Coordinate expression of anticoagulant heparan sulfate proteoglycans and serine protease inhibitors in the rat ovary: a potent system of proteolysis control.

During the reproductive cycle, ovarian follicles undergo major tissue-remodeling involving vascular changes and proteolysis. Anticoagulant heparan sulfate proteoglycans (aHSPGs) are expressed by granulosa cells during the development of the ovarian follicle. The function of aHSPGs in the ovary is unknown, but they might be involved in proteolysis control through binding and activation of serine protease inhibitors. To identify functional interactions between aHSPGs and heparin-binding protease inhibitors in the follicle, we have coordinately localized aHSPGs, antithrombin III, protease nexin-1, and plasminogen activator inhibitor-1 in the rat ovary during natural and gonadotropin-stimulated cycles. Anticoagulant HSPGs were visualized by autoradiography of cryosections incubated with 125I-antithrombin III, and protease inhibitors were assessed by immunohistochemistry and Northern blot hybridization. Anticoagulant HSPGs were expressed in follicles before ovulation, were transiently decreased in postovulatory follicles, and were abundant in the corpus luteum, mainly on capillaries. Anticoagulant HSPGs were colocalized with protease nexin-1 in follicles from the early antral stage until ovulation, with antithrombin III in the preovulatory stage and after ovulation, and with plasminogen activator inhibitor-1 in the corpus luteum. These data demonstrate that aHSPGs are critically expressed in the ovary to interact sequentially with protease nexin-1, antithrombin III, and plasminogen activator inhibitor-1 during the cycle. The specificity of these inhibitors is shifted toward thrombin inhibition in the presence of heparin, suggesting that aHSPGs direct their action to control fibrin deposition in the follicle. The occupation of aHSPGs antithrombin-binding sites by mutant R393C antithrombin III, injected in the ovarian bursa, decreased ovulation efficiency, further supporting the involvement of aHSPGs in the ovulation process.     

Investigation of Adhesion and Mechanical Properties of Human Glioma Cells by Single Cell Force Spectroscopy and Atomic Force Microscopy

Active cell migration and invasion is a peculiar feature of glioma that makes this tumor able to rapidly infiltrate into the surrounding brain tissue. In our recent work, we identified a novel class of glioma-associated-stem cells (defined as GASC for high-grade glioma -HG- and Gasc for low-grade glioma -LG-) that, although not tumorigenic, act supporting the biological aggressiveness of glioma-initiating stem cells (defined as GSC for HG and Gsc for LG) favoring also their motility. Migrating cancer cells undergo considerable molecular and cellular changes by remodeling their cytoskeleton and cell interactions with surrounding environment. To get a better understanding about the role of the glioma-associated-stem cells in tumor progression, cell deformability and interactions between glioma-initiating stem cells and glioma-associated-stem cells were investigated. Adhesion of HG/LG-cancer cells on HG/LG-glioma-associated stem cells was studied by time-lapse microscopy, while cell deformability and cell-cell adhesion strengths were quantified by indentation measurements by atomic force microscopy and single cell force spectroscopy. Our results demonstrate that for both HG and LG glioma, cancer-initiating-stem cells are softer than glioma-associated-stem cells, in agreement with their neoplastic features. The adhesion strength of GSC on GASC appears to be significantly lower than that observed for Gsc on Gasc. Whereas, GSC spread and firmly adhere on Gasc with an adhesion strength increased as compared to that obtained on GASC. These findings highlight that the grade of glioma-associated-stem cells plays an important role in modulating cancer cell adhesion, which could affect glioma cell migration, invasion and thus cancer aggressiveness. Moreover this work provides evidence about the importance of investigating cell adhesion and elasticity for new developments in disease diagnostics and therapeutics.     

Homogentisic acid induces morphological and mechanical aberration of ochronotic cartilage in alkaptonuria

Alkaptonuria (AKU) is a disease caused by a deficient homogentisate 1,2-dioxygenase activity leading to systemic accumulation of homogentisic acid (HGA), that forms a melanin-like polymer that progressively deposits onto connective tissues causing a pigmentation called “ochronosis” and tissue degeneration. The effects of AKU and ochronotic pigment on the biomechanical properties of articular cartilage need further investigation. To this aim, AKU cartilage was studied using thermal (thermogravimetry and differential scanning calorimetry) and rheological analysis. We found that AKU cartilage had a doubled mesopore radius compared to healthy cartilage. Since the mesoporous structure is the main responsible for maintaining a correct hydrostatic pressure and tissue homoeostasis, drastic changes of thermal and rheological parameters were found in AKU. In particular, AKU tissue lost its capability to enhance chondrocytes metabolism (decreased heat capacity) and hence the production of proteoglycans. A drastic increase in stiffness and decrease in dissipative and lubricant role ensued in AKU cartilage. Multiphoton and scanning electron microscopies revealed destruction of cell–matrix microstructure and disruption of the superficial layer. Such observations on AKU specimens were confirmed in HGA-treated healthy cartilage, indicating that HGA is the toxic responsible of morphological and mechanical alterations of cartilage in AKU.     

The phlebotomine fauna (Diptera: Psychodidae) of Guaraí, state of Tocantins,with an emphasis on the putative vectors of American cutaneous leishmaniasis in rural settlement and periurban areas

Phlebotomine sandflies were captured in rural settlement and periurban areas of the municipality of Guaraí in the state of Tocantins (TO), an endemic area of American cutaneous leishmaniasis (ACL). Forty-three phlebotomine species were identified, nine of which have already been recognised as ACL vectors. Eleven species were recorded for the first time in TO. Nyssomyia whitmani was the most abundant species, followed by Evandromyia bourrouli, Nyssomyia antunesi and Psychodopygus complexus. The Shannon-Wiener diversity index and the evenness index were higher in the rural settlement area than in the periurban area. The evaluation of different ecotopes within the rural area showed the highest frequencies of Ev. bourrouli and Ny. antunesi in chicken coops, whereas Ny. whitmani predominated in this ecotope in the periurban area. In the rural settlement area, Ev. bourrouli was the most frequently captured species in automatic light traps and Ps. complexus was the most prevalent in Shannon trap captures. The rural settlement environment exhibited greater phlebotomine biodiversity than the periurban area. Ps. complexus and Psychodopygus ayrozai naturally infected with Leishmania (Viannia) braziliensis were identified. The data identified Ny. whitmani as a potential ACL vector in the periurban area, whereas Ps. complexus was more prevalent in the rural environment associated with settlements.     

A Stable Bacterial Peroxidase with Novel Halogenating Activity and an Autocatalytically Linked Heme Prosthetic Group

Reconstructing the phylogenetic relationships of the main evolutionary lines of the mammalian peroxidases lactoperoxidase and myeloperoxidase revealed the presence of novel bacterial heme peroxidase subfamilies. Here, for the first time, an ancestral bacterial heme peroxidase is shown to possess a very high bromide oxidation activity (besides conventional peroxidase activity). The recombinant protein allowed monitoring of the autocatalytic peroxide-driven formation of covalent heme to protein bonds. Thereby, the high spin ferric rhombic heme spectrum became similar to lactoperoxidase, the standard reduction potential of the Fe(III)/Fe(II) couple shifted to more positive values (−145 ± 10 mV at pH 7), and the conformational and thermal stability of the protein increased significantly. We discuss structure-function relationships of this new peroxidase in relation to its mammalian counterparts and ask for its putative physiological role.     

Difference of two dependent sensitivities and specificities: Comparison of various approaches

In diagnostic studies, a new diagnostic test is often compared with a standard test and both tests are applied on the same patients, called paired design. The true disease state is in general given by the so‐called gold standard (most reliable method for classification), which has to be known for all patients. The benefit of the new diagnostic test can be evaluated by sensitivity and specificity, which are in fact proportions. This means, for the comparison of two diagnostic tests, confidence intervals for the difference of the dependent estimated sensitivities and specificities are calculated. In the literature, many comparisons of different approaches can be found, but none explicitly for diagnostic studies. For this reason we compare 13 approaches for a set of scenarios that represent data of diagnostic studies (e.g., with sensitivity and specificity ?0.8). With simulation studies, we show that the nonparametric interval with normal approximation can be recommended for the difference of two dependent sensitivities or specificities without restriction, the Wald interval with the limitation of slightly anti‐conservative results for small sample sizes, and the nonparametric intervals with t‐approximation, and the Tango interval with the limitation of conservative results for high correlations.     

Pockmarks enhance deep‐sea benthic biodiversity: a case study in the western Mediterranean Sea

Aim Pockmarks are craters on the sea floor formed by sub‐sea‐floor fluid expulsions, which occur world‐wide at all ocean depths. These habitats potentially host a highly specialized fauna that can exploit the hydrocarbons released. Pockmarks at relatively shallow depths can be easily destroyed by human activities, such as bottom trawling. In the present study, we investigated the combined effects of sea‐floor heterogeneity, rate of fluid emission and trophic conditions of different pockmarks on the biodiversity of the deep‐sea assemblages. Location Continental slope of the Gulf of Lions, western Mediterranean Sea, at water depths from 265 to 434 m. Methods We investigated the biodiversity associated with sea‐floor pockmarks that are both inactive and that have active gas emissions. Control sites were selected on the sea floor outside the influence of the gas seepage, both within and outside the pockmark fields. We examined the combined effects of: (i) sea‐floor heterogeneity; (ii) variable levels of fluid (gas) emissions; and (iii) trophic characteristics of the meiofaunal assemblage structure and nematode diversity. Results Sediments within the pockmark fields had lower meiofaunal abundance and biomass when compared with the surrounding sediments that were not influenced by the gas seepage. Although several higher taxa were absent in the pockmarks (e.g. Turbellaria, Tardigrada, Cumacea, Isopoda, Tanaidacea, Nemertina and Priapulida, which were present in the control areas), the richness of the nematode species within all of these pockmarks was very high. About 25% of the total species encountered in the deep‐sea sediments of the investigated areas was exclusively associated with these pockmarks. Main conclusions We conclude that both active and inactive pockmarks provide significant contributions to the regional (gamma) diversity of the continental slope in the western Mediterranean Sea, and thus the protection of these special and fragile habitats is highly relevant to the conservation of deep‐sea biodiversity.     

Antagonism of the Prokineticin System Prevents and Reverses Allodynia and Inflammation in a Mouse Model of Diabetes

Neuropathic pain is a severe diabetes complication and its treatment is not satisfactory. It is associated with neuroinflammation-related events that participate in pain generation and chronicization. Prokineticins are a new family of chemokines that has emerged as critical players in immune system, inflammation and pain. We investigated the role of prokineticins and their receptors as modulators of neuropathic pain and inflammatory responses in experimental diabetes. In streptozotocin-induced-diabetes in mice, the time course expression of prokineticin and its receptors was evaluated in spinal cord and sciatic nerves, and correlated with mechanical allodynia. Spinal cord and sciatic nerve pro- and anti-inflammatory cytokines were measured as protein and mRNA, and spinal cord GluR subunits expression studied. The effect of preventive and therapeutic treatment with the prokineticin receptor antagonist PC1 on behavioural and biochemical parameters was evaluated. Peripheral immune activation was assessed measuring macrophage and T-helper cytokine production. An up-regulation of the Prokineticin system was present in spinal cord and nerves of diabetic mice, and correlated with allodynia. Therapeutic PC1 reversed allodynia while preventive treatment blocked its development. PC1 normalized prokineticin levels and prevented the up-regulation of GluN2B subunits in the spinal cord. The antagonist restored the pro-/anti-inflammatory cytokine balance altered in spinal cord and nerves and also reduced peripheral immune system activation in diabetic mice, decreasing macrophage proinflammatory cytokines and the T-helper 1 phenotype. The prokineticin system contributes to altered sensitivity in diabetic neuropathy and its inhibition blocked both allodynia and inflammatory events underlying disease.