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Objective: To investigate whether there is any relationship between otological as well as vestibular symptoms, audiological findings and type of temporomandibular disorder (articular, muscular and mixed); and to check the distribution of the temporomandibular disorders (TMD) dysfunction degree in the research population. Methods: A retrospective study involving 30 patients of both sexes, aged between 18 and 49 years old, diagnosed with TMD and dentofacial deformities, who were subject to clinical evaluation (muscle palpation, auscultation of temporomandibular joint during mandibular motion and measurement of jaw movement), audiological testing (pure tone audiometry and immittance testing) and two questionnaires, one on otological and vestibular symptoms and the other on TMD anamnesis. Based on both the anamnesis questionnaire and the clinical assessment, the subjects were divided according to the type and degree of TMD dysfunction (mild, moderate and severe), and compared regarding the occurrence of auditory signs and symptoms, vestibular symptoms and audiological findings according to TMD type. Results: The anamnesis questionnaire demonstrated higher prevalence (83.33%) of severe TMD. Subjects with mixed TMD had more complaints about hypoacusis than those with muscular TMD (p < 0.05). The results showed no change in either audiological and immittance testing for all assessed individuals. Conclusion: Otological symptoms are present in subjects with TMD and dentofacial deformities, regardless of the classification of TMD (articular, muscular or mixed). Those with mixed TMD may have higher incidence of complaints about hypoacusis than subjects with muscular TMD. Further studies are needed to investigate the relationship between otological symptoms and the different types of TMD. [ABSTRACT FROM AUTHOR]

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17β-estradiol (E2) regulates diverse physiological effects, including cell proliferation, by binding to estrogen receptor α (ERα). ERα is both a transcription factor that drives E2-sensitive gene expression and an extra-nuclear localized receptor that triggers the activation of diverse kinase cascades. While E2 triggers cell proliferation, it also induces ERα degradation in a typical hormone-dependent feedback loop. Although ERα breakdown proceeds through the 26S proteasome, a role for lysosomes and for some endocytic proteins in controlling ERα degradation has been reported. Here, we studied the role of the endocytic protein dynamin II in E2-dependent ERα signaling and degradation. The results indicate that dynamin II siRNA-mediated knock-down partially prevents E2-induced ERα degradation through the inhibition of an autophagy-based pathway and impairs E2-induced cell proliferation signaling. Altogether, these data demonstrate that dynamin II is required for the E2:ERα signaling of physiological functions and uncovers a role for autophagy in the control of ERα turnover.

The homeostatic control of the cellular proteome steady-state is dependent either on the 26S proteasome activity or on the lysosome function. The sex hormone 17β-estradiol (E2) controls a plethora of biological functions by binding to the estrogen receptor α (ERα), which is both a nuclear ligand-activated transcription factor and also an extrinsic plasma membrane receptor. Regulation of E2-induced physiological functions (e.g., cell proliferation) requires the synergistic activation of both transcription of estrogen responsive element (ERE)-containing genes and rapid extra-nuclear phosphorylation of many different signalling kinases (e.g., ERK/MAPK; PI3K/AKT). Although E2 controls ERα intracellular content and activity via the 26S proteasome-mediated degradation, biochemical and microscopy-based evidence suggests a possible cross-talk among lysosomes and ERα activities. Here, we studied the putative localization of endogenous ERα to lysosomes and the role played by lysosomal function in ERα signalling. By using confocal microscopy and biochemical assays, we report that ERα localizes to lysosomes and to endosomes in an E2-dependent manner. Moreover, the inhibition of lysosomal function obtained by chloroquine demonstrates that, in addition to 26S proteasome-mediated receptor elimination, lysosome-based degradation also contributes to the E2-dependent ERα breakdown. Remarkably, the lysosome function is further involved in those ERα activities required for E2-dependent cell proliferation while it is dispensable for ERα-mediated ERE-containing gene transcription. Our discoveries reveal a novel lysosome-dependent degradation pathway for ERα and show a novel biological mechanism by which E2 regulates ERα cellular content and, as a consequence, cellular functions.

17β-estradiol (E2)-dependent estrogen receptor (ER) α intracellular concentration is a well recognized critical step in the pleiotropic effects elicited by E2 in several target tissues. Beside E2, a class of synthetic and plant-derived chemicals collectively named endocrine disruptors (EDs) or xenoestrogens bind to and modify both nuclear and extra-nuclear ERα activities. However, at the present no information is available on the ability of EDs to hamper ERα intracellular concentration. Here, the effects of bisphenol A (BPA) and naringenin (Nar), prototypes of synthetic and plant-derived ERα ligands, have been evaluated on ERα levels in MCF-7 cells. Both EDs mimic E2 in triggering ERα Ser118 phosphorylation and gene transcription. However, only E2 or BPA induce an increase of cell proliferation; whereas 24 hrs after Nar stimulation a dose-dependent decrease in cell number is reported. E2 or BPA treatment reduces ERα protein and mRNA levels after 24 hrs. Contrarily, Nar stimulation does not alter ERα content but reduces ERα mRNA levels like other ligands. Co-stimulation experiments indicate that 48 hrs of Nar treatment prevents the E2-induced ERα degradation and hijacks the physiological ability of E2:ERα complex to regulate gene transcription. Mechanistically, Nar induces ERα protein accumulation by preventing proteasomal receptor degradation via persistent activation of p38/MAPK pathway. As a whole these data demonstrate that ERα intracellular concentration is an important target through which EDs hamper the hormonal milieu of E2 target cells driving cells to different outcomes or mimicking E2 even in the absence of the hormone.

Background: Supplements to support clinical-grade cultures of mesenchymal stem cells (MSC) are required to promote growth and expansion of these cells. Platelet lysate (PL) is a human blood component which may replace animal serum in MSC cultures being rich in various growth factors. Here, we describe a plasma poor pathogen-free platelet lysate obtained by pooling 12 platelet (PLT) units, to produce a standardized and safe supplement for clinical-grade expansion of MSC., Methods: PL lots were obtained by combining 2 6-unit PLT pools in additive solution (AS) following a transfusional-based procedure including pathogen inactivation (PI) by Intercept technology and 3 cycles of freezing/thawing, followed by membrane removal. Three PI-PL and 3 control PL lots were produced to compare their ability to sustain bone marrow derived MSC selection and expansion. Moreover, two further PL, subjected to PI or not, were also produced starting from the same initial PLT pools to evaluate the impact of PI on growth factor concentration and capacity to sustain cell growth. Additional PI-PL lots were used for comparison with fetal bovine serum (FBS) on MSC expansion. Immunoregulatory properties of PI-PL-generated MSC were documented in vitro by mixed lymphocyte culture (MLC) and peripheral blood mononuclear cells (PBMC) mitogen induced proliferation., Results: PI-PL and PL control lots had similar concentrations of 4 well-described growth factors endowed with MSC stimulating ability. Initial growth and MSC expansion by PI-PL and PL controls were comparable either using different MSC populations or in head to head experiments. Moreover, PI-PL and PL control sustained similar MSC growth of frozen/thawed MSC. Multilineage differentiation of PI-derived and PI-PL-derived MSC were maintained in any MSC cultures as well as their immunoregulatory properties. Finally, no direct impact of PI on growth factor concentration and MSC growth support was observed, whereas the capacity of FBS to sustain MSC expansion in basic medium was irrelevant as compared to PL and PI-PL., Conclusion: The replacement of animal additives with human supplements is a basic issue in MSC ex vivo production. PI-PL represents a standardized, plasma-poor, human preparation which appears as a safe and good candidate to stimulate MSC growth in clinical-scale cultures.

Background: Highly Expressed in Cancer protein 1 (Hec1) is a constituent of the Ndc80 complex, a kinetochore component that has been shown to have a fundamental role in stable kinetochore-microtubule attachment, chromosome alignment and spindle checkpoint activation at mitosis. HEC1 RNA is found up-regulated in several cancer cells, suggesting a role for HEC1 deregulation in cancer. In light of this, we have investigated the consequences of experimentally-driven Hec1 expression on mitosis and chromosome segregation in an inducible expression system from human cells., Methodology/principal Findings: Overexpression of Hec1 could never be obtained in HeLa clones inducibly expressing C-terminally tagged Hec1 or untagged Hec1, suggesting that Hec1 cellular levels are tightly controlled. On the contrary, a chimeric protein with an EGFP tag fused to the Hec1 N-terminus accumulated in cells and disrupted mitotic division. EGFP- Hec1 cells underwent altered chromosome segregation within multipolar spindles that originated from centriole splitting. We found that EGFP-Hec1 assembled a mutant Ndc80 complex that was unable to rescue the mitotic phenotypes of Hec1 depletion. Kinetochores harboring EGFP-Hec1 formed persisting lateral microtubule-kinetochore interactions that recruited the plus-end depolymerase MCAK and the microtubule stabilizing protein HURP on K-fibers. In these conditions the plus-end kinesin CENP-E was preferentially retained at kinetochores. RNAi-mediated CENP-E depletion further demonstrated that CENP-E function was required for multipolar spindle formation in EGFP-Hec1 expressing cells., Conclusions/significance: Our study suggests that modifications on Hec1 N-terminal tail can alter kinetochore-microtubule attachment stability and influence Ndc80 complex function independently from the intracellular levels of the protein. N-terminally modified Hec1 promotes spindle pole fragmentation by CENP-E-mediated plus-end directed kinetochore pulling forces that disrupt the fine balance of kinetochore- and centrosome-associated forces regulating spindle bipolarity. Overall, our findings support a model in which centrosome integrity is influenced by the pathways regulating kinetochore-microtubule attachment stability.

In diabetic patients and animal models of diabetes mellitus (DM), circulating endothelial progenitor cell (EPC) number is lower than in normoglycaemic conditions and EPC angiogenic properties are inhibited. Stromal cell derived factor-1 (SDF-1) plays a key role in bone marrow (BM) c-kit(+) stem cell mobilization into peripheral blood (PB), recruitment from PB into ischemic tissues and differentiation into endothelial cells. The aim of the present study was to examine the effect of DM in vivo and in vitro, on murine BM-derived c-kit(+) cells and on their response to SDF-1. Acute hindlimb ischemia was induced in streptozotocin-treated DM and control mice; circulating c-kit(+) cells exhibited a rapid increase followed by a return to control levels which was significantly faster in DM than in control mice. CXCR4 expression by BM c-kit(+) cells as well as SDF-1 protein levels in the plasma and in the skeletal muscle, both before and after the induction of ischemia, were similar between normoglycaemic and DM mice. However, BM-derived c-kit(+) cells from DM mice exhibited an impaired differentiation towards the endothelial phenotype in response to SDF-1; this effect was associated with diminished protein kinase phosphorylation. Interestingly, SDF-1 ability to induce differentiation of c-kit(+) cells from DM mice was restored when cells were cultured under normoglycaemic conditions whereas c-kit(+) cells from normoglycaemic mice failed to differentiate in response to SDF-1 when they were cultured in hyperglycaemic conditions. These results show that DM diminishes circulating c-kit(+) cell number following hindlimb ischemia and inhibits SDF-1-mediated AKT phosphorylation and differentiation towards the endothelial phenotype of BM-derived c-kit(+) cells.

Thrombin generation increases in several pathological conditions, including cancer, thromboembolism, diabetes and myeloproliferative syndromes. During tumor development, thrombin levels increase along with several other molecules, including cytokines and angiogenic factors. Under such conditions, it is reasonable to predict that thrombin may recognize new low-affinity substrates that usually are not recognized under low-expression levels conditions. In the present study, we hypothesized that fibroblast growth factor (FGF)-2 may be cleaved by thrombin and that such action may lead to an impairment of its biological activity. The evidence collected in the present study indicates that FGF-2-induced proliferation and chemotaxis/invasion of SK-MEL-110 human melanoma cells were significantly reduced when FGF-2 was pre-incubated with active thrombin. The inhibition of proliferation was not influenced by heparin. Phe-Pro-Arg-chloromethyl ketone, a specific inhibitor of the enzymatic activity of thrombin, abolished the thrombin-induced observed effects. Accordingly, both FGF-2-binding to cell membranes as well as FGF-2-induced extracellular signal-regulated kinase phosphorylation were decreased in the presence of thrombin. Finally, HPLC analyses demonstrated that FGF-2 is cleaved by thrombin at the peptide bond between residues Arg42 and Ile43 of the mature human FGF-2 sequence. The apparent k(cat)/K(m) of FGF-2 hydrolysis was 1.1 x 10(4) M(-1) x s(-1), which is comparable to other known low-affinity thrombin substrates. Taken together, these results demonstrate that thrombin digests FGF-2 at the site Arg42-Ile43 and impairs FGF-2 activity in vitro, indicating that FGF-2 is a novel thrombin substrate.