Regulation and Release of Vasoactive Endoglin by Brain Endothelium in Response to Hypoxia/Reoxygenation in Stroke

In large vessel occlusion stroke, recanalization to restore cerebral perfusion is essential but not necessarily sufficient for a favorable outcome. Paradoxically, in some patients, reperfusion carries the risk of increased tissue damage and cerebral hemorrhage. Experimental and clinical data suggest that endothelial cells, representing the interface for detrimental platelet and leukocyte responses, likely play a crucial role in the phenomenon referred to as ischemia/reperfusion (I/R)-injury, but the mechanisms are unknown. We aimed to determine the role of endoglin in cerebral I/R-injury; endoglin is a membrane-bound protein abundantly expressed by endothelial cells that has previously been shown to be involved in the maintenance of vascular homeostasis. We investigated the expression of membranous endoglin (using Western blotting and RT-PCR) and the generation of soluble endoglin (using an enzyme-linked immunosorbent assay of cell culture supernatants) after hypoxia and subsequent reoxygenation in human non-immortalized brain endothelial cells. To validate these in vitro data, we additionally examined endoglin expression in an intraluminal monofilament model of permanent and transient middle cerebral artery occlusion in mice. Subsequently, the effects of recombinant human soluble endoglin were assessed by label-free impedance-based measurement of endothelial monolayer integrity (using the xCELLigence DP system) and immunocytochemistry. Endoglin expression is highly inducible by hypoxia in human brain endothelial monolayers in vitro, and subsequent reoxygenation induced its shedding. These findings were corroborated in mice during MCAO; an upregulation of endoglin was displayed in the infarcted hemispheres under occlusion, whereas endoglin expression was significantly diminished after transient MCAO, which is indicative of shedding. Of note is the finding that soluble endoglin induced an inflammatory phenotype in endothelial monolayers. The treatment of HBMEC with endoglin resulted in a decrease in transendothelial resistance and the downregulation of VE-cadherin. Our data establish a novel mechanism in which hypoxia triggers the initial endothelial upregulation of endoglin and subsequent reoxygenation triggers its release as a vasoactive mediator that, when rinsed into adjacent vascular beds after recanalization, can contribute to cerebral reperfusion injury.     

The P2X7R-NLRP3 and AIM2 Inflammasome Platforms Mark the Complexity/Severity of Viral or Metabolic Liver Damage

P2X7R-NLRP3 and AIM2 inflammasomes activate caspase-1 and the release of cytokines involved in viral-related liver disease. Little is known about their role in non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steato-hepatitis (NASH). We characterized the role of inflammasomes in NAFLD, NASH, and HCV. Gene expression and subcellular localization of P2X7R/P2X4R-NLRP3 and AIM2 inflammasome components were examined in histopathological preparations of 46 patients with biopsy-proven viral and metabolic liver disease using real-time PCR and immunofluorescence. P2X7R, P2X4R, and Caspase-1 are two- to five-fold more expressed in patients with NAFLD/NASH associated with chronic HCV infection than those with metabolic damage only (p ≤ 0.01 for all comparisons). The AIM2 inflammasome is 4.4 times more expressed in patients with chronic HCV infection, regardless of coexistent metabolic abnormalities (p = 0.0006). IL-2, a cytokine playing a pivotal role during chronic HCV infection, showed a similar expression in HCV and NASH patients (p = 0.77) but was virtually absent in NAFLD. The P2X7R-NLRP3 complex prevailed in infiltrating macrophages, while AIM2 was localized in Kupffer cells. Caspase-1 expression correlated with elastography-based liver fibrosis (r = 0.35, p = 0.02), whereas P2X7R, P2X4R, NRLP3, Caspase-1, and IL-2 expression correlated with circulating markers of disease severity. P2X7R and P2X4R play a major role in liver inflammation accompanying chronic HCV infection, especially when combined with metabolic damage, while AIM2 is specifically expressed in chronic viral hepatitis. We describe for the first time the hepatic expression of IL-2 in NASH, so far considered a peculiarity of HCV-related liver damage.     

Inference and uncertainty quantification of stochastic gene expression via synthetic models

Estimating uncertainty in model predictions is a central task in quantitative biology. Biological models at the single-cell level are intrinsically stochastic and nonlinear, creating formidable challenges for their statistical estimation which inevitably has to rely on approximations that trade accuracy for tractability. Despite intensive interest, a sweet spot in this trade-off has not been found yet. We propose a flexible procedure for uncertainty quantification in a wide class of reaction networks describing stochastic gene expression including those with feedback. The method is based on creating a tractable coarse-graining of the model that is learned from simulations, a synthetic model, to approximate the likelihood function. We demonstrate that synthetic models can substantially outperform state-of-the-art approaches on a number of non-trivial systems and datasets, yielding an accurate and computationally viable solution to uncertainty quantification in stochastic models of gene expression.     

Adipose Tissue Dysfunction and Obesity-Related Male Hypogonadism

Obesity is a chronic illness associated with several metabolic derangements and comorbidities (i.e., insulin resistance, leptin resistance, diabetes, etc.) and often leads to impaired testicular function and male subfertility. Several mechanisms may indeed negatively affect the hypothalamic–pituitary–gonadal health, such as higher testosterone conversion to estradiol by aromatase activity in the adipose tissue, increased ROS production, and the release of several endocrine molecules affecting the hypothalamus–pituitary–testis axis by both direct and indirect mechanisms. In addition, androgen deficiency could further accelerate adipose tissue expansion and therefore exacerbate obesity, which in turn enhances hypogonadism, thus inducing a vicious cycle. Based on these considerations, we propose an overview on the relationship of adipose tissue dysfunction and male hypogonadism, highlighting the main biological pathways involved and the current therapeutic options to counteract this condition.     

A Novel Morphological Parameter Predicting Fibrotic Evolution in Myeloproliferative Neoplasms: New Evidence and Molecular Insights

Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) represent a group of hematological disorders that are traditionally considered as indistinct slow progressing conditions; still, a subset of cases shows a rapid evolution towards myelofibrotic bone marrow failure. Specific abnormalities in the megakaryocyte lineage seem to play a central role in this evolution, especially in the bone marrow fibrosis but also in the induction of myeloproliferation. In this review, we analyze the current knowledge of prognostic factors of MPNs related to their evolution to myelofibrotic bone marrow failure. Moreover, we focused the role of the megakaryocytic lineage in the various stages of MPNs, with updated examples of MPNs in vitro and in vivo models and new therapeutic implications.     

Morphological and Biological Evaluations of Human Periodontal Ligament Fibroblasts in Contact with Different Bovine Bone Grafts Treated with Low-Temperature Deproteinisation Protocol

Several types of deproteinised bovine bone mineral (DBBM) are available on the market, and each one is obtained with a thermic and chemical process that can differ, achieving different results. Currently, several protocols using low temperature are suggested to reduce the possible particle crystallisation during the production process. This study aimed to evaluate the biomorphological reaction of periodontal fibroblast cultures in contact with different DBBM particles treated with a low-temperature protocol (Thermagen^®) and without exposure to sodium hydroxide (NaOH). Morphological evaluation was performed using light, confocal laser, and scanning electron microscopy, and the biological reaction in terms of proliferation was performed using an XTT proliferation assay at 24 h (T1), 72 h (T2), and 7 days (T3). The morphological analysis highlighted how the presence of the materials stimulated a change in the morphology of the cells into a polygonal shape, surface reactions with the thickening of the membrane, and expression of actin. In particular, the morphological changes were appreciable from T1, with a progressive increase in the considered morphological characteristics at T2 and T3 follow-ups. The proliferation assay showed a statistical significance between the different experimental materials and the negative control in T2 and T3 follow-ups. The post hoc analysis did not reveal any differences between the materials. In conclusion, the grafts obtained with the low-temperature extractions protocol and not exposed to NaOH solution showed positive morphological reactions with no differences in the sizes of particles.     

Measurement involving human perception and interpretation

The measurement of features related to human perception and interpretation is discussed. After a brief review of the historical framework, the state of the art is presented on the basis of the authors’ experience in the field and their participation in related European projects, with development of experiments, and co-ordination activities. Future research needs and challenges are then addressed.     

An optimal decision-making approach for the management of radiotherapy patients

In this paper, novel integer programming formulations are developed for solving the optimal scheduling of patients waiting for radiotherapy treatment. In this specific clinical domain, the suitable management and control of a patients’ waiting list strongly affect both the quality of the therapeutical outcome, in terms of effectiveness, and the cost-saving use of the overall therapeutical resources, in terms of efficiency. The proposed models allow the best scheduling strategy to be devised by taking into account the quality of the health care service offered to the patient as well as the status and the preferences of the patient. The computational experiments, carried out on realistic scenarios and considering real data, are very promising and show the efficiency and robustness of the proposed models to address the problem under consideration.     

Finite element model updating and structural damage identification using OMAX data

The main limitations in the finite element (FE) model updating technique lie in the ability of the FE model to represent the true behavior of the structure (modelling problem), and in the ability to identify enough modal parameters with sufficient accuracy, especially for large structures that are tested in operational conditions (identification problem). In this paper, the identification problem is solved with an OMAX approach, where an artificial force is used in operational conditions and a structural model is identified that takes both the forced and the ambient excitation into account. From an extensive case study on a real three-span bridge, it is observed that, while updating the FE model using the experimental output-only data yields a good fit, discrepancies show up when the more extensive set of OMAX data is used for validation, or even for updating. It can be concluded that an OMAX approach not only increases the well-posedness of the updating problem, it also allows to detect potential inaccuracies in the FE model.