TET2 overexpression in chronic lymphocytic leukemia is unrelated to the presence of TET2 variations

This is an open access article distributed under the Creative Commons Attribution License.TET2 is involved in a variety of hematopoietic malignancies, mainly in myeloid malignancies. Most mutations of TET2 have been identified in myeloid disorders, but some have also recently been described in mature lymphoid neoplasms. In contrast to the large amount of data about mutations of TET2, some data are available for gene expression. Moreover, the role of TET2 in chronic lymphocytic leukemia (CLL) is unknown. This study analyzes both TET2 expression and mutations in 48 CLL patients. TET2 expression was analyzed by exon arrays and quantitative real-time polymerase chain reaction (qRT-PCR). Next-generation sequencing (NGS) technology was applied to investigate the presence of TET2 variations. Overexpression of TET2 was observed in B-cell lymphocytes from CLL patients compared with healthy donors (P = 0.004). In addition, in CLL patients, an overexpression of TET2 was also observed in the clonal B cells compared with the nontumoral cells (P = 0.002). However, no novel mutations were observed. Therefore, overexpression of TET2 in CLL seems to be unrelated to the presence of genomic TET2 variations.This work was partially supported by Grants from the Spanish Fondo de Investigaciones Sanitarias FIS 09/01543, PI12/00281, Proyectos de Investigacion del SACYL 355/A/09, COST Action “EuGESMA” (BM0801), Fundación “Manuel Solorzano,” Obra Social Banca Cívica (Caja Burgos), Fundacion Española de Hematología y Hemoterapia (FEHH), and by a Grant (RD12/0036/0069) from Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Economy and Competitiveness and European Regional Development Fund (ERDF) “Una manera de hacer Europa”, and NGS-PTL no. 306242. Maríıa Hernandez-Sánchez is fully suported by an “Ayuda predoctoral de la Junta de Castilla y Leon” by the “Fondo Social Europeo.”Peer Reviewe

Non-conventional yeasts as hosts for heterologous protein production

Creative Commons-Attribution-Non-Commercial-Share Alike 3.0 Spain.-- et al.Yeasts are an attractive group of lower eukaryotic microorganisms, some of which are used in several industrial processes that include brewing, baking and the production of a variety of biochemical compounds. More recently, yeasts have been developed as host organisms for the production of foreign (heterologous) proteins. Saccharomyces ccrevisiae has usually been the yeast of choice, but an increasing number of alternative non-Saccharomyces yeasts has now become accessible for modern molecular genetics techniques. Some of them exhibit certain favourable traits such as high-level secretion or very strong and tightly regulated promoters, offering significant advantages over traditional bakers' yeast. In the present work, the current status of Kluyveromyces lactis, Yarrowia lipolytica, Hansennla polymorpha and Picliia pastoris (the best-known alternative yeast systems) is reviewed. The advantages and limitations of these systems are discussed in relation to S. cerevisiae. © Springer-Verlag 1998.This work was partially supported by grants from the CICYT (BIO92-0304 and BIO 95-0518) and EU (BIO4-CT96-0003).Peer Reviewe

Transcriptome analysis reveals molecular profiles associated with evolving steps of monoclonal gammopathies

This is an open-access paper.-- et al.A multistep model has been proposed of disease progression starting in monoclonal gammopathy of undetermined significance continuing through multiple myeloma, sometimes with an intermediate entity called smoldering myeloma, and ending in extramedullary disease. To gain further insights into the role of the transcriptome deregulation in the transition from a normal plasma cell to a clonal plasma cell, and from an indolent clonal plasma cell to a malignant plasma cell, we performed gene expression profiling in 20 patients with monoclonal gammopathy of undetermined significance, 33 with high-risk smoldering myeloma and 41 with multiple myeloma. The analysis showed that 126 genes were differentially expressed in monoclonal gammopathy of undetermined significance, smoldering myeloma and multiple myeloma as compared to normal plasma cell. Interestingly, 17 and 9 out of the 126 significant differentially expressed genes were small nucleolar RNA molecules and zinc finger proteins. Several proapoptotic genes (AKT1 and AKT2) were down-regulated and antiapoptotic genes (APAF1 and BCL2L1) were up-regulated in multiple myeloma, both symptomatic and asymptomatic, compared to monoclonal gammopathy of undetermined significance. When we looked for those genes progressively modulated through the evolving stages of monoclonal gammopathies, eight snoRNA showed a progressive increase while APAF1, VCAN and MEGF9 exhibited a progressive downregulation. In conclusion, our data show that although monoclonal gammopathy of undetermined significance, smoldering myeloma and multiple myeloma are not clearly distinguishable groups according to their gene expression profiling, several signaling pathways and genes were significantly deregulated at different steps of the transformation process.This study was partially supported by Spanish FIS (PI080568, PS09/01450 and PS0901897), “Gerencia Regional de Salud, Junta de Castilla y León” (GRS 702/A/11) grant, and the Spanish Myeloma Network Program (RD06/0020/0006, RD12/0036/0058 and RD12/0036/0046).Peer Reviewe

Integrative analysis of DNA copy number, DNA methylation and gene expression in multiple myeloma reveals alterations related to relapse

Multiple myeloma (MM) remains incurable despite the introduction of novel agents, and a relapsing course is observed in most patients. Although the development of genomic technologies has greatly improved our understanding of MM pathogenesis, the mechanisms underlying relapse have been less thoroughly investigated. In this study, an integrative analysis of DNA copy number, DNA methylation and gene expression was conducted in matched diagnosis and relapse samples from MM patients. Overall, the acquisition of abnormalities at relapse was much more frequent than the loss of lesions present at diagnosis, and DNA losses were significantly more frequent in relapse than in diagnosis samples. Interestingly, copy number abnormalities involving more than 100 Mb of DNA at relapse significantly affect the gene expression of these samples, provoking a particular deregulation of the IL-8 pathway. On the other hand, no significant modifications of gene expression were observed in those samples with less than 100 Mb affected by chromosomal changes. Although several statistical approaches were used to identify genes whose abnormal expression at relapse was regulated by methylation, only two genes that were significantly deregulated in relapse samples (SORL1 and GLT1D1) showed a negative correlation between methylation and expression. Further analysis revealed that DNA methylation was involved in regulating SORL1 expression in MM. Finally, relevant changes in gene expression observed in relapse samples, such us downregulation of CD27 and P2RY8, were most likely not preceded by alterations in the corresponding DNA. Taken together, these results suggest that the genomic heterogeneity described at diagnosis remains at relapse.This work was partially supported by the Instituto de Salud Carlos III-Cofinanciación con fondos FEDER (PI080568, PS0901897 and PI13/00111), the Gerencia Regional de Salud, Junta de Castilla y León (GRS202/A08 and GRS 702/A/11), the Spanish Myeloma Network Program (RD06/0020/0006) and the Asociación Española Contra el Cáncer (AECC, GCB120981SAN).Peer Reviewe

Non-conventional yeasts as hosts for heterologous protein production

Yeasts are an attractive group of lower eukaryotic microorganisms, some of which are used in several industrial processes that include brewing, baking and the production of a variety of biochemical compounds. More recently, yeasts have been developed as host organisms for the production of foreign (heterologous) proteins. Saccharomyces cerevisiae has usually been the yeast of choice, but an increasing number of alternative non-Saccharomyces yeasts has now become accessible for modern molecular genetics techniques. Some of them exhibit certain favourable traits such as high-level secretion or very strong and tightly regulated promoters, offering significant advantages over traditional bakers’ yeast. In the present work, the current status of Kluyveromyces lactis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris (the best-known alternative yeast systems) is reviewed. The advantages and limitations of these systems are discussed in relation to S. cerevisiae

Non-conventional yeasts as hosts for heterologous protein production.

Yeasts are an attractive group of lower eukaryotic microorganisms, some of which are used in several industrial processes that include brewing, baking and the production of a variety of biochemical compounds. More recently, yeasts have been developed as host organisms for the production of foreign (heterologous) proteins. Saccharomyces cerevisiae has usually been the yeast of choice, but an increasing number of alternative non-Saccharomyces yeasts has now become accessible for modern molecular genetics techniques. Some of them exhibit certain favourable traits such as high-level secretion or very strong and tightly regulated promoters, offering significant advantages over traditional bakers' yeast. In the present work, the current status of Kluyveromyces lactis, Yarrowia lipolytica, Hansenula polymorpha and Pichia pastoris (the best-known alternative yeast systems) is reviewed. The advantages and limitations of these systems are discussed in relation to S. cerevisiae.Spanish Society for Microbiolog

Deslignificación de pasta kraft de Pinus radiata con una levadura genéticamente modificada para producir lacasa

Una posible aplicación biotecnológica de las lacasas fúngicas es el blanqueo de pasta de papel mediante la degradación de la lignina en la pasta cruda. Para ello es necesaria su producción a bajo coste y a concentraciones superiores a las secretadas por los hongos de forma natural. La expresión heteróloga en levaduras es una alternativa para su producción a mayor escala. En el presente trabajo se ha realizado la expresión heteróloga del gen cglcc1, responsable de la producción de lacasa en el hongo basidiomiceto Coriolopsis gallica, en la levadura Kluyveromyces lactis. Posteriormente y para verificar si la capacidad deslignificante se mantiene en la levadura, se ha tratado una pasta kraft de Pinus radiata con la cepa recombinante. Tras el tratamiento, el número kappa disminuyó en un 13%, mientras que el contenido en lignina lo hizo en un 22%, corroborando así el efecto deslignificador. Se concluye que el empleo de microorganismos carentes de actividades celulolíticas y genéticamente modificados para producir altos niveles de actividad lacasa constituye una opción para deslignificar aplicable a procesos de blanqueo de pasta de celulosa.Peer Reviewe

Heterologous protein secretion directed by a repressible acid phosphatase system of Kluyveromyces lactis: Characterization of upstream region-activating sequences in the KIPH05 gene

Transcription of the repressible acid phosphatase gene (KIPH05) in Kluyveromyces lactis is strongly regulated in response to the level of inorganic phosphate (P(i)) present in the growth medium. We have begun a study of the promoter region of this gene in order to identify sequences involved in the phosphate control of KIPH05 expression and to design new expression-secretion systems in K. lactis. Deletion analysis and directed mutagenesis revealed two major identical upstream activating sequences (UAS) CACGTG at positions -430 (UAS1) and -192 (UAS2) relative to the ATG initiation codon. These sequences are identical to those described for Saccharomyces cerevisiae for the binding of Pho4p. Deletion or directed mutagenesis of either one or both UAS reduce KIPH05 expression by the same amount (approximately 80%). When fused to the coding region of trout growth hormone cDNA (tGH-II), the promoter and signal peptide-encoding region of the phosphate-repressible KIPH05 gene drives the expression of this gene and the secretion of the tGHH protein. Synthesis of tGHIIp in K. lactis transformants carrying this construct was found to be regulated by the P(i) present in the medium; derepression of heterologous protein expression can therefore be achieved by lowering the P(i) concentration.This work was partially supported by grants from the CICYT (BIO92-0304 and BIO95-0518) and EU (BIO4-CT96-0003).Peer Reviewe

Disruption of six Saccharomyces cerevisiae genes from chromosome IV and basic phenotypic analysis of deletion mutants

We report here the construction of six deletion mutants and the analysis of their basic phenotype. Deletion cassettes containing the KanMX4 marker module and long flanking regions homologous to the target locus were constructed for each of the six open reading-frames (ORFs YDL088c, YDL087c, YDL086w, YDL085w, YDL084w and YDL082w) located on chromosome IV. Sporulation and tetrad analysis of heterozygous deletant strains revealed that, in the FY1679 genetic background, ORFs YDL088c, YDL087c and YDL084w are essential genes for vegetative growth whereas YDL086w, YDL085w and YDL082w are non-essential. ydl088cΔ and ydl084wΔ haploid strains are viable in the CEN. PK2 genetic background although ydl084wΔ grows at a slower rate than the wild type. Complementation tests by corresponding cognate genes confirmed that gene inactivation was responsible for these growth defects.Contract/grant sponsor: European Commission (EUROFAN I programme). Contract/grant sponsor: CICYT. Contract/grant number: BIO96–2055-CE. This work was supported by the EUROFAN I programme of the European Commission and by a grant from the CICYT (BIO96–2055-CE).Peer Reviewe

Molecular characterization of chronic lymphocytic leukemia patients with a high number of losses in 13q14

This is an open-access article distributed under the terms of the Creative Commons Attribution License.-- et al.[Background]: Patients with chronic lymphocytic leukemia and 13q deletion as their only FISH abnormality could have a different outcome depending on the number of cells displaying this aberration. Thus, cases with a high number of 13q- cells (13q-H) had both shorter overall survival and time to first therapy. The goal of the study was to analyze the genetic profile of 13q-H patients. [Design and Methods]: A total of 102 samples were studied, 32 of which served as a validation cohort and five were healthy donors. [Results]: Chronic lymphocytic leukemia patients with higher percentages of 13q- cells (>80%) showed a different level of gene expression as compared to patients with lower percentages (<80%, 13q-L). This deregulation affected genes involved in apoptosis and proliferation (BCR and NFkB signaling), leading to increased proliferation and decreased apoptosis in 13q-H patients. Deregulation of several microRNAs, such as miR-15a, miR-155, miR-29a and miR-223, was also observed in these patients. In addition, our study also suggests that the gene expression pattern of 13q-H cases could be similar to the patients with 11q- or 17p-. [Conclusions]: This study provides new evidence regarding the heterogeneity of 13q deletion in chronic lymphocytic leukemia patients, showing that apoptosis, proliferation as well as miRNA regulation are involved in cases with higher percentages of 13q- cells.The study was partially supported by grants from the Spanish Fondo de Investigaciones Sanitarias 02/1041 and FIS 09/01543; Caja de Burgos-Banca Cívica, Proyectos de Investigación del SACYL 106/A/06 and by the Acción Transversal del Cáncer project, through an agreement between the Instituto de Salud Carlos III (ISCIII), the Spanish Ministry of Science and Innovation, the Cancer Research Foundation of Salamanca University and the Redes de Investigación RTIIC (FIS). AR is fully supported by an Ayuda Predoctoral FIS de Formación en Investigación by the Spanish Fondo de Investigaciones Sanitarias.Peer Reviewe