RESUMEN

BACKGROUND: Cannabidiol (CBD) is one of the main cannabinoids present in Cannabis sativa female flowers. Previous investigation has already provided insights into the CBD molecular mechanism; however, there is no transcriptome data for CBD effects on hippocampal subfields. Here, we investigate transcriptomic changes in dorsal and ventral CA1 of adult mice hippocampus after 100 mg/kg of CBD administration (i.p.) for one or seven consecutive days. METHODS: C57BL/6JUnib mice were treated with either vehicle or CBD for 1 or 7 days. The collected brains were sectioned, and the hippocampal sub-regions were laser microdissected for RNA-Seq analysis. RESULTS: The transcriptome analysis following 7 days of CBD administration indicates the differential expression of 1559 genes in dCA1 and 2924 genes in vCA1. Furthermore, GO/KEGG analysis identified 88 significantly enriched biological process and 26 significantly enriched pathways for dCBD7, whereas vCBD7 revealed 128 enriched BPs and 24 pathways. CONCLUSION: This dataset indicates a widespread decrease of electron transport chain and ribosome biogenesis transcripts in CA1, while chromatin modifications and synapse organization transcripts were increased following CBD administration for 7 days.

Asunto(s)

Cannabidiol , Hipocampo , Ratones Endogámicos C57BL , Mitocondrias , Ribosomas , Sinapsis , Cannabidiol/farmacología , Animales , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Ratones , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Ribosomas/genética , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/genética , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Transcriptoma/efectos de los fármacos , Masculino , Cromatina/efectos de los fármacos , Cromatina/metabolismo , Cromatina/genética , Región CA1 Hipocampal/efectos de los fármacos , Región CA1 Hipocampal/metabolismo , Perfilación de la Expresión Génica/métodos

RESUMEN

The hippocampus comprises several neuronal populations such as CA1, CA2, CA3, and the dentate gyrus (DG), which present different neuronal origins, morphologies, and molecular mechanisms. Laser capture microdissection (LCM) allows selectively collecting samples from target regions and eliminating unwanted cells to obtain more specific results. LCM of hippocampus neuronal populations coupled with RNA-seq analysis has the potential to allow the exploration of the molecular machinery unique to each of these subfields. Previous RNA-seq investigation has already provided a molecular blueprint of the hippocampus, however, there is no RNA-seq data specific for each of the rat hippocampal regions. Serial tissue sections covering the hippocampus were produced from frozen brains of adult male Wistar rats, and the hippocampal subfields CA1, CA2, CA3, and DG were identified and isolated by LCM. We found evident segregation of the transcriptomic profile from different regions of the hippocampus and the expression of known, as well as novel, specific marker genes for each region. Gene ontology enrichment analysis of CA1 subfield indicates an enrichment of actin regulation and postsynaptic membrane AMPA receptors genes indispensable for long-term potentiation. CA2 and CA3 transcripts were found associated with the increased metabolic processes. DG expression was enriched for ribosome and spliceosome, both required for protein synthesis and maintenance of cell life. The present findings contribute to a deeper understanding of the differences in the molecular machinery expressed by the rat hippocampal neuronal populations, further exploring underlying mechanisms responsible for each subflied specific functions.

RESUMEN

Laser Capture Microdissection (LCM) is a method that allows one to select and dissect well-defined structures, specific cell subpopulations, or even single cells from different types of tissue for subsequent extraction of DNA, RNA, or proteins. Its precision allows the dissection of specific groups of cells, avoiding unwanted cells. However, despite its efficiency, several steps can affect the sample RNA integrity. RNA instability represents a challenge in the LCM method, and low RNA integrity can introduce biases, as different transcripts often have different degradation rates. Here we describe an optimized protocol to provide good-concentration and high-quality RNA from specific structures: dentate gyrus and CA1 in the hippocampus, basolateral amygdala, and anterior cingulate cortex of mouse brain tissue. However, the protocol is applicable to other areas of interest. © 2022 Wiley Periodicals LLC. Basic Protocol: Laser capture microdissection of mouse brain tissue.

Asunto(s)

Encéfalo , ARN , Animales , Captura por Microdisección con Láser/métodos , Ratones , ARN/genética , Estabilidad del ARN

RESUMEN

Hepatocellular carcinoma expressing hepatobiliary progenitor markers, is considered of poor prognosis. By using a hepatocarcinogenesis model, laser capture microdissection, and RNA-Sequencing analysis, we identified an expression profile in GGT/KRT19-positive experimental tumors; 438 differentially expressed genes were found in early and late nodules along with increased collagen deposition. Dysregulated genes were involved in Fatty Acid Metabolism, RXR function, and Hepatic Stellate Cells Activation. Downregulation of Slc27a5, Acsl1, and Cyp2e1, demonstrated that Retinoid X Receptor α (RXRα) function is compromised in GGT/KRT19-positive nodules. Since RXRα controls NRF2 pathway activation, we determined the expression of NRF2 targeted genes; Akr1b8, Akr7a3, Gstp1, Abcc3, Ptgr1, and Txnrd1 were upregulated, indicating NRF2 pathway activation. A comparative analysis in human HCC showed that SLC27A5, ACSL1, CYP2E1, and RXRα gene expression is mutually exclusive with KRT19 gene expression. Our results indicate that the downregulation of Slc27a5, Acsl1, Rxrα, and Cyp2e1 genes is an early event within GGT/KRT19-positive HCC.

Asunto(s)

Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/metabolismo , Ácidos Grasos , Humanos , Neoplasias Hepáticas/metabolismo , Receptor alfa X Retinoide/genética , Receptor alfa X Retinoide/metabolismo , Transcriptoma

RESUMEN

Mesial temporal lobe epilepsy (MTLE) is a chronic neurological disorder characterized by the occurrence of seizures, and histopathological abnormalities in the mesial temporal lobe structures, mainly hippocampal sclerosis (HS). We used a multi-omics approach to determine the profile of transcript and protein expression in the dorsal and ventral hippocampal dentate gyrus (DG) and Cornu Ammonis 3 (CA3) in an animal model of MTLE induced by pilocarpine. We performed label-free proteomics and RNAseq from laser-microdissected tissue isolated from pilocarpine-induced Wistar rats. We divided the DG and CA3 into dorsal and ventral areas and analyzed them separately. We performed a data integration analysis and evaluated enriched signaling pathways, as well as the integrated networks generated based on the gene ontology processes. Our results indicate differences in the transcriptomic and proteomic profiles among the DG and the CA3 subfields of the hippocampus. Moreover, our data suggest that epileptogenesis is enhanced in the CA3 region when compared to the DG, with most abnormalities in transcript and protein levels occurring in the CA3. Furthermore, our results show that the epileptogenesis in the pilocarpine model involves predominantly abnormal regulation of excitatory neuronal mechanisms mediated by N-methyl D-aspartate (NMDA) receptors, changes in the serotonin signaling, and neuronal activity controlled by calcium/calmodulin-dependent protein kinase (CaMK) regulation and leucine-rich repeat kinase 2 (LRRK2)/WNT signaling pathways.

Asunto(s)

Epilepsia del Lóbulo Temporal , Animales , Epilepsia del Lóbulo Temporal/patología , Hipocampo/metabolismo , Pilocarpina/toxicidad , Proteómica , Ratas , Ratas Wistar

RESUMEN

To make accurate determinations regarding potential and actual impact of HPV vaccine programs, precise estimates of genotype-specific contributions to disease are required for pre- and post-vaccine populations. Definitive determination of lesion-specific genotypes, particularly where multiple genotypes are detected in a sample, can be technically demanding and resource intensive; therefore, most prevalence studies use mathematical algorithms to adjust for multiple genotype detections. There are currently several algorithms, which can produce genotype estimates within a wide range of variability. The use of these for cervical cytology samples has recently been assessed for accuracy against a definitive reference standard, but none have yet been assessed for multiple-genotype-containing whole biopsy specimens. Using laser capture microdissection (LCM) on biopsy samples, lesion-specific genotype prevalence data were generated for a cohort of 516 young Australian women (aged 18-32 years) with cervical intraepithelial neoplasia grade 3 or adenocarcinoma in situ. Using whole tissue section genotype data from the same cohort, including 71 (13.7%) with multiple genotypes, lesion-associated genotype prevalence was estimated using four different attribution algorithms. The proportion of lesions attributable to HPV16 and HPV18 by LCM were 58.4% and 5%, respectively; hierarchical, proportional, single type/minimum and any type/maximum attribution estimates were comparable across genotypes. For analyses utilising whole tissue biopsy cervical specimens, attribution estimates are appropriate for estimating the proportional contribution of individual genotypes to lesions in a population.

Asunto(s)

Infecciones por Papillomavirus , Displasia del Cuello del Útero , Neoplasias del Cuello Uterino , Adolescente , Adulto , Algoritmos , Australia , Biopsia , Femenino , Genotipo , Humanos , Captura por Microdisección con Láser , Papillomaviridae/genética , Infecciones por Papillomavirus/epidemiología , Prevalencia , Estándares de Referencia , Neoplasias del Cuello Uterino/epidemiología , Adulto Joven

RESUMEN

INTRODUCTION: Ischemia-reperfusion (I/R) injury of the liver is a common area of interest to transplant and hepatic surgery. Nevertheless, most of the current knowledge of I/R of the liver derives from the hepatocyte and little is known of what happens to the cholangiocytes. Herein, we assess the sequence of early events involved in the I/R injury of the cholangiocytes. METHODS: Sixty Wistar rats were randomized in a SHAM group and I/R group. Serum biochemistry, histopathology, immunohistochemistry, transmission electron microscopy (TEM) and laser capture microdissection (LCM) were used for group comparison. RESULTS: There was peak of alkaline phosphatase 24 h after IR injury, and an increase of aspartate aminotransferase and alanine aminotransferase after 6 h of reperfusion, followed by a return to normal levels 24 h after injury. The I/R group presented the liver parenchyma with hepatocellular degeneration up to 6 h, followed by hepatocellular necrosis at 24 h. TEM showed cholangiocyte injury, including a progressive nuclear degeneration and cell membrane rupture, beginning at 6 h and peaking at 24 h after reperfusion. Cytokeratin-18 and caspase-3-positive areas were observed in the I/R group, peaking at 24-h reperfusion. Anti-apoptotic genes Bcl-2 and Bcl-xl activity were expressed from 6 through 24 h after reperfusion. BAX expression showed an increase for 24 h. CONCLUSIONS: I/R injury to the cholangiocyte occurs from 6 through 24 h after reperfusion and a combination of TEM, immunohistochemistry and LCM allows a better isolation of the cholangiocyte and a proper investigation of the events related to the I/R injury. Apoptosis is certainly involved in the I/R process, particularly mediated by BAX.

RESUMEN

Arbuscular mycorrhizal fungi (AMF) colonization in plants promotes both local and systemic changes in the gene expression profiles of the host that might be relevant for drought-stress perception and response. Drought-tolerant common bean plants (cv. BAT 477), colonized by a mixture of AMF (Glomus clarum, Acaulospora scrobiculata, and Gigaspora rosea), were exposed to a water deprivation regime of 96 h during pre-flowering. Root transcriptomes were accessed through RNA-Seq revealing a set of 9,965 transcripts with significant differential regulation in inoculated plants during a water deficit event, and 10,569 in non-inoculated. These data include 1,589 transcripts that are exclusively regulated by AMF-inoculation, and 2,313 under non-inoculation conditions. Relative gene expression analyses of nine aquaporin-related transcripts were performed in roots and leaves of plants harvested at initial stages of treatment. Significant shifts in gene expression were detected in AM water deficit-treated roots, in relation to non-inoculated, between 48 and 72 h. Leaves also showed significant mycorrhizal influence in gene expression, especially after 96 h. Root cortical cells, harboring or not arbuscules, were collected from both inoculation treatments through a laser microdissection-based technique. This allowed the identification of transcripts, such as the aquaporin PvPIP2;3 and Glucan 1,3 ß-Glucosidase, that are unique to arbuscule-containing cells. During the water deficit treatment, AMF colonization exerted a fine-tune regulation in the expression of genes in the host. That seemed to initiate in arbuscule-containing cells and, as the stressful condition persisted, propagated to the whole-plant through secondary signaling events. Collectively, these results demonstrate that arbuscular mycorrhization leads to shifts in common bean's transcriptome that could potentially impact its adaptation capacity during water deficit events.

RESUMEN

Several observational studies have showed a combination of lower testosterone (T) to LH ratio and higher estradiol (E2 ) to T ratio in secretory infertile men compared to men with normal spermatogenesis, suggesting a steroidogenic dysfunction of Leydig cells (Lc) that may involve increased aromatase activity. Low T/LH ratio is associated with Lc hyperplasia, which together with LH hyperstimulation may represent compensation for impaired T production. Aromatase expression and oestrogen production are mainly detected in Lc of the testis, although Sertoli and germ cells also contribute to testicular aromatase activity. The aim of this study was to assess the transcriptional expression of CYP19A1 (aromatase) in isolated Lc of subjects with Sertoli cell-only syndrome (SCOS) and signs of Lc impairment. Nineteen patients with SCOS and 10 controls with normal spermatogenesis who had medical indication of testicular biopsy for sperm retrieval were studied. Leydig cells were isolated by Laser Capture Microdissection (LCM) and CYP19A1 mRNA expression was quantified by SYBR® Green-based qPCR. In addition, testicular T and E2 and serum hormonal levels were measured. Relative to control group, CYP19A1 was overexpressed more than twofold in 10/19 cases (2.3-12.2-fold increase), showing a significant increment in cases with low T/LH ratio (T/LH < 2) compared to cases with T/LH ≥ 2 (p = 0.038, REST® ). Moreover, Rq data for CYP19A1 had a direct correlation with testicular levels of E2 and the E2 /T ratio (r = 0.869; p < 0.001 and r = 0.633; p = 0.005). In summary, Lc from infertile patients with signs of Lc dysfunction overexpressed aromatase and showed an increment of testicular E2 . Our results suggest that increased expression of aromatase in Lc leads to higher E2 production and may account for the functional impairment of the Lc in patients with SCOS.

Asunto(s)

Aromatasa/genética , Células Intersticiales del Testículo/metabolismo , Síndrome de Sólo Células de Sertoli/genética , Testículo/metabolismo , Adulto , Aromatasa/metabolismo , Estradiol/sangre , Hormona Folículo Estimulante/sangre , Humanos , Captura por Microdisección con Láser , Hormona Luteinizante/sangre , Masculino , Síndrome de Sólo Células de Sertoli/metabolismo , Espermatogénesis/genética , Testosterona/sangre

RESUMEN

Whole-transcriptome evaluation by next-generation sequencing (NGS) has been widely applied in the investigation of diverse transcriptional scenarios. In many clinical situations, including needle biopsy samples or laser microdissected cells, limited amounts of RNA are usually available for the assessment of the whole transcriptome. Here, we describe an mRNA amplification protocol based on in vitro T7 transcription for transcriptome evaluation by NGS. Initially, we performed RNAseq from two human mammary epithelial cell lines and evaluated several aspects of the transcriptomes generated by linear amplification of Poly (A)(+) mRNA species, including transcript representation, variability and abundance. Our protocol showed to be efficient with respect to full-length transcript coverage and quantitative expression levels. We then evaluated the applicability of using this protocol in a more realistic research scenario, analyzing tumor tissue samples microdissected by laser capture. In order to increase the quantification power of the libraries only the 3' end of transcripts were sequenced. We found highly reproducible RNAseq data among amplified tumor samples, with a median Spearman's correlation of 80%, strongly suggesting that the amplification step and library protocol preparation lead to a consistent transcriptional profile. Altogether, we established a robust protocol for assessing the polyadenylated transcriptome derived from limited amounts of total RNA that is applicable to all NGS platforms.

Asunto(s)

Perfilación de la Expresión Génica/métodos , Análisis de Secuencia de ARN/métodos , Adenocarcinoma/genética , Adenocarcinoma/patología , Neoplasias de la Mama/genética , Línea Celular , Humanos , Masculino , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , ARN Mensajero/análisis , ARN Mensajero/genética

RESUMEN

γ-Glutamyl transferase (GGT) is useful as a marker in pathological conditions, including several types of cancer. We optimized the histochemical detection of GGT to assay the gene expression profiles of phenotype-specific cells selected by laser capture microdissection (LCM). For optimization, we used the livers of rats subjected to hepatocarcinogenesis. This model induced nodules of hepatocytes and tumors with GGT activity. To obtain sufficient high-quality RNA after histochemistry and LCM, we included an RNase inhibitor and air-dried the tissue sections. This optimization allowed the visualization of GGT activity in situ and a yield of 1.4 to 2.0 µg of total RNA from 15 to 18 mm² of microdissected tissue (20 µm thickness). The average RNA integrity number in GGT-positive tissue, determined by chip-capillary electrophoresis, was 6.9, and the 28S/18S ribosomal RNA (rRNA) ratio was 1.4. The RNAs were processed for the Rat Gene 1.0 ST Array (Affymetrix). Comparable quality control metrics, such as signal intensity and RNA degradation plots, were found between the LCM samples and non-LCM tissue. The increased expression of Ggt1 expected in GGT-positive tissue was confirmed by microarrays and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). This optimization provided a suitable method for whole-transcript analysis of GGT-positive tissue isolated using LCM.

Asunto(s)

Perfilación de la Expresión Génica/métodos , Inmunohistoquímica/métodos , Captura por Microdisección con Láser/métodos , gamma-Glutamiltransferasa/metabolismo , Animales , Hígado/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , gamma-Glutamiltransferasa/genética

RESUMEN

Fibropapillomatosis (FP), a transmissible neoplastic disease of marine turtles characterized by a likely herpesviral primary etiology, has emerged as an important disease in green sea turtles (Chelonia mydas) over the past three decades. The objectives of this study were to determine the suitability of three different chelonid fibropapilloma-associated herpesvirus (CFPHV) gene targets in polymerase chain reaction (PCR) assays of affected tissues; to explore the presence of CFPHV in non-affected skin from turtles with and without tumors; and to better understand tissue localization of the CFPHV genome in a tumor-free turtle by evaluating CFPHV presence in microanatomic tissue sites. Two aggregations of green sea turtles (Chelonia mydas) in Puerto Rico were evaluated, with six sampling intervals over the three-year period 2004-2007. Primary and nested PCR for three different herpesviral gene targets- DNA polymerase, capsid maturation protease, and membrane glycoprotein B- were performed on 201 skin biopsies taken from 126 turtles with and without external tumors. Laser capture microdissection and nested PCR were used to identify tissue localizations of CFPHV in skin from a normal turtle. Of the turtles sampled in Manglar Bay, 30.5% had tumors; at the relatively more pristine Culebrita, 5.3% of turtles sampled had tumors. All three PCR primer combinations successfully amplified CFPHV from tumors, and from normal skin of both tumored and tumor-free turtles. Via nested PCR, the polymerase gene target proved superior to the other two gene targets in the positive detection of CFPHV DNA. CFPHV infection may be common relative to disease incidence, supporting the idea that extrinsic and/or host factors could play a transforming role in tumor expression. Laser capture microdissection revealed CFPHV in skin from a tumor-free turtle, harbored in both epidermal and dermal tissues. Identification of CFPHV harbored in a non-epidermal site (dermis) of a tumor-free turtle indicates that virus is latent in a non-tumored host.