The Effect of HMGB1 and HMGB2 on Transcriptional Regulation Differs in Neuroendocrine and Adenocarcinoma Models of Prostate Cancer.

Human high-mobility group-B (HMGB) proteins regulate gene expression in prostate cancer (PCa), a leading cause of oncological death in men. Their role in aggressive PCa cancers, which do not respond to hormonal treatment, was analyzed. The effects of HMGB1 and HMGB2 silencing upon the expression of genes previously related to PCa were studied in the PCa cell line PC-3 (selected as a small cell neuroendocrine carcinoma, SCNC, PCa model not responding to hormonal treatment). A total of 72% of genes analyzed, using pre-designed primer panels, were affected. HMGB1 behaved mostly as a repressor, but HMGB2 as an activator. Changes in SERPINE1, CDK1, ZWINT, and FN1 expression were validated using qRT-PCR after HMGB1 silencing or overexpression in PC-3 and LNCaP (selected as an adenocarcinoma model of PCa responding to hormonal treatment) cell lines. Similarly, the regulatory role of HMGB2 upon SERPINE1, ZWINT, FN1, IGFPB3, and TYMS expression was validated, finding differences between cell lines. The correlation between the expression of HMGB1, HMGB2, and their targets was analyzed in PCa patient samples and also in PCa subgroups, classified as neuroendocrine positive or negative, in public databases. These results allow a better understanding of the role of HMGB proteins in PCa and contribute to find specific biomarkers for aggressive PCa.

Thanksgiving to Yeast, the HMGB Proteins History from Yeast to Cancer.

Yeasts have been a part of human life since ancient times in the fermentation of many natural products used for food. In addition, in the 20th century, they became powerful tools to elucidate the functions of eukaryotic cells as soon as the techniques of molecular biology developed. Our molecular understandings of metabolism, cellular transport, DNA repair, gene expression and regulation, and the cell division cycle have all been obtained through biochemistry and genetic analysis using different yeasts. In this review, we summarize the role that yeasts have had in biological discoveries, the use of yeasts as biological tools, as well as past and on-going research projects on HMGB proteins along the way from yeast to cancer.

HMG Proteins from Molecules to Disease.

High Mobility Group (HMG) proteins are today the focus of interest due to their participation in human degenerative diseases and inflammatory responses [...].

Performance evaluation of the QIAstat-Dx® Respiratory SARS-CoV-2 Panel.

OBJECTIVE: The aim of this study was to evaluate the QIAstat-Dx® Respiratory SARS-CoV-2 Panel (QIAstat-SARS-CoV-2), which is a closed, fully automated, multiplex polymerase chain reaction (PCR) assay that detects severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and 21 other pathogens that cause respiratory disease. METHODS: Nasopharyngeal swabs from patients with or suspected of having coronavirus disease 2019 were collected and tested at Bichat-Claude Bernard Hospital, Paris, France. Using the World Health Organisation-approved real-time-PCR assay developed by the Charité Institute of Virology as the reference, positive percent agreement (PPA) and negative percent agreement (NPA) were calculated. RESULTS: In total, 189 negative and 88 positive samples were analyzed. QIAstat-SARS-CoV-2 had an NPA of 90.48% (95% confidence interval (CI), 85.37%, 94.26%) and a PPA of 94.32% (95% CI, 87.24%, 98.13%). Co-infections were detected by QIAstat-SARS-CoV-2 in 4/277 specimens. The methods exhibited comparable failure rates (23/307 [7.5%] vs. 6/298 [2.0%] for QIAstat-SARS-CoV-2 and reference methods, respectively). The turnaround time was shorter for QIAstat-SARS-CoV-2 compared with the reference method (difference in mean -14:30 h [standard error, 0:03:23; 95% CI, -14:37, -14:24]; P < 0.001). CONCLUSIONS: QIAstat-SARS-CoV-2 shows good agreement with the reference assay, providing faster and accurate results for detecting SARS-CoV-2.

The HMGB1-2 Ovarian Cancer Interactome. The Role of HMGB Proteins and Their Interacting Partners MIEN1 and NOP53 in Ovary Cancer and Drug-Response.

High mobility group box B (HMGB) proteins are overexpressed in different types of cancers such as epithelial ovarian cancers (EOC). We have determined the first interactome of HMGB1 and HMGB2 in epithelial ovarian cancer (the EOC-HMGB interactome). Libraries from the SKOV-3 cell line and a primary transitional cell carcinoma (TCC) ovarian tumor were tested by the Yeast Two Hybrid (Y2H) approach. The interactome reveals proteins that are related to cancer hallmarks and their expression is altered in EOC. Moreover, some of these proteins have been associated to survival and prognosis of patients. The interaction of MIEN1 and NOP53 with HMGB2 has been validated by co-immunoprecipitation in SKOV-3 and PEO1 cell lines. SKOV-3 cells were treated with different anti-tumoral drugs to evaluate changes in HMGB1, HMGB2, MIEN1 and NOP53 gene expression. Results show that combined treatment of paclitaxel and carboplatin induces a stronger down-regulation of these genes in comparison to individual treatments. Individual treatment with paclitaxel or olaparib up-regulates NOP53, which is expressed at lower levels in EOC than in non-cancerous cells. On the other hand, bevacizumab diminishes the expression of HMGB2 and NOP53. This study also shows that silencing of these genes affects cell-viability after drug exposure. HMGB1 silencing causes loss of response to paclitaxel, whereas silencing of HMGB2 slightly increases sensitivity to olaparib. Silencing of either HMGB1 or HMGB2 increases sensitivity to carboplatin. Lastly, a moderate loss of response to bevacizumab is observed when NOP53 is silenced.

The Challenges and Opportunities of LncRNAs in Ovarian Cancer Research and Clinical Use.

Ovarian cancer is one of the most lethal gynecological malignancies worldwide because it tends to be detected late, when the disease has already spread, and prognosis is poor. In this review we aim to highlight the importance of long non-coding RNAs (lncRNAs) in diagnosis, prognosis and treatment choice, to make progress towards increasingly personalized medicine in this malignancy. We review the effects of lncRNAs associated with ovarian cancer in the context of cancer hallmarks. We also discuss the molecular mechanisms by which lncRNAs become involved in cellular physiology; the onset, development and progression of ovarian cancer; and lncRNAs' regulatory mechanisms at the transcriptional, post-transcriptional and post-translational stages of gene expression. Finally, we compile a series of online resources useful for the study of lncRNAs, especially in the context of ovarian cancer. Future work required in the field is also discussed along with some concluding remarks.

Characterization of HMGB1/2 Interactome in Prostate Cancer by Yeast Two Hybrid Approach: Potential Pathobiological Implications.

High mobility group box B (HMGB) proteins are pivotal in the development of cancer. Although the proteomics of prostate cancer (PCa) cells has been reported, the involvement of HMGB proteins and their interactome in PCa is an unexplored field of considerable interest. We describe herein the results of the first HMGB1/HMGB2 interactome approach to PCa. Libraries constructed from the PCa cell line, PC-3, and from patients' PCa primary tumor have been screened by the yeast 2-hybrid approach (Y2H) using HMGB1 and HMGB2 baits. Functional significance of this PCa HMGB interactome has been validated through expression and prognosis data available on public databases. Copy number alterations (CNA) affecting these newly described HMGB interactome components are more frequent in the most aggressive forms of PCa: those of neuroendocrine origin or castration-resistant PCa. Concordantly, adenocarcinoma PCa samples showing CNA in these genes are also associated with the worse prognosis. These findings open the way to their potential use as discriminatory biomarkers between high and low risk patients. Gene expression of a selected set of these interactome components has been analyzed by qPCR after HMGB1 and HMGB2 silencing. The data show that HMGB1 and HMGB2 control the expression of several of their interactome partners, which might contribute to the orchestrated action of these proteins in PCa.

Structural determination of Enzyme-Graphene Nanocomposite Sensor Material.

State-of-the-art ultra-sensitive blood glucose-monitoring biosensors, based on glucose oxidase (GOx) covalently linked to a single layer graphene (SLG), will be a valuable next generation diagnostic tool for personal glycemic level management. We report here our observations of sensor matrix structure obtained using a multi-physics approach towards analysis of small-angle neutron scattering (SANS) on graphene-based biosensor functionalized with GOx under different pH conditions for various hierarchical GOx assemblies within SLG. We developed a methodology to separately extract the average shape of GOx molecules within the hierarchical assemblies. The modeling is able to resolve differences in the average GOx dimer structure and shows that treatment under different pH conditions lead to differences within the GOx at the dimer contact region with SLG. The coupling of different analysis methods and modeling approaches we developed in this study provides a universal approach to obtain detailed structural quantifications, for establishing robust structure-property relationships. This is an essential step to obtain an insight into the structure and function of the GOx-SLG interface for optimizing sensor performance.

Optimization of Saccharomyces cerevisiae α-galactosidase production and application in the degradation of raffinose family oligosaccharides.

BACKGROUND: α-Galactosidases are enzymes that act on galactosides present in many vegetables, mainly legumes and cereals, have growing importance with respect to our diet. For this reason, the use of their catalytic activity is of great interest in numerous biotechnological applications, especially those in the food industry directed to the degradation of oligosaccharides derived from raffinose. The aim of this work has been to optimize the recombinant production and further characterization of α-galactosidase of Saccharomyces cerevisiae. RESULTS: The MEL1 gene coding for the α-galactosidase of S. cerevisiae (ScAGal) was cloned and expressed in the S. cerevisiae strain BJ3505. Different constructions were designed to obtain the degree of purification necessary for enzymatic characterization and to improve the productive process of the enzyme. ScAGal has greater specificity for the synthetic substrate p-nitrophenyl-α-D-galactopyranoside than for natural substrates, followed by the natural glycosides, melibiose, raffinose and stachyose; it only acts on locust bean gum after prior treatment with ß-mannosidase. Furthermore, this enzyme strongly resists proteases, and shows remarkable activation in their presence. Hydrolysis of galactose bonds linked to terminal non-reducing mannose residues of synthetic galactomannan-oligosaccharides confirms that ScAGal belongs to the first group of α-galactosidases, according to substrate specificity. Optimization of culture conditions by the statistical model of Response Surface helped to improve the productivity by up to tenfold when the concentration of the carbon source and the aeration of the culture medium was increased, and up to 20 times to extend the cultivation time to 216 h. CONCLUSIONS: ScAGal characteristics and improvement in productivity that have been achieved contribute in making ScAGal a good candidate for application in the elimination of raffinose family oligosaccharides found in many products of the food industry.

Genomic analysis and lactose transporter expression in Kluyveromyces marxianus CCT 7735.

Kluyveromyces marxianus CCT 7735 has been used to produce ethanol, aromatic compounds, enzymes and heterologous proteins besides assimilates lactose as carbon source. Its genome has 10.7 Mb and encodes 4787 genes distributed in 8 nuclear chromosomes and one mitochondrial. Contrary to Kluyveromyces lactis, which has a unique LAC12 gene (encodes lactose permease), K. marxianus possesses four. The presence of degenerated copies and Solo-LTRs related to retrotransposon TKM close to the LAC12 genes in K. marxianus indicates ectopic recombinations. The Lac12 permeases of K. marxianus and K. lactis are conserved, however the conservation is higher between the copy of the left side of the chromosome three and the unique copy of K. lactis, indicating that this copy is the ancestor. The expression of the four LAC12 genes occurred in aerobiosis and hypoxia. Notably, the high lactose consumption in hypoxia seems to be related to the high expression of the LAC12 genes.

Bioconversion of Beet Molasses to Alpha-Galactosidase and Ethanol.

Molasses are sub-products of the sugar industry, rich in sucrose and containing other sugars like raffinose, glucose, and fructose. Alpha-galactosidases (EC. 3.2.1.22) catalyze the hydrolysis of alpha-(1,6) bonds of galactose residues in galacto-oligosaccharides (melibiose, raffinose, and stachyose) and complex galactomannans. Alpha-galactosidases have important applications, mainly in the food industry but also in the pharmaceutical and bioenergy sectors. However, the cost of the enzyme limits the profitability of most of these applications. The use of cheap sub-products, such as molasses, as substrates for production of alpha-galactosidases, reduces the cost of the enzymes and contributes to the circular economy. Alpha-galactosidase is a specially indicated bioproduct since, at the same time, it allows to use the raffinose present in molasses. This work describes the development of a two-step system for the valuation of beet molasses, based on their use as substrate for alpha-galactosidase and bioethanol production by Saccharomyces cerevisiae. Since this yeast secretes high amounts of invertase, to avoid congest the secretory route and to facilitate alpha-galactosidase purification from the culture medium, a mutant in the SUC2 gene (encoding invertase) was constructed. After a statistical optimization of culture conditions, this mutant yielded a very high rate of molasses bioconversion to alpha-galactosidase. In the second step, the SUC2 wild type yeast strain fermented the remaining sucrose to ethanol. A procedure to recycle the yeast biomass, by using it as nitrogen source to supplement molasses, was also developed.

Valuation of agro-industrial wastes as substrates for heterologous production of α-galactosidase.

BACKGROUND: The recycling of agro-industrial wastes is at present limited by the availability of efficient and low-cost enzyme cocktails. The use of these materials as culture media to produce the enzymes can contribute to the profitability of the recycling process and to the circular economy. The aim of this work is the construction of a recombinant yeast strain efficient to grow in mixed whey (residue of cheese making) and beet molasses (residue of sugar manufacture) as culture medium, and to produce heterologous α-galactosidase, an enzyme with varied industrial applications and wide market. RESULTS: The gene MEL1, encoding the α-galactosidase of Saccharomyces cerevisiae, was integrated (four copies) in the LAC4 locus of the Kluyveromyces lactis industrial strain GG799. The constructed recombinant strain produces high levels of extracellular α-galactosidase under the control of the LAC4 promoter, inducible by lactose and galactose, and the native MEL1 secretion signal peptide. K. lactis produces natively beta-galactosidase and invertase thus metabolizing the sugars of whey and molasses. A culture medium based on whey and molasses was statistically optimized, and then the cultures scaled-up at laboratory level, thus obtaining 19 U/mL of heterologous α-galactosidase with a productivity of 0.158 U/L h, which is the highest value reported hitherto from a cheap waste-based medium. CONCLUSIONS: A K. lactis recombinant strain was constructed and a sustainable culture medium, based on a mixture of cheese whey and beet molasses, was optimized for high productivity of S. cerevisiae α-galactosidase, thus contributing to the circular economy by producing a heterologous enzyme from two agro-industrial wastes.

Heat-Loving ß-Galactosidases from Cultured and Uncultured Microorganisms.

ß-galactosidases (EC.3.2.1.23), which hydrolyze lactose to glucose and galactose, have two main applications in the food industry: the production of low-lactose milk and dairy goods for lactose intolerant people, and the generation of galacto-oligosaccharides by transgalactosylation reactions. Due to their thermostability, ß-galactosidases from thermophilic microorganisms are very interesting for industrial processes, as high temperatures can increase the initial productivity of the enzyme, provide higher solubility of substrates, and prevent microbial contamination. In the past, it was necessary to cultivate and grow thermophilic microorganisms to discover novel thermozymes, but the development of metagenomic techniques has allowed researchers to access the genomic potential of uncultivated microbes and their enzymes. The present review gives a brief outline of thermophilic ß-galactosidases, with a special focus on those obtained through metagenomics. Additionally, the sequences of ß-galactosidases found in some public metagenomes from hot springs were studied and compared to other known thermostable ß-galactosidases.

Cellulases from Thermophiles Found by Metagenomics.

Cellulases are a heterogeneous group of enzymes that synergistically catalyze the hydrolysis of cellulose, the major component of plant biomass. Such reaction has biotechnological applications in a broad spectrum of industries, where they can provide a more sustainable model of production. As a prerequisite for their implementation, these enzymes need to be able to operate in the conditions the industrial process requires. Thus, cellulases retrieved from extremophiles, and more specifically those of thermophiles, are likely to be more appropriate for industrial needs in which high temperatures are involved. Metagenomics, the study of genes and gene products from the whole community genomic DNA present in an environmental sample, is a powerful tool for bioprospecting in search of novel enzymes. In this review, we describe the cellulolytic systems, we summarize their biotechnological applications, and we discuss the strategies adopted in the field of metagenomics for the discovery of new cellulases, focusing on those of thermophilic microorganisms.

Delineating the HMGB1 and HMGB2 interactome in prostate and ovary epithelial cells and its relationship with cancer.

High Mobility Group B (HMGB) proteins are involved in cancer progression and in cellular responses to platinum compounds used in the chemotherapy of prostate and ovary cancer. Here we use affinity purification coupled to mass spectrometry (MS) and yeast two-hybrid (Y2H) screening to carry out an exhaustive study of HMGB1 and HMGB2 protein interactions in the context of prostate and ovary epithelia. We present a proteomic study of HMGB1 partners based on immunoprecipitation of HMGB1 from a non-cancerous prostate epithelial cell line. In addition, HMGB1 and HMGB2 were used as baits in yeast two-hybrid screening of libraries from prostate and ovary epithelial cell lines as well as from healthy ovary tissue. HMGB1 interacts with many nuclear proteins that control gene expression, but also with proteins that form part of the cytoskeleton, cell-adhesion structures and others involved in intracellular protein translocation, cellular migration, secretion, apoptosis and cell survival. HMGB2 interacts with proteins involved in apoptosis, cell motility and cellular proliferation. High confidence interactors, based on repeated identification in different cell types or in both MS and Y2H approaches, are discussed in relation to cancer. This study represents a useful resource for detailed investigation of the role of HMGB1 in cancer of epithelial origins, as well as potential alternative avenues of therapeutic intervention.

Transcriptome analysis of the thermotolerant yeast Kluyveromyces marxianus CCT 7735 under ethanol stress.

The thermotolerant yeast Kluyveromyces marxianus displays a potential to be used for ethanol production from both whey and lignocellulosic biomass at elevated temperatures, which is highly alluring to reduce the cost of the bioprocess. Nevertheless, contrary to Saccharomyces cerevisiae, K. marxianus cannot tolerate high ethanol concentrations. We report the transcriptional profile alterations in K. marxianus under ethanol stress in order to gain insights about mechanisms involved with ethanol response. Time-dependent changes have been characterized under the exposure of 6% ethanol and compared with the unstressed cells prior to the ethanol addition. Our results reveal that the metabolic flow through the central metabolic pathways is impaired under the applied ethanol stress. Consistent with these results, we also observe that genes involved with ribosome biogenesis are downregulated and gene-encoding heat shock proteins are upregulated. Remarkably, the expression of some gene-encoding enzymes related to unsaturated fatty acid and ergosterol biosynthesis decreases upon ethanol exposure, and free fatty acid and ergosterol measurements demonstrate that their content in K. marxianus does not change under this stress. These results are in contrast to the increase previously reported with S. cerevisiae subjected to ethanol stress and suggest that the restructuration of K. marxianus membrane composition differs in the two yeasts which gives important clues to understand the low ethanol tolerance of K. marxianus compared to S. cerevisiae.

Viabilidad y resultados de un cuestionario breve de calidad dietética en atención primaria: estudio EMAP / Feasibility and results of the short Diet Quality Screener in Primary Care: EMAP study

OBJETIVO: Estudiar la viabilidad y resultados del cuestionario autoadministrado sDQS en consultas de atención primaria y las variables asociadas a su dificultad y a una dieta inadecuada. MÉTODO: Estudio descriptivo transversal. Participaron 4 centros de salud de Barcelona. Se incluyeron consecutivamente 196 personas >18 años con diabetes mellitus (DM), hipertensión arterial (HTA) o hipercolesterolemia mediante muestreo consecutivo. Las variables principales fueron: edad, sexo, nivel educacional, factores de riesgo cardiovascular, índice de masa corporal, tiempo, dificultad en rellenar el cuestionario y puntuación: dieta inadecuada ≤18, adecuada en algunos aspectos 19-27, adecuada > 27. RESULTADOS: La edad media fue de 48,8 años (52% varones). El 50% tenían estudios primarios o más. El 54,6% tenían HTA, el 23,5% DM, el 56,6% hipercolesterolemia y el 27,5% obesidad. El tiempo medio para completar el cuestionario fue 2,3min y > 80% lo consideraron fácil o muy fácil. Tenían dieta inadecuada el 21,4%, dieta adecuada en algunos aspectos el 76,5% y dieta adecuada el 2%. La edad > 49 años (OR 2,0; IC95%: 1,0-4,3) y la dieta inadecuada (OR 2,3; IC95%: 1,1-5,1) se asociaron a un tiempo ≥2min para hacer el cuestionario. La edad Ͱ 4;49 años (OR 2,9; IC 95%: 1,2-6,8) y no realizar dieta hipolipemiante (OR 2,2; IC 95%: 1,1- 4,5) se asociaron a dieta inadecuada. CONCLUSIONES: El cuestionario autoadministrado sDQS es fácil de aplicar y no representa un aumento de tiempo importante en atención primaria. Una importante proporción de personas realiza una dieta de baja calidad

OBJECTIVE: To study the feasibility and results of the self-reported short diet quality screener (sDQS) in Primary Care. The variables associated with difficulty and inadequate diet are also determined. METHOD: Cross-sectional descriptive study conducted with 196 participants aged >18 years with diabetes mellitus, hypertension, or hypercholesterolaemia, consecutively included from 4 Primary Health Care Centres in Barcelona. The main variables collected were, age, sex, educational level, cardiovascular risk factors, body mass index, time to complete the sDQS, degree of difficulty, and diet score: inadequate diet ≤18, adequate in some aspects 19-27, adequate >27. RESULTS:The mean age was 48.8 years (52% males). The analysis of the variables showed that the prevalence of having higher than a primary education level, hypertension, diabetes, hypercholesterolemia, and obesity was 50%, 54.6%, 23.5%, 56.6%, and 27.5%, respectively. The mean time to complete the questionnaire was 2.3min. More than 80% considered it easy or very easy. An inadequate diet was reported by 21.4%, adequate in some aspects by 76.5%, and an adequate diet only by 2%. To be older than 49 years and a low diet quality increased the risk of needing ≥2 min to complete the sDQS (OR 2.0, 95% CI; 1.0-4.3, and OR 2.3, 95% CI; 1.1-5.1, respectively). Not following a low cholesterol diet and age less than 49 years increased the risk of a low diet quality (OR 2.2; 95% CI: 1.1-4.5, and OR 2.9; 95% CI: 1.2-6.8, respectively). CONCLUSIONS: The completion of the sDQS is easy and was not a significant time-burden in Primary Care. A significant proportion of participants with cardiovascular risk reported a low diet quality

[Feasibility and results of the short Diet Quality Screener in Primary Care: EMAP study]. / Viabilidad y resultados de un cuestionario breve de calidad dietética en atención primaria: estudio EMAP.

OBJECTIVE: To study the feasibility and results of the self-reported short diet quality screener (sDQS) in Primary Care. The variables associated with difficulty and inadequate diet are also determined. METHOD: Cross-sectional descriptive study conducted with 196 participants aged >18 years with diabetes mellitus, hypertension, or hypercholesterolaemia, consecutively included from 4 Primary Health Care Centres in Barcelona. The main variables collected were, age, sex, educational level, cardiovascular risk factors, body mass index, time to complete the sDQS, degree of difficulty, and diet score: inadequate diet ≤18, adequate in some aspects 19-27, adequate >27. RESULTS: The mean age was 48.8 years (52% males). The analysis of the variables showed that the prevalence of having higher than a primary education level, hypertension, diabetes, hypercholesterolemia, and obesity was 50%, 54.6%, 23.5%, 56.6%, and 27.5%, respectively. The mean time to complete the questionnaire was 2.3min. More than 80% considered it easy or very easy. An inadequate diet was reported by 21.4%, adequate in some aspects by 76.5%, and an adequate diet only by 2%. To be older than 49 years and a low diet quality increased the risk of needing ≥2min to complete the sDQS (OR 2.0, 95% CI; 1.0-4.3, and OR 2.3, 95% CI; 1.1-5.1, respectively). Not following a low cholesterol diet and age less than 49 years increased the risk of a low diet quality (OR 2.2; 95% CI: 1.1-4.5, and OR 2.9; 95% CI: 1.2-6.8, respectively). CONCLUSIONS: The completion of the sDQS is easy and was not a significant time-burden in Primary Care. A significant proportion of participants with cardiovascular risk reported a low diet quality.