Evolution of lysine-specific demethylase 1 and REST corepressor gene families and their molecular interaction.

Lysine-specific demethylase 1A (LSD1) binds to the REST corepressor (RCOR) protein family of corepressors to erase transcriptionally active marks on histones. Functional diversity in these complexes depends on the type of RCOR included, which modulates the catalytic activity of the complex. Here, we studied the duplicative history of the RCOR and LSD gene families and analyzed the evolution of their interaction. We found that RCOR genes are the product of the two rounds of whole-genome duplications that occurred early in vertebrate evolution. In contrast, the origin of the LSD genes traces back before to the divergence of animals and plants. Using bioinformatics tools, we show that the RCOR and LSD1 interaction precedes the RCOR repertoire expansion that occurred in the last common ancestor of jawed vertebrates. Overall, we trace LSD1-RCOR complex evolution and propose that animal non-model species offer advantages in addressing questions about the molecular biology of this epigenetic complex.

Type 1 Corticotropin-Releasing Factor Receptor Differentially Modulates Neurotransmitter Levels in the Nucleus Accumbens of Juvenile versus Adult Rats.

Adversity is particularly pernicious in early life, increasing the likelihood of developing psychiatric disorders in adulthood. Juvenile and adult rats exposed to social isolation show differences in anxiety-like behaviors and significant changes in dopamine (DA) neurotransmission in the nucleus accumbens (NAc). Brain response to stress is partly mediated by the corticotropin-releasing factor (CRF) system, composed of CRF and its two main receptors, CRF-R1 and CRF-R2. In the NAc shell of adult rats, CRF induces anxiety-like behavior and changes local DA balance. However, the role of CRF receptors in the control of neurotransmission in the NAc is not fully understood, nor is it known whether there are differences between life stages. Our previous data showed that infusion of a CRF-R1 antagonist into the NAc of juvenile rats increased DA levels in response to a depolarizing stimulus and decreased basal glutamate levels. To extend this analysis, we now evaluated the effect of a CRF-R1 antagonist infusion in the NAc of adult rats. Here, we describe that the opposite occurred in the NAc of adult compared to juvenile rats. Infusion of a CRF-R1 antagonist decreased DA and increased glutamate levels in response to a depolarizing stimulus. Furthermore, basal levels of DA, glutamate, and γ-Aminobutyric acid (GABA) were similar in juvenile animals compared to adults. CRF-R1 protein levels and localization were not different in juvenile compared to adult rats. Interestingly, we observed differences in the signaling pathways of CRF-R1 in the NAc of juveniles compared to adult rats. We propose that the function of CRF-R1 receptors is differentially modulated in the NAc according to life stage.

Revealing RCOR2 as a regulatory component of nuclear speckles.

BACKGROUND: Nuclear processes such as transcription and RNA maturation can be impacted by subnuclear compartmentalization in condensates and nuclear bodies. Here, we characterize the nature of nuclear granules formed by REST corepressor 2 (RCOR2), a nuclear protein essential for pluripotency maintenance and central nervous system development. RESULTS: Using biochemical approaches and high-resolution microscopy, we reveal that RCOR2 is localized in nuclear speckles across multiple cell types, including neurons in the brain. RCOR2 forms complexes with nuclear speckle components such as SON, SRSF7, and SRRM2. When cells are exposed to chemical stress, RCOR2 behaves as a core component of the nuclear speckle and is stabilized by RNA. In turn, nuclear speckle morphology appears to depend on RCOR2. Specifically, RCOR2 knockdown results larger nuclear speckles, whereas overexpressing RCOR2 leads to smaller and rounder nuclear speckles. CONCLUSION: Our study suggests that RCOR2 is a regulatory component of the nuclear speckle bodies, setting this co-repressor protein as a factor that controls nuclear speckles behavior.

Cross-talk between dopamine D1 and corticotropin releasing factor type 2 receptors leads to occlusion of their ERK1/2 signaling.

One manner in which G protein-coupled receptors potentiate, increase, and change their functionality is through the formation of heteromers in a specific cellular context. Previously, we have shown that dopamine D1 receptor (D1R) and the corticotropin releasing factor receptor type-2α (CRF2α) heteromerize in HEK293T cells, enabling D1R to mobilize intracellular calcium in response to D1R agonists. In this study, we further investigated the pharmacological properties of the CRF2α-D1R heteromer and the consequences of the heteromerization in their signaling and subcellular localization when both receptors are co-expressed in HEK293T cells. Using immunoprecipitation assays, we observed that the addition of 10 µM dopamine in the incubation medium significantly decreased the amount of CRF2α on the cell surface of cells expressing both receptors. The presence of agonists of both receptors increased the interaction between CRF2α and D1R as assessed by co-immunoprecipitation. However, the presence of agonists of both receptors resulted in a lesser efficient activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase. Using a synaptosomal preparation of rat prefrontal cortex devoid of post-synaptic elements, we found that CRF2α and D1R co-localize in synaptic terminals of the rat medial prefrontal cortex and that the simultaneous activation of both receptors also occluded phosphorylation of extracellular signal-regulated kinase. These results strengthen the idea that the heteromer CRF2a-D1R is an entity functionally different from each receptor that composes it and suggests that its formation is enhanced by CRF and dopamine co-transmission, as occurs in stress and addiction.

[Estimation of time detection limit for human cytochrome b in females of Lutzomyia evansi].

INTRODUCTION: Molecular biology techniques have allowed a better knowledge of sources of blood meals in vector insects. However, the usefulness of these techniques depends on both the quantity of ingested blood and the digestion process in the insect. OBJECTIVE: To identify the time limit for detection of the human cytochrome b (Cyt b) gene in experimentally fed females of Lutzomyia evansi. MATERIALS AND METHODS: Eight groups of L. evansi females were fed on human blood and sacrificed at intervals of 24 hours post-ingestion. Total DNA was extracted from each female and a segment of 358 bp of Cyt b was amplified. In order to eliminate false positives, amplification products were subjected to a restriction fragment length polymorphism (RFLP) analysis. RESULTS: The human Cyt b gene segment was detected in 86% (49/57) of the females of L. evansi, from 0 to 168 hours after blood ingestion. In 7% (4/57) of the individuals we amplified insect DNA, while in the remaining 7%, the band of interest was not amplified. We did not find any statistical differences between groups of females sacrificed at different times post-blood meal regarding the amplification of the human Cyt b gene segment or the number of samples amplified. CONCLUSION: The human Cyt b gene segment was detectable in L. evansi females up to 168 hours after blood ingestion.

Estimación del tiempo límite de detección del gen citocromo b de humanos en hembras de Lutzomyia evansi / Estimation of time detection limit for human cytochrome b in females of Lutzomyia evansi

Resumen Introducción. Las técnicas de biología molecular han permitido ampliar el conocimiento sobre las fuentes de ingestión de sangre de los insectos vectores. Sin embargo, la utilidad de estas técnicas depende de la cantidad de sangre ingerida y del proceso de digestión en el insecto. Objetivo. Determinar el tiempo límite de detección del gen citocromo b (Cyt b) de humanos en hembras de Lutzomyia evansi alimentadas experimentalmente. Materiales y métodos. Se evaluaron ocho grupos de hembras de L. evansi alimentadas con sangre humana, las cuales fueron sacrificadas en intervalos de 24 horas desde el momento de la ingestión sanguínea. Se extrajo el ADN total de cada hembra y se amplificó un segmento de 358 pb del gen Cyt b. Los productos amplificados fueron sometidos a un análisis de polimorfismos en la longitud de los fragmentos de restricción (Restriction Fragment Length Polymorphism, RFLP), con el fin de descartar falsos positivos. Resultados. El segmento del gen Cyt b de humanos fue detectado en 86 % (49/57) de las hembras de L. evansi a partir de las 0 horas y hasta 168 horas después de la ingestión de sangre. En 7 % (4/57) de los individuos se amplificó el ADN del insecto y en el 7 % restante no se amplificó la banda de interés. No se encontraron diferencias estadísticas en cuanto a la amplificación del segmento del gen Cyt b de humanos ni al número de muestras amplificadas entre los grupos de hembras sacrificadas a distintas horas después de la ingestión. Conclusión. El segmento del gen Cyt b de humanos fue detectable en hembras de L. evansi hasta 168 horas después de la ingestión de sangre.

Abstract Introduction: Molecular biology techniques have allowed a better knowledge of sources of blood meals in vector insects. However, the usefulness of these techniques depends on both the quantity of ingested blood and the digestion process in the insect. Objective: To identify the time limit for detection of the human cytochrome b (Cyt b) gene in experimentally fed females of Lutzomyia evansi. Materials and methods: Eight groups of L. evansi females were fed on human blood and sacrificed at intervals of 24 hours post-ingestion. Total DNA was extracted from each female and a segment of 358 bp of Cyt b was amplified. In order to eliminate false positives, amplification products were subjected to a restriction fragment length polymorphism (RFLP) analysis. Results: The human Cyt b gene segment was detected in 86% (49/57) of the females of L. evansi, from 0 to 168 hours after blood ingestion. In 7% (4/57) of the individuals we amplified insect DNA, while in the remaining 7%, the band of interest was not amplified. We did not find any statistical differences between groups of females sacrificed at different times post-blood meal regarding the amplification of the human Cyt b gene segment or the number of samples amplified. Conclusion: The human Cyt b gene segment was detectable in L. evansi females up to 168 hours after blood ingestion.

Comparison of 16S and COX1 genes mitochondrial regions and their usefulness for genetic analysis of ticks (Acari: Ixodidae).

INTRODUCTION: In recent decades the analysis of mitochondrial genes has been used for population and phylogenetic studies of ticks allowing many advances in their systematics. Mitochondrial ribosomal 16S (16S) subunit is one of the most frequently used among those genes available for tick analysis, whereas cytochrome oxidase gene 1 (COX1) has recently been used and proposed as an alternative to the traditional 16S gene marker.  OBJECTIVE: To evaluate the usefulness of 16S and COX1 in genetic studies of ticks by analyzing sequences of three species commonly found in the Caribbean region of Colombia.  RESULTS: The analysis of both genes sequences allowed us to identify the three species with high levels of confidence and interspecific genetic divergence (19-22%), although only COX1 allowed us to detect intraspecific genetic variability (up to ~0.8%). A substitution saturation analysis indicated that the 16S gene was not saturated with transitions while the COX1 gene showed saturation distances starting at ~17%.  CONCLUSION: Our results indicated that the 16S gene seems to have better features for interspecific phylogenetic analyses because of its high level of genetic divergence and low saturation pattern, while the COX1 gene appears to be more useful for intraspecific genetic variability studies. However, as our study was conducted at a local scale, future studies at different biogeographical scales would help to establish its usefulness in wider and more complex scenarios.

Comparación y utilidad de las regiones mitocondriales de los genes 16S y COX1 para los análisis genéticos en garrapatas (Acari: Ixodidae) / Comparison of 16S and COX1 genes mitochondrial regions and their usefulness for genetic analysis of ticks (Acari: Ixodidae)

Introducción. En las últimas décadas, el análisis de los genes mitocondriales se ha utilizado en los estudios poblacionales y filogenéticos de garrapatas, lo cual ha permitido numerosos avances en la sistemática de estos ácaros. El gen mitocondrial de la subunidad 16S del ARN ribosómico ( 16S ) es uno de los más usados, mientras que el gen mitocondrial de la citocromo oxidasa 1 ( COX1 ) se ha empleado recientemente y se propone como un marcador genético alternativo frente al 16S . Objetivo. Evaluar la utilidad de los genes 16S y COX1 en los estudios genéticos de las garrapatas mediante el análisis de secuencias en tres especies de la región Caribe de Colombia. Resultados. El análisis de secuencias mostró que los dos genes permitieron identificar las tres especies con mucha confiabilidad y con niveles de divergencia genética interespecífica relativamente similares (19 a 22 %), aunque solo el gen COX1 permitió detectar la variabilidad genética intraespecífica (hasta de ˜0,8 %). El análisis de saturación de sustituciones indicó que el gen 16S no se saturó con transiciones, mientras que el COX1 mostró saturación a partir de distancias de ˜17 %. Conclusión. Los resultados indicaron que el gen 16S parece tener mejores características para los análisis filogenéticos interespecíficos dada su alta divergencia genética y baja saturación de transiciones, mientras que el gen COX1 parece ser más útil para estudios de variabilidad genética intraespecífica. Sin embargo, dado que el estudio se hizo a escala local, se requieren más investigaciones en diferentes escalas biogeográficas para establecer su utilidad en circunstancias más amplias y complejas.

Introduction: In recent decades the analysis of mitochondrial genes has been used for population and phylogenetic studies of ticks allowing many advances in their systematics. Mitochondrial ribosomal 16S ( 16S ) subunit is one of the most frequently used among those genes available for tick analysis, whereas cytochrome oxidase gene 1 ( COX1 ) has recently been used and proposed as an alternative to the traditional 16S gene marker. Objective: To evaluate the usefulness of 16S and COX1 in genetic studies of ticks by analyzing sequences of three species commonly found in the Caribbean region of Colombia. Results: The analysis of both genes sequences allowed us to identify the three species with high levels of confidence and interspecific genetic divergence (19-22%), although only COX1 allowed us to detect intraspecific genetic variability (up to ˜0.8%). A substitution saturation analysis indicated that the 16S gene was not saturated with transitions while the COX1 gene showed saturation distances starting at ˜17%. Conclusion: Our results indicated that the 16S gene seems to have better features for interspecific phylogenetic analyses because of its high level of genetic divergence and low saturation pattern, while the COX1 gene appears to be more useful for intraspecific genetic variability studies. However, as our study was conducted at a local scale, future studies at different biogeographical scales would help to establish its usefulness in wider and more complex scenarios.

Evidence for anthropophily in five species of phlebotomine sand flies (Diptera: Psychodidae) from northern Colombia, revealed by molecular identification of bloodmeals.

Identification of the bloodmeal sources of phlebotomine sand flies is fundamental to determining which species are anthropophilic and understanding the transmission of Leishmania parasites in natural epidemiological settings. The objective of this study was to identify sand fly bloodmeals in the mixed leishmaniasis focus of the department of Sucre, northern Colombia. In all 141 engorged female sand flies were analyzed, after being captured in intradomiciliary, peridomiciliary and extradomiciliary habitats with Shannon and CDC traps and by active searching in diurnal resting sites. Bloodmeals were identified by sequencing and analysis of a 358bp fragment of the mitochondrial gene Cytochrome b (CYB) and a 330bp fragment of the nuclear gene prepronociceptin (PNOC). Using both genes 105 vertebrate bloodmeals were identified, with an efficiency of 72% for CYB but only 7% for PNOC. Ten species of vertebrates were identified as providing bloodmeal sources for 8 sand fly species: Homo sapiens (Lutzomyia evansi, Lutzomyia panamensis, Lutzomyia micropyga, Lutzomyia shannoni and Lutzomyia atroclavata), Equus caballus (L. evansi, L. panamensis and Lutzomyia cayennensis cayennensis), Equus asinus (L. evansi and L. panamensis), Bos taurus (L. evansi, L. panamensis and L. c. cayennensis), Tamandua mexicana (L. shannoni and Lutzomyia trinidadensis), Proechimys guyanensis (L. evansi, L. panamensis and L. c. cayennensis), Mabuya sp. (Lutzomyia micropyga), Anolissp. (L. micropyga), Sus scrofa (L. evansi and Lutzomyia gomezi) and Gallus gallus (L. evansi). Cattle, donkeys, humans and pigs were significantly more important than other animals (P=0.0001) as hosts of L. evansi, this being the most abundant sand fly species. The five Lutzomyia species in which blood samples of human origin were detected included L. micropyga and L. atroclavata, constituting the first evidence of anthropophily in both species.