An enhancer-AAV toolbox to target and manipulate distinct interneuron subtypes.

In recent years, we and others have identified a number of enhancers that, when incorporated into rAAV vectors, can restrict the transgene expression to particular neuronal populations. Yet, viral tools to access and manipulate fine neuronal subtypes are still limited. Here, we performed systematic analysis of single cell genomic data to identify enhancer candidates for each of the cortical interneuron subtypes. We established a set of enhancer-AAV tools that are highly specific for distinct cortical interneuron populations and striatal cholinergic neurons. These enhancers, when used in the context of different effectors, can target (fluorescent proteins), observe activity (GCaMP) and manipulate (opto- or chemo-genetics) specific neuronal subtypes. We also validated our enhancer-AAV tools across species. Thus, we provide the field with a powerful set of tools to study neural circuits and functions and to develop precise and targeted therapy.

Serum Levels of Galectin-9 are Increased in Cervical Cancer Patients and are Higher in Advanced Clinical Stages.

Purpose: Cervical cancer (CC) is the second most frequent cancer in undeveloped countries. Serum biomarkers could be useful for evaluation of the treatment response and as a complementary means to improve diagnosis. The expression of galectin-9 is altered in cancer tissue, and higher concentrations are found in the serum of cancer patients. The objectives of this study were (a) to determine the serum galectin-9 concentration in patients with intraepithelial lesions and CC, (b) to determine if the concentration was related to the clinicopathological characteristics and (c) to determine if the galectin-9 concentration was related to its expression level in tumour tissue. Patients and Methods: In all, 222 serum samples from women with different diagnoses, including premalignant lesions and CC, as well as samples from women with normal cytology were included in the study. The serum galectin-9 concentration was determined by ELISA. To evaluate the expression level of galectin-9 in CC tissue, immunohistochemistry was performed in 34 CC biopsy specimens. Results: The galectin-9 concentration in the serum of CC patients (8.171 ng/mL) was increased compared with serum from women with normal epithelia (4.654 ng/mL) and those with low-grade (4.806 ng/mL) and high-grade (5.354 ng/mL) intraepithelial lesions (p value < 0.0001). The area under the ROC curve considering the CC group and the control group was 0.882. The optimal cut-off value was ≥6.88 ng/mL, the specificity obtained was 100%, and the sensitivity was 68.2%. In the CC group, the analysis of the clinical stage showed an increase of galectin-9 in the advanced stage IV group. Serum galectin-9 was not related to the level of galectin-9 expression in tissue, which suggests that galectin-9 is not secreted by tumour cells. Conclusion: The serum galectin-9 concentration is related to cancer progression, as the level of this protein is higher in patients with advanced-stage disease.

Identification and characterization of the V3 promoter of the ST3GAL4 gene.

The ST3GAL4 gene encodes the enzyme Galß1-4GlcNAc α2,3 sialyltransferase (ST3Gal IV). This enzyme participates in the synthesis of the sialyl Lewis x antigen. In different cancer types altered expression of this antigen has been reported. The transcriptional regulation of this gene is very complex, different mRNA variants (V1-V10) have been reported and are originated by the activity of different promoters and alternative splicing. Only the promoter that gives rise to the V3 variant has not been previously reported. The objective of this work was to identify and characterize the V3 promoter of the ST3GAL4 gene. For this, the putative V3 promoter of the ST3GAL4 gene was delimited by in silico analysis. The complete promoter and smaller versions were cloned in a reporter plasmid. The constructs were transfected in the HaCaT cells and the promoter activity was evaluated by luciferase reporter assays. The cloned region showed promoter activity, and the basal activity was not dependent on TATA boxes. However, the GC boxes, an initiator element (Inr) and downstream promoter element (DPE) could contribute to basal activity. The promoter contains several binding sites for the nuclear factor of activated T-cells (NFAT) that could participate in inducible activity during the immune response. The minimal promoter corresponds to a fragment of approximately 300 bp, located in the position -347 b to -40 b. The characterization of the V3 promoter of the ST3GAL4 gene completes the study of the four promoters of this gene, this contributes to the understanding of its complex transcription regulation.

Histone H3K9 and H3K14 acetylation at the promoter of the LGALS9 gene is associated with mRNA levels in cervical cancer cells.

Galectin-9 levels have been reported to be altered in several cancer types, but the mechanism that regulates the expression of Galectin-9 has not been clarified. Galectin-9 is encoded by the LGALS9 gene, which gives rise to eight mRNA variants. The aims of this study were: (a) to identify the mRNA variants of LGALS9, (b) to characterize CpG methylation and H3K9 and H3K14 histone acetylation at the promoter of the LGALS9 gene, and (c) to characterize the relationship between these modifications and LGALS9 expression level in cervical cancer cells. All mRNA variants were detected in HaCaT (nontumoural keratinocytes) and SiHa cells, and seven were observed in HeLa cells. The promoter region of LGALS9 contains eight CpG dinucleotides. No hypermethylation pattern related to low LGALS9 expression was identified in tumour cells. Chromatin immunoprecipitation analysis demonstrated higher acetylation of H3K9ac and H3K14ac in HaCaT cells, which was related to higher mRNA levels. The presence of the mRNA variants suggests that alternative splicing may regulate the expression of galectin-9 isoforms. The results of this study suggest that histone acetylation, but not promoter CpG methylation, may be involved in the transcriptional regulation of the LGALS9 gene.