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
, 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 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.
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.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Colorectal cancer (CRC) cells undergo the remodeling of intracellular Ca
homeostasis, which contributes to cancer hallmarks such as enhanced proliferation, invasion and survival. Ca
remodeling ...includes critical changes in store-operated Ca
entry (SOCE) and Ca
store content. Some changes have been investigated at the molecular level. However, since nearly 100 genes are involved in intracellular Ca
transport, a comprehensive view of Ca
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 Ca
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 Ca
channels and store-operated Ca
entry players, transient receptor potential (TRP) channels, Ca
release channels, Ca
pumps, Na⁺/Ca
exchanger isoforms and genes involved in mitochondrial Ca
transport. These data provide the first comprehensive transcriptomic analysis of Ca
remodeling in CRC.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Potato (
) is considered one of the most widely consumed crops worldwide, due to its high yield and nutritional profile, climate change-related environmental threats and increasing food demand. This ...scenario highlights the need of sustainable agricultural practices to enhance potato productivity, while preserving and maintaining soil health. Plant growth-promoting bacteria (PGPB) stimulate crop production through biofertilization mechanisms with low environmental impact. For instance, PGPB promote biological nitrogen fixation, phosphate solubilization, production of phytohormones, and biocontrol processes. Hence, these microbes provide a promising solution for more productive and sustainable agriculture. In this study, the effects of
QST713 based-product (MINUET™, Bayer) were assessed in terms of yield, soil microbiome, potato peel and petiole nutrient profile as a promising PGPB in a wide range of potato cultivars across the United States of America. Depending on the location, potato yield and boron petiole content increased after biostimulant inoculation to maximum of 24% and 14%, respectively. Similarly, nutrient profile in potato peel was greatly improved depending on the location with a maximum of 73%, 62% and 36% for manganese, zinc and phosphorus. Notably, fungal composition was shifted in the treated group. Yield showed strong associations with specific microbial taxa, such as
. Moreover, local networks strongly associated with yield, highlighting the important role of the native soil microbiome structure in indirectly maintaining soil health. Our results showed that treatment with
based product correlated with enhanced yield, with minor impacts on the soil microbiome diversity. Further studies are suggested to disentangle the underlying mechanisms of identified patterns and associations.
Genetic testing of
and
identifies a large number of variants of uncertain clinical significance whose functional and clinical interpretations pose a challenge for genetic counseling. Interestingly, a ...relevant fraction of DNA variants can disrupt the splicing process in cancer susceptibility genes. We have tested more than 200 variants throughout 19 BRCA2 exons mostly by minigene assays, 54% of which displayed aberrant splicing, thus confirming the utility of this assay to check genetic variants in the absence of patient RNA. Our goal was to investigate
exon 16 with a view to characterizing spliceogenic variants recorded at the mutational databases. Seventy-two different BIC and UMD variants were analyzed with NNSplice and Human Splicing Finder, 12 of which were selected because they were predicted to disrupt essential splice motifs: canonical splice sites (ss; eight variants) and exonic/intronic splicing enhancers (four variants). These 12 candidate variants were introduced into the BRCA2 minigene with seven exons (14-20) by site-directed mutagenesis and then transfected into MCF-7 cells. Seven variants (six intronic and one missense) induced complete abnormal splicing patterns: c.7618-2A>T, c.7618-2A>G, c.7618-1G>C, c.7618-1G>A, c.7805G>C, c.7805+1G>A, and c.7805+3A>C, as well as a partial anomalous outcome by c.7802A>G. They generated at least 10 different transcripts: Δ16p
(alternative 3'ss 44-nt downstream; acceptor variants), Δ16 (exon 16-skipping; donor variants), Δ16p
(alternative 3'ss 55-nt downstream), Δ16q
(alternative 5'ss 4-nt upstream), Δ16q
(alternative 5'ss 4-nt upstream), ▾16q
(alternative 5'ss 20-nt downstream), as well as minor (Δ16p
and Δ16,17p
) and uncharacterized transcripts of 893 and 954 nucleotides. Isoforms Δ16p
, Δ16, Δ16p
, Δ16q
, Δ16q
, and ▾16q
introduced premature termination codons which presumably inactivate BRCA2. According to the guidelines the American College of Medical Genetics and Genomics these eight variants could be classified as pathogenic or likely pathogenic whereas the Evidence-based Network for the Interpretation of Germline Mutant Alleles rules suggested seven class 4 and one class 3 variants. In conclusion, our study highlights the relevance of splicing functional assays by hybrid minigenes for the clinical classification of genetic variations. Hence, we provide new data about spliceogenic variants of BRCA2 exon 16 that are directly correlated with breast cancer susceptibility.
DANAGEN-BIOTED S.L has developed a method for the collection, stabilization, transportation and purification of DNA from saliva samples using the DANASALIVA Sample Collection Kit, a cost-effective ...collection and transportation device that effectively stabilizes buccal cells and white blood cells found in saliva over 1 year at room temperature. Then, saliva DNA is isolated from the preserved saliva samples via DANAGENE Saliva Kit. Here, it is demonstrated the efficiency genomic DNA samples extracted with DANAGENE Saliva System to prepare a custom library (HC-Gen Test) designed by AC-Gen Reading Life Inc. for Hereditary Cancer Diagnostic (http://www.ac-aen.com/hereditarv-cancer).
Agroecosystems are human-managed ecosystems subject to generalized ecological rules. Understanding the ecology behind the assembly and dynamics of soil fungal communities is a fruitful way to improve ...management practices and plant productivity. Thus, monitoring soil health would benefit from the use of metrics that arise from ecological explanations that can also be informative for agricultural management. Beyond traditional biodiversity descriptors, community-level properties have the potential of informing about particular ecological situations. Here we assess the impact of different farming practices in a survey of 350 vineyard soils from the United States and Spain by estimating network properties based on spatial associations. Our observations using traditional approaches show results concurring with previous literature: the influence of geographic and climatic factors on sample distributions, or different operational taxonomic unit (OTU) compositions depending on agricultural managements. Furthermore, using network properties, we observe that fungal communities ranged from dense arrangements of associations to a sparser structure of associations, indicating differential levels of niche specialization. We detect fungal arrangements capable of thriving in wider or smaller ranges of temperature, revealing that niche specialization may be a critical soil process impacting soil health. Low-intervention practices (organic and biodynamic managements) promoted densely clustered networks, describing an equilibrium state based on mixed collaborative communities. In contrast, conventionally managed vineyards had highly modular sparser communities, supported by a higher coexclusion proportion. Thus, we hypothesize that network properties at the community level may help to understand how the environment and land use can affect community structure and ecological processes in agroecosystems.
Soil fungal communities play a key role in agroecosystem sustainability. The complexity of fungal communities, at both taxonomic and functional levels, makes it difficult to find clear patterns connecting community composition with ecosystem function and to understand the impact of biotic (interspecies interactions) and abiotic (e.g., climate or anthropogenic disturbances) factors on it. Here we combine network analysis methods and properties, proposing a novel analytical approach: to infer ecological properties from local networks, which we apply to the study of fungal communities in vineyard soils. We conclude that different levels of farming intensification may lead to different ecological strategies in soil fungal communities settled by particular association arrangements.
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.
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.
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.
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.
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