Moreover, DESS showed comparable performance to the commercially available preservation kit (R2 of 0.98), and samples consistently clustered by subject across each method. These data support that the DESS wipe method can be used for stable, room temperature collection and transport of human stool specimens.While a range of methods for stool collection exist, many require complicated, self-directed protocols and stool transfer. In this study, we introduce and validate a novel, wipe-based approach to fecal sample collection and stabilization for metagenomics analysis. A total of 72 samples were collected across four different preservation types: freezing at -20°C, room temperature storage, a commercial DNA preservation kit, and a dissolvable wipe used with DESS  solution. These samples were sequenced and analyzed for taxonomic abundance metrics, bacterial metabolic pathway classification, and diversity analysis. Overall, the DESS wipe results validated the use of a wipe-based capture method to collect stool samples for microbiome analysis, showing an R There is a complex interplay between diet, the microbiome, and the metabolome that plays a central role in human biology and health –5. The dFor human gut microbiome analysis, recent advances in sequencing techniques and bioinformatics have increased our knowledge of the complex microbial communities and their interactions. It is now well established that these microbes play important roles in relation to inflammation , metabolPreviously, authors have examined various collection methods and stabilization reagents . And although the stability of all these methods and the technical reproducibility were relatively high, the intraclass correlation coefficients were below 0.6 for metrics related to relative abundance . In anotIn addition to these technical limitations and potential loss of data analysis accuracy after sequencing, the stool specimen collection itself can be challenging, and many individuals find the process difficult and not user-friendly , limitinA 2016 study, spanning 15 individuals and over 1,200 samples, provided the most comprehensive view to date of storage and stabilization effects on stool. It suggested that 95% ethanol can preserve samples sufficiently well at ambient temperatures for periods of up to 8 weeks, and includes the types of variation often encountered under field conditions, such as freeze-thaw cycles and high-temperature fluctuations. In addition, a solution containing dimethyl sulphoxide, disodium EDTA, and saturated NaCl (DESS) was originally used for various applications in the preservation entire soil/sediment samples or as a storage medium for microbial community analysis –34. SuchTo address such technical errors and biases in sample collection methods, as well as to enhance the user experience of stool sample collection, we have designed a practical and user-friendly fecal sample collection kit that includes a dissolvable wipe , in which the biological sample is dissolved in a DNA stabilizing solution (DESS). The film, solution, and biological sample are disposed of in a sealable container. Therefore, people can collect their fecal samples as easily as using toilet paper after defecation. The DNA stabilizing solution ensures that the microbiomic community structure is well-preserved during ambient temperature transportation and storage, and the microbiome DNA is extracted from the fixed microbes and used for further laboratory analysis when the samples arrive at the laboratory. In addition, the dissolvable characters of the wipe ensure that all the microbes contact the stabilizing solution adequately when the sample is collected. The primary objective of this study is to assess the extent to which our novel approach to fecal sample collection and stabilization could maintain microbiota composition compared to the following methods: immediate freezing (-20°C), preservation with a commercially available kit, and storage at room temperature (RT).2. Four preservation methods were used to process the samples for metagenomics sequencing: freezing (-20°C), room temperature storage, a commercial preservation kit , and DESS DNA preservation (room temperature). Three replicates per subject for each preservative were collected for a total of 72 samples. A total of 71 samples were successfully sequenced with an average of 8 million sequencing reads and a range of 3 to 38 million reads. One sample from the commercial preservation kit cohort was removed from the analysis due to sequencing failure (no library). The amount of DNA captured by wipe was found to be comparable to other collection and extraction methods. The average DNA yields from extraction were 98.2 ± 49.1ng/uL for DESS, 44.6 ± 42.7ng/uL for the commercial preservation kit, 286 ± 175.8ng/uL for the -20°C samples, and 122.3 ± 95.1ng/uL for the room temperature samples , and negative controls (room temperature without any stabilization) Figure 1A t-SNE analysis across sample types and subjects showed clear clustering by each individual across the cohort, wherein each subject was isolated and separated from one another based on their unique microbiome signature Figure 2Firmicutes, Bacteroidetes, and Actinobacteria, were the top phyla across all samples, with some room-temperature samples also having Proteobacteria, particularly in Subject 4. Overall, no significant changes were found in phylum, class, order, family, genus ranks for any of the collection methods compared to the -20°C samples after correcting for subject and taxa in question . On species level only the room temperature samples showed significant difference compared to the frozen sample (p = 0.033), with the wipe sample and commercial sample not showing any significant differences (p > 0.5 for both). Generalized unifrac distance within the subject’s -20°C samples (intra-group) to their samples collected by the wipe or commercial kit (inter-group) were not statistically significantly different (p > 0.1), whereas the room temperature samples had significant differences (p = 2.18x10-18).Taxonomic assignments of reads to each domain of life were then examined for their relative distributions across the sample types. As expected, Bacteria was the predominant domain (>99% relative abundance) captured by the microbiome analysis across all subjects (-16 for the sample Pearson correlation based on a t distribution). There is an increased relative abundance of human DNA seen in wipe samples compared to the commercial kit , as well as strong inter-sample correlation between the wipe and frozen samples (p < 2.2x10-22). These correlations are even comparable to the correlation found between the commercial preservation and frozen samples , functional pathway classification , and had no significant differences of alpha diversity (p > 0.5) nor beta diversity . Both the DESS and the commercial preservation method showed significantly higher Shannon diversity compared to the room temperature negative control (p < 0.05).This study demonstrates the use of a wipe-based capture method to collect stool samples for microbiome/metagenomics taxonomic and functional metabolite analysis, making collection from large numbers of people simpler and more user-friendly. The DESS wipe preservation method showed comparable performance to a commercial DNA/RNA preservation kit, and was also very similar to the gold standard frozen samples for most metrics, namely the taxonomic classification abundances had strong correlation  where human DNA is more present in the stool , 36, to  studies . Finally studies  and futuThis study provides validation evidence for a wipe-based collection and RT transport method for gut microbiome sampling and metagenomics sequencing analysis. Such a method may enable easier access to sampling, testing, and metagenomics implementation in clinical trials, home use, or even in remote environments, especially given the stability of the method when shipped at room temperature. Indeed, wipe-based collection and processing offers a more user-friendly approach to collecting stool samples for microbiome analysis. Its ease-of-use design and simple instructions (just wipe and place into the tube) should enable easy integration with commercial stool collection kits and future biomedical studies and trials. Importantly, simplified collection protocols that eliminate tasks that most people do not like (such as scooping their own feces for sampling) provides the opportunity to greatly increase adoption of microbiome testing in clinics and at-home testing. Increasing adoption generates enhanced data resources to the scientific community for discovery. Indeed, tools and methods such as these can be applied to help deploy metagenomics analysis approaches for a wide range of both research and clinical applications to learn more about the interplay between diets, microbiota, bacterial metabolites and host to elucidate the role this complex system plays in intestinal health and disease.A total of 6 healthy subjects were enrolled in this pilot study. The inclusion criteria to be enrolled in the study included: age >18; Bristol Stool Scale type 3 and 4 , agree to collect and donate the feces, and the ability to understand and write English. Exclusion criteria included people with constipation, slightly dry, or diarrhea feces , pregnant or breastfeeding females, history of alcohol, drug, or medication abuse, known allergies to any substance in the study product, current diagnosis of inflammatory bowel disease (Crohn’s Disease or Ulcerative Colitis), and currently taking any medication that may interfere with defecation.Fecal collection kits were created and mailed to enrolled subjects with clear instructions on sample collection. A total of 12 samples were collected by each subject yielding a total of 72 samples to be processed. Four preservation methods were used to process the samples for metagenomics sequencing: freezing (snap frozen at -20°C), room temperature storage, Zymo DNA shield kit (room temperature), and DESS DNA preservation (room temperature) . In anotThe wipe sheet is a polyvinyl alcohol film, 8cm x 8cm, similar size as/a little bit smaller than a piece of toilet paper. The volume of preservative is 30 mL. We advised participants to ensure that the size of each sample is around 1 peanut size and shake well until the wipe and stool particles are fully dissolved/dispersed. All the samples are shipped at room temperature except the samples meant for freezing which were shipped on dry ice. It took several days to up to a week for the shipment. When the samples arrived in the lab, all the samples were put into a fridge (4°C) except for the frozen samples which were put in a -20°C freezer.DNA was extracted from all samples using QIAgen PowerSoil Pro Kit. 150 ng of genomic DNA input was used for library preparation with the Illumina DNA Prep kit following manufacturer’s protocol. Indexed libraries were pooled at equimolar concentration and sequenced as 150bp single end reads in an Illumina NextSeq 550 platform. Samples were sequenced as paired-end 150bp for a mean depth of 8.0 million reads per sample . 5 positive plate controls that contain accurately characterized microbial communities  and 5 plate negative controls  were sequenced alongside the samples to control. Resulting sequences were trimmed by Trimmomatic , and theThe study protocol was approved by the Institutional Review Board of the University of Maryland Baltimore (Protocol # HP-00087571). The study participants provided their written informed consent before enrolling in the study.All shotgun metagenomics data associated with the study are deposited into NCBI Sequencing Read Archive under the project accession PRJNA824068.The study protocol was approved by the Institutional Review Board of the University of Maryland Baltimore (Protocol # HP-00087571). The patients/participants provided their written informed consent to participate in this study.HH, CM, BZ, JD, and CM designed the study. HH, BZ, and CD’A coordinated the IRB and subject recruitment. HH, CM, EA, LL, NR, NP, BZ, CM were involved in data analysis, interpretation, and preparation of the manuscript. All authors approved and contributed to writing the manuscript. All authors contributed to the article and approved the submitted version.All authors except for CD’A are employees of Thorne HealthTech.The remaining author declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The preferred method of fixation and surgical treatment for ligamentous Lisfranc injuries is controversial. Transarticular screws, bridge plating, fusion, and flexible fixation have been described, yet none have demonstrated superiority. Furthermore, screw fixation and plating often require secondary surgery to remove implants, leading surgeons to seek alternative fixation methods.To compare transarticular screws and a fiber tape construct under a spectrum of biomechanical loads by evaluating the diastasis at 3 joints in the Lisfranc complex.Controlled laboratory study.Eight matched pairs of fresh, previously frozen lower extremity cadaveric specimens were fixed with either 2 cannulated transarticular crossed screws or a fiber tape construct with a supplemental intercuneiform limb. The diastasis between bones was measured at 3 midfoot joints in the Lisfranc complex: the Lisfranc articulation, the second tarsometatarsal joint, and the intercuneiform joint. Measurements were obtained for the preinjured, injured, and fixation conditions under static loading at 50% donor body weight. Specimens then underwent cyclic loading performed at 1 Hz and 100 cycles, based on 100-N stepwise increases in ground-reaction force from 100 to 2000 N, to simulate postoperative loading from the partial weightbearing stage to high-energy activities. Failure of fixation was defined as diastasis ≥2 mm at the Lisfranc articulation .There were no significant differences in diastasis detected at the Lisfranc articulation or the intercuneiform joint throughout all loading cycles between groups. All specimens endured loading up to 50% body weight + 1400 N. Up to and including this stage, there were 2 failures in the cannulated transarticular crossed-screw group and none in the fiber tape group.The fiber tape construct with a supplemental intercuneiform limb, which does not require later removal, may provide comparable biomechanical stability to cannulated transarticular crossed screws, even at higher loads.Ligamentous Lisfranc injuries are common among athletes. Therefore, biomechanical evaluations are necessary to determine stable constructs that can limit the time to return to play. This study compares the biomechanical stability of 2 methods of fixation for ligamentous injury through a wide spectrum of loading, including those experienced by athletes. The relatively limited stability of this region, lacking transverse stabilizers between the first and second metatarsals, is highly dependent on the oblique Lisfranc ligaments traversing from the medial cuneiform (MC) to the second metatarsal (2MT).46 These ligamentous ruptures may present subtly without subluxation, dislocation, or fracture but will include pain, swelling, and tenderness over the area and are often misdiagnosed as a “foot sprain.” If left untreated, the ligamentous injury can lead to increased pain, instability, osteoarthritis, and morbidity.46 Furthermore, younger patients and athletes, eager to return to physical activity, may have prolonged periods of disability even with less severe ligamentous injuries.Isolated ligamentous Lisfranc injuries often occur because of trauma during sports or recreational activities.19 The goal of surgical intervention is primary reduction, with the most common technique utilizing cannulated transarticular crossed (CTC) screws. Multiple modes of fixation have been proposed as alternatives to CTC screws for open reduction and internal fixation procedures.With recent systematic reviews showing opposing outcomes between primary arthrodesis and open reduction and internal fixation procedures, the controversy regarding ligamentous Lisfranc injury management continues.(IC) adjunct vs (2) traditional CTC screws. We compared reduction and stability at the Lisfranc joint (MC-2MT) and the 2 adjacent joints (IC-2MT and IC-MC) after being exposed to a wide spectrum of loading, including that experienced during sports.The purpose of this study was to compare 2 forms of internal fixation: (1) a fiber tape device passing intraosseously through the Lisfranc articulation with a dorsal intercuneiform 24 The cohorts consisted of 5 male and 3 female donors with an average age of 50 years , an average height of 69 inches , and an average weight of 158 lb . All specimens were thawed overnight. The proximal tibia and fibula were dissected, and the subcutaneous fat and soft tissue were rigorously removed. The bones were dried with paper towels and sanded to create an adhering surface for the potting material. The tibia and fibula were fixed using syndesmotic screw fixation and potted in polyvinyl chloride piping using a urethane filler . The Achilles tendon and distal gastrocnemius muscle were isolated and kept intact during dissection and then whipstitched using a heavy Kevlar braided line for tensile loading (Emma Kites). Careful dissection involved the removal of the dorsal subcutaneous tissue while preserving the capsular attachments of the midfoot and maintaining the tie rod mechanism of the arch, which was performed according to previously published work.38 Miniature screws , used as coordinate markers for diastasis measurements, were inserted into the 2MT, MC, and IC, which were predrilled with a Kirschner wire for pilot holes  without preexisting foot deformity were obtained (Anatomy Gifts Registry) and prepared according to previous work.ot holes . Two scrot holes . Specimens were placed under a static load equal to 50% of their donor body weight (BW) to simulate weightbearing radiographs, which are the most common and preferred method of identifying Lisfranc injuries  located below the plate that supported the bearings. The polyvinyl chloride tube, encompassing the tibia and fibula, was fastened to the vertical actuator of a servohydraulic test frame , as performed in previous work.A linear bone clamp was used to reduce the Lisfranc diastasis, and the clamp was left in place during the surgical repair. Guide wires were utilized with fluoroscopy to ensure proper trajectory of the fiber tape device limbs and CTC screws for placement across the Lisfranc interval and the intercuneiform space. The 2MT-MC limb of the fiber tape repair (Arthrex Inc) utilized a 4.75-mm locking suture anchor in the medial aspect of the MC with an oblong button on the lateral side of the 2MT, while the supplemental limb was created using the same suture and a 3.5-mm anchor in the dorsal aspect of the IC . The CTCDiastasis measurements for the 3 joints  were determined using a 3-dimensional (3D) coordinate digitizer . Three-dimensional coordinate measurements were obtained for the aforementioned 2 dorsal screws in each bone crossing each joint . The tip of the digitizer was inserted into the head of the screw, which helped to fix the axis of the stylus perpendicular to the top face of the screw. The diastasis for each stage of loading was calculated as the 3D Euclidean distance between 2 markers minus the distance measured at the state before cyclic loads were applied (“fixation”). The error of diastasis measurements was characterized by examining the absolute value of changes in intermarker distances between 2 markers in the same bone, which averaged 0.20 mm . The diastasis was measured with the tibia statically compressed, resulting in ground-reaction force equal to 50% of the donor BW. The Achilles was loaded under tension with a force equal to 75% of this ground-reaction force load (37.5% BW).Diastasis measurements were longitudinally recorded at the following sequential conditions as in previous research: (1) “preinjury” with the native anatomy, (2) “injury” after Lisfranc ligament sectioning, (3) “fixation” with either the fiber tape device or CTC screws, and (4) after cyclic loading at the increments specified as follows. Loading was performed at 100 cycles at 1 Hz and a peak magnitude of 50% BW + 100-N (compressive) ground-reaction force, followed by 100 cycles at 50% BW + 200 N, with the force increasing by 100 N every 100 cycles. For all cycles, the “valley” magnitude of load was 50% BW. Specimens were tested until the end of the “50% BW + 2000 N” cycles to evaluate the devices at a wide spectrum of loading that could simulate partial weightbearing to running. The Achilles tendon was held in constant tension at the aforementioned force. Three-dimensional coordinate measurements to evaluate the diastases, as described earlier, were taken after every 200 cycles. The diastasis measurements were made in the following order: proximal 2MT, distal 2MT, distal MC, proximal MC, proximal IC, and distal IC. Measurements were made during a 60-second dwell period immediately after the last cycle every 200 N.Cyclic loading, using 100-N stepwise increases in ground-reaction force, was performed similarly to previous work.48Diastasis at the Lisfranc joint (MC-2MT) ≥2 mm was indicative of failure based on previous studies evaluating this structure and other ligaments in the foot.24 A random specimen donor effect accounted for 2 repeated factors per donor: method of fixation and peak force during cycles. The method of Kenward and Roger was utilized to determine the denominator degrees of freedom, and the estimation method used within the linear mixed-effect models was the restricted maximum likelihood. The set inequality for statistical significance was P < .05. Analyses were performed in SAS software Version 9.4 (SAS Institute Inc), and all hypotheses were tested as 2-sided. In addition, the censored paired data were compared using a paired Prentice-Wilcoxon test.Linear mixed-effects models were used to compare the diastasis measurements for each fixation type  for each of the 3 measured joints at each interval of cyclic loading.P = .55; IC-MC, P = .41; IC-2MT, P = .17). The fiber tape device group had an average MC-2MT diastasis decrease of −2.81 ± 0.52 mm (2.44 to 3.71) while the CTC screw group had an average decrease of −2.61 ± 0.32 mm (2.23 to 3.17), which was nonsignificant between groups (P = .34).The injury model created diastasis mean ± SE values of 2.71 ± 0.44 mm , 0.27 ± 0.56 mm (−0.56 to 0.68), and −0.05 ± 0.44 mm (−0.68 to 0.88) for the MC-2MT, IC-MC, and IC-2MT joints, respectively, across all specimens from all groups before fixation, and there were no significant differences between fixation groups , 50% BW + 1400 N (P = .022), and all subsequent stages through 50% BW + 2000 N (P < .024) (The IC-2MT and IC-MC joint diastases are shown in  < .024) , A-C. CoAt the intercuneiform joint (IC-MC), the average diastasis was 0.01 ± 0.49 mm for the fiber tape group and 0.04 ± 0.55 mm for the CTC screw group at 50% BW + 1400 N including all 8 pairs . At 50% The MC-2MT Lisfranc joint and IC-MC diastases showed no significant differences between the fixation groups. Up to and including this stage, there were 2 failures (≥2-mm MC-2MT diastasis) in the CTC screw group and none in the fiber tape group , B and C Yet, disadvantages including damage to the articular surfaces of joints, retained intra-articular threads, and the need for additional surgery to remove hardware have led surgeons to seek alternatives. Flexible fixation devices have recently been proposed as alternatives, and a fiber tape device with a supplemental intercuneiform limb attached to the dorsal aspect of the IC maintained reduction at high loads. On the basis of these data, we sought to compare the commonly performed CTC screw technique with what we believe is the most relevant and adequate flexible fixation. To our knowledge, this is the first cadaveric biomechanical study comparing a fiber tape device with a supplemental intercuneiform limb to CTC screws. Additionally, no study of the Lisfranc complex has evaluated the joint spaces at loads based on the BWs of donors to simulate diastasis during a weightbearing radiograph or computed tomography. Finally, the model utilized is only the second to increase the ground-reaction force up to loads that simulate those experienced by active patients and athletes, greatly exceeding that applied in most biomechanical models of this injury.32CTC screw fixation is the most common repair of ligamentous Lisfranc injuries to maintain rigid fixation after reduction.The data demonstrate that fiber tape and CTC screws provide biomechanical stability in repairing ligamentous Lisfranc injuries while generally withstanding high loads . Three sWhile elongation is the most intuitive cause of diastasis in the fiber tape group, the reason behind the CTC screw diastasis is less clear. Modes of screw fixation include screw cutout or loosening, screw fracture, and bony fracture. As no implant failures and no gross bony fractures were observed, a plausible cause may be the screws cutting interosseously through the bones. In addition, the 2 specimen failures within the CTC screw group were both female .35 By performing this careful sectioning under fluoroscopy in the anteroposterior plane, we ensured that the intercuneiform joint was not disrupted. Furthermore, the injury model was confirmed through 3D coordinate measurements displaying relatively low diastasis at the IC-MC and IC-2MT joints after sectioning.The injury model in this study involved sectioning all 3 components  of the Lisfranc articulation to yield the most instability from the isolated injury. An advantage of this injury model was that the ligaments were sectioned under 50% BW loading that did not overconstrain the foot and could reproduce at least 2 mm of diastasis at the MC-2MT joint for all specimens. compared a single screw and a suture button, without intercuneiform stabilization in either procedure. Nery et al compared CTC screws and a fiber tape device, also without a supplemental intercuneiform limb. Both these studies reported no significant differences between the flexible fixation methods and their comparator screws. Unlike in our study, both these studies utilized a biomechanical model that constrained the ankle and the forefoot and loaded specimens to a maximum axial load of 343 and 400 N. In a separate recent study of fiber tape reconstructions from our laboratory, we determined significantly less MC-2MT diastasis at higher loads for the fiber tape device with a supplemental intercuneiform limb, when compared with fiber tape alone, under similar loading conditions to those tested in this study.There have been 2 studies to date that have biomechanically compared screws with flexible fixation devices for Lisfranc injuries. Panchbhavi et al45 Some studies have loaded the foot in a fixed plantarflexed position based on reports of this being a common injury mechanism.39 These models, which involve “locking” the foot in place through the ankle using Steinman pins or screws, assess the Lisfranc joint with applied axial loads as low as 100 N and a maximum of 400 N.45 The plantarflexed position may increase shear force between bones and bending moment at the location of the Lisfranc complex; however, the loads tested represent only those experienced during partial weightbearing.40 This is likely due to inherent instability in this setup, which we observed in our pilot testing. A recent study evaluating different screws potted the hindfoot, which allowed for static loads up to 1100 N to be tested. The present biomechanical model allowed for external rotation and pronation without constraint, with the use of the bearing plate.While ligamentous evaluation through 3D diastasis has become a standard approach, controversy exists over the exact mechanism of injury and ideal biomechanical models to test Lisfranc reconstruction. Ten years of National Football League (NFL) Combine data indicated that the average NFL offensive lineman weight was approximately 315 lb (~1400 N). In the stance phase of gait alone, which produces loads slightly larger than full BW, this would provide much larger stresses than most studies, which tested between 250 and 400 N. In addition, a video analysis study involving Lisfranc injuries in NFL players determined that 90% occurred while engaging another player, further increasing these loads. Loads that greatly exceed those involved in gait are required to simulate these types of instances for athletes.Previous literature stated that the position with the highest percentage of Lisfranc injuries in football players is offensive lineman.27 While both fixations withstood relatively high loads, the fiber tape device does not require the planned second surgery, which may allow for faster return to play. By way of a cadaveric model being used in the present study, there is no healing present, nor can healing time be evaluated. However, this study does support the need for a clinical prospective study as the loads tested may provide some evidence for allowing earlier weightbearing and recovery. Although we believe that this study may set the framework for a clinical outcome study, the purpose of the present study was to evaluate biomechanical efficacy and safety.Studies evaluating postoperative protocols for professional athletes have recommended 6 to 8 months before engaging in limited activities, when hardware removal is necessary, and current management outcomes have shown that return to play is nearly a year for Lisfranc injuries.24 A limitation of our study attributed to budgetary restrictions was use of 8 matched pairs of lower extremity cadaveric specimens, similar to other studies.38The main limitation of this study is that measurements were obtained for only the dorsal aspect of the joint. Moreover, after each interval of cyclic loading, measurements were obtained at a static load of 50% BW rather than at the peak loading force . These measurements were obtained immediately after the cessation of cyclic loading in the same order of markers for all specimens to reduce the variability in creep; yet, the direct effects of creep were not analyzed. Given that measurements were obtained in the same order and within a relatively small time window, we expect that there could be small systematic effects of creep among the various diastasis outcomes. Nevertheless, since we had matched pairs, any creep effects would be similar between treatment groups. Specimens were loaded consistently for approximately 1 hour, and we obtained measurements at a static load that was less than the peak loads during our cyclic loading. The cyclic loading can be viewed partly as a preconditioning that should reduce the effects of creep during measurements. Additionally, measurements at loads <800 N, for the reason of smaller loading magnitude, could have been more influenced by system errors. Additionally, the range of donor ages may provide differences in bone and tissue quality, although this issue is often encountered in cadaveric studies. The simplification of loading and standardized failure criteria were also limitations; however, these methods have been previously published.In this cadaveric biomechanical study, the use of CTC screws or a fiber tape device with a supplemental intercuneiform augmentation appeared to provide similar biomechanical stability. The fiber tape device may serve as a viable alternative to screw fixation, while obviating the need for hardware removal. Future work should evaluate clinical outcomes of patients, especially in regard to their rehabilitation protocols and potentially with the use of weightbearing computed tomography scans.


Purified cannabinoids have been shown to prevent proliferation and induce apoptosis in colorectal carcinoma cell lines. To assess the cytotoxic effect of cannabinoid extracts and purified cannabinoids on both colorectal polyps and normal colonic cells, as well as their synergistic interaction. Various blends were tested to identify the optimal synergistic effect. Methods: Biopsies from polyps and healthy colonic tissue were obtained from 22 patients undergoing colonic polypectomies. The toxicity of a variety of cannabinoid extracts and purified cannabinoids at different concentrations was evaluated. The synergistic effect of cannabinoids was calculated based on the cells’ survival. Isolated cannabinoids illustrated different toxic effects on the viability of cells derived from colorectal polyps. THC-d8 and THC-d9 were the most toxic and exhibited persistent toxicity in all the polyps tested. CBD was more toxic to polypoid cells in comparison to normal colonic cells at a concentration of 15 µM. The combinations of the cannabinoids CBDV, THCV, CBDVA, CBCA, and CBGA exhibited a synergistic inhibitory effect on the viability of cells derived from colon polyps of patients. Isolated cannabinoid compounds interacted synergistically against colonic polyps, and some also possessed a differential toxic effect on polyp and adjacent colonic tissue, suggesting possible future therapeutic value. Colorectal cancer (CRC) is the third most common cancer affecting both males and females in the United States  despite Lesions ≥ 20 mm, whether sessile or flat and laterally spreading, are forms of advanced mucosal neoplasia and are considered high-risk precursors of CRC ,7,8. SurCannabinoids are a group of compounds extracted from the cannabis plant. They include endocannabinoids, phytocannabinoids, and synthetic cannabinoids . The phaIn a previous study, tetrahydrocannabinol (THC) treatment induced apoptosis in a CB1-dependent pathway in CRC cells by inhibiting various survival signaling cascades, while activating the proapoptotic BCL-2 family member BAD . AdditioDespite the accumulating literature regarding the anticancer action of cannabinoids, there is little data regarding their cytotoxic activity on colorectal polyps , and theP001-P022 designate the various patients’ polyps. Two patients were excluded from the study analysis due to borderline-positive hepatitis A virus (HAV) serology [patients’ polyps’ number 11 and 15 (P011 and P015)]. Cannabinoid extracts, as well as isolated cannabinoids, were tested on patients’ polyps and adjacent colonic mucosa numbered 1 and 2 (P001-P002). Isolated cannabinoids alone were tested on patients’ polyps and adjacent mucosa numbered 3–10 (P003–P010). P003 was used for calibration. Patients’ polyps numbered 12–22 (P012–P014 and P016–P022) were evaluated for synergistic interaction in inhibiting the viability of colon-polyp-derived cells by various isolated cannabinoids. Patient and polyp characteristics are presented in The isolated cannabinoids’ effect on the viability of the polyp-derived cells was evaluated and is presented in p < 0.03). CBD-rich extract had a similar effect as synthetic CBD.Polyp and adjacent normal colonic tissues were obtained following polyp resection. The differential toxic effect of the various cannabinoids extracts as well as of the isolated cannabinoids was examined. Most of the cannabinoids showed similar toxicity to polyp or adjacent colonic tissue. However, cannabidiol (CBD) exerted a differential toxic effect on polypoid cells and adjacent normal colonic cells as shown in p < 0.0001, Bliss equation calculation = 0.6), CBCA + THCV , CBDV + CBGA , and THCV + CBGA  presented a synergistic effect. However, the cannabinoid combinations THCV+CBDVA and THCV+ CBDV had no synergistic effect, as presented in p > 5, Bliss equation calculation = 0.01 and p > 5, Bliss equation calculation = −0.15, respectively). Notably, the cannabinoids CBCA and CBGA exhibited weak toxicity when administered as a single active compound  were the most toxic and exhibited persistent toxicity in all polyps tested. Additionally, several of the cannabinoids exhibited a broad variability in their capacity to elicit toxicity when different polyps were tested.In view of the differential effect of the various cannabinoids on different polyps, it may be necessary to initially test the effect of the extracts on polyps, prior to using them as therapeutic agents. Cells of a resected polyp could potentially be incubated with various cannabinoids to identify and select the agents that most effectively inhibit the viability of the lesion’s cells. Those toxic agents could be then prescribed for subsequent treatments by applying a pharmaceutical composition composed of one or more of the effective agents. Moreover, such personalized combinations, specifically those with differential activity against polyp-derived cells, could be administered for preventive purposes.CBD was shown to have a differential cytotoxic effect in the presented study. It appeared to be more toxic to polypoid cells than to adjacent colonic cells at a concentration of 15 µM. Since nearly every carcinoma begins with a polyp , CBD couCombinations of the cannabinoids CBDV, THCV, CBDVA, CBCA, and CBGA exhibited a synergistic inhibitory effect on the viability of cells derived from colon polyps of patients. Previous studies have demonstrated a synergistic interaction of cytotoxic activity against colorectal polyps for several combinations . SynergiTo the best of our knowledge, in vivo studies assessing the effect of local administration of cannabinoids to polyp resection beds have not been published to date. Two theoretical measures of safety, the use of non-psychotropic purified cannabinoids and synergism, should be provided for optimized general and local safety profiles. Moreover, the concentrations of cannabinoids used in the cell experiments  were orders of magnitude lower than those used in clinical trials , as shown in a recent publication . CertainTo conclude, our study results support the potential cytotoxic effect of cannabinoid extracts on colorectal polyps, as well as their synergistic and differential interactions. Further studies examining this postulation and the ultimate combination of cannabinoids for inhibiting/decreasing the recurrence rate of neoplastic polyps, and for preventing their malignant transformation into adenocarcinoma, are needed. Furthermore, the combined activity of selected cannabinoids with conventional chemotherapeutic agents, as well as biological selective agents, should be explored.Cells were isolated from polyp specimens and from normal colon specimens. Biopsies from polyps and healthy colonic tissue from the same patient were obtained from twenty-two patients scheduled for polyp resection. Only patients with a large polyp that appeared benign endoscopically, was a granular lateral spreading tumor with 0-Is or 0-IIa morphology as per the Paris endoscopic classification , and wasThe study was conducted according to the guidelines of the Declaration of Helsinki and was approved by the Institutional Ethics Committee of Tel Aviv medical center . Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.Biopsies taken during each colonoscopy were placed in tissue culture media preservation media  and immediately transported to the laboratory. Dissociation of the biopsies’ tissue into single-cell suspensions for subsequent use was performed by using the MACS Technology by Miltenyi Biotec (Human Tumor Dissociation Kit cat. 130-095-929 and gentle MACS octo Dissociator) according to manufacturer’s instructions. After dissociation, cell viability was determined by using cell-permeable dye  and analyzed by flow cytometric analysis.1 μL PI was diluted in 1 mL of 0.5%BSA/PBS buffer. A total of 90 μL diluted PI were added to 10 μL cells and was mixed well. Cell viability was analyzed by flow cytometric analysis .Dry inflorescences of cannabis plants  were used in this experiment for the extraction of cannabis tinctures. The inflorescences were pulverized with liquid nitrogen and then extracted by press at 75 °C for 120 s. The decarboxylation of extracts was performed by heating for 2 h at 140 °C. The heated extracts contained mainly CBD (76%). The tincture was resuspended in dimethyl sulfoxide (DMSO) for a stock solution of 100 mM (control DMSO). For the treatments, the stock solution was diluted accordingly for cell cultures and biopsies in all experiments. Data regarding types of the CBD-rich extract as well as the percentage of cannabinoids as measured by HPLC chromatogram are presented in 50, as measured in our laboratory on many cell lines, ranged from 15–100 µM. Based on this measurement, the starting concentrations for polyp experiments were determined. Later, these concentrations were adjusted to obtain the concentrations defined as 1X and 2X for each cannabinoid. Due to the very small number of cells obtained from one polyp, the ability to perform experiments with different concentrations was limited, as well as the variation from polyp to polyp. Therefore, an IC50 test was not performed for these cannabinoids on polyps; hence, we performed toxicity experiments at uniform cannabinoid concentrations and synergy experiments. The cannabinoids were dissolved in DMSO for a stock solution of 20 mM. The tested cannabinoid concentration did not exceed 60 µM and the DMSO concentration did not exceed 0.3%. This concentration of DMSO showed no toxic effect on the cells in these experiments. The cannabinoids and extracts were analyzed by HPLC .Isolated cannabinoids were purchased from RESTEK for analytical purposes as HPLC markers and for research. The cannabinoid purity was > 99%. The ICCTG  was used to check the cytotoxic effect of the cannabinoids and extracts. CTG was added to each well of the treatments and incubated at room temperature for 10 min. The luminescence was then recorded. The percentage of live cells was calculated relative to the nontreated control wells.To evaluate cannabinoids’ toxicity effects, about 4000–5000 polyp-derived cells were plated into each well in a 384-well plate. Cells were incubated for 48 h at 37 °C with various purified cannabinoids or extracts at different concentrations. The experiment sets were calibrated extensively on many cell lines and different types of cancerous and adjacent normal tissue. Time from 12–72 h was evaluated. The time point of 48 h of incubation was defined as the preferred time in terms of assay sensitivity and allowed the cells to perform normal behavior, such as cell division attempts, protein synthesis, etc. Cell viability (toxicity) was performed by CTG assay.Polyp and adjacent normal colonic tissues were obtained following polyp resection. The differential toxic effect of the extracts on polyp cells as compared to normal colonic tissue cells was evaluated using an assessment of overall cell viability by CTG assay.To determine whether the interaction of different cannabinoids was synergistic, that is, their combined activity was greater than the sum of their separate activities, the extent of activity in different combined concentrations of cannabinoids was examined. To examine synergy in cytotoxic activity between cannabinoids, a CTG assay was used on biopsy cells as described above. For the synergy experiments, cannabinoids were used at a concentration of 1× (viability above 85%) and 2×. For the synergic assay, we used 1× of A, 1× of B, and 1× of A + B. The synergistic effect was calculated according to cells’ survival experimental results according to the Bliss Equations (1) and (2) as follows:S designates survival, wherein SAB  is a calculation of survival, e.g., of cells exposed to a combination of cannabinoid A and cannabinoid B, SA is the survival of cells exposed to cannabinoid A only, and SB is the survival of cells exposed to cannabinoid B only. SAB (Exp) designates the observed survival of cells exposed to the combination of cannabinoid A and cannabinoid B.t-test was used for p value assessment. Statistical analysis was performed on the results by performing Student’s t-Test .Student’s https://patents.google.com/patent/WO2021245677A1/en, accessed on 5 June 2020).International Patent Application, publication No.: WO/2011/110866 discloses the use of phytocannabinoids, either in an isolated form or in the form of a botanical drug substance (BDS) in the treatment of cancer (


Adherence to dietary interventions is a significant barrier in the treatment of childhood obesity. Time-limited eating (TLE) is a simple dietary approach that limits food intake to a given number of consecutive hours per day, but parental and youth acceptability of TLE in youth with obesity is unknown. This study explored the feasibility of utilizing TLE among parents and youth attending pediatric weight management (PWM).via REDCap or manually to parents of patients between the ages of 8-17 years old and to patients 11-17 years old attending one of five PWM practices in the COMPASS network.Members of COMPASS  developed a survey to assess the acceptability of TLE in families attending PWM, which included patient characteristics, current diet and sleep schedules, and interests in trying TLE. The survey was administered electronically Patients (n=213) were 13.0 ± 2.5 years old, 58% female, 52% White, 22% Black, 17% Hispanic/Latino, and 47% reported a diagnosed psychological disorder. On average, parents reported their child’s daily eating spanned 12.5 ± 1.9 hours (7:35am - 8:05pm) and included 5.6 ± 1.6 eating bouts . Most parents reported being likely to try TLE ≤12 hours/d (TLE12: 66%), which was similar to the likelihood of following a nutrient-balanced diet (59%). Likelihood was lower for TLE ≤10 hours/d (TLE10: 39%) or ≤8 hours/d (TLE8: 26%) (p<0.001 for both). Interest in TLE was not consistently related to patient age, sex, or ethnicity, but was lower in patients with a psychiatric diagnosis vs. no diagnosis . Patients of parents who reported being likely to try TLE, compared to those unlikely to try TLE, had shorter eating windows (p<0.001) and ate fewer snacks (p=0.006).Two-thirds of parents with children attending PWM programs report interest in TLE ≤12 hours/d regardless of demographic characteristics, but interest wanes when limiting eating to ≤10 or ≤8 hours per day. Time-limited eating appears to be a feasible option in PWM settings provided treatment options are individualized based on the interests and barriers of patients and their families. Traditionally, obesity was viewed as a simple behavioral problem involving an imbalance in caloric intake and energy expenditure. However, current evidence has revealed obesity to be a multifactorial disease that involves multiple organ systems as well as behavioral, physiological, environmental, genetic and epigenetic components. One such component includes the central circadian system involved in regulating sleep/wake cycles, hormones, and metabolism. Evidence has now demonstrated a reciprocal link between the circadian system and the timing and quantity of food intake . For exaConversely, research in mice where food intake is limited to a particular time window each day, known as time-limited eating (TLE), reduces obesity and related metabolic disorders such as impaired glucose tolerance –10. A reFindings from human research corroborate results from animal models, albeit with less pronounced outcomes . In studTo date, no TLE studies have been conducted in human youth; however, TLE has recently been evaluated in adolescent mice with obesity with promising results  and at lFive pediatric weight management programs from across the United States participated in the study. Programs were recruited through the Childhood Obesity Multi-Program Analysis and Study System (COMPASS), a practice-based national research network with a focus on better understanding the etiology of childhood obesity and improving its treatment through translational research. Participants for the current study were parents of patients 8 to 17 years of age who were invited to participate in the study by completing a survey while attending a treatment visit at one of the programs. If parents agreed to participate and their child was 11 to 17 years of age, the child/patient was asked to complete a similar patient version of the survey. Each program received approval from their respective Institutional Review Board (IRB) before participating.All data for the current study was collected between January and November 2018 from a parent and patient/child survey administered at a PWM treatment visit. The survey used in the current study was developed by a team of clinicians and researchers specializing in childhood obesity, child psychology, and behavior analysis. The parent and child instruments contained items that assessed patient factors as they relate to TLE, including typical meal and snacking patterns, sleep habits, and acceptability of TLE, including different TLE lengths, TLE days of the week, and TLE barriers. Only the parent version of the survey included questions regarding the child’s demographic characteristics, but all other questions were identical between parent and child version. Time-of-day questions provided response options in half-hour increments, and TLE barriers were provided as a select-all-that-apply list. Acceptability was assessed using questions that asked about hypothetical TLE eating plans that differed by day of the week (school days vs. school days and weekends) and number of TLE hours per day. Assessments for TLE interest included how likely the child would be to follow four plans: 1) TLE limited eating to 12 hours per day on school days only (TLE12), 2) TLE limited to 12 hours per day on school days and weekends (TLE12week), 3) TLE limited to 10 hours per day on school days only (TLE10), and 4) TLE limited to 8 hours per day on school days only (TLE8). Response options for TLE acceptability questions used a Likert scale with 5 options ranging from “extremely unlikely” to “extremely likely”.The parent survey included questions concerning their child’s demographic and anthropometric characteristics and psychological disorders and medications for these disorders. Height and weight were parent-reported with the option for metric or imperial units, and parents were asked to use the height and weight recorded during the child’s current treatment visit, if possible. Parents were asked if their child had ever been diagnosed with the following psychological disorders: Attention Deficit/Hyperactivity Disorder (ADHD/ADD), mood disorder, such as depression or bipolar, anxiety disorder, Autism Spectrum Disorder (ASD), including Asperger’s Syndrome, and learning disabilities or delays. Any medications taken for these disorders were recorded and categorized as stimulant or non-stimulant medications. The parent survey was prefaced by a brief paragraph indicating that the completion of the survey constituted parental consent to participate and let their child participate. The patient portion of the survey was then offered to patients 11 to 17 years of age after the parent portion was completed. Both parent and patient surveys were available in English and Spanish.th BMI percentile was assessed for each child based on his/her age, height and weight using CDC growth curves (th to <95th), class 1 obesity (percent of the 95th BMI percentile: 100% to <120%), and severe (class 2 + class 3) obesity (percent of the 95th BMI percentile: ≥120%) Due to the low number of patients defined as overweight, this group was combined with obesity for analyses. Descriptive statistics were presented as means and standard deviations for continuous variables and as frequencies and proportions for categorical data. Parent and patient characteristics were compared across programs using Pearson Chi Square as was the frequencies of survey responses across nominal groupings, such as sex. Mantel Hanzel chi square was used to evaluate linear relationships in survey responses across ordinal groups . Likert responses were dichotomized as “unlikely”  and “likely” (including “Likely” or “Very Likely”). Wilcoxon rank sum test (Mann-Whitney U test) was used to compare differences in the ordinal Likert responses across groups, and multiple logistic regression was used to compare group differences based on dichotomized patient responses, while adjusting for potential confounders.In order to determine weight status, BMI percentile and percent of the 95h curves . Then, wA total of 222 parents agreed to review the survey’s consent page and 213 consented to participate (96%), with respondents of any single PWM program representing 8% to 44% of the total sample (median: 11%). Of participating parents, 167 had a child patient between 11-17 years of age and 159 (95%) assented to participate. Patient characteristics for each pediatric weight management program studied are displayed in Among children aged 8-10 years, parent-reported mean (± SD) sleep duration was 9.6 (0.8) hours based on an average wake time of 6:52am and an average bedtime of 9:19pm. Mean breakfast, lunch, and dinner times were 7:54am, 11:57am, and 6:24pm, respectively. In addition to meals, parents reported their child ate an average of 3.3 (2.0) snacks per day. Meals and snacks were consumed across an eating time window of 12.1 (1.3) hours. No previous dieting was reported among the majority (60%) of 8 to 10-year-olds, but those who had dieted most frequently reported calorie tracking  (10%), My Plate (10%), or calorie restricted (7.5%) diets.Parent-reported interest in TLE was high for TLE12 (71%) but decreased for TLE10 (46%) and TLE8 (27%) and was also lower for TLE12week (59%). For all TLE options, parents of 8–10-year-olds reported similar interest as parents of older children and adolescents.Parents estimated wake times for 11 to 17-year-olds as 6:18am, on average, and bedtimes as 10:01pm (1.1) hours for a total sleep length of 8.3 (1.2) hours. Youth reported similar wake times (6:14am) but later bedtimes (10:18pm) than parents (p<0.001), resulting in a shorter mean sleep length (7.9 (1.4) hours; p<0.001).Parent-reported mealtimes for breakfast, lunch, and dinner for the adolescents were 7:09am, 11:39am, and 6:17pm, respectively, and were similar to adolescent-reported mealtimes. Parents reported mean first daily food intake as 7:31am (due to some youth skipping breakfast) and last daily food intake as 8:06pm for a total daily eating length of 12.6 (2.0) hours. Adolescents reported similar first food intake times (7:37am) and non-significantly earlier last food intake times (7:57pm), resulting in a shorter eating length (12.2 (2.0) hours) when compared to parent-reported data (p=0.026). The number of non-meal snacks reported by parents and youth were 2.7 (1.7) and 2.6 (1.6) snacks per day, respectively.Interest in TLE did not differ between adolescents aged 11 to 13y and teens aged 14 to 17y; therefore, TLE results were combined for all youth 11 to 17y. Parent- and adolescent-reported interest in TLE is shown in Interest in TLE did not differ between parents and adolescents when youth were grouped by demographic characteristics. However, TLE interest did differ across some patient characteristics, including race/ethnicity and the presence of a diagnosed psychiatric disorder Table 2.Associations between parent interest in TLE and their child’s eating habits are presented in When testing for linear associations between TLE and daily snack intake, parent interest in TLE12 was negatively associated with the number of snacks their child consumed daily (p=0.018) such that 79% of parents reporting 0-1 snacks per day were interested in TLE12, but interest dropped among parents reporting 2-3 snacks (69%), 4-5 snacks (51%), and 6+ snacks per day (55%). Parent interest in TLE10 and TLE8 showed similar, though non-significant, inverse trends (p<0.1) with snacking. For adolescents, interest in TLE was not associated with the number of snacks they reported.Perceived barriers to TLE for parents and youth are shown in Snacking while watching TV was perceived as a barrier less frequently for those interested in TLE12  and TLE10 . Similarly, family schedule was seen as a barrier less frequently for those interested in TLE10  and TLE8  as was parent work for TLE8 . Among adolescents, interest in TLE12 was associated with a lower frequency of the following barriers: late bedtime , snacking at night , snacking while watching TV , and snacking without permission . School schedule was viewed as a barrier less frequently for those interested in TLE10 , while no differences in barrier frequency were seen for TLE8.Those interested in TLE perceived some barriers at different frequencies than those not interested in TLE. This acceptability study included 213 patients ages 8-17 years old and their caretakers participating in five tertiary pediatric weight management programs across the United States to determine the interest in a TLE program for their child. The results show that TLE appears to be an acceptable treatment option among some pediatric patients with obesity who are seen in a pediatric weight management setting, provided that treatment options are individualized and based on the interests and barriers of patients and their families.In this study, two-thirds of parents and children attending these programs reported interest in TLE12, regardless of demographic characteristics. This finding was similar to the likelihood of following a nutrient-balanced diet (59%). High acceptability of TLE12 is not surprising considering parents of 11-17 year-olds reported that their child’s eating spanned 12.6 hours, on average, and parents of 8-10 year-olds reported an eating time window spanning 12.1 hours. Unlike TLE10 and TLE8, interest TLE12 did not differ by the typical daily length of eating in youth, suggesting this interval may not have felt restrictive based on current habits. Therefore, despite the increased feasibility of a 12-hour eating window when compared to shorter eating intervals, TLE12 may not meaningfully influence eating timing in youth seeking obesity treatment. Therefore, determining the hours of eating for a TLE plan in youth would need to be strategic, previously agreed upon, and may need to be reduced in the middle of a TLE plan to have beneficial health outcomes.Our findings showed a substantial decrease in interest in TLE in both parents and children when questioned about TLE10 and TLE8. This is not surprising considering these diets are more restrictive compared to current eating habits. We hypothesized that interest in shorter TLE plans would be higher among older youth when compared to younger children, since adolescents may be better able to manage their hunger; however, TLE interest did not differ across age groups for any time interval. Perhaps the increased maturation and potential dietary restraint of older youth was offset by their later bedtimes and longer daily eating windows. This is evidenced by the fact that, regardless of patient age, parents who reported being likely to try TLE10 and TLE8 had eating windows that were almost an hour shorter than those unlikely to try these plans. In addition, parents may perceive less control over the food choices of older adolescents, which may reduce their interest in attempting a TLE plan.Though no data are available in youth, adult TLE research supports relatively high levels of acceptability and adherence. In a recent 21-day randomized controlled trial , women wAccording to our findings, both parents and youth often foresee schedule-related barriers as well as behavior-based challenges to adopting a TLE plan. When asked about anticipated barriers to TLE, the top two concerns described by parents were the parent’s work schedule (35%) and the family schedule (33%), while youth most frequently reported school schedule (36%) and after-school schedule (28%) as anticipated barriers. However, the third most frequent barrier was behavior-based for both parents (unsupervised snacking: 30%) and youth (hunger: 28%). Therefore, when determining whether TLE would be an appropriate treatment plan for a pediatric patient with obesity, the family schedule, including work, school and after-school arrangements, including accommodation of different eating schedules, should be a part of the conversation, as should potential strategies to tailor behaviors and environments to bolster TLE compliance. Such strategies may include eliminating tempting foods from the home, meal planning, minimizing boredom and providing structure during fasting periods, and adjusting sleep schedules to avoid late-night hunger and snacking.In our study, interest in TLE was not consistently related to the patient’s age, sex or weight status, but was lower in participants with a psychiatric diagnosis compared to participants without a psychiatric diagnosis for both TLE10 and TLE8. Given the high association between attending a pediatric weight management program and having a psychiatric comorbidity (47% in this study though somewhat lower in others) , a patieIn addition, the amount of patient snacking per day was associated with interest in TLE. Parents reported an average of 2.8 snacks daily with approximately one-quarter of the children eating four or more snacks per day. We found that patients of parents who reported being likely to try TLE ate fewer snacks than youth of parents unlikely to try TLE such that 79% of parents reporting interest in TLE12 if their child ate 0-1 snacks per day compared to 69% for children eating 2-3 snacks per day and 51% for children eating 4-5 snacks per day. Therefore, lower rates of snacking, was a sign of improved interest in a TLE plan; however, families with higher snacking levels may benefit more from TLE. Thus, motivation to reduce snacking intake may be necessary for successful implementation of a TLE plan among youth with frequent bouts of eating.For those children and parents who are interested in TLE, successful implementation may provide health gains and quality of life benefits. For instance, adolescents who socialize and snack with friends would not have to disclose that they are “on a diet” and would be able to adhere to a TLE plan without social stigma. For younger children, creating set times and expectations of a meal and snack schedule may allow for easier implementation to reduce calorie intake and adherence to reducing snacks later in the day. Lastly, mealtime eating and snacking often changes during the summertime, causing an increased rate of BMI gain for children , 31. BasStrengths of this study include the assessment of both parent and adolescent perspectives on TLE feasibility among families attending pediatric weight management. This research topic is unique, given the limited information in the current literature as to whether children and adolescents would be interested in attempting a TLE program. In addition, this research study includes a cohort of 213 patients from 5 different pediatric weight management programs nationwide and includes patients with diverse demographic characteristics which provides broad generalizability for this treatment-seeking population. A limitation of this study includes self-reported nature of youths’ daily habits such as sleep, meal timing and snacking; however, providing both parent and youth responses allows for greater confidence in the validity of these estimates. Also, parent finances and work schedules likely influence perceived barriers to TLE, yet we did not collect these data as we were concerned the sensitivity of such questions may have reduced response rates. Another limitation is that patient behaviors likely vary depending on the time of the year due to seasonal factors like weather and school schedules. While time of year was not accounted for in this study, the majority of data were collected during the academic school year  in order to provide comparable results.Based on our findings, TLE would likely be well-received by many parents and youth enrolled in pediatric weight management. Future research is needed to implement a TLE protocol to youth participating in clinically supervised obesity treatment. Optimally, a TLE intervention could be compared to patients not undergoing TLE  or perhaps even a different TLE duration . Patients who snack frequently, snack without parental permission, have parents or families with busy work and after-school schedules, or have a psychiatric diagnosis should be invited to participate with caution as these groups may perceive more barriers to TLE. Ultimately, a “shared decision making” approach should be used to guide obesity treatment for patients and their families , 34. ThiThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Spectrum Health Human Research Protection Program, Cincinnati Children’s Hospital Medical Center Institutional Review Board, West Virginia University Office of Research Integrity and Compliance, Penn State Human Research Protection Program, The University of Tennessee Health Science Center Institutional Review Board. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.JT, MN, PM, JH, and RS contributed to the conception and design of the study. All authors contributed to the methodology and data collection. JT organized the database and performed the statistical analysis. JT and MN wrote the first draft of the manuscript. All authors contributed to manuscript revision, read and approved the final 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


P < 0.05. Bioinformatics analysis indicated 27 DEPs were mainly enriched in “regulation of synaptic plasticity and structure” and “calmodulin-dependent protein kinase activity,” which implicate synaptic remodeling may play a vital role in the pathogenesis of TLE. Consequently, the synaptic plasticity-related proteins and synaptic structure were investigated to verify it. It has been demonstrated that CaMKII-α, CaMKII-β, and GFAP were significant upregulated coincidently with proteomic analysis in the hippocampus of TLE rats. Moreover, the increased dendritic spines and hippocampal sclerosis further proved that synaptic plasticity involves in the development of TLE. The present study may help to understand the molecular mechanisms underlying epileptogenesis and provide a basis for further studies on synaptic plasticity in TLE.Temporal lobe epilepsy (TLE) is a chronic neurological disorder that is often resistant to antiepileptic drugs. The pathogenesis of TLE is extremely complicated and remains elusive. Understanding the molecular mechanisms underlying TLE is crucial for its diagnosis and treatment. In the present study, a lithium-pilocarpine-induced TLE model was employed to reveal the pathological changes of hippocampus in rats. Hippocampal samples were taken for proteomic analysis at 2 weeks after the onset of spontaneous seizure (a chronic stage of epileptogenesis). Isobaric tag for relative and absolute quantization (iTRAQ) coupled with liquid chromatography-tandem mass spectrometry (LC–MS/MS) technique was applied for proteomic analysis of hippocampus. A total of 4173 proteins were identified from the hippocampi of epileptic rats and its control, of which 27 differentially expressed proteins (DEPs) were obtained with a fold change > 1.5 and  Epilepsy is a devastating and complex neurological disease characterized with spontaneous recurrent seizures (SRS) . It affeIn recent decades, with the rapid development of proteomic-based techniques, iTRAQ has been employed in a large range of studies for the relative quantification of proteins –12. TherMale Sprague Dawley rats (200 ± 20 g) were obtained from Experimental Animal Center of Guangzhou University of Chinese Medicine (SCXK(YUE) 2013-0034). The animals were housed in cages under standard environmental conditions . This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health . All protocols were approved by the Animal Research Committee, Guangzhou Medical University, Guangzhou, China.−1, Sigma-Aldrich) and pilocarpine hydrochloride . Atropine  was used to reduce the peripheral cholinergic effects. The evoked seizures were scored according to Racine's scale. Only those rats that attained stage IV or V were employed in our experiment. Diazepam  was injected to stop seizures 60 min after the onset of status epilepticus (SE). Rats received intensive care including keeping warm and sugar saline oral administration. SE is followed by a latent period and later by the appearance of SRS. The rats were sacrificed at two weeks after the onset of spontaneous seizure, and hippocampi were collected for proteomic analysis. All 8 rats received EEG monitoring and Morris water maze test. For immunohistochemistry analysis and western blotting assay, five rats were included. Three rats were applied to proteomic detection.Pilocarpine-induced epilepsy rat model was considered as TLE according to a previous report . Rats weThe rats were anaesthetized with isoflurane, and then, electrodes were implanted by surgery after two weeks of SE induction. The electrodes were fixed to rat's skull at a position of 4.2 mm behind the coronal suture and 2.5 mm from the sagittal suture. Recovery period between surgery and EEG recording lasted for one week. The EEG was recorded in free moving rats for 7 days. The Spike 2 software  was used to monitor and analyze EEG.Morris water maze tests were performed to evaluate learning and memory at one week after the onset of spontaneous seizures in rats. In the acquisition phase, each rat was given four trials per day for five consecutive days, during which rats were allowed to swim to search the underwater platform. In each test, the distance travelled, time reaching to underwater platform, and mean swimming speed were recorded by a computerized video tracking system. After five days of acquisition training, a probe trial was performed on the sixth day. The time spent in the target quadrant was calculated within 120 s.Rats were deeply anaesthetized using 1.5% isoflurane, and the thoracic cavity was opened rapidly to expose the heart. A catheter was inserted into the ascending aorta, and rats were perfused with heparin saline, followed by cold 4% paraformaldehyde (PFA). The brains were removed and postfixed in 4% PFA overnight at 4°C. Brains were dehydrated, transparentized, and embedded in paraffin. Serial coronal sections containing hippocampus were cut and collected sequentially.Nissl staining was employed to assess hippocampus formation. Briefly, paraffin sections were deparaffinized, rehydrated, and stained with 1% toluidine blue. Every 15th staining section was chosen for quantitative analysis (five sections per rat). Images of CA1, CA3, and hilus of hippocampus sections were obtained with 40x magnification. The neurons were measured and quantified by using ImageJ/NIH image analysis system.Immunohistochemical analysis was carried out according to standard protocol. Hippocampal slices were incubated overnight with NeuN primary antibody  and GFAP primary antibody  at 4°C. After washed, the slices were incubated with IgG-alexa fluor-555  and IgG-alexa fluor-647  in the dark for 2 hours. Subsequently, the DAPI dye solution (CST) was used to stain nucleus for 10 min. The slices were observed with confocal microscope, and images of CA1, CA3, and hilus of hippocampus sections were obtained with 40x magnification.μm thick and mounted with glycerin gelatin. Images of hippocampal sections were obtained via a digital slice scanner. There is evidence that terminal branches are more plastic than nonterminal branches, so numbers of terminal branches of dendrite were used to evaluate the localized dendritic remodeling [μm in length each) of 10 neurons per animal were selected in the region of dentate gyrus for counting numbers of dendritic spines. Densities of dendritic spines were expressed as spines per unit length.Golgi staining was used to analyze the structure of dendritic spines according to our previous research . The bramodeling . Dendritα and II-β , rabbit anti-GFAP , rabbit anti-F-actin , and mouse anti-GAPDH  at 4°C overnight. After washed with TBST, the membranes were incubated with goat anti-rabbit  for 1 h at room temperature. Blots were developed using a chemiluminescent substrate and analyzed with the Quantity One software (version 4.4).The hippocampal samples were separated using 10% SDS-PAGE and transferred to PVDF membranes. Then, membranes were probed with mouse anti-CaMKII-μm) with Agilent 1100 HPLC (USA). The mobile phase is composed of buffer A  and buffer B . The elusion was performed at 2-40% linear gradient within 55 minutes. The flow rate of column was maintained at 0.3 mL/min, and the detection wavelength was set at 215 nm. The effluents were collected every 1 minute within the gradient range. Subsequently, separated peptides were further analyzed using Eksigent nanoLC-Ultra™ liquid chromatography and Triple TOF LC-MS/MS 5600 plus Mass Spectrometer .Proteolysis of hippocampus was in accordance with the FASP method . The prohttps://www.uniprot.org/, April 2017), including 37,686 protein entries. The following criteria were used for the identification and quantification of proteins: false positive rate (FPR) was set at 1%; the error of the first-order mass spectrum was 20 PPM, and the error of the second-order mass spectrum was 0.1 Da. Quality control of data was evaluated by the Protein Pilot Descriptive Statistics Template (PDST) 3.0 software . Labelling efficiency of iTRAQ 8 plex@N-term and iTRAQ 8plex (K) was 97.55% and 99.97%, respectively. The missed cleavages for single site and dual sites were 11.3% and 0.3%, respectively. The mass accuracy of parent ion was within 5 ppm. Target-decoy search strategy and Bonferroni method were used to control global FPR. Only protein differences with P value < 0.05 and fold change > 1.5 (or <0.67), which corresponded to 99% of confidence, were considered as differential expression. Volcano plot, principal components analysis, and hierarchical cluster analysis were performed with software PDST 3.0.The tandem mass spectra map was processed using SCIEX's Protein Pilot software (v4.5), and data was searched in the Rattus norvegicus protein database. The FASTA files of all Rattus norvegicus protein data were downloaded from UniProt database  and pathway-protein network were constructed by STRING database. The minimum required PPI score was set at a 90% of confidence.The OmicsBean software was used to conduct the enrichment analysis of DEPs by which gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathway were analyzed. The query proteins were conducted for blast P search before enrichment analysis. Quick GO database was used to annotate the GO classification, which consisted of biological processes (BP), molecular functions (MF), and cellular components (CC). t-test using the GraphPad Prism software. Differences were considered statistically significant at P < 0.05. Bonferroni correction was applied to control the global FPR in proteomic analysis, and Benjamini method was used to compare the significance of DEPs in bioinformatics analysis.Behavioral score, immunohistochemistry, and western blotting analysis are expressed as mean ± SD. The differences between two groups were evaluated by Student's Rat TLE model was validated with EEG monitoring. SRS occurred at an average of 12 days after SE induction in TLE rats, which is in agreement with our previous studies , 17. FreThe impact on behavior was evaluated since cognitive function is known to be altered in TLE. Learning and memory in epileptic rats were assessed using the Morris water maze. The travel distance, latency to target, and mean swimming speed for 5 training days were recorded Figures . EpileptHippocampal neuronal injury is the primary neuropathological feature of TLE. In order to observe the changes, Nissl staining was used to examine neuronal damage in epileptic rats. Our results revealed that pyramidal neurons were arranged regularly and exhibited structural integrity with clear nucleoli in the CA1 and CA3 regions of the hippocampus of control rats . The CA1P value < 0.05 and fold change > 1.5 or <0.67), including 18 upregulated and 9 downregulated proteins . Detailed information of 27 DEPs is listed in P value of each protein were provided as supplementary data (shown in Tab The rat TLE model has been validated as showing the characteristic signs of TLE. The cellular differences during epileptogenesis will be investigated in the following study. iTRAQ proteomic analysis was used to gain insight into the molecular mechanisms of pathogenesis of TLE rats, by which a total of 4173 proteins were identified. Hierarchical clustering analysis revealed the data between TLE rats and control rats could be completely distinguished (shown in supplementary Fig proteins . Among 2Bioinformatics analysis was used to analyze the DEPs, which were categorized according to GO terms and KEGG pathway. Enriched proteins count mainly concentrated in biological process based on GO terms and KEGG pathway analysis Figures . For theKEGG pathways analysis revealed that 28 pathways including “ErbB signaling pathway,” “HIF-1 signaling pathway,” “Wnt signaling pathway,” “calcium signaling pathway,” and “cAMP signaling pathway” were related to epileptogenesis . Among tα and histone H3. KEGG pathway-protein network was enriched into two distinct modules. The proteins interacted with “systemic lupus erythematosus” and “alcoholism” pathway centralized in histone family, while the proteins interacted with the other 8 KEGG pathways such as “ErbB signaling pathway” and “long-term potentiation” centralized in CaMKII family (27 DEPs and the top ten KEGG pathways were mapped to STRING database for the analysis of PPI and pathway-protein interaction, respectively. In the PPI network constructed by 27 DEPs, there was a strong linkage among the members of CaMKII family besides the members of histone family . In addiI family .α, and II-β in the hippocampus of TLE rats. Western blotting was used as the method of validation, and blots were the representative of 5 replicates. It has been revealed that the increased expression of GFAP, CaMKII-α, and II-β was detected in whole lysates, consistent with the upregulation detected in the proteomic analysis (Figures Proteomic analysis revealed that the most significantly altered proteins were GFAP and the members of CaMKII family, highlighting their implication in epileptogenesis. Therefore, to further confirm this, we verified the expression of GFAP, CaMKII- Figures . These rBioinformatics analysis indicated that the regulation of synaptic plasticity is the most important biological process, implying its vital role in the pathogenesis of TLE. F-Actin is the major cytoskeletal protein in spine, and the structural modification of dendritic spines plays a critical role in synaptic plasticity . In ordeHippocampus sclerosis is the major pathological feature of TLE, in which gliosis may contribute to its development. In present experiment, both proteomic analysis and validation of protein expression revealed that the level of GFAP increased in the hippocampus of TLE rats. In order to observe the alteration of hippocampal structure, immunohistochemical stain was conducted to detect the manifestation of GFAP and gliosis in TLE rats. It has been demonstrated an upregulated expression of GFAP in the CA1 region of hippocampus was accompanied with gliosis and neuronal loss .In the present study, iTRAQ proteomic analysis was employed to investigate the underlying mechanisms of epileptogenesis in rat TLE model. Abnormal EEG, cognitive and memory impairments, and hippocampal damage have been shown in epileptic rat, which is in accordance with the known pattern of TLE, thereby confirming that the TLE model in our present study is reliable for the subsequent investigation. iTRAQ coupled with LC-MS/MS proteomic technique could profile the protein patterns and identify the variation of protein expression in various disease states, which has been widely used to analyze biological samples –12. HippP value. The most pronounced proteins were GFAP and CaMKII family, suggesting their implication in epileptogenesis. According to GO analysis of 27 DEPs, the annotation of biological processes has revealed that the top 10 items mainly involved in the regulation of synaptic plasticity and structure, cellular component organization, and glia cell differentiation. As far as molecular function was concerned, CaMKII family is the most significant molecule, which is in correspondence with the proteomic analysis. For the analysis of cell component, cytoskeletal part ranked in the top ten items, implying its essential role in the pathogenesis of TLE. Briefly, GO term analysis demonstrated that synaptic plasticity is the major biological processes of epileptogenesis, and CaMKII family and cytoskeletal components involve in the pathogenesis of TLE.With iTRAQ-based quantitative proteomic analysis, 4173 proteins were identified from the hippocampus of TLE rats, of which 27 DEPs with the significant difference were obtained through the filtration of fold change and KEGG pathways analysis of 27 DEPs demonstrated that 28 pathways are related with epileptogenesis, of which “glioma” and “long-term potentiation” were included in the top ten items. Glioma in the present circumstance means that the abnormal proliferation of glia cell occurs in the hippocampus of TLE rats. Similarly, glia cell differentiation was identified to be the important event during epileptogenesis according to GO analysis. Long-term potentiation is an important form of synaptic plasticity , which iA variety of research has explored the pathogenesis of epilepsy based on proteomic analysis with different epileptic model and samples. Studies on epileptic patients in multiple brain regions have shown a dysregulation in pathways associated with mitochondrial function, protein synthesis, synaptic transmission, and remodeling of the neuronal network architecture . Investi2+ regulatory network show an evident alterations with different epileptic animal model [Studies on epileptic animal model have shown the differences by proteomic analysis. DEPs are focused on the modulators of disease-associated inflammatory signaling at different stages of epiletogenesis in electrically induced rat model . Similaral model –35. A coal model . AlthougIn the present study, proteomic analysis has shown that the pronounced proteins were GFAP and CaMKII family. Furthermore, bioinformatics analysis revealed that synaptic plasticity is the major biological processes, and CaMKII is a vital molecular during epileptogenesis. On the basis of this, we make a further validation on the proteins involving in gliosis and synaptic plasticity, including GFAP, the members of CaMKII and F-actin. The functional role of GFAP in astrocytes has been widely accepted, which is involved in the process of regeneration, synaptic remodeling, and reactive gliosis , 38. Hipα is essential for synaptic plasticity [β, a structural subunit, possesses F-actin binding and bundling properties, providing essential functions for synapse formation and dendritic morphology [α and II-β and F-actin and the alteration of synaptic structure have been detected in the hippocampus of TLE rats. The upregulated expression of CaMKII-α, CaMKII-β, and F-actin and the increased density of dendritic spine suggest a potential link between CaMKII and synaptic remodeling within hippocampus. A significant upregulation of CaMKII has been observed in the hippocampus of TLE patients and different epileptic models [CaMKII is the major postsynaptic protein at excitatory synapses, which is fundamentally important for synaptic plasticity . The actasticity , whereasrphology . In the rphology . Therefoc models , 45, 46,c models , 46. Thec models . Despiteα and II-β, and synaptic plasticity were involved in the pathogenesis of TLE by proteomic analysis and further investigation provided an anatomical evidence for the role of synaptic plasticity during epileptogenesis. However, it must be mentioned that iTRAQ-based proteomic analysis has the advantages in both high throughput and the precision in the abundance measurements, whereas the accuracy of the estimations of differential expression may be affected due to ratio compression [In conclusion, our study demonstrated that GFAP, CaMKII-pression . As a re


Hip fracture and surgery are associated with moderate to severe pain, which hampers early mobilization and extends the hospital stay. Femoral nerve block and fascia iliaca compartment block could provide effective postoperative pain relief. Unfortunately, they could weaken the strength of the quadriceps muscle and increase the risk of falls. Iliopsoas plane block (IPB) is a novel motor-sparing regional technique, which targets the sensory branches of the hip joint originating from the femoral nerve. However, the analgesic effect of IPB has not been confirmed yet.In the present case series, IPB and lateral femoral cutaneous nerve block were implemented under the guidance of ultrasound for eight patients with hip fractures. The median (IQR) visual analog scale (VAS) score  decreased from 1.5 (0.25–2) before IPB to 0 (0–0) 0.5h after IPB at rest. The median (IQR) VAS score decreased from 8 (7–8) before IPB to 2 (1–2) 0.5h after IPB during flexion of hip 30°. Pain score was no more than one at rest and three during flexion of the hip 30° within 48h after surgery. Furthermore, the MMT grades of quadriceps strength were no less than four after IPB.Our case series firstly highlights that IPB might be an effective analgesic technique for hip fracture and surgery, while retaining motor function. It is recognized that hip fracture and surgery are associated with moderate to severe pain, which hampers early mobilization and extends the hospital stay. Peripheral nerve block technologies, such as femoral nerve block (FNB), fascia iliaca compartment block (FICB), and 3-in-1 femoral nerve block, have been used for analgesia perioperative analgesia in patients undergoing hip surgery for a long time , a novel motor-sparing technique described by Nielsen et al., targets selectively the sensory branches of the hip joint originating from the femoral nerve and accessory obturator nerve  with femoral neck fracture were scheduled for surgery, including an internal screw fixation, three total hip arthroplasties, and four hip hemiarthroplasties. The demographics of patients were shown in Table Prior to general anesthesia, IPB was implemented under the guidance of ultrasound as reported by Nielsen et al. . In ordeAnesthesia induction was performed after confirming the effect of the nerve block. Propofol, remifentanil, sevoflurane, and cisatracurium were used for anesthesia induction and maintenance. Ventilation by laryngeal mask, end-tidal carbon dioxide was maintained at 35 to 40mmHg. During the operation, the bispectral index was maintained at 45 to 55, and the fluctuation of mean artery pressure and heart rate was not more than ±10% of the baseline value.The operations took 60–130 min. After the operation, flurbiprofen 50 mg was administrated by intravenous injection for 3 days, twice a day. Opioids  were used for rescue analgesia. The postoperative pain and quadriceps strength were assessed respectively by the visual analog scale (VAS)  and manual muscle testing grades (MMT grades)  in post-anesthesia care unit (PACU), at 0.5h after block and 2, 4, 6, 24, and 48 h after surgery. The VAS score, MMT grades, and opioid consumption were shown in Table In the present case series, the median (IQR) VAS score decreased from 1.5 (0.25–2) before IPB to 0 (0–0) 0.5h after IPB at rest. The median (IQR) VAS score decreased from 8 (7–8) before IPB to 2 (1–2) 0.5h after IPB during flexion of hip 30°. Moreover, the pain score was no more than one at rest and three during flexion of the hip at 30° within 48h after surgery. These results suggested IPB could improve pain effectively of hip fracture and surgery, which were consistent with our assumptions. Furthermore, the MMT grades of quadriceps strength were no less than four after IPB. All patients were able to fully participate in physiotherapy, and there were no falls happened in the hospital. The above results suggested IPB could provide good pain relief, while retaining motor function. Although a recent volunteer study indicated that IPB did not weaken the strength of the quadriceps muscle (Nielsen et al. The innervation of the hip joint is complicated. The great majority of nociceptors are located in the anterior part of the capsule of the hip joint rather than the posterior capsule, which indicates that the anterior capsule is the main target of postoperative analgesia after hip surgery (Simons et al. In conclusion, IPB may be an effective analgesic technique for hip fracture and surgery, while retaining motor function. More studies are needed to further confirm the validity of IPB and its optimum volume of local anesthetic.