New transcriptional-based insights into the pathogenesis of desmoplastic small round cell tumors (DSRCTs).

To gain new insights into desmoplastic small round cell tumors (DSRCTs) by means of gene expression profiling (GEP). Formalin-fixed, paraffin-embedded surgical specimens obtained from seven pretreated DSRCT patients were interrogated using GEP complemented by immunohistochemistry, a cancer stem cell array, and miRNA in situ hybridisation, including the combined chimera modules miRNA-200/ZEB1 and miRNA-34/SLUG. The chimera modules divided the cases into three classes that respectively recapitulated the traits of mesenchymal epithelial reverse transition (MErT), epithelial mesenchymal transition (EMT), and hybrid/partial EMT. This indicates a close correlation between the reprogramming governed by EMT regulators and DSRCT biology, which was further confirmed by miRNA-21 and is consistent with the broad morphological spectrum of DSRCTs. Starting from the miRNA-200/ZEB1 axis, we also found that DSRCTs carry a signature of immunological ignorance that is not responsive to PD--L1 blockade. Evidence that the up-regulation of miRNA-200 and E-cadherin, and quite a high level of miRNA-21 expression segregate with the MErT supports the idea that, in addition to the hybrid/partial state, MErT is also enriched in stemness: the androgen-positive cases, whose stemness traits were confirmed by stem cell arrays, all fell into these two classes. Our findings also confirmed that tumoral cell PDGFRA expression correlates with desmoplasia, and demonstrated the co-expression of PDGFRA and ISLR/Meflin, another marker of pluripotency. Despite the limited number of cases, these findings provide unexpectedly relevant information concerning the pathogenesis of DSRCTs, and prove the validity of miRNA-based chimera circuit modelling in the clinico-pathological setting

Models and methods to characterise levonorgestrel release from intradermally administered contraceptives

Microneedle (MN)-based technologies have been proposed as a means to facilitate minimally invasive sustained delivery of long-acting hormonal contraceptives into the skin. Intradermal administration is a new route of delivery for these contraceptives and therefore no established laboratory methods or experimental models are available to predict dermal drug release and pharmacokinetics from candidate MN formulations. This study evaluates an in vitro release (IVR) medium and a medium supplemented with ex vivo human skin homogenate (SH) as potential laboratory models to investigate the dermal release characteristics of one such hormonal contraceptive that is being tested for MN delivery, levonorgestrel (LNG), and provides details of an accompanying novel two-step liquid–liquid drug extraction procedure and sensitive reversed-phase HPLC–UV assay. The extraction efficiency of LNG was 91.7 ± 3.06% from IVR medium and 84.6 ± 1.6% from the medium supplemented with SH. The HPLC–UV methodology had a limit of quantification of 0.005 µg/mL and linearity between 0.005 and 25 µg/mL. Extraction and detection methods for LNG were exemplified in both models using the well-characterised, commercially available sustained-release implant (Jadelle®). Sustained LNG release from the implant was detected in both media over 28 days. This study reports for the first time the use of biologically relevant release models and a rapid, reliable and sensitive methodology to determine release characteristics of LNG from intradermally administered long-acting drug delivery systems

A Novel Form of Chondrocyte Stress is Triggered by a COMP Mutation Causing Pseudoachondroplasia

Pseudoachondroplasia (PSACH) results from mutations in cartilage oligomeric matrix protein (COMP) and the p.D469del mutation within the type III repeats of COMP accounts for approximately 30% of PSACH. To determine disease mechanisms of PSACH in vivo, we introduced the Comp D469del mutation into the mouse genome. Mutant animals were normal at birth but grew slower than their wild-type littermates and developed short-limb dwarfism. In the growth plates of mutant mice chondrocyte columns were reduced in number and poorly organized, while mutant COMP was retained within the endoplasmic reticulum (ER) of cells. Chondrocyte proliferation was reduced and apoptosis was both increased and spatially dysregulated. Previous studies on COMP mutations have shown mutant COMP is co-localized with chaperone proteins, and we have reported an unfolded protein response (UPR) in mouse models of PSACH-MED (multiple epiphyseal dysplasia) harboring mutations in Comp (T585M) and Matn3, Comp etc (V194D). However, we found no evidence of UPR in this mouse model of PSACH. In contrast, microarray analysis identified expression changes in groups of genes implicated in oxidative stress, cell cycle regulation, and apoptosis, which is consistent with the chondrocyte pathology. Overall, these data suggest that a novel form of chondrocyte stress triggered by the expression of mutant COMP is central to the pathogenesis of PSACH. Hum Mutat 33:218–231, 2012. © 2011 Wiley Periodicals, Inc

A diastrophic dysplasia sulfate transporter (SLC26A2) mutant mouse: morphological and biochemical characterization of the resulting chondrodysplasia phenotype.

Mutations in the diastrophic dysplasia sulfate transporter (DTDST or SLC26A2) cause a family of recessively inherited chondrodysplasias including, in order of decreasing severity, achondrogenesis 1B, atelosteogenesis 2, diastrophic dysplasia (DTD) and recessive multiple epiphyseal dysplasia. The gene encodes a widely distributed sulfate/chloride antiporter of the cell membrane whose function is crucial for the uptake of inorganic sulfate, which is needed for proteoglycan sulfation. To provide new insights in the pathogenetic mechanisms leading to skeletal and connective tissue dysplasia and to obtain an in vivo model for therapeutic approaches to DTD, we generated a Dtdst knock-in mouse with a partial loss of function of the sulfate transporter. In addition, the intronic neomycine cassette in the mutant allele contributed to the hypomorphic phenotype by inducing abnormal splicing. Homozygous mutant mice were characterized by growth retardation, skeletal dysplasia and joint contractures, thereby recapitulating essential aspects of the DTD phenotype in man. The skeletal phenotype included reduced toluidine blue staining of cartilage, chondrocytes of irregular size, delay in the formation of the secondary ossification center and osteoporosis of long bones. Impaired sulfate uptake was demonstrated in chondrocytes, osteoblasts and fibroblasts. In spite of the generalized nature of the sulfate uptake defect, significant proteoglycan undersulfation was detected only in cartilage. Chondrocyte proliferation and apoptosis studies suggested that reduced proliferation and/or lack of terminal chondrocyte differentiation might contribute to reduced bone growth. The similarity with human DTD makes this mouse strain a useful model to explore pathogenetic and therapeutic aspects of DTDST-related disorders

How Covid-19 changed the epidemiology of febrile urinary tract infections in children in the emergency department during the first outbreak

Background: The first Covid-19 pandemic affected the epidemiology of several diseases. A general reduction in the emergency department (ED) accesses was observed during this period, both in adult and pediatric contexts. Methods: This retrospective study was conducted on the behalf of the Italian Society of Pediatric Nephrology (SINePe) in 17 Italian pediatric EDs in March and April 2020, comparing them with data from the same periods in 2018 and 2019. The total number of pediatric (age 0–18 years) ED visits, the number of febrile urinary tract infection (UTI) diagnoses, and clinical and laboratory parameters were retrospectively collected. Results: The total number of febrile UTI diagnoses was 339 (73 in 2020, 140 in 2019, and 126 in 2018). During the first Covid-19 pandemic, the total number of ED visits decreased by 75.1%, the total number of febrile UTI diagnoses by 45.1%, with an increase in the UTI diagnosis rate (+ 121.7%). The data collected revealed an increased rate of patients with two or more days of fever before admission (p = 0.02), a significant increase in hospitalization rate (+ 17.5%, p = 0.008) and also in values of C reactive protein (CRP) (p = 0.006). In 2020, intravenous antibiotics use was significantly higher than in 2018 and 2019 (+ 15%, p = 0.025). Urine cultures showed higher Pseudomonas aeruginosa and Enterococcus faecalis percentages and lower rates of Escherichia coli (p = 0.02). Conclusions: The first wave of the Covid-19 pandemic had an essential impact on managing febrile UTIs in the ED, causing an absolute reduction of cases referring to the ED but with higher clinical severity. Children with febrile UTI were more severely ill than the previous two years, probably due to delayed access caused by the fear of potential hospital-acquired Sars-Cov-2 infection. The possible increase in consequent kidney scarring in this population should be considered

Causes and Consequences of miR-150-5p Dysregulation in Myasthenia Gravis

Autoimmune Myasthenia gravis (MG) is a chronic neuromuscular disease mainly due to antibodies against the acetylcholine receptor (AChR) at the neuromuscular junction that induce invalidating muscle weaknesses. In early-onset MG, the thymus is the effector organ and is often characterized by B-cell infiltrations leading to ectopic germinal center (GC) development. The microRNA miR-150-5p has been previously characterized as a biomarker in MG due to its increase in the serum of patients and its decrease after thymectomy, correlated with an improvement of symptoms. Here, we investigated the causes and consequences of the miR-150 increase in the serum of early-onset MG patients. We observed that miR-150 expression was upregulated in MG thymuses in correlation with the presence of thymic B cells and showed by in situ hybridization experiments, that miR-150 was mainly expressed by cells of the mantle zone of GCs. However, we did not observe any correlation between the degree of thymic hyperplasia and the serum levels in MG patients. In parallel, we also investigated the expression of miR-150 in peripheral blood mononuclear cells (PBMCs) from MG patients. We observed that miR-150 was down-regulated, especially in CD4+ T cells compared to controls. These results suggest that the increased serum levels of miR-150 could result from a release from activated peripheral CD4+ T cells. Next, we demonstrated that the in vitro treatment of PBMCs with miR-150 or antimiR-150 oligonucleotides, respectively, decreased or increased the expression of one of its major target gene: the proto-oncogene MYB, a well-known actor of hematopoiesis. These results revealed that increased serum levels of miR-150 in MG patients could have a functional effect on PBMCs. We also showed that antimiR-150 caused increased cellular death of CD4+ and CD8+ T cells, along with the overexpression of pro-apoptotic genes targeted by miR-150 suggesting that miR-150 controlled the survival of these cells. Altogether, these results showed that miR-150 could play a role in MG both at the thymic level and in periphery by modulating the expression of target genes and peripheral cell survival

Tumor-derived microRNAs induce myeloid suppressor cells and predict immunotherapy resistance in melanoma

The accrual of myeloid-derived suppressor cells (MDSCs) represents a major obstacle to effective immunotherapy in cancer patients, but the mechanisms underlying this process in the human setting remain elusive. Here, we describe a set of microRNAs (miR-146a, miR-155, miR-125b, miR-100, let-7e, miR-125a, miR-146b, miR-99b) that are associated with MDSCs and with resistance to treatment with immune checkpoint inhibitors in melanoma patients. The miRs were identified by transcriptional analyses as being responsible for the conversion of monocytes into MDSCs (CD14+HLA-DRneg cells) mediated by melanoma extracellular vesicles (EVs) and were shown to recreate MDSC features upon transfection. In melanoma patients, these miRs are increased in circulating CD14+ monocytes, plasma and tumor samples, where they correlate with the myeloid cell infiltrate. In plasma, their baseline level clusters with the clinical efficacy of CTLA-4 or PD-1 blockade. Hence, MDSC-related miRs represent an indicator of MDSC activity in cancer patients and a potential blood marker of a poor immunotherapy outcome

Increased classical endoplasmic reticulum stress is sufficient to reduce chondrocyte proliferation rate in the growth plate and decrease bone growth

Copyright: © 2015 Kung et al. Mutations in genes encoding cartilage oligomeric matrix protein and matrilin-3 cause a spectrum of chondrodysplasias called multiple epiphyseal dysplasia (MED) and pseudoachondroplasia (PSACH). The majority of these diseases feature classical endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) as a result of misfolding of the mutant protein. However, the importance and the pathological contribution of ER stress in the disease pathogenesis are unknown. The aim of this study was to investigate the generic role of ER stress and the UPR in the pathogenesis of these diseases. A transgenic mouse line (ColIITgcog) was generated using the collagen II promoter to drive expression of an ER stress-inducing protein (Tgcog) in chondrocytes. The skeletal and histological phenotypes of these ColIITgcog mice were characterised. The expression and intracellular retention of Tgcog induced ER stress and activated the UPR as characterised by increased BiP expression, phosphorylation of eIF2á and spliced Xbp1. ColIITgcog mice exhibited decreased long bone growth and decreased chondrocyte proliferation rate. However, there was no disruption of chondrocyte morphology or growth plate architecture and perturbations in apoptosis were not apparent. Our data demonstrate that the targeted induction of ER stress in chondrocytes was sufficient to reduce the rate of bone growth, a key clinical feature associated with MED and PSACH, in the absence of any growth plate dysplasia. This study establishes that classical ER stress is a pathogenic factor that contributes to the disease mechanism of MED and PSACH. However, not all the pathological features of MED and PSACH were recapitulated, suggesting that a combination of intra- and extra-cellular factors are likely to be responsible for the disease pathology as a whole

An Overview of Warships Damage Data from 1967 to 2013

In the last decade, in the field of merchant ships, a long harmonization process has taken place at IMO, resulting in the enforcement of the so called probabilistic SOLAS2009 for the residual buoyancy and stability assessment of a ship in a damaged condition. In the warships design process, the probabilistic methodology might represent a consistent approach to complement the fundamental overall ship survivability assessment. Nevertheless among the most critical issues, while discussing the possible implementation of this innovative approach, are the lack of a damage database and the significantly different threat typology. In this perspective, significant damage cases in the field of warships are investigated and critically analyzed. The observed time period will regard the period from 1967 (sinking of the Eilat) to 2013