Sistema electrónico para cuantificar poblaciones de insectos voladores y evaluar atrayentes y repelentes

Número de publicación: ES2235575 A1 (01.07.2005) También publicado como: ES2235575 B1 (01.11.2006) Número de Solicitud: Consulta de Expedientes OEPM (C.E.O.) P200202622 (07.11.2002)Esta invención se refiere a un sistema electrónico que permite hacer una evaluación cuantitativa de la cantidad de insectos voladores que intentan atravesar una determina zona del espacio. La zona en donde se realiza el seguimiento de los insectos que intentan atravesarla es una superficie preferentemente plana, donde se ha situado una serie de electrodos conductores, conectados a una fuente de alto voltaje. La finalidad del sistema es proporcionar, un pulso eléctrico cada vez que sea electrocutado un insecto y este pulso eléctrico tiene unas características que permiten que pueda ser aprovechado y procesado por otros sistemas electrónicos, tales como contadores, autómatas programables, microcontroladores, o ser acoplado a los buses de entrada de un ordenador.Universidad de Almerí

Transgenic Overexpression of Myocilin Leads to Variable Ocular Anterior Segment and Retinal Alterations Associated with Extracellular Matrix Abnormalities in Adult Zebrafish

Myocilin is an enigmatic glaucoma-associated glycoprotein whose biological role remains incompletely understood. To gain novel insight into its normal function, we used transposon-mediated transgenesis to generate the first zebrafish line stably overexpressing myocilin [Tg(actb1:myoc-2A-mCherry)]. qPCR showed an approximately four-fold increased myocilin expression in transgenic zebrafish embryos (144 hpf). Adult (13 months old) transgenic animals displayed variable and age-dependent ocular anterior segment alterations. Almost 60% of two-year-old male, but not female, transgenic zebrafish developed enlarged eyes with severe asymmetrical and variable abnormalities in the anterior segment, characterized by corneal limbus hypertrophy, and thickening of the cornea, iris, annular ligament and lens capsule. The most severe phenotype presented small or absent ocular anterior chamber and pupils, due to iris overgrowth along with dysplastic retinal growth and optic nerve hypertrophy. Immunohistochemistry revealed increased presence of myocilin in most altered ocular tissues of adult transgenic animals, as well as signs of retinal gliosis and expanded ganglion cells and nerve fibers. The preliminary results indicate that these cells contributed to retinal dysplasia. Visual impairment was demonstrated in all old male transgenic zebrafish. Transcriptomic analysis of the abnormal transgenic eyes identified disrupted expression of genes involved in lens, muscular and extracellular matrix activities, among other processes. In summary, the developed transgenic zebrafish provides a new tool to investigate this puzzling protein and provides evidence for the role of zebrafish myocilin in ocular anterior segment and retinal biology, through the influence of extracellular matrix organization and cellular proliferation.This research was funded by research grants from the “Instituto de Salud Carlos III/European Regional Development Fund (ERDF)” (PI19/00208 and RD16/0008/0019, OFTARED), the Regional Ministry of Science and Technology of the Board of the Communities of “Castilla-La Mancha” (SBPLY/17/180501/000404; http://www.educa.jccm.es/idiuniv/es, accessed on 3 March 2022) and research funds from Universidad de Castilla-La Mancha (2019-GRIN-26945). A.T. was recipient of a predoctoral contract from Castilla-La Mancha University (Ref.: 2020-PREDUCLM-16605)

Role of GUCA1C in Primary Congenital Glaucoma and in the Retina: Functional Evaluation in Zebrafish

Primary congenital glaucoma (PCG) is a heterogeneous, inherited, and severe optical neuropathy caused by apoptotic degeneration of the retinal ganglion cell layer. Whole-exome sequencing analysis of one PCG family identified two affected siblings who carried a low-frequency homozygous nonsense GUCA1C variant (c.52G > T/p.Glu18Ter/rs143174402). This gene encodes GCAP3, a member of the guanylate cyclase activating protein family, involved in phototransduction and with a potential role in intraocular pressure regulation. Segregation analysis supported the notion that the variant was coinherited with the disease in an autosomal recessive fashion. GCAP3 was detected immunohistochemically in the adult human ocular ciliary epithelium and retina. To evaluate the ocular effect of GUCA1C loss-of-function, a guca1c knockout zebrafish line was generated by CRISPR/Cas9 genome editing. Immunohistochemistry demonstrated the presence of GCAP3 in the non-pigmented ciliary epithelium and retina of adult wild-type fishes. Knockout animals presented up-regulation of the glial fibrillary acidic protein in Müller cells and evidence of retinal ganglion cell apoptosis, indicating the existence of gliosis and glaucoma-like retinal damage. In summary, our data provide evidence for the role of GUCA1C as a candidate gene in PCG and offer new insights into the function of this gene in the ocular anterior segment and the retina.This research was funded by research grants from the “Instituto de Salud Carlos III/European Regional Development Fund (ERDF)” (PI15/01193, PI19/00208 and RD16/0008/0019, OFTARED), the Regional Ministry of Science and Technology of the Board of the Communities of “Castilla-La Mancha” (SBPLY/17/180501/000404; http://www.educa.jccm.es/idiuniv/es). SA-M was sponsored by the Regional Ministry of Science and Technology of the Board of the Communities of “Castilla-La Mancha” (PREJCCM2016/28)

The Role of hsa-miR-548l Dysregulation as a Putative Modifier Factor for Glaucoma-Associated FOXC1 Mutations

Mutations of the FOXC1 transcription factor are involved in a variety of autosomal dominant ocular anterior segment defects, ranging from Axenfeld-Rieger malformations to isolated glaucoma in some patients. In this study we have evaluated the possible role of the c.*734A>T FOXC1 variant as a modifier factor of the activity of two FOXC1 mutations previously identified in families primarily affected by dominant glaucoma (haplotypes p.G447_G448insDG-c.*734A>T and p.I126S-c.*734A>T). Previous bioinformatic analyses indicated that the c.*734A>T variant is located in a potential target sequence for hsa-miR-548l. Co-expression of this miRNA with a reporter cDNA construct in which the wild-type 3’UTR sequence of FOXC1 was fused to the 3’-end of the firefly luciferase coding region, led to approximately 20% decreased luciferase activity compared to the controls, confirming the presence of a target sequence for hsa-miR-548l. In contrast, this miRNA did not show any effect on the luciferase activity associated with the mutant 3’UTR FOXC1 sequence, showing that it resulted in a loss-of-function of the has-miR-548l target sequence. In addition, functional evaluation of the two glaucoma-associated haplotypes revealed increased protein levels and transactivation, compared to the corresponding individual coding mutations (approximately 1.2-fold on average). These data support the role of hsa-miR-548l as a regulator of FOXC1 translation and provide evidence for the c.*734A>T variant as a modifier factor for the activity of coding glaucoma-associated FOXC1 mutations

Knockout of myoc Provides Evidence for the Role of Myocilin in Zebrafish Sex Determination Associated with Wnt Signalling Downregulation

Myocilin is a secreted glycoprotein with a poorly understood biological function and it is mainly known as the first glaucoma gene. To explore the normal role of this protein in vivo we developed a myoc knockout (KO) zebrafish line using CRISPR/Cas9 genome editing. This line carries a homozygous variant (c.236_239delinsAAAGGGGAAGGGGA) that is predicted to result in a loss-of-function of the protein because of a premature termination codon p.(V75EfsX60) that resulted in a significant reduction of myoc mRNA levels. Immunohistochemistry showed the presence of myocilin in wild-type embryonic (96 h post-fertilization) anterior segment eye structures and caudal muscles. The protein was also detected in different adult ocular and non-ocular tissues. No gross macroscopic or microscopic alterations were identified in the KO zebrafish, but, remarkably, we observed absence of females among the adult KO animals and apoptosis in the immature juvenile gonad (28 dpf) of these animals, which is characteristic of male development. Transcriptomic analysis showed that adult KO males overexpressed key genes involved in male sex determination and presented differentially expressed Wnt signalling genes. These results show that myocilin is required for ovary differentiation in zebrafish and provides in vivo support for the role of myocilin as a Wnt signalling pathway modulator. In summary, this myoc KO zebrafish line can be useful to investigate the elusive function of this protein, and it provides evidence for the unexpected function of myocilin as a key factor in zebrafish sex determination

CPAMD8 loss-of-function underlies non-dominant congenital glaucoma with variable anterior segment dysgenesis and abnormal extracellular matrix

Abnormal development of the ocular anterior segment may lead to a spectrum of clinical phenotypes ranging from primary congenital glaucoma (PCG) to variable anterior segment dysgenesis (ASD). The main objective of this study was to identify the genetic alterations underlying recessive congenital glaucoma with ASD (CG-ASD). Next-generation DNA sequencing identified rare biallelic CPAMD8 variants in four patients with CG-ASD and in one case with PCG. CPAMD8 is a gene of unknown function and recently associated with ASD. Bioinformatic and in vitro functional evaluation of the variants using quantitative reverse transcription PCR and minigene analysis supported a loss-of-function pathogenic mechanism. Optical and electron microscopy of the trabeculectomy specimen from one of the CG-ASD cases revealed an abnormal anterior chamber angle, with altered extracellular matrix, and apoptotic trabecular meshwork cells. The CPAMD8 protein was immunodetected in adult human ocular fluids and anterior segment tissues involved in glaucoma and ASD (i.e., aqueous humor, non-pigmented ciliary epithelium, and iris muscles), as well as in periocular mesenchyme-like cells of zebrafish embryos. CRISPR/Cas9 disruption of this gene in F0 zebrafish embryos (96 hpf) resulted in varying degrees of gross developmental abnormalities, including microphthalmia, pharyngeal maldevelopment, and pericardial and periocular edemas. Optical and electron microscopy examination of these embryos showed iridocorneal angle hypoplasia (characterized by altered iris stroma cells, reduced anterior chamber, and collagen disorganized corneal stroma extracellular matrix), recapitulating some patients’ features. Our data support the notion that CPAMD8 loss-of-function underlies a spectrum of recessive CG-ASD phenotypes associated with extracellular matrix disorganization and provide new insights into the normal and disease roles of this gene

Identificación de genes candidatos en glaucoma congénito mediante secuenciación masiva de exomas: papel del gen GPATCH3 en glaucoma congénito y en desarrollo craneofacial

El glaucoma congénito es una neuropatía óptica severa y hereditaria que se origina por un desarrollo anómalo del segmento anterior del ojo. Genéticamente, esta enfermedad es heterogénea y su etiología apenas se conoce. Con el objetivo de identificar nuevos genes implicados en la enfermedad, se realizó la secuenciación del exoma de 26 pacientes sin parentesco entre ellos. Se identificaron tres genes candidatos (LRP2, GUCA1C y GPATCH3) en un total de 4 pacientes. De los tres genes candidatos identificados, GPATCH3, un gen de función desconocida, fue seleccionado como el mejor candidato para realizar ensayos funcionales. Se identificaron dos variantes raras, presentes en heterocigosis compuesta y localizadas en la región codificante del este gen en uno de los pacientes. En este trabajo se ha comprobado que la proteína GPATCH3 recombinante activa in vitro el promotor proximal del gen CXCR4, que codifica un receptor de quimiocinas involucrado en la migración de células embrionarias de la cresta neural. Las mutaciones identificadas en el paciente resultaron ser hipermorfas, aumentando en aproximadamente un 17% la transactivación de CXCR4. La proteína GPATCH3 se detectó en tejidos del segmento anterior del ojo humano relevantes en glaucoma, como el cuerpo ciliar. La búsqueda de variaciones en la secuencia de GPATCH3 reveló la presencia de variantes raras de este gen en el 5% de los sujetos de una cohorte formada por 170 pacientes de glaucoma congénito sin parentesco entre ellos. Mediante hibridación in situ e inmunohistoquímica en embriones tempranos de pez cebra se comprobó que el gen ortólogo en pez cebra gpatch3 se expresa en la dermis, musculo esquelético, células del mesénquima periocular y endotelio corneal. Tanto la inhibición transitoria de la expresión de este gen mediante morfolinos como su sobreexpresión mediante la microinyección de mRNA, produjeron diferentes grados de goniodisgenesis, así como anomalías oculares y craneofaciales similares a las encontradas en los peces cebra con expresión reducida de los genes implicados en glaucoma pitx2 y foxc1. En conclusión, los resultados de este trabajo indican la existencia de una alta heterogeneidad genética en glaucoma congénito, sugieren que una alteración funcional leve de GPATCH3 podría producir glaucoma por posibles defectos en la migración de las células derivadas de la cresta neural y revelan que este gen codifica una nueva proteína implicada en el desarrollo embrionario ocular y craneofacial. Los resultados de este trabajo también sugieren fuertemente que el glaucoma congénito no es una enfermedad monogénica simple

Role of FOXC2 and PITX2 rare variants associated with mild functional alterations as modifier factors in congenital glaucoma.

Congenital glaucoma (CG) is a severe and inherited childhood optical neuropathy that leads to irreversible visual loss and blindness in children. CG pathogenesis remains largely unexplained in most patients. Herein we have extended our previous studies to evaluate the role of FOXC2 and PITX2 variants in CG. Variants of the proximal promoter and transcribed sequence of these two genes were analyzed by Sanger sequencing in a cohort of 133 CG families. To investigate possible oligogenic inheritance involving FOXC2 or PITX2 and CYP1B1, we also analyzed FOXC2 and PITX2 variants in a group of 25 CG cases who were known to carry CYP1B1 glaucoma-associated genotypes. The functional effect of three identified variants was assessed by transactivation luciferase reporter assays, protein stability and subcellular localization analyses. We found eight probands (6.0%) who carried four rare FOXC2 variants in the heterozygous state. In addition, we found an elevated frequency (8%) of heterozygous and rare PITX2 variants in the group of CG cases who were known to carry CYP1B1 glaucoma-associated genotypes, and one of these PITX2 variants arose de novo. To the best of our knowledge, two of the identified variants (FOXC2: c.1183C>A, p.(H395N); and PITX2: c.535C>A, p.(P179T)) have not been previously identified. Examination of the genotype-phenotype correlation in this group suggests that the presence of the infrequent PITX2 variants increase the severity of the phenotype. Transactivation reporter analyses showed partial functional alteration of three identified amino acid substitutions (FOXC2: p.(C498R) and p.(H395N); PITX2: p.(P179T)). In summary, the increased frequency in PCG patients of rare FOXC2 and PITX2 variants with mild functional alterations, suggests they play a role as putative modifier factors in this disease further supporting that CG is not a simple monogenic disease and provides novel insights into the complex pathological mechanisms that underlie CG

Hypo- and Hypermorphic <i>FOXC1</i> Mutations in Dominant Glaucoma: Transactivation and Phenotypic Variability

<div><p>Dominant glaucoma, a heterogeneous, infrequent and irreversible optic neuropathy, is often associated with elevated intraocular pressure and early-onset. The role of <i>FOXC1</i> in this type of glaucoma was investigated in twelve Spanish probands via nucleotide variation screening of its proximal promoter and unique exon. Functional evaluations of the identified variants included analyses of the transcriptional activity, protein stability, DNA binding ability and subcellular localization. Four different mutations that were identified in four probands (33.3%) were associated with remarkable phenotypic variability and were functionally classified as either hypermorphic (p.Y47X, p.Q106X and p.G447_G448insDG) or hypomorphic (p.I126S) alleles. To the best of our knowledge, three of the variants are novel (p.Y47X, p.I126S and p.G447_G448insDG) and, in addition, hypermorphic <i>FOXC1</i> mutations are reported herein for the first time. The presence of an intact N-terminal activation domain in the truncated proteins p.Y47X and p.Q106X may underlie their associated transactivation hyperactivity by a gain-of-function mechanism involving dysregulated protein-protein interactions. Similarly, altered molecular interactions may also lead to increased p.G447_G448insDG activity. In contrast, the partial loss-of-function associated with p.I126S was due to impaired protein stability, DNA binding, protein phosphorylation and subcellular distribution. These results support that moderate and variable <i>FOXC1</i> transactivation changes are associated with moderate goniodysgenesis, dominant glaucoma and remarkable phenotypic variability.</p></div

<i>FOXC1</i> mutation segregation in families with autosomal dominant glaucoma.

<p><b>(A, B, C and D)</b> Pedigrees of the families. The numbers in the inferior left side of the symbols indicate age at diagnosis. The oblique lines represent dead subjects. Black, dark grey and light grey symbols indicate glaucoma, Axenfeld-Rieger anomaly and ocular hypertension, respectively. The arrows in the pedigrees show the index cases. +: wild-type allele. The insets in each panel correspond to the electropherograms of the <i>FOXC1</i> mutations identified in each family. To facilitate the comparison, the wild-type sequence is shown above the corresponding mutant sequence. All the mutations were detected in the heterozygous state. Arrows in the electropherograms indicate the location of mutations. Duplicated nucleotides in panel <b>D</b> inset are indicated by boxes.</p