Prenatal Δ9-Tetrahydrocannabinol Exposure in Males Leads to Motivational Disturbances Related to Striatal Epigenetic Dysregulation.

BACKGROUND: Cannabis remains one of the most widely abused drugs during pregnancy. In utero exposure to its principal psychoactive component, Δ9-tetrahydrocannabinol (THC), can result in long-term neuropsychiatric risk for the progeny. This study investigated epigenetic signatures underlying these enduring consequences. METHODS: Rat dams were exposed daily to THC (0.15 mg/kg) during pregnancy, and adult male offspring were examined for reward and depressive-like behavioral endophenotypes. Using unbiased sequencing approaches, we explored transcriptional and epigenetic profiles in the nucleus accumbens (NAc), a brain area central to reward and emotional processing. An in vitro CRISPR (clustered regularly interspaced short palindromic repeats) activation model coupled with RNA sequencing was also applied to study specific consequences of epigenetic dysregulation, and altered molecular signatures were compared with human major depressive disorder transcriptome datasets. RESULTS: Prenatal THC exposure induced increased motivation for food, heightened learned helplessness and anhedonia, and altered stress sensitivity. We identified a robust increase specific to males in the expression of Kmt2a (histone-lysine N-methyltransferase 2A) that targets H3K4 (lysine 4 on histone H3) in cellular chromatin. Normalizing Kmt2a in the NAc rescued the motivational phenotype of prenatally THC-exposed animals. Comparison of RNA- and H3K4me3-sequencing datasets from the NAc of rat offspring with the in vitro model of Kmt2a upregulation revealed overlapping, significant disturbances in pathways that mediate synaptic plasticity. Similar transcriptional alterations were detected in human major depressive disorder. CONCLUSIONS: These studies provide direct evidence for the persistent effects of prenatal cannabis exposure on transcriptional and epigenetic deviations in the NAc via Kmt2a dysregulation and associated psychiatric vulnerability.

Walter Thiel's embalming method: review of solutions and applications in different fields of biomedical research / Método de embalsamamiento de Walther Thiel: revisión de las soluciones y sus aplicaciones en diferentes campos de investigación biomédica

Walter Thiel developed the method that enables preservation of the body with natural colors in 1992. It consists in the application of an intravascular injection formula, and maintaining the corps submerged for a determinate period of time in the immersion solution in the pool. After immersion, it is possible to maintain the corps in a hermetically sealed container, thus avoiding dehydration outside the pool. The aim of this work was to review the Thiel method, searching all scientific articles describing this technique from its development point of view, and application in anatomy and morphology teaching, as well as in clinical and surgical practice. Most of these studies were carried out in Europe. We used PubMed, Ebsco and Embase databases with the terms "Thiel cadaver", "Thiel embalming", "Thiel embalming method" and we searched for papers that cited Thiel`s work. In comparison with methods commonly used with high concentrations of formaldehyde, this method lacks the emanation of noxious or irritating gases; gives the corps important passive joint mobility without stiffness; maintaining color, flexibility and tissue plasticity at a level equivalent to that of a living body. Furthermore, it allows vascular repletion at the capillary level. All this makes for great advantage over the formalin-fixed and fresh material. Its multiple uses are applicable in anatomy teaching and research; teaching for undergraduates (prosection and dissection) and for training in surgical techniques for graduates and specialists (laparoscopies, arthroscopies, endoscopies).

En 1992, Walter Thiel desarrolló el método que permite la preservación del cuerpo con colores naturales. Consiste en la aplicación de una fórmula de inyección intravascular y el mantenimiento del cuerpo sumergido en pileta, en una solución de inmersión específica, durante un período determinado de tiempo. Después de la inmersión, es posible mantener el cuerpo en un recipiente herméticamente sellado, evitando así la pérdida del líquido fijador, fuera de la pileta. El objetivo de este trabajo fue revisar el método de Thiel, buscando todos los artículos científicos que describen esta técnica desde el punto de vista de su desarrollo, y su aplicación en la enseñanza de la anatomía y morfología, así como en la práctica clínica y quirúrgica. La mayoría de estos estudios se realizaron en Europa. Utilizamos las bases de datos PubMed, Ebsco y Embase con los términos "Thiel cadaver", "Thiel embalsamamiento", "método de embalsamamiento de Thiel" y se buscaron los documentos que citan el trabajo de Thiel. En comparación con los métodos comúnmente utilizados con altas concentraciones de formaldehído, este método carece de emanación de gases nocivos o irritantes; Da al cuerpo una movilidad articular pasiva importante sin rigidez; Manteniendo el color, la flexibilidad y la plasticidad del tejido a un nivel equivalente al de un cuerpo vivo. Además, permite la repleción vascular a nivel capilar. Todo esto hace una gran ventaja sobre el material fijado con formalina y fresco. Sus usos múltiples son aplicables en la enseñanza y la investigación de la anatomía; (prosección y disección) y para la formación en técnicas quirúrgicas para graduados y especialistas (laparoscopias, artroscopias, endoscopias).

Angiotensin-II acute regulation of rapid response genes in human, bovine, and rat adrenocortical cells.

Angiotensin-II (Ang-II) regulates adrenal steroid production and gene transcription through several signaling pathways. Changes in gene transcription occur within minutes after Ang-II stimulation, causing an increase in aldosterone production and subsequent increase in the overall capacity to produce aldosterone. Our goal was to compare the Ang-II regulation of early gene expression and confirm the up-regulation of selected genes using quantitative real-time RT-PCR (qPCR) across three species, such as, human, bovine, and rat. Microarray analyses were performed using samples from control and Ang-II (10 nM)-treated (1 h) cells from human adrenocortical tumor cell line H295R, and primary adrenal glomerulosa cells from bovine and rat, applied respectively to human, bovine, and rat chips. qPCR was performed to confirm up-regulation of selected genes using mRNA. The microarray comparison revealed 18% similarity among the top 50 up-regulated genes, with human/rat, 20%; human/bovine, 36%; and rat/bovine, 26% similarity. The gene list generated by this comparison included: activating transcription factor 3, B-cell translocation gene (BTG2), Nuclear receptor subfamily 4, group A, member 1 (NR4A1), NR4A2, NR4A3, early growth response 1, v-fos FBJ murine osteosarcoma viral oncogene homolog (c-FOS), FOSB, and Jun family member B (JUNB). Pretreatment of H295R cells with cycloheximide had no effect on Ang-II induction of these genes, suggesting that they are direct targets of Ang-II signaling. The Ang-II gene targets have been defined in three different adrenocortical model systems. Several of the listed genes have previously been described as being key regulators of adrenocortical function. The presence of adrenal cell common genes in such distinct cell models strengthens the hypothesis that these genes are regulators of aldosterone production.

Epimerase-deficiency galactosemia is not a binary condition.

Epimerase-deficiency galactosemia results from the impairment of UDP-galactose 4'-epimerase (GALE), the third enzyme in the Leloir pathway of galactose metabolism. Originally identified as a clinically benign "peripheral" condition with enzyme impairment restricted to circulating blood cells, GALE deficiency was later demonstrated also to exist in a rare but clinically severe "generalized" form, with enzyme impairment affecting a range of tissues. Isolated cases of clinically and/or biochemically intermediate cases of epimerase deficiency have also been reported. We report here studies of 10 patients who, in the neonatal period, received the diagnosis of hemolysate epimerase deficiency. We have characterized these patients with regard to three parameters: (1) GALE activity in transformed lymphoblasts, representing a "nonperipheral" tissue, (2) metabolic sensitivity of those lymphoblasts to galactose challenge in culture, and (3) evidence of normal versus abnormal galactose metabolism in the patients themselves. Our results demonstrate two important points. First, whereas some of the patients studied exhibited near-normal levels of GALE activity in lymphoblasts, consistent with a diagnosis of peripheral epimerase deficiency, many did not. We detected a spectrum of GALE activity levels ranging from 15%-64% of control levels, demonstrating that epimerase deficiency is not a binary condition; it is a continuum disorder. Second, lymphoblasts demonstrating the most severe reduction in GALE activity also demonstrated abnormal metabolite levels in the presence of external galactose and, in some cases, also in the absence of galactose. These abnormalities included elevated galactose-1P, elevated UDP-galactose, and deficient UDP-glucose. Moreover, some of the patients themselves also demonstrated metabolic abnormalities, both on and off galactose-restricted diet. Long-term follow-up studies of these and other patients will be required to elucidate the clinical significance of these biochemical abnormalities and the potential impact of dietary intervention on outcome.

Both KH and non-KH domain sequences are required for polyribosome association of Scp160p in yeast.

Scp160p is a 160 kDa RNA-binding protein in yeast previously demonstrated to associate with specific messages as an mRNP component of both soluble and membrane-bound polyribosomes. Although the vast majority of Scp160p sequence consists of 14 closely spaced KH domains, comparative sequence analyses also demonstrate the presence of a potential nuclear localization sequence located between KH domains 3 and 4, as well as a 110 amino acid non-KH N-terminal region that includes a potential nuclear export sequence (NES). As a step toward investigating the structure/function relationships of Scp160p, we generated two truncated alleles, FLAG.SCP160DeltaN1, encoding a protein product that lacks the first 74 amino acids, including the potential NES, and FLAG.SCP160DeltaC1, encoding a protein product that lacks the final KH domain (KH14). We report here that the N-truncated protein, expressed as a green fluorescent protein fusion in yeast, remains cytoplasmic, with no apparent nuclear accumulation. Biochemical studies further demonstrate that although the N-truncated protein remains competent to form RNPs, the C-truncated protein does not. Furthermore, polyribosome association is severely compromised for both truncated proteins. Perhaps most important, both truncated alleles appear only marginally functional in vivo, as demonstrated by the inability of each to complement scp160/eap1 synthetic lethality in a tester strain. Together, these data challenge the notion that Scp160p normally shuttles between the nucleus and cytoplasm, and further implicate polyribosome association as an essential component of Scp160p function in vivo. Finally, these data underscore the vital roles of both KH and non-KH domain sequences in Scp160p.

Genetic and biochemical interactions between SCP160 and EAP1 in yeast.

Scp160p is a multiple KH-domain RNA-binding protein in yeast known to associate with polyribosomes as an mRNP component, although its biological role remains unclear. As a genetic approach to examine Scp160p function, we applied an ethyl methanesulfonate (EMS) screen for loci synthetically lethal with scp160 loss, and identified a single candidate gene, EAP1, whose protein product functions in translation as an eIF4E-binding protein, with additional uncharacterized spindle pole body functions. To reconfirm scp160/eap1 synthetic lethality, we constructed a strain null for both genes, supported by an SCP160 maintenance plasmid. We used this strain to establish a quantitative assay for both Scp160p and Eap1p functions in vivo, and applied this assay to demonstrate that Y109A EAP1, a previously described allele of EAP1 that cannot bind eIF4E, is markedly impaired with regard to its SCP160-related activity. In addition, we explored the possibility of physical interaction between Eap1p and Scp160p, and discovered that Eap1p associates with Scp160p-containing complexes in an RNA-dependent manner. Finally, we probed the impact of EAP1 loss on Scp160p, and vice versa, and found that loss of each gene resulted in a significant change in either the complex associations or subcellular distribution of the other protein. These results clearly support the hypothesis that Scp160p plays a role in translation, demonstrate that the interaction of SCP160 and EAP1 is biologically significant, and provide important tools for future studies of the in vivo functions of both genes.