ASACO: Automatic and Serial Analysis of CO-expression to discover gene modifiers with potential use in drug repurposing.

Massive gene expression analyses are widely used to find differentially expressed genes under specific conditions. The results of these experiments are often available in public databases that are undergoing a growth similar to that of molecular sequence databases in the past. This now allows novel secondary computational tools to emerge that use such information to gain new knowledge. If several genes have a similar expression profile across heterogeneous transcriptomics experiments, they could be functionally related. These associations are usually useful for the annotation of uncharacterized genes. In addition, the search for genes with opposite expression profiles is useful for finding negative regulators and proposing inhibitory compounds in drug repurposing projects. Here we present a new web application, Automatic and Serial Analysis of CO-expression (ASACO), which has the potential to discover positive and negative correlator genes to a given query gene, based on thousands of public transcriptomics experiments. In addition, examples of use are presented, comparing with previous contrasted knowledge. The results obtained propose ASACO as a useful tool to improve knowledge about genes associated with human diseases and noncoding genes. ASACO is available at http://www.bioinfocabd.upo.es/asaco/.

Ribonucleotide reductase inhibition improves the symptoms of a Caenorhabditis elegans model of Alzheimer's disease.

Alzheimer's disease is the main cause of aging-associated dementia, for which there is no effective treatment. In this work, we reanalyze the information of a previous genome wide association study, using a new pipeline design to identify novel potential drugs. With this approach, ribonucleoside-diphosphate reductase gene (RRM2B) emerged as a candidate target and its inhibitor, 2', 2'-difluoro 2'deoxycytidine (gemcitabine), as a potential pharmaceutical drug against Alzheimer's disease. We functionally verified the effect of inhibiting the RRM2B homolog, rnr-2, in an Alzheimer's model of Caenorhabditis elegans, which accumulates human Aß1-42 peptide to an irreversible paralysis. RNA interference against rnr-2 and also treatment with 200 ng/ml of gemcitabine, showed an improvement of the phenotype. Gemcitabine treatment increased the intracellular ATP level 3.03 times, which may point to its mechanism of action. Gemcitabine has been extensively used in humans for cancer treatment but at higher concentrations. The 200 ng/ml concentration did not exert a significant effect over cell cycle, or affected cell viability when assayed in the microglia N13 cell line. Thus, the inhibitory drug of the RRM2B activity could be of potential use to treat Alzheimer's disease and particularly gemcitabine might be considered as a promising candidate to be repurposed for its treatment.

Influence of Oak Species, Toasting Degree, and Aging Time on the Differentiation of Brandies Using a Chemometrics Approach Based on Phenolic Compound UHPLC Fingerprints.

Oak wood is the main material used by coopers to manufacture casks for the aging of spirits or wines. Phenolic compounds are the main components extracted from the wood during spirit aging. In the present study, a chemometric approach based on unsupervised (PCA) and supervised (PLS-DA) pattern recognition techniques has been applied to the chromatographic instrumental fingerprints, obtained by ultra-high-performance liquid chromatography (UHPLC) at 280 nm, of the phenolic profiles of brandies aged in casks made of different oak wood species. The resulting natural data groupings and the PLS-DA models have revealed that the oak wood species, the toasting level, and the aging time are the most influential factors on the phenolic profile of the final products. Fingerprinting should be considered as a very useful feature, as it represents a considerable advantage, in terms of internal and quality control, for brandy producers.

A Functional Pipeline of Genome-Wide Association Data Leads to Midostaurin as a Repurposed Drug for Alzheimer's Disease.

Genome-wide association studies (GWAS) constitute a powerful tool to identify the different biochemical pathways associated with disease. This knowledge can be used to prioritize drugs targeting these routes, paving the road to clinical application. Here, we describe DAGGER (Drug Repositioning by Analysis of GWAS and Gene Expression in R), a straightforward pipeline to find currently approved drugs with repurposing potential. As a proof of concept, we analyzed a meta-GWAS of 1.6 × 107 single-nucleotide polymorphisms performed on Alzheimer's disease (AD). Our pipeline uses the Genotype-Tissue Expression (GTEx) and Drug Gene Interaction (DGI) databases for a rational prioritization of 22 druggable targets. Next, we performed a two-stage in vivo functional assay. We used a C. elegans humanized model over-expressing the Aß1-42 peptide. We assayed the five top-scoring candidate drugs, finding midostaurin, a multitarget protein kinase inhibitor, to be a protective drug. Next, 3xTg AD transgenic mice were used for a final evaluation of midostaurin's effect. Behavioral testing after three weeks of 20 mg/kg intraperitoneal treatment revealed a significant improvement in behavior, including locomotion, anxiety-like behavior, and new-place recognition. Altogether, we consider that our pipeline might be a useful tool for drug repurposing in complex diseases.

Analytical and Chemometric Characterization of Sweet Pedro Ximénez Sherry Wine during Its Aging in a Criaderas y Solera System.

Pedro Ximénez is a naturally sweet sherry wine produced in southern Spain from raisined Pedro Ximénez grape must and aged using a traditional Criaderas y Solera system. Complete analytical characterization has been useful in determining which parameters are the most influential in the aging of this wine. The organic acids, volatile compounds (higher alcohols, esters, aldehydes, and acetals), and phenolic compounds of this wine evolve during its aging, mainly through physico-chemical reactions and the contributions of wood compounds. During their aging, Pedro Ximénez sherry wines develop their organoleptic profiles, as tasting sessions have confirmed. A strong correlation between the aging of a wine and the parameters analyzed has also been corroborated through an MLR analysis. This allowed for the development of a model that, by using just 8 of the variables considered in the study, led to the determination of wine samples' ages at over 97% confidence. This constitutes a rather useful tool for wineries to control Pedro Ximénez sherry wine aging processes.

Influence of the use of sulfur dioxide, the distillation method, the oak wood type and the aging time on the production of brandies.

Brandies are spirits produced from wine spirit and wine distillates. The original wines selected to be distilled to produce the wine spirits as well as the distillation method used determine, to a large extent, the organoleptic characteristics of the final products. The young wine spirits evolve during their aging in oak casks, this being another key stage that affects the chemical and sensorial characteristics of the final brandy. In this work, seven different brandies have been studied. They were obtained from wine produced with and without the addition of sulfur dioxide, during their fermentation, using different distillation methods (single, double or serial distillation using pot stills and continuous column distillation) and aged for 14 or 28 months in three different types of oak wood (Quercus alba, Quercus robur and Quercus petraea) previously toasted to two different grades (medium or light). The use of unsupervised pattern recognition methods (HCA and FA) determined that the addition of sulfur dioxide during the fermentation of the base wine has a major influence on the aromatic and phenolic profile of the aged distillates. On the other hand, by means of supervised pattern recognition methods such as LDA and ANNs, the most significant variables that would allow to discriminate between the classes of brandies identified in the study were evaluated. Thus, the results obtained should cast some light on the most significant variables to be taken into account regarding Brandy production processes if a better control over these production processes is to be achieved, so that more exclusive and better quality products are obtained.

Analytical, Chemometric and Sensorial Characterization of Oloroso and Palo Cortado Sherries during Their Ageing in the Criaderas y Solera System.

Oloroso and Palo Cortado are two types of sherry wines, produced in the Sherry Wine Region in Southern Spain, known as Marco de Jerez, where it is aged following the traditional Criaderas y Solera system. All of them are aged through oxidative ageing, even though the peculiar Palo Cortado Sherry wine is also aged biologically under a veil of flor yeasts in the first stage. Total dry extract, organic acids, aldehydes, esters, higher alcohols and phenolic compounds in these sherry wines evolve during their ageing as a consequence of evaporation and/or perspiration processes, chemical reactions, extraction of compounds from oakwood and microbiological activity. Sherry wines develop their characteristic organoleptic profile during their ageing, as could be proven through their tasting sessions. According to the sherry type, some natural groupings of the wines could be observed after their principal component analysis. Furthermore, by multiple linear regression methods, an important correlation between the parameters that were analyzed and the ageing of each specific wine has been confirmed, which allowed us to establish two different models, each corresponding to the sherry type in question. Only five of the variables that were investigated were required to successfully estimate each wine's age at over 99% confidence. This represents a rather convenient tool for wineries to monitor the ageing of these sherry wines.

A Study on the Influence of the Use of Sulphur Dioxide, the Distillation System and the Aging Conditions on the Final Sensory Characteristics of Brandy.

Brandy is a unique alcoholic beverage obtained from wine distillates. Numerous studies have been published on its physicochemical traits and the effect of certain elaboration variables on them, but not many studies have been carried out from a sensory point of view or that have followed standardized methods applicable to this discipline. This study intends to determine the effect that certain production variables have on the sensory characteristics of brandy. These variables are the following: The use of sulphur dioxide during the fermentation of the base wine, the subsequent distillation system, the alcohol content during aging, the botanical origin of the aging casks, and their toasting degree. For this purpose, the guidelines provided by the ISO standards for sensory analysis have been followed, and chromatic parameters have also been determined. Heavy extractions from Quercus petraea casks resulted in brandies with widely varying colors, although these were hard to distinguish using the olfactory and gustatory properties associated with the factors under study. Conversely, those brandies aged in Quercus alba casks presented very consistent greenish shades that are not traditionally associated with aged brandy. This lower extraction could explain why the aromatic traits that are found in the fresh spirit are better preserved when this type of oak is used. The spirit obtained through the distillation of SO2-free wines aged in Quercus robur presented average sensory characteristics: Good color, smooth in the mouth, and medium-intensity oak notes. The distillates that were aged at 55% ABV were later perceived as more aromatically intense with a greater oak note on the palate. On the other hand, the brandies obtained from wines without SO2 added were perceived as less alcoholic, sweeter, and more balanced, with a softer oak note.

DNA Damage-Induced RNAPII Degradation and Its Consequences in Gene Expression.

RPB1, the major and catalytic subunit of human RNA Polymerase II (RNAPII), is specifically degraded by the ubiquitin-proteasome system upon induction of DNA damage by different agents, such as ultraviolet (UV) light. The "last resort" model of RNAPII degradation states that a persistently stalled RNAPII is degraded at the site of the DNA lesion in order to facilitate access to Nucleotide Excision Repair (NER) factors, thereby promoting repair in template strands of active genes. Recent identification and mutation of the lysine residue involved in RPB1 ubiquitylation and degradation unveiled the relevance of RNAPII levels in the control of gene expression. Inhibition of RNAPII degradation after UV light exposure enhanced RNAPII loading onto chromatin, demonstrating that the mere concentration of RNAPII shapes the gene expression response. In this review, we discuss the role of RNAPII ubiquitylation in NER-dependent repair, recent advances in RPB1 degradation mechanisms and its consequences in gene expression under stress, both in normal and repair deficient cells.

Analytical and Chemometric Characterization of Fino and Amontillado Sherries during Aging in Criaderas y Solera System.

Fino and Amontillado are Sherry wines, produced in Marco de Jerez area (southern Spain), and aged in Criaderas y Solera system. Fino Sherry wine follows a biological aging process, under a veil of flor yeasts, while Amontillado Sherry wine shares the same biological aging firstly, followed by oxidative aging, which gives them special features. Organic acids, esters, higher alcohols, phenolic compounds and total dry extract of Sherries evolve during aging due to evaporation processes, physical-chemical reactions, wood contributions and microbiological activity. During aging, Sherry wines improve their organoleptic profile, as could be proved in the tasting sessions. Hierarchical Cluster Analysis and Factor Analysis with factor extraction using Principal Components of Sherry wines studied were carried out and natural groupings of the wines according to the type of aging and their age were observed. A strong correlation between the parameters analyzed and the aging of each wine has been seen in the Multiple Linear Regression studies, establishing two different models, one for each type of Sherry wine, that, with only four of all the variables studied estimated the wine age with more than 99% of confidence. This constitutes a useful tool to control the age of these Sherry wines in the winery.

Early redox homeostasis disruption contributes to the differential cytotoxicity of imiquimod on transformed and normal endothelial cells.

The off-label use of imiquimod (IQ) for hemangioma treatment has shown clinical benefits. We have previously reported a selective direct IQ-cytotoxic effect on transformed (H5V) vs. normal (1G11) endothelial cells (EC). In the present study, we investigated the mechanism underlying this selective cytotoxicity in terms of TLR7/8 receptor expression, NF-κB signalling and time-dependent modifications of oxidative stress parameters (ROS: reactive oxygen species, catalase and superoxide dismutase activities, GSH/GSSG and lipid peroxidation). TLR7/8 level was extremely low in both cell lines, and IQ did not upregulate TLR7/8 expression or activate NF-κB signalling. IQ significantly induced ROS in H5V after 2 h and strongly affected antioxidant defenses. After 12 h, enzyme activities were restored to baseline levels but a robust drop in GSH/GSSG persisted together with increased lipid peroxidation levels and a marked mitochondrial dysfunction. Although in normal IQ-treated EC some oxidative stress parameters were affected after 4 h, mitochondrial health and GSH/GSSG ratio remained notably unaffected after 12 h. Therefore, the early alterations (0-2 h) in transformed EC breached redox homeostasis as strongly as to enhance their susceptibility to IQ. This interesting facet of IQ as redox disruptor could broaden its therapeutic potential for other skin malignancies, alone or in adjuvant schemes.

A DNA intercalating dye-based RT-qPCR alternative to diagnose SARS-CoV-2.

Early detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been proven crucial during the efforts to mitigate the effects of the COVID-19 pandemic. Several diagnostic methods have emerged in the past few months, each with different shortcomings and limitations. The current gold standard, RT-qPCR using fluorescent probes, relies on demanding equipment requirements plus the high costs of the probes and specific reaction mixes. To broaden the possibilities of reagents and thermocyclers that could be allocated towards this task, we have optimized an alternative strategy for RT-qPCR diagnosis. This is based on a widely used DNA-intercalating dye and can be implemented with several different qPCR reagents and instruments. Remarkably, the proposed qPCR method performs similarly to the broadly used TaqMan-based detection, in terms of specificity and sensitivity, thus representing a reliable tool. We think that, through enabling the use of vast range of thermocycler models and laboratory facilities for SARS-CoV-2 diagnosis, the alternative proposed here can increase dramatically the testing capability, especially in countries with limited access to costly technology and reagents.

Comparative Evaluation of Brandy de Jerez Aged in American Oak Barrels with Different Times of Use.

Brandy de Jerez is a European Geographical Indication for grape-derived spirits aged in oak casks that have previously contained any kind of Sherry wine and, therefore, are known as Sherry Casks®. Wood compounds have a substantial influence in the quality of the brandies that are aged in the barrels. In the cellar, the barrels that have been used for many years to keep Sherry wine or other wine spirits are often used for this purpose. When wooden barrels are used for the first time, they release a large amount of compounds into the liquid contained in them. Such amount decreases over time but casks life cycle has remained unexplored until now. The present work has the aim to study the brandies obtained from the same wine spirit after two years ageing in three differently oak casks: namely new, 7 years of use (4 years containing Oloroso wine and 3 years containing wine spirits) and 32 years of use (8 years containing Oloroso wine and 24 years containing wine spirits). According to the results from our experiments, even after 32 years of use, the wood barrels still contribute to modify the organoleptic characteristics of brandy. Moreover, the brandies aged in used barrels were judged more balanced than those aged in new barrels.

Steroid hormones sulfatase inactivation extends lifespan and ameliorates age-related diseases.

Aging and fertility are two interconnected processes. From invertebrates to mammals, absence of the germline increases longevity. Here we show that loss of function of sul-2, the Caenorhabditis elegans steroid sulfatase (STS), raises the pool of sulfated steroid hormones, increases longevity and ameliorates protein aggregation diseases. This increased longevity requires factors involved in germline-mediated longevity (daf-16, daf-12, kri-1, tcer-1 and daf-36 genes) although sul-2 mutations do not affect fertility. Interestingly, sul-2 is only expressed in sensory neurons, suggesting a regulation of sulfated hormones state by environmental cues. Treatment with the specific STS inhibitor STX64, as well as with testosterone-derived sulfated hormones reproduces the longevity phenotype of sul-2 mutants. Remarkably, those treatments ameliorate protein aggregation diseases in C. elegans, and STX64 also Alzheimer's disease in a mammalian model. These results open the possibility of reallocating steroid sulfatase inhibitors or derivates for the treatment of aging and aging related diseases.

Serial co-expression analysis of host factors from SARS-CoV viruses highly converges with former high-throughput screenings and proposes key regulators.

The current genomics era is bringing an unprecedented growth in the amount of gene expression data, only comparable to the exponential growth of sequences in databases during the last decades. This data allow the design of secondary analyses that take advantage of this information to create new knowledge. One of these feasible analyses is the evaluation of the expression level for a gene through a series of different conditions or cell types. Based on this idea, we have developed Automatic and Serial Analysis of CO-expression, which performs expression profiles for a given gene along hundreds of heterogeneous and normalized transcriptomics experiments and discover other genes that show either a similar or an inverse behavior. It might help to discover co-regulated genes, and common transcriptional regulators in any biological model. The present severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is an opportunity to test this novel approach due to the wealth of data that are being generated, which could be used for validating results. Thus, we have identified 35 host factors in the literature putatively involved in the infectious cycle of SARS-CoV viruses and searched for genes tightly co-expressed with them. We have found 1899 co-expressed genes whose assigned functions are strongly related to viral cycles. Moreover, this set of genes heavily overlaps with those identified by former laboratory high-throughput screenings (with P-value near 0). Our results reveal a series of common regulators, involved in immune and inflammatory responses that might be key virus targets to induce the coordinated expression of SARS-CoV-2 host factors.

Characterization of the Aromatic and Phenolic Profile of Five Different Wood Chips Used for Ageing Spirits and Wines.

Wooden barrels and wood chips are usually used in the ageing of spirits and wines to improve their sensorial profile. Oak wood is the most popular material used in cooperage, but there are other interesting woods, such as cherry or chestnut, that could be considered for this purpose. In this study, a novel method for the determination of the aromatic profile of wood powder by Direct Thermal Desorption-Gas Chromatography-Mass Spectrometry (DTD-GC-MS) was optimized by experimental design. The volatile composition of five different types of wood chips was determined by direct analysis of wood powder by DTD-GC-MS method developed. Thirty-one compounds from wood were identified through this analysis, allowing the differentiation between woods. The aromatic and phenolic compound profile of the 50% hydroalcoholic extract of each type of wood studied was analyzed by Stir-bar Sorptive Extraction-Gas Chromatography-Mass Spectrometry (SBSE-GC-MS) and Ultra-High-Performance Liquid Chromatography (UHPLC) to determine which wood compounds are transferred to spirits and wine after ageing. Different phenolic profiles were found by UHPLC in each wood extract, allowing their differentiation. However, results obtained by SBSE-GC-MS did not allow distinguishing between wood extracts. The analysis of wood in solid state, without any type of previous treatment except grinding, by DTD-GC-MS does not imply any loss of information of the aromatic compounds present in wood as other techniques. This is a potential method to identify aromas in wood that, in addition, allows different types of wood to be differentiated.

GSK-3 is an RNA polymerase II phospho-CTD kinase.

We have previously found that UV-induced DNA damage causes hyperphosphorylation of the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), inhibition of transcriptional elongation and changes in alternative splicing (AS) due to kinetic coupling between transcription and splicing. In an unbiased search for protein kinases involved in the AS response to DNA damage, we have identified glycogen synthase kinase 3 (GSK-3) as an unforeseen participant. Unlike Cdk9 inhibition, GSK-3 inhibition only prevents CTD hyperphosphorylation triggered by UV but not basal phosphorylation. This effect is not due to differential degradation of the phospho-CTD isoforms and can be reproduced, at the AS level, by overexpression of a kinase-dead GSK-3 dominant negative mutant. GSK-3 inhibition abrogates both the reduction in RNAPII elongation and changes in AS elicited by UV. We show that GSK-3 phosphorylates the CTD in vitro, but preferentially when the substrate is previously phosphorylated, consistently with the requirement of a priming phosphorylation reported for GSK-3 efficacy. In line with a role for GSK-3 in the response to DNA damage, GSK-3 inhibition prevents UV-induced apoptosis. In summary, we uncover a novel role for a widely studied kinase in key steps of eukaryotic transcription and pre-mRNA processing.

Reciprocal regulation between alternative splicing and the DNA damage response.

Splicing, the process that catalyzes intron removal and flanking exon ligation, can occur in different ways (alternative splicing) in immature RNAs transcribed from a single gene. In order to adapt to a particular context, cells modulate not only the quantity but also the quality (alternative isoforms) of their transcriptome. Since 95% of the human coding genome is subjected to alternative splicing regulation, it is expected that many cellular pathways are modulated by alternative splicing, as is the case for the DNA damage response. Moreover, recent evidence demonstrates that upon a genotoxic insult, classical DNA damage response kinases such as ATM, ATR and DNA-PK orchestrate the gene expression response therefore modulating alternative splicing which, in a reciprocal way, shapes the response to a damaging agent.

Serial co-expression analysis of host factors from SARS-CoV viruses highly converges with former high-throughput screenings and proposes key regulators and co-option of cellular pathways.

The current genomics era is bringing an unprecedented growth in the amount of gene expression data, only comparable to the exponential growth of sequences in databases during the last decades. This data now allows the design of secondary analyses that take advantage of this information to create new knowledge through specific computational approaches. One of these feasible analyses is the evaluation of the expression level for a gene through a series of different conditions or cell types. Based on this idea, we have developed ASACO, Automatic and Serial Analysis of CO-expression, which performs expression profiles for a given gene along hundreds of normalized and heterogeneous transcriptomics experiments and discover other genes that show either a similar or an inverse behavior. It might help to discover co-regulated genes, and even common transcriptional regulators in any biological model, including human diseases or microbial infections. The present SARS-CoV-2 pandemic is an opportunity to test this novel approach due to the wealth of data that is being generated, which could be used for validating results. In addition, new cell mechanisms identified could become new therapeutic targets. Thus, we have identified 35 host factors in the literature putatively involved in the infectious cycle of SARS-CoV and/or SARS-CoV-2 and searched for genes tightly co-expressed with them. We have found around 1900 co-expressed genes whose assigned functions are strongly related to viral cycles. Moreover, this set of genes heavily overlap with those identified by former laboratory high-throughput screenings (with p-value near 0). Some of these genes aim to cellular structures such as the stress granules, which could be essential for the virus replication and thereby could constitute potential targets in the current fight against the virus. Additionally, our results reveal a series of common transcription regulators, involved in immune and inflammatory responses, that might be key virus targets to induce the coordinated expression of SARS-CoV-2 host factors. All of this proves that ASACO can discover gene co-regulation networks with potential for proposing new genes, pathways and regulators participating in particular biological systems. Highlights: ASACO identifies regulatory associations of genes using public transcriptomics data.ASACO highlights new cell functions likely involved in the infection of coronavirus.Comparison with high-throughput screenings validates candidates proposed by ASACO.Genes co-expressed with host's genes used by SARS-CoV-2 are related to stress granules.

Redox-dependent and redox-independent functions of Caenorhabditis elegans thioredoxin 1.

Thioredoxins (TRX) are traditionally considered as enzymes catalyzing redox reactions. However, redox-independent functions of thioredoxins have been described in different organisms, although the underlying molecular mechanisms are yet unknown. We report here the characterization of the first generated endogenous redox-inactive thioredoxin in an animal model, the TRX-1 in the nematode Caenorhabditis elegans. We find that TRX-1 dually regulates the formation of an endurance larval stage (dauer) by interacting with the insulin pathway in a redox-independent manner and the cGMP pathway in a redox-dependent manner. Moreover, the requirement of TRX-1 for the extended longevity of worms with compromised insulin signalling or under calorie restriction relies on TRX-1 redox activity. In contrast, the nuclear translocation of the SKN-1 transcription factor and increased LIPS-6 protein levels in the intestine upon trx-1 deficiency are strictly redox-independent. Finally, we identify a novel function of C. elegans TRX-1 in male food-leaving behaviour that is redox-dependent. Taken together, our results position C. elegans as an ideal model to gain mechanistic insight into the redox-independent functions of metazoan thioredoxins, overcoming the limitations imposed by the embryonic lethal phenotypes of thioredoxin mutants in higher organisms.