Genetic dissection of Hoxb1 function in the developing mouse auditory and vestibular system rhombomere 4-derived

Rhombomere (r4) and Hox associated genes Hoxb1 and Hoxb2 contribute to the formation of specific auditory and vestibular subcircuits. In particular, sensory and motor components of the sound transmission pathway, sensorimotor reflex circuits, as well as the hindlimb vestibule-spinal reflex, derive from r4 and are strongly affected in Hoxb1 mutants (Di Bonito et al., 2013). Inner ear efferent (IEE) neurons also originate from r4 and form vestibular (VEN) and cochlear (CEN) efferent neuron subpopulations. The CEN is further subdivided into medial (MOC) and lateral (LOC) olivo-cochlear motoneurons. MOC neurons inhibit the motility of outer hair cells (OHCs), which amplify low intensity sounds, while LOCs innervate the afferent terminations on the inner hair cells (IHC) modulating the excitability of the cochlear nerve, thus protecting the cochlea from acoustic damage. Hoxb1null mutants lack MOC and LOC efferent neurons leading to defects in OHCs and in cochlear amplification, and mice have increasing auditory thresholds (Di Bonito et al., 2013). Our hypothesis is that MOC neuron endings play a trophic function on OHCs and that the physical interaction between MOC efferents and OHCs is essential for proper maturation and functioning of OHCs. To exclude a possible contribute of sensory auditory neurons at this phenotype, we performed analysis of conditional Hoxb1 mutants (Ptf1acre Hoxb1 flox/flox, Atoh1cre Hoxb1 flox/flox) where Hoxb1 expression were eliminated in the dorsal region of rhombomere 4 and the sensory structures involved in the acoustic pathway, were selective affected. Our data show that when the ventral domain, where the motoneurons MOC and LOC develop, normally expresses Hoxb1 the OHCs show a regular morphology, even if Hoxb1 expression is affected in the dorsal sensory auditory neurons. So, our data suggest that MOCs could play an important postnatal role but to confirm this hypothesis further investigations are needed using a Cre specific line that selectively eliminate the Hoxb1 expression in the ventral domain of motoneurons. Regarding the vestibular system, Hoxb1null mutant mice also fail to form the VEN at early developmental stages (Di Bonito et al., 2015). However, transmission electron microscopy (TEM) in adult mice reveals the presence of both afferent and efferent neuronal endings on receptor cells. To understand whether projections are missing at birth and new connections gradually appear during the first month by compensatory plasticity mechanisms, we analyzed newborn mutant pups for the presence of efferent endings on hair cells by TEM. Our analysis showed that this mutant fail to develop efferent terminations corroborating our hypothesis that these endings derive from other district and could act as a compensatory mechanism. So, to further confirm this hypothesis we aim to use retrograde labelling in 3-month old mutant mice to assess their eventual presence and identify their origin

Primera comunciación del virus israelí de la parálisis aguda en colmenas asintomáticas de la República Argentina

Honey bee mortality has recently been associated with Israeli acute paralysis virus (IAPV), a proposed etiological agent for a new syndrome known as Colony Collapse Disorder. Bees infected with this virus show shivering wings, progress into paralysis, and finally die outside the hive. During the last years, honey bee mortality became a serious problem for Argentinean beekeepers. We herein report the preliminary results of a survey carried out to detect IAPV in samples taken from several Argentine provinces, by using a reverse transcription Polymerase Chain Reaction assay. Our data indicate the existence of high frequency of IAPV in asymptomatic hives of Argentina.Recientemente la mortalidad de las abejas melíferas ha sido asociada al virus israelí de la parálisis aguda (IAPV), propuesto como agente etiológico del denominado síndrome de despoblamiento de las colmenas. Las abejas infectadas con este virus presentan temblores en las alas que progresan hasta convertirse en parálisis, y finalmente mueren fuera de la colmena. Durante los últimos años, la mortalidad de las abejas melíferas se ha transformado en un serio problema para los productores de miel de la Argentina. Nosotros informamos aquí los resultados preliminares de un estudio realizado para detectar IAPV en muestras de colmenas provenientes de varias provincias argentinas utilizando la técnica de transcripción reversa-reacción en cadena de la polimerasa. Nuestros datos indican la presencia de IAPV en un alto porcentaje de las colonias estudiadas.Fil: Reynaldi, Francisco José. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Agrarias y Forestales. Departamento de Ciencias Biológicas. Centro de Investigaciones de Fitopatología. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Centro de Investigaciones de Fitopatología; ArgentinaFil: Sguazza, Guillermo Hernán. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; ArgentinaFil: Tizzano, Marco Antonio. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; ArgentinaFil: Fuentealba, Nadia Analia. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Galosi, Cecilia Monica. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; ArgentinaFil: Pecoraro, Marcelo Ricardo. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Gross anatomy and ultrastructure of Moorish Gecko, Tarentola mauritanica skin

The epidermis of Tarentola mauritanica in the skin regions of back, flank and belly has been described using light and electron microscopy. This animal model was useful to give an insight of the functional pattern involved in pigmentation, cryptism and photosensitivity. Skin from back and flanks, in electron microscopy, shows a high concentration of chromatophores, among those melanophores, xanthophores and iridophores have been reported. Interestingly, in the flank-back transition region electron microscopy reveals the presence of nerve endings. Our contribution adds new knowledge about the skin of this species, and it could be useful to study in deep the mechanism of cryptic colour change in reptiles

HOXB1 AND HOXB2 ARE INVOLVED IN THE DETERMINATION OF THE RHOMBOMERE 4-DERIVED VESTIBULAR SISTEM

The central auditory pathway consists of sensory nuclei that transmit the ascending acoustic information, and efferent motor neurons that modulate primary afferent responses. We have previously shown that rhombomere 4 (r4) contributes to structurally and functionally linked sensory afferent and motor efferent components of the central auditory system, and that in the absence of Hoxb1 mutant mice have severe hearing problems. We subsequently demonstrated that Hoxb2 and Hoxa2 and their genetic interactions are also necessary for the proper development of r4. The vestibular nuclear complex, in part derives from r4 and consists of a collection of sensory nuclei that integrates and relays information for the coordination of eye movements, balance and posture. We still used Hoxb1 mutant mice to investigate the contribution of r4 in the developmental patterning of vestibular projection neurons, with particular focus on the lateral vestibulo spinal tract (LVST). Retrograde labelling and marker analysis on Hoxb1 mutant embryos and postnatal pups confirmed specific absence of the LVST and of the vestibular efferent neurons (VEN), in accordance with loss of r4 identity and ectopic production of r3 neurons. However, transmission electron microscopy experiments in adult mice show the presence of both afferent and efferent nervous endings. It is thus plausible that in the adult mouse mutant a compensatory mechanism overtaken by other neuronal tracts is able to compensate for the early absence of the vestibular nuclei, in line with a partial rescue of the vestibulo-spinal reflex.2We also analyzed vestibular afferences and efferences of Hoxb2 and Hoxa2 mutant mice and demonstrated presence of afferent and efferent endings. To this purpose, we are in the process of using newborn mutant pups to assess whether these projections are already lost at birth and whether new connections gradually appear during the first month of life

Expresión de la subunidad HA1 del virus de influenza equina utilizando un sistema de expresión en baculovirus

Equine influenza virus is a leading cause of respiratory disease in horses worldwide. Disease prevention is by vaccination with inactivated whole virus vaccines. Most current influenza vaccines are generated in embryonated hens´ eggs. Virions are harvested from allantoic fluid and chemically inactivated. Although this system has served well over the years, the use of eggs as the substrate for vaccine production has several well-recognized disadvantages (cost, egg supply, waste disposal and yield in eggs). The aim of this study was to evaluate a baculovirus system as a potential method for producing recombinant equine influenza hemagglutinin to be used as a vaccine. The hemagglutinin ectodomain (HA1 subunit) was cloned and expressed using a baculovirus expression vector. The expression was determined by SDS-PAGE and immunoblotting. A high yield, 20μg/ml of viral protein, was obtained from recombinant baculovirus-infected cells. The immune response in BALB/c mice was examined following rHA1 inoculation. Preliminary results show that recombinant hemagglutinin expressed from baculovirus elicits a strong antibody response in mice; therefore it could be used as an antigen for subunit vaccines and diagnostic tests.El virus de la infl uenza equina es una de las principales causas de enfermedad respiratoria en caballos de todo el mundo. La prevención de la enfermedad es a través de la vacunación con vacunas a virus inactivado. La mayoría de las vacunas se producen en huevos embrionados, de los cuales los viriones son cosechados del líquido alantoideo e inactivados químicamente. Aunque este sistema ha servido bien durante años, el uso de huevos como sustrato para la producción de vacuna presenta varias desventajas bien reconocidas (costo, provisión de huevos, manejo de los residuos, rinde por huevo). El objetivo del presente trabajo fue evaluar preliminarmente un sistema de expresión en baculovirus como método de producción de hemoaglutinina recombinante (rHA) para ser utilizada como vacuna para la prevención de la infl uenza equina. Para ello el ectodominio de la hemaglutinina (la subunidad HA1) del virus de la infl uenza equina se expresó en células de insecto infectadas con un baculovirus recombinante. La expresión fue demostrada por SDS-PAGE e inmunoblotting. El método empleado fue capaz de producir gran cantidad de rHA1. En este estudio se obtuvieron 20 μg/ml (200 μg de HA1 purifi cada de 2,5x107 células infectadas). La respuesta inmune fue evaluada mediante la inmunización de ratones BALB/c. Los resultados preliminares demostraron que la proteína recombinante expresada en baculovirus genera una fuerte respuesta inmune en ratones, por lo tanto podría ser utilizada como antígeno para la producción de una vacuna a subunidades y en pruebas diagnósticas.Fil: Sguazza, Guillermo Hernán. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Fuentealba, Nadia Analia. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Tizzano, Marco Antonio. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; ArgentinaFil: Galosi, Cecilia Monica. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; ArgentinaFil: Pecoraro, Marcelo Ricardo. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Identification of cellular, functional and genetic causes of Hoxb1null sensorineural hearing loss

The assembly of central auditory subcircuits depends on the spatially and temporally ordered sequence of specification, migration and connectivity. Alterations of one of these events will lead to abnormal circuit formation and different types of deafness. Recessive mutations in the human homeobox HOXB1 gene cause sensorineural hearing impairments characterized by increased auditory threshold and loss of distortion product otoacoustic emissions, mainly due to abnormal cochlear activity of outer hair cells (OHCs) and absence of sound amplification Our studies in mouse show that Hoxb1 is essential for the specification of rhombomere 4 (r4)-derived auditory sensory and motor neurons contributing to the sound transmission pathway and to the establishment of sensorimotor reflex circuits. Hoxb1null mice have abnormal r4-derived sensory nuclei, lack inner ear efferent motor neurons and show degeneration of OHCs. This leads to defective cochlear amplification and altered auditory thresholds that well replicate the hearing loss observed in patients with mutations in the HOXB1 gene. However, the exact mechanisms leading to the auditory impairments described in mice and patients are not known. Hoxb1 is expressed in the medial olivocochlear (MOC) neurons that innervate the OHCs, but not in cochlear hair cells. In Hoxb1null mice, MOC neurons fail to be generated, but morphology of OHCs is not affected until P8, when MOC/OHC interactions normally occur; however, OHCs start to degenerate later when these interactions fail to be established, with consequent appearance of threshold impairments. Interesting, the persistence of few MOC neurons in Hoxb1flox b1r4-Cre mutants, in which Hoxb1 is inactivated only in r4, correlates well with less severe OHC morphology and threshold defects. To directly assess whether the degeneration of OHCs and the increased hearing threshold observed in Hoxb1null mutants depends on the absence of MOC innervation, or alternatively is due to affected sensory r4-derived components, we independently altered Hoxb1 function in r4 motor or sensory neurons. The OC efferent bundles were genetically deleted by using Hoxb1flox Nkx2.2-Cre mutants, in which Hoxb1 is exclusively inactivated in r4 motor neurons, whereas the sensory glutamatergic and GABAergic/glycinergic cochlear populations were affected in Hoxb1flox Atoh1-Cre and Hoxb1flox Ptf1a-Cre mice. By combining anatomical, electrophysiological and molecular analyses, we demonstrated a key role for MOC neurons on OHC survival and sound amplification as the major cause for the Hoxb1 phenotype. Overall, these data show that during a critical postnatal period interactions between MOC neurons and OHCs are crucial for establishing the correct sound amplification

An effective and simplified DO-stat control strategy for production of rabies glycoprotein in Pichia pastoris

The glycoprotein (G-protein) of rabies virus is responsible for viral attachment to the host cell surface and induces virus neutralization antibodies. In the present study, the G-protein gene of rabies virus CVS strain was cloned, sequenced and expressed in the yeast, Pichia pastoris, as a secreted protein, using a simplified DO-stat control feeding strategy. This strategy involves the addition of methanol when the dissolved oxygen (DO) level rises above the setpoint avoiding methanol accumulation and oxygen limitation. The G-protein expression was evaluated by SDS-PAGE, ELISA, and western blot assays. Like native G-protein, the recombinant G-protein was found reactive when it was challenged against specific antibodies. The data indicate that the recombinant G-protein can be easily expressed and isolated, and may be useful as a safe source in the production of diagnostic kits and subunit vaccines to prevent rabies.Fil: Picotto, Leandro Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Sguazza, Guillermo Hernán. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; ArgentinaFil: Tizzano, Marco Antonio. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; ArgentinaFil: Galosi, Cecilia Monica. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; ArgentinaFil: Cavalitto, Sebastian Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Pecoraro, Marcelo Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Microbiología. Cátedra de Virología; Argentin

Abnormal outer hair cell efferent innervation in Hoxb1-dependent sensorineural hearing loss.

Autosomal recessive mutation of HOXB1 and Hoxb1 causes sensorineural hearing loss in patients and mice, respectively, characterized by the presence of higher auditory thresholds; however, the origin of the defects along the auditory pathway is still unknown. In this study, we assessed whether the abnormal auditory threshold and malformation of the sensory auditory cells, the outer hair cells, described in Hoxb1null mutants depend on the absence of efferent motor innervation, or alternatively, is due to altered sensory auditory components. By using a whole series of conditional mutant mice, which inactivate Hoxb1 in either rhombomere 4-derived sensory cochlear neurons or efferent motor neurons, we found that the hearing phenotype is mainly reproduced when efferent motor neurons are specifically affected. Our data strongly suggest that the interactions between olivocochlear motor neurons and outer hair cells during a critical postnatal period are crucial for both hair cell survival and the establishment of the cochlear amplification of sound

Long term exposure to cadmium: Pathological effects on kidney tubules cells in Sparus aurata juveniles

The effects of an exposure to cadmium chloride 0.47 μM for 150 days were studied in kidneys of juveniles Sparus aurata by a multidisciplinary approach so to correlate uptake and detoxification potential to changes in brush border and glycocalyx sugar composition. Results demonstrated that cadmium concentration in kidney significantly increased from day 30 reaching a plateau on day 120 while metallothioneins reached a peak on day 90 and by day 120 were already decreasing to control values. Cytological damage was extensive on day 90, clearly detectable at both structural and ultrastructural levels, in tubular cells and brush-border. Staining with a panel of four lectins revealed a significant increase in N-Ac-Gal and a decrease in mannose in the glycocalyx and the tubular basal membranes. From day 120, when cadmium concentration was high and metallothionein concentration decreasing, a clear recovery was observed in tubular cells morphology and sugar composition. Possible significance of these apparently contrasting data are discussed

Conditional <i>Hoxb1</i> inactivation in r4-derived motor and sensory neurons and recorded audiograms.

(A) Schematics of half of rhombomere 4 (r4) in the developing hindbrain showing DV domain of the activity of the different Cre-recombinase lines used in this study. While the Nkx2.2-Cre line is expressed in ventral r4 from which MOC, LOC, and FMN efferent motor neurons originated, the Atoh1- and Ptf1-Cre lines are expressed in distinct dorsal domains from which the sensory glutamatergic or GABAergic/glycinergic populations, respectively, of the cochlear nucleus (CN) are generated. (B) The genetic strategy used to inactivate Hoxb1 in different domains. In the null group, Hoxb1 is inactivated from the onset of expression; in the conditional CKOr4 mice, Hoxb1 expression is exclusively abolished in r4 from E8.5 onwards. DV-restricted inactivation is obtained by using the CKOAtoh1 and CKOPtf1a mice for CN sensory components or the CKONkx2.2 mice for the motor efferent structures. (C-H) Audiograms from distinct mouse groups are indicated above each graph. Empty circles: mean ABR ± SEM, dashed lines: individual cochleae. n indicates the number of cochleae recorded. See also S1 Data.</p