Differential expression of espin isoforms during epithelial morphogenesis, stereociliogenesis and postnatal maturation in the developing inner ear

AbstractThe espins are a family of multifunctional actin cytoskeletal proteins. They are present in hair cell stereocilia and are the target of mutations that cause deafness and vestibular dysfunction. Here, we demonstrate that the different espin isoforms are expressed in complex spatiotemporal patterns during inner ear development. Espin 3 isoforms were prevalent in the epithelium of the otic pit, otocyst and membranous labyrinth as they underwent morphogenesis. This espin was down-regulated ahead of hair cell differentiation and during neuroblast delamination. Espin also accumulated in the epithelium of branchial clefts and pharyngeal pouches and during branching morphogenesis in other embryonic epithelial tissues, suggesting general roles for espins in epithelial morphogenesis. Espin reappeared later in inner ear development in differentiating hair cells. Its levels and compartmentalization to stereocilia increased during the formation and maturation of stereociliary bundles. Late in embryonic development, espin was also present in a tail-like process that emanated from the hair cell base. Increases in the levels of espin 1 and espin 4 isoforms correlated with stereocilium elongation and maturation in the vestibular system and cochlea, respectively. Our results suggest that the different espin isoforms play specific roles in actin cytoskeletal regulation during epithelial morphogenesis and hair cell differentiation

Strategies for the Accurate Measurement of the Resonance Frequency in QCM-D Systems via Low-Cost Digital Techniques

In this paper, an FPGA (Field Programmable Gate Array)-based digital architecture for the measurement of quartz crystal microbalance (QCM) oscillating frequency of transient responses, i.e., in QCM-D (QCM and Dissipation) applications, is presented. The measurement system is conceived for operations in liquid, with short QCM transient responses due to the large mechanical load. The proposed solution allows for avoiding the complex processing systems typically required by the QCM-D techniques and grants frequency resolutions better than 1 ppm. The core of the architecture is a reciprocal digital frequency meter, combined with the preprocessing of the QCM signal through mixing operations, such as a step-down of the input frequency and reducing the measurement error. The measurement error is further reduced through averaging. Different strategies are proposed to implement the proposed measurement solution, comprising an all-digital circuit and mixed analog/digital ones. The performance of the proposed architectures is theoretically derived, compared, and analyzed by means of experimental data obtained considering 10 MHz QCMs and 200 μs long transient responses. A frequency resolution of about 240 ppb, which corresponds to a Sauerbrey mass resolution of 8 ng/cm2, is obtained for the all-digital solution, whereas for the mixed solution the resolution halves to 120 ppb, with a measurement time of about one second over 100 repetitions

Espin cross-links cause the elongation of microvillus-type parallel actin bundles in vivo

The espin actin-bundling proteins, which are the target of the jerker deafness mutation, caused a dramatic, concentration-dependent lengthening of LLC-PK1-CL4 cell microvilli and their parallel actin bundles. Espin level was also positively correlated with stereocilium length in hair cells. Villin, but not fascin or fimbrin, also produced noticeable lengthening. The espin COOH-terminal peptide, which contains the actin-bundling module, was necessary and sufficient for lengthening. Lengthening was blocked by 100 nM cytochalasin D. Espin cross-links slowed actin depolymerization in vitro less than twofold. Elimination of an actin monomer-binding WASP homology 2 domain and a profilin-binding proline-rich domain from espin did not decrease lengthening, but made it possible to demonstrate that actin incorporation was restricted to the microvillar tip and that bundles continued to undergo actin treadmilling at ∼1.5 s−1 during and after lengthening. Thus, through relatively subtle effects on actin polymerization/depolymerization reactions in a treadmilling parallel actin bundle, espin cross-links cause pronounced barbed-end elongation and, thereby, make a longer bundle without joining shorter modules

The novel mu-opioid antagonist, GSK1521498, reduces ethanol consumption in C57BL/6J mice.

RATIONALE Using the drinking-in-the-dark (DID) model, we compared the effects of a novel mu-opioid receptor antagonist, GSK1521498, with naltrexone, a licensed treatment of alcohol dependence, on ethanol consumption in mice. OBJECTIVE We test the ability of GSK1521498 to reduce alcohol consumption and compare its intrinsic efficacy to that of naltrexone by comparing the two drugs at doses matched for equivalent receptor occupancy. METHODS Thirty-six C57BL/6J mice were tested in a DID procedure. In 2-day cycles, animals experienced one baseline, injection-free session, and one test session when they received two injections, one of test drug and one placebo. All animals received GSK1521498 (0, 0.1, 1 and 3 mg/kg, i.p., 30 min pre-treatment) and naltrexone (0, 0.1, 1 and 3 mg/kg, s.c. 10 min pre-treatment) in a cross-over design. Receptor occupancies following the same doses were determined ex vivo in separate groups by autoradiography, using [3H]DAMGO. Binding in the region of interest was measured integrally by computer-assisted microdensitometry and corrected for non-specific binding. RESULTS Both GSK1521498 and naltrexone dose-dependently decreased ethanol consumption. When drug doses were matched for 70-75 % receptor occupancy, GSK1521498 3 mg/kg, i.p., caused a 2.5-fold greater reduction in alcohol consumption than naltrexone 0.1 mg/kg, s.c. Both GSK1521498 and naltrexone significantly reduced sucrose consumption at a dose of 1 mg/kg but not 0.1 mg/kg. In a test of conditioned taste aversion, GSK1521498 (3 mg/kg) reduced sucrose consumption 24 h following exposure to a conditioning injection. CONCLUSIONS Both opioid receptor antagonists reduced alcohol consumption but GK1521498 has higher intrinsic efficacy than naltrexone

Guidelines for incorporating scientific knowledge and practice on rare diseases into higher education: neuronal ceroid lipofuscinoses as a model disorder model disorder.

This article addresses the educational issues associated with rare diseases (RD) and in particular the Neuronal Ceroid Lipofuscinoses (NCLs, or CLN diseases) in the curricula of Health Sciences and Professional's Training Programs. Our aim is to develop guidelines for improving scientific knowledge and practice in higher education and continuous learning programs. Rare diseases (RD) are collectively common in the general populationwith 1 in 17 people affected by a RDin their lifetime. Inherited defects in genes involved in metabolism are the commonest group of RD with over 8000 known inborn errors of metabolism. The majority of these diseases are neurodegenerative including the NCLs. Any professional training program on NCL must take into account the medical, social and economic burdens related to RDs. To address these challenges and find solutions to themit is necessary that individuals in the government and administrative authorities, academia, teaching hospitals and medical schools, the pharmaceutical industry, investment community and patient advocacy groups all work together to achieve these goals. The logistical issues of including RD lectures in university curricula and in continuing medical education should reflect its complex nature. To evaluate the state of education in the RD field, a summary should be periodically up dated in order to assess the progress achieved in each country that signed up to the international conventions addressing RD issues in society. It is anticipated that auditing current practice will lead to higher standards and provide a framework for those educators involved in establishing RD teaching programs world-wide.publishedVersio

Prevalence of Toxoplasma gondii infection in Myocastor coypus in a protected Italian wetland

<p>Abstract</p> <p>Background</p> <p><it>Toxoplasma gondii </it>is the causative agent for a major zoonosis with cosmopolitan distribution. Water has been implicated in outbreaks of toxoplasmosis in recent years. Coypus (<it>Myocastor coypus</it>), commonly nutria, are large semi-aquatic invasive rodents, naturalized throughout European countries, including most wetlands of Central Italy. The habitat of these animals is both terrestrial and aquatic, making them a species highly exposed to the parasite.</p> <p>Findings</p> <p>The occurrence of the infection was evaluated using a modified agglutination test (MAT) in 74 adult coypus from a naturalized population living in a wetland of Central Italy. Nested PCR (n-PCR) assay was carried out on some of them. Positive <it>T. gondii </it>MAT results were found in 44 animals (59·4%), 30 males (68·2%) and 14 females (31·8%). Antibody titers were ranging from 20 to 40960, while 12 out of 23 (52·2%), examined animals, 8 males (66·7%) and 4 females (33·3%), resulted positive to n-PCR. All n-PCR positive animals were seropositive, showing antibody titers ranging from 640 to 40960.</p> <p>Conclusions</p> <p>Our results indicate that examined animals are heavily parasitized with <it>Toxoplasma</it>. This suggests that coypus could be a reservoir of this parasite, because they can be eaten both by scavenger animals and by humans, and that these animals would play a role in maintaining the cycle of <it>T. gondii</it>.</p