Investigation and comparison of the regulation of ATOH1 in the mammalian and avian inner ear

Atonal homolog 1 (Atoh1) is a basic helix-loop-helix (bHLH) transcription factor required for the formation of sensory hair cells in the inner ear (Bermingham et al. 1999). Understanding the Atoh1 regulatory network is crucial for the development of new therapies to treat hearing loss. However, to date, little is known about the mechanisms controlling ATOH1 expression. The loss of sensory hair cells can cause deafness and balance disorders. In the mammalian inner ear, the loss of ATOH1 expression in adults has been linked with a limited capacity to regenerate hair cells (Wang et al. 2010). However, in non-mammalian vertebrates, ATOH1 expression re-activates spontaneously after hair cell damage and new hair cells can be formed throughout life (Cafaro et al. 2007) (Daudet et al. 2009). In this thesis, I aimed to identify regulatory elements responsible for ATOH1 expression through a comparative analysis of avian and mammalian ATOH1 gene loci. A bioinformatic approach was used to identify evolutionary conserved non-coding ATOH1 DNA sequences including those that are conserved between avian and mammals and those that are specific to both groups. Putative transcription factor binding sites predicted within these conserved elements were then investigated using EMSA analysis and reporter gene assays. These experiments suggest that the E2F1 transcription factor activates the chick ATOH1 gene and that this activation occurs predominantly via a direct interaction with a regulatory region that is conserved between avian species but absent from mammals. E2F transcription factors control cell cycle progression so the identification of this family as novel regulators of the chick ATOH1 expression links avian ATOH1 re-activation to cell proliferation. If confirmed, this would provide a possible mechanism to explain the different regenerative capabilities of mammalian and non-mammalian sensory cells and therefore contribute to the design of therapies for the regeneration of hair cells after damage

Differential regulation of mammalian and avian ATOH1 by E2F1 and its implication for hair cell regeneration in the inner ear

The mammalian inner ear has a limited capacity to regenerate its mechanosensory hair cells. This lack of regenerative capacity underlies the high incidence of age-related hearing loss in humans. In contrast, non-mammalian vertebrates can form new hair cells when damage occurs, a mechanism that depends on re-activation of expression of the pro-hair cell transcription factor Atoh1. Here, we show that members of the E2F transcription factor family, known to play a key role in cell cycle progression, regulate the expression of Atoh1. E2F1 activates chicken Atoh1 by directly interacting with a cis-regulatory region distal to the avian Atoh1 gene. E2F does not activate mouse Atoh1 gene expression, since this regulatory element is absent in mammals. We also show that E2F1 expression changes dynamically in the chicken auditory epithelium during ototoxic damage and hair cell regeneration. Therefore, we propose a model in which the mitotic regeneration of non-mammalian hair cells is due to E2F1-mediated activation of Atoh1 expression, a mechanism which has been lost in mammals

Record of Cletocamptus sinaloensis (COPEPODA: HARPACTICOIDA: CANTHOCAMPTIDAE) from the caribbean coast of Colombia

Objetivos: Reportar el primer registro de C. sinaloensis para Colombia. Alcance. Presentar una breve descripción de este material para apoyar la identificación del nuevo material encontrado. Metodología: Se tomaron muestras de agua en Ciénaga Grande de Santa Marta Colombia utilizando un balde de 25 L. Las muestras se filtraron con una red estándar de zooplancton (malla con diámetro de poro de 45 μm) y se fijaron y conservaron en etanol al 70%. Los copépodos harpacticoides se separaron con un pincel. Las muestras disecadas y los apéndices se montaron en glicerina. Los apéndices disecados se fotografiaron usando una cámara digital Kodak Easy Share C140 adaptada a un microscopio compuesto con un aumento de 1000X. Resultados principales: Cletocamptus sinaloensis parece estar estrechamente relacionado con C. levis Gómez 2005, conocido en Brasil. La cercanía de estas especies se asumió en función de la forma general y el número de setas del palpo mandibular (un segmentado con tres setas), P2-P4 EXP3 y ENP2 con 5-5-4 y 3-3-2 elementos respectivamente, relación longitud: anchura de la rama caudal, y ornamentación del opérculo anal con dos filas de espínulas fuertes. Sin embargo, pueden separarse por 1) el complemento de armadura del exopodo antenal, 2) la longitud del lóbulo exopodal de la P5 de la hembra, 3) la longitud relativa del P3ENP, 4) el número de setas en la P6 de la hembra. Conclusiones: El espécimen de Colombia tiene las características diagnósticas de C. sinaloensis como se describió originalmente, pero muestra diferencias sutiles en la longitud de P1 ENP, en la forma del receptáculo seminal y en la longitud relativa de P6.Objectives: to report the first record of C. sinaloensis for Colombia. Scope: to present a brief description of this material in order to support the identification of the newly found material. Methodology: Water samples were taken at Ciénaga Grande de Santa Marta Colombia using a 25 L bucket. Samples were filtered with a standard zooplankton net (45 μm mesh) and fixed and preserved in 70% ethanol. Harpacticoid copepods were separated with a brush. Dissected specimens and appendages were mounted in glycerine. The dissected appendages were photographed using a Kodak Easy Share C140 digital camera adapted to a compound microscope at a magnification of 1000X. Main results: Cletocamptus sinaloensis seems to be closely related to C. levis Gómez 2005, known from Brazil. The closeness of these species was assumed based on the general shape and number of setae of the mandibular palp (one-segmented with three setae), P2-P4 EXP3 and ENP2 with 5-5-4 and 3-3-2 elements respectively, length: width ratio of the caudal rami, and ornamentation of the anal operculum with two rows of strong spinules. Nevertheless, they can be separated by 1) the armature complement of the antennal exopod, 2) length of the exopodal lobe of the female P5, 3) relative length of the P3ENP, 4) number of setae on the female P6. Conclusions: The specimen from Colombia bears the diagnostic features of C. sinaloensis as originally described, but shows subtle differences in the length of P1ENP, shape of seminal receptacle and relative length of P6

Flavonoid Phloretin Inhibits Adipogenesis and Increases OPG Expression in Adipocytes Derived from Human Bone-Marrow Mesenchymal Stromal-Cells

Phloretin (a flavonoid abundant in apple), has antioxidant, anti-inflammatory, and glucose-transporter inhibitory properties. Thus, it has interesting pharmacological and nutraceutical potential. Bone-marrow mesenchymal stem cells (MSC) have high differentiation capacity, being essential for maintaining homeostasis and regenerative capacity in the organism. Yet, they preferentially differentiate into adipocytes instead of osteoblasts with aging. This has a negative impact on bone turnover, remodeling, and formation. We have evaluated the effects of phloretin on human adipogenesis, analyzing MSC induced to differentiate into adipocytes. Expression of adipogenic genes, as well as genes encoding OPG and RANKL (involved in osteoclastogenesis), protein synthesis, lipid-droplets formation, and apoptosis, were studied. Results showed that 10 and 20 µM phloretin inhibited adipogenesis. This effect was mediated by increasing beta-catenin, as well as increasing apoptosis in adipocytes, at late stages of differentiation. In addition, this chemical increased OPG gene expression and OPG/RANKL ratio in adipocytes. These results suggest that this flavonoid (including phloretin-rich foods) has interesting potential for clinical and regenerative-medicine applications. Thus, such chemicals could be used to counteract obesity and prevent bone-marrow adiposity. That is particularly useful to protect bone mass and treat diseases like osteoporosis, which is an epidemic worldwide

Coronary and carotid artery dysfunction and KV7 overexpression in a mouse model of Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disease caused by expression of progerin, a lamin A variant that is also expressed at low levels in non-HGPS individuals. Although HGPS patients die predominantly from myocardial infarction and stroke, the mechanisms that provoke pathological alterations in the coronary and cerebral arteries in HGPS remain ill defined. Here, we assessed vascular function in the coronary arteries (CorAs) and carotid arteries (CarAs) of progerin-expressing LmnaG609G/G609G mice (G609G), both in resting conditions and after hypoxic stimulus. Wire myography, pharmacological screening, and gene expression studies demonstrated vascular atony and stenosis, as well as other functional alterations in progeroid CorAs and CarAs and aorta. These defects were associated with loss of vascular smooth muscle cells and overexpression of the KV7 family of voltage-dependent potassium channels. Compared with wild-type controls, G609G mice showed reduced median survival upon chronic isoproterenol exposure, a baseline state of chronic cardiac hypoxia characterized by overexpression of hypoxia-inducible factor 1α and 3α genes, and increased cardiac vascularization. Our results shed light on the mechanisms underlying progerin-induced coronary and carotid artery disease and identify KV7 channels as a candidate target for the treatment of HGPS.Work supported by Ministerio de Ciencia e Innovación (MCIN) and Agencia Estatal de Investigación (AEI) (MCIN/AEI/https:// doi. org/ 10. 13039/ 50110 00110 33 grants SAF2016-79490-R and PID2019-108489RB-I00), with cofunding from Fondo Social Europeo (“El FSE invierte en tu futuro”), and a donation from Asociación Progeria Alexandra Peraut. Microscopy was conducted at the Microscopy & Dynamic Imaging Unit, CNIC, ICTS-ReDib, co-funded by MCIN/AEI/https:// doi. org/ 10. 13039/ 50110 00110 33. R.M.N was supported by the Ministerio de Educación, Cultura y Deporte (pre-doctoral contract FPU16/05027), and I.B. is supported by the Comunidad Autónoma de Madrid (grants 2017- T1/BMD-5247 and 2021-5A/BMD-20944), and the Ramón y Cajal contract (RYC2021-033805-I) funded by MCIN/ AEI/10.13039/501100011033 and the European Union “Next- GenerationEU”/PRTR. The CNIC is supported by the MCIN, the Instituto de Salud Carlos III, the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant number CEX2020-001041-S funded by MCIN/AEI/https:// doi. org/ 10. 13039/ 50110 00110 33).N

Factors Affecting Embryo Recovery Rate, Quality, and Diameter in Andalusian Donkey Jennies

Embryo transfer and the vitrification of embryos could be used for the conservation and recovery of endangered donkey breeds. It is important to develop techniques that optimize recovery rates and the cryotolerance of donkey embryos. This study evaluates factors affecting the recovery rate, quality, and diameter of embryos obtained from donor jennies as a starting point for the use of vitrification and embryo transfer in the conservation of the Andalusian donkey. A total of 100 embryos were recovered out of 124 estrous cycles (80.6%). The donor jenny affected the rates of positive flushings (PFR; p = 0.040) and embryo recovery (ERR; p < 0.05) as well as embryo quality (p = 0.004). ERR was also affected by the number of flushings (p < 0.001), donor age (p < 0.05), successive cycle within donor (p < 0.001), and jacks (p < 0.05). Number of flushings (p < 0.001) and jack (p < 0.05) had a significant effect on PFR, whereas the day of flushing influenced the developmental stage (p < 0.001), embryo quality (p < 0.05), and diameter of embryos (p < 0.001). The number of flushings significantly influenced the diameter (p = 0.038) and embryo developmental stage (p = 0.001), whereas the developmental stage was statistically different between herds (p = 0.020). The factors influencing the success of this assisted reproductive technique were donor jenny, donor age, successive cycle within donor, day of flushing, number of flushings, and jack. The identification of these key points is crucial to achieve a higher efficiency of embryo transfer and vitrification processes, before considering their application in the conservation of endangered donkey breeds

Effect of single layer centrifugation using Androcoll-E-Large on the sperm quality parameters of cooled-stored donkey semen doses

The aim of this study was to determine the effect of single layer centrifugation (SLC) using Androcoll-E-Large on donkey sperm quality parameters after 24 h of cool-storage. Ejaculates were collected from Andalusian donkeys and then cooled at 5°C. SLC was carried out after 24 h of cool-storage using Androcoll-E-Large. In the first experiment, all sperm parameters assessed (total and progressive sperm motility, viability, sperm morphology and sperm kinematics VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF) were statistically compared between semen samples processed or not with Androcoll-E-Large. Significant differences ( P<0.05) were found between SLC-selected and unselected semen samples for all parameters assessed, obtaining better results after SLC. In the second experiment, semen samples were classified in two groups according to their sperm progressive motility (PM) before SLC. Then, the increments obtained in semen quality parameters after SLC were compared between groups. No significant differences were found between groups, indicating that SLC improved the sperm quality parameters of entire set of semen samples processed with independence to their original PM. In conclusion, SLC with Androcoll-E-Large can be used in donkeys, increasing the sperm quality of cooled-stored donkey semen doses after 24 h of cool storag

The Myxococcus xanthus Two-Component System CorSR Regulates Expression of a Gene Cluster Involved in Maintaining Copper Tolerance during Growth and Development

Myxococcus xanthus is a soil-dwelling member of the δ–Proteobacteria that exhibits a complex developmental cycle upon starvation. Development comprises aggregation and differentiation into environmentally resistant myxospores in an environment that includes fluctuations in metal ion concentrations. While copper is essential for M. xanthus cells because several housekeeping enzymes use it as a cofactor, high copper concentrations are toxic. These opposing effects force cells to maintain a tight copper homeostasis. A plethora of paralogous genes involved in copper detoxification, all of which are differentially regulated, have been reported in M. xanthus. The use of in-frame deletion mutants and fusions with the reporter gene lacZ has allowed the identification of a two-component system, CorSR, that modulates the expression of an operon termed curA consisting of nine genes whose expression slowly increases after metal addition, reaching a plateau. Transcriptional regulation of this operon is complex because transcription can be initiated at different promoters and by different types of regulators. These genes confer copper tolerance during growth and development. Copper induces carotenoid production in a ΔcorSR mutant at lower concentrations than with the wild-type strain due to lack of expression of a gene product resembling subunit III of cbb3-type cytochrome c oxidase. This data may explain why copper induces carotenoid biosynthesis at suboptimal rather than optimal growth conditions in wild-type strains.This work has been funded by the Spanish Government (grants CSD2009-00006 and BFU2012-33248, 70% funded by FEDER). This work was also supported by the National Institute of General Medical Science of the National Institutes of Health under award number R01GM095826 to LJS, and by the National Science Foundation under award number MCB0742976 to LJS. JMD and JP received a fellowship from Junta de Andalucía to do some work at University of Georgia

The relative effects of upwelling and river flow on the phytoplankton diversity patterns in the ria of A Coruña (NW Spain)

Phytoplankton species assemblages in estuaries are connected to those in rivers and marine environments by local hydrodynamics leading to a continuous flow of taxa. This study revealed differential effects of upwelling and river flow on phytoplankton communities observed in 2011 along a salinity gradient from a river reservoir connected to the sea through a ria-marine bay system in A Coruña (NW Spain, 43° 16-21’ N, 8° 16-22’ W). With 130 phytoplankton taxa identified, the assemblages were dominated in general by diatoms, particularly abundant in the bay and in the estuary, but also by chlorophycea and cyanobacteria in the reservoir. Considering the entire seasonal cycle, the local assemblages were mainly characterized by changes in cryptophytes and diatoms, small dinoflagellates and some freshwater chlorophycea. Salinity, nitrate, and organic matter variables, were the main environmental factors related to the changes in the phytoplankton communities through the system, while phosphate and nitrite were also important for local communities in the estuary and the bay, respectively. The corresponding local phytoplankton assemblages showed moderate levels of connectivity. The estuarine community shared a variable number of taxa with the adjacent zones, depending on the relative strength of upwelling (major influence from the bay) and river flow (major influence of the reservoir) but had on average 35% of unique taxa. Consequently, local and zonal diversity patterns varied seasonally and were not simply related to the salinity gradient driven by the river flow.ANILE (CTM2009-08396 and CTM2010-08804-E), FIOME (CTM2011-28792-C02-01-MAR), and MEFIO (CTM2011-28792-C02-02-MAR) of the Plan Nacional de I+D+i (Spain), and RADIALES of the Instituto Español de Oceanografía (IEO, Spain).Versión del editor2,01

Exacerbated atherosclerosis in progeria is prevented by progerin elimination in vascular smooth muscle cells but not endothelial cells

Hutchinson-Gilford progeria syndrome (HGPS) is a rare disease caused by the expression of progerin, a mutant protein that accelerates aging and precipitates death. Given that atherosclerosis complications are the main cause of death in progeria, here we investigated whether progerin-induced atherosclerosis is prevented in HGPSrev-Cdh5-CreERT2 and HGPSrev-SM22α-Cre mice with progerin suppression in endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), respectively. HGPSrev-Cdh5-CreERT2 mice were undistinguishable from HGPSrev mice with ubiquitous progerin expression, in contrast with the ameliorated progeroid phenotype of HGPSrev-SM22α-Cre mice. To study atherosclerosis, we generated atheroprone mouse models by overexpressing a PCSK9 gain-of-function mutant. While HGPSrev-Cdh5-CreERT2 and HGPSrev mice developed a similar level of excessive atherosclerosis, plaque development in HGPSrev-SM22α-Cre mice was reduced to wild-type levels. Our studies demonstrate that progerin suppression in VSMCs, but not in ECs, prevents exacerbated atherosclerosis in progeroid mice