasting properties, thermal properties, retrogradation, freeze–thaw stability, and in vitro digestibility. The improved physicochemical, structural, and functional properties and digestibility of modified PMS are discussed in terms of their suitability for specific applications. The potential applications of native and modified PMS in food and nonfood products are presented. Future prospects for research and commercial use of PMS in the food industry are also highlighted. Global production of cereal grains has reached record levels. Cereal grains play an important role in the human diet as a primary source of energy. The Food and Agriculture Organization (FAO)  reportedPanicum miliaceum), pearl millet (Pennisetum glaucum), and finger millet  [Millet belongs to the Poaceae family and is cultivated in subtropical and tropical regions of marginal drylands. Over 10,000 years ago, prior to the widespread consumption of wheat, maize, barley, and rice, this food item served as a staple for the people of that era. Currently, the most commonly cultivated species include proso millet  . Millet italica) . Nutritiitalica) . In addiitalica) .Panicum miliaceum L.) is also known as common millet, hog millet, Russian millet, and broomcorn millet in certain areas [Proso millet  and waxy (low amylose content) . Yang etA number of researchers conducted an analysis comparing various types of millet starches, but, unfortunately, they did not provide a thorough study of proso millet starch (PMS) ,16,17. AOur goal is to fill this information gap by conducting a comprehensive study that includes the latest and detailed information on the isolation, physicochemical, functional, and morphological properties, modifiability, and in vitro digestibility of PMS and to investigate its potential applications in novel foods. Our research will provide detailed insights and contribute to the existing body of knowledge on this topic.The starch granules within proso millet grains exhibit strong binding affinity to the surrounding protein matrix. Various methods and chemical reagents are used to extract starch and solubilize the proteins in the grain . General2) solution for 48 h at 52 °C. The soaked grains are then crushed with a blender, sieved through a 40-mesh sieve, and washed with water. The residual material is then crushed using a mortar and pestle and filtered through a 200-mesh sieve and then through a 270-mesh sieve. The residue is again washed with water, filtered through a Buchner funnel with No. 2 Whatman filter paper, centrifuged, and dried overnight at 45 °C [In the alkaline steeping method, the grains are soaked with 0.3% sodium hydroxide (NaOH) solution for 24 h at 4 °C. The soaked grains are then ground to a wet slurry using a mill and then sieved through a 100-mesh sieve. After this, the samples are centrifugated at 3000 rpm for 15 min, the supernatant is removed, and the remaining contents are resuspended in water. This washing step is repeated for a total of 3 cycles; the slurry is then neutralized with hydrochloric acid (HCl). After washing/neutralization, the starch cake is dehydrated at 40 °C for 48 h . In the at 45 °C .2S2O5 (0.5%) are used to isolate starch from these treatments. The use of these isolating solutions can significantly impact the chemical composition and characteristics of the extracted starch. In a comparative study between acid and alkaline steeping, acid steeping has a higher residual protein content (4.3%) than alkaline steeping (0.7%) [To extract starch from proso, pearl, kodo, foxtail, little, and barnyard millets, a practically neutral solution pH 6.5) containing a minute amount of either sodium azide (0.01%) or mercury (II) chloride (0.01%) is used to prevent bacterial growth and inhibit amylase activity [.5 contaig (0.7%) .The amount of millet starch obtained and the resulting chemical compositions differ significantly in the studies presented in The size of starch granules in millet varies depending on the plant species. Despite being generally spherical and polygonal in shape  was observed for native starch (2.62–34.88%), acid-modified starch (18.97–86.17%), and hydrothermally modified starch (1.71–12.45%) . The solIn the majority of cases, rheological evaluation of starch was carried out using both the Rapid Visco Analyzer (RVA) and the Brabender Visco-Amylograph (BVA), and the findings are presented in To), peak (Tp), and conclusion (Tc) gelatinization temperatures, as well as the enthalpy change (ΔH), are regularly recorded [To (64.6–71.1 °C); Tp (70.5–77.9 °C); Tc (77.4–82.3 °C); and ΔH (9.6–10.8 J/g). A higher gelatinization temperature indicates a perfect crystal structure of starch, while a higher enthalpy indicates that the gelatinization of starch requires more energy [The process of gelatinization occurs when starch granules are subjected to a specific temperature range and a sufficient quantity of water, leading to an order–disorder phase transition. Gelatinization is characterized by radial swelling of the granules, water absorption by the amorphous region, leaching of starch molecules, and collapse of the crystalline region with breakup of the double helices . Differerecorded . Table 4recorded . Variousrecorded . Gelatinrecorded . Thermalrecorded  in both e energy .To) and gelatinization enthalpy (ΔH) values than foxtail and pearl millets, but lower than finger millet. Retrograded millet starch, including retrograded pearl millet starch, typically exhibits reduced enthalpies and melting temperatures. This could be due to the fact that the stored gel exhibits unstable recrystallization of the branched chains of amylopectin compared to native gels [After gelatinization, when starch is cooled, the amylose and amylopectin molecules bind to each other, leading to recrystallization and forming a more structured organization than previously. Retrogradation is a process, which demonstrates the ability of starch to thicken and create rigid gels. Amylose content, water content, amylopectin’s molecular structure, storage parameters , and the presence of other elements  are all factors that influence retrogradation . Annor eive gels . Yang etive gels  stated tive gels . Li et aive gels  reportedive gels  conducteive gels .Starchy foods are exposed to multiple cycles of freezing and thawing during different stages of their preparation and storage. The freeze–thaw stability of starch is evaluated by measuring the quantity of water that separates from starch gels as a result of freeze–thaw cycles (syneresis) . Freeze–Starch digestibility is an important nutritional indicator that influences consumer perception of the acceptability or unacceptability of a product . The ratThe structure and functions of native starch depend on the plant from which the starch is obtained. There are some limitations of native starch that restrict its direct application in the food and nonfood industry, especially the low thermal stability and shear strength and the high degree of retrogradation . Notwith2SO4, H3PO4, and HNO3, resulting in an increase in the number of short starch chains, as found in amylose. Despite being commonly employed, the primary limitation of this technology is its heavy reliance on chemical agents, which can result in adverse environmental impacts [Acid modification is a chemical process used to modify starch that involves hydrolysis of the α-glucan chains through the use of mineral acids. This process results in the breaking of glycosidic bonds, leading to the alteration of the structure and properties of native starch . Acid hy impacts . In addi impacts . Compare impacts .Heat–moisture treatment (HMT) is a hydrothermal processing method carried out under high temperatures ranging from 90–120 °C, and low moisture levels of 35% or below. The process of HMT involves subjecting starch to a controlled temperature above its glass transition temperature for a specified duration, typically between 1 to 24 h, to achieve a low moisture content. The period of the procedure is dependent on the desired outcome and specific treatment process . AnnealiTo) and peak (Tp) gelatinization temperatures and a decrease in gelatinization enthalpy (ΔH). A decrease in ΔH could be attributed to a decrease in crystallinity in the starch granule after DHT, which was determined by X-ray diffraction [DHT is a form of physical modification that induces alterations in the physicochemical properties of starch, while preserving its granular structure. Dry heat represents a simple, nontoxic, and healthful substitute for chemical-based techniques. The new properties that occur with thermally treated starch are similar to the results of the chemical crosslinking process. Dry heat improves the pasting and functional properties of starch as effectively as the chemical alternative . In the fraction .UHP is a nonthermal modification approach that can be employed to induce gelatinization or physical modification of diverse starch types. The degree of gelatinization attained through UHP, as an alternative to conventional thermal processing, is influenced by several factors, including starch type, pressure levels, water content, temperature ranges, and duration of treatment. UHP completely gelatinizes starch when it is under sufficient pressure at specified ambient temperatures . The useH) of the fermented PMS increased compared to that of the nonfermented PMS, reflecting the increase in crystallinity after fermentation.Through the process of fermentation, it is feasible to alter the chemical and physical characteristics of starch. This methodology serves to augment the structural attributes of starch. Bian et al.  conducteDual modification of starches is gaining popularity in current scientific research. This is because single modification methods may not always fulfill the necessary functional requirements for food and industrial applications. Dual modification of starch offers the ability to customize starch properties for specific applications and to enhance the functionality of single modified starch, thereby increasing the range of applications for starch. Sun et al.  studied Starch is a versatile biomaterial with a wide range of applications across the globe in industries such as food, pharmaceuticals, textiles, and various other industrial sectors. The physicochemical and functional properties of starch determine its role in several industries. Both native and modified forms of millet starch are utilized in several industries, especially in the food industry. However, native millet starch has limited functionality and, as a result, has fewer industrial applications. Native and modified starches are commonly employed in the food industry as thickeners, binders, fat substitutes, gelling agents, and stabilizers for emulsions and foams . The impMillet is a crop that is typically grown in semiarid regions where traditional cereals struggle to grow. It possesses a wealth of health-promoting properties that are unfortunately neglected, resulting in millet not being widely accepted as a food in its own right by the commercial food industry. Starch is a key component of millet, and yet this too is underutilized as a source in preference to other conventional sources of starch. The functional properties of millet starch are similar to those of other starches, as it can act as a structure provider, texture modifier, binder, and viscosity regulator.PMS has been underutilized and under-researched in comparison to major cereal and tuber starches that have been extensively studied. Research carried out on the modifications of starch depends on the wide range of targeted consumer products by for-profit companies and is motivated by desired functionalities rather than commercial distribution of the starch. Research on the modification of PMS using such methods as chemical, physical, and enzymatic treatments (or a combination of these methods) still have areas that remain to be investigated; due to this, its commercial application is disproportionately limited given its equivalent biological performance. Physical modification of all starches has gained popularity in recent years because it is nontoxic, hygienic, and environmentally friendly. In the search for better methods, novel physical modifications such as pulsed electric field processing, ultrasonication, cold plasma treatment, and irradiation can be used. This provides the opportunity to explore the physicochemical properties of PMS  and categorize them using laboratory research methods and the full range of our advanced scientific instruments to reveal the unique properties of this starch. While it is acknowledged that PMS can be used as a stabilizer, thickener, and fat substitute in various food products, there is still room for further exploration of its potential applications in the food industry. In particular, there is potential for increased use of PMS in the formulation of functional foods as well as in products such as bakery items and pasta. Beyond the food sector, PMS can also be used in other industries, and a comparative study with the predominantly used conventional starches will certainly open up new areas of functionality.Therefore, PMS is an excellent alternative for the many industrial applications currently covered by the more widely used starches . Due to its initial poor functionality and lack of known modifications, PMS is currently rarely used in its original form. However, various agencies and research studies have found that starch modification can produce the desired properties and characteristics in the targeted foods. All starches are rarely used in their native form due to their low functionality, and most starches must be modified prior to commercial use . This is similar to the circumstances surrounding PMS and cannot explain the clear bias against PMS. At this point, it can also be noted that all future research will bring newly discovered modifications that can make new contributions to structural changes in starch. This, in turn, could change a negative starch property for the better.These treatments, past, present, and future, have the common denominator of making structural changes to starch and bringing benefits to our modern lifestyle. The connection between the native starch properties and the modified starch structure can be better understood with the use of several techniques such as spectroscopy, microscopy, XRD, and calorimetry. Currently, access to these scientific tools for PMS is limited. The information that will result from greater access to scientific methods will contribute to the development of PMS-based foods that are beneficial to both lifestyle and health. This review provided a comprehensive overview of the properties of PMS; by highlighting the value-added uses of PMS, its equivalent properties, and its potential future advances, PMS can be developed as a highly utilized starch in related industries.


Intestinal transplantation (ITx) remains a lifesaving option for patients suffering from irreversible intestinal failure and complications from total parenteral nutrition. Since its inception, it became obvious that intestinal grafts are highly immunogenic, due to their high lymphoid load, the abundance in epithelial cells and constant exposure to external antigens and microbiota. This combination of factors and several redundant effector pathways makes ITx immunobiology unique. To this complex immunologic situation, which leads to the highest rate of rejection among solid organs (>40%), there is added the lack of reliable non-invasive biomarkers, which would allow for frequent, convenient and reliable rejection surveillance. Numerous assays, of which several were previously used in inflammatory bowel disease, have been tested after ITx, but none have shown sufficient sensibility and/or specificity to be used alone for diagnosing acute rejection. Herein, we review and integrate the mechanistic aspects of graft rejection with the current knowledge of ITx immunobiology and summarize the quest for a noninvasive biomarker of rejection. Intestinal transplantation (ITx) remains a lifesaving option for patients suffering from irreversible intestinal failure and complications from total parenteral nutrition. Since the technique was established and became a clinical option in the late 1990s, it has been recognized that one of the outstanding features of this procedure was the strong allogenic response triggered by the graft, which has to be contained using more aggressive immunosuppressive regimes compared to other types of solid organ transplantations . ConsideTraditionally, it has been considered that there are several possibilities to elicit an allogenic immune response that will contribute to graft rejection . The so-Some years later, the semi-direct pathway was described ,17,18, wIrrespective of the pathway that is eliciting the allogenic response, one feature that has been clearly established in experimental models of organ transplantation is the importance of the secondary lymphoid organs as sites of allorecognition and T cell activation. In the absence of secondary lymphoid organs in mouse models, the kinetics of alloresponse are much slower or absent, depending on the type of solid organ considered . ITx hasAdditionally, in human ITx, it has also been shown a fast turnover (more than 50% of the population at days 3 to 5 post-transplant) of the recipient-derived lymphocytes emerging through the mesenteric lymph  and alsoRecent evidence indicates the existence of a new activation pathway, the so-called inverted direct pathway, that is responsible for early de novo DSA formation. This pathway operates by the interaction of donor-derived CD4+ T lymphocytes with recipient B cells that may present in the context of recipient MHCII molecules and consequently act as direct alloactivators of donor T lymphocytes, operating in a way similar to the direct pathway with the particular feature that, in this case, donor vs. recipient cell roles are inverted . From anIn the past few years, with the possibility of performing sequencing techniques that allow the identification of individual T cell clones and a clever combination of allostimulation experiments , pioneerResident memory T cells (Trm) are a subset of memory cells phenotypically and transcriptionally different from circulating effector and central memory T cells, which are mainly characterized by their non-circulating behavior, remaining long term, particularly in barrier organs such as skin, gut, lungs and reproductive tract, with the subset numerically the largest T cell memory subset. In recent years, it has been clearly established that in addition to its role in infectious immunity, this subset can be particularly relevant in the process of graft rejection . InteresAuthors have also transcriptionally and functionally characterized the profile of the alloreactive CD4+ Trm, which are mainly Th17-like cells in this model. In the field of clinical ITx, Kroemer et al. (2021) recently described Th17 cells as central players in keeping the progress of thymoglobulin-resistant graft rejection . The chaFurthermore, considering the above-mentioned two-way alloreactivity pathway, it is important to consider that the intestinal graft harbors an important population of resident memory T cells ,42 that Although the main drivers of alloresponse have been characterized, as described above, there are several mechanisms that have been described on homeostatic/physiopathogenic circuits in the intestinal mucosa that have not been explored in the context of intestinal transplantation.MHC class II expression by intestinal epithelial cells (IECs) was described in the 1980s ,44, and As of today, there are no noninvasive biomarkers showing adequate sensitivity and specificity to stand alone as diagnostic assays for intestinal acute cellular rejection (ACR). However, a number of biomolecules have been suggested as potential rejection markers, with some of them, such as plasma citrulline or fecal calprotectin, which are in clinical use. These biomolecules show altered expression during ACR, either due to tissue injury, the accompanying immune response activation and/or bystander inflammation or the ensuing metabolic changes in the rejecting intestinal graft . UnfortuBelow, we summarize the most significant findings and hypotheses explored so far according to three main pathophysiological mechanisms: direct graft injury secondary to the acute rejection process, local and systemic inflammation triggered by the alloimmune response and altered local metabolism following damage to the intestinal graft, explored via high throughput technologies. Many molecular events and subsequent cellular and molecular alterations are intertwined and dependent on the intensity and gravity of the immune response.Intestinal mucosa is the main target of the rejection process, and ultimately enterocyte loss ensues. Circulating levels of citrulline, a nonprotein amino acid produced mostly by enterocytes as the end product of glutamine metabolism, have been suggested as a measure of functional enterocyte mass. Citrulline is stable and relatively easy to analyze in plasma and dried blood spot samples . In ITx Several other biomolecules involved in enterocyte metabolism have been explored in the setting of intestinal ACR. Rodent studies have indicated that the intestinal fatty-acid binding protein (I-FABP), present primarily in mature, villus enterocytes which are normally undetectable in the serum, increased during ACR (196 ± 24 ng/mL), and rejection treatment with cyclosporine consistently reversed rejection and decreased I-FABP in rats . HoweverThe activity of histamine-degrading enzymes diamine oxidase and histamine N-methyltransferase (HNMT) was found to decrease in the mucosa of rejecting rat allografts and revealed a strong negative correlation with the histological rejection score, whereas isografts showed rather constant tissue enzyme activity up to eight days after ITx . These tEnterocyte apoptosis is a central mechanism of tissue injury during intestinal ACR as one of the key effector mechanisms of cytotoxic T lymphocytes (CTL). Its increased occurrence is ubiquitous in different species, and its magnitude correlates with the severity of rejection ,63,64. IIncreased serum levels of suppressor of tumorigenicity 2 (ST2), a regulatory decoy receptor of IL-33, have been observed during inflammatory bowel diseases . In a smA recent murine study using proteomics revealed significant alterations in the tissue expression of ninety-four proteins in the intestinal grafts that developed moderate rejection, compared to non-rejecting isografts . InteresThe initial inflammation in the early stages of allorecognition initiates multiple phenotypic changes in the graft. Earlier experimental studies have indicated numerous alterations, including progressive mucosal pro-inflammatory activation ,72, heatCytokines and their receptors play key roles in the activation and propagation of the alloimmune response through their chemotactic effects, inducing the expression of adhesion molecules, promoting the proliferation and differentiation of specific alloreactive T and B cell clones or having a direct cytotoxic effect on the allograft cells ,76. SeveIn spite of the early enthusiasm around the potential of various cytokines to diagnose intestinal ACR, there seems to be a gap between the experimental and clinical data, and only a few clinical reports have been published and are based on a small number of analytes and patients ,82,83,84A small pilot study found an increase in plasma regenerating islet-derived 3-alpha , a C-type lectin antimicrobial peptide synthesized by enterocytes and Paneth cells, during intestinal acute rejection . As the A relatively recent and still evolving paradigm in the follow-up of ITx patients is the monitoring of the development of humoral immunity against the graft. De novo production of antibodies against the graft’s human leukocyte antigens (HLA), the so-called donor-specific HLA antibodies (DSA), has been linked with a higher risk of ACR in several types of organ transplants, including ITx ,92,93,94A different and more pragmatic approach has been represented in the analysis of the ostomy effluent. Whereas this approach is less used in rodents, it allows easy, non-invasive access to abundant biological material originating in or having direct contact with the intestinal mucosa, the same site where rejection occurs. Fecal calprotectin levels (FCL) have been routinely used in practice over the last few decades to initially differentiate between inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), as well as to assess mucosal healing or the recurrence of inflammation in the follow-up of IBD patients. Calprotectin is a calcium- and zinc-binding protein of the S-100 protein family, mainly found within neutrophils, and its presence in feces is due to neutrophil infiltration into the gastrointestinal tissue secondary to an infection or an inflammatory process. Fecal calprotectin is homogenously distributed in stools and is very resistant to in vivo degradation  and in vitro degradation . The first article on the analysis of calprotectin in the stomal fluid of intestinal transplant recipients reported increased FCL both during ACR rejection and non-specific enteritis and found an optimal cut-off level for calprotectin in predicting the presence or absence of intestinal allograft rejection at 92 mg/L with 77% specificity and 83% sensitivity . This inA combined approach using a multiplexed analysis of 17 cytokines and high-throughput proteomics of the ostomal effluent of 16 intestinal transplant recipients found increased levels of five innate immune cytokines early post-transplantation -8, tumor necrosis factor (TNF)-alpha, IL-1beta and IFNγ), but only IFNγ levels were significantly higher in samples with rejection . ProteomThe presence of a very large, complex and dynamic microbial microenvironment makes the intestinal graft unique. The recent advances in sequencing technology have allowed the high-resolution analysis of the intestinal microbiome, unraveled the profound effects of intestinal microbiota on their hosts and involved the occurrence of multiple, highly diverse diseases, such as asthma , diabeteIn a clinical study on 19 ITx recipients, comparisons between samples from non-rejecting ITx and ITx with ACR revealed major alterations in the proportions of multiple bacterial taxa associated with active rejection . At the Although a direct cause–effect relationship between the microbiome alterations (including the enterococcus predominance) and the ACR and its role in graft monitoring remains to be established, these initial findings have important potential clinical implications in terms of patient management, as future targeted strategies to control the degree of intestinal Enterococcus colonization could have a beneficial impact on graft and patient outcome.The last four decades of experimental and clinical research have identified hundreds of molecules that may be altered during the intestinal ACR, with some having the potential to assist with its diagnosis, prognosis and follow-up. Unfortunately, very few, if any, have shown true value in a clinical setting. This may be due both to the differences between experimental and clinical settings, the small size and inherent heterogeneity of clinical studies, as well as the technical and biological limitations of the approaches used. Nowadays, it is believed that either an entirely new, intestine-specific assay or a panel of less specific tests would be necessary to increase the accuracy of non-invasive rejection monitoring. The application of new analytical and bioinformatics techniques would allow a comprehensive analysis of complex biological samples and the simultaneous detection of a large number of chemically diverse analytes. This has already been applied in kidney transplantation, where a composite metabolite–mRNA signature in urine was diagnostic of ACR with high accuracy ,112. In In 36 ITx patients receiving different types of visceral allografts, metabolomics of the stomal effluent or feces demonstrated different metabolomic profiles between rejection and nonrejection . A totalA second high-throughput analysis using quantitative proteomics with iTRAQ-labeling and mass spectrometry performed in rejecting murine intestines and mentioned earlier  identifiThe intestinal graft appears to have a unique immunology due to its contact with external antigens and the exposure to the intestinal microbiota, the gut-associated lymphoid tissue following with the graft, as well as the abundance of epithelial cells. Graft rejection occurs more frequently than in any other type of organ transplant, in spite of potent immunosuppression, and may rapidly result in the loss of the mucosal barrier, life-threatening sepsis and patient death. At the same time, the transplanted intestine lacks reliable, specific, noninvasive biomarkers, allowing non-invasive rejection surveillance. As most of the non-invasive assays tested to date in ITx are the same as those used in the management of inflammatory bowel disease, they all lack specificity by default.Future research should continue the quest to improve our understanding of two-way allogenicity, with an emphasis on the generation of regulatory populations that may reduce the burden of immunosuppression. Additionally, further research efforts should continue to explore the development of assays capable of determining the involvement of memory T cells in the graft rejection process and their potential translational use. Likewise, advances in the monitoring of other types of allografts, such as circulating cell-free donor  DNA or the alterations in the microbiota that may herald or favor ACR, should be tested in adequately sized, multicenter studies on either prospectively collected material or on existing biobank samples. The continuous quest for the identification of well-defined alterations occurring during rejection in the main targets of the immune response, that is, the intestinal mucosa and its microenvironment, could ultimately reveal the long-sought and badly needed rejection biomarker.


This study presents an initial study on the adaptive bistable stiffness of the hair cell bundle structure in a frog cochlea, and aims to harness its bistable nonlinearity that features a negative stiffness region for broadband vibration applications such as vibration-based energy harvesters. To this end, the mathematical model for describing the bistable stiffness is first formulated based on the modeling concept of piecewise type nonlinearities. The harmonic balance method was then employed to examine the nonlinear responses of bistable oscillator, mimicking hair cells bundle structure under the frequency sweeping condition, and their dynamic behaviors induced by bistable stiffness characteristics are projected on phase diagrams, and Poincare maps concerning the bifurcation. In particular, the bifurcation mapping at the super- and sub-harmonic regimes provides a better perspective to examine the nonlinear motions which occur in the biomimetic system. The use of bistable stiffness characteristics of hair cell bundle structure in frog cochlea thus offers physical insights to harness the adaptive bistable stiffness for metamaterial-like potential engineering structures such as vibration-based energy harvester, and isolator etc. The primary function of the hair bundle structure is to send biologically induced electrical impulse signals to the brain in response to the vertical oscillation produced by the traveling wave propagation on the basilar membrane of the cochlea, as shown in Fig. Stereocilia that lean on each other with tip links in the hair cell, as shown in Fig. 2. Furthermore, calcium ion (Ca2+) concentration plays an essential role in impulse signal transduction because the channel’s open probability is determined by the hair bundle displacement and calcium ion concentration. As illustrated in Fig. Stereocilia of the inner hair cells are linearly arranged, whereas outer hair bundles are arranged in a V-shaped pattern. These different morphologies are likely to reflect the distinct functions of inner and outer hair cells. Outer hair cells can amplify small oscillations, and therefore, significantly enhance the sensitivity and dynamic range of hearing. In contrast, inner hair cells do not amplify but transmit electrical signals to the auditory-nerve fibers4.The cochlea present inside the inner ear is one of the primary auditory organs in which sound is transduced from acoustic energy into an electrical signal. Its sensory receptors are called hair cells and feature densely bundled hair structures7. The dynamic oscillator with this nonlinearity is referred to as a bistable oscillator, and it has a unique double-well restoring force potential and provides three distinct operating regions depending on the input amplitude. This bistable oscillator may be excited to exhibit aperiodic or nonlinear vibrations between the two wells . Thus, numerous studies have attempted to harness the bistability featuring a snap-through  action in various engineering applications, such as energy harvesting devices, acoustic transducers, and fluidic sensors11.As shown in Fig. 12. Hence, even though a sinusoidal stimulus is applied in a specific frequency range, the transferred sound signals can be amplified owing to the mechanical adaptation ability that shifts the region of highest sensitivity toward the active operation range of the hair cell structure. Although the detailed mechanism by which mechanical energy is amplified by the nonlinearity of hair cells describes well5, some relevant studies have still attempted to further identify the amplification mechanism15. In this study, we mimic only its mechanical property featuring a negative stiffness region and adaptation ability. The primary objective of this study is thus to investigate the nonlinear dynamic behavior of a bistable oscillator with adaptive bistable stiffness, mimicking hair cell bundle structure in a frog cochlea, and aims to harness its bistable nonlinearity that features a negative stiffness region for broadband vibration applications.To date, it has been hypothesized that bistability is harnessed to amplify mechanical auditory stimuli in hair bundle structures. The sensitivity of hearing can be enhanced by the mechanoelectrical phenomenon observed in the outer hair bundle structures, which can amplify hair cell motion over broad frequency ranges. This high sensitivity is also hypothesized to exist because of the combination of an adaptation and a negative stiffness property inherent in the hair bundle structuresS-shaped) function has been proved in many prior research16.kb(i), such as kb1 and kb2, stiffness values for each stage; xbpr = xb − xpr; xpr, preload location; FS, total spring force; FS1, spring force without the preload; FSpr, preload; Fbp(i), spring force on the positive side; Fbn(i), spring force on the negative side; νp(i), transition displacement on the positive side; νn(i), transition displacement on the negative side; σb, smoothing factor. Thus, arbitrary piecewise-type nonlinearities of the bistable stiffness profiles can be determined by employing their numerical values. Case 2 in Fig. 11. For different piecewise-type nonlinearities, multiple profiles can be produced using Eq. (2), as shown in Fig. σb is set as 5 × 103. Table FSpr and xpr based on the symmetric case, such as in Case 2. For example, Table FSpr and xpr to determine three different cases of nonlinear stiffness profiles, as shown in Fig. m = 0.015 kg; damping coefficient c = 51.6 N s m−1, assuming that the employed modal damping ratio, ζ, and natural frequency, ωn (fn), are 5% and 516.4 rad s−1 (82.2 Hz) respectively. Additionally, the natural frequency ωn is obtained using the positive stiffness value kb2 = 4 × 103 N m−1.In this study, the nonlinear stiffness characteristic of the hair cell bundle structure is represented by the nonlinear spring element of a 1-DOF vibrating system , as shown in Fig. entclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minimaentclass1pt{minima19. First, the system response xb(t) and the input Fb(t) can be considered as follows:xm, xak, and xbk are the mean and alternating parts of the cosine and sine functions for the system responses, respectively; Fm is the average force (0.1 N); k are the sub- and super-harmonic indices, respectively; Nmax is the maximum number of harmonics correlated with the harmonic index of the HBM. Fpk is the magnitude of the sinusoidal input force and selected to be 7 N such that it can induce the chaotic interwell vibrations8; Assuming that the system is in a steady state, the Galerkin scheme in Eq.  was implemented in this study based on the Galerkin schemes because all the information of the system responses including the stability conditions in both time and frequency domains under the steady state conditions is efficiently obtained by employing the HBM and it has been known as one of the powerful tools to analyze the strongly nonlinear stiffness systemHere, its relevant terms are defined as follows.Furthermore, its nonlinear and input functions are defined as follows.k and l represent the incremental index where The relevant variables used are: n of Eq.  is expre16.To determine the solutions of Nmax for the HBM are 2 and 12, respectively.The HBM results with the root mean square (RMS) values of the displacement along with three different bistable stiffness curves are compared in Fig. 22. To reveal the sub-harmonic responses in greater detail, the values for Nmax of the HBM were 6 and 12, respectively. A greater number of complex sub-harmonic responses could be obtained with an increase in the number of sub-harmonic indices 25. The details of the super- and sub-harmonic responses are shown in Fig. 22. Thus, the NS can reflect all possible dynamic behaviors with time variations. However, the HBM determines its solutions efficiently by estimating the frequency and time domain information with the integer-based Fourier expansion, even though it cannot include all the possible time histories, such as transient responses24. Based on two different analytical approaches, the super- and sub-harmonic responses induced by bistable nonlinearities can be investigated in detail.Figure 18. To analyze the dynamic responses in detail, the time histories can be reviewed in terms of two specific locations, The nonlinear dynamic characteristics of the NS and HBM were compared with the bifurcations in the super-harmonic regime, as shown in Fig. mentclass2pt{minimIn this regime, the time histories exhibit significant complexities, as shown in Fig. In addition, the complexities of the dynamic behaviors could be efficiently examined from the phase diagrams and Poincare maps. Figure The nonlinear dynamic behaviors in the sub-harmonic regime are shown in Fig. The dynamic characteristics of the phase diagrams and Poincaré maps are shown in Fig. 27. Once we fabricate a prototype that mimics the adaptive bistable stiffness of the hair cell bundle, this new adaptive structure enables the development of metamaterial-like broadband vibration applications, such as vibration-based energy harvesters, despite some technical issues that need to be further examined.Based on the nonlinear frequency response analysis, we propose the desired frequency response function (gray dashed line) tuned by adaptively changing the bistable stiffness, as shown in Fig. First, the bistable nonlinearity using the hyperbolic tangent function was effective to avoid instability induced when the discontinuity is connected with the piecewise linear functions when it combines with the harmonic balance method.Secondly, we investigated all possible nonlinear dynamic characteristics of bistable oscillator by examining the nonlinear frequency responses, phase diagrams, and Poincare maps.Lastly, we report for the first time that it is possible to achieve a new means of producing broadband  frequency response functions by mimicking the adaptive bistable stiffness of hair cell bundles.In this study, the dynamic behavior of bistable oscillator with adaptive bistable stiffness, which is similar to the hair cell bundle structure in a frog cochlea, was successfully investigated. The main contributions of this study are summarized as follows.Overall, it is necessary to implement the proposed bistable oscillator, primarily focusing with adaptive stiffness switching mechanism although our study provides the initial information through the simulation for designing broadband vibration applications such as vibration-based energy harvesters. The adaptive bistable stiffness will be explored by combining different preload adjustment mechanisms with smart-material-based actuators. For the future research direction, we will continue to address some ongoing issues. In particular, further studies such as in-depth bifurcation analysis by mapping the Floquet multipliers are required to complete the nonlinear frequency–response analysis.