Wine originally emerged as a serendipitous mix of chemistry and biology, where microorganisms played a decisive role. From these ancient fermentations to the current monitored industrial processes, winegrowers and winemakers have been continuously changing their practices according to scientific knowledge and advances. A new enology direction is emerging and aiming to blend the complexity of spontaneous fermentations with industrial safety of monitored fermentations. In this context, wines with distinctive autochthonous peculiarities have a great acceptance among consumers, causing important economic returns. The concept of terroir, far from being a rural term, conceals a wide range of analytical parameters that are the basis of the knowledge-based enology trend. In this sense, the biological aspect of soils has been underestimated for years, when actually it contains a great microbial diversity. This soil-associated microbiota has been described as determinant, not only for the chemistry and nutritional properties of soils, but also for health, yield, and quality of the grapevine. Additionally, recent works describe the soil microbiome as the reservoir of the grapevine associated microbiota, and as a contributor to the final sensory properties of wines. To understand the crucial roles of microorganisms on the entire wine making process, we must understand their ecological niches, population dynamics, and relationships between ‘microbiome- vine health’ and ‘microbiome-wine metabolome’. These are critical steps for designing precision enology practices. For that purpose, current metagenomic techniques are expanding from laboratories, to the food industry. This review focuses on the current knowledge about vine and wine microbiomes, with emphasis on their biological roles and the technical basis of next-generation sequencing (NGS) pipelines. An overview of molecular and informatics tools is included and new directions are proposed, highlighting the importance of –omics technologies in wine research and industry.

The microbial biodiversity found in different vitivinicultural regions is an important determinant of wine terroir. It should be studied and preserved, although it may, in the future, be subjected to manipulation by precision agriculture and oenology. Here, we conducted a global survey of vineyards’ soil microbial communities. We analysed soil samples from 200 vineyards on four continents to establish the basis for the development of a vineyard soil microbiome’s map, representing microbial biogeographical patterns on a global scale. This study describes vineyard microbial communities worldwide and establishes links between vineyard locations and microbial biodiversity on different scales: between continents, countries, and between different regions within the same country. Climate data correlates with fungal alpha diversity but not with prokaryotes alpha diversity, while spatial distance, on a global and national scale, is the main variable explaining beta-diversity in fungal and prokaryotes communities. Proteobacteria, Actinobacteria and Acidobacteria phyla, and Archaea genus Nitrososphaera dominate prokaryotic communities in soil samples while the overall fungal community is dominated by the genera Solicoccozyma, Mortierella and Alternaria. Finally, we used microbiome data to develop a predictive model based on random forest analyses to discriminate between microbial patterns and to predict the geographical source of the samples with reasonable precision.

Mutation screening of the breast cancer genes BRCA1 and BRCA2 identifies a large fraction of variants of uncertain clinical significance (VUS) whose functional and clinical interpretations pose a challenge for genomic medicine. Likewise, an increasing amount of evidence indicates that genetic variants can have deleterious effects on pre-mRNA splicing. Our goal was to investigate the impact on splicing of a set of reported variants of BRCA2 exons 17 and 18 to assess their role in hereditary breast cancer and to identify critical regulatory elements that may constitute hotspots for spliceogenic variants. A splicing reporter minigene with BRCA2 exons 14 to-20 (MGBR2_ex14-20) was constructed in the pSAD vector. Fifty-two candidate variants were selected with splicing prediction programs, introduced in MGBR2_ex14-20 by site-directed mutagenesis and assayed in triplicate in MCF-7 cells. Wild type MGBR2_ex14-20 produced a stable transcript of the expected size (1,806 nucleotides) and structure (V1-[BRCA2_exons_14–20]–V2). Functional mapping by microdeletions revealed essential sequences for exon recognition on the 3’ end of exon 17 (c.7944-7973) and the 5’ end of exon 18 (c.7979-7988, c.7999-8013). Thirty out of the 52 selected variants induced anomalous splicing in minigene assays with >16 different aberrant transcripts, where exon skipping was the most common event. A wide range of splicing motifs were affected including the canonical splice sites (15 variants), novel alternative sites (3 variants), the polypyrimidine tract (3 variants) and enhancers/silencers (9 variants). According to the guidelines of the American College of Medical Genetics and Genomics (ACMG), 20 variants could be classified as pathogenic (c.7806-2A>G, c.7806-1G>A, c.7806-1G>T, c.7806-1_7806-2dup, c.7976+1G>A, c.7977-3_7978del, c.7977-2A>T, c.7977-1G>T, c.7977-1G>C, c.8009C>A, c.8331+1G>T and c.8331+2T>C) or likely pathogenic (c.7806-9T>G, c.7976G>C, c.7976G>A, c.7977-7C>G, c.7985C>G, c.8023A>G, c.8035G>T and c.8331G>A), accounting for 30.8% of all pathogenic/likely pathogenic variants of exons 17–18 at the BRCA Share database. The remaining 8 variants (c.7975A>G, c.7977-6T>G, c.7988A>T, c.7992T>A, c.8007A>G, c.8009C>T, c.8009C>G, and c.8072C>T) induced partial splicing anomalies with important ratios of the full-length transcript (≥70%), so that they remained classified as VUS. Aberrant splicing is therefore especially prevalent in BRCA2 exons 17 and 18 due to the presence of active ESEs involved in exon recognition. Splicing functional assays with minigenes are a valuable strategy for the initial characterization of the splicing outcomes and the subsequent clinical interpretation of variants of any disease-gene, although these results should be checked, whenever possible, against patient RNA. A significant proportion of disease-causing mutations of inherited disorders impair splicing. Massive sequencing projects of genetic diseases generate thousands of sequence variations that require functional and clinical interpretations. We have shown that splicing reporter minigenes of the breast cancer genes BRCA1 and BRCA2 are useful tools to functionally test DNA variants. In this work, we have constructed a 7-exon BRCA2 minigene (exons 14 to 20) where we mapped critical splicing regulatory sequences and tested 52 selected variants of exons 17 and 18 detected in breast cancer patients. We finely located three DNA segments on both exons that presumably contain splicing enhancer sequences. We observed that a total of 30 variants of any type disrupted the splicing patterns and, given the severity of their outcomes, we classified 20 of them as pathogenic or likely pathogenic. We also showed that a wide range of splicing elements were affected including canonical and novel 5’ and 3’ splice sites, the polypyrimidine tract and enhancer and silencer sequences. We concluded that splicing aberrations are frequent in Hereditary Breast and Ovarian Cancer and that minigenes are valuable tools to functionally classify DNA variants of any human disease gene under the splicing viewpoint.

Numerous pathogenic DNA variants impair the splicing mechanism in human genetic diseases. Minigenes are optimal approaches to test variants under the splicing viewpoint without the need of patient samples. We aimed to design a robust minigene construct of the breast cancer gene BRCA2 in order to investigate the impact of variants on splicing. BRCA2 exons 19–27 (MGBR2_ex19–27) were cloned in the new vector pSAD. It produced a large transcript of the expected size (2,174 nucleotides) and exon structure (V1-ex19-27-V2). Splicing assays showed that 18 (17 splice-site and 1 silencer variants) out of 40 candidate DNA variants induced aberrant patterns. Twenty-four anomalous transcripts were accurately detected by fluorescent-RT-PCR that were generated by exon-skipping, alternative site usage, and intron-retention events. Fourteen variants induced major anomalies and were predicted to disrupt protein function so they could be classified as pathogenic. Furthermore, minigene mimicked previously reported patient RNA outcomes of seven variants supporting the reproducibility of minigene assays. Therefore, a relevant fraction of variants are involved in breast cancer through splicing alterations. MGBR2_ex19–27 is the largest reported BRCA2 minigene and constitutes a valuable tool for the functional and clinical classification of sequence variations.

Introduction: The underlying pathogenic mechanism of a large fraction of DNA variants of disease-causing genes is the disruption of the splicing process. We aimed to investigate the effect on splicing of the BRCA2 variants c.8488-1G > A (exon 20) and c.9026_9030del (exon 23), as well as 41 BRCA2 variants reported in the Breast Cancer Information Core (BIC) mutation database. Methods: DNA variants were analyzed with the splicing prediction programs NNSPLICE and Human Splicing Finder. Functional analyses of candidate variants were performed by lymphocyte RT-PCR and/or hybrid minigene assays. Forty-one BIC variants of exons 19, 20, 23 and 24 were bioinformatically selected and generated by PCR-mutagenesis of the wild type minigenes. Results: Lymphocyte RT-PCR of c.8488-1G > A showed intron 19 retention and a 12-nucleotide deletion in exon 20, whereas c.9026_9030del did not show any splicing anomaly. Minigene analysis of c.8488-1G > A displayed the aforementioned aberrant isoforms but also exon 20 skipping. We further evaluated the splicing outcomes of 41 variants of four BRCA2 exons by minigene analysis. Eighteen variants presented splicing aberrations. Most variants (78.9%) disrupted the natural splice sites, whereas four altered putative enhancers/silencers and had a weak effect. Fluorescent RT-PCR of minigenes accurately detected 14 RNA isoforms generated by cryptic site usage, exon skipping and intron retention events. Fourteen variants showed total splicing disruptions and were predicted to truncate or eliminate essential domains of BRCA2. Conclusions: A relevant proportion of BRCA2 variants are correlated with splicing disruptions, indicating that RNA analysis is a valuable tool to assess the pathogenicity of a particular DNA change. The minigene system is a straightforward and robust approach to detect variants with an impact on splicing and contributes to a better knowledge of this gene expression step.

Colorectal cancer (CRC) cells undergo the remodeling of intracellular Ca²⁺ homeostasis, which contributes to cancer hallmarks such as enhanced proliferation, invasion and survival. Ca²⁺ remodeling includes critical changes in store-operated Ca²⁺ entry (SOCE) and Ca²⁺ store content. Some changes have been investigated at the molecular level. However, since nearly 100 genes are involved in intracellular Ca²⁺ transport, a comprehensive view of Ca²⁺ remodeling in CRC is lacking. We have used Next Generation Sequencing (NGS) to investigate differences in expression of 77 selected gene transcripts involved in intracellular Ca²⁺ transport in CRC. To this end, mRNA from normal human colonic NCM460 cells and human colon cancer HT29 cells was isolated and used as a template for transcriptomic sequencing and expression analysis using Ion Torrent technology. After data transformation and filtering, exploratory analysis revealed that both cell types were well segregated. In addition, differential gene expression using R and bioconductor packages show significant differences in expression of selected voltage-operated Ca²⁺ channels and store-operated Ca²⁺ entry players, transient receptor potential (TRP) channels, Ca²⁺ release channels, Ca²⁺ pumps, Na⁺/Ca²⁺ exchanger isoforms and genes involved in mitochondrial Ca²⁺ transport. These data provide the first comprehensive transcriptomic analysis of Ca²⁺ remodeling in CRC.