Polyploidy and hybridization in the Mediterranean: unravelling the evolutionary history of Centaurium (Gentianaceae).

BACKGROUND AND AIMS: Polyploidy is considered one of the main mechanisms of plant evolution and speciation. In the Mediterranean basin, polyploidy has contributed to making this region a biodiversity hotspot, along with its geological and climatic history and other ecological and biogeographic factors. The Mediterranean genus Centaurium Hill (Gentianaceae) comprises ca. 25 species, of which 60% are polyploids, including tetraploids and hexaploids. To date, the evolutionary history of centauries has been studied using Sanger sequencing phylogenies, which have been insufficient to fully understand the phylogenetic relationships in this lineage. The goal of this study is to gain a better understanding of the evolutionary history of Centaurium by exploring the mechanisms that have driven its diversification, specifically hybridization and polyploidy. We aim at identifying the parentage of hybrid species, at the species or clade level, as well as assessing whether morphological traits are associated with particular ploidy levels. METHODS: We sequenced RADseq markers from 42 samples of 28 Centaurium taxa, and performed phylogenomic analyses using maximum likelihood, summary coalescent SVDquartets and NeighborNet approaches. To identify hybrid taxa, we used Phylonetworks and the fastStructure algorithm. To infer the putative parental species of the allopolyploids, we employed genomic analyses (SNIPloid). The association between different traits and particular ploidy levels was explored with NMDS. KEY RESULTS: Our phylogenetic analyses confirmed the long-suspected occurrence of recurrent hybridization. The allopolyploid origin of the tetraploid C. serpentinicola and the hexaploids C. mairei, C. malzacianum and C. centaurioides were also confirmed, unlike that of C. discolor. We inferred additional signatures of hybridization events within the genus and identified morphological traits differentially distributed in different ploidy levels. CONCLUSIONS: This study highlights the important role that hybridization has played in the evolution of a Mediterranean genus such as Centaurium, leading to a polyploid complex, which facilitated its diversification and may exemplify that of other Mediterranean groups.

Molecular Mechanisms Driven by MT4-MMP in Cancer Progression.

MT4-MMP (or MMP-17) belongs to the membrane-type matrix metalloproteinases (MT-MMPs), a distinct subset of the MMP family that is anchored to the cell surface, in this case by a glycosylphosphatidylinositol (GPI) motif. Its expression in a variety of cancers is well documented. However, the molecular mechanisms by which MT4-MMP contributes to tumor development need further investigation. In this review, we aim to summarize the contribution of MT4-MMP in tumorigenesis, focusing on the molecular mechanisms triggered by the enzyme in tumor cell migration, invasiveness, and proliferation, in the tumor vasculature and microenvironment, as well as during metastasis. In particular, we highlight the putative substrates processed and signaling cascades activated by MT4-MMP that may underlie these malignancy processes and compare this with what is known about its role during embryonic development. Finally, MT4-MMP is a relevant biomarker of malignancy that can be used for monitoring cancer progression in patients as well as a potential target for future therapeutic drug development.

Acute Stress Regulates Sex-Related Molecular Responses in the Human Jejunal Mucosa: Implications for Irritable Bowel Syndrome.

Irritable bowel syndrome (IBS) is a prevalent gastrointestinal disorder linked to intestinal barrier dysfunction and life stress. We have previously reported that female sex per se determines an increased susceptibility to intestinal barrier dysfunction after cold pain stress (CPS). We aimed to identify sex-related molecular differences in response to CPS in healthy subjects to understand the origin of sex bias predominance in IBS. In 13 healthy males and 21 females, two consecutive jejunal biopsies were obtained using Watson's capsule, at baseline, and ninety minutes after CPS. Total mucosal RNA and protein were isolated from jejunal biopsies. Expression of genes related to epithelial barrier (CLDN1, CLDN2, OCLN, ZO-1, and ZO-3), mast cell (MC) activation (TPSAB1, SERPINA1), and the glucocorticoid receptor (NR3C1) were analyzed using RT-qPCR. NR3C1, ZO-1 and OCLN protein expression were evaluated through immunohistochemistry and western blot, and mucosal inflammation through MC, lymphocyte, and eosinophil numbering. Autonomic, hormonal, and psychological responses to CPS were monitored. We found an increase in jejunal MCs, a reduced CLDN1 and OCLN expression, and an increased CLDN2 and SERPINA1 expression 90 min after CPS. We also found a significant decrease in ZO-1, OCLN, and NR3C1 gene expression, and a decrease in OCLN protein expression only in females, when compared to males. CPS induced a significant increase in blood pressure, plasma cortisol and ACTH, and subjective stress perception in all participants. Specific and independent sex-related molecular responses in epithelial barrier regulation are unraveled by acute stress in the jejunum of healthy subjects and may partially explain female predominance in IBS.

Mucosal Plasma Cell Activation and Proximity to Nerve Fibres Are Associated with Glycocalyx Reduction in Diarrhoea-Predominant Irritable Bowel Syndrome: Jejunal Barrier Alterations Underlying Clinical Manifestations.

Irritable bowel syndrome (IBS) is a disorder of brain-gut interaction characterised by abdominal pain and changes in bowel habits. In the diarrhoea subtype (IBS-D), altered epithelial barrier and mucosal immune activation are associated with clinical manifestations. We aimed to further evaluate plasma cells and epithelial integrity to gain understanding of IBS-D pathophysiology. One mucosal jejunal biopsy and one stool sample were obtained from healthy controls and IBS-D patients. Gastrointestinal symptoms, stress, and depression scores were recorded. In the jejunal mucosa, RNAseq and gene set enrichment analyses were performed. A morphometric analysis by electron microscopy quantified plasma cell activation and proximity to enteric nerves and glycocalyx thickness. Immunoglobulins concentration was assessed in the stool. IBS-D patients showed differential expression of humoral pathways compared to controls. Activation and proximity of plasma cells to nerves and IgG concentration were also higher in IBS-D. Glycocalyx thickness was lower in IBS-D compared to controls, and this reduction correlated with plasma cell activation, proximity to nerves, and clinical symptoms. These results support humoral activity and loss of epithelial integrity as important contributors to gut dysfunction and clinical manifestations in IBS-D. Additional studies are needed to identify the triggers of these alterations to better define IBS-D pathophysiology.

Emerging roles of MT-MMPs in embryonic development.

Membrane-type matrix metalloproteinases (MT-MMPs) are cell membrane-tethered proteinases that belong to the family of the MMPs. Apart from their roles in degradation of the extracellular milieu, MT-MMPs are able to activate through proteolytic processing at the cell surface distinct molecules such as receptors, growth factors, cytokines, adhesion molecules, and other pericellular proteins. Although most of the information regarding these enzymes comes from cancer studies, our current knowledge about their contribution in distinct developmental processes occurring in the embryo is limited. In this review, we want to summarize the involvement of MT-MMPs in distinct processes during embryonic morphogenesis, including cell migration and proliferation, epithelial-mesenchymal transition, cell polarity and branching, axon growth and navigation, synapse formation, and angiogenesis. We also considered information about MT-MMP functions from studies assessed in pathological conditions and compared these data with those relevant for embryonic development.

Isolation and characterization of microsatellites markers in Centaurium grandiflorum ssp. boissieri.

BACKGROUND: Estimating outcrossing/selfing rates and characterizing genetic diversity with microsatellite markers are crucial to understanding the evolution of plant mating systems. METHODS AND RESULTS: We developed, optimized and characterized eight new primer pairs for Centaurium grandiflorum ssp. boissieri and transferred them to three subspecies of Centaurium quadrifolium. Two SSR loci were transferred from Sabatia campestris to the four Centaurium taxa. Polymorphisms, He, Ho and H-W deviations were estimated in two populations of C. grandiflorum ssp. boissieri and in seven individuals each of C. quadrifolium ssp. barrelieri, C. quadrifolium ssp. parviflorum and C. quadrifolium ssp. quadrifolium. A total of 80 individuals was used in these experiments. The number of polymorphic loci varied among species from one to ten. A total of 127 alleles was scored. The average number of alleles per locus was 12.7. He was higher than Ho in all sampled populations. Hardy-Weinberg equilibrium was found for some loci in different species. CONCLUSIONS: This is the first report of microsatellites successfully amplified in the whole Centaurium genus. They will be valuable for estimating mating system parameters and genetic diversity and exploring their relationships with the wide variation in flower morphology in the genus, especially anther-stigma separation.

Dynamic Expression of Membrane Type 1-Matrix Metalloproteinase (Mt1-mmp/Mmp14) in the Mouse Embryo.

MT1-MMP/MMP14 belongs to a subgroup of the matrix metalloproteinases family that presents a transmembrane domain, with a cytosolic tail and the catalytic site exposed to the extracellular space. Deficient mice for this enzyme result in early postnatal death and display severe defects in skeletal, muscle and lung development. By using a transgenic line expressing the LacZ reporter under the control of the endogenous Mt1-mmp promoter, we reported a dynamic spatiotemporal expression pattern for Mt1-mmp from early embryonic to perinatal stages during cardiovascular development and brain formation. Thus, Mt1-mmp shows expression in the endocardium of the heart and the truncus arteriosus by E8.5, and is also strongly detected during vascular system development as well as in endothelial cells. In the brain, LacZ reporter expression was detected in the olfactory bulb, the rostral cerebral cortex and the caudal mesencephalic tectum. LacZ-positive cells were observed in neural progenitors of the spinal cord, neural crest cells and the intersomitic region. In the limb, Mt1-mmp expression was restricted to blood vessels, cartilage primordium and muscles. Detection of the enzyme was confirmed by Western blot and immunohistochemical analysis. We suggest novel functions for this metalloproteinase in angiogenesis, endocardial formation and vascularization during organogenesis. Moreover, Mt1-mmp expression revealed that the enzyme may contribute to heart, muscle and brain throughout development.

Polyploidy Expands the Range of Centaurium (Gentianaceae).

The Mediterranean region is one of the most important worldwide hotspots in terms of number of species and endemism, and multiple hypotheses have been proposed to explain how diversification occurred in this area. The contribution of different traits to the diversification process has been evaluated in different groups of plants. In the case of Centaurium (Gentianaceae), a genus with a center of diversity placed in the Mediterranean region, polyploidy seems to have been an important driver of diversification as more than half of species are polyploids. Moreover, ploidy levels are strongly geographically structured across the range of the genus, with tetraploids distributed towards more temperate areas in the north and hexaploids in more arid areas towards the south. We hypothesize that the diversification processes and biodiversity patterns in Centaurium are explained by the coupled formation of polyploid lineages and the colonization of different areas. A MCC tree from BEAST2 based on three nuclear DNA regions of a total of 26 taxa (full sampling, of 18 species and 8 subspecies) was used to perform ancestral area reconstruction analysis in "BioGeoBEARS." Chromosome evolution was analyzed in chromEvol and diversification in BAMM to estimate diversification rates. Our results suggest that two major clades diverged early from the common ancestor, one most likely in the western Mediterranean and the other in a widespread area including west and central Asia (but with high uncertainty in the exact composition of this widespread area). Most ancestral lineages in the western clade remained in or around the western Mediterranean, and dispersal to other areas (mainly northward and eastward), occurred at the tips. Contrarily, most ancestral lineages in the widespread clade had larger ancestral areas. Polyploidization events in the western clade occurred at the tips of the phylogeny (with one exception of a polyploidization event in a very shallow node) and were mainly tetraploid, while polyploidization events occurred in the widespread clade were at the tips and in an ancestral node of the phylogeny, and were mainly hexaploid. We show how ancestral diploid lineages remained in the area of origin, whereas recent and ancestral polyploidization could have facilitated colonization and establishment in other areas.

Overexpression of corticotropin-releasing factor in intestinal mucosal eosinophils is associated with clinical severity in Diarrhea-Predominant Irritable Bowel Syndrome.

Corticotropin-releasing factor (CRF) has been identified in intestinal mucosal eosinophils and associated with psychological stress and gut dysfunction. Irritable bowel syndrome (IBS) is commonly characterized by altered intestinal motility, immune activation, and increased gut barrier permeability along with heightened susceptibility to psychosocial stress. Despite intensive research, the role of mucosal eosinophils in stress-associated gut dysfunction remains uncertain. In this study, we evaluated eosinophil activation profile and CRF content in the jejunal mucosa of diarrhea-predominant IBS (IBS-D) and healthy controls (HC) by gene/protein expression and transmission electron microscopy. We also explored the association between intestinal eosinophil CRF and chronic stress, and the potential mechanisms underlying the stress response by assessing eosinophil response to neuropeptides. We found that mucosal eosinophils displayed higher degranulation profile in IBS-D as compared to HC, with increased content of CRF in the cytoplasmic granules, which significantly correlated with IBS clinical severity, life stress background and depression. Eosinophils responded to substance P and carbachol by increasing secretory activity and CRF synthesis and release, without promoting pro-inflammatory activity, a profile similar to that found in mucosal eosinophils from IBS-D. Collectively, our results suggest that intestinal mucosal eosinophils are potential contributors to stress-mediated gut dysfunction through CRF production and release.

Peripheral Corticotropin-Releasing Factor Triggers Jejunal Mast Cell Activation and Abdominal Pain in Patients With Diarrhea-Predominant Irritable Bowel Syndrome.

INTRODUCTION: To determine the effect of peripheral CRF on intestinal barrier function in diarrhea-predominant IBS (IBS-D). Irritable bowel syndrome (IBS) pathophysiology has been linked to life stress, epithelial barrier dysfunction, and mast cell activation. Corticotropin-releasing factor (CRF) is a major mediator of stress responses in the gastrointestinal tract, yet its role on IBS mucosal function remains largely unknown. METHODS: Intestinal response to sequential i.v. 5-mL saline solution (placebo) and CRF (100 µg) was evaluated in 21 IBS-D and 17 healthy subjects (HSs). A 20-cm jejunal segment was perfused with an isosmotic solution and effluents collected at baseline, 30 minutes after placebo, and 60 minutes after CRF. We measured water flux, albumin output, tryptase release, stress hormones, cardiovascular and psychological responses, and abdominal pain. A jejunal biopsy was obtained for CRF receptor expression assessment. RESULTS: Water flux did not change after placebo in IBS-D and HS but significantly increased after CRF in IBS-D (P = 0.007). Basal luminal output of albumin was higher in IBS-D and increased further after CRF in IBS-D (P = 0.042). Basal jejunal tryptase release was higher in IBS-D, and CRF significantly increased it in both groups (P = 0.004), the response being higher in IBS-D than in HS (P = 0.0023). Abdominal pain worsened only in IBS-D after CRF and correlated with jejunal tryptase release, water flux, and albumin output. IBS-D displayed jejunal up-regulation of CRF2 and down-regulation of CRF1 compared with HS. DISCUSSION: Stress via CRF-driven mast cell activation seems to be relevant in the pathophysiology of IBS-D.

What are we discarding during the life cycle of a building? Case studies of social housing in Andalusia, Spain.

The paper evaluates for the first time the embodied impact in CDW during the buildings life cycle by means of the bill of quantities of construction projects. The main objective is to be able to predict the future CDW to be generated by a project in the design stage, by means of the bill of quantities of the urbanization, construction, renovation, rehabilitation and demolition projects. The tools already in place for cost control can be used as an instrument for the introduction of sustainability considerations in construction projects. The methodology proposes a connection between the different stages of a building's life cycle, more precisely its budget. The latter is linked to other future budgets for building renovations or retrofitting projects. The result shows that urbanization and demolition generate 90% of CDW, the former is caused by earthworks and the latter is due to the elimination of all building materials. The building is removed 1.3 times, in terms of material weight, energy and water. Finally, traditional models for economic control and waste management in construction projects can be the vector which introduce environmental assessment through the building life cycle.

Mucosal RNA and protein expression as the next frontier in IBS: abnormal function despite morphologically intact small intestinal mucosa.

Irritable bowel syndrome (IBS) is one of the commonest gastrointestinal disorders. Although long-time considered a pure functional disorder, intense research in past years has rendered a very complex and varied array of observations indicating the presence of structural and molecular abnormalities underlying characteristic motor and sensitive changes and clinical manifestations. Analysis of gene and protein expression in the intestinal mucosa has shed light on the molecular mechanisms implicated in IBS physiopathology. This analysis uncovers constitutive and inductive genetic and epigenetic marks in the small and large intestine that highlight the role of epithelial barrier, immune activation, and mucosal processing of foods and toxins and several new molecular pathways in the origin of IBS. The incorporation of innovative high-throughput techniques into IBS research is beginning to provide new insights into highly structured and interconnected molecular mechanisms modulating gene and protein expression at tissue level. Integration and correlation of these molecular mechanisms with clinical and environmental data applying systems biology/medicine and data mining tools emerge as crucial steps that will allow us to get meaningful and more definitive comprehension of IBS-detailed development and show the real mechanisms and causality of the disease and the way to identify more specific diagnostic biomarkers and effective treatments.

Tracing Gene Expression Through Detection of ß-galactosidase Activity in Whole Mouse Embryos.

The Escherichia coli LacZ gene, encoding ß-galactosidase, is largely used as a reporter for gene expression and as a tracer in cell lineage studies. The classical histochemical reaction is based on the hydrolysis of the substrate X-gal in combination with ferric and ferrous ions, which produces an insoluble blue precipitate that is easy to visualize. Therefore, ß-galactosidase activity serves as a marker for the expression pattern of the gene of interest as the development proceeds. Here we describe the standard protocol for the detection of ß-galactosidase activity in early whole mouse embryos and the subsequent method for paraffin sectioning and counterstaining. Additionally, a procedure for clarifying whole embryos is provided to better visualize X-gal staining in deeper regions of the embryo. Consistent results are obtained by performing this procedure, although optimization of reaction conditions is needed to minimize background activity. Limitations in the assay should be also considered, particularly regarding the size of the embryo in whole mount staining. Our protocol provides a sensitive and a reliable method for ß-galactosidase detection during the mouse development that can be further applied to the cryostat sections as well as whole organs. Thus, the dynamic gene expression patterns throughout development can be easily analyzed by using this protocol in whole embryos, but also detailed expression at the cellular level can be assessed after paraffin sectioning.

Late G2 vagina toxicity in post-operative endometrial carcinoma is associated with a 68 Gy dose equivalent to 2 Gy per fraction(α/ß=3Gy) at 2 cm3 of vagina.

PURPOSE: To evaluate if the dose equivalent to 2 Gy per fraction (EQD2)(α/ß=3Gy) at 0.1 cm3, 1 cm3, and 2 cm3 of vagina in vaginal-cuff-brachytherapy (VBT) (high-dose-rate [HDR] 192Ir-source) ± external-beam-irradiation (EBRT) is associated with toxicity in post-operative endometrial carcinoma (P-EC). MATERIAL AND METHODS: From June 2014 till November 2015, 67 consecutive P-EC patients underwent VBT ± EBRT; 44 patients received EBRT (median, 45 Gy; range, 44-50.4) + VBT (7 Gy), and 23 exclusive-VBT (6 Gy x 3 fractions). The upper 2.5 cm of vagina was delineated on computed tomography (CT). The active-length source was 2.5 cm, and the brachytherapy dose was prescribed at 5 mm from the applicator. D90, V100, and EQD2(α/ß=3Gy) at 0.1 cm3, 1 cm3, and 2 cm3 of the most exposed part of the vagina were calculated. Vaginal toxicity assessment was completed with a LENT-SOMA-objective-criteria. Statistics were done with the use of χ2 and Student's-t test. RESULTS: The mean follow-up was 23.2 months (7.6-46.8). Median D90 was 7.8 Gy(α/ß=3Gy). Late toxicity: 8 G1 and 9 G2. Median EQD2(α/ß=3Gy) in vagina was 88.6 Gy (62.8-177.6) for 0.1 cm3, 72.4 Gy (57.1-130.4) for 1 cm3, and 69 Gy (53-113.4) for 2 cm3. Exclusive VBT vs. EBRT+VBT showed no differences in vaginal toxicity. There was no relationship between EQD2(α/ß=3Gy) at 0.1 cm3 and 1 cm3 of vagina with G1-G2 toxicity (p = 0.62 and p = 0.58, respectively). G2 toxicity was related to EQD2(α/ß=3Gy) at 2 cm3 (p = 0.03). EQD2(α/ß=3Gy) > 68 Gy caused G2 late toxicity in 20.5% patients. All patients presenting G2 toxicity received > 68 Gy EQD2(α/ß=3Gy). CONCLUSIONS: More than 68 Gy EQD2(α/ß=3Gy) at 2 cm3 was related to G2 toxicity in P-EC-VBT. Further studies including larger number of patients are needed to confirm these results. Patients receiving these doses should be informed of the risk of toxicity, with individualized treatment planning and follow-up to reduce G2 toxicity.

Decreased TESK1-mediated cofilin 1 phosphorylation in the jejunum of IBS-D patients may explain increased female predisposition to epithelial dysfunction.

Disturbed intestinal epithelial barrier and mucosal micro-inflammation characterize irritable bowel syndrome (IBS). Despite intensive research demonstrating ovarian hormones modulation of IBS severity, there is still limited knowledge on the mechanisms underlying female predominance in this disorder. Our aim was to identify molecular pathways involved in epithelial barrier dysfunction and female predominance in diarrhea-predominant IBS (IBS-D) patients. Total RNA and protein were obtained from jejunal mucosal biopsies from healthy controls and IBS-D patients meeting the Rome III criteria. IBS severity was recorded based on validated questionnaires. Gene and protein expression profiles were obtained and data integrated to explore biological and molecular functions. Results were validated by western blot. Tight junction signaling, mitochondrial dysfunction, regulation of actin-based motility by Rho, and cytoskeleton signaling were differentially expressed in IBS-D. Decreased TESK1-dependent cofilin 1 phosphorylation (pCFL1) was confirmed in IBS-D, which negatively correlated with bowel movements only in female participants. In conclusion, deregulation of cytoskeleton dynamics through TESK1/CFL1 pathway underlies epithelial intestinal dysfunction in the small bowel mucosa of IBS-D, particularly in female patients. Further understanding of the mechanisms involving sex-mediated regulation of mucosal epithelial integrity may have significant preventive, diagnostic, and therapeutic implications for IBS.

Epithelial immunity: priming defensive responses in the intestinal mucosa.

As the largest interface between the outside and internal milieu, the intestinal epithelium constitutes the first structural component facing potential luminal threats to homeostasis. This single-cell layer is the epicenter of a tightly regulated communication network between external and internal factors that converge to prime defensive responses aimed at limiting antigen penetration and the maintenance of intestinal barrier function. The defensive role developed by intestinal epithelial cells (IEC) relies largely on the variety of receptors they express at both extracellular (apical and basolateral) and intracellular compartments, and the capacity of IEC to communicate with immune and nervous systems. IEC recognize pathogen-associated molecules by innate receptors that promote the production of mucus, antimicrobial substances, and immune mediators. Epithelial cells are key to oral tolerance maintenance and also participate in adaptive immunity through the expression of immunoglobulin (Ig) receptors and by promoting local Ig class switch recombination. In IEC, different types of antigens can be sensed by multiple immune receptors that share signaling pathways to assure effective responses. Regulated defensive activity maintains intestinal homeostasis, whereas a breakdown in the control of epithelial immunity can increase the intestinal passage of luminal content and microbial invasion, leading to inflammation and tissue damage. In this review, we provide an updated overview of the type of immune receptors present in the human intestinal epithelium and the responses generated to promote effective barrier function and maintain mucosal homeostasis.

Downregulation of mucosal mast cell activation and immune response in diarrhoea-irritable bowel syndrome by oral disodium cromoglycate: A pilot study.

BACKGROUND AND GOAL: Diarrhoea-predominant irritable bowel syndrome (IBS-D) exhibits intestinal innate immune and mucosal mast cell (MC) activation. MC stabilisers have been shown to improve IBS symptoms but the mechanism is unclear. Our primary aim was to investigate the effect of oral disodium cromoglycate (DSCG) on jejunal MC activation and specific innate immune signalling pathways in IBS-D, and secondarily, its potential clinical benefit. STUDY: Mucosal MC activation (by ultrastructural changes, tryptase release and gene expression) and innate immune signalling (by protein and gene expression) were quantified in jejunal biopsies from healthy (HS; n = 16) and IBS-D subjects after six months of either treatment with DSCG (600 mg/day, IBS-D-DSCG group; n = 18) or without treatment (IBS-D-NT group; n = 25). All IBS-D patients recorded abdominal pain and bowel habits at baseline and in the last 10 days prior to jejunal sampling. RESULTS: IBS-D-NT exhibited significant MC activation and over-expression of immune-related genes as compared to HS, whereas in IBS-D-DSCG MC activity and gene expression were similar to HS. Furthermore, DSCG significantly reduced abdominal pain and improved stool consistency. CONCLUSION: Oral DSCG modulates mucosal immune activity and improves gut symptoms in IBS-D patients. Future placebo-controlled clinical trials are needed for confirmation of clinical benefit of DSCG for IBS-D.

Developmental expression of membrane type 4-matrix metalloproteinase (Mt4-mmp/Mmp17) in the mouse embryo.

Matrix metalloproteinases (MMPs) constitute a large group of endoproteases that play important functions during embryonic development, tumor metastasis and angiogenesis by degrading components of the extracellular matrix. Within this family, we focused our study on Mt4-mmp (also called Mmp17) that belongs to a distinct subset that is anchored to the cell surface via a glycosylphosphatidylinositol (GPI) moiety and with the catalytic site exposed to the extracellular space. Information about its function and substrates is very limited to date, and little has been reported on its role in the developing embryo. Here, we report a detailed expression analysis of Mt4-mmp during mouse embryonic development by using a LacZ reporter transgenic mouse line. We showed that Mt4-mmp is detected from early stages of development to postnatal stages following a dynamic and restricted pattern of expression. Mt4-mmp was first detected at E8.5 limited to the intersomitic vascularization, the endocardial endothelium and the dorsal aorta. Mt4-mmpLacZ/+ cells were also observed in the neural crest cells, somites, floor plate and notochord at early stages. From E10.5, expression localized in the limb buds and persists during limb development. A strong expression in the brain begins at E12.5 and continues to postnatal stages. Specifically, staining was observed in the olfactory bulb, cerebral cortex, hippocampus, striatum, septum, dorsal thalamus and the spinal cord. In addition, LacZ-positive cells were also detected during eye development, initially at the hyaloid artery and later on located in the lens and the neural retina. Mt4-mmp expression was confirmed by quantitative RT-PCR and western blot analysis in some embryonic tissues. Our data point to distinct functions for this metalloproteinase during embryonic development, particularly during brain formation, angiogenesis and limb development.

Comparative results of three short brachytherapy schedules as exclusive treatment in postoperative endometrial carcinoma.

PURPOSE: To compare vaginal control and treatment toxicity of three different high-dose-rate brachytherapy schedules as exclusive treatment in postoperative endometrial carcinoma. METHODS AND MATERIALS: From 2003 to 2015, three different schedules were used as postoperative treatment for 146 patients (p) with intermediate-risk endometrial carcinoma. Group 1 (41 p): six fractions of 4-6 Gy, 3-4 fractions per week; Group 2 (59 p): four fractions of 5-6 Gy administered daily; Group 3 (46 p): 6 Gy × 3 fractions in three consecutive days. The dose was prescribed at 5 mm of applicator surface using an active treatment length of 2.5 cm. Toxicity scores were evaluated using the Radiation Therapy Oncology Group scores for bladder and rectum and the objective criteria of late effects of normal tissues-subjective, objective, management, analytic for vagina. Statistics used were group descriptions calculating their means, medians, and ranges. Bivariate analysis was evaluated using variance models and χ2 tests. RESULTS: The mean followup was as follows: Group 1: 88 months, Group 2: 75 months, and 41 months in Group 3. No vaginal relapses were found. Late toxicity ≥ G2: rectum: 0 p in the three groups (0%). Bladder: Group 1: 1 p (2.4%), Group 2: 0%, and Group 3: 0%. Vagina: Group 1: 4 p (9.5%); Group 2: 9 p (15.3%); and Group 3:10 p (21.8%). There were no differences in late toxicity among the three groups of patients for rectum (p = 0.83), bladder (p = 0.58), and vagina (p = 0.67); the expected global risk of complications for rectum, bladder, and vagina is 0.8%, 0.8%, and 28.8%, respectively. CONCLUSIONS: Similar results in vaginal control and complications were achieved with the three schedules. The use of three fractions of 6 Gy administered daily is the best option for patient comfort and convenience and use of resources. Nonetheless, specific studies are needed to demonstrate the best cost-efficacy regime.

Mucosal pathobiology and molecular signature of epithelial barrier dysfunction in the small intestine in irritable bowel syndrome.

Irritable bowel syndrome (IBS) is one of the most prevalent gastrointestinal disorders in developed countries. Its etiology remains unknown; however, a common finding, regardless of IBS subtype, is the presence of altered intestinal barrier. In fact, signaling and location of cell-to-cell adhesion proteins, in connection with increased immune activity, seem abnormal in the intestinal epithelium of IBS patients. Despite that most research is performed on distal segments of the intestine, altered permeability has been reported in both, the small and the large bowel of all IBS subtypes. The small intestine carries out digestion and nutrient absorption and is also the site where the majority of immune responses to luminal antigens takes place. In fact, the upper intestine is more exposed to environmental antigens than the colon and is also a site of symptom generation. Recent studies have revealed small intestinal structural alterations of the epithelial barrier and mucosal immune activation in association with intestinal dysfunction, suggesting the commitment of the intestine as a whole in the pathogenesis of IBS. This review summarizes the most recent findings on mucosal barrier alterations and its relationship to symptoms arising from the small intestine in IBS, including epithelial structural abnormalities, mucosal immune activation, and microbial dysbiosis, further supporting the hypothesis of an organic origin of IBS.