The results showed that waxy corn starch (WCS) and corn starch (CS) have the highest digestibility, while high amylose corn starch (HACS) has a higher content of resistant starch (RS). Besides being related to amylose content, RS content is also closely related to particle shape, debranched fine structure, molecular structure, and semi-crystalline structure. HACS can maintain a complete particle structure after gelatinization and enzymolysis; differential scanning calorimetry showed a positive correlation between gelatinization enthalpy and RS content. As the amylose content increased, the content of fa (DP 6–12) decreased, while the content of fb2 (DP 25–36) and fb3 (DP ≥ 37) increased, which in-turn decreased the cluster polymer formed by short branch chains, and the formation of more hydrogen bonds between long chain branches improved starch stability. D, which characterizes the compactness of starch semi-crystalline structure, increased with the increase of RS content. HACS 60 with the highest RS content had a unique surface fractal structure between 7.41 < d (2π/q) < 10.58 nm, indicating that the dense structure is effective in maintaining the RS content.In this study, the  Starch is one of the most important carbohydrates in human life, with different applications in industries such as the food, petroleum, pharmaceutical, paint, and cosmetic industries, among others . Corn isRS has been classified into five general subtypes: RS1–RS5 . Differein vitro digestibility, provide accurate target structures for gene breeding, and reduce the difficulty of gene breeding to regulate the RS content of corn starch.HACS has always been an important RS, but the principle of amylose increasing starch RS content needs further study. This experiment studied the changes in particle shape, molecular structure, crystal structure and fractal structure brought about by amylose, and its influence on RS content. The results obtained from these studies were used to establish the correlation between structural features and Aspergillus niger, ≥ 300 U/mL), pepsin , invertase , and isoamylase  were purchased from Sigma-Aldrich ; The glucose oxidase-peroxidase (GOPOD) kit was purchased from Beijing Leadman Biochemistry Co., Ltd. ; Other reagents were purchased from China National Medicines Co., Ltd.  and were of analytical grade.Waxy corn starch  was purchased from COFCO Corporation ; Corn starch , HACS 70 , and HACS 80  were purchased from AnHui King Corn Agriculture Science and Technology Development Co., Ltd. ; HACS 50  was purchased from Cargill Investment Co., Ltd. ; HACS 60  was purchased from Yiruian Food Ingredients Co., Ltd. ; Pancreatin , amyloglucosidase ; V is the total volume of the test solution; D is the dilution factor; As is the absorbance of the standard solution at 520 nm; w is the weight of the sample used for analysis (mg), which can be corrected for moisture content using Eqs.  of the sample.Samples  were weighed, 2 mL of water was added, and the samples were gelatinized at 100°C for 30 min. The gelatinized samples were freeze-dried and dispersed in an aqueous solution (glycerin:water = 1:1). The polarized light birefringence cross was observed under a polarized light microscope .in vitro was performed. The products digested for 120 min were collected, ethanol precipitated and freeze-dried. According to the reported method  using a Tech Master analyzer   using the method already reported . The temThe debranched chain length distributions of samples were determined using the method already reported , with sow) of the samples.The weight-average molecular weight of the samples were measured using gel permeation chromatography (GPC) according to a previous method . After pAccording to the method already reported , the cryThe semi-crystalline structure was determined by Small angle X-ray scattering (SAXS) analysis . SAXS meThe data were statistically analyzed using IBM SPSS Statistics v20 software . Results were expressed as mean ± standard deviation.in vitro digestive properties of corn starch with different amylose content are shown in in vitro digestibility of HACS ranged from 45 to 80%, indicating that HACS has strong digestion resistance. The RS content values of RDS, SDS, and RS for the samples are listed in The results of the It is worth noting that the sample with the highest RS content is HACS 60, which has an RS content of 49.10%. This result shows that besides the amylose content, some factors affect the RS content of starch , peak gelatinization temperature (Tp), and conclusion (termination) temperature (Tc) of HACS were significantly delayed, and the gelatinization temperature range (Tc–To) increased significantly. These results concur with those reported for WCS (H) of HACS increased, the maximum ΔH was 18.24 J/g. Comparing the content of resistant starch in To, Tp, Tc, and ΔH of HACS showed a consistent increasing trend, which indicates that the RS content of starch is closely related to the thermal stability of starch. Besides amylose content, starch digestibility is still affected by the orderly spiral structure between amylose molecules  and low degree of branching [fa/(fb1 + fb2 + fb3)] in amylopectin are associated with the digestibility of starch. The long chains in amylopectin can form longer double helices and strengthen the hydrogen bonding force between the chain segments, making the structure more stabilized and increasing the RS content of starch. However, the short branches of amylopectin make the layered structure of the crystal unstable.According to existing report , the debw) of the samples continued to decrease. The maximum and minimum Mw of the samples were 2.23 × 107 and 5.03 × 105, respectively. HACS obviously had a narrow Mw  was lower, and the content of fb3 (DP ≥ 37) was higher, which is consistent with the results in I = q−α, where I is the SAXS intensity and α is the index which can calculate the D value of the surface/mass fractal structure, and can be obtained from the slope of the log-log SAXS diagram. When 1 < α < 3, the scatterer is a mass fractal, which is relatively loose. For fractal index Dm = α, the closer Dm is to 1, the looser it is, and the closer to 3 the denser; when 3 < α < 4, the scatterer is a surface fractal. Relatively dense, rough surface, the fractal index Ds = 6–α is generally between 2 and 3. The closer Ds is to 2, the smoother the surface, whereas the closer to 3, the rougher the surface. According to the Woolf-Bragg formula d = 2π/q, the scattering peak of about 0.6 nm−1 in the SAXS curve can calculate the semi-crystalline layer thickness d of starch granules. The scattering object of the surface fractal is more compact than the scattering object of the mass fractal.The double logarithmic SAXS pattern of corn starch with different linear contents is shown in q = 0.2–0.4 nm−1, which may be caused by the tight rearrangement of amylose  and CS (Dm1 = 1.01) were characterized between 17.20 < d (2π/q) < 43.77 nm. HACS 50 (Dm1 = 1.28), HACS 70 (Dm1 = 1.11), and HACS 80 (Dm1 = 1.16) were characterized between 17.20 < d (2π/q) < 37.04 nm. HACS 60 (Dm1 = 1.44) was characterized between 12.67 < d (2π/q) < 57.09 nm. These data indicate that the scattering objects in HACS 60 samples are more compact and the mass fractal structure is formed in a large range. Interestingly, for HACS 60, the surface fractal structure is observed to be between ~7.41 < d (2π/q) < 10.58 nm, and the fractal dimension Ds2 = 2.46, which indicates that the scattering objects at the lower scale level are dense and smooth surfaces. As shown in q = 3.5, which is a unique phenomenon of type B crystals (HACS 60 has a shoulder-like structure at  amylose , which mh scales . As prescrystals . This foin vitro digestibility of corn starch with different amylose content. In addition, the factors affecting digestibility were discussed, including particle morphology, debranched chain fine structure, molecular structure, and semi-crystalline structure. The results showed that the higher the amylose content, the higher the RS content. The lower the fa content, the higher the fb2 and fb3 content, and the more stable the starch structure, the higher the RS content. In addition to amylose content, the arrangement of amylose also has an important influence on RS content. In the quality fractal, the larger the Dm, the higher the RS content. The shoulder structure is the special structural factor contributing to the highest resistance of HACS 60, and the dense surface fractal is also one of the biggest factors for resistance in starch. Understanding the relationship between the structural factors affecting HACS resistance and RS content will help us to further cultivate corn starch with specific RS content.In this study, we determined the The original contributions generated for the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.XL: experimental arrangement and writing. YH: conceptualization, methodology, and supervision. QZ and CJ: analysis and literature review.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.


There were no statistically significant differences between tacrolimus C/D ratios of patients carried with SLCO1B1 rs2291075 CT, TT genotype donors, and those carried with SLCO1B1 rs2291075 CC genotype donors. A model consisting of tacrolimus daily dose, total bilirubin, FIS classification, and recipient SLCO1B1 rs2291075 could predict tacrolimus C/D ratios in the convalescence phase by multivariate analysis. However, recipient SLCO1B1 rs2291075 genotype failed to enter forecast model for C/D ratios in stationary phase. Recipient SLCO1B1 rs2291075 genotype had significant effect on tacrolimus C/D ratios in convalescence phase (p = 0.0300) and stationary phase (p = 0.0400) in subgroup, which excluded the interference come from donor and recipient CYP3A5 rs776746.The combined analysis of donor and recipient CYP3A5 rs776746 could distinguish the metabolic phenotype of tacrolimus into three groups: fast elimination (FE), intermediate elimination (IE), and slow elimination (SE), which was entitled the FIS classification system. Tacrolimus C/D ratios of recipient SLCO1B1 rs2291075 CT and TT carriers were very close and were significantly lower than those of recipient SLCO1B1 rs2291075 CC genotype carriers in convalescence phase (SLCO1B1 rs2291075 could be a novel genetic locus associated with tacrolimus metabolism. The combined analysis of donor and recipient CYP3A5 rs776746, recipient SLCO1B1 rs2291075 genotypes, could be helpful to guide the personalized administration of tacrolimus in early period after liver transplantation.The online version contains supplementary material available at 10.1007/s00228-020-03058-w. Liver transplantation is the method of choice in the treatment of patients with end-stage liver failure and selected group of patients with hepatocellular carcinoma (HCC) , 2. TacrTherapeutic drug monitoring (TDM) is the most common strategy for immunosuppressive therapy in daily clinical practice and effectively ameliorates drug efficacy and safety , 8. At oIn recent years, pharmacogenomics research had provided an active strategy that can forecast drug metabolism phenotypes according to genotype , 10. Enzp = 0.0349, and p = 0.0041, respectively). There were no statistically significant differences between tacrolimus C/D ratios of donors with SLCO1B1 rs2291075 CT, TT genotype, and those with CC genotype carriers in week 1 and week 2 post-operation.In our previous study, 115 donors and recipient liver samples in our hospital were genotyped using the Affymetrix DMET Plus microarray (including 1936 genetic variants over 225 related genes) for investigating broad coverage of pharmacogenomic markers and screening unknown genes may be involved in tacrolimus metabolism. Tacrolimus trough blood concentration by the corresponding weight-adjusted dosage (C/D ratio) was used as an indicator of drug elimination [The SLCO1B1 gene which is located on chromosome 12p12.1 encodes the protein of organic anion transporting polypeptide 1B1 (OATP1B1), an influx transmembrane protein transporter. Several groups of the influx transporters uptake a variety of drugs and organic compounds from the blood into the cell, especially the organic anion transporting polypeptides (OATPs) which are expressed in many organs such as the intestine, liver, and kidneys. OATPs have shown an important role in clinical implications for the pharmacokinetics of many drugs, including drug absorption, distribution, and elimination. Evidence of changes in OATP transport function has been found to affect the efficacy and safety of many drugs and leading to instability in drug disposition and response. SLCO1B1 was expressed in both the liver and intestine –18. SLCOIn this study, we examined the CYP3A5 rs776746 and SLCO1B1 rs2291075 genotypes of both recipients and donors to clarify the influence of these genetic variants on tacrolimus dose requirements and pharmacokinetics in the early period after liver transplantation.n = 115) and First Affiliated Hospital of Zhengzhou University (n = 95) were enrolled in this retrospective study . The donor liver and recipient liver tissues were deposited − 80 °C low temperature refrigerator. All patients received tacrolimus, mycophenolate mofetil, and steroids administered at a standard dosage regimen. The initial oral daily dose of tacrolimus was 0.05~0.10 mg/(kg/day) on the first postoperative day. Tacrolimus  was orally administered twice daily. The dosage was adjusted to achieve the target trough level, which was set at between 7 and 10 ng/ml during the first month. Clinical parameters including daily drug dose, serum albumin concentration, hemoglobin, glutamate pyruvate transaminase, and total bilirubin were also recorded at 1–28 days after operation in this study.210 patients who received orthotopic liver transplantations between January 2015 and December 2019 in Shanghai General Hospital Affiliated to Shanghai Jiao Tong University .Liver grafts were obtained from donation after cardiac death (DCD). No donor livers were harvested from executed prisoners. The participants or the next of kin gave their informed consent for the study. The research was approved by the Ethics Committee of Shanghai General Hospital Affiliated to Shanghai Jiao Tong University, the First Affiliated Hospital of Zhengzhou University. The methods were carried out in accordance with the Declaration of Helsinki and its later amendments or comparable ethical standards. This study had register on −1 mg−1 kg−1) was used as an index of tacrolimus pharmacokinetics and was calculated by dividing the tacrolimus trough blood concentration (ng/ml) by the corresponding weight-adjusted total daily dosage (mg/kg body weight) [The drug trough blood concentration was measured in whole blood in laboratories at each participating center by the Pro-TracTMII tacrolimus ELISA kit  with a microparticle enzyme immunoassay . The tacrolimus C/D ratio (ng ml weight) , 22.Both donor and recipient genomic DNA were extracted from liver tissues using an AllPrepDNA/RNA Mini Kit . PCR amplification was done for the CYP3A5 and SLCO1B1 genes containing the polymorphisms. Primers were designed as follows. CYP3A5 rs776746: forward 5′-TCTCCCCTCAAGTCCTCAG-3′ and reverse 5′-CCATACCCCTAGTTGTACGAC-3′; SLCO1B1 rs2291075: forward 5′-GTTCATGGGTAATATGCTTCGTGGAATA-3′ and reverse 5′-GCCAAGAATGCATGGTTCTTATTCAC-3′.The PCR mixture contained 250 ng of genomic DNA. Final volume of 50 μl in reaction buffer contained 5 μl of 10 × PCR buffer, 5 μl of MgCl2 (1 mmol/l), 1 μl of dNTP (10 mM), 2 μl of each primer (10 μm/ml), and 3 U of Taq gold DNA polymerase. Cycling used a GeneAmp PCR system  as follows: initial denaturation at 94 °C for 3 min; 35 cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min; and a final step at 72 °C for 10 min. The amplified PCR products were sequenced using an ABI 3730-XL Automated Sequencer .U test was used to investigate the difference between the two groups, and a Kruskal–Wallis test was used to assess the statistical significance among several groups. Multivariate analysis was conducted using the logistic regression model. Statistical analysis was performed using SPSS version 17.0 . Hardy–Weinberg equilibrium, allele frequency, linkage disequilibrium was analyzed using SHEsis software . p < 0.05 was considered statistically significant. The statistical graphs were made by Graphpad Prism version 5.00 software .The early postoperative period after liver transplantation was divided into convalescence phase (1–14 days) and stationary phase (15–28 days) according to the change of liver function and tacrolimus C/D values . A chi-sp > 0.05). No statistically significant differences in genotype and allele frequencies were observed between recipients and donors for any of CYP3A5 rs776746 and SLCO1B1 rs2291075 polymorphisms.The distributions of CYP3A5 rs776746 and SLCO1B1 rs2291075 genotypes and alleles of recipients and donors are shown in Supplementary Table p < 0.0001)   Fig. . Tacroli01) Fig. .Fig. 1Inp = 0.0501) and there were statistically significant differences in stabilizing period (p = 0.0318). Tacrolimus C/D ratios of recipient SLCO1B1 rs2291075 CT and TT carriers were very close and were significantly lower than those of CC genotype carriers in convalescence period (p = 0.0195) and in stabilizing period (p = 0.0152) .Tacrolimus C/D ratios of recipient SLCO1B1 rs2291075 CC, CT, and TT genotype carriers were 172.8 ± 183.2, 118.4 ± 106.3, and 125.8 ± 88.70 in convalescence period and 121.0 ± 108.5, 84.00 ± 70.84, and 88.65 ± 75.08 in stabilizing period, respectively. The differences were borderline significant in convalescence period (52) Fig. . There w52) Fig.  at each p < 0.0001, Supplementary Fig. p < 0.0001, Supplementary Fig. p = 0.5917 and 0.7164, respectively, Supplementary Fig. p < 0.0001)  and stabilizing period (p = 0.040)  subgroup multivariate analysis explored the influence of recipient SLCO1B1 rs2291075 genotype on tacrolimus C/D ratios excluded the interference that comes from donor and recipient CYP3A5 rs776746. Variables of SLCO1B1 rs2291075, total bilirubin, and daily dose constituted prediction model of tacrolimus elimination in SE subgroup. Recipient SLCO1B1 rs2291075 was an independent predictor of C/D ratios of SE subgroup in convalescence period . Thirdly, the influence of recipient SLCO1B1 rs2291075 genotype on tacrolimus C/D ratios was confirmed in the slow elimination subgroup, which excluded the interference come from donor and recipient CYP3A5 rs776746. Two studies of renal transplant patients illustrated that SLCO1B1 polymorphisms were associated with tacrolimus concentrations, and genotyping for SLCO1B1 SNPs may be useful for individualized medicine of tacrolimus , 24. TheDuring recent years, increasing pieces of evidence showed that drug transporters played important roles in absorption and distribution of multiple drugs. Organic anion transporting polypeptides (OATPs) are central membrane transporters involved in the cellular uptake of drugs in tissues , which is important for pharmacokinetics. SLCO1B1 gene produces the protein of organic anion transporting polypeptide 1B1 (OATP1B1) and is involved in various kinds of drug disposition , 26. SLCAccording to the report by Shu Liu et al., SLCO1B1 rs2306283 polymorphisms were associated with tacrolimus concentrations in Chinese renal transplant recipients [Rs2291075, a genetic variant in SLCO1B1, has been reported as a tag SNP to distinguish the three major SLCO1B1 haplotypes . Additionally, rs2291075, among several other variants in the SLCO1B3-SLCO1B1 genomic region, is significantly associated with SLCO1B1 protein expression. The transporter SLCO1B1 has an important role in the system pharmacokinetics of multiple drugs used in the treatment of acute myeloid leukemia (AML). AML patients harboring two copies of the rs2291075 variant (CC) had a significantly more favorable survival outcome compared to individuals who are heterozygous (CT) or noncarriers (TT) . In anotp = 0.0195) and in stabilizing period (p = 0.0152). However, there were no statistically significant differences between tacrolimus C/D ratios of donors with SLCO1B1 rs2291075 CT, TT genotype, and those with CC genotype carriers at each period. The expression of SLCO1B1 was often thought to be liver-specific. However, SLCO1B1 mRNA could be expressed in a variety of tissues and cells according to data of GENECARDS (https://www.genecards.org/cgi-in/carddisp.pl?gene=SLCO1B1&keywords=SLCO1B1). A study showed that SLCO1B1 mRNA was expressed in the islets in very low quantities (< 1% relative to the liver control sample). The quantity of SLCO1B1 in the islets was significantly reduced by the presence of sirolimus, and tacrolimus antagonizes the sirolimus inhibition of OATP1B1 expression. SLCO1B1 in the islets might involve in the drug–drug interaction of tacrolimus and sirolimus [Tacrolimus was metabolized in the recipient intestine and donor liver in liver transplantation patients . In mostirolimus . The SLCirolimus . We infeIn this study, both donor and recipient CYP3A5 rs776746 genotypes were confirmed influencing tacrolimus elimination in the early period after liver transplantation . MoreoveA model consisting of tacrolimus daily dose, total bilirubin, FIS classification, and recipient SLCO1B1 rs2291075 could predict tacrolimus C/D ratios in convalescence phase by multivariate analysis. However, recipient SLCO1B1 rs2291075 genotype failed to enter forecast model for C/D ratios in stationary phase. Recipient SLCO1B1 rs2291075 genotype had significant effect on tacrolimus C/D ratios in convalescence phase and stationary phase in SE subgroup, which excluded the interference come from donor and recipient CYP3A5 rs776746. The CYP3A5 protein expression quantity was reduced obviously in donor liver and recipient intestine in convalescence phase, which resulted in high tacrolimus C/D ratios. And the CYP3A5 protein produced by donor liver and recipient intestine cells returned to nearly normal levels in stationary phase [Tacrolimus daily dose and total bilirubin were the main clinical influencing factors of tacrolimus C/D ratios in multivariate analysis. This result was consistent with previous studies. Tacrolimus was metabolized by the liver microsomal cytochrome P450 enzyme system and the metabolite of tacrolimus was excreted with bile. High total bilirubin level indicted reducing drug excretion and slow drug clearance rate. There was a positive feedback in tacrolimus metabolism: drug elimination rate accelerated with the increase of dose , 11, 37.There are some limitations to the study: Firstly, microparticle enzyme immunoassay was used to detect tacrolimus metabolites. The cross-reactivity with various tacrolimus metabolites of the antibody might have a potential impact on the testing result. Secondly, the drug–drug interaction (DDI) and indexes of inflammation, which had some effects on tacrolimus pharmacokinetics, should be included in multivariate analysis in further study. Thirdly, the large sample multicenter study was needed to confirm the relationships between recipient SLCO1B1 rs2291075 polymorphism and tacrolimus dose–corrected trough concentration in the early period after liver transplantation.In conclusion, SLCO1B1 rs2291075 could be a novel genetic locus associated with tacrolimus metabolism. Recipient SLCO1B1 rs2291075 was an important genetic factor under the circumstances of poor CYP3A5 protein enzyme activity . The combined analysis of donor and recipient CYP3A5 rs776746, recipient SLCO1B1 rs2291075 genotypes, could be helpful to guide the personalized administration of tacrolimus in the early period after liver transplantation.ESM 1(PDF 101 kb)ESM 2(DOCX 189 kb)


Case Summary. This patient is a 75-year-old male with a history of a prior aortobifemoral bypass graft in 2005. He presented with complaints of right paraspinal pain and chills. He had no symptoms of gastrointestinal bleeding or abdominal pain. His white blood cell count was 25,600/mcl  with left shift. The erythrocyte sedimentation rate was 11 mm/hr (0-38 mm/hr), and C-reactive protein was 95.5 mg/L (<=10.0 mg/L). Blood cultures were obtained and eventually grew Candida glabrata. A computed tomography angiogram (CTA) of abdomen and pelvis demonstrated inflammation surrounding the graft concerning for graft infection with additional inflammatory changes tracking down both femoral limbs. He underwent staged bilateral femoralaxillary bypass followed by the excision of aortobifemoral bypass.  An aortoenteric fistula (AEF) describes a communication of the aorta or aortic graft with an adjacent loop of the bowel. Aortic graft erosion is a rare complication of abdominal aortic aneurysm repair. We describe a case of a patient presenting with sepsis from Candida glabrata fungemia secondary to aortoenteric erosion without any symptoms or signs of gastrointestinal bleeding. This is a unique case of Candida glabrata fungemia from aortoenteric graft erosion.  Patients with aortoenteric erosion can present with sepsis in absence of gastrointestinal bleeding. Emergent computed tomography angiogram (CTA) of abdomen and pelvis should be performed to assess for aortic graft erosion or fistula. Empiric treatment with antibiotics should include antifungal agent like micafungin until the final culture is reported. The definite management is an extra anatomic bypass, followed by graft excision. An aortoenteric fistula (AEF) describes a communication of aorta or aortic graft with an adjacent loop of the bowel . They arA 75-year-old male with history of aortoiliac atherosclerosis requiring aortobifemoral bypass graft at an outside hospital in 2005 presented with complaints of right paraspinal pain for 2 weeks with worsening of pain during the past 24 hours associated with chills. He had no other systemic symptoms of infection, gastrointestinal bleeding, or abdominal pain. His other medical comorbidities were hypertension and hyperlipidemia, and his prior abdominal surgeries included laparoscopic cholecystectomy, right inguinal hernia repair, and open appendectomy. The patient had stable vitals on presentation . The white blood cell count was 25,600/mcl  with left shift. The erythrocyte sedimentation rate was 11 mm/hr (0-38 mm/hr), and C-reactive protein was 95.5 mg/L (<=10.0 mg/L). Lactic acid was 1.8 mmol/L (<2 mmol/L). Blood cultures were obtained and eventually grew Candida glabrata. A computed tomography angiogram (CTA) of abdomen and pelvis was performed as it is the diagnostic modality of choice to assess for aortoenteric fistula. It demonstrated inflammation surrounding the graft concerning for a graft infection with additional inflammatory changes tracking down both femoral limbs . The patient underwent cardiac catherization which showed no significant epicardial coronary stenosis; therefore, no intervention was performed. An upper GI series performed on postoperative day 4 after the graft excision demonstrated no evidence of contrast extravasation from the duodenal repair; therefore, tube feeding was initiated. However, the drainage from both Jackson Pratt (JP) drains was noted to turn a milky color after the initiation of tube feeding. The fluid triglyceride level was 640 mg/dL concerning for chylous ascites. Therefore, the patient was switched to low fat tube feed and slowly transitioned to a low-fat diet which led to the resolution of chylous ascites. Transthoracic echocardiogram and ophthalmoscopy were performed to rule out endocarditis and endophthalmitis in setting of fungemia and both were negative. The final tissue culture of the aortic graft grew Lactobacillus platarum and Candida glabrata. The patient had a slow recovery; however, there were no further complications. He was discharged to acute rehabilitation on postoperative day 18 after the graft excision. He was prescribed 6 weeks of IV micafungin and piperacillin/tazobactam.The patient was seen in the outpatient clinic where he continued to do well. He had evidence of arterial compromise on the right leg with an ankle brachial index (ABI) of 0.55. The ABI for the left leg was 0.82. He was asymptomatic at this time, and he was treated conservatively with regular follow-up every year using ankle brachial index and pulse volume recordings.Aortoenteric fistula (AEF) represents one the most challenging complication of abdominal aortic aneurysm repair. Both primary and secondary fistulas are extremely rare. The literature shows that primary AEFs have an incidence of 0.04% to 0.07%, whereas secondary aortoenteric fistulas are a complication of 0.36% to 1.6% of all aortic operations [Secondary aortoenteric fistula can occur months to years after the aortic surgery. The most common theory suggests that the combination of continuous friction between the intestine and the aortic graft and the local inflammation leads to formation of aortoenteric fistula . The duoSecondary aortoenteric fistula is further classified as type 1 or type 2 and has different presentations. Type 1 involves a fistula formation between the aortic graft and the adjacent bowel, commonly involving the proximal aortic suture line. Type 1 typically presents as massive gastrointestinal hemorrhage in 76% of cases. Sepsis and abdominal pain are relatively rare with this type of fistula. Type 2, or a paraprosthetic enteric fistula, involves erosion of the graft into the bowel without formation of a fistula. This generally manifests as sepsis and has been the presenting complaint in 75% of the cases. Bleeding is uncommon but can occur from the edges of the bowel eroded by the aortic graft [One of the largest case series by Schoell et al. comprising of 32 patients revealed that aortoenteric fistula and aortoenteric erosion accounted for 10 and 22 patients, respectively. The most common presentation was bacteremia (53%) followed by sepsis (41%), graft thrombosis (31%), and inguinal abscess (21%). The infection was polymicrobial in 18 patients and monobacterial in 1 patient with E. coli being the most common organism. Candida fungemia was identified in 8 patients; however, individual candida species were not mentioned .Prompt diagnosis of aortoenteric fistula is essential to reduce mortality and morbidity. A case report including 31 patients showed that 17 patients presented with herald bleed while 14 patients presented with massive GI bleeding. The time interval between herald bleed and subsequent massive hemorrhage was unpredictable and ranged from 4 to 92 days . It is eThe management of aortoenteric fistula or erosion requires excision of the infected graft and revascularization. Placement of an extra anatomic bypass, followed by graft excision, has been the usual treatment . EndovasCandida fungemia is rare presentation of aortoenteric erosion. Vegunta et al. reported a case of aortoiliac graft erosion in the duodenum presenting as Candida albicans fungemia and bloody bowel movements . The prePatients with aortoenteric erosion can present with sepsis in the absence of gastrointestinal bleeding. Emergent computed tomography angiogram (CTA) of abdomen and pelvis should be performed to assess for aortic graft erosion or fistula. Empiric treatment with antibiotics should include antifungal agent like micafungin until the final culture is reported. Endovascular aortic stent graft placement and coil embolization are safe and effective methods of rapidly controlling hemorrhage from aortoenteric fistula. The definite management is an extra anatomic bypass, followed by graft excision.


In well-developed countries, people have started to pay additional attention to preserving healthy dietary habits, as it has become common knowledge that neglecting them may easily lead to severe health impairments, namely obesity, malnutrition, several cardiovascular diseases, type-2 diabetes, cancers, hypertensions, and inflammations. Various types of functional foods were developed that are enriched with vitamins, probiotics, prebiotics, and dietary fibers in order to develop a healthy balanced diet and to improve the general health of consumers. Numerous kinds of fiber are easily found in nature, but they often have a noticeable undesired impact on the sensory features of foods or on the digestive system. This led to development of modified dietary fibers, which have little to no impact on taste of foods they are added to. At the same time, they possess all the benefits similar to those of prebiotics, such as regulating gastrointestinal microbiota composition, increasing satiety, and improving the metabolic parameters of a human. In the following review, the evidence supporting prebiotic properties of modified starches, particularly resistant starches and their derivatives, resistant dextrins, was assessed and deliberated, which allowed drawing an interesting conclusion on the subject. One of the first definitions of prebiotics stems from the mid-1990s, when they were described as “the non-digestible components of the food that facilitate growth and/or activity of a beneficial microorganisms inhabiting the gastrointestinal tract of a host”. In 2007, the World Health Organization (WHO) decided to update said definition by describing prebiotics as “non-viable food components that have a health benefit on the host by modulation of the microbiota” [Prebiotics are usually plant-derived oligosaccharides, essentially fructans, galactans, or similar compounds ,4,5. In Nonetheless, it should be noted that not all fiber sources are considered prebiotics. The European Food Safety Authority (EFSA) strongly differentiates the terms dietary fiber and prebiotic by specifying that distinctive products cannot be classified as prebiotics, but only as a dietary fiber, if “a cause-and-effect relationship has not been established between the consumption of the food constituents, which are the subject of the health claims and a beneficial physiological effect related to increasing numbers of gastrointestinal microbiota” . MoreovePrebiotics per se do not possess many outstanding health-promoting properties; instead, they are an important substrate of fermentation for the beneficial species of microorganisms present in the host’s gastrointestinal tract and thus induce their growth . Then, tBifodobacteria and Lactobacillus spp., are able to utilize saccharides as a carbon source by means of fermentation. Desired byproducts of their metabolism include SCFAs, which are associated with various beneficial effects noticeable in the host’s organism, i.e., control of appetite by decreasing the concentrations of ghrelin [Valuable microbial genera present in the intestines, namely  ghrelin . Moreove ghrelin ,13.Lacticaseibacillus casei DG® on the treatment of chronic bacterial prostatitis with exceptionally promising results [L. casei DG® significantly decreases the usage of antibiotics during the treatment, which is likewise economically positive, given that it potentially leads to lower costs of therapies.Furthermore, a recent study by T. Cai et al. (2021) investigated the effects of  results . Over 70Lactobaliccus and Bifidobacterium spp.) and the production of SCFAs, which modulate the intestinal pH, thus improving the solubility of minerals, making them easier to assimilate [Several studies on prebiotics highlighted their promising potential for the absorption of microelements such as calcium, magnesium, zinc, copper, iron, and phosphorus. This effect is associated with an increased count of gut microbes  or indirectly through the production of SCFA, which contributes to an increased activity of immune cells  ,19,20. Aer cells ,24. Proper cells . Prebioter cells .An important role of prebiotics is the inhibition of toxins in the intestines. Bacteria grown during the prebiotic treatment were shown to protect the colon lumen by adhering to its surface and reducing the effect of toxic carcinogenic compounds such as N-methyl-N′-nitro-N-nitrosoguanidin or 1,2-dimethylhydrazine. The exact effect of the latter is not fully described yet, but it is suspected that lactic acid bacteria (LAB) may secrete metabolites that inactivate or scavenge some of the carcinogenic intermediates ,28. It iThe definition of dietary fiber (DF) and its effects on health have been widely discussed topics over the years. In 2008, the most recent and uniform definition was issued after the Codex Alimentarius ,31. AccoWhen properly extracted  and purified, the generally recognized scientific evidence of health benefits must be proved to define a polymer as DF. Respective definitions often include other ‘associated substances’ that are not carbohydrates, e.g., lignin .These views are evident in the worldwide dietary recommendations, which strongly emphasize the consumption of DFs naturally occurring in food, whereas added and nondigested carbohydrate polymers are much less recommended, due to the limited evidence of health benefits of these specific types of fiber.Regarding the benefits for health, it is indicated that DF must generally present one or more of the following properties:➢reduce the stool transit time, while increasing its bulk ;➢be used as a carbon source by colonic microbiota (as fermentation substrate) ;➢reduce the levels of cholesterol  ; and➢support glucose and insulin metabolism (reduce the levels of blood glucose and insulin) .These, among other related criteria, were presented in the EU Directive 2008/100/EC and were recently used for the evaluation of the health-promoting properties of a large number of fibers by the FDA  and Heal➢non-starch polysaccharides including cellulose, hemicelluloses, mannans, pectins, and other hydrocolloids , inulin, and fructans;➢resistant oligosaccharides including fructo-oligosaccharides (FOS), galactooligosaccharides (GOS);➢resistant starch and dextrins .Purportedly, the number of approved fibers will rise over time, as more studies will provide sufficient evidence for the health benefits of dietary fiber’s candidate compounds. Considering the role of the colonic fermentation of fiber as the source of its health-promoting properties, some differences exist among countries. According to the FDA, the applicable fermentation-related criterium is “increased mineral absorption”, which is associated with the increased solubility and bioavailability through the process of production of SCFA. Moreover, the “reduction of energy intake” strongly correlated with the colonic fermentation of fibers is likewise considered a valid criterium .In the EU, the process of evaluation and recognition of dietary fiber is usually conducted using analytical criteria through the commonly acknowledged AOAC 2009.01 standard method or other similar methods .In most of these methods, thermostable bacterial α-amylase, protease, and amyloglucosidase are used. However, due to the high temperature of amylase incubation (95 °C), there are notable losses in resistant starch since it undergoes hydrolysis in such conditions. On the other hand, AOAC methods 2009.01 and 2011.25 both employ incubation with pancreatic α-amylase under specific conditions that are comparable to physiological ones ; thus, they offer a much more accurate estimation of resistant starch . A generFor these methods , it is rIt was proven that several types of dietary fibers are completely fermentable, or at least to a certain degree . The maiDietary fibers have varying characteristics; therefore, various classifications are used to appropriately characterize them. They include origin, chemical composition, and physicochemical properties with subcategorization including the degree of polymerization. Notably, these properties can have a significant impact on the fermentation processes .According to their origin, plant-originated fibers can be divided into the ones derived from fruit and vegetables, cereals and grains, nuts, and legumes. Nevertheless, it must be noted that the different types of plant origin will result in significantly distinct chemical compositions of dietary fibers and different physicochemical properties ,44. In tThe main physicochemical properties of dietary fibers consist of fermentability, viscosity, and solubility. All of them can alter both fermentation processes and the overall effects of fiber product consumption .Insoluble fibers are usually less used for microbial fermentation , but their presence in the gastrointestinal tract elevates the food transit rate and prevents constipation .Another non-fermentable fiber is psyllium, which is known for its increased solubility and high viscosity. Combining these properties proved to be useful in the treatment of high blood cholesterol and in improving the glycemic control of patients ,52,53.β-glucans and pectins are fibers that are easily fermentable by gut microbiota while possessing high solubility and viscosity, similarly to psyllium . These fFibers with properties such as low viscosity and high solubility can be rapidly used by the gut microbiota for fermentation purposes: this includes resistant starch and dextrins, inulin, polydextrose, and absoluble type of corn fiber . Inulin-Although properties including degree of polymerization or the origin of inulin-type fructans have proven to influence the fermentation processes in patients, there is still not enough evidence confirming their physiological or metabolic benefits . On the The solubility of carbohydrates impacts the place in the gastrointestinal tract where the microbial fermentation occurs. Fibers such as FOS or pectins that possess relatively high solubility can be used by the bacteria residing in the gastrointestinal tract . On the other hand, less-soluble fibers such as cellulose are often metabolized partially in the distal colon because of their significantly slower transit time and the higher densities of bacteria present there ,60.A number of studies highlighted that many positive effects attributed to high-fiber diets are actually directly linked to the short-chain fatty acids, which are produced by the gastrointestinal microbiota via the fermentation of ingested oligosaccharides ,62,63,64It was proven that there is a direct correlation between the consumption of prebiotics and dietary fibers with elevated SCFAs production in the intestines and high peripheral circulating SCFAs . VariousSome of the main benefits of prebiotics are presented in The idea that the SCFAs and energy homeostasis are correlated has been investigated thoroughly over the past years. Recent studies have provided an abundance of evidence for the multi-level network in which SCFAs can utilize their beneficial actions such as improving lipid, glucose, and cholesterol metabolism ,70. The + and Mg2+ cations is increased by butyrate and propionate, respectively [In the human organism, three main SCFAs are produced by fermenting gut microbes, namely acetate, propionate, and butyrate, which constitute about 95% of SCFA content in the gastrointestinal tract ,73. The ectively . Acetateectively . Moreoveectively . Additioectively .In the early 21st century, the SCFAs sensing receptors (GPR41 and GPR43) were discovered and described , SCFAs wIt was discovered that free SCFAs are able to use monocarboxylate transporters to transmit the current state of the intestines to the brain similarly to signaling molecules .Research performed by Frost et al. (2014) indicated that SCFAs produced by gut microbiota can act as appetite-regulating agents . The mecThe intestine is capable of the secretion of hormones (formed in the gut) that can convey information about the nutritional and energy status of the gut directly to the brain . The disInterestingly, a direct administration of propionate to the human colon led to similar results (decreased ghrelin and elevated PYY), which further confirmed the role of SCFA in the regulation of appetite hormones .The liver is a vital organ, especially for the absorption of propionate and butyrate ,97. The w/w), a significant increase in the glycogen, citrate, and lower xylulose-5-phosphate concentrations in the liver was observed, which suggests that the inactivation of synthesis of fructose-2,6-bisphosphate induces processes such as acetate-activated gluconeogenesis and acetate-inactivated glycolysis [SCFAs can also influence glucose metabolism in the liver . In an aycolysis .SCFAs were also able to significantly reduce the synthesis of total cholesterol in the liver . SupplemNevertheless, studies mentioned in this section were conducted on animals (mostly rodents), so they only give a view of how SCFAs could potentially have similar metabolic effects on humans. Studies on humans in this area are limited, but as the potential applications are visible, this topic should be investigated thoroughly in the upcoming years .Over the last decade, the topic of new fiber preparations has received great attention. As a result, many prebiotic candidate products were developed by physical and chemical modification of starch, such as type 4 resistant starch (RS4) or resistant dextrins (RDs).By definition, the RDs are short-chain glucose polymers that lack a sweet taste but exhibit increased resistance to the enzymatic hydrolysis by the digestive enzymes of human .The basic methods of RD production include starch dextrinization, which replaces default 1,4- and 1,6- glycosidic bonds in starch with 1,2- and 1,3- glycosidic bonds . This phBacteroides while decreasing Clostridium spp. [Nutriose is supposedly one of the most popular commercially available resistant dextrins concluding from the number of studies using it. It is a soluble fiber of low viscosity made from wheat, maize, or pea starch through a highly regulated dextrinization process accompanied by chromatographic fractionation . It exhiium spp. ,112,113.Nutriose has a variety of applications in the food and pharmaceutical industry, as a valid ingredient of drinks and supplements enriched with fiber , granulaAnother commercially available dextrin preparation is Fibersol-2, which is a resistant maltodextrin with prebiotic properties produced from corn-originated starch. Due to specific treatment, it becomes highly resistant to digestion in the small intestine, contrary to the typical maltodextrin .The main production procedure of Fibersol-2 includes pyrolysis and enzymatic treatment of corn starch detailed in US Patent Nos. 5620873 and 5358729. In the first step, the hydrochloric acid is used to perform a transglucosidation reaction in corn starch. Then, the obtained mixture is hydrolyzed by an amylase, purified, and analyzed in order to confirm the proper quality. Finally, it is spray dried to obtain the final product.The abovementioned steps lead to conversion of part of the original α-1,4 glycosidic bonds to random α-1,2- or 1,3- bonds. Thus, the final product contains not only α-1,4 and 1,6 glycosidic bonds typical for starch but also α-1,2- or 1,3- bonds .w/w at 20 °C); it does not produce any kind of clods, only clear and transparent water-like solutions; therefore, it is a perfect additive to several types of healthy drinks. Moreover, Fibersol-2 has no flavor or odor and minimal-to-no impact on the sweetness of products. It is stable in a variety of food processing conditions such as high temperatures or acidity and therefore can be used in products such as sauces, milk drinks, yoghurts, sports drinks, puddings, juices, and other similar products. Other features of Fibersol-2 include low hygroscopicity, very low viscosity, and high freezing–thawing stability [According to the manufacturer, Fibersol-2 is well-soluble in water  . HoweverRS—resistant starchRS1, RS2, RS3—resistant starch type 1, 2, 3, respectivelyTS—total starchRDS—quickly digestible starchSDS—slowly digestible starchResistant starch has been defined in five forms . RS1 is Type 1 resistant starch (RS1) refers to starch molecules inside plant cells with undamaged cell walls. This starch cannot be reached by amylolytic enzymes, since the enzymes of the gastrointestinal tract are unable to degrade cellulose, hemicelluloses, and lignins, which are common components of plant cell walls. That is why RS1 can pass the small intestine without being digested .Resistant starch of the second type (RS2) constitutes granules of raw starch of certain plant species, e.g., potatoes or green bananas. Its resistance to the activity of digestive enzymes has not been completely described yet.It appears, however, that the reason for different susceptibility of potato starch to amylolytic enzymes to starches of other cereals can be caused by significantly lower ability of the enzymes to attach to the potato starch granules compared to that of cereal starches ,149.Another feature of potato starch is a relatively high amount of amylopectin together with rather high degree of crystallization. Since the amylolytic enzymes target the amorphous regions first, their activity might become significantly impaired if they encounter the crystalline structure of starch granules. Nevertheless, the reason underlying the resistance of starch to activity of amylases is not only linked to the degree of starch crystallization. For example, cereal starches, which are characterized by a type A crystallization of relatively high degree, are less resistant to the enzymatic activity than potato starch of type B with even a two-times-lower crystallization degree. Interestingly, the starches of legumes, which possess a mixed crystalline pattern , likewise present a high resistance to enzymatic hydrolysis .The resistance of starch granules can be increased by annealing, which happens when the starch is kept in water with a temperature lower than that of gelatinization. Generally, when the water temperature around the starch granules reaches 20 °C, they increase their volume (by approximately 30%). When starch is exposed to an increased temperature, water starts to penetrate the interior of the granule, thus enlarging it. As a result, hydrogen bonds are altered, and water molecules can then bind to the released hydroxyl groups. However, if the temperature is lower than that of the starch gelatinization, the granules are not being damaged but develop new properties, which are dependent on the specific botanical origin of the starch, temperature of annealing, and the concentration of starch in water solution ,151.During the starch annealing, their crystallization degree increases (together with the strength of granules structure), which in turn causes the increase in temperature of gelatinization ,152.It was further discovered that double helices of potato starch can undergo elongation after annealing, whereas in maize starch, it can induce the formation of new double helices ,154.According to the presented data, it can be concluded that the resistance of some types of starch to the enzymatic activity of amylases is determined by the crystalline structure of starch granules, especially the crystallinity of type B.Resistant starch of the third type (RS3) is created during the precipitation of starch paste or gel in the retrogradation process . During Contrary to the amylose, the crystalline structures of amylopectin display lesser stability, which is the primary reason for the lower length of their chains and the dissolving temperature (range from 30–80 °C). Nevertheless, the resistance to the amylolytic enzymes remains only a minor part of the retrograded starch that can undergo enzymatic hydrolysis ,158,159.Physical  and chemical modifications (or both) of starch result in the formation of resistant starch of the fourth type (RS4). Resistance of these modified starches increases with a higher degree of their substitution with different functional groups ,160. SucAnother process that occurs during the physical and chemical treatment of starch is dextrinization . It takeInteractions of starch molecules with certain substances (penetration of the amylose helices or formation of complexes) such as lipids or fatty acids may also result in the formation of resistant starch—RS5—in this case. It was observed that complexes of starch with monoglycerides of fatty acids in starch paste exhibit higher resistance to the activity of amylases . Fatty aData presented in In recent years, the surge in lifestyle-originated health problems, such as the pandemic of obesity and other diseases directly or indirectly linked to metabolic disorders, has started to become severely evident. For this reason, it has become particularly important to investigate every possible solution that can limit or prevent such a rapid spread. Seeing an increasing number of studies that associate intestinal microbiota with metabolic disorders, it is obvious that it is not a neglectable factor. Neglected balance of gut microbiome can lead to various diseases, i.e., obesity, type 2 diabetes, irritated bowel syndrome, chronic bacterial prostatitis, and various types of cancers, and can also significantly impair the immune system. In order to achieve a proper balance of intestinal microbiota, a well-thought-out diet is necessary. Probiotics, prebiotics, and certain dietary fibers are proven to be a valid way to support the microbial balance. These bacterial strains and specific compounds possess several health-promoting properties, e.g., anti-obesity or immunological effects. Both resistant starch and dextrins were proven in various independent clinical trials to have positive effects on the subjects such as reduction of BMI, total body fat, and markers of metabolic disorders. Moreover, they increased counts of beneficial gut microorganisms and increased concentrations of short-chain fatty acids and various others highlighted in this review. These outcomes of RS and RD consumption are greatly similar to those of prebiotics. Nevertheless, the number and quality of studies in this field is still not sufficient. The important matter that also must addressed is the influence of gender on the effectiveness of prebiotic and dietary fiber supplementation, which is omitted in the vast majority of studies. Such knowledge may potentially be of great importance in the field of improving the efficacies of prebiotic treatments. Moreover, during research, the number of tested subjects is usually relatively low, mostly due to legal requirements and lack of volunteers. When combined with the restricted ability of supervision of the participants and the long time required for the studies, a high probability of errors is created. There is a real potential in the usage of RS and RD supplements, which are easily produced and accessible to the public. Accordingly, additional research is required to fill the void of knowledge on RS and RD and their association with human health to fully utilize their capabilities.


Arterial stiffness is an important measure of vascular aging and atherosclerosis. Though it is measured in many well-known epidemiologic cohort studies, arterial stiffness is often overlooked in routine clinical practice for a number of reasons including difficulties in measurement, variations in definition, and uncertainties surrounding treatment. Central arterial stiffness, a surrogate for aortic stiffness, is the most commonly measured marker of arterial stiffness. In addition to central stiffness, there are also a number of ultrasound based techniques to measure local vascular stiffness, including carotid stiffness. There is evidence that both local carotid stiffness and central arterial stiffness measures are associated with multiple cerebrovascular processes, including stroke and cognitive dysfunction. Mechanistic explanations supporting this association include increased flow load experienced by the cerebral microvasculature leading to cerebral parenchymal damage. In this article, we review definitions of carotid artery stiffness measures and pathophysiologic mechanisms underpinning its association with plaque development and downstream cerebral pathology. We will review the evidence surrounding the association of carotid stiffness measures with downstream manifestations including stroke, cerebral small vessel disease detected on brain MR such as white matter hyperintensities and covert brain infarctions, brain atrophy, and cognitive dysfunction. With consistent definitions, measurement methods, and further scientific support, carotid stiffness may have potential as an imaging-based risk factor for stroke and cognitive decline. Diseases of the carotid artery are a major cause of worldwide morbidity and mortality, with carotid atherosclerosis accounting for nearly 1 in 5 acute ischemic strokes . Though Though arterial stiffness is not commonly measured in routine clinical practice, it has been well-studied in part because of its inclusion in multiple large epidemiologic cohort studies –9. Most In this article, we will review common methods for measuring arterial stiffness including definitions of the most common measures of stiffness. We will also review mechanisms by which arterial stiffness may contribute to cerebrovascular ischemia, cognitive function, and carotid plaque formation.Arterial stiffness is a general term to describe rigidity of the arterial walls. It is frequently assessed by measuring the velocity of a given pulse-wave in a specific vessel segment , though Pulse wave velocity (PWV) is perhaps the most commonly used and most well-known technique to evaluate arterial stiffness. PWV is a measure that evaluates the propagation of a wave through the vasculature by taking simultaneous recordings at two separate sites. The PWV increases as arterial stiffness increases. Carotid-femoral PWV (cfPWV) is often considered the gold standard of measuring central artery stiffness and is recommended by the American Heart Association with the highest level of supporting evidence as a method for evaluating arterial stiffness . This usOne major limitation of cfPWV is that it is based on models for wave propagation through the arterial tree, which may not accurately reflect true stiffness, especially in aging arteries . cfPWV iCardio-Ankle Vascular Index (CAVI) is another type of systemic measure of arterial stiffness which differs from PWV in that it is not dependent on blood pressure measurement . CAVI isThough PWV is the most commonly performed measure of central arterial stiffness, there are several measures for direct, local measurement of stiffness. Most of these measures of local arterial stiffness are determined via ultrasound techniques. Ultrasound is frequently performed throughout the cardiac cycle to determine changes in vessel diameter in diastole and systole. In addition to measuring luminal diameter to determine changes in volume throughout the cardiac cycle, ultrasound can also be used to measure wall thickness which can be used to determine the elastic properties of the arterial wall. Since most local stiffness measurement calculations require pressure measurements, local blood pressure measurements obtained via applanation tonometers are often calibrated to brachial pressures measured with standard sphygmomanometer. Given the lack of precise measurements on ultrasound, some studies utilize specific echotracking techniques to measure the lumen diameters which can provide more detailed measurements than ultrasound techniques . Local mCompliance is a local measure of stiffness and can be used to directly measure carotid arterial wall stiffness. Compliance is defined as the ratio of any volume change  for a given pressure change. (ΔD/ ΔP cm/mm Hg). Compliance is an inverse measure of stiffness, meaning the greater the compliance, the lower the arterial stiffness. Larger arteries with higher cross-sectional volumes can general accommodate more volume for a given pressure change compared to smaller arteries.Distensibility is another local measure of stiffness. It is similar to compliance but can be more useful when comparing arteries of different sizes across the arterial tree. It is defined as the relative change in arterial lumen diameter or area for given pressure change [ΔD/ (ΔP x ΔD)].Elastic modulus is another measure of stiffness that is defined as the pressure change necessary for theoretical 100% stretch (ΔP x ΔD)/ (ΔD). Young's elastic modulus is more commonly used and is defined as elastic modulus per unit area. Young's elastic modulus is used to measure elastic properties of a given vessel and can take the arterial wall thickness into account, including intima-media thickness in the carotid artery. Accounting for wall thickness is important, especially in the setting of atherosclerosis and plaque.One of the reasons that arterial stiffness measures are not commonly performed in clinical practice is the difficulty in identifying the ideal measure for stiffness. While PWV is currently the simplest non-invasive method for measuring arterial stiffness, it has several limitations as discussed above, including inability to provide local stiffness measurement. In order for measures of arterial stiffness to become more mainstream, more uniformity in measurement techniques, definitions, and clinical application are necessary.Arterial stiffness is a prominent feature of aging, but also a result of many systemic processes including atherosclerosis, diabetes, and even chronic renal disease. Stiffening is the product of complex interactions on the vessel wall, including hemodynamic forces, extrinsic effects such as hormonal changes and salt intake, and structural changes to the wall itself . VariatiThe exact role that carotid stiffness plays in the development of cerebrovascular ischemia and atherosclerosis is still under investigation. Given the shared risk factors for arterial stiffness and stroke, it is difficult to disentangle the complex relationship between stiffness, cardiovascular risk factors, and stroke. Still, one widely proposed link between arterial stiffness and cerebrovascular ischemia may be that arterial stiffness causes large arteries to become less responsive to normal pulsatile flow. This inability to respond to fluctuations of blood pressure throughout the cardiac cycle may result in more continuous perfusion, rather than pulsatile perfusion. Ultimately, more continuous perfusion results in increased flow load experienced by end-organs, especially the cerebral parenchyma , 20. TheAnother potential mechanism by which arterial stiffness leads to stroke is through the development of plaque formation, specifically the formation of vulnerable plaque features . EmerginSimilarly, arterial stiffness may contribute to cerebral small vessel disease. Given the increased flow load experienced by the cerebral microvasculature in the setting of increased arterial stiffness, there is evidence of cerebral microvascular dysfunction and damage which can be detected on brain magnetic resonance (MR) imaging. The sequelae of cerebral microvascular damage can be seen as white matter hyperintensities, covert brain infarctions and cerebral microbleeds on MR. In addition, the increased flow load from altered cerebral microvascular flow leading to microstructural damage is thought to contribute to brain parenchymal atrophy, which is considered another marker of cerebral small vessel disease .In addition to overt ischemia such as stroke, there is also evidence of downstream cognitive dysfunction in those with increased arterial stiffness. The microvascular damage from constant high flow loads leading to cerebral small vessel disease may in turn lead to cognitive impairment and dementia, given the very strong association between markers of cerebral small vessel disease and cognitive dysfunction –34. In aArterial stiffness may lead to stroke both because of increased flow load on the cerebral vasculature and due to accelerated development of rupture-prone atherosclerotic plaque in the carotid artery. Meta-analyses and individual-participant level data from epidemiologic cohorts demonstrate strong evidence of an association between central arterial stiffness and incident stroke , 39, incAnother study using a large epidemiologic cohort found that the addition of carotid distensibility measures improved ischemic stroke risk prediction . AccountArterial stiffness also has a complex association with atrial fibrillation, which is the most common chronic arrhythmia and a strong predictor of stroke and mortality , 44. SevGeneralized cerebral parenchymal volume loss and hippocampal volume loss are commonly encountered findings on routine brain imaging. In addition to being closely associated with advancing age, low cerebral parenchymal volume is also associated with Alzheimer's disease, stroke, and generalized cognitive dysfunction . MultiplThere are also a number of studies correlating aortic and central arterial stiffness with decreased parenchymal brain volume –54 thougSeveral markers of cerebral small vessel disease evident on routine brain imaging, including white matter hyperintensities (WMH), covert brain infarctions (CBI), and cerebral microbleeds (CMBs) are associated with mortality, stroke, cognitive dysfunction, and dementia , 55, 56.Despite the uncertainty surrounding the contribution of local carotid stiffness to markers of small vessel disease, there is stronger evidence of central arterial stiffness being associated with markers of cerebral small vessel disease, including white matter hyperintensities , 54. TheSeveral studies show that markers of central arterial stiffness are independent predictors of cognitive dysfunction. A recent cohort study found that higher central arterial stiffness (cfPWV) was associated with cognitive dysfunction and that association was in part mediated by markers of cerebral small vessel disease on brain MR . Data frThe data supporting a link between carotid stiffness is less clear than for central arterial stiffness. For example, several studies have shown that carotid stiffness via distensibility or compliance measures is associated with cognitive dysfunction –75. OtheOne of the possible explanations for the association between central arterial stiffness and cognitive dysfunction, specifically Alzheimer's dementia is the thought that there is decreased clearance of amyloid protein, leading to disproportionate accumulation and the development of Alzheimer's dementia . Those iArterial stiffness, a specific measure of wall rigidity, can be measured both centrally in the aorta and locally in certain vascular beds such as the carotid artery. Having increased carotid stiffness is associated with multiple downstream effects including cardiovascular disease, stroke, and cognitive dysfunction. One of the leading pathophysiologic mechanisms underpinning these associations is the loss of vascular regulation from stiffness leading to increased flow load experienced by the cerebral parenchymal microvasculature. Both local measurements of carotid stiffness and measures of central arterial stiffness are variably associated with stroke, cognitive dysfunction, markers of cerebral small vessel disease, and low cerebral parenchymal volume. Currently, the lack of validated measurements precludes the routine use of measures of arterial stiffness in clinical practice. Future studies including those using optical With more standardized methods of measurements and continued validation, carotid stiffness measures may achieve greater clinical adoption as imaging-based risk factors for the development of stroke and dementia.HB and AG designed the overall manuscript with the collection and assembly of data by HB. HB drafted manuscript with critical revision and feedback from AG. Both AG and HB gave final approval of the manuscript. All authors contributed to the article and approved the submitted version.HB was supported by a grant from the Association of University Radiologists- GE Radiology Research Academic Fellowship Award. AG was in part supported by National Institutes of Health grants R01HL144541 and R21HL145427. Research by HB was in part supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Number UL1TR002538.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.


Current commercial PPL-dependent clotting assays use chemically phospholipid depleted plasma to measure PPL activity. The purpose of our study was to modify the PPL assay by substituting the chemically phospholipid depleted plasma with PPL depleted plasma obtained by ultracentrifugation This in order to get readily access to a sensitive and reliable assay to measure PPL activity in human plasma and cell supernatants. The performance of the assay was tested, including the influence of individual coagulation factors and postprandial lipoproteins and compared to a commercial PPL assay (STA-Procoag-PPL). The two PPL assays displayed similar sensitivity to exogenously added standardized phospholipids. The PPL activity measured by the modified assay strongly correlates with the results from the commercial assay. The intraday- and between-days coefficients of variation ranged from 2–4% depending on the PPL activity in the sample. The modified PPL assay was insensitive to postprandial lipoprotein levels in plasma, as well as to tissue factor (TF) positive EVs from stimulated whole blood. Our findings showed that the modified assay performed equal to the comparator, and was insensitive to postprandial lipoproteins and TF As early as 1946, Chargaff and West observed that the clotting time of plasma was prolonged by applying high-speed centrifugation to remove “thromboplastic substances”3. Accordingly, Connor and colleagues demonstrated that the amount of annexin A5 positive EVs, measured by flow cytometry, showed a significant and inverse correlation with clotting time4. These findings suggest that EVs play a significant role in coagulation, apparently due to exposure of phospholipids, and phosphatidylserine (PS) in particular, on their surface. The increase in surface expression of negatively charged PPL will facilitate the assembly of coagulation factors upon cell activation or apoptosis5. This is crucial for several stages of the coagulation pathway, namely the formation of the intrinsic and extrinsic tenase complexes, as well as the conversion of prothrombin to thrombin by coagulation factor Xa (FXa)6. The activity of the extrinsic tenase complex, the tissue factor (TF)—factor VIIa (FVIIa) complex, is increased by several orders of magnitude in the presence of negatively charged membrane phospholipids7.Procoagulant phospholipid (PPL) activity has regained interest in recent years, mainly due to the increased understanding of the role of extracellular vesicles (EVs) in thrombosis and haemostasis2+8, others are clot-based, utilizing the ability of PPL to accelerate the conversion of prothrombin to thrombin. Annexin A5-based assays are widely used, often in a flow cytometry setting, a method that is time consuming, requires expensive equipment and experienced personnel. In addition, annexin A5 is commonly used in chromogenic FXa assays, where the activity measured is based on the EVs exposing PS that are bound to the microplate. These EVs are then able to accelerate the cleavage of the chromogenic substrate by FXa9.Several assays have been developed to measure the PPL activity in human plasma. While some are based on the ability of annexin A5 to bind PS in the presence of Ca9. To the best of our knowledge, there are currently two clot-based assays commercially available, the STA-Procoag-PPL assay from Diagnostica Stago  and the XACT assay from Haematex . Both assays use chemical phospholipase treatment to deplete phospholipids from plasma, but differ with regard to the phospholipase used, plasma origin, and the use of a phospholipid calibrator. The XACT assay uses a snake phospholipase10 and porcine plasma, while an unspecified phospholipase and human plasma is used for the Stago assay.Compared to FXa chromogenic assays, which measure procoagulant activity of EVs in a purified system, clotting assays involve a more complex reaction and a physiological end-point as they measure the PPL activity of plasma, and not only captured PS-positive EVsIn this study, we aimed to develop a modified PPL-dependent clotting assay, capable of measuring the PPL activity in human plasma and cell supernatants of in vitro experiments, by removing PPL from plasma by sequential centrifugation, including final ultracentrifugation. The performance of the modified assay was then validated against the commercially available Stago STA-Procoag-PPL assay.CT) of plasma samples (n = 6) subjected to sequential centrifugation procedures . A second centrifugation step of 2500×g for 15 min resulted in a prolongation of the clotting times to 92.5 ± 6.3 s (mean ± 1 SD). Pelleting larger EVs (e.g. microvesicles) from platelet free plasma (PFP) by an additional spin of 20,000×g for 30 min at room temperature (RT) further prolonged the clotting times to 127.8 ± 16.9 s (mean ± 1 SD). The final spin at 100,000×g for 60 min to remove the smallest and lightest EVs (e.g. exosomes) further prolonged the clotting times to 159.3 ± 7.1 s (mean ± 1 SD).The removal of PPL from plasma is fundamental for the clot-based assays. As EVs are the main source of PPL, we first tested whether sequential centrifugation reliably depleted EVs from plasma. To achieve this, we compared the PPL clotting times  by ultracentrifugation  resulted in a mean clotting time of 163.2 ± 7.5 s (mean ± 1 SD)  prepared from pooled PFP  and prothrombin time (PT). The aPTT was 30.2  and PT 21.3 s, corresponding to a PT-INR of 1.00 .In order to demonstrate that PPL depleted plasma prepared by ultracentrifugation was comparable to enzymatic depletion of PPL, we tested the sensitivity of the STA-Procoag-PPL assay and the modified PPL assay using serial dilutions of a standardized phospholipid reagent (UPTT reagent). The addition of UPTT shortened the clotting times in a concentration dependent manner Fig. . The twoCT measured by the modified assay were consistently longer than for the STA-Procoag-PPL assay.To compare the performance of the modified PPL and STA-Procoag-PPL assays, plasma samples from ten healthy blood donors were tested on both assays Fig. . A stronCT of 16 s. and the other PPLCT of 54 s, as well as a pooled PFP sample with PPLCT of 85 s. The day-to-day CV for the Stago standards (n = 30) was 3.9% and 3.2%, respectively, whereas the within-a-day CV (n = 20) was 3.7% and 2.8%, respectively. Pooled PFP had a day-to-day CV of 4.1% and a within-a-day CV of 3.3%.To assess the coefficients of variation (CV) of the modified PPL assay, we used the two plasma standards provided by the STA-Procoag-PPL assay, one resulting in PPLCT measurements without the addition of UPTT and with the addition of two different concentrations of UPTT   affected PPLCT .We wanted to investigate to what extent two different centrifugation protocols  and 4 h after a standardized high fat meal (1 g fat/kg body weight) . This indicates that the modified PPL assay was insensitive to postprandial lipemia measured in PPP samples subjected to a second high speed centrifugations after thawing, that generate mean CT values around 50 s.To test the sensitivity of the modified PPL assay to postprandial lipemia, we compared the PPLht) Fig. . This caCT . As seen in Fig. Addition of recombinant relipidated TF to PPL depleted plasma resulted in a dose-dependent shortening of clotting time Fig. A. Since The procoagulant potential of plasma phospholipids is modified under pathological conditions. Currently, two commercial assays, namely XACT and STA-Procoag PPL are widely used to monitor PPL activity plasma. We thoroughly validated a modified and easy to use PPL assay for the measurement of procoagulant phospholipids in test specimens (plasma samples or isolated EVs) and compared its performance with the STA-Procoag-PPL assay. Our primary modification included preparation of PPL-depleted plasma by ultracentrifugation to remove EVs, assumed to be the main source of negatively charged phospholipids in plasma. This modification allowed for the establishment of an accessible and in-house assay with a comparable performance to that of the commercial assay. The addition of a standardized phospholipid reagent (UPTT) allows for clotting times to be converted into a standardized unit of phospholipids.13, plasma EVs15, and monitoring of plasma EV levels in various disease states19. Assuming that the vast majority of procoagulant phospholipids in plasma are EV-bound, we examined whether sequential centrifugation, including final ultracentrifugation, might substitute phospholipase treatment for production of a suitable assay plasma. Indeed, subjecting plasma to sequential centrifugation resulted in prolonged clotting times (i.e. reduced PPL activity), thus supporting that the major part of PPL activity in plasma is mediated by EVs, as originally suggested by Chargaff and West3.The commercially available PPL assays are well established in research and provide a reliable tool for assessing PPL activity in human plasma. Such assays are dependent on chemically phospholipid depleted plasma provided by the assay as a reagent. Their performance has been assessed under different pre-analytical and experimental settings such as quality control of cell storageCT. These findings support that the modified assay provide comparable performance to the STA-Procoag PPL assay with regard to sensitivity and measurement of levels of PPL in plasma.Since ultracentrifugation may leave residual amounts of PPL in the assay plasma, it was crucial to compare the properties of our PPL depleted plasma with the phospholipase-depleted plasma. Therefore, the performance of the modified PPL assay and the STA-Procoag-PPL assay were compared using a standardized UPTT reagent. The results revealed a high degree of coherence between assays. Further, measurement of endogenous plasma PPL in PFP obtained from 10 volunteers demonstrated strong correlations between assays assessed by PPLCT14. They found that PFP prepared according to the International Society of Thrombosis and Haemostasis (ISTH) recommended protocol (2500×g for 15 min twice) yielded similar PPLCT to another common centrifugation protocol  in fresh PFP. However, PFP obtained by the second, less intensive centrifugation protocol, displayed a substantial shortening of the PPLCT after a freeze–thaw cycle (− 154%). A minor effect on PPL activity (− 5%) was observed when analyzing frozen-thawed PFP using the ISTH recommended centrifugation protocol14. Accordingly, we found that a routine protocol for PFP preparation  yielded a 24% shorter PPLCT than PFP prepared as recommended by ISTH. Hence, the plasma centrifugation protocol should be taken into consideration when comparing plasma PPLCT between studies. As plasma depleted of PPL is an essential reagent in the modified assay, the importance of not introducing assay variations is critical. We demonstrated that three independent batches of PPL depleted plasma prepared from the same donors performed similarly, supporting the use of ultracentrifugation as a method for PPL depletion.It is important to note the effects of pre-analytical parameters on the performance of the modified PPL assay. Recently, Lacroix and colleagues used the STA-Procoag PPL assay to investigate the effect of different centrifugation protocols for PFP preparation on plasma PPLCT was unchanged in fasting and postprandial plasma, suggesting that PPLCT is independent of postprandial lipemia and that there is no need to use fasting blood samples to obtain reliable PPL activity in plasma samples. Similar studies on the effect of lipemia should be repeated using more stringent pre-analytical conditions of plasma collection to see whether lipemia has a slight albeit detectable effect on the procoagulant properties of EVs. However, our findings are supported by Silveira et. al. who reported no effect of postprandial lipemia on the overall PPL measures, using the STA-Procoag PPL assay20. Using the same assay, Mørk and colleagues showed that even a non-standardized meal and a shorter time interval between the ingestion of the meal and blood draw (75 min) resulted in no change in PPL between fasting and postprandial samples21.An additional pre-analytical challenge for population based studies is the availability of fasting blood samples as the PPL activity in plasma may be affected by plasma levels of triglyceride-rich lipoproteins. We therefore tested the PPL activity in plasma isolated before (0 h) and 4 h (4 h) after a high fat meal on the modified PPL assay. PPL6. The inter-individual variability may affect the degree of activation of several coagulation factors in the test samples. We therefore tested a wide range of FVIIa and FVa concentrations, where high concentrations of coagulation factors proved to shorten the clotting time in the modified PPL assay. However, these effects occurred only at supra-physiological concentrations significantly higher than those observed in vivo. Another aspect is the presence of TF. While only minute quantities are normally present in human plasma, TF is thought to be a major procoagulant factor found in EVs22. Previously, Connor and coworkers showed that increasing concentrations (0–0.1%) of TF added to whole blood were insensitive to the XACT assay4. Accordingly, we found that monocyte-derived EVs expressing TF after LPS stimulation  didn’t affect the clotting time in our assay. However, we observed a dose-dependent decrease in the clotting times with increasing supra-physiological concentrations of relipidated TF. Taken together, our findings suggest that the modified PPL assay is not influenced by physiological concentrations of the clotting factors in the test samples.Procoagulant phospholipids, and PS in particular, affect the activity of both the intrinsic and extrinsic tenase and prothrombinase complexes, as well as the activation of FXI by thrombin25, arterial cardiovascular diseases26, cancer28, atherosclerosis30 and diabetes31. In vascular disorders, the procoagulant properties of EVs are of particular interest. It has been shown that PS is the main phospholipid contributing to the procoagulant function32.Over the last decade the interest in assays measuring the negatively charged phospholipid fraction of plasma has increased along with the growing interest and knowledge about EVs. Elevated levels of EVs, most frequently measured by flow cytometry, were found in venous thromboembolism33. It has been demonstrated that lactadherin is an effective anticoagulant blocking the activity of PS, and inhibits the procoagulant activity of blood cells, endothelial cells and extracellular vesicles by 80%34. Here we showed that PPLCT was prolonged with increasing concentrations of lactadherin to the extent that it was no longer measurable in the assay, implying that the measured PPL activity is largely dependent on PS in the test samples. Similarly, using the XACT assay Aung et al. showed that pre-treatment of packed red blood cell supernatants by lactadherin prolonged clotting times13.The abundance of PS on the EV surface is often used to characterize EVs, by exploiting the ability of annexin A5 or lactadherin to bind PS on the outer leaflet of the membrane. Lactadherin is a small glycoprotein that binds PS in a calcium-independent manner and with higher affinity than annexin A535. Further, our modified PPL assay displayed minor variation in the assay performance. CVs obtained with either the standards from the Stago STA-Procoag-PPL or in-house pooled PFP range from 2.8 to 4.1%, well within recommended acceptable limits for within-day and between-day variability. Similar results were shown by van Dreden and colleagues in the XACT assay, with intra-assay CVs of 3.3% and 3.1% for normal pooled plasma and patient plasma, respectively, and inter-assay CVs of 3.9% and 4.2%10.Any assay designed for large-scale applications should be reproducible over time, and it should be possible to compare the results between different laboratories. To solve the latter challenge we propose the introduction of the UPTT reagent—an inexpensive standardized preparation of rabbit brain cephalin, which allows for clotting times to be converted into a standardized unit of phospholipids. The XACT assay solves this problem by inclusion of a synthetic PPL calibrator, while the Stago assay leaves it up to the users to create a reference range and standards for the clotting time35. Second, pre-analytical conditions and inter-individual variations might impact plasma concentrations of coagulation factors in PPL depleted plasma. However, our modified assay seems to be unaffected by variations within the pathophysiological range.There are two main considerations with the modified PPL assay. First, the results will be influenced by the presence of lupus anticoagulants as well as high concentrations of coagulation factors which may lead to falsely prolonged or shortened clotting times. This is common for all plasma-based assays, and should be accounted for when interpreting the results. The creators of the XACT assay tried to overcome this issue by using plasma of porcine origin. However, while it significantly decreased the assay sensitivity to some of the lupus anticoagulants, it failed to completely eliminate the problemIn conclusion, the use of sequential centrifugation, including final ultracentrifugation, to deplete plasma of procoagulant phospholipids performed equal to enzymatic depletion of phospholipids from plasma in a FXa-based clotting assay to determine PPL clotting times. In addition, we introduced a standardized PPL reagent (UPTT) which allows for clotting times to be converted into a standardized unit of phospholipids. These modifications allowed us to establish an accessible and convenient in-house assay.®, Greiner Bio-One, Kremsmünster, Austria), 3 mL K2EDTA Vacuette® tubes  for cell count or Fragmin  for the Ca-ionophore experiments (preparation of EVs from PRP). The first 3 mL of blood were discarded. Blood was mixed with anticoagulant by gentle inversions of the tube. The samples were kept at room temperature (20–24 °C) and processed within 15 min of collection. Blood was centrifuged twice at 2500×g for 15 min to obtain platelet free plasma (PFP). Both individual PFP samples and pooled PFP (n = 11) were prepared. PFP samples were aliquoted and stored at − 80 °C until use.For the calibration of the modified PPL assay, blood was drawn from healthy volunteers (n = 25) aged 25–79 years old, by venipuncture of an antecubital vein using a 21-gauge needle and minimal stasis. Blood was collected into 3 mL tubes containing 3.2% sodium citrate  . Supernatants were pooled, aliquoted and stored at − 80 °C until further use.Procoagulant phospholipid depleted plasma (PPL depleted plasma) was prepared by sequentially centrifuging citrated blood (n = 18) using the following protocol: 2500×Activated partial thromboplastin time (aPTT) and prothrombin time (PT-INR) were determined for the pooled PPL depleted plasma used in the modified assay. Both aPTT and PT-INR were measured on ACL TOP 750 CTS , using the kits SynthASil , and STA-SPA + , respectively.g for 60 min at 16 °C, pooled, aliquoted and frozen at − 80 °C. PPLCT was measured using the modified PPL assay. Clotting tests were perform in PPL depleted plasma alone, or with UPTT added to mimic normal CT (1:3200 U/ml UPTT) and short CT (1:100 U/ml UPTT).The reproducibility of three independent preparations of PPL depleted plasma from the same six donors were tested. Citrated PFP from six volunteers were centrifuged at 100,000×11. Briefly, blood was drawn from an antecubital vein using a 19-gauge needle in a Vacutainer system with minimal stasis in the morning after 12 h fasting and then 2, 4, 6, and 8 h after a standardized high fat meal (1 g fat/kg body weight). Blood for plasma preparation was collected into 4.5-mL Vacutainers  containing 0.129 M sodium citrate (1:9 v/v). Serum was prepared by letting blood clot for 1 h in a glass tube at room temperature. Plasma and serum were centrifuged at 2000×g for 15 min at 22 °C, transferred into cryovials  and stored at − 80 °C until further analysis. Blood samples collected before (fasting) and 4 h after the meal were selected for the present study. For analysis, samples were thawed, centrifuged 13,500×g for 2 min and measured on the modified PPL assay. Informed written consent was obtained from all participants, and the regional committee for medical and health research ethics (REC North) approved the study. The study was conducted in accordance with relevant guidelines and regulations.In order to investigate the impact of postprandial lipemia, forty study participants donated blood for plasma and serum analysis in a previously described studyThe UPTT reagent was purchased from BioData Corporation . Bovine FXa (bFXa), FVIIa, bovine FV/Va (bFV/Va) were purchased from Enzyme Research Laboratory . Human recombinant tissue factor was purchased from Sekisui Diagnostics, LLC  and lactadherin from Haematologic Technologies Inc. . The STA-Procoag-PPL assay was purchased from Diagnostica Stago Inc. . All other chemicals were from Sigma-Aldrich .Clotting tests were carried out in duplicate by using a Start Max instrument from Diagnostica Stago . Twenty five µl of test plasma or EV suspension was mixed with 25 µl of PPL depleted plasma in a Start-cuvette containing a steel ball, and pre-warmed for 2 min to 37 °C. The reaction was initiated by the addition of 100 µl of pre-warmed assay buffer with a cabled pipette that automatically starts the timer upon pipetting, and clotting time was measured. The assay buffer contains bFXa (0.1 U/ml) in 15 mM calcium chloride, 100 mM sodium chloride and 20 mM HEPES buffer (pH 7.0). The STA-Procoag-PPL assay  was performed according to the manufacturer’s protocol.g for 15 min, 2500×g for 15 min twice, 20,000×g for 30 min and 100,000×g for 60 min. Clotting time was measured after each centrifugation step.Citrated blood samples from six individuals were subjected to sequential centrifugation in order to remove an increasing amount of EVs. The centrifugation protocol was as following; 2500×Concentration of bFXa for the modified assay was determined by serially diluting bFXa from 0.01 to 2 U/ml. Clotting time was measured using the modified PPL assay. Pooled PFP added to PPL depleted plasma or PPL depleted plasma alone were used as test plasma.Standardized UPTT reagent containing 0.1% of rabbit brain cephalin in a buffered solution was used as a calibrator. The UPTT reagent was reconstituted in milliQ water according to the manufacturer’s protocol, and set as 1 U/ml. To obtain a standard curve, serial dilutions of the UPTT reagent  in the assay buffer were added to phospholipid-depleted plasma. UPTT dilution curves were measured for both the modified PPL assay and STA-Procoag-PPL assay.g for 15 min twice  and the second fraction 3000×g for 10 min, followed by 13,500×g for 2 min . Clotting time was measured using the modified PPL assay.The effect of PFP preparation was tested using two different centrifugation protocols. Citrated blood samples from ten volunteers were split in two, one fractions was centrifuged 2500×The effect of varying levels of coagulation factors Va and VIIa were tested on the modified PPL assay. The coagulation factors were added to the reaction buffer in concentrations from 0.02 to 3.0 nM for FVa, and from 5 to 80 nM for FVIIa, and tested in pooled PFP.2+ and Mg2+) 1:3 v/v and centrifuged at 20,000×g for 30 min at 22 °C. The supernatant was discarded and the EV-pellet was resuspended in PPL depleted plasma. PRP-derived EVs were prepared by incubation of PRP (anticoagulated with Fragmin) with 10 µM of calcium ionophore A23187 for 15 min at 37 °C, followed by the isolation procedure described above. The concentrated EV suspension was serially diluted with PPL depleted plasma and assayed within 1 h.The effect of lactadherin pretreatment of PRP-derived EVs on PPL activity was tested on the modified PPL assay. Plasma EVs were isolated from pooled citrated PFP, diluted with PBS  and PMA  at 37 °C for 4 h with gentle agitation. EVs were isolated as described above. TF activity was blocked with an inhibitory antibody at a final concentration of 26 µg/ml . Clotting time was measured on the modified PPL assay.Serum triglycerides were measured by the use of an enzymatic photometric method on the ABX Pentra 400 instrument .Statistical analysis were performed in Graph Pad Prism 9.0.0 . ANOVA was used to test for differences in performance between the modified PPL assay and the STA-Procoag- PPL assay.Supplementary Information


Retraction: LncRNA LINC01305 silencing inhibits cell epithelial‐mesenchymal transition in cervical cancer by inhibiting TNXB‐mediated PI3K/Akt signalling pathway. Shu‐Ping Yan, Dan‐Xia Chu, Hai‐Feng Qiu, Ya Xie, Chun‐Fang Wang, Jian‐Ying Zhang, Wen‐Cai Li and Rui‐Xia Guo. J Cell Mol Med. 2019; 23: 2656– 2666.https://doi.org/10.1111/jcmm.14161), has been retracted by agreement between the journal Editor‐in‐Chief and John Wiley & Sons Ltd. The retraction has been agreed following an investigation based on allegations raised by a third party. A detailed investigation revealed that several image elements of the experimental data were published elsewhere in a different scientific context, while duplications were found in two images in the paper. Based on the findings, the editors consider the conclusions invalid. Despite several attempts of contact with all authors of the paper, a response was not received.The above article from Journal of Cellular and Molecular Medicine, published online on 29 January 2020 on Wiley Online Library (


International Journal of Telerehabilitation (IJT) is a biannual journal dedicated to advancing telerehabilitation by disseminating information about current research and practices. This open-source journal is indexed by PubMed and Scopus. IJT requires no readership or author fees.The The website for the International Journal of Telerehabilitation (IJT) was recently upgraded to OJS 3 which offers significant enhancements over the previous version of the software, especially for accessibility. The conversion features an updated look and feel and improved functionality that will benefit the journal and its work. We are grateful to Michael Johnston who created an updated banner graphic and a new journal format, and Vanessa Gabler, who spear-headed the OJS3 upgrade.This issue was produced during an historic time. The issue's development paradoxically spanned the darkest days of the 2020 COVID-19 pandemic both in the United States and around the world, as well as the miraculously rapid development and distribution of life preserving vaccines. Yet, as we write this, our hearts hurt for our authors and readership whose locales and families are still severely challenged by the virus. We posit that both telerehabilitation and our larger society will forever be changed.Despite the difficult circumstances and the need for most to work remotely, submissions to the journal increased dramatically during the pandemic, hence the current larger than typical issue. COVID19 apparently turned on the spigot of telerehabilitation, full force! Prospective authors and experienced reviewers met the challenge; the latter generously completed extra reviews.International Journal of Telerehabilitation (IJT) features impactful research across rehabilitation disciplines. Among the curated articles, several detail how telerehabilitation is strategically deployed in underserved regions. Essential research on group-based telerehabilitation is presented as the lead article.The current issue of the multi-disciplinary 1 is as relevant for this issue as it was for the previous issue:A portion of a prior Editors' noteWe cannot over-state the contributions that volunteer reviewers make to a journal, its authors, and to current and future readers. Discerning reviewers are essential to the quality control of every journal. The purpose of an IJT review is not just to advise the editors on whether to publish an article. Most importantly, reviewers dig deep inside an article and if needed, advise authors how to elevate the quality of their work. We are most grateful to an assiduous group of new and returning anonymous reviewers, about whose efforts our authors routinely offer appreciation. One experienced scholar recently wrote: “Again, we want to pass on our sincere thank you for considering this submission and what a reviewer this is! They are exceptional and have provided great feedback.”Sections Editor, William E. Janes, OTD, MSCI, OTR/L continues to be a very impactful reviewer who renders supports to many IJT authors.2 We applaud the expertise and the professionalism of Vanessa Gabler, Electronic Publications Manager, Office of Scholarly Communication and Publishing University Library System, University of Pittsburgh, for her long-term work with IJT.Thank you also to the publisher of IJT, the University Library System, University of Pittsburgh.IJT is currently sponsored by the Rehabilitation Engineering Research Center on Information and Communication Technology Access at the University of Pittsburgh. The RERC is funded by the National Institute on Disability, Independent Living, and Rehabilitation Research (NIDILRR). Volumes 1–7 were sponsored by the Rehabilitation Engineering Research Center (RERC) on Telerehabilitation at the University of Pittsburgh.We cordially invite submissions to the Fall 2021 issue beginning on July 1, 2021. IJT accepts original research, case studies, viewpoints, technology reviews, book reviews, and country reports that detail the status of telerehabilitation. References should adhere to the conventions of APA version 7.Sincerely,Ellen R. Cohn, PhD, CCC-SLP, ASHA-F, IJT EditorJana Cason, DHSc, OTR/L, FAOTA, Senior Associate Editor