Identification of lncRNAs Deregulated in Epithelial Ovarian Cancer Based on a Gene Expression Profiling Meta-Analysis.

Epithelial ovarian cancer (EOC) is one of the deadliest gynecological cancers worldwide, mainly because of its initially asymptomatic nature and consequently late diagnosis. Long non-coding RNAs (lncRNA) are non-coding transcripts of more than 200 nucleotides, whose deregulation is involved in pathologies such as EOC, and are therefore envisaged as future biomarkers. We present a meta-analysis of available gene expression profiling (microarray and RNA sequencing) studies from EOC patients to identify lncRNA genes with diagnostic and prognostic value. In this meta-analysis, we include 46 independent cohorts, along with available expression profiling data from EOC cell lines. Differential expression analyses were conducted to identify those lncRNAs that are deregulated in (i) EOC versus healthy ovary tissue, (ii) unfavorable versus more favorable prognosis, (iii) metastatic versus primary tumors, (iv) chemoresistant versus chemosensitive EOC, and (v) correlation to specific histological subtypes of EOC. From the results of this meta-analysis, we established a panel of lncRNAs that are highly correlated with EOC. The panel includes several lncRNAs that are already known and even functionally characterized in EOC, but also lncRNAs that have not been previously correlated with this cancer, and which are discussed in relation to their putative role in EOC and their potential use as clinically relevant tools.

Characterisation of clays from Alicante province (SE Spain) for use in the recovery of degraded soils.

The goal of this paper is the characterisation of seven clays of the province of Alicante (SE Spain) and their possible use to improve the fertility, water absorption and contaminant-retaining capacity of degraded soils. Three soils affected by the dumping of construction debris were also studied to diagnose the problems and possible recovery strategies. Several physicochemical properties were measured, such as the water holding capacity, soil organic matter, lime, pH, EC and CEC. A high correlationship between mineralogical and elemental composition was obtained. Illite was present in all clays and soils. Some of the samples also contained kaolinite and significant amounts of lime. The CEC, as expected, was more closely related to the organic matter content. Soil organic matter was detected in the second derivative of the FTIR spectra by the signals of the CH2 groups at 2850 and 2919. This way, the FTIR spectrum for the soils of the area would make it possible to estimate both the organic matter content and the CEC. Despite their origin, soils did not show heavy metal pollution; however, salinisation risk seemed to be the most probable cause of degradation. According to the organic matter, lime and illite content, two clays were selected as the most suitable for soil degradation recovery. Furthermore, organic matter additions may help to improve the self-depurative ability of the soil.

HMGB proteins involved in TOR signaling as general regulators of cell growth by controlling ribosome biogenesis.

The number of ribosomes and their activity need to be highly regulated because their function is crucial for the cell. Ribosome biogenesis is necessary for cell growth and proliferation in accordance with nutrient availability and other external and intracellular signals. High-mobility group B (HMGB) proteins are conserved from yeasts to human and are decisive in cellular fate. These proteins play critical functions, from the maintenance of chromatin structure, DNA repair, or transcriptional regulation, to facilitation of ribosome biogenesis. They are also involved in cancer and other pathologies. In this review, we summarize evidence of how HMGB proteins contribute to ribosome-biogenesis control, with special emphasis on a common nexus to the target of rapamycin (TOR) pathway, a signaling cascade essential for cell growth and proliferation from yeast to human. Perspectives in this field are also discussed.

Characterization of mussel H2A.Z.2: a new H2A.Z variant preferentially expressed in germinal tissues from Mytilus.

Histones are the fundamental constituents of the eukaryotic chromatin, facilitating the physical organization of DNA in chromosomes and participating in the regulation of its metabolism. The H2A family displays the largest number of variants among core histones, including the renowned H2A.X, macroH2A, H2A.B (Bbd), and H2A.Z. This latter variant is especially interesting because of its regulatory role and its differentiation into 2 functionally divergent variants (H2A.Z.1 and H2A.Z.2), further specializing the structure and function of vertebrate chromatin. In the present work we describe, for the first time, the presence of a second H2A.Z variant (H2A.Z.2) in the genome of a non-vertebrate animal, the mussel Mytilus. The molecular and evolutionary characterization of mussel H2A.Z.1 and H2A.Z.2 histones is consistent with their functional specialization, supported on sequence divergence at promoter and coding regions as well as on varying gene expression patterns. More precisely, the expression of H2A.Z.2 transcripts in gonadal tissue and its potential upregulation in response to genotoxic stress might be mirroring the specialization of this variant in DNA repair. Overall, the findings presented in this work complement recent reports describing the widespread presence of other histone variants across eukaryotes, supporting an ancestral origin and conserved role for histone variants in chromatin.

Sky1 regulates the expression of sulfur metabolism genes in response to cisplatin.

Cisplatin is commonly used in cancer therapy and yeast cells are also sensitive to this compound. We present a transcriptome analysis discriminating between RNA changes induced by cisplatin treatment, which are dependent on or independent of SKY1 function--a gene whose deletion increases resistance to the drug. Gene expression changes produced by addition of cisplatin to W303 and W303-Δsky1 cells were recorded using DNA microarrays. The data, validated by quantitative PCR, revealed 122 differentially expressed genes: 69 upregulated and 53 downregulated. Among the upregulated genes, those related to sulfur metabolism were over-represented and partially dependent on Sky1. Deletions of MET4 or other genes encoding co-regulators of the expression of sulfur-metabolism-related genes, with the exception of MET28, did not modify the cisplatin sensitivity of yeast cells. One of the genes with the highest cisplatin-induced upregulation was SEO1, encoding a putative permease of sulfur compounds. We also measured the platinum, sulfur and glutathione content in W303, W303-Δsky1 and W303-Δseo1 cells after cisplatin treatment, and integration of the data suggested that these transcriptional changes might represent a cellular response that allowed chelation of cisplatin with sulfur-containing amino acids and also helped DNA repair by stimulating purine biosynthesis. The transcription pattern of stimulation of sulfur-containing amino acids and purine synthesis decreased, or even disappeared, in the W303-Δsky1 strain.

Proteomic analyses reveal that Sky1 modulates apoptosis and mitophagy in Saccharomyces cerevisiae cells exposed to cisplatin.

Sky1 is the only member of the SR (Serine-Arginine) protein kinase family in Saccharomyces cerevisiae. When yeast cells are treated with the anti-cancer drug cisplatin, Sky1 kinase activity is necessary to produce the cytotoxic effect. In this study, proteome changes in response to this drug and/or SKY1 deletion have been evaluated in order to understand the role of Sky1 in the response of yeast cells to cisplatin. Results reveal differential expression of proteins previously related to the oxidative stress response, DNA damage, apoptosis and mitophagy. With these precedents, the role of Sky1 in apoptosis, necrosis and mitophagy has been evaluated by flow-cytometry, fluorescence microscopy, biosensors and fluorescence techniques. After cisplatin treatment, an apoptotic-like process diminishes in the ∆sky1 strain in comparison to the wild-type. The treatment does not affect mitophagy in the wild-type strain, while an increase is observed in the ∆sky1 strain. The increased resistance to cisplatin observed in the ∆sky1 strain may be attributable to a decrease of apoptosis and an increase of mitophagy.

Structural basis of specificity in tetrameric Kluyveromyces lactis ß-galactosidase.

ß-Galactosidase or lactase is a very important enzyme in the food industry, being that from the yeast Kluyveromyces lactis the most widely used. Here we report its three-dimensional structure both in the free state and complexed with the product galactose. The monomer folds into five domains in a pattern conserved with the prokaryote enzymes of the GH2 family, although two long insertions in domains 2 and 3 are unique and related to oligomerization and specificity. The tetrameric enzyme is a dimer of dimers, with higher dissociation energy for the dimers than for its assembly. Two active centers are located at the interface within each dimer in a narrow channel. The insertion at domain 3 protrudes into this channel and makes putative links with the aglycone moiety of docked lactose. In spite of common structural features related to function, the determinants of the reaction mechanism proposed for Escherichia coli ß-galactosidase are not found in the active site of the K. lactis enzyme. This is the first X-ray crystal structure for a ß-galactosidase used in food processing.

Ixr1p and the control of the Saccharomyces cerevisiae hypoxic response.

In Saccharomyces cerevisiae, adaptation to hypoxia/anaerobiosis requires the transcriptional induction or derepression of multiple genes organized in regulons controlled by specific transcriptional regulators. Ixr1p is a transcriptional regulatory factor that causes aerobic repression of several hypoxic genes (COX5B, TIR1, and HEM13) and also the activation of HEM13 during hypoxic growth. Analysis of the transcriptome of the wild-type strain BY4741 and its isogenic derivative Δixr1, grown in aerobic and hypoxic conditions, reveals differential regulation of genes related not only to the hypoxic and oxidative stress responses but also to the re-adaptation of catabolic and anabolic fluxes in response to oxygen limitation. The function of Ixr1p in the transcriptional regulation of genes from the sulfate assimilation pathway and other pathways producing α-keto acids is of biotechnological importance for industries based on yeast-derived fermentation products.

Two proteins with different functions are derived from the KlHEM13 gene.

Two proteins that differ at the N terminus (l-KlCpo and s-KlCpo) are derived from KlHEM13, a single-copy-number gene in the haploid genome of Kluyveromyces lactis. Two transcriptional start site (tss) pools are detectable using primer extension, and their selection is heme dependent. One of these tss pools is located 5' of the first translation initiation codon (TIC) in the open reading frame of KlHEM13, while the other is located between the first and second TICs. In terms of functional significance, only s-KlCpo complements the heme deficiency caused by the Δhem13 deletion in K. lactis. Data obtained from immune detection in subcellular fractions, directed mutagenesis, chromatin immunoprecipitation (ChIP) assays, and the functional relevance of ΔKlhem13 deletion for KlHEM13 promoter activity suggest that l-KlCpo regulates KlHEM13 transcription. A hypothetical model of the evolutionary origins and coexistence of these two proteins in K. lactis is discussed.

SKY1 and IXR1 interactions, their effects on cisplatin and spermine resistance in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has been previously used as a model eukaryotic system to identify genes related to drug resistance. Deletion of the IXR1 gene increases resistance to cisplatin, and deletion of the SKY1 gene increases resistance to cisplatin and spermine. Three S. cerevisiae strains and their derivatives, carrying single Δixr1 and Δsky1 and double Δixr1Δsky1 deletions, were compared in terms of resistance against these compounds. We found that the effects of these deletions are highly dependent on the genetic background of the selected strains. These results are valuable in the selection of yeast strains to be used in genetic screenings of compounds with putative pharmacological interest.

The yeast hypoxic responses, resources for new biotechnological opportunities.

Recent advances in the knowledge of molecular mechanisms that control the adaptation to low oxygen levels in yeast and their biotechnological applications, including bioproduct synthesis, such as ethanol, glutathione or recombinant proteins, as well as pathogenic virulence, are reviewed. Possible pathways and target genes, which might be of particular interest for the improvement of biotechnological applications, are evaluated.

Role of the E3 ubiquitin-ligase Hakai in intestinal inflammation and cancer bowel disease.

The E3 ubiquitin-ligases are important for cellular protein homeostasis and their deregulation is implicated in cancer. The E3 ubiquitin-ligase Hakai is involved in tumour progression and metastasis, through the regulation of the tumour suppressor E-cadherin. Hakai is overexpressed in colon cancer, however, the implication in colitis-associated cancer is unknown. Here, we investigated the potential role of Hakai in intestinal inflammation and cancer bowel disease. Several mouse models of colitis and associated cancer were used to analyse Hakai expression by immunohistochemistry. We also analysed Hakai expression in patients with inflamed colon biopsies from ulcerative colitis and Crohn's disease. By Hakai interactome analysis, it was identified Fatty Acid Synthase (FASN) as a novel Hakai-interacting protein. Moreover, we show that Hakai induces FASN ubiquitination and degradation via lysosome, thus regulating FASN-mediated lipid accumulation. An inverse expression of FASN and Hakai was detected in inflammatory AOM/DSS mouse model. In conclusion, Hakai regulates FASN ubiquitination and degradation, resulting in the regulation of FASN-mediated lipid accumulation, which is associated to the development of inflammatory bowel disease. The interaction between Hakai and FASN may be an important mechanism for the homeostasis of intestinal barrier function and in the pathogenesis of this disease.

Structural analysis of Saccharomyces cerevisiae alpha-galactosidase and its complexes with natural substrates reveals new insights into substrate specificity of GH27 glycosidases.

Alpha-galactosidases catalyze the hydrolysis of terminal alpha-1,6-galactosyl units from galacto-oligosaccharides and polymeric galactomannans. The crystal structures of tetrameric Saccharomyces cerevisiae alpha-galactosidase and its complexes with the substrates melibiose and raffinose have been determined to 1.95, 2.40, and 2.70 A resolution. The monomer folds into a catalytic (alpha/beta)(8) barrel and a C-terminal beta-sandwich domain with unassigned function. This pattern is conserved with other family 27 glycosidases, but this enzyme presents a unique 45-residue insertion in the beta-sandwich domain that folds over the barrel protecting it from the solvent and likely explaining its high stability. The structure of the complexes and the mutational analysis show that oligomerization is a key factor in substrate binding, as the substrates are located in a deep cavity making direct interactions with the adjacent subunit. Furthermore, docking analysis suggests that the supplementary domain could be involved in binding sugar units distal from the scissile bond, therefore ascribing a role in fine-tuning substrate specificity to this domain. It may also have a role in promoting association with the polymeric substrate because of the ordered arrangement that the four domains present in one face of the tetramer. Our analysis extends to other family 27 glycosidases, where some traits regarding specificity and oligomerization can be formulated on the basis of their sequence and the structures available. These results improve our knowledge on the activity of this important family of enzymes and give a deeper insight into the structural features that rule modularity and protein-carbohydrate interactions.

Production and characterization of two N-terminal truncated esterases from Thermus thermophilus HB27 in a mesophilic yeast: effect of N-terminus in thermal activity and stability.

Two N-terminally truncated variants of the esterase E34Tt from Thermus thermophilus HB27 (YP_004875.1) were expressed in Kluyveromyces lactis. Production and biochemical properties of both recombinant proteins were investigated. The esterase activity was greatly increased compared to the wild-type strain. In particular, the extracellular production of the ΔN16 variant (KLEST-3S) was 50-fold higher than that obtained with T. thermophilus HB27. Response surface methodology was applied to describe the pH and temperature dependence of both activity and stability. When compared with the wild type esterase, the optimal temperature of reaction decreased 35 and 15 °C for ΔN16 and ΔN26, respectively. KLEST-3S showed a maximum of activity at pH 7.5 and 47.5 °C, and maximal stability at pH 8.1 and 65 °C. KLEST-5A (ΔN26) did not show an absolute maximum of activity. However, best results were obtained at 40 °C and pH 8.5. KLEST-5A showed also a lower stability. In the presence of a surfactant, both proteins showed lower stability at 85 °C (t(½)< 5 min) than the wild-type enzyme (t(½)=135 min). However, in the absence of detergent, the stability of KLEST-3S was higher (t(½)=230 min, at 85 °C) than that of the mutant KLEST-5A (12 min) or the wild type enzyme (19 min). Minor differences were observed in the substrate specificity. Our results suggest that the N-terminal segment is critical for maintaining the hyperthermophilic function and stability.

Comparative transcriptome analysis of yeast strains carrying slt2, rlm1, and pop2 deletions.

The function of the genes SLT2 (encoding the Mpk1 protein), RLM1, and POP2 have previously been related to several stress responses in yeasts. DNA arrays have been used to identify differences among the transcriptomes of a Saccharomyces cerevisiae wild type strain and its derivative Δslt2, Δrlm1, and Δpop2 mutants. Correspondence analyses indicate that the vast majority of genes that show lower expression in Δrlm1 also show lower expression in Δslt2. In contrast, there is little overlap between the results of the transcriptome analyses of the Δpop2 strain and the Δslt2 or Δrlm1 strains. The DNA array data were validated by reverse Northern blotting and chromatin immunoprecipitation (ChIp). ChIp assays demonstrate Rlm1p binding to specific regions of the promoters of two genes that show expression differences between the Δrlm1 mutant and wild type strains. Interestingly, RLM1 deletion decreases the transcription of SLT2, encoding the Mpk1p kinase that phosphorylates Rlm1p, suggesting a feedback control in the signal transduction pathway. Also, deletion of RLM1 causes a decrease in the mRNA level of KDX1, which is paralogous to SLT2. In contrast, deletion of POP2 is accompanied by an increase of both SLT2 and KDX1 levels. We show that SLT2 mRNA increase in the Δpop2 strain is due to a decrease in RNA turnover, consistent with the expected loss of RNA-deadenylase activity in this strain.

The HMGB Protein KlIxr1, a DNA Binding Regulator of Kluyveromyces lactis Gene Expression Involved in Oxidative Metabolism, Growth, and dNTP Synthesis.

In the traditional fermentative model yeast Saccharomyces cerevisiae, ScIxr1 is an HMGB (High Mobility Group box B) protein that has been considered as an important regulator of gene transcription in response to external changes like oxygen, carbon source, or nutrient availability. Kluyveromyces lactis is also a useful eukaryotic model, more similar to many human cells due to its respiratory metabolism. We cloned and functionally characterized by different methodologies KlIXR1, which encodes a protein with only 34.4% amino acid sequence similarity to ScIxr1. Our data indicate that both proteins share common functions, including their involvement in the response to hypoxia or oxidative stress induced by hydrogen peroxide or metal treatments, as well as in the control of key regulators for maintenance of the dNTP (deoxyribonucleotide triphosphate) pool and ribosome synthesis. KlIxr1 is able to bind specific regulatory DNA sequences in the promoter of its target genes, which are well conserved between S. cerevisiae and K. lactis. Oppositely, we found important differences between ScIrx1 and KlIxr1 affecting cellular responses to cisplatin or cycloheximide in these yeasts, which could be dependent on specific and non-conserved domains present in these two proteins.

HMGB1 Protein Interactions in Prostate and Ovary Cancer Models Reveal Links to RNA Processing and Ribosome Biogenesis through NuRD, THOC and Septin Complexes.

This study reports the HMGB1 interactomes in prostate and ovary cancer cells lines. Affinity purification coupled to mass spectrometry confirmed that the HMGB1 nuclear interactome is involved in HMGB1 known functions such as maintenance of chromatin stability and regulation of transcription, and also in not as yet reported processes such as mRNA and rRNA processing. We have identified an interaction between HMGB1 and the NuRD complex and validated this by yeast-two-hybrid, confirming that the RBBP7 subunit directly interacts with HMGB1. In addition, we describe for the first time an interaction between two HMGB1 interacting complexes, the septin and THOC complexes, as well as an interaction of these two complexes with Rab11. Analysis of Pan-Cancer Atlas public data indicated that several genes encoding HMGB1-interacting proteins identified in this study are dysregulated in tumours from patients diagnosed with ovary and prostate carcinomas. In PC-3 cells, silencing of HMGB1 leads to downregulation of the expression of key regulators of ribosome biogenesis and RNA processing, namely BOP1, RSS1, UBF1, KRR1 and LYAR. Upregulation of these genes in prostate adenocarcinomas is correlated with worse prognosis, reinforcing their functional significance in cancer progression.

Study of the prevalence of the risk of malnutrition in the non-institutionalized population over 65 years old attended in a health center in Barcelona. / Estudio de la prevalencia del riesgo de desnutrición en la población mayor de 65 años no institucionalizada atendida en un centro de salud de Barcelona.

OBJECTIVE: To know the prevalence of risk of malnutrition in community-dwelling elderly (defined as aged >65) attended in a Primary Care Center, to find the main factors associated to malnutrition risk and to evaluate the Mini Nutritional Assessment Questionnaire (MNA) MNA Short Form vs. MNA Full Test. METHOD: Design: Cross-Sectional study. SETTING: Primary Care Center. SUBJECTS: 337 participants visited in the Community Care Center. Mini Nutritional Assessment Questionnaire (MNA) was applied; sociodemographic and Health variables were collected as well as functional evaluation tests (Short Portable Mental Status Questionnaire and Lawton & Brody Instrumental Activities of Daily Living Scale). Clinical history information was taken from the Medical Records. Using MNA Full Test (MNA-FT) as the gold standard, sensitivity, specificity and predictive values of MNA Short Form (MNA-SF) were evaluated. RESULTS: prevalence according MNA-FT was 0.6% for malnutrition and 7.7% for malnutrition risk. No gender differences were found. The average age was higher in the population with malnutrition or at risk for malnutrition (p=0.016). Significant association of malnutrition with having carer (p<0.0001) or being more dependent (p<0.0001) was found. MNA-SF showed an acceptable sensitivity (67.9%) and good specificity (92.6%). CONCLUSIONS: Compared with other studies this data showed a low prevalence of malnutrition risk in community-living elderly using the MNA test. It is recommended to use the MNA-FT in order to avoid under diagnosing malnutrition with MNA-SF.

Proteomic analysis of the oxidative stress response in Kluyveromyces lactis and effect of glutathione reductase depletion.

Yeasts are unicellular eukaryotes that provide useful models for studying the oxidative stress (OS) response. Most investigations to date have been performed on the fermentative Saccharomyces cerevisiae. The respiratory Kluyveromyces lactis is emerging as an alternative model. Our previous studies showed that glutathione reductase (Glr1) is an interesting point of difference in the OS response between the two yeasts. In the present study, using extensive proteomic analyses, the response to H(2)O(2) and its relationship to Glr1 were investigated in wild-type and glr1-deletion mutant K. lactis strains. We identified 46 proteins that showed modified expression after H(2)O(2) addition and 42 for which the change was Glr1-dependent. As expected, these proteins include a variety of antioxidant enzymes, chaperones, and oxidoreductases related to defense against OS and damage repair. They also include a number of proteins necessary for energy production and carbohydrate and amino acid metabolism. H(2)O(2) addition causes down-regulation of enzymes from the glycolytic pathway and Krebs cycle in wild-type K. lactis, whereas glr1-deletion prevents this effect and actually causes up-regulation of the glycolytic, Krebs cycle, and oxidative pentose phosphate pathways. To our knowledge, this is the first global proteomic analysis performed on K. lactis.

A functional analysis of Kluyveromyces lactis glutathione reductase.

Glutathione reductase (GLR) null mutants of the yeast Kluyveromyces lactis, a model eukaryotic respiratory cell, were created and phenotypically analysed. We found that cells lacking GLR show decreased resistance to oxidative stress and higher levels of reactive oxygen species and catalase activity than the wild-type strain. However, glutathione redox levels (GSH : GSSG ratio) were similar in the DeltaKlglr1 mutant and wild-type strains. The thioredoxin-thioredoxin reductase system is proposed as an alternative system for maintaining the GSH : GSSG ratio in the DeltaKlglr1 mutant. The involvement of GLR in glucose metabolism in K. lactis is suggested by the improved growth on glucose caused by the DeltaKlglr1 mutation and by the increased GLR activity in the DeltaKlgcr1 strain, KlGcr1 being a transcriptional activator of glycolytic genes. We also studied the subcellular location of GLR in K. lactis, showing that it is present in mitochondria; however, the DeltaKlglr1 mutation does not affect mitochondrial morphology. Genomic DNA integrity and life span are also unaffected by the DeltaKlglr1 mutation, at least under the conditions tested in this study.