Quantification of fibrosis extend and airspace availability in lung: A semi-automatic ImageJ/Fiji toolbox.

The evaluation of the structural integrity of mechanically dynamic organs such as lungs is critical for the diagnosis of numerous pathologies and the development of therapies. This task is classically performed by histology experts in a qualitative or semi-quantitative manner. Automatic digital image processing methods appeared in the last decades, and although immensely powerful, tools are highly specialized and lack the versatility required in various experimental designs. Here, a set of scripts for the image processing software ImageJ/Fiji to easily quantify fibrosis extend and alveolar airspace availability in Sirius Red or Masson's trichrome stained samples is presented. The toolbox consists in thirteen modules: sample detection, particles filtration (automatic and manual), border definition, air ducts identification, air ducts walls definition, parenchyma extraction, MT-staining specific pre-processing, fibrosis detection, fibrosis particles filtration, airspace detection, and visualizations (tissue only or tissue and airspace). While the process is largely automated, critical parameters are accessible to the user for increased adaptability. The modularity of the protocol allows for its adjustment to alternative experimental settings. Fibrosis and airspace can be combined as an evaluation of the structural integrity of the organ. All settings and intermediate states are saved to ensure reproducibility. These new analysis scripts allow for a rapid quantification of fibrosis and airspace in a large variety of experimental settings.

Effect of the accordion technique on bone regeneration during distraction osteogenesis: A computational study.

BACKGROUND AND OBJECTIVE: Distraction osteogenesis (DO), a bone lengthening technique, is widely employed to treat congenital and acquired limb length discrepancies and large segmental bone defects. However, a major issue of DO is the prolonged consolidation phase (10-36 months) during which patients must wear a cumbersome external fixator. Attempts have been made to accelerate the healing process of DO by an alternating distraction and compression mode (so-called "accordion" technique or AT). However, it remains unclear how varied AT parameters affect DO outcomes and what the most effective AT mode is. METHODS: Based on an experimentally-verified mechanobiological model, we performed a parametric analysis via in silico simulation of the bone regeneration process of DO under different AT modes, including combinations of varied application times (AT began at week 1-8 of the consolidation phase), durations (AT was used continuously for 1 week, 2 weeks or 4 weeks) and rates (distraction or compression at 0.25, 0.5, 0.75, and 1 mm/12 h). The control group had no AT applied during the consolidation phase. RESULTS: Compared with the control group (no AT), AT applied at an early consolidation stage (e.g. week 1 of the consolidation phase) significantly enhanced bone formation and reduced the overall healing time. However, the effect of AT on bone healing was dependent on its duration and rate. Specifically, a moderate rate of AT (e.g. 0.5 mm/12 h) lasting for two weeks promoted blood perfusion recovery and bone regeneration, ultimately shortening the healing time. Conversely, over-high rates (e.g. 1 mm/12 h) and longer durations (e.g. 4 weeks) of AT adversely affected bone regeneration and blood perfusion recovery, thereby delaying bone bridging. CONCLUSIONS: These results suggest that the therapeutic effects of AT on DO are highly dependent of the AT parameters of choice. Under appropriate durations and rates, the AT applied at an early consolidation phase is beneficial for blood recovery and bone regeneration. These results may provide a basis for selecting effective AT modes to accelerate consolidation and reduce the overall treatment period of DO.

A novel agonist for the HGF receptor MET promotes differentiation of human pluripotent stem cells into hepatocyte-like cells.

Hepatocyte growth factor (HGF) is the natural ligand of the MET receptor tyrosine kinase. This ligand-receptor couple is essential for the maturation process of hepatocytes. Previously, the rational design of a synthetic protein based on the assembly of two K1 domains from HGF led to the production of a potent and stable MET receptor agonist. In this study, we compared the effects of K1K1 with HGF during the differentiation of hepatocyte progenitors derived from human induced pluripotent stem cells (hiPSCs). In vitro, K1K1, in the range of 20 to 200 nM, successfully substituted for HGF and efficiently activated ERK downstream signaling. Analysis of the levels of hepatocyte markers showed typical liver mRNA and protein expression (HNF4α, albumin, alpha-fetoprotein, CYP3A4) and phenotypes. Although full maturation was not achieved, the results suggest that K1K1 is an attractive candidate MET agonist suitable for replacing complex and expensive HGF treatments to induce hepatic differentiation of hiPSCs.

Rethinking Weight Decay for Efficient Neural Network Pruning.

Introduced in the late 1980s for generalization purposes, pruning has now become a staple for compressing deep neural networks. Despite many innovations in recent decades, pruning approaches still face core issues that hinder their performance or scalability. Drawing inspiration from early work in the field, and especially the use of weight decay to achieve sparsity, we introduce Selective Weight Decay (SWD), which carries out efficient, continuous pruning throughout training. Our approach, theoretically grounded on Lagrangian smoothing, is versatile and can be applied to multiple tasks, networks, and pruning structures. We show that SWD compares favorably to state-of-the-art approaches, in terms of performance-to-parameters ratio, on the CIFAR-10, Cora, and ImageNet ILSVRC2012 datasets.

In vivo and in silico monitoring bone regeneration during distraction osteogenesis of the mouse femur.

BACKGROUND AND OBJECTIVE: Distraction osteogenesis (DO) is a mechanobiological process of producing new bone by gradual and controlled distraction of the surgically separated bone segments. Mice have been increasingly used to study the role of relevant biological factors in regulating bone regeneration during DO. However, there remains a lack of in silico DO models and related mechano-regulatory tissue differentiation algorithms for mouse bone. This study sought to establish an in silico model based on in vivo experimental data to simulate the bone regeneration process during DO of the mouse femur. METHODS: In vivo micro-CT, including time-lapse morphometry was performed to monitor the bone regeneration in the distraction gap. A 2D axisymmetric finite element model, with a geometry originating from the experimental data, was created. Bone regeneration was simulated with a fuzzy logic-based two-stage (distraction and consolidation) mechano-regulatory tissue differentiation algorithm, which was adjusted from that used for fracture healing based on our in vivo experimental data. The predictive potential of the model was further tested with varied distraction frequencies and distraction rates. RESULTS: The computational simulations showed similar bone regeneration patterns to those observed in the micro-CT data from the experiment throughout the DO process. This consisted of rapid bone formation during the first 10 days of the consolidation phase, followed by callus reshaping via bone remodeling. In addition, the computational model predicted a faster and more robust bone healing response as the model's distraction frequency was increased, whereas higher or lower distraction rates were not conducive to healing. CONCLUSIONS: This in silico model could be used to investigate basic mechanobiological mechanisms involved in bone regeneration or to optimize DO strategies for potential clinical applications.

Enhancing the Efficiency of Distraction Osteogenesis through Rate-Varying Distraction: A Computational Study.

Distraction osteogenesis (DO) is a mechanobiological process of producing new bone and overlying soft tissues through the gradual and controlled distraction of surgically separated bone segments. The process of bone regeneration during DO is largely affected by distraction parameters. In the present study, a distraction strategy with varying distraction rates (i.e., "rate-varying distraction") is proposed, with the aim of shortening the distraction time and improving the efficiency of DO. We hypothesized that faster and better healing can be achieved with rate-varying distractions, as compared with constant-rate distractions. A computational model incorporating the viscoelastic behaviors of the callus tissues and the mechano-regulatory tissue differentiation laws was developed and validated to predict the bone regeneration process during DO. The effect of rate-varying distraction on the healing outcomes (bony bridging time and bone formation) was examined. Compared to the constant low-rate distraction, a low-to-high rate-varying distraction provided a favorable mechanical environment for angiogenesis and bone tissue differentiation, throughout the distraction and consolidation phase, leading to an improved healing outcome with a shortened healing time. These results suggest that a rate-varying clinical strategy could reduce the overall treatment time of DO and decrease the risk of complications related to the external fixator.

Human cardiac fibroblasts expressing VCAM1 improve heart function in postinfarct heart failure rat models by stimulating lymphangiogenesis.

Cardiovascular diseases are a leading cause of death worldwide. After an ischemic injury, the myocardium undergoes severe necrosis and apoptosis, leading to a dramatic degradation of function. Numerous studies have reported that cardiac fibroblasts (CFs) play a critical role in heart function even after injury. However, CFs present heterogeneous characteristics according to their development stage (i.e., fetal or adult), and the molecular mechanisms by which they maintain heart function are not fully understood. The aim of this study is to explore the hypothesis that a specific population of CFs can repair the injured myocardium in heart failure following ischemic infarction, and lead to a significant recovery of cardiac function. Flow cytometry analysis of CFs defined two subpopulations according to their relative expression of vascular cell adhesion molecule 1 (VCAM1). Whole-transcriptome analysis described distinct profiles for these groups, with a correlation between VCAM1 expression and lymphangiogenesis-related genes up-regulation. Vascular formation assays showed a significant stimulation of lymphatic cells network complexity by VCFs. Injection of human VCAM1-expressing CFs (VCFs) in postinfarct heart failure rat models (ligation of the left anterior descending artery) led to a significant restoration of the left ventricle contraction. Over the course of the experiment, left ventricular ejection fraction and fractional shortening increased by 16.65% ± 5.64% and 10.43% ± 6.02%, respectively, in VCF-treated rats. Histological examinations revealed that VCFs efficiently mobilized the lymphatic endothelial cells into the infarcted area. In conclusion, human CFs present heterogeneous expression of VCAM1 and lymphangiogenesis-promoting factors. VCFs restore the mechanical properties of ventricular walls by mobilizing lymphatic endothelial cells into the infarct when injected into a rat heart failure model. These results suggest a role of this specific population of CFs in the homeostasis of the lymphatic system in cardiac regeneration, providing new information for the study and therapy of cardiac diseases.

Analysis of the transcription factors and their regulatory roles during a step-by-step differentiation of induced pluripotent stem cells into hepatocyte-like cells.

We investigated the human induced pluripotent stem cells (hiPSCs) during a sequential in vitro step-by-step differentiation into hepatocyte-like cells (HLCs) using nanoCAGE, a method for promoters, transcription factors, and transcriptome analysis. Specific gene clusters reflected the different steps of the hepatic differentiation. The proliferation step was characterized by a typical cell cycle and DNA replication. The hepatic endoderm and the HLC steps were marked by a common signature including cell interactions with extracellular matrix (ECM), lipoproteins and hepatic biomarkers (such as albumin and alpha-fetoprotein). The specific HLC profile was characterized by important transcription factors such as HIF1A, JUN, MAF, KLF6, BMP4 and with a larger expression of genes related to Wnt signaling, extracellular matrix, lipid metabolism, urea cycle, drugs, and solute transporters. HLC profile was also characterized by the activation of upstream regulators such as HNF1A, MEIS2, NFIX, WRNIP1, SP4, TAL1. Their regulatory networks highlighted HNF4a as a bridge and linked them to important processes such as EMT-MET transitions, ECM remodeling and liver development pathways (HNF3, PPARA signaling, iron metabolism) along the different steps of differentiation.

Alterations of redox dynamics and desmin post-translational modifications in skeletal muscle models of desminopathies.

Desminopathies are a type of myofibrillar myopathy resulting from mutations in DES, encoding the intermediate filament protein desmin. They display heterogeneous phenotypes, suggesting environment influences. Patient muscle proteins show oxidative features linking oxidative stress, protein aggregation, and abnormal protein deposition. To improve understanding of redox balance in desminopathies, we further developed cellular models of four pathological mutants localized in 2B helical domain (the most important region for desmin polymerization) to explore desmin behavior upon oxidative stress. We show that the mutations desQ389P and desD399Y share common stress-induced aggregates, desR406W presents more scattered cytoplasmic aggregative pattern, and pretreatment with N-acetyl-l-cysteine (NAC), an antioxidant molecule, prevents all type of aggregation. Mutants desD399Y and desR406W had delayed oxidation kinetics following H2O2 stress prevented by NAC pretreatment. Further, we used AAV-injected mouse models to confirm in vivo effects of N-acetyl-l-cysteine. AAV-desD399Y-injected muscles displayed similar physio-pathological characteristics as observed in patients. However, after 2 months of NAC treatment, they did not have reduced aggregates. Finally, in both models, stress induced some post-translational modifications changing Isoelectric Point, such as potential hyperphosphorylations, and/or molecular weight of human desmin by proteolysis. However, each mutant presented its own pattern that seemed to be post-aggregative. In conclusion, our results indicate that individual desmin mutations have unique pathological molecular mechanisms partly linked to alteration of redox homeostasis. Integrating these mutant-specific behaviors will be important when considering future therapeutics.

N-acetyl-L-cysteine prevents stress-induced desmin aggregation in cellular models of desminopathy.

Mutations within the human desmin gene are responsible for a subcategory of myofibrillar myopathies called desminopathies. However, a single inherited mutation can produce different phenotypes within a family, suggesting that environmental factors influence disease states. Although several mouse models have been used to investigate organ-specific desminopathies, a more general mechanistic perspective is required to advance our knowledge toward patient treatment. To improve our understanding of disease pathology, we have developed cellular models to observe desmin behaviour in early stages of disease pathology, e.g., upon formation of cytoplasmic desmin aggregates, within an isogenic background. We cloned the wildtype and three mutant desmin cDNAs using a Tet-On Advanced® expression system in C2C12 cells. Mutations were selected based on positioning within desmin and capacity to form aggregates in transient experiments, as follows: DesS46Y (head domain; low aggregation), DesD399Y (central rod domain; high aggregation), and DesS460I (tail domain; moderate aggregation). Introduction of these proteins into a C2C12 background permitted us to compare between desmin variants as well as to determine the role of external stress on aggregation. Three different types of stress, likely encountered during muscle activity, were introduced to the cell models-thermal (heat shock), redox-associated (H2O2 and cadmium chloride), and mechanical (stretching) stresses-after which aggregation was measured. Cells containing variant DesD399Y were more sensitive to stress, leading to marked cytoplasmic perinuclear aggregations. We then evaluated the capacity of biochemical compounds to prevent this aggregation, applying dexamethasone (an inducer of heat shock proteins), fisetin or N-acetyl-L-cysteine (antioxidants) before stress induction. Interestingly, N-acetyl-L-cysteine pre-treatment prevented DesD399Y aggregation during most stress. N-acetyl-L-cysteine has recently been described as a promising antioxidant in myopathies linked to selenoprotein N or ryanodin receptor defects. Our findings indicate that this drug warrants further study in animal models to speed its potential development as a therapy for DesD399Y-linked desminopathies.

Diagnostic performance of computed tomography coronary angiography (from the Prospective National Multicenter Multivendor EVASCAN Study).

Computed tomographic coronary angiography (CTCA) has been proposed as a noninvasive test for significant coronary artery disease (CAD), but only limited data are available from prospective multicenter trials. The goal of this study was to establish the diagnostic accuracy of CTCA compared to coronary angiography (CA) in a large population of symptomatic patients with clinical indications for coronary imaging. This national, multicenter study was designed to prospectively evaluate stable patients able to undergo CTCA followed by conventional CA. Data from CTCA and CA were analyzed in a blinded fashion at central core laboratories. The main outcome was the evaluation of patient-, vessel-, and segment-based diagnostic performance of CTCA to detect or rule out significant CAD (≥50% luminal diameter reduction). Of 757 patients enrolled, 746 (mean age 61 ± 12 years, 71% men) were analyzed. They underwent CTCA followed by CA 1.7 ± 0.8 days later using a 64-detector scanner. The prevalence of significant CAD in native coronary vessels by CA was 54%. The rate of nonassessable segments by CTCA was 6%. In a patient-based analysis, sensitivity, specificity, positive and negative predictive values, and positive and negative likelihood ratios of CTCA were 91%, 50%, 68%, 83%, 1.82, and 0.18, respectively. The strongest predictors of false-negative results on CTCA were high estimated pretest probability of CAD (odds ratio [OR] 1.97, p <0.001), male gender (OR 1.5, p <0.002), diabetes (OR 1.5, p <0.0001), and age (OR 1.2, p <0.0001). In conclusion, in this large multicenter study, CTCA identified significant CAD with high sensitivity. However, in routine clinical practice, each patient should be individually evaluated, and the pretest probability of obstructive CAD should be taken into account when deciding which method, CTCA or CA, to use to diagnose its presence and severity.

First experience of percutaneous radio-frequency ablation for atrial flutter and atrial fibrillation in a patient with HeartMate II left ventricular assist device.

We report the first case of percutaneous radio-frequency (RF) ablation procedure in a patient implanted with a HeartMate II left ventricular assist device for refractory heart failure. This procedure was performed for poorly tolerated recurrent atrial arrhythmias. No harmful consequence happened during or after the procedure despite the potential electromagnetic interferences existing between the RF delivery and the functioning of the device.

Cardiac magnetic resonance demonstrates myocardial oedema in remote tissue early after reperfused myocardial infarction.

BACKGROUND: Cardiac magnetic resonance can detect myocardial oedema using myocardial transverse relaxation time (T2)-weighted sequences but quantitative data are lacking in patients evaluated early after acute myocardial infarction. AIM: To assess the spatial distribution of T2 in patients with recent acute myocardial infarction. METHODS: Twenty-four consecutive patients (mean age 60+/-11 years) with acute myocardial infarction (anterior, n=12; inferior, n=12) were evaluated prospectively. T2 was determined using a series of breath-hold T2-weighted segmented half-Fourier turbo-spin echo sequences. No-reflow was defined as the association of early hypoenhancement and delayed enhancement in an akinetic region after a bolus injection of DOTA-Gd (0.2 mmol/kg). RESULTS: No-reflow was present in 13 (54%) patients and absent in 11 (46%) patients. Mean T2 was increased in the infarct region (84.9+/-23.7 ms) compared with in the remote myocardium (62.8+/-10.3 ms, p=0.0001) and in control subjects (55.7+/-4.6 ms, p<0.0001), but also in the remote myocardium compared with control subjects (p<0.02). In patients with no-reflow, T2 was further increased within the infarcted subendocardium compared with in patients without no-reflow (97.9+/-24.8 ms vs 76.3+/-24.7 ms, p<0.03). Peak troponin correlated with T2 (r=0.47, p<0.02) and was higher in patients with no-reflow (297.9+/-249.7 microg/L) than in patients without no-reflow (42.4+/-43.1 microg/L, p=0.003). CONCLUSION: T2 was lengthened in both infarcted and remote myocardium and was influenced by the occurrence of no-reflow.

[Indications for MRI in coronary disease]. / Indications de l'IRM dans la maladie coronaire.

Magnetic resonance imaging (MRI) has become a useful, even essential, examination in recent years, for the exploration of patients with coronary disease. MRI has several different roles, even though it remains insufficiently requested because insufficiently available. Today it is the reference examination for assessing indicators of ventricular function (volume, ejection fraction, ventricular mass); their prognostic value and importance in determining treatment are well recognized. In the postinfarction period, MRI using late enhancement techniques allows a precise analysis of the extent of necrosis, in terms of segments and transmural involvement. MRI is indicated, especially preoperatively, in cases of ventricular remodeling and its consequences (functional impairment, aneurysms, parietal thrombus). MRI with pharmacological stress may also be used as a tool for detecting myocardial ischemia; in this case, perfusion or first-pass sequences should be used. On the other hand, cardiac MRI for morphologic exploration of the coronary network and measurement of stenosis is not yet routine.