Unlike the ePTFE graft, CD31-positive endothelial cells covered the whole luminal surface of the SF vascular graft at 4 weeks, indicating better endothelialization. SF vascular grafts may be a promising tissue-engineered scaffold material for abdominal venous system replacement.No alternative tissue-engineered vascular grafts for the abdominal venous system are reported. The present study focused on the development of new tissue-engineered vascular graft using a silk-based scaffold material for abdominal venous system replacement. A rat vein, the inferior vena cava, was replaced by a silk fibroin , tissue-engineered vascular graft . The 1 and 4 -week patency rates and histologic reactions were compared with those of expanded polytetrafluoroethylene vascular grafts . The patency rate at 1 and 4 weeks after replacement in the SF group was 100.0% and 94.7%, and that in the ePTFE group was 100.0% and 80.0%, respectively. There was no significant difference between groups ( These surgical procedures depend on the ability of the surgeon to obtain a size-matched graft to replace the venous defect.An R0 resection, in which the resection margin is free of cancer cells microscopically, is the only curative treatment for hepato-biliary-pancreatic malignant tumors, such as hepatocellular carcinoma, colorectal liver metastasis, biliary tract cancer, and pancreatic cancer. These types of malignant tumors often invade the nearby major abdominal venous system, including the hepatic vein6, great saphenous vein7, renal vein8, and internal jugular vein9 has been reported, but smaller graft size, additional surgical incisions, and longer operation time are disadvantages of autologous venous grafts. Although cryopreserved homologous veins produce better results10, the donor shortage and higher preservation cost remain problematic. Synthetic vascular grafts, expanded polytetrafluoroethylene (ePTFE) and polyethylene terephthalate, are used mainly for arterial replacement and their application for reconstruction of the venous system has been attempted11. In hepato-biliary-pancreatic surgery, however, surgeons are reluctant to use artificial vascular grafts because of low-flow thrombogenicity without endothelialization and higher graft infection rates in contaminated tissue beds under digestive fluid12. Furthermore, these synthetic vascular grafts permanently remain in the human body and the long-term outcome is unclear. To date, no alternative tissue-engineered venous grafts have been reported. A new tissue-engineered venous graft with better endothelialization and a more tolerable risk of infection using absorbable material is desirable.Unlike arterial replacement, venous replacement is rarely reported and there are several inherent limitations. The use of tailor-made autologous venous grafts utilizing the iliac vein13. Silk fibers comprise silk fibroin (SF) and silk sericin14. Silk sericin is an antigenic gum-like protein that surrounds the SF core fibers13 and can be removed through a degumming process15. SF biomaterial has biologic advantages, such as better biocompatibility, high affinity for cells, and susceptibility to proteolytic degradation in vivo without antigenicity17. Recently, experimental artery replacement using double-raschel knitted SF vascular grafts coated with an SF sponge was reported in animal models19. Enomoto et al. first reported rat aorta replacement using an SF graft with a 1-year patency rate of 85%. Anti-CD31 and anti-smooth muscle actin immunostaining revealed that endothelial cells and smooth muscle cells migrate into the SF graft early after implantation and become organized into endothelial and medial layers18. No venous replacement model has yet been reported.Silk fiber is a natural protein fiber and silk thread has long been used in surgery for suturing and ligatureWe evaluated the patency rate of double-raschel knitted SF grafts coated with an SF sponge as an abdominal venous system replacement for the IVC in a rat model. In addition, we investigated the histologic reaction to the SF graft. We hypothesized that SF grafts would have a better patency rate than ePTFE grafts.2, respectively. Porosity was 82%.A schematic figure of the double-raschel knitting pattern is shown in Fig. The number of rats, age, and mean body weight were as follows; SF group: n = 19, 13 weeks, 450 g, and the ePTFE group: n = 10, 12 weeks, 405 g, respectively. The surgical time did not differ significantly between groups , additional studies are needed to evaluate the long-term patency rate of SF grafts for venous replacement in an animal model.In present study, the SF graft had a 95% patency rate at 4 weeks, whereas the ePTFE graft had an 80% patency rate, although the difference was not statistically significant. Unlike reports on arterial replacement, there are few reports of animal models of abdominal venous replacement. In a canine IVC replacement model, an ePTFE graft had a 100% patency rate at 4 weeks, but more than half of each ePTFE graft lumen was filled with a thrombus22. Endothelialization of remodeling processes contributes antithrombogenic and antiatherogenic properties for better patency23, and was reported for an SF vascular graft model of artery replacement16. Endothelial cells covered most of the luminal surface of the SF graft walls at 12 weeks after artery replacement18. Haga and colleagues reported the early completion of endothelialization at 3 weeks after rat aorta replacement with an SF graft16. Furthermore, endothelialization protects the graft from infection23, but endothelialization is not observed in ePTFE grafts24. In present study, CD31-positive endothelial cells covered the whole luminal surface of the SF vascular grafts at 4 weeks. These findings suggest the potential application of the SF vascular graft for abdominal venous replacement in a contaminated surgical field, such as in hepato-biliary-pancreatic surgery.The remodeling processes is key to developing tissue-engineered vascular graft using various scaffold materials. Of these, SF is a biodegradable protein derived from silk that provides an ideal scaffold for various cell types in tissue engineering because of their favorable biocompatibility24. Although SF fibers are coated with glycerin to prevent blood leakage between the fibers during replacement25, in the present study, the SF vascular grafts quickly absorbed blood after recanalization without bleeding  in general. If the outer surface of the SF graft was framed by mesothelial-like cells, the SF graft would have tolerance against infection because mesothelial cells may provide a non-adhesive and protective abilities for the replacement SF graft31. In the present study, however, the inside wall of the SF and the fibrin-like tissue of the ePTFE graft also showed podoplanin staining. The significance of podoplanin for venous remodeling in the present experiment model requires further investigation.In the present study, podoplanin-positive cells were observed on the outer surface of the SF vascular graft, indicating that mesothelial cells were established. Podoplanin is a mesothelial marker32. Endothelialization and cell infiltration were similar in the SF vascular graft compared with a previously reported artery replacement model18, but the thickness of the elastic fibers and smooth muscle, and the remodeling speed may differ. Further studies are needed to elucidate the differences.Although the wall structure of the rat native aorta is similar to that of the inferior vena cava, the elastic fibers and smooth muscle are thickerA limitation of the present study is that we did not assess infection tolerability and inflammatory response to the SF graft. Further experiments with not only a 4-week time-point, but also a shorter and longer observation periods are needed to clarify the remodeling processes, the infection tolerability and the differences between venous and artery replacement animal models.In conclusion, SF vascular grafts may be a promising tissue-engineered scaffold material for replacement in the abdominal venous system.Bombyx mori SF threads was prepared using a double needle bar raschel with ten 30-guage needles and 20 courses on a computer-controlled double-raschel knit machine . In our previous report33, the permeability of SF grafts with a double bar-cord knitting pattern based on the international organization for standardization (ISO) 7198 procedure was 4 ml/cm2/min and its density was 10.96 mg/cm2. The double bar-cord knitting pattern produced by a double-raschel machine has better strength to avoid coming apart at the seams and a thicker structure to allow cell infiltration inside the knitting pattern as a scaffold.The SF double-raschel knit tube made of 33. To make the SF sponge, an aqueous solution of SF was prepared. The remaining degummed SF fibers were dissolved by adding CaCl2-H2O-EtOH solution (molar ratio: 1:8:2) to SF at a concentration of 10% w/v and boiling them at 70 °C for 1 h. This solution was filtered to remove residual solid components, and then dialyzed against a cellulose dialysis membrane  at 4 °C for 3 days. An approximately 4% (w/v) SF aqueous solution was obtained and centrifuged for 30 min at 4 °C at 18,000 rpm to remove impurities. The knitted SF tube was then coated with the SF sponge was as follows. The SF tube was immersed into a pipe filled with the mixed aqueous solution at a ratio of 1 : 1 (w/w) SF and glycerin, which was used as a porogen for coating. The pipe was placed in a desiccator where the interior was kept under reduced pressure of 100 hPa, and the SF graft was removed from desiccator and frozen at − 20 °C overnight. The graft was then immersed in distilled water for 3 days with several water changes to remove the glycerin. The graft was placed a bag with distilled water and sterilized in an autoclave at 120 °C for 20 min.The inner diameter of the SF tube was 3 mm. To remove silk sericin, the knitted SF tube was degummed in a mixture of sodium carbonate (0.08% w/v) and Marseille soap (0.12% w/v) solution at 95 °C for 2 h, as previously reportedThe morphology of the outer surface and the cross-section of the knit SF grafts coated with SF sponges were observed by scanning electron microscopy .34 . For the longitudinal suture retention test, a sample tube was cut to a length of 20 mm and then the suture was passed through 2 mm from the end., pulled by a clamp (3 mm/min.) using a 100 N operator cell until the breaking point, and analyzed. To determine the circumferential tensile strength and circumferential compressive elastic modulus, we prepared dry, short, ring-shaped specimens with an axial length of 10 mm for each tube. The load cell was set to 5 N, and the rate of stretching was 2 mm/min. The tensile strength was measured as a function of the stroke distance and the circumferential compressive elastic modulus was measured when the specimen was compressed using 10% of the inner diameter. All specimens were hydrated for 1 h before testing. Porosity, ε (%), was calculated by following the formula, ε (%) = (V2 − V1)/(V3 − V1) × 100. An SF graft was immersed in hexane solution (V1) for 10 min  under reduced pressure (0.05 MPa). The volume of the hexane solution (V3) was measured again after removing the SF graft filled with hexane.The longitudinal suture retention strength, circumferential tensile strength, and circumferential compressive elastic modulus of the SF grafts coated with SF sponges were determined using a tensile testing machine, table-top material tester  according to the previously reported methodsThe study protocol (I-P16-034) was approved by the University of Tokyo Animal Ethics Committee in accordance with the Japanese and ARRIVE  guidelines. Male Sprague–Dawley rats  were used. All rats were kept for 1 to 4 weeks in micro-isolator cages with a 12-h light/dark cycle and fed a certified diet . The rats were fasted overnight before undergoing the surgical procedure.All surgical procedures were performed by hepato-biliary-pancreatic surgeons . The rats were anesthetized by intraperitoneal injection of pentobarbital (30 mg/kg body weight). The IVC was exposed from the left renal vein at the cranial side to the bifurcation on the caudal side, and all branches of the IVC were ligated and divided with 6-0 nylon  knotted sutures using an electric scalpel. After intravenous injection of unfractionated heparin (100 IU/kg), the proximal and distal portions of the infra-renal IVC were clamped with vascular clips. A 10-mm segment of the IVC was removed and replaced with an SF vascular graft  by end-to-end anastomosis using 10-0 nylon (Keisei Med.) sutures, starting with 2 stay sutures at 180° apart at both the cranial and caudal sides, then suturing the front wall, followed by the back wall. The cranial and caudal side anastomosis each required 12 stitches. The caudal and cranial sides of the vascular clamps were slowly removed, and vessel flow was restored through the SF vascular graft. Graft patency was confirmed by gross inspection. In another group of rats, the IVC was replaced with an ePTFE graft  in the same manner. No anticoagulant, antiplatelet, or antibiotic agents were administered postoperatively.The patency of the grafts in the SF and ePTFE groups was monitored by Doppler ultrasonography with a diagnostic ultrasound system  at 1 day and every 3 days for 1 to 4 weeks after venous replacement. Graft occlusion was defined as the absence of a color Doppler signal. When we found the absence of a color Doppler signal, the rat was anesthetized with an intraperitoneal injection of 30 mg/kg body weight pentobarbital and the graft was grossly and pathologically evaluated.At 4-weeks after replacement, rats underwent a general physical examination to evaluate their condition. Following induction of general anesthesia with an intraperitoneal injection of pentobarbital (50 mg/kg body weight), the rats were perfused with 0.9% saline solution through the left ventricle. The grafts were carefully removed together with the surrounding tissue. Tissue cross-sections were prepared at the middle of the SF and ePTFE vascular grafts and fixed in 10% formalin and snap-frozen in Tissue Tek O.C.T. compound  for histologic analysis. The tissues were fixed in 10% formalin, embedded in paraffin, sectioned , and then processed for hematoxylin and eosin staining. Elastica van Gieson stain was also applied to detect elastic and collagen fibers.35.Semi-quantitative analysis was used to determine the ratio of the SF fibers and infiltrated tissue area of the representative cross-section of the SF graft wall. SF fibers, seen as aggregations of transparent dots on a representative cut surface were encircled by yellow lines on a histologic image. At the same time, infiltrated native cells, the other area of the whole SF graft wall, was encircled with a blue line. The ratio of the remaining SF fiber area (yellow) to native cells (blue) was determined to calculate the area of one cut surface of the SF graft that was replaced by native cells at 4 weeks. The analysis was performed using Image J software 29. The sections were incubated with primary antibodies, including alkaline phosphatase-conjugated anti-αSMA , anti-rat CD 31 antibody , and anti-podoplanin antibody  followed by incubation with biotinylated anti-mouse immunoglobulin (Ig) G secondary antibody . Histologic analysis was performed by one pathologist (M. T. ) without knowledge of the patency outcome. All pathological figures were made using Digital Sight camera control unit with software .Immunohistochemical staining was performed as previously reportedp-value less than 0.05 was considered statistically significant. IBM SPSS Statistics for Windows, version 25.0  was used for the analysis.Factors of both groups were compared using the Mann–Whitney U test. The log rank test was used to assess the patency rate in both groups. A


Persistent neck-pain disability (PNPD) is common following traumatic stress exposures such as motor vehicle collision (MVC). Substantial literature indicates that fat infiltration into neck muscle (MFI) is associated with post-MVC PNPD. However, little is known about the molecular mediators underlying this association. In the current study, we assessed whether microRNA expression signatures predict PNPD and whether microRNA mediate the relationship between neck MFI and PNPD. A nested cohort of 43 individuals from a longitudinal study of MVC survivors, who provided blood (PAXgene RNA) and underwent magnetic resonance imaging (MRI), were included in the current study. Peritraumatic microRNA expression levels were quantified via small RNA sequencing, neck MFI via MRI, and PNPD via the Neck Disability Index two-weeks, three-months, and twelve-months following MVC. Repeated measures regression models were used to assess the relationship between microRNA and PNPD and to perform mediation analyses. Seventeen microRNA predicted PNPD following MVC. One microRNA, let-7i-5p, mediated the relationship between neck MFI and PNPD. Peritraumatic blood-based microRNA expression levels predict PNPD following MVC and let-7i-5p might contribute to the underlying effects of neck MFI on persistent disability. In conclusion, additional studies are needed to validate this finding. Recent studies have identified psychosocial and demographic predictors of PNPD and co-occurring neuropsychiatric disorders9, yet little is known regarding molecular and blood-based predictors10. Additionally, the underlying pathogenesis that drives the transition from acute to persistent neck-pain related disability is not fully understood. This lack of understanding of biological variation in the early peritraumatic period impedes the discovery of blood-based risk biomarkers and tractable targets for preventative interventions.While most individuals recover following traumatic stress exposures such as motor vehicle collision (MVC), a substantial subset develop adverse posttraumatic neuropsychiatric sequelae such as persistent neck pain and persistent neck-pain related disability (PNPD)13. Additionally, microRNAs are also highly stable in blood16 and serve as promising blood-based biomarkers of disease17. Recent cross-sectional human cohort studies indicate that blood microRNAs are associated with PNPD related outcomes of trauma20 and prospective studies indicate that microRNAs can predict similar posttraumatic sequelae23. Such biomarker discovery has also led to the increased understanding, via back-translational and animal model studies, of the role of microRNAs in the pathogenesis of PNPD and related post-trauma outcomes26.Since their discovery, microRNAs have been shown to be powerful regulators of the mammalian transcriptome, play highly influential roles in disease pathogenesis, and represent a promising new class of therapeutic targets33. Recent longitudinal studies have demonstrated that MFI is predictive of28 and associated with34 PNPD, yet the molecular mechanisms mediating this association are not yet clear.In addition to microRNAs as blood-based predictors of adverse posttraumatic outcomes, substantial research efforts have focused on understanding the contribution of skeletal muscle changes to post-trauma recovery, including how fatty infiltration of cervical muscle (MFI) influences tissue/stress injuries in the neckIn this preliminary study, we combined advanced fat/water magnetic resonance imaging (MRI) data of neck muscle and fat composition with blood-based peritraumatic microRNA expression levels to gain insights into microRNA predictors of PNPD (per NDI % scores). In addition, we used in silico analyses to gain insight into shared pathways through which the collective set of PNPD-predictive microRNAs might influence post-trauma recovery. Finally, we assessed how microRNAs might mediate the relationship between neck MFI and PNPD development.All analyses were performed using biological, sociodemographic, neuroimaging, and longitudinal outcome data from a prospective observational cohort of multiethnic men and women enrolled following MVC trauma.30. The current study is a secondary analysis of data investigating the neuromuscular mechanisms underlying poor recovery following MVC . Ninety-seven participants were recruited in the full cohort, were consented, and enrolled via an urban academic emergency medicine department and were eligible provided they both reported MVC-related neck pain  and were within the Quebec Task Force Classification category of Whiplash Associated Disorder Grade II (movement restriction with no radicular symptoms). Exclusion criteria included participant age younger than 18 or older than 65, one or more previous MVC in their lifetime, treatment for neck pain disorders in the past ten years, any nervous system disorder , metabolic system disorder (e.g. diabetes), or those who, by standard emergency medical service protocols were deemed to be at risk for multi-system trauma. The Institutional Review Board of Northwestern University, Feinberg School of Medicine granted approval (STU00090769) and all participants provided informed written consent. All methods  were performed in accordance with the relevant guidelines and regulations set forth by this Board. As part of this longitudinal parent study, all enrolled participants, underwent serial MRI examination at < 1 week, 2-weeks, 3-months, and 12-months post injury to quantify MFI in select bodily muscles. All participants completed a suite of questionnaires including one pertaining to neck-related disability. Only participants without any injury other than whiplash from the MVC were included in the study. Based on initial assessments, participants were screened and enrolled in the study if they did not demonstrate other comorbidities or mental health diagnoses within the last decade. For this sub-study, blood RNA samples were collected from forty-three participants at the time of enrollment (i.e. < 1 week following MVC), and all collected samples were included in the present analysis. In the nested cohort used for the current set of analyses, MRI and blood collection occurred in the same visit and on average was 4.8 ± 1.9 days following MVC trauma.The details of this longitudinal observational cohort have been described previously5. This 10-item questionnaire measures a patient's self-reported neck pain disability on a scale of 0–50 via individual statements about neck pain intensity, headaches, personal care, lifting heavy objects, reading, concentrating, working, driving, sleeping, and recreation. The total score can be multiplied by 2 to produce a percentage score. Percentage scores ≥ 30% have been reported to indicate moderate/severe neck-related disability31 and extensive psychometric testing has indicated adequate measurement invariance to be used as a reliable indicator of self-reported PNPD over time35.Self-reported neck-pain disability  was measured using the Neck Disability Index (NDI)36. Higher initial pain (> 5.5/10) intensity has been associated with worse outcomes36.The Numeric Pain Rating Scale was used as a unidimensional measure of pain intensity in which the respondent selects a whole number (0–10 integers) that best reflects the intensity of their pain37. Higher scores (out of 51) indicate more severe symptoms. Previous work supports using the hyperarousal subscale score as a measure of distress38. Accordingly, we used the hyperarousal subscale score as a way to measure emotional-mental distress37.Symptoms of distress were measured using total symptom severity score of the Posttraumatic Stress Diagnostic Scale (PDS)30. All post-MVC MRI data were collected with a 3.0 T Prisma scanner . A localizer scan and a T2-weighted sagittal turbo spin echo sequence was performed to determine the location of the fat–water scan. High-resolution axial 3D fat–water images of the cervical spine area were acquired using a dual-echo gradient-echo sequence . The scanner outputs the in- and opposed-phased data as well as the water and fat images. A 64-channel head/neck coil was used as a receiver coil to improve signal-to-noise. This scan covered the cephalad portion of C3 through the caudal portion of the C7 vertebral end plate. To address the current set of study aims, MFI was calculated using MRI data from the initial timepoint (< 1 week) only.Muscle fat determination from magnetic resonance imaging (MRI) has been described previouslyhttps://www.osirix-viewer.com/), regions of interest were manually drawn within the fascial borders of the multifidus-semispinalis cervicis from C4 to C7, on the co-registered fat and water images. The software obtains the mean signal intensity within each region of interest, for fat and water. The MFI (%) from 3D water-fat imaging was calculated as the mean pixel intensity of fat-only (Fat) and the mean pixel intensity of water-only (Water) images using the following equation: MFI (%) = Fat/(Fat + Water)*10030. MFI for the left and right multifidus-semispinalis cervicis were measured for each participant. Total MFI was defined as the mean of the left and right MFI at that level. Average of total MFI of all the cervical levels for each patient was calculated.Using OsiriX MD image processing software  was isolated using the PAXgene blood microRNA kit , and RNA was stored at − 80 °C until use.22 and were normalized using upper quartile normalization. microRNAs with an average read count of five counts or less were removed from the dataset, leaving a total of 278 microRNAs for the current analyses. Of note, blood expression levels of let-7i-5p were consistent with previous reports 39.Template libraries for microRNA next-generation sequencing were produced from 1.0-µg total RNA. The samples were prepped using TruSeq Small RNA library prep kits according to manufacturer's specifications . Twelve barcoded libraries were combined per lane and sequenced on a HiSeq 2000 (Illumina). Raw sequencing reads were processed using a custom bioinformatics pipeline as described previously40. Stem-loop RT primers and TaqMan probes for let-7i-5p and endogenous control RNU48 were obtained from ThermoFisher . MiRNA expression was quantified on a QuantStudio QS3, data uploaded to the ThermoFisher Cloud, and analyzed using the Applied Biosystems visualization portal. Relative quantitation (RQ) values were calculated via the 2-ΔΔϹt methodology using RNU48 as the normalizing control. RQ values were then compared to RNA sequencing values using Pearson’s correlation.We used the stem-loop RT-qPCR method described by Chen et al. to quantify let-7i-5p levels in the same samples used for RNA sequencingp < 0.05 and effect size of β =  ± 5 were considered significantly associated with PNPD. These thresholds were selected based previously defined thresholds41, sample size estimates, and post-hoc analyses of the distribution of β-coefficients such that microRNAs to the left and right of the inflection point in the distribution of coefficients were selected (i.e. β =  ± 5). The relationship between MFI and PNPD was also assessed using repeated measures mixed models adjusting for the same potential confounders as above. Statistical analyses were carried out using R 3.5.342.Of the total 97 individuals enrolled in the parent study, the only individuals that were included in the current set of analyses were those from which an RNA PAXgene blood tube was collected . Cohort sociodemographic characteristics were summarized using standard descriptive statistics. Generalized Estimating Equation (GEE) models including random effects were used to evaluate the association between each of the peri-traumatically expressed blood microRNAs and persistent PNPD outcomes for all time points combined . Models were adjusted for potential confounding by age, sex, time since the traumatic event, and participant Body Mass Index (BMI). microRNAs with a significance threshold of 43. Mediation model analyses were performed independently for each microRNA transcript. Briefly, in step 1 of mediation analyses, we assessed the relationship between MFI and PNPD; in step 2, we assessed the relationship between MFI and microRNA transcripts; in step 3 we assessed the relationship between MFI + microRNA and PNPD. In step 2, microRNA transcripts were filtered based on a statistically significant relationship with MFI. In step 3, if the effect of MFI on PNPD was no longer significant with the addition of a microRNA transcript from step 2, that microRNA transcript was considered a mediator.To explore specific microRNA transcripts that might mediate the relationship between MFI and PNPD, mediation modeling procedures were employed44 rank and significance level based on the relative number of targets in that pathway. DIANA miRPath results have been validated and its predictive binding algorithm has been shown to have high concordance with actual microRNA binding. MicroRNAs that have been experimentally validated to bind to the predicted mRNA were identified using TarBase v8.045.A web-based computational tool, DIANA miRPath v3.0, was used to identify molecular pathways overrepresented in predicted targeting by the seventeen differentially expressed microRNAs. DIANA miRPath uses its predictive binding algorithm, DIANA-microT-CDS, to define a list of potential targets for each microRNA, then assigns a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathwayCharacteristics of the 43 participants who provided blood samples at enrollment are shown in Table Neck pain related disability scores varied widely in the week following MVC Fig. , with anp = 0.002), miR-3913–1-5p , let-7b-3p , and let-7i-5p  . Further, using the values derived from RT-qPCR, we were able to replicate the association between let-7i-5p and PNPD trajectories .Seventeen of 278 (6%) microRNAs detected in blood samples predicted PNPD outcomes  in the neck was measured using 3D fat/water images  and that with the addition of let-7i-5p in the model assessing the relationship between neck MFI and PNPD (Table p = 0.366). Such results indicate full mediation by let-7i-5p.Of the seventeen microRNAs identified to predict PNPD Table , let-7i-71, we assessed for molecular pathways (KEGG pathways44) overrepresented in predicted targeting by the seventeen microRNAs that predict PNPD. The pathways with the highest number of gene transcripts targeted by the seventeen microRNAs in Table p = 1.0 × 10−325), the TGF-β signaling pathway (p = 1.40 × 10−6), Amphetamine addiction (p = 1.8 × 10−5) and Axon guidance pathway (p = 1.8 × 10−4). Let-7i-5p is a predicted regulator of the three most statistically significant KEGG pathways presented in Table 78.Using DIANA miRPath v3.032, and persistent neck-pain disability. In this preliminary proof-of-concept study, we combined blood-based microRNA expression data with MFI data to gain increased understanding of the interplay between physiological and molecular biological influences on the development of PNPD following MVC. We first identified seventeen microRNAs that predict PNPD and identified biological pathways in which these microRNAs are involved. We then provided a small-sample validation of the association between neck MFI and PNPD, and finally, we discovered preliminary evidence that let-7i-5p mediates the relationship between fatty infiltrates in neck muscle and PNPD following MVC.Currently, little is known about the molecular mediators predicting PNPD following traumatic stress exposures such as MVC. Additionally, little is known about whether molecular regulatory events mediate the relationship between fatty infiltrates in neck muscle, a validated predictor of PNPD22 and has been shown to be associated with other types of pain, anxiety, and physiological distress79. miR-106-3p has been shown to be involved in neuronal repair following injury80, is affected by physiological stress in animals and humans81, and is a negative regulator of adipocyte differentiation56. Let-7 microRNAs including let-7i-5p and let-7b-3p have been shown to be differentially expressed after stress exposure81, and multiple members of the let-7 family have been shown to be associated with chronic pain85. Consistent with our findings indicating that let-7i-5p mediates the relationship between fatty infiltrates in neck muscle and neck-pain disability, let-7i-5p has been shown to influence adipocyte function in both mice and humans53. Future studies are needed to better understand the contribution of let-7i-5p and other microRNAs to PNPD following trauma exposure and to identify whether let-7i-5p might play a role in the underlying molecular pathogenesis connecting fatty infiltration of neck muscle to the development of PNPD.A number of the microRNAs identified as predictors of neck-pain disability in the current study have been shown previously to be associated with both neuropsychiatric disorders that are related to neck-pain disability and biological processes involved in the pathogenesis of adverse posttraumatic outcomes. For instance, miR-19b-3p has been shown to predict persistent widespread pain and posttraumatic stress symptoms following MVC and sexual assault traumas72) and hippocampal plasticity78, and through the generation and transmission of nociceptive signals77. Additional biological pathways identified through pathway enrichment analyses included the TGF-β signaling pathway, the amphetamine addiction pathway and the axon guidance pathway. Each of these pathways have been shown to influence persistent pain and/or PNPD previously78. Of note, despite not being identified via in silico pathway analyses, it was interesting to find via a structured literature search that over half of the seventeen microRNAs identified as predictors of neck-pain disability following MVC have previously been shown to be associated with adipose tissue biology  receptor interaction” was the most highly enriched KEGG pathway. Components of this pathway have previously been shown to play a role in pain persistence and related disability through its effect on intervertebral disc stability (i.e. degradation of ECM by inflammatory mediators leads to spinal instability and subsequent pain10. This study design also enables the discovery of promising candidates for preventative therapeutics. The limitations of this study include the small sample size, the use of a cohort with predominately women, the lack of adjustment for medication use before, at the time of, or after MVC, and the lack of external replication of microRNA findings. Therefore, the generalizability of our results to additional populations is currently unknown. Future studies should validate this proof-of-concept work in larger and more diverse cohorts.The strengths of this study include the use of a longitudinal cohort study design, paired microRNA and MRI data, and the use of mediation analyses to determine whether microRNAs transmit the effect between our independent and dependent variables. The longitudinal study design in which we measured both microRNA expression levels and fatty infiltrates of muscle in the early peritraumatic period enabled the preliminary identification of biological predictors of a persistent posttraumatic outcome. Such type of discovery is currently in its infancy but holds promise for identifying susceptibility/risk biomarkers of at-risk individualsIn conclusion, in this preliminary study, we identified a set of microRNAs that predict PNPD following MVC trauma. While modest in size and scope, these findings demonstrate promise for future large-scale work that could identify blood-based risk biomarkers and therapeutic targets for preventive intervention. Through such work, we can hope to decrease the global burden of adverse posttraumatic outcomes such as persistent neck-pain disability.


To present the anatomical and functional outcomes of autologous surgical transplantation of a free neurosensory retinal graft in three cases of recurrent and chronic full thickness macular hole (MH).A retrospective case series, reporting the profile, preoperative presentation, surgical technique, and postoperative outcome of three consecutive eyes of three patients who had autologous retina transplantation (ART) surgery for recurrent and chronic MHs, and had a minimum of six months follow-up. The technique involved excision of a free neurosensory graft after laser demarcation of the harvest site. The graft was slid under perfluorocarbon liquid (PFCL) into the MH. A five-day tamponade with PFCL was used to secure the graft within the MH and then exchanged with air.The patients were one female and two males aged 60, 44, and 67 years, respectively. All eyes had successful surgery. Postoperative vision improved from 6/36 to 6/18 in patient 1 and remained same as preoperative vision in the other two eyes. No eye lost vision postoperatively. The main complication of surgery was occurrence of retinal and vitreous hemorrhage in one eye (this did not appear to jeopardize the outcome) and retraction of graft tissue in two eyes.ART appears to be a safe and effective treatment for difficult MHs. Our results are comparable to previous studies. Short-term use of PFCL can be useful to secure the graft within the MH. Methods of improving visual function should be the focus of further research in this promising area. It is hoped that the piece of neurosensory retinal tissue will remain within the hole and eventually get integrated into the surrounding retina and that the surviving graft tissue will provide vision. Initial cases reported dislocation of the neurosensory retinal patch from within the MH intra- and postoperatively.There are few reports on the techniques for autologous retinal transplantation (ART). ART has shown to be promising for the treatment of recurrent, chronic, and myopic macular hole (MH).We report our experience of three consecutive eyes that had ART with short-term perfluorocarbon liquid (PFCL) tamponade and a minimum of six months follow-up.Our technique was a standard 23G unimanual vitrectomy, for all three cases. The Constellation vitrectomy unit  was used. PFCL injection was followed by a laser demarcation of the chosen site for the free graft harvest. Neurosensory retinal tissue was excised within the laser-demarcated area, using a 23G intraocular vertical scissors. The free graft was then slid under the PFCL into the MH with the use of intraocular forceps. The harvested piece of retinal tissue was teased open and spread over the MH (but not tucked into it) to cover the MH using the soft edge of a silicone-tipped cannula. PFCL was left in the eye for five days to secure graft stability and prevent graft dislocation from the MH. On the fifth day, air-fluid exchange was performed.There was no positioning of the patient within this five-day period. The patient was allowed to assume any face or head position they chose during this five-day period.The fluid-air exchange was performed in the operating theatre and took approximately 15 min to do. The procedure involved the use of a 23G system. Air was infused into the eye, while silicon-tipped cannula was utilized to aspirate the PFCL. After all the PFCL bubble was removed, a fluid rinse of the vitreous cavity using balanced salt solution (BSS) was done to ensure that all possibly trapped bubbles of the PFCL in the vitreous base and elsewhere were rinsed out into the vitreous-filled BSS, and this was removed with a repeat fluid-air exchange. This fluid rinse was repeated several times to ensure the vitreous cavity was free of PFCL droplets. An irrigation of the anterior chamber (AC) with saline was also performed to ensure that there were no PFCL bubbles in the AC.All the eyes were commenced on frequent topical steroids (Pred Forte eye drops) and antibiotics as a standard protocol. All three patients gave a written informed consent before the procedure.There was one female and two males aged 60, 44, and 67 years, respectively. Patient 3 was diabetic, but achieved a good control of blood sugar. The other two patients had no systemic comorbidities. All three eyes had successful surgery with retention of the free neurosensory retinal graft within the MH. Integration of the graft with surrounding retinal tissue was evident on OCT after three months in patient 1, as shown in Figure 3. In patients 2 and 3, the free neurosensory graft plugged the MH as seen in Figures 5 and 8, which show the appearance of tissue plugging the MH. Postoperative visual acuity improved from 6/36 to 6/18 in one eye and remained same as preoperative vision in two eyes. No eye suffered a loss of vision. All three eyes were pseudophakic and maintained normal intraocular pressures postoperatively. There was no complication or excessive inflammation noticed from the five-day use of PFCL as tamponade. Full details of the cases are described below.The only intraoperative complication of note, which occurred in patient 3, was intraoperative retinal hemorrhage that happened during the time of neurosensory retina harvest and continued as a postoperative vitreous hemorrhage. He had been on anticoagulants prior to the surgery. This hemorrhage was limited by the PFCL and an adjacent fresh site of harvest was then chosen, and the procedure was completed as planned. Two eyes had retraction of graft tissue, which was evident on postoperative OCT.The first case is of a 60-year-old female, who had a reopened right eye MH after an initial internal limiting membrane (ILM) peel procedure in November 2014. Visual acuity was 6/36, and she complained of a persistent central scotoma due to the recurrent MH . The pre-ART MH base diameter was 1200 microns . ART surgery was performed in July 2018. Site of neurosensory retinal harvest was anterior to the superotemporal arcade . Post ART surgery, visual acuity improved to 6/18 at two months postoperative visit and had remained so till her last clinic visit in December 2019. The MH was closed, and the retinal graft remained within the MH . Her left eye macula was normal . There was preservation of some of the ellipsoid zone (EZ) in the free retinal patch, and this appeared to be continuous with the EZ of the adjacent host retina as seen on OCT .The second case is of a 44-year-old male, who presented with a chronic total retinal detachment and proliferative vitreoretinopathy of greater than six months duration, with multiple peripheral retinal breaks and a MH. Right eye was blind from rubeotic glaucoma.He had a vitrectomy with silicone-oil tamponade in January 2017 followed by removal of the silicone oil in August 2017 with successful retinal reattachment. He regained a postoperative vision of 6/36, however, the MH persisted as shown in the OCT image .The pre-ART MH base diameter was 1060 microns. ART surgery was performed in January 2019. Site of neurosensory retinal harvest was anterior to the inferotemporal arcade.Post ART surgery, visual acuity remained 6/36, however, he gave a subjective impression of visual benefit since he claimed to now see facial details better than preoperatively .There was anatomical MH closure, with retention of the retinal graft within the MH in the early postoperative period as seen on OCT . However, there were persistent intraretinal cystic spaces. At the third postoperative month, the graft tissue was noticed to have retracted as there was now a gap between the edge of the tissue graft and the edge of MH as seen on OCT . Despite this, the outer retina was noted to be present in the graft tissue on OCT .The third case is of a 67-year-old male who presented with a chronic MH and emulsified silicone oil in the right eye . He had a history of having had combined vitreoretinal and cataract surgery in November 2014 during which silicone oil tamponade was used. Visual acuity was 6/36. The MH base diameter was 790 microns and there was emulsified silicone oil within the MH as seen on OCT.Silicone oil removal combined with ART was performed in January 2019. The site of neurosensory retinal harvest was anterior to the inferotemporal arcade. Intraoperatively, at the time of graft tissue harvest there was a significant retinal hemorrhage. This hemorrhage occurred because the patient was on anticoagulant therapy prior to surgery. The hemorrhage was however limited by the PFCL. This hemorrhage necessitated the abandoning of the harvest site and moving to an adjacent site. Postoperative vision remained 6/36. Postoperative OCT revealed that the retinal graft remained within the MH as shown in Figure 8. However, retraction of the graft as happened in patient 2 was evident, and outer retinal layers or intraretinal structures were not preserved. Prior to this, several authors have published works on transplantation of retinal pigment epithelium for the treatment of neovascular age-related macular degeneration (AMD).,5,6 Since this first report, there have been few case reports on the use of ART for treatment of difficult to treat MHs, such as those associated with retinal detachment.,8 Recently, an international collaboration published the largest series on the use of ART for the repair of refractory, large MHs. The findings by this group could provide a yardstick against which future outcomes can be measured. Our consecutive series adds to the growing number of cases and seems to agree with current reports. We found that ART can be performed with relative safety and that it was effective for achieving anatomical MH closure. However, improvements in visual acuity are possibly not yet optimized and may revolve around graft size and harvest site.Grewal and Mahmoud first reported the successful transplantation of extramacular retinal tissue into a refractory MH, with good anatomical and functional outcome.Graft size may be an important factor as it was for patients 2 and 3; there was a postoperative retraction of the graft tissue with a reduction in graft size. This suggests that the size of the free retinal graft should be larger than the MH to ensure MH closure even after the anticipated graft retraction, as was suggested in the original report.,9Improving visual acuity remains a challenge. In our study, one eye had a Snellen acuity improvement in vision, but vision remained the same in the other two eyes. In the collaborative study, vision remained unchanged in 41.5% of eyes and worsened in 21.9% of eyes. None of our patients had a worse postoperative vision, including patient 3 who suffered intraoperative hemorrhage. However, our sample size is much smaller than the reference study. Understanding the functionality of the graft is important to be able to determine visual outcomes in future. Microperimetry can be a useful tool in assessing functionality of the graft tissue, when testing response to light. Other studies reported retinal graft tissue response to microperimetric light testing, but we were not able to perform this in our study. In all three cases, PFCL was used as tamponade for only five days and was then replaced with air. No complications of PFCL tamponade were noticed. In particular, no exaggerated intraocular inflammation due to the use of PFCL was seen within the period of follow-up. Short-term use of PFCL tamponade has been reported previously without significant complications and was also used in the collaborative study.,11,2 PFCL served as a good tool to ensure the free graft covered the MH. Furthermore, it is possible that the PFCL may provide oxygen for graft survival in the early stages of transplantation.In terms of complications, the major postoperative complication encountered was in patient 3 who suffered a retinal and vitreous hemorrhage. The collaborative study also reported one case of the vitreous hemorrhage. Our patient had been on anticoagulants. We therefore recommend considering discontinuation of anticoagulant use before performing this procedure. In our patient, this hemorrhage was limited by the PFCL. Stopping and limiting intra- and postoperative hemorrhage is another useful function of PFCL, as was demonstrated, in this case.To conclude, this study appears to concur with previous reports, suggesting that ART is a relatively safe technique in the management of refractory, chronic MHs. The visual outcome, which may be unpredictable, requires further research to determine optimum graft to host (MH) size and functionality at the macula.Nil.There are no conflicts of interest.


Journal of Clinical and Translational Sciences in 2019. The editors, authors and readers are immensely grateful for their thoughtfulness and expertise that have served our journal well.Stephanie AbbuhlTerry AinsworthMyles AkabasTabia AkintobiBianca AlbersEmily AndersonJoseph E. AndrewsJudith AronsonJane AtkinsonMona AuYoungLorena BaccagliniLaura BalisMakenzie L. BarrMark BauerAna BaumannLiza BehrensMiriam BenderL. Michelle BennettJesse David BermanNazleen BharmalArlene BiermanBeatrice A. BoatengKathleen T. BradyPatrick BrandtDonna BrassilTabetha A. BrockmanChad BrummettCheryl BushnellNancy A. Calvin-NaylorJose CancelasMarjory CharlotMichael CharltonPeggy ChenJames J. CiminoRobert A. ClarkBarry CollerLinda CottlerMara CoyleJennifer CrokerOrianna DamasColin A. DeppMegan DoerrAalap DoshiAnn M. DozierMilton Mickey EderVicki L. EllingrodKolaleh EskandanianEstela EstapeAlecia FairStephanie FreelJanice L. GabriloveRussell E. GlasgowMelody GoodmanMary GorfineShelly GrayAlexandra Greenberg-WorisekMatthew GrossmanKristie B. HaddenHeidi HansonBrett HarnettPaul A. HarrisMatthew HartJ.R. HaywoodWilliam R. HoganMike HogarthLianna IshiharaRebecca D. JacksonJason JohnsonRachel JonesYvonne JoostenFelichism W. KaboAllison KarpynHeidi KeelerMatthew KeenerPhilip KernRoohi KharofaDana KingAgnes KiraggaJackie KnapkeRon KoenigH. Robert KolbRhonda G. KostSunil KripalaniJack KuesDanielle LavalleeColleen E. LawrenceScott P. LayneScott LeischowAaron L. LeppinVivian LewisZ.N. LiMike LinkeKanchan LotaMike LyonsJane MahoneyElizabeth MalcolmCamille Anne MartinaGeorge A. MashourPhoenix A. MatthewsColleen A. MayowskiWayne T. McCormackRachel McGarrigleEmma Anne MeagherTara G. MehtaPaul MeissnerJonathan MerrellFrederick J. MeyersPeter MeyersLloyd MichenerChristopher MillerRachel MoonSean MooneyChristopher MorleyCynthia MorrisGia Mudd–MartinDonald E. NeaseJohn OetzelChristian OhmannJanet M. OkamotoJulie OzierDinesh PalAngela L. Palmer–WackerlyCecilia Patino SuttonSusan M PerkinsSharon PlonByron PowellLori Lyn PriceEnola ProctorJill M. PulleyBoriska RabinJulie RainwaterMarie RapeJennifer ReedRobert L. RhyneSuzanne RiveraBrenda RoblesStephen Joseph RosenfeldKristie RossDoris RubioPatrick RyanMaritza Salazar CampoElias SamuelsElaine SchattnerMichael SchembriCari SchmidtEllie SchoenbaumLinda M. SchollHarry SelkerDana ShafferJackilen ShannonAnantha ShekharGreg SimonChristine SorknessKathleen R. StevensWilliam StratbuckerAlisa SurkisAlan TaitEllen TamborVelma ThompsonJonathan TobinJoel TsevatKatherine R. TuttleJason UmansLaurie Van EgerenMelissa Van DykeBoris VolkovRahma WarsameKevin J. WeatherwaxKaren WeaversFern WebbMary Beth WeberTerrence WittHenry Nolan YoungWe are indebted to the expert referees who have volunteered their time to review submissions for the


A 61-year-old man presented with corneal perforation of 1.0 mm in diameter in his right eye caused by a metallic foreign body fragment. We used the “one-bite mini-keratoplasty” technique, which uses a cornea patch with a single host-graft-host suture to stop aqueous humor leakage. Postoperatively, the graft was completely epithelialized. The suture was removed and the use of soft contact lens was discontinued. Postoperative best-corrected visual acuity (BCVA) recovered to 180/200 and corneal astigmatism was 0.6 diopters. The postoperative course was unremarkable, but corneal perforation recurred due to an ocular contusion at 17 months. He was reoperated using the same technique. His BCVA was 160/200 and corneal astigmatism was 1.1 diopters after reoperation. Despite performing this surgical technique twice for corneal perforation, optimal visual function was maintained even after 2 years. For paracentral corneal perforations, our simple technique may reduce astigmatism and maintain high visual function. Corneal perforation is an emergency condition that may occur due to eye trauma or infectious corneal disorders and leads to hypotony. Although it is necessary to close the perforation, strong astigmatism is likely to occur with simple sutures and conventional lamellar keratoplasty for paracentral corneal perforation. To improve visual acuity and prevent corneal irregular astigmatism as much as possible, we performed “one-bite mini-keratoplasty,” in which insertion of a very small corneal graft into the paracentral corneal perforation was achieved using a single suture to the host cornea. Using this new approach, we successfully treated the patient’s corneal perforation and also minimized astigmatism.A 61-year-old man consulted an ophthalmologist with a foreign body in his right eye. Corneal foreign body was detected. During the removal of the foreign body, corneal perforation was observed. Thus, he consulted our hospital with a soft contact lens (SCL). At the initial visit, the best-corrected visual acuity (BCVA) in his right eye was 20/200. Slit-lamp examination and anterior segment optical coherence tomography revealed a flat anterior chamber  and a paAfter sub-Tenon’s anesthesia of the right eye with 2% lidocaine, we manually excised a portion of the endothelial stroma of a preserved cornea and prepared a graft of approximately the same size as the corneal perforation. Without inserting viscoelastic material into the anterior chamber, we passed a single 10-0 nylon suture  through the host cornea , then thThe following day, the anterior chamber had reformed and Seidel test result was negative. Postoperatively, the patient was treated with 0.1% fluorometholone and 1.5% levofloxacin 4 times daily. One month postoperatively, the graft was completely epithelialized. Two months postoperatively, we removed the suture and discontinued use of the SCL. Postoperative BCVA recovered to 180/200, and corneal astigmatism was 0.6 diopters. However, at 17 months, corneal perforation recurred because of ocular trauma, and a flat anterior chamber and partial laceration at the graft-host junction were observed. We attempted to reform the anterior chamber using an SCL but were unsuccessful. Because the original graft was damaged, the patient underwent a repeat one-bite mini-keratoplasty with a new graft. The postoperative course was similar to that of his first surgery, and the graft was completely epithelialized 2 weeks postoperatively. We removed the suture and discontinued the use of the SCL 4 months postoperatively. Subsequently, graft adhesion was good  and the 1 However, large corneal perforations cannot be prevented using SCL; furthermore, simple suturing causes visual function deterioration due to strong corneal irregular astigmatism. Amniotic 2 and fibrin glue-assisted amniotic membrane transplant methods 4 have been reported to alleviate corneal astigmatism, but these procedures are more complex than our technique. Lamellar keratoplasty is another procedure that is effective against perforation.5 However, this procedure is associated with a possibility of negatively affecting visual function when using a graft larger than the perforated area in cases where the perforation is near the center of the cornea. We performed one-bite mini-keratoplasty in a patient in whom aqueous humor leakage could not be stopped using an SCL. Because the diameter of the corneal defect was 1.0 mm, we initially considered conventional lamellar keratoplasty; however, due to the difficulty of applying several sutures radially around the graft, we adopted the single-suture method using as a filler a corneal graft of approximately the same size as that of the corneal defect. Previous studies have reported the use of “small-diameter keratoplasty”7 or “small-diameter graft,”8 but the use of a small-diameter graft approximately 1.0 mm in size or a suturing method with a single suture has not been previously reported. Chern et al.6 reported severe irregular astigmatism after a “small-diameter, eccentric penetrating keratoplasty” was performed for paracentral corneal perforation. In our patient, the first surgery using this technique showed successful results with little astigmatism. Also, we observed corneal astigmatism of 1.1 diopters and BCVA of 160/200 despite the paracentral corneal re-perforation secondary to ocular trauma that occurred after performing one-bite mini-keratoplasty.Use of SCL is an effective approach to suppress aqueous humor leakage occurring as a consequence of corneal perforation.Corneal tissue is sturdier than amniotic material, and suturing tangentially to the cornea can help minimize corneal astigmatism. In addition, because corneal grafts can be secured without gaps, our technique enables plugging the aqueous humor leakage more reliably than using an amniotic membrane. The corneal tissue provides a scaffold for promoting corneal epithelialization in addition to filling the defect site. In this case, the graft epithelialized and remained stable over the long term. Therefore, in our patient, one-bite mini-keratoplasty resulted in the maintenance of high visual function by suppressing aqueous humor leakage, helping attain corneal epithelialization, and maintaining good visual acuity. This technique is useful in settings where donor cornea is available. Therefore, one-bite mini-keratoplasty could help preserve long-term corneal stability and good visual function including minimize corneal astigmatism for the treatment of paracentral corneal perforation.


DS by stoichiometric calculation was shown to be in agreement with FTIR and was comparable with DS obtained from Proton nuclear magnetic resonance (1H-NMR). Based on this study, stoichiometric calculation allows rapid and direct determination of substitution levels and acyl content without the loss of samples, which provides efficiency and optimization of manufacturing procedures in producing the desired level of esterified starches.Rice and quinoa starch esters were prepared by acylation using short-chain fatty acid anhydrides with different chain lengths . A direct stoichiometric method based on the acylation reaction was used to determine the degree of substitution (DS) and acyl content (AC). In addition, Fourier-transform infrared spectroscopy (FTIR) was used to validate the conformational changes of acylated starch and  Starch is one of the major carbohydrates in the biosphere and the main source of energy in green plants. It is found as granules in grains, tubers, roots, leaves, fruits, and stems. Starch is composed of two main macromolecules, amylose and amylopectin. Starch granules have a wide range of sizes, categorized as large granule: 30–100 µm ; medium granule: 5–30 µm  ; small gIn order to improve the functionality of starch, starch in its native form can undergo modification. Such improvement techniques include chemical , physical , enzymatic, and genetic modifications. Chemical modification of starch by esterification/acylation is most extensively investigated technique. Examples of starch esterification include: modification with octenyl succinic anhydride (OSA) ,9,10, faEsterification with fatty acid anhydrides, as shown in 1H-NMR, 13C-NMR, 31P-NMR, and 17O-NMR) and solid-state NMR (13C CP/MAS-NMR) [Chemical modification by acylation is quantified by the determination of the degree of substitution (DS). Several analytical techniques have been introduced for DS determination such as titration-based methods (acidification and saponification) , FourierMAS-NMR) ,26. FTIRMAS-NMR) . The samThus, the purpose of this study was to compare different techniques in determining the DS of acylated starches modified at different fatty acid alkyl chain lengths  and concentrations. The determination of DS is crucial as a primary stage to validate the effectiveness of starch modification. In this respect, the development and comparison of efficient and reliable DS determination techniques would be highly appreciated by researchers in seeking the precise and accurate DS quantification.Oryza sativa was obtained from Hermann Kröner GmbH, Ibbenbüren, Germany. Starch from Chenopodium quinoa was isolated according to a previous procedure [2SO4 in order to establish approximately 0.7 M of NaOH. Potassium bromide for IR spectroscopy Uvasol® was purchased from Merck . Deuterated dimethyl sulfoxide d-6 (DMSO-d6) (Cas no: P2206-27-1) was purchased from Sigma Aldrich .Commercial rice starch from rocedure . Starche(−) groups, respectively. After the completion of the reaction, the pH was again adjusted from about 5.8–6.3 by the addition of the 0.7 M NaOH solution to 6.5. The molar amount of NaOH was determined throughout the duration of the reaction by monitoring the mass of the consumed NaOH solution using the electronic balance (Starch (70.0 g) was dispersed in 280 g of distilled water for 20 min by stirring with a magnetic bar and plate at 500 rpm. The pH of the dispersion was adjusted to 6.5 by titration with 0.7 M NaOH. At room temperature, the anhydride was gradually added (within approx. 20–40 min) with a burette parallel with 0.7 M NaOH to maintain the pH value of the suspension constant at pH 8.5 ± 0.2. When the desired amount of anhydride was added, the suspension was continuously stirred to allow the acylation reaction to continue under the controlled pH. The pH remained constant after 10–60 min, indicating that the entire amount of added anhydride had reacted with starch or OH balance . Knowledg) and was washed three times. Two washes were done by re-suspending the product in 420 mL of distilled water (15 min) and centrifugation . The final washing step was done by re-suspending the product in 400 mL of acetone and centrifugation. The final product of SCFA starch was dried at room temperature and conditioned for two days until it reached a constant weight.The product was then isolated by centrifugation  with the anhydride. Meanwhile, a side reaction is a reaction of the anhydride with OH(−)– ions. The acylation reaction is stated as below:During the reaction of starch with fatty acid anhydrides  in an aqueous weak alkaline solution, a main and a side reaction occurs. The main reaction of acylation is the reaction of the alkaline activated starch St–OMain reaction:St–OH = starch,3CO)2O = acetic anhydride,(CH3 = starch acetate.St–OCOCHSide reaction:(−) is needed for one mole of acetic anhydride. On the other hand, for the side reaction, two moles of OH(−) are needed for pH neutrality. Therefore, the ratio of the molar amount of OH(−) to the molar amount of anhydride must be between 1 (only main reaction) and 2 (only side reaction). The molar amounts of anhydride and NaOH, which were determined stoichiometrically in this section, are presented in In the main reaction, one mole of OHData from The DS and AC were calculated stoichiometrically from the derived equation by considering the molar amount of educts. From the molar amounts of anhydride (n (Anhyd.)) and sodium hydroxide (n (NaOH)) presented in In order to determine the number of moles of anhydroglucose, the following Equation (3) was used:From the DS value, the acyl content (AC) was calculated by the Equation (4) below:Molar mass of acetyl, M (acetyl) = 43.04 g/mol; Molar mass of propionyl, M (propionyl) = 57.07 g/mol; Molar mass of butyryl, M (butyryl) = 71.10 g/mol; Molar mass of hydrogen = 1 g/mol.Reaction efficiency (RE) of starch modification was calculated using the following Equation (5) based on the starch modification reaction:−1 at a resolution of 2 cm−1. Modified starch, 1.5 ± 0.1 mg, was mixed thoroughly with a mortar and pestle with 250 mg of KBr. One hundred and fifty milligrams of the mixture was first evacuated for 2 min and then compressed with a force of 115 kN (8700 bar) for 3 min. The resulting pellets with a diameter of 13 mm and a thickness of 0.50 mm were then used for measuring the spectra in the transmission mode. The transmittance spectra were baseline corrected and converted into absorbance spectra. KBr pellets were prepared in triplicate and were used for the determination of the mean value ± standard deviation of the integrals.The acyl content of starch esters was determined by a Tensor 37 FTIR spectrometer . FTIR spectra of KBr pellets were recorded in the range of 4000–400 cm1H-NMR spectroscopy. Experiments were performed on an Agilent UNITY Inova  operating at 500 MHz for 1H-NMR. Samples were prepared by following a procedure from [6 at 85 °C with constant stirring until clear solutions were obtained. Then, 500 µL of clear solutions was transferred to 5 mm NMR tubes. Samples were analyzed at 45 °C, with 4.8 s relaxation delay and 128 scans. Due to the hygroscopic nature of both DMSO-d6 and starch, samples in triplicates were freshly prepared before the analysis. The 1H-NMR spectra were analyzed using MestReNova 14.0.1 software , and the DS of modified starch was calculated using the following Equation (6):The chemical structure of starch and DS of modified starches were identified by ure from  with sliThe AC and DS of SCFA starches calculated from stoichiometric equation are shown in −1 of all spectra to an absorbance of 2.000. By calculation of the differences between spectra , a well-separated ester band was obtained. The carbonyl stretching vibration (C=O) band which ranged from 1781 to 1690 cm−1, with a maximum at approximately 1730 cm−1, was determined as shown in A Fourier transform infrared spectroscopy study was conducted in order to determine and validate the conformational changes of SCFA starches compared to their native form. For the DS determination of SCFA starches by FTIR, the transmittance spectra were baseline corrected and converted into absorbance spectra. The absorbance spectra were normalized against the mass of the starch ester by setting the large absorption band of starch at 1020.3 cm−1. This shows that the acylation process has taken place and the starch was successfully esterified. The absorbance increased with the increase of DS or AC. In addition, a positive correlation was identified between integral calculated from the FTIR spectra and AC obtained by stoichiometric calculation as demonstrated in From the FTIR spectra depicted in 1H-NMR was conducted as a reference method to determine the DS values of esterified starches. The resonance peaks observed in the 1H-NMR spectra were identified from their chemical shifts, and the four protons of the anhydroglucose part of the molecule have signals at 5.50 ppm (OH-3), 5.40 ppm (OH-2), 4.58 ppm (OH-6), 4.90 ppm (OH-4). The three methyl protons of the acyl chain of the acyl anhydride were assigned to the chemical shifts shown in the 1H-NMR spectra in 1H-NMR spectra also showed the increasing intensity of assigned methyl and methylene peaks with increasing DS. The 1H-NMR analysis showed that the DS values obtained in this work were in close agreement with the DS obtained in the stoichiometric method as shown in Analysis of 1H-NMR can be used in DS determination, however, challenges in determining the appropriate solvents for the best H shift or minimizing impurities influence the DS determination by 1H-NMR [Advanced techniques such as y 1H-NMR . For thi1H-NMR and are in positive agreement with both methods. The stoichiometric calculation provides an efficient determination with no loss of sample and prevents sampling errors. An important aspect is that the DS is performed simultaneously during the acylation reaction on the entire amount of starch undergoing modification, thereby reducing error, as well as with the possibility for control of the reaction.In this work, a new direct method for the quantification of DS values of SCFA starches has been developed by using stoichiometric calculations. The acylation and DS values obtained in this way were verified by FTIR and


Carboxymethyl cellulose, a hydrophobic derivative from cellulose that can be prepared from different biomass, has been widely applied in food, medicine, chemical, and other industries. In this work, carboxymethyl cellulose was used as the additive to improve the hydrophobicity and strength of carboxylated starch film, which is prepared from starch catalyzed by bio-α-amylase. This study investigated the effects of different bio-α-amylase dosages  and different activation times  on starch to prepare the carboxylated starch. The effects of different carboxymethyl cellulose content on the carboxylated starch film were investigated by analysis viscosity, fourier-transform infrared spectroscopy, thermogravimetric analysis, differential scanning calorimetry, x-ray powder diffraction, scanning electron microscope, and contact angle. The results showed that preparing carboxylated starch using activated starch increased the carboxyl content, which could improve the effectiveness of the activated enzyme compared to prolonging the activation time. The carboxyl starch prepared by enzyme catalysis had a lower gelatinization temperature, and enzyme activation destroyed the crystallization area of the starch, thus facilitating the carboxylation reaction. The addition of 15% carboxymethyl cellulose improved the mechanical properties of the prepared film with maximum tensile strength of 44.8 MPa. Carboxymethyl cellulose effectively improved the hydrophobicity of the starch film with the addition amount of 10–30%, while hydrophobic property was stable at 66.8° when the addition amount was exceeded to 35%. In this work, it can be found that carboxymethyl cellulose improve the mechanical and hydrophobic properties of starch film, laying the foundation for the application of carboxylated starch materials. Cellulose has abundant reserves, low price, environmental protection, and wide application. Carboxymethyl cellulose (CMC), a derivative of cellulose, is a natural anionic water-soluble polysaccharide that is used in food, medicine, chemical, and other industries , thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), x-ray powder diffraction (XRD), scanning electron microscope, contact angle, mechanical properties, and other parameters were used to characterize the prepared carboxylated starch and film. For the first time, the effect of enzyme pretreatment on the performance and application of carboxylated starch was studied. At the same time, the effect of CMC on starch film was discussed to establish a foundation for the industrial application of carboxylated starch film.2 ≥ 98.0%. According to GB/ T601-2002 chemical reagent solution standard, 0.4% copper sulfate solution, 4% sodium hydroxide solution, 3% hydrochloric acid solution, 0.1 mol/L hydrochloric acid solution, 0.085 mol/L sodium hydroxide solution and 10% sodium sulfite were prepared.Starch-Unmodified was purchased from Sigma-Aldrich, USA. Sodium hypochlorite, active chlorine≥7.5%, AR grade, purchased from China Shanghai Titan Technology Co., Ltd. Potassium iodide test paper was purchased from China Shanghai Sanaisi Reagent Co., Ltd, and coppe sulfate pentahydrate was purchased from China Shanghai Titan Technology Co., Ltd., AR grade. Sodium hydroxide was purchased from China Shanghai Titan Technology Co., Ltd., AR grade. Hydrochloric acid sodium hydroxide was purchased from China Shanghai Titan Technology Co., Ltd., AR grade, purity 36~38%. Carboxymethyl cellulose hydroxide was purchased from China Shanghai Titan Technology Co., Ltd., M.W.90000(DS = 0.7), AR grade. Sodium sulfite was purchased from Shanghai Titan Technology Co., Ltd., China, AR grade, SOAccording to the data and preliminary test results, the optimized process was: Toke 60 g of native starch in a round bottom flask and added 90 g of water to made 4 groups of 40% starch milk, put the flask in a water bath and heated to 55°C and adjusted pH = 6. This study investigated the effects of different bio-α-amylase dosages and different activation times on starch to prepare the carboxylated starch. After the enzymolysis reaction was completed, used sodium hydroxide to adjusted the pH=9 to kill the enzyme. After the sample is dried and crushed, waiting to be used in the next step. The samples 1: enzyme content 0 g, activation 0 min; sample 2: enzyme content 0.3 g, activation 10 min; sample 3: enzyme content 0.6 g, activation 10 min; sample 4: enzyme content 0.3 g, activation 30 min; sample 5: enzyme content 0.6 g, activation 30 min.The starch milk pretreated by the first step enzyme was added into a round-bottom flask with agitator and temperature control device, and the pH was adjusted to 9 by 3% sodium hydroxide and 3% hydrogen chloride. Increased the temperature to 45°C, added 15 g NaClO (15% of dry starch), kept the constant temperature for reaction, after reaction for a period of time, determined the pH value in the reactor, and neutralized with 3% HCl, the reaction time was 6 h. After the reaction, potassium iodide test paper was used to determine whether the reaction of the modifier NaClO was complete. Added an appropriate amount of 10% sodium sulfite solution to stopped the reaction, toke out the reaction flask, washed the product with distilled water for three times, then put it in the oven for 4 h at 45°C, and then grinded the carboxylated starch.Weighed 4 g carboxylated starch, preparation of 4% starch aqueous solution. and weighed 0 g, 0.2 g, 0.4 g, 0.6 g, 0.8 g, 1 g, 1.2 g, 1.4 g, 1.4 g, 1.4, g and 1.6 g carboxymethyl cellulose in conical flask , and dissolved with water. The solution was mixed in a conical flask and placed on a magnetic agitator. The rotating speed was adjusted to make the rotor rotate at a constant speed. After dissolution, vacuum degassing, and defogging were carried out. After completion, the solution was poured into the glass culture dish, drying in 45°C drying oven for 6 h to peel off the film.−1, and the number of scans was 64; DSC used DSC204 differential scanning calorimeter manufactured by NETZSCH, Germany. The sample was 3.5, and 5 mg of water was added with a micro sampler. The program was set at 5°C/min, 200°C was the termination temperature, and argon gas was blown and protected at 30 mL /min; TG-DTG was used by TGA209 F3, NETZSCH, Germany. The samples were dried in a drying oven at 50°C for 24 h, weighed 3~5 mg, and the heating rate was 10°C/min. Argon was used as protective gas, and thermogravimetric analysis was performed on the samples at a gas flow rate of 30 ml/min; XRD test adopted Japanese Neo-D/MAX220, the optical tube was made by Philips and the target material was Cu. The sample was 120 mesh powder, dried at 100°C for 8 h. The test conditions were as follows: voltage 40 kV, current 30 mA, starting angle of 10°, termination angle of 60°, step width of 0.02°, curve fitting peak parting method was adopted for calculation . It can be seen from Five starch samples were analyzed using DSC, the starch gelatinization start temperature (To/°C), starch gelatinization peak temperature (Tp/°C), gelatinization end temperature (Tc/°C), and heat absorbed by gelatinization  graph was developed to differentiate the TG data of the thin film materials. In It can be seen from In conclusion, carboxymethyl cellulose was used as the additive to improve the hydrophobicity and strength of carboxylated starch film, which is prepared from starch catalyzed by bio-α-amylase. Activated enzyme was more effective at increasing the carboxyl content than was prolonging the activation time of the enzyme. Enzyme activation destroyed the crystalline structure of starch and effectively promoted the carboxylation reaction. Carboxymethyl cellulose effectively improved the hydrophobicity of the starch film with the addition of >10% carboxymethyl cellulose. The contact angle was 66.8° when the additive amount was 35%. The addition of carboxymethyl cellulose improved the mechanical properties of the membrane material, and the maximum membrane performance was 44.8 MPa when 15% was added. The addition of carboxymethyl cellulose can form an aliphatic aromatic structure partly through an esterification reaction, which can improve the thermal stability of thin film materials. The modified carboxylated starch film can be used at higher temperature and humidity, expanding the application range of the film.The original contributions presented in the study are included in the article/CL, HK, and ZS conceived the project and contributed to the concept of the manuscript. SQ, JX, and XL synthesized and characterized the carboxylated starch, and performed experimental works. JY and YZ synthesized and characterized the composites. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


HCR/Tol) present with improved vascular function. Conversely, HCR-mtLCR/Tol rats displayed indices for cardiac dysfunction. The outcome of this study suggests that the interplay between the nuclear genome and the maternally inherited mitochondrial genome with high intrinsic exercise capacity is a significant factor for improved vascular physiology, and animal models developed on an interaction between nuclear and mtDNA are valuable new tools for probing vascular risk factors in the offspring.Exercise capacity is a strong predictor of all-cause morbidity and mortality in humans. However, the associated hemodynamic traits that link this valuable indicator to its subsequent disease risks are numerable. Additionally, exercise capacity has a substantial heritable component and genome-wide screening indicates a vast amount of nuclear and mitochondrial DNA (mtDNA) markers are significantly associated with traits of physical performance. A long-term selection experiment in rats confirms a divide for cardiovascular risks between low- and high-capacity runners , equipping us with a preclinical animal model to uncover new mechanisms. Here, we evaluated the LCR and HCR rat model system for differences in vascular function at the arterial resistance level. Consistent with the known divide between health and disease, we observed that LCR rats present with resistance artery and perivascular adipose tissue dysfunction compared to HCR rats that mimic qualities important for health, including improved vascular relaxation. Uniquely, we show by generating conplastic strains, which LCR males with mtDNA of female HCR (LCR-mt  Near the close of the 20th century, the HERITAGE family study established that our untrained capacity to exercise, or our intrinsic exercise capacity, is genetically heritable2max, myocardial function, energy expenditure, and a lean body mass, as well as greater mitochondrial biogenesis.,,It is well-established that exercise brings about a host of benefits to the health of an individual,8–11 Given that intrinsic exercise capacity, in addition to cardiovascular disease risk, is genetically heritable, we questioned if deleterious vascular plasticity would be present in LCR, while beneficial vascular plasticity would be present in HCR. Our first aim was to determine whether intrinsic (untrained) exercise capacity induces divergent vascular plasticity. Here we observed that the arteries from HCR have increased vascular relaxation and anticontractile effect of perivascular vascular adipose tissue (PVAT). On the contrary, LCR presents vascular atrophy and a procontractile PVAT. By contrast, studies with closely apposed PVAT from HCR and arteries from LCR improved vascular function in arteries from LCR. These data reveal a novel, dichotomous mechanism for vascular homeostasis in inherited exercise capacity.Low intrinsic exercise capacity and physical inactivity lead to cardiovascular diseases.,Humans are the result of billions of years of coevolution between mitochondrial and nuclear genomes. Studies in the 21st century began demonstrating the interplay between these two genomes and how mitochondrial variations can have significant impact on gene expressions and phenotype.,,https://rgd.mcw.edu/rgdweb/report/strain/main.html?id=10402167; https://rgd.mcw.edu/rgdweb/report/strain/main.html?id=10402163). In this study, we used male LCR and HCR rats (20–26-weeks-old) from generation 42 of selection; male LCR-mtHCR/Tol and male HCR-mtLCR/Tol Toledo strain (Tol) rats (30–36-weeks-old) . Given that the selectively bred rats present with a background genetic variability which could impact the interpretation of the data, we also performed a key experiment (vascular function), which is pertinent to this study, in arteries from inbred LCR/Tol and HCR/Tol rats (26–28-weeks-old). In all cases, no more than two males from the same litters were used in the current investigation.LCR and HCR rats were generated from genetically heterogenous N:NIH stock.HCR/Tol (RGD: 39457682), HCR-mtLCR/Tol (RGD: 39457683), LCR/Tol (RGD: 39457699), and HCR/Tol (RGD: 39457701) are new strains that are registered in the RGD. These are named according to the guidelines by the international committee on standardized genetic nomenclature for inbred rats (http://www.informatics.jax.org/mgihome/nomen/strains.shtml#rats). Tol: designates the origin of these rats as Toledo. LCR-mtHCR and LCR-mtLCR are standard nomenclatures for a congenic strain, and in this case, because it is mitochondrial transfer, the name is “conplastic” instead of congenic. Usually, the number of the chromosome follows in the name of the congenic. In this case, it is replaced by the suffix “mt” referring to the transfer of mitochondria.LCR-mtHCR/Tol strainLCR/Tol strain and maintained in subsequent generations by brother–sister mating.HCR/Tol and HCR-mtLCR/Tol rats were 30–36 weeks old when studied. The conplastic rats were from +30 generations of inbreeding.The full report about conplastic animals is described in our previous study.LCR/Tol mtDNA were identical to the mtDNA reported from Wistar Kyoto inbred strain, while HCR/Tol and LCR-mtHCR/Tol mtDNA were identical to the mtDNA reported from Fischer 344 Brown Norway F1-hybrid strain. Sequence differences in mtDNA from conplastic animals are available in our previous study.We previouslyAll rats were maintained on a 12-h light cycle with normal chow and water ad libitum. All procedures were performed as per NIH guidelines and were in accordance with the guidelines by the Institutional Animal Care and Use Committee of the University of Toledo.Left ventricular (LV) function and geometry were evaluated by echocardiography using a Sequoia C512 System  with a 15-MHz linear array transducer as previously described.2 administered via nose cone). Mesenteric resistance arteries (MRAs), aortas, mesenteric PVAT (M-PVAT), whole hearts, and tibias were harvested from all rats. The left and right ventricles were dissected and weighed. The following tissues were flash-frozen in liquid nitrogen to be used for molecular biology experiments: MRA, M-PVAT, aortas, and heart.Rats were weighed and euthanized by thoracotomy and exsanguination via cardiac puncture under isoflurane anesthesia (5% in 100% O3 (cubed).3 showed that LV weight crossed the x-axis at TL3 −26.73  −29.83; −23.43). Therefore, TL and LV weight were indexed by dividing the weights by 26.73 + TL3.It has been shown that body weight correlated nonlinearly  with tibia length (TL), but linearly with TLPhotographs of M-PVAT were taken prior to vascular function experiments using an iPhone 8 . The images are presented in Microsoft PowerPoint .−6 mol/L) and relaxed with acetylcholine .Third-order MRAs were mounted on DMT wire myographs . The MRAs were normalized to their optimal lumen diameter for active tension development as described previously by our group.−6 mol/L PE. All curves were from 10−9 to 3 × 10−5 mol/L. Relaxation responses to ACh and SNP are shown as a percent of the initial PE contraction (3 × 10−6 mol/L). Vascular relaxation was also evaluated in the presence of M-PVAT from different strains. For this, 2 cm long strands of M-PVAT were placed inside of each wire myograph chamber prior to the first cumulative concentration-response curve to ACh. Given that we also evaluated the effect of “sandwich” bioassay studies with closely apposed PVAT from LCR with arteries from HCR and vice versa, we deattached the PVAT from all arteries to eliminate divergences in the results between non- and native PVAT. We ensured that the M-PVAT was resting near the mounted artery but was neither compressing the artery nor contacting the force transducers of the wire myograph. Therefore, any changes in the vascular function performed in the presence of PVAT were due to PVAT-derived factors.Vasodilation was evaluated by performing cumulative concentration-response curves to ACh and nitric oxide (NO) donor sodium nitroprusside (SNP) after initially contracting them with 3 × 103, 4.7 KCl, 1.2 MgSO4.7H2O, 2.5 CaCl2, 1.2 KH2PO4, 11.1 glucose, and 0.01 Na2EDTA). Intraluminal pressure was raised to 160 mmHg and the artery was adjusted to ensure the walls remained parallel. The MRA was equilibrated at 60 mmHg in normal Krebs for 30 min. After testing VSMC integrity with 120 mmol/L KCl, the extraluminal Krebs was exchanged with high Ca2+ Krebs (2.5 mM). Intraluminal pressure was dropped to 3 mmHg and a pressure curve was obtained by increasing the intraluminal pressure in 20 mmHg steps to 160 mmHg. After this curve, the extraluminal buffer was exchanged with zero Ca2+ Krebs (plus EGTA) and equilibrated at 60 mmHg for 10 min. After this period, another pressure curve was obtained by increasing the intraluminal pressure in 20 mmHg steps from 3 mmHg to 160 mmHg. Videos and/or images were captured using the DMT data capture software and subsequently analyzed with the VasoTracker Offline Diameter Analyzer for accurate inner and outer diameter measurements.Fifth to seventh order MRAs were mounted on DMT culture myographs  with intraluminal HEPES physiologic buffer and extraluminal Krebs physiologic buffer  containing 10% fetal bovine serum and 1% penicillin/streptomycin solution . In this study, cells of passage four through six were used. We used the Agilent Seahorse XF Cell Mito Stress Test to measure key mitochondrial function parameters by direct measurement of the oxygen consumption rate (OCR) of the VSMCs on the Seahorse XF96 Analyzer. The day prior to the assay, cells were plated at a density of 8000 cells per 80 µL into a 96 well Seahorse XF Cell Culture Microplate and the XF96 sensor cartridge was hydrated in Seahorse XF Calibrant at 37°C in a non-CO2 incubator overnight. The plate was mapped to have triplicates per animal. The day of the assay, Seahorse XF DMEM was supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose. The final well concentration of oligomycin, an ATP synthase inhibitor, was 1.5 µM, FCCP, an ionophore that shuttles hydrogen ions, was 1 µM, and Rotenone/Antimycin A, an electron transport chain complex I and III inhibitor, respectively, was 0.5 µM. These drugs were added sequentially added by the Seahorse XF96 analyzer to assess mitochondrial respiration. Hoechst 33342 (20 µM) nuclear staining dye was mixed with the Rotenone/Antimycin A mixture to allow for post-assay fluorescent image captures with the BioTek Cytation 5  and subsequent cell counting for data normalization using Fiji,VSMCs were harvested from the collected thoracic aortas. Cells were then grown in a humidified chamber at 37°C, with 5% COP < 0.05. Specific procedures used include Student’s unpaired t-test, to compare the means between two samples; one-way or two-way analysis of variance (ANOVA) to compare more than two conditions and concentration-response curves, respectively. Tukey’s post hoc testing and the Bonferroni post hoc testing were used in one-way ANOVA and two-way ANOVAs, respectively. Data are normally distributed. For this, we used normality test (D’Agostino and Person and/or Shapiro–Wilk test). The sample size indicated per experiment is the number of independent rats used.All statistical analyses were performed using GraphPad Prism 8.2 . Data are presented as mean ± SEM and statistical significance was set at To confirm that inbred (Tol) and selectively bred strains preset similar phenotypes, we performed vascular function in MRAs from HCR/Tol and LCR/Tol and compared with selectively bred HCR and LCR. Arteries from inbred (Tol) and selectively bred LCR present with comparable vascular relaxation . LikewisHCR/Tol rats when compared to LCR .Pressure myography enables us to examine the passive structural characteristics of fifth to seventh order MRAs. HCR/Tol  or HCR-m and decreased RWT, reflecting changes similar to those seen in the development of dilated cardiomyopathy. These results suggest that mtDNA is indeed a heritable and important factor in determining cardiac phenotype and function.An “athlete’s heart” is a concept that has been extensively used to characterize the changes that occur to cardiac function and structure due to long-term physical exercise.HCR/Tol and HCR-mtLCR/Tol may not be associated with food intake. Accordingly, it has been shown that lean, HCR rats consumed more calories after mass correction compared to overweight, LCR rats.Changes in the body weight from these animals were directly correlated with changes in epididymal fat pad. Epididymal fat is a visceral fat and excess of this fat brings on a host of consequences including increases in cardiovascular and metabolic disease risk.HCR/Tol. Given that resistance arteries are important to control blood pressure, here we investigated if deleterious functional and structural changes would be present in arteries from LCR and if LCR-mtHCR/Tol would improve these parameters. We observed that LCR MRA presented a significant dysfunction in the endothelium-induced relaxation when compared to HCR. Endothelial dysfunction was also correlated with mtDNA, since LCR-mtHCR/Tol restored relaxation response of LCR MRA. These findings suggest that mtDNA is important to maintain vascular function and subsequently, blood pressure homeostasis. Mitochondria within VSMC are implicated in maintaining vascular tone and energy production for vascular cell secretion.35–37 Given that LCR-mtHCR/Tol also had improved endothelium-independent relaxation, it is possible to suggest that the restoration in vascular relaxation is, at least in part, mediated by intrinsic changes in VSMCs. In this study, mitochondrial function, measured by OCR of the VSMCs, was not different between LCR and HCR. However, we observed that nonmitochondrial respiration was elevated in LCR. In leucocytes, nonmitochondrial OCR is typically attributed to enzymes associated with inflammation, including cyclooxygenases, lipoxygenases, and NADPH oxidases, and are regarded as negative indicators of bioenergetic health. Therefore, an increase in nonmitochondrial respiration is due to an increase in reactive oxygen species generation. In the last decade, the importance of bioenergetic health has increased, suggesting that the measurement could become the next major predictor of mortality and disease, like BMI.As described in our previous study,HCR/Tol rats.Examining passive, mechanical properties of MRA led us to apply the label of “inward hypotrophic remodeling” (vascular atrophy) to both the LCR and LCR-mtHCR/Tol presented a PVAT with ridged appearance while HCR-mtLCR/Tol abolished the ridged appearance of HCR PVAT. The macrostructure of M-PVAT is rarely mentioned in the current literature because typically, small sections of PVAT or PVAT supernatant are used to study the biochemical characteristics of the tissue. Presently, there is no standard method for quantifying visible differences in PVAT macrostructure. We still do not know why these morphology changes occurred in HCR M-PVAT. However, it is possible to infer that with a greater surface area, the HCR M-PVAT can exert a greater net vasodilatory influence via increased paracrine communication between the M-PVAT, MRA, and the surrounding environment.In this study, we observed macrostructural change of M-PVAT in HCR animals. Specifically, HCR M-PVAT presented a ridged and bulbous appearance when compared to LCR. These changes could be due to mtDNA, since LCR-mt,,HCR/Tol restored endothelial cell-mediated impaired relaxation in LCR MRA, there is promise in further examining the characteristics of HCR M-PVAT and its potential therapeutic capabilities in vascular diseases. A limitation of this study was that the specific adipose-derived contracting or relaxation factors were not investigated. The identification of these factors is fundamental for understanding mechanisms of vascular plasticity in divergent models of untrained intrinsic exercise capacity.In health, PVAT secretes anticontractile factors that relax the underlying artery.In conclusion, our results are the first to demonstrate that the interplay between the nuclear genome and the maternally inherited mitochondrial genome with high intrinsic exercise capacity decreases vascular dysfunction in the offspring, while mitochondria DNA of mothers with low intrinsic exercise capacity increases vascular risk.HCR/Tol and HCR-mtLCR/Tol to their outbred progenitors, there is a chance to reduce power and increase variation in the experimental models due to high levels of genetic variation. Such comparisons are not equal to comparisons of conplastic strains because these outbred parental strains and conplastic strains differ in many genes in nuclear DNA in addition to mtDNA, especially when outbred LCR and HCR strains were bred by a system that minimized inbreeding.Nature Methods comparing phenotypic variation between inbred and outbred animals. Traditionally, inbred mice are preferred over outbred mice because it is assumed that they display less trait variability. However, Tuttle et al.By comparing inbred LCR-mtF344 vs. SHR, presented multiple differences in the mitochondria genome.LCR/Tol mtDNA were identical to the mtDNA reported from Wistar Kyoto inbred strain, while HCR/Tol and LCR-mtHCR/Tol mtDNA were identical to the mtDNA reported from Fischer 344 Brown Norway F1-hybrid strain.In a previous study, Houštěk et al.n = 3) for the LCR arteries to evaluate vascular mechanics (HCR/Tol and HCR vs. HCR-mtLCR/Tol)  .zqaa029_Supplementary_DataClick here for additional data file.


The journal retracts the article, “pH Dependent Molecular Self-Assembly of Octaphosphonate Porphyrin of Nanoscale Dimensions: Nanosphere and Nanorod Aggregates” , cited aInternational Journal of Molecular Sciences has approved this retraction. Following publication, concerns were brought to the attention of the publisher regarding the figures. Compared with the raw data, Figure 5a appears to have been manipulated. This has brought about uncertainty regarding the scientific conclusions. In addition, this paper  was submWe apologize to our readership that this went undetected until now.