Proteomics and cytokine analyses distinguish myalgic encephalomyelitis/chronic fatigue syndrome cases from controls.

BACKGROUND: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex, heterogenous disease characterized by unexplained persistent fatigue and other features including cognitive impairment, myalgias, post-exertional malaise, and immune system dysfunction. Cytokines are present in plasma and encapsulated in extracellular vesicles (EVs), but there have been only a few reports of EV characteristics and cargo in ME/CFS. Several small studies have previously described plasma proteins or protein pathways that are associated with ME/CFS. METHODS: We prepared extracellular vesicles (EVs) from frozen plasma samples from a cohort of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) cases and controls with prior published plasma cytokine and plasma proteomics data. The cytokine content of the plasma-derived extracellular vesicles was determined by a multiplex assay and differences between patients and controls were assessed. We then performed multi-omic statistical analyses that considered not only this new data, but extensive clinical data describing the health of the subjects. RESULTS: ME/CFS cases exhibited greater size and concentration of EVs in plasma. Assays of cytokine content in EVs revealed IL2 was significantly higher in cases. We observed numerous correlations among EV cytokines, among plasma cytokines, and among plasma proteins from mass spectrometry proteomics. Significant correlations between clinical data and protein levels suggest roles of particular proteins and pathways in the disease. For example, higher levels of the pro-inflammatory cytokines Granulocyte-Monocyte Colony-Stimulating Factor (CSF2) and Tumor Necrosis Factor (TNFα) were correlated with greater physical and fatigue symptoms in ME/CFS cases. Higher serine protease SERPINA5, which is involved in hemostasis, was correlated with higher SF-36 general health scores in ME/CFS. Machine learning classifiers were able to identify a list of 20 proteins that could discriminate between cases and controls, with XGBoost providing the best classification with 86.1% accuracy and a cross-validated AUROC value of 0.947. Random Forest distinguished cases from controls with 79.1% accuracy and an AUROC value of 0.891 using only 7 proteins. CONCLUSIONS: These findings add to the substantial number of objective differences in biomolecules that have been identified in individuals with ME/CFS. The observed correlations of proteins important in immune responses and hemostasis with clinical data further implicates a disturbance of these functions in ME/CFS.

Anti-adhesive bioresorbable elastomer-coated composite hernia mesh that reduce intraperitoneal adhesions.

Intraperitoneal adhesions (IAs) are a major complication arising from abdominal repair surgeries, including hernia repair procedures. Herein, we fabricated a composite mesh device using a macroporous monofilament polypropylene mesh and a degradable elastomer coating designed to meet the requirements of this clinical application. The degradable elastomer was synthesized using an organo-base catalyzed thiol-yne addition polymerization that affords independent control of degradation rate and mechanical properties. The elastomeric coating was further enhanced by the covalent tethering of antifouling zwitterion molecules. Mechanical testing demonstrated the elastomer forms a robust coating on the polypropylene mesh does not exhibit micro-fractures, cracks or mechanical delamination under cyclic fatigue testing that exceeds peak abdominal loads (50 N/cm). Quartz crystal microbalance measurements showed the zwitterionic functionalized elastomer further reduced fibrinogen adsorption by 73% in vitro when compared to unfunctionalized elastomer controls. The elastomer exhibited degradation with limited tissue response in a 10-week murine subcutaneous implantation model. We also evaluated the composite mesh in an 84-day study in a rabbit cecal abrasion hernia adhesion model. The zwitterionic composite mesh significantly reduced the extent and tenacity of IAs by 94% and 90% respectively with respect to uncoated polypropylene mesh. The resulting composite mesh device is an excellent candidate to reduce complications related to abdominal repair through suppressed fouling and adhesion formation, reduced tissue inflammation, and appropriate degradation rate.

A semiparametric risk score for physical activity.

We develop a generalized partially additive model to build a single semiparametric risk scoring system for physical activity across multiple populations. A score comprised of distinct and objective physical activity measures is a new concept that offers challenges due to the nonlinear relationship between physical behaviors and various health outcomes. We overcome these challenges by modeling each score component as a smooth term, an extension of generalized partially linear single-index models. We use penalized splines and propose two inferential methods, one using profile likelihood and a nonparametric bootstrap, the other using a full Bayesian model, to solve additional computational problems. Both methods exhibit similar and accurate performance in simulations. These models are applied to the National Health and Nutrition Examination Survey and quantify nonlinear and interpretable shapes of score components for all-cause mortality.

Poly(lactide-co-ε-caprolactone) scaffold promotes equivalent tissue integration and supports skin grafts compared to a predicate collagen scaffold.

Dermal scarring from motor vehicle accidents, severe burns, military blasts, etc. is a major problem affecting over 80 million people worldwide annually, many of whom suffer from debilitating hypertrophic scar contractures. These stiff, shrunken scars limit mobility, impact quality of life, and cost millions of dollars each year in surgical treatment and physical therapy. Current tissue engineered scaffolds have mechanical properties akin to unwounded skin, but these collagen-based scaffolds rapidly degrade over 2 months, premature to dampen contracture occurring 6-12 months after injury. This study demonstrates a tissue engineered scaffold can be manufactured from a slow-degrading viscoelastic copolymer, poly(ι-lactide-co-ε-caprolactone), with physical and mechanical characteristics to promote tissue ingrowth and support skin-grafts. Copolymers were synthesized via ring-opening polymerization. Solvent casting/particulate leaching was used to manufacture 3D porous scaffolds by mixing copolymers with particles in an organic solvent followed by casting into molds and subsequent particle leaching with water. Scaffolds characterized through SEM, micro-CT, and tensile testing confirmed the required thickness, pore size, porosity, modulus, and strength for promoting skin-graft bioincorporation and dampening fibrosis in vivo. Scaffolds were Oxygen Plasma Treatment and collagen coated to encourage cellular proliferation. Porosity ranging from 70% to 90% was investigated in a subcutaneous murine model and found to have no clinical effect on tissue ingrowth. A swine full-thickness skin wound model confirmed through histology and Computer Planimetry that scaffolds promote skin-graft survival, with or without collagen coating, with equal safety and efficacy as a commercially available tissue engineered scaffold. This study validates a scalable method to create poly(ι-lactide-co-ε-caprolactone) scaffolds with appropriate characteristics and confirms in mouse and swine wound models that the scaffolds are safe and effective at supporting skin-grafts. The results of this study have brought us closer towards developing an alternative technology that supports skin grafts with the potential to investigate long-term hypertrophic scar contractures.

Osteogenic benefits of low-intensity pulsed ultrasound and vibration in a rodent osseointegration model.

OBJECTIVES: Osseointegrated prostheses are increasingly used for amputees, however, the lengthy rehabilitation time of these prostheses remains a challenge to their implementation. The aim of this study was to investigate the ability of locally applied vibration or low-intensity pulsed ultrasound (LIPUS) to accelerate osseointegration and increase peri-implant bone volume. METHODS: A 4-week and 8-week rodent study were conducted in a femoral intramedullary implant model (control, vibration, LIPUS, and combined treatment) to determine effects on healing. Osseointegration was evaluated quantitatively through mechanical, µCT and histological evaluations. RESULTS: Maximum pushout load at 4 weeks increased with LIPUS relative to control (37.7%, P=0.002). Histologically, LIPUS and vibration separately increased peri-implant bone formation after 4 weeks relative to control. Vibration resulted in greater peri-implant bone after 8 weeks than all other groups (25.7%, P<0.001). However, no significant group differences in pushout load were noted at 8 weeks. CONCLUSIONS: Although vibration increased bone around implants, LIPUS was superior to vibration for accelerating osseointegration and increasing bone-implant failure loads at 4 weeks. However, the LIPUS benefits on osseointegration at 4 weeks were not sustained at 8 weeks.

Application of a novel suture anchor to abdominal wall closure.

BACKGROUND: Mesh suture used in high-tension wound closures produces large knots susceptible to increased palpability, infection, and foreign body response; yet has superior tensile strength and increased resistance to cutting through tissue compared to standard suture. This study investigates mesh suture fixation in abdominal tissue with a knotless novel, low-profile anchor-clip. METHODS: Single and double end fixation of mesh suture in swine rectus abdominus fascia with an anchor-clip, a knot, and predicate device fixation underwent cyclic testing followed by pull-to-failure testing. RESULTS: Failure load of standard knot, single corkscrew and double anchor-clip were not statistically different, but were significantly greater than single anchor-clip and double corkscrew fixation (p > 0.05). CONCLUSIONS: The anchor-clip is ∼60% smaller than a standard knot while maintaining fixation strength when exposed to physiologic forces using double anchor-clip fixation in abdominal wall closure.

Modeling and Prediction of Multiple Correlated Functional Outcomes.

We propose a copula-based approach for analyzing functional data with correlated multiple functional outcomes exhibiting heterogeneous shape characteristics. To accommodate the possibly large number of parameters due to having several functional outcomes, parameter estimation is performed in two steps: first, the parameters for the marginal distributions are estimated using the skew t family, and then the dependence structure both within and across outcomes is estimated using a Gaussian copula. We develop an estimation algorithm for the dependence parameters based on the Karhunen-Loève expansion and an EM algorithm that significantly reduces the dimension of the problem and is computationally efficient. We also demonstrate prediction of an unknown outcome when the other outcomes are known. We apply our methodology to diffusion tensor imaging data for multiple sclerosis (MS) patients with three outcomes and identify differences in both the marginal distributions and the dependence structure between the MS and control groups. Our proposed methodology is quite general and can be applied to other functional data with multiple outcomes in biology and other fields.

Soft Tissue Anchoring Performance, Biomechanical Properties, and Tissue Reaction of a New Hernia Mesh Engineered to Address Hernia Occurrence and Recurrence.

One opportunity to reduce hernia occurrence and recurrence rates (currently estimated to be 30% at 10 years postoperatively) is by enhancing the ability of hernia meshes to anchor into tissue to prevent mesh migration, mesh contraction, and mesh tearing away from tissue. To address this, we developed a novel moderate-weight, macroporous, polypropylene mesh (termed the T-line mesh) with mesh extensions to optimize anchoring. We examined the physical properties, biomechanical performance, and biocompatibility of this novel mesh versus a predicate mesh anchored with #0-suture. The physical properties of the T-line mesh and predicate mesh were measured using American Society for Testing and Materials (ASTM) standards. Meshes were implanted into a swine hernia model and harvested after one day to determine anchoring strength of the meshes in the perioperative period. A separate group was implanted into a swine hernia model and harvested at 30 days and 90 days for semiquantitative histological analysis of biocompatibility. T-line mesh physical properties were similar to commonly used moderate-weight meshes in thickness and areal density. The T-line mesh outperformed the predicate mesh in all mechanical testing (P < 0.05). In the perioperative period, the T-line mesh was ∼275% stronger (P < 0.001) than the standard of care. Histological analysis of biocompatibility demonstrated no significant difference between the T-line mesh and predicate mesh (P > 0.05). The T-line mesh is a novel hernia mesh that outperforms a predicate mesh in mechanical and biomechanical performance testing while exhibiting similar biocompatibility. The T-line mesh has the potential to reduce hernia occurrence and recurrence caused by mechanical failure.

Prospective Biomarkers from Plasma Metabolomics of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome Implicate Redox Imbalance in Disease Symptomatology.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a disease of enigmatic origin with no established cure. Its constellation of symptoms has silently ruined the lives of millions of people around the world. A plethora of hypotheses have been vainly investigated over the past few decades, so that the biological basis of this debilitating condition remains a mystery. In this study, we investigate whether there is a disturbance in homeostasis of metabolic networks in the plasma of a female 32-patient cohort compared to 19 healthy female controls. Extensive analysis of the 832-metabolite dataset generated by Metabolon®, covering eight biological classes, generated important insight into metabolic disruptions that occur in ME/CFS. We report on 14 metabolites with differences in abundance, allowing us to develop a theory of broad redox imbalance in ME/CFS patients, which is consistent with findings of prior work in the ME/CFS field. Moreover, exploration of enrichment analysis using www.MetaboAnalyst.ca provides information concerning similarities between metabolite disruptions in ME/CFS and those that occur in other diseases, while its biomarker analysis unit yielded prospective plasma biomarkers for ME/CFS. This work contributes key elements to the development of ME/CFS diagnostics, a crucial step required for discovering a therapy for any disease of unknown origin.

Bone changes after short-term whole body vibration are confined to cancellous bone.

OBJECTIVE: This study assessed femur properties in 80 adult female rats exposed to a range of whole body vibration amplitudes at 45 Hz over five weeks. Our hypothesis was that an optimal amplitude for whole body vibration would be apparent and would result in increased bone strength. METHODS: Animals were treated in five amplitude groups (0 g, 0.15 g, 0.3 g, 0.6 g, and 1.2 g peak), for 15 minutes per day, five days per week, for five weeks. Femur strength was assessed via: (1) three-point bending of the shaft, (2) cantilever bending of the neck, and (3) indentation of distal cancellous bone. Femoral bone mineral density, plasma prostaglandin E2 (PGE2) concentrations, cartilage thickness, and histopathologic properties were measured. RESULTS: Vibration doubled (P=0.039) cancellous bone stiffness in the 0.6 g and 1.2 g groups and induced a 74% increase in PGE2 concentrations (P=0.007). However, femoral densitometry and strength of the neck and shaft were unchanged and the cancellous bone indentation strength did not differ statistically (P=0.084). Cartilage thickness of vibrated groups at the medial condyle did not increase significantly (P=0.142) and the histopathologic grade did not change. There was no definitive optimal vibration amplitude. CONCLUSION: The benefits of vibration therapy over five weeks were confined to cancellous bone.

Local delivery of a zoledronate solution improves osseointegration of titanium implants in a rat distal femur model.

This study aimed to determine whether locally applied anti-resorptive agents acetazolamide or zoledronic acid would improve mechanical stability in implant osseointegration when applied as a solution within the medullary canal. Thirty-three rats received titanium-implants bilaterally in their intramedullary femoral canals. Prior to implantation, animals received 0.1 ml saline, 1 mM acetazolamide solution, or 0.7 mM zoledronic acid solution directly into the medullary cavity. The control group only received saline within the medullary canal while the treatment groups only received the respective treatment to which they were randomized. Animals were allowed to heal 4 weeks, at which time they were euthanized and femurs isolated for mechanical and radiographic evaluation. Push-out force to failure increased 152% in the zoledronic acid group relative to the control. There was no significant difference in push-out force with acetazolamide relative to control. Also, zoledronic acid increased metaphyseal bone volume fraction 46% and increased metaphyseal bone-implant contact 58% relative to the control. Recent research exploring local injection of medications to improve implant osseointegration and minimize systemic-effects has failed to quantitatively evaluate implant fixation strength on non-hydroxyapatite coated implants or implants without previous bone compaction. This study demonstrated that a simple injection of zoledronic acid into the medullary canal, rather than coatings or commercial gels, can increase fixation strength of an uncoated titanium-implant. Our findings indicate simple injection of zoledronic acid in saline solution has the potential for improving fixation of uncemented joint implants. Clinical Significance: Intramedullary injection of local bisphosphonate solutions could be implemented to improve osseointegration in cementless arthroplasty. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:3294-3298, 2018.

Improved osseointegration with as-built electron beam melted textured implants and improved peri­implant bone volume with whole body vibration.

Transcutaneous osseointegrated prostheses provide stable connections to the skeleton while eliminating skin lesions experienced with socket prosthetics. Additive manufacturing can create custom textured implants capable of interfacing with amputees' residual bones. Our objective was to compare osseointegration of textured surface implants made by electron beam melting (EBM), an additive manufacturing process, to machine threaded implants. Whole body vibration was investigated to accelerate osseointegration. Two cohorts of Sprague-Dawley rats received bilateral, titanium implants (EBM vs. threaded) in their tibiae. One cohort comprising five groups vibrated at 45 Hz: 0.0 (control), 0.15, 0.3, 0.6 or 1.2 g was followed for six weeks. Osseointegration was evaluated through torsional testing and bone volume fraction (BV/TV). A second cohort, divided into two groups (control and 0.6 g), was followed for 24 days and evaluated for resonant frequency, bone-implant contact (BIC) and fluorochrome labeling. The EBM textured implants exhibited significantly improved mechanical stability independent of vibration, highlighting the benefits of using EBM to produce custom textured surfaces. Bone formation on and around the EBM textured implants increased compared to machined implants, as seen by BIC and fluorescence. No difference in torque, BIC or fluorescence among vibration levels was detected. BV/TV significantly increased at 0.6 g compared to control for both implant types.

Additive Function-on-Function Regression.

We study additive function-on-function regression where the mean response at a particular time point depends on the time point itself, as well as the entire covariate trajectory. We develop a computationally efficient estimation methodology based on a novel combination of spline bases with an eigenbasis to represent the trivariate kernel function. We discuss prediction of a new response trajectory, propose an inference procedure that accounts for total variability in the predicted response curves, and construct pointwise prediction intervals. The estimation/inferential procedure accommodates realistic scenarios, such as correlated error structure as well as sparse and/or irregular designs. We investigate our methodology in finite sample size through simulations and two real data applications. Supplementary Material for this article is available online.

Hydrology and Soil Manipulations of Iron-Rich Ditch Mesocosms Provide Little Evidence of Phosphorus Capture within the Profile.

Agricultural drainage ditches function as first-order streams and affect nutrient management. Soil mesocosms from a ditch featuring a vertical (increasing upward) gradient in iron (Fe) and phosphorus (P) were subjected to hydraulic and soil treatments. These manipulations mimicked aspects of dredging and controlled drainage and inspected the soil release and retention of P. Treatments did not remove P from simulated groundwater. Throughput water either gained in P (lack of dredging, especially under Fe-reducing conditions) or had P concentrations indistinguishable from input water (dredging). Undredged mesocosms, when Fe-reducing, released Fe and P simultaneously. Simultaneous release of P and Fe from our Fe-reducing mesocosms indicates a mechanism whereby P capture occurs by Fe precipitation upon emergence to aerated surficial waters. Upwelling and surficial phases of ditch hydrology and the lowering of the ditch surface on dredging complicate interpretation of traditional means of describing ditch P retention and release.

Metabolic profiling of a myalgic encephalomyelitis/chronic fatigue syndrome discovery cohort reveals disturbances in fatty acid and lipid metabolism.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) remains a continuum spectrum disease without biomarkers or simple objective tests, and therefore relies on a diagnosis from a set of symptoms to link the assortment of brain and body disorders to ME/CFS. Although recent studies show various affected pathways, the underlying basis of ME/CFS has yet to be established. In this pilot study, we compare plasma metabolic signatures in a discovery cohort, 17 patients and 15 matched controls, and explore potential metabolic perturbations as the aftermath of the complex interactions between genes, transcripts and proteins. This approach to examine the complex array of symptoms and underlying foundation of ME/CFS revealed 74 differentially accumulating metabolites, out of 361 (P < 0.05), and 35 significantly altered after statistical correction (Q < 0.15). The latter list includes several essential energy-related compounds which could theoretically be linked to the general lack of energy observed in ME/CFS patients. Pathway analysis points to a few pathways with high impact and therefore potential disturbances in patients, mainly taurine metabolism and glycerophospholipid metabolism, combined with primary bile acid metabolism, as well as glyoxylate and dicarboxylate metabolism and a few other pathways, all involved broadly in fatty acid metabolism. Purines, including ADP and ATP, pyrimidines and several amino acid metabolic pathways were found to be significantly disturbed. Finally, glucose and oxaloacetate were two main metabolites affected that have a major effect on sugar and energy levels. Our work provides a prospective path for diagnosis and understanding of the underlying mechanisms of ME/CFS.

Osseointegration of Coarse and Fine Textured Implants Manufactured by Electron Beam Melting and Direct Metal Laser Sintering.

Osseointegrated implants transfer loads from native bone to a synthetic joint and can also function transdermally to provide a stable connection between the skeleton and the prostheses, eliminating many problems associated with socket prostheses. Additive manufacturing provides a cost-effective means to create patient-specific implants and allows for customized textures for integration with bone and other tissues. Our objective was to compare the osseointegration strength of two primary additive manufacturing methods of producing textured implants: electron beam melting (EBM) (mean Ra = 23 µm) and direct metal laser sintering (DMLS) (mean Ra = 10 µm). Due to spatial resolution, DMLS can produce surfaces with a roughness comparable to EBM. Two cohorts of Sprague-Dawley rats received bilateral, titanium implants in their distal femurs and were followed for 4 weeks. The first-cohort animals received EBM implants transcortically in one femur and a DMLS implant in the contralateral femur. The second cohort received DMLS implants (either fine textured or coarse textured to mimic EBM) in the intramedullary canal of each femur. Osseointegration was evaluated through mechanical testing and micro-computed tomography (bone volume fraction [BV/TV] and bone-implant contact [BIC]). The fixation strength of coarse textured implants provided superior interlocking relative to fine textured implants without affecting BV/TV or BIC in both cohorts. Coarse EBM implants in a transcortical model demonstrated an 85% increase in removal torque relative to the fine DMLS textured implants. The thrust load in the intramedullary model saw a 35% increase from fine to coarse DMLS implants.

Spatiotemporal mixed modeling of multi-subject task fMRI via method of moments.

Estimating spatiotemporal models for multi-subject fMRI is computationally challenging. We propose a mixed model for localization studies with spatial random effects and time-series errors. We develop method-of-moment estimators that leverage population and spatial information and are scalable to massive datasets. In simulations, subject-specific estimates of activation are considerably more accurate than the standard voxel-wise general linear model. Our mixed model also allows for valid population inference. We apply our model to cortical data from motor and theory of mind tasks from the Human Connectome Project (HCP). The proposed method results in subject-specific predictions that appear smoother and less noisy than those from the popular single-subject univariate approach. In particular, the regions of motor cortex associated with a left-hand finger-tapping task appear to be more clearly delineated. Subject-specific maps of activation from task fMRI are increasingly used in pre-surgical planning for tumor removal and in locating targets for transcranial magnetic stimulation. Our findings suggest that using spatial and population information is a promising avenue for improving clinical neuroimaging.

Fast Covariance Estimation for High-dimensional Functional Data.

We propose two fast covariance smoothing methods and associated software that scale up linearly with the number of observations per function. Most available methods and software cannot smooth covariance matrices of dimension J > 500; a recently introduced sandwich smoother is an exception but is not adapted to smooth covariance matrices of large dimensions, such as J = 10, 000. We introduce two new methods that circumvent those problems: 1) a fast implementation of the sandwich smoother for covariance smoothing; and 2) a two-step procedure that first obtains the singular value decomposition of the data matrix and then smoothes the eigenvectors. These new approaches are at least an order of magnitude faster in high dimensions and drastically reduce computer memory requirements. The new approaches provide instantaneous (a few seconds) smoothing for matrices of dimension J = 10,000 and very fast (< 10 minutes) smoothing for J = 100, 000. R functions, simulations, and data analysis provide ready to use, reproducible, and scalable tools for practical data analysis of noisy high-dimensional functional data.

Bayesian Models for Multiple Outcomes in Domains with Application to the Seychelles Child Development Study.

The Seychelles Child Development Study (SCDS) examines the effects of prenatal exposure to methylmercury on the functioning of the central nervous system. The SCDS data include 20 outcomes measured on 9-year old children that can be classified broadly in four outcome classes or "domains": cognition, memory, motor, and social behavior. Previous analyses and scientific theory suggest that these outcomes may belong to more than one of these domains, rather than only a single domain as is frequently assumed for modeling. We present a framework for examining the effects of exposure and other covariates when the outcomes may each belong to more than one domain and where we also want to learn about the assignment of outcomes to domains. Each domain is defined by a sentinel outcome which is preassigned to that domain only. All other outcomes can belong to multiple domains and are not preassigned. Our model allows exposure and covariate effects to differ across domains and across outcomes within domains, and includes random subject-specific effects which model correlations between outcomes within and across domains. We take a Bayesian MCMC approach. Results from the Seychelles study and from extensive simulations show that our model can effectively determine sparse domain assignment, and at the same time give increased power to detect overall, domain-specific and outcome-specific exposure and covariate effects relative to separate models for each endpoint. When fit to the Seychelles data, several outcomes were classified as partly belonging to domains other than their originally assigned domains. In retrospect, the new partial domain assignments are reasonable and, as we discuss, suggest important scientific insights about the nature of the outcomes. Checks of model misspecification were improved relative to a model that assumes each outcome is in a single domain.