Transcriptomic Profiling Identifies Ferroptosis-Related Gene Signatures in Ischemic Muscle Satellite Cells Affected by Peripheral Artery Disease-Brief Report.

BACKGROUND: We hypothesized that transcriptomic profiling of muscle satellite cells in peripheral artery disease (PAD) would identify damage-related pathways contributing to skeletal muscle myopathy. We identified a potential role for ferroptosis-a form of programmed lytic cell death by iron-mediated lipid peroxidation-as one such pathway. Ferroptosis promotes myopathy in ischemic cardiac muscle but has an unknown role in PAD. METHODS: Muscle satellite cells from donors with PAD were obtained during surgery. cDNA libraries were processed for single-cell RNA sequencing using the 10X Genomics platform. Protein expression was confirmed based on pathways inferred by transcriptomic analysis. RESULTS: Unsupervised cluster analysis of over 25 000 cells aggregated from 8 donor samples yielded distinct cell populations grouped by a shared unique transcriptional fingerprint. Quiescent cells were diminished in ischemic muscle while myofibroblasts and apoptotic cells were prominent. Differential gene expression demonstrated a surprising increase in genes associated with iron transport and oxidative stress and a decrease in GPX4 (glutathione peroxidase 4) in ischemic PAD-derived cells. Release of the danger signal HMGB1 (high mobility group box-1) correlated with ferroptotic markers including surface transferrin receptor and were higher in ischemia. Furthermore, lipid peroxidation in muscle satellite cells was modulated by ferrostatin, a ferroptosis inhibitor. Histology confirmed iron deposition and lipofuscin, an inducer of ferroptosis in PAD-affected muscle. CONCLUSIONS: This report presents a novel finding that genes known to be involved in ferroptosis are differentially expressed in human skeletal muscle affected by PAD. Targeting ferroptosis may be a novel therapeutic strategy to reduce PAD myopathy.

Attenuated cell-cycle division protein 2 and elevated mitotic roles of polo-like kinase 1 characterize deficient myoblast fusion in peripheral arterial disease.

INTRODUCTION: We propose that MuSC-derived myoblasts in PAD have transcriptomic differences that can highlight underlying causes of ischemia-induced myopathy. METHODS: Differentiation capacity among perfused and ischemic human myoblasts was compared. Following next generation sequencing of mRNA, Ingenuity Pathway Analysis (IPA) was performed for canonical pathway enrichment. Live cell imaging and immunofluorescence were performed to determine myocyte fusion index and protein expression based on insights from IPA, specifically concerning cell cycle regulators including cell-division cycle protein 2 (CDC2) and polo-like kinase 1 (PLK1). RESULTS: Ischemic myoblasts formed attenuated myotubes indicative of reduced fusion. Additionally, myoblasts from ischemic segments showed significant differences in canonical pathways associated with PLK1 (upregulated) and G2/M DNA damage checkpoint regulation (downregulated). PLK1 inhibition with BI2536 did not affect cell viability in any group over 24 h but deterred fusion more significantly in PAD myoblasts. Furthermore, PLK1 inhibition reduced the expression of checkpoint protein CDC2 in perfused but not ischemic cells. CONCLUSION: Differentiating myoblasts derived from ischemic muscle have significant differences in gene expression including those essential to DNA-damage checkpoint regulation and cell cycle progress. DNA-damage checkpoint dysregulation may contribute to myopathy in PAD.

Visitors' perceptions of zoo-housed lesser anteater (Tamandua tetradactyla) welfare: Observation plays a larger role than a brief informative talk.

There is a growing ethical concern in modern society about animals' quality of life. We hypothesize that zoo visitors' perception of zoo animal welfare, particularly in the case of lesser anteaters, changes positively after listening to scientific information. Visitors observing active lesser anteaters in their enclosures at Córdoba Zoo (Argentina) were asked to respond to a questionnaire about animal welfare. The treatment group (T) answered the questionnaire after listening to a brief informative talk based on local scientific studies on lesser anteaters. The control group (C) answered the questionnaire without hearing the informative talk. Visitors (87.2%) considered biological, sanitary, and sociocultural aspects to be necessary conditions for optimum wild zoo-housed animal welfare. The majority of visitors considered that natural surroundings provide the highest level of welfare for wild animals. Visitors in the T group ranked the zoo as providing a higher level of animal welfare than those in group C. In reference to management measurements, the T group agreed on the positive effect of the application of environmental enrichment (Likert Medians: C = 4 and T = 5; p = .0443). On the basis of their perception, most visitors in both groups stated that the lesser anteaters at Córdoba Zoo appeared to be in a good state of welfare. We interpret this as meaning that, what these Córdoba zoo visitors personally perceived while observing the lesser anteaters carried greater weight than what they learned from the informative talk, though the talk did slightly affect their opinion.

Caspase1/11 signaling affects muscle regeneration and recovery following ischemia, and can be modulated by chloroquine.

BACKGROUND: We previously showed that the autophagy inhibitor chloroquine (CQ) increases inflammatory cleaved caspase-1 activity in myocytes, and that caspase-1/11 is protective in sterile liver injury. However, the role of caspase-1/11 in the recovery of muscle from ischemia caused by peripheral arterial disease is unknown. We hypothesized that caspase-1/11 mediates recovery in muscle via effects on autophagy and this is modulated by CQ. METHODS: C57Bl/6 J (WT) and caspase-1/11 double-knockout (KO) mice underwent femoral artery ligation (a model of hind-limb ischemia) with or without CQ (50 mg/kg IP every 2nd day). CQ effects on autophagosome formation, microtubule associated protein 1A/1B-light chain 3 (LC3), and caspase-1 expression was measured using electron microscopy and immunofluorescence. Laser Doppler perfusion imaging documented perfusion every 7 days. After 21 days, in situ physiologic testing in tibialis anterior muscle assessed peak force contraction, and myocyte size and fibrosis was also measured. Muscle satellite cell (MuSC) oxygen consumption rate (OCR) and extracellular acidification rate was measured. Caspase-1 and glycolytic enzyme expression was detected by Western blot. RESULTS: CQ increased autophagosomes, LC3 consolidation, total caspase-1 expression and cleaved caspase-1 in muscle. Perfusion, fibrosis, myofiber regeneration, muscle contraction, MuSC fusion, OCR, ECAR and glycolytic enzyme expression was variably affected by CQ depending on presence of caspase-1/11. CQ decreased perfusion recovery, fibrosis and myofiber size in WT but not caspase-1/11KO mice. CQ diminished peak force in whole muscle, and myocyte fusion in MuSC and these effects were exacerbated in caspase-1/11KO mice. CQ reductions in maximal respiration and ATP production were reduced in caspase-1/11KO mice. Caspase-1/11KO MuSC had significant increases in protein kinase isoforms and aldolase with decreased ECAR. CONCLUSION: Caspase-1/11 signaling affects the response to ischemia in muscle and effects are variably modulated by CQ. This may be critically important for disease treated with CQ and its derivatives, including novel viral diseases (e.g. COVID-19) that are expected to affect patients with comorbidities like cardiovascular disease.

Arsenic Promotes NF-Κb-Mediated Fibroblast Dysfunction and Matrix Remodeling to Impair Muscle Stem Cell Function.

Arsenic is a global health hazard that impacts over 140 million individuals worldwide. Epidemiological studies reveal prominent muscle dysfunction and mobility declines following arsenic exposure; yet, mechanisms underlying such declines are unknown. The objective of this study was to test the novel hypothesis that arsenic drives a maladaptive fibroblast phenotype to promote pathogenic myomatrix remodeling and compromise the muscle stem (satellite) cell (MuSC) niche. Mice were exposed to environmentally relevant levels of arsenic in drinking water before receiving a local muscle injury. Arsenic-exposed muscles displayed pathogenic matrix remodeling, defective myofiber regeneration and impaired functional recovery, relative to controls. When naïve human MuSCs were seeded onto three-dimensional decellularized muscle constructs derived from arsenic-exposed muscles, cells displayed an increased fibrogenic conversion and decreased myogenicity, compared with cells seeded onto control constructs. Consistent with myomatrix alterations, fibroblasts isolated from arsenic-exposed muscle displayed sustained expression of matrix remodeling genes, the majority of which were mediated by NF-κB. Inhibition of NF-κB during arsenic exposure preserved normal myofiber structure and functional recovery after injury, suggesting that NF-κB signaling serves as an important mechanism of action for the deleterious effects of arsenic on tissue healing. Taken together, the results from this study implicate myomatrix biophysical and/or biochemical characteristics as culprits in arsenic-induced MuSC dysfunction and impaired muscle regeneration. It is anticipated that these findings may aid in the development of strategies to prevent or revert the effects of arsenic on tissue healing and, more broadly, provide insight into the influence of the native myomatrix on stem cell behavior.

Face-to-face and telerehabilitation delivery of circuit training have similar benefits and acceptability in patients with knee osteoarthritis: a randomised trial.

QUESTION: Is periodised circuit training delivered via a telerehabilitation model of care as effective as the same training applied face-to-face for improving pain intensity, physical function, muscle strength, pain catastrophising, body composition, intermuscular adipose tissue and muscle architecture in people with knee osteoarthritis (OA)? DESIGN: Randomised controlled, non-inferiority trial with concealed allocation, blinded assessors and intention-to-treat analysis. PARTICIPANTS: One hundred adults aged ≥ 40 years with knee OA and pain for ≥ 3 months, with current pain ≥ 40 mm on a 100-mm visual analogue scale (VAS). INTERVENTION: The experimental group received 14 weeks of circuit training delivered via telerehabilitation using video recordings, followed by periodic phone calls in order to motivate and instruct participants. The control group received the same circuit training program in a face-to-face format. OUTCOME MEASURES: The primary outcomes were pain VAS and the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) physical function subscale, measured at 14 weeks. Secondary outcomes included objective physical function, strength, pain catastrophising and morphological measures (muscle architecture and thigh and body composition). Outcomes were measured at 14 and 26 weeks. RESULTS: Periodised circuit training delivered via telerehabilitation had equivalent effects to face-to-face delivery for pain intensity, physical function, muscle strength, pain catastrophising, thigh composition, intermuscular adipose tissue and muscle architecture. Whole body composition did not change appreciably in either group. Adherence to the training was excellent and participants in each group reported good perceptions of their randomised intervention. CONCLUSION: A periodised circuit training protocol can be delivered to people with knee OA in their own homes, using available technology while maintaining high levels of acceptability. More importantly, telerehabilitation appears to cause non-inferior physical and functional outcomes to face-to-face rehabilitation programs. TRIAL REGISTRATION: RBR-662hn2.

Automated detection, selection and classification of hippocampal landmark points for the diagnosis of Alzheimer's disease.

BACKGROUND AND OBJECTIVE: Alzheimer's disease (AD) is a neurodegenerative, progressive, and irreversible disease that accounts for up to 80% of all dementia cases. AD predominantly affects older adults, and its clinical diagnosis is a challenging evaluation process, with imprecision rates between 12 and 23%. Structural magnetic resonance (MR) imaging has been widely used in studies related to AD because this technique provides images with excellent anatomical details and information about structural changes induced by the disease in the brain. Current studies are focused on detecting AD in its initial stage, i.e., mild cognitive impairment (MCI), since treatments for preventing or delaying the onset of symptoms is more effective when administered at the early stages of the disease. This study proposes a new technique to perform MR image classification in AD diagnosis using discriminative hippocampal point landmarks among the cognitively normal (CN), MCI, and AD populations. METHODS: Our approach, based on a two-level classification, first detects and selects discriminative landmark points from two diagnosis populations based on their matching distance compared to a probabilistic atlas of 3-D labeled landmark points. The points are classified using attributes computed in a spherical support region around each point using information from brain probability image tissues of gray matter, white matter, and cerebrospinal fluid as sources of information. Next, at the second level, the images are classified based on a quantitative evaluation obtained from the first-level classifier outputs. RESULTS: For the CN×MCI experiment, we achieved an AUC of 0.83, an accuracy of 75.58%, with 72.9% of sensitivity and 77.81% of specificity. For the MCI×AD experiment, we achieved an AUC value of 0.73, an accuracy of 69.8%, a sensitivity of 74.09% and specificity of 64.57%. Finally, for the CN×AD, we achieved an AUC of 0.95, an accuracy of 89.24%, with 85.58% of sensitivity and 92.71% of specificity. CONCLUSIONS: The obtained classification results are similar to (or even higher than) other studies that classify AD compared to CN individuals and comparable to those classified patients with MCI.

Inflammatory Caspase Activity Mediates HMGB1 Release and Differentiation in Myoblasts Affected by Peripheral Arterial Disease.

Introduction: We previously showed that caspase-1 and -11, which are activated by inflammasomes, mediate recovery from muscle ischemia in mice. We hypothesized that similar to murine models, inflammatory caspases modulate myogenicity and inflammation in ischemic muscle disease. Methods: Caspase activity was measured in ischemic and perfused human myoblasts in response to the NLRP3 and AIM2 inflammasome agonists (nigericin and poly(dA:dT), respectively) with and without specific caspase-1 or pan-caspase inhibition. mRNA levels of myogenic markers and caspase-1 were assessed, and protein levels of caspases-1, -4, -5, and -3 were measured by Western blot. Results: When compared to perfused cells, ischemic myoblasts demonstrated attenuated MyoD and myogenin and elevated caspase-1 mRNA. Ischemic myoblasts also had significantly higher enzymatic caspase activity with poly(dA:dT) (p < 0.001), but not nigericin stimulation. Inhibition of caspase activity including caspase-4/-5, but not caspase-1, blocked activation effects of poly(dA:dT). Ischemic myoblasts had elevated cleaved caspase-5. Inhibition of caspase activity deterred differentiation in ischemic but not perfused myoblasts and reduced the release of HMGB1 from both groups. Conclusion: Inflammatory caspases can be activated in ischemic myoblasts by AIM2 and influence ischemic myoblast differentiation and release of pro-angiogenic HMGB1. AIM2 inflammasome involvement suggests a role as a DNA damage sensor, and our data suggest that caspase-5 rather than caspase-1 may mediate the downstream mediator of this pathway.

Detecting cells in intravital video microscopy using a deep convolutional neural network.

The analysis of leukocyte recruitment in intravital video microscopy (IVM) is essential to the understanding of inflammatory processes. However, because IVM images often present a large variety of visual characteristics, it is hard for an expert human or even conventional machine learning techniques to detect and count the massive amount of cells and extract statistical measures precisely. Convolutional neural networks are a promising approach to overcome this problem, but due to the difficulty of labeling cells, large data sets with ground truth are rare. The present work explores an adaptation of the RetinaNet model with a suite of augmentation techniques and transfer learning for detecting leukocytes in IVM data. The augmentation techniques include simulating the Airy pattern and motion artifacts present in microscopy imaging, followed by traditional photometric, geometric and smooth elastic transformations to reproduce color and shape changes in cells. In addition, we analyzed the use of different network backbones, feature pyramid levels, and image input scales. We have found that even with limited data, our strategy not only enables training without overfitting but also boosts generalization performance. Among several experiments, the model reached a value of 94.84 for the average precision (AP) metric as our best outcome when using data from different image modalities. We also compared our results with conventional image processing techniques and open-source tools. The results showed an outstanding precision of the method compared with other approaches, presenting low error rates for cell counting and centroid distances. Code is available at: https://github.com/brunoggregorio/retinanet-cell-detection.

Robust texture features for response monitoring of glioblastoma multiforme on T1-weighted and T2-FLAIR MR images: a preliminary investigation in terms of identification and segmentation.

PURPOSE: Image texture has recently attracted much attention in providing quantitative features that are unique to various different tissue types, in particular, in MR images of the brain. Such image features may be useful for tumor response quantification. As a first step, one needs to establish if these features are sensitive to different tissues of clinical relevance. Here, a novel method of texture analysis based on the Hartley transform has been investigated and applied to MR images of glioblastoma multiforme (GBM). METHODS: Contrast-enhanced T1-weighted gradient-echo and T2-FLAIR spin-echo MR images of 27 GBM patients acquired prior to radiation therapy were available for analysis. Before computing texture features on these images, a novel image transformation was employed in the form of a power map computed from the localized Hartley transform of the image. Haralick statistical texture features were then computed based on the power map. This method was compared to the standard approach of obtaining texture features directly from the image. Twelve different features were computed on different resolution levels. On a regional resolution level, image texture features were identified that are able to correctly classify entire regions within T1-weighted and T2-FLAIR brain MR images of GBM patients into abnormal (containing contrast-enhancing GBM tumor) and brain tissue. Various metrics [area under the ROC curve (AUC), maximum accuracy, and Canberra distance] have been computed to quantify the usefulness of these features. On a local resolution level, it was investigated which of these features are able to provide a voxel-by-voxel enhancement that could be used for assisting the segmentation of the gross tumor volume on T1 images. The "gold standard" for this analysis was a gross tumor volume corresponding to the contrast-enhancing lesion visualized on T1-weighted images as segmented by a radiation oncologist. RESULTS: The Sum-mean and Variance features demonstrated the best performance overall. For the T1-weighted images, the identification performance of Sum-mean and Variance features computed from the power map was higher (AUC = 0.9959 and AUC = 0.9918, respectively) and with higher Canberra distances as compared to features computed directly from the images (AUC = 0.8930 and AUC = 0.9163, respectively). These results in T2-FLAIR images were even superior. The features computed from the power map showed an unequivocal identification (AUC = 1) with higher Canberra distances, whereas the performance of the features from the original images was slightly lower (AUC = 0.9739 and AUC = 0.9904, respectively). The same features computed on the power map of the T1-weighted images also provided superior enhancement in individual tumor voxels as compared to the features computed on the original images. CONCLUSIONS: The Sum-mean and Variance features are both useful for identifying and segmenting GBM tumors on localized Hartley transformed MR images.

Detector of 3-D salient points based on the dual-tree complex wavelet transform for the positioning of hippocampi meshes in magnetic resonance images.

BACKGROUND: Brain Magnetic Resonance (MR) image segmentation methods based on deformable models depend on the initial positioning to maximize the chances of successful segmentation. To minimize this limitation, salient-point based registration is used to perform the initial positioning of brain structure meshes close to their image target representation. The analysis of brain structures (such as the hippocampus) can help in the diagnosis and follow-up of neurodegenerative diseases like Alzheimer's. METHODS: We present a technique for detection and description of 3-D salient points, which combines filter response maps estimated for different scales and orientations of the dual-tree complex wavelet transform (DT-CWT). We apply our technique to detect salient points in volumetric brain MR images and use the detected points in a positioning methodology. To illustrate the applicability, we applied our method for the positioning of hippocampi meshes in 3-D brain MR images and assessed the results by overlapping the positioned meshes with manual annotations made by medical specialists. RESULTS: Our method yielded mean values of normalized Dice Similarity Coefficient (nDSC) of 0.74/0.68 and Hausdorff Average Distance (HAD) of 0.73/0.75 for the left and right hippocampus, respectively. COMPARISON WITH OTHER METHODS: The mean nDSC and HAD results of our detector were significantly better than the ones achieved by an Affine and a Phase Congruency (PC) guided positioning. CONCLUSIONS: The detection via DT-CWT decomposition is computationally less demanding than the detection via PC and represents a robust alternative for the positioning of mesh models.

Fibrosis Distinguishes Critical Limb Ischemia Patients from Claudicants in a Transcriptomic and Histologic Analysis.

Most patients with critical limb ischemia (CLI) from peripheral arterial disease (PAD) do not have antecedent intermittent claudication (IC). We hypothesized that transcriptomic analysis would identify CLI-specific pathways, particularly in regards to fibrosis. Derivation cohort data from muscle biopsies in PAD and non-PAD (controls) was obtained from the Gene Expression Omnibus (GSE120642). Transcriptomic analysis indicated CLI patients (N = 16) had a unique gene expression profile, when compared with non-PAD controls (N = 15) and IC (N = 20). Ninety-eight genes differed between controls and IC, 2489 genes differed between CLI and controls, and 2783 genes differed between CLI and IC patients. Pathway enrichment analysis showed that pathways associated with TGFß, collagen deposition, and VEGF signaling were enriched in CLI but not IC. Receiver operating curve (ROC) analysis of nine fibrosis core gene expression revealed the areas under the ROC (AUC) were all >0.75 for CLI. Furthermore, the fibrosis area (AUC = 0.81) and % fibrosis (AUC = 0.87) in validation cohort validated the fibrosis discrimination CLI from IC and control (all n = 12). In conclusion, transcriptomic analysis identified fibrosis pathways, including those involving TGFß, as a novel gene expression feature for CLI but not IC. Fibrosis is an important characteristic of CLI, which we confirmed histologically, and may be a target for novel therapies in PAD.

A periodized training attenuates thigh intermuscular fat and improves muscle quality in patients with knee osteoarthritis: results from a randomized controlled trial.

OBJECTIVE: To analyze the influence of a 14-week periodized circuit training (CT) protocol on thigh intermuscular fat and muscle quality (force per unit area of lean tissue) in patients with knee osteoarthritis (KOA). DESIGN: Randomized controlled trial METHODS: Sixty-one selected participants with KOA grades 2 and 3, 40-65 years old, and BMI < 30 kg/m2 were randomized into three groups: CT, conventional strength training (ST), and educational protocol (EP). The CT and ST protocols consisted of 14-week training protocols conducted 3 times a week. The CT group performed exercises stratified as light, moderate, and intense, arranged progressively in a circuit model. The ST group performed conventional strength exercises, and the EP group participated in lectures twice a month about healthy lifestyles. Baseline and follow-up (week 0 and week 14) evaluations were conducted for thigh intermuscular fat (computed tomography), knee extension maximal isometric voluntary contraction (MIVC), and muscle quality (knee extension MIVC/muscle mass cross-sectional area). RESULTS: Only the CT group presented significant reductions in thigh intermuscular fat (p = 0.003) and significantly lower values in week 14 compared with the EP (p = 0.032). Both trained groups presented significant increases in muscle mass area (p=0.002 for CT and p=0.008 for ST) and increments in knee extension MIVC (p=0,033 for CT nd p=0.019 for ST) in week 14 compared with the EP and increases in muscle quality (p = 0.004 and 0.042). CONCLUSION: It can be concluded that a 14-week periodized CT protocol attenuates thigh intermuscular fat and improves muscle quality in patients with KOA. TRIAL REGISTRATION: ClinicalTrials.gov, NCT02761590; https://clinicaltrials.gov/ct2/show/NCT02761590Key Points•Fourteen weeks of periodized circuit training attenuates thigh intermuscular fat in patients with knee osteoarthritis.•Circuit training is as effective as strength training for improving muscle mass, strength, and quality.

Functional overloading of dystrophic mice enhances muscle-derived stem cell contribution to muscle contractile capacity.

OBJECTIVES: To evaluate the effect of functional overloading on the transplantation of muscle derived stem cells (MDSCs) into dystrophic muscle and the ability of transplanted cells to increase dystrophic muscle's ability to resist overloading-induced weakness. DESIGN: Cross-sectional. SETTING: Laboratory. ANIMALS: Male mice (N=10) with a dystrophin gene mutation. INTERVENTIONS: MDSCs were intramuscularly transplanted into the extensor digitorum longus muscle (EDL). Functional overloading of the EDL was performed by surgical ablation of the EDL's synergist. MAIN OUTCOME MEASURES: The total number of dystrophin-positive fibers/cross-section (as a measure of stem cell engraftment), the average number of CD31+ cells (as a measure of capillarity), and in vitro EDL contractile strength. Independent t tests were used to investigate the effect of overloading on engraftment, capillarity, and strength. Paired t tests were used to investigate the effect of MDSC engraftment on strength and capillarity. RESULTS: MDSC transplantation protects dystrophic muscles against overloading-induced weakness (specific twitch force: control 4.5N/cm2+/-2.3; MDSC treated 7.9N/cm2+/-1.4) (P=.02). This improved force production following overloading is concomitant with an increased regeneration by transplanted MDSCs (MDSC: 26.6+/-20.2 dystrophin-positive fibers/cross-section; overloading + MDSC: 170.6+/-130.9 dystrophin-positive fibers/cross-section [P=.03]). Overloading-induced increases in skeletal muscle capillarity is significantly correlated with increased MDSC engraftment (R2=.80, P=.01). CONCLUSIONS: These findings suggest that the functional contribution of transplanted MDSCs may rely on activity-dependent mechanisms, possibly mediated by skeletal muscle vascularity. Rehabilitation modalities may play an important role in the development of stem cell transplantation strategies for the treatment of muscular dystrophy.

Automatic iterative segmentation of multiple sclerosis lesions using Student's t mixture models and probabilistic anatomical atlases in FLAIR images.

Multiple sclerosis (MS) is a demyelinating autoimmune disease that attacks the central nervous system (CNS) and affects more than 2 million people worldwide. The segmentation of MS lesions in magnetic resonance imaging (MRI) is a very important task to assess how a patient is responding to treatment and how the disease is progressing. Computational approaches have been proposed over the years to segment MS lesions and reduce the amount of time spent on manual delineation and inter- and intra-rater variability and bias. However, fully-automatic segmentation of MS lesions still remains an open problem. In this work, we propose an iterative approach using Student's t mixture models and probabilistic anatomical atlases to automatically segment MS lesions in Fluid Attenuated Inversion Recovery (FLAIR) images. Our technique resembles a refinement approach by iteratively segmenting brain tissues into smaller classes until MS lesions are grouped as the most hyperintense one. To validate our technique we used 21 clinical images from the 2015 Longitudinal Multiple Sclerosis Lesion Segmentation Challenge dataset. Evaluation using Dice Similarity Coefficient (DSC), True Positive Ratio (TPR), False Positive Ratio (FPR), Volume Difference (VD) and Pearson's r coefficient shows that our technique has a good spatial and volumetric agreement with raters' manual delineations. Also, a comparison between our proposal and the state-of-the-art shows that our technique is comparable and, in some cases, better than some approaches, thus being a viable alternative for automatic MS lesion segmentation in MRI.

Automatic detection of motion blur in intravital video microscopy image sequences via directional statistics of log-Gabor energy maps.

Intravital microscopy is an important experimental tool for the study of cellular and molecular mechanisms of the leukocyte-endothelial interactions in the microcirculation of various tissues and in different inflammatory conditions of in vivo specimens. However, due to the limited control over the conditions of the image acquisition, motion blur and artifacts, resulting mainly from the heartbeat and respiratory movements of the in vivo specimen, will very often be present. This problem can significantly undermine the results of either visual or computerized analysis of the acquired video images. Since only a fraction of the total number of images are usually corrupted by severe motion blur, it is necessary to have a procedure to automatically identify such images in the video for either further restoration or removal. This paper proposes a new technique for the detection of motion blur in intravital video microscopy based on directional statistics of local energy maps computed using a bank of 2D log-Gabor filters. Quantitative assessment using both artificially corrupted images and real microscopy data were conducted to test the effectiveness of the proposed method. Results showed an area under the receiver operating characteristic curve (AUC) of 0.95 (AUC = 0.95; 95 % CI 0.93-0.97) when tested on 329 video images visually ranked by four observers.

Arsenic induces sustained impairment of skeletal muscle and muscle progenitor cell ultrastructure and bioenergetics.

Over 4 million individuals in the United States, and over 140 million individuals worldwide, are exposed daily to arsenic-contaminated drinking water. Human exposures can range from below the current limit of 10 µg/L to over 1mg/L, with 100 µg/L promoting disease in a large portion of those exposed. Although increased attention has recently been paid to myopathy following arsenic exposure, the pathogenic mechanisms underlying clinical symptoms remain poorly understood. This study tested the hypothesis that arsenic induces lasting muscle mitochondrial dysfunction and impairs metabolism. Compared to nonexposed controls, mice exposed to drinking water containing 100 µg/L arsenite for 5 weeks demonstrated impaired muscle function, mitochondrial myopathy, and altered oxygen consumption that were concomitant with increased mitochondrial fusion gene transcription. There were no differences in the levels of inorganic arsenic or its monomethyl and dimethyl metabolites between controls and exposed muscles, confirming that arsenic does not accumulate in muscle. Nevertheless, muscle progenitor cells isolated from exposed mice recapitulated the aberrant myofiber phenotype and were more resistant to oxidative stress, generated more reactive oxygen species, and displayed autophagic mitochondrial morphology, compared to cells isolated from nonexposed mice. These pathological changes from a possible maladaptive oxidative stress response provide insight into declines in muscle functioning caused by exposure to this common environmental contaminant.

Off-line determination of the optimal number of iterations of the robust anisotropic diffusion filter applied to denoising of brain MR images.

Although anisotropic diffusion filters have been used extensively and with great success in medical image denoising, one limitation of this iterative approach, when used on fully automatic medical image processing schemes, is that the quality of the resulting denoised image is highly dependent on the number of iterations of the algorithm. Using many iterations may excessively blur the edges of the anatomical structures, while a few may not be enough to remove the undesirable noise. In this work, a mathematical model is proposed to automatically determine the number of iterations of the robust anisotropic diffusion filter applied to the problem of denoising three common human brain magnetic resonance (MR) images (T1-weighted, T2-weighted and proton density). The model is determined off-line by means of the maximization of the mean structural similarity index, which is used in this work as metric for quantitative assessment of the resulting processed images obtained after each iteration of the algorithm. After determining the model parameters, the optimal number of iterations of the algorithm is easily determined without requiring any extra computation time. The proposed method was tested on 3D synthetic and clinical human brain MR images and the results of qualitative and quantitative evaluation have shown its effectiveness.

Neuromuscular electrical stimulation as a method to maximize the beneficial effects of muscle stem cells transplanted into dystrophic skeletal muscle.

Cellular therapy is a potential approach to improve the regenerative capacity of damaged or diseased skeletal muscle. However, its clinical use has often been limited by impaired donor cell survival, proliferation and differentiation following transplantation. Additionally, functional improvements after transplantation are all-too-often negligible. Because the host microenvironment plays an important role in the fate of transplanted cells, methods to modulate the microenvironment and guide donor cell behavior are warranted. The purpose of this study was to investigate whether the use of neuromuscular electrical stimulation (NMES) for 1 or 4 weeks following muscle-derived stem cell (MDSC) transplantation into dystrophic skeletal muscle can modulate the fate of donor cells and enhance their contribution to muscle regeneration and functional improvements. Animals submitted to 4 weeks of NMES after transplantation demonstrated a 2-fold increase in the number of dystrophin+ myofibers as compared to control transplanted muscles. These findings were concomitant with an increased vascularity in the MDSC+NMES group when compared to non-stimulated counterparts. Additionally, animals subjected to NMES (with or without MDSC transplantation) presented an increased maximal specific tetanic force when compared to controls. Although cell transplantation and/or the use of NMES resulted in no changes in fatigue resistance, the combination of both MDSC transplantation and NMES resulted in a faster recovery from fatigue, when compared to non-injected and non-stimulated counterparts. We conclude that NMES is a viable method to improve MDSC engraftment, enhance dystrophic muscle strength, and, in combination with MDSC transplantation, improve recovery from fatigue. These findings suggest that NMES may be a clinically-relevant adjunct approach for cell transplantation into skeletal muscle.

Acute toxicity profile in prostate cancer with conventional and hypofractionated treatment.

PURPOSE: To compare the acute toxicities in radical treatment of prostate cancer between conventional schedule (C-ARM) with 78 Gy/39 fractions and hypofractionation conformal treatment (H-ARM) with 69 Gy/23 fractions. METHODS AND MATERIAL: This prospective double arm study consisted of 217 patients with prostate cancer, 112 in H-ARM and 105 in C-ARM arm. C-ARM received conventional six- field conformal radiotherapy with 78 Gy in 39 fractions while H-ARM received hypofractionation with 69 Gy in 23 fractions. Weekly assessment of acute reactions was done during treatment and with one, and 3 months using RTOG scale. Univariated analysis was performed to evaluate differences between the incidences of acute reaction in the treatment arms. Variables with p value less than 0.1 were included in the multivariated logistic regression. RESULTS: There was no difference between H-ARM versus C-ARM for severity and incidence in genitourinary (GU) and gastrointestinal (GI) acute toxicity. During the treatment comparing H-ARM with C-ARM no differences was observed for GI toxicity (grade 0-3; H-ARM=45.5%, 34%, 18.7% and 1.8% versus C-ARM=47.6%, 35.2%, 17.2% and 0). For acute GU toxicity no difference was detected between H-ARM (grade 0-3; 22.3%, 54.5%, 18.7% and 4.5%) and C-ARM (grade 0-3; 25.8%, 53.3%, 17.1% and 3.8%).At the 3- months follow-up, persistent Grade> =2 acute GU and GI toxicity were 2.5% and 1.8% in H-ARM versus 5.7% and 3% in C-ARM (p>0.05). In univariated and multivariated analyses, there was not any dosimetric predictor for GI and GU toxicity. CONCLUSIONS: Our data demonstrate that hypofractionated radiotherapy achieving high biological effective dose using conformal radiotherapy is feasible for prostate cancer, being well tolerated with minimal severe acute toxicity.