Mild hypothermia upregulates myc and xbp1s expression and improves anti-TNFα production in CHO cells.

Chinese hamster ovary (CHO) cells are the most frequently used host for commercial production of therapeutic proteins. However, their low protein productivity in culture is the main hurdle to overcome. Mild hypothermia has been established as an effective strategy to enhance protein specific productivity, although the causes of such improvement still remain unclear. The self-regulation of global transcriptional regulatory factors, such as Myc and XBP1s, seems to be involved in increased the recombinant protein production at low temperature. This study evaluated the impact of low temperature in CHO cell cultures on myc and xbp1s expression and their effects on culture performance and cell metabolism. Two anti-TNFα producing CHO cell lines were selected considering two distinct phenotypes: i.e. maximum cell growth, (CN1) and maximum specific anti-TNFα production (CN2), and cultured at 37, 33 and 31°C in a batch system. Low temperature led to an increase in the cell viability, the expression of the recombinant anti-TNFα and the production of anti-TNFα both in CN1 and CN2. The higher production of anti-TNFα in CN2 was mainly associated with the large expression of anti-TNFα. Under mild hypothermia myc and xbp1s expression levels were directly correlated to the maximal viable cell density and the specific anti-TNFα productivity, respectively. Moreover, cells showed a simultaneous metabolic shift from production to consumption of lactate and from consumption to production of glutamine, which were exacerbated by reducing culture temperature and coincided with the increased anti-TNFα production. Our current results provide new insights of the regulation of myc and xbp1s in CHO cells at low temperature, and suggest that the presence and magnitude of the metabolic shift might be a relevant metabolic marker of productive cell line.

A temperature-responsive network links cell shape and virulence traits in a primary fungal pathogen.

Survival at host temperature is a critical trait for pathogenic microbes of humans. Thermally dimorphic fungal pathogens, including Histoplasma capsulatum, are soil fungi that undergo dramatic changes in cell shape and virulence gene expression in response to host temperature. How these organisms link changes in temperature to both morphologic development and expression of virulence traits is unknown. Here we elucidate a temperature-responsive transcriptional network in H. capsulatum, which switches from a filamentous form in the environment to a pathogenic yeast form at body temperature. The circuit is driven by three highly conserved factors, Ryp1, Ryp2, and Ryp3, that are required for yeast-phase growth at 37°C. Ryp factors belong to distinct families of proteins that control developmental transitions in fungi: Ryp1 is a member of the WOPR family of transcription factors, and Ryp2 and Ryp3 are both members of the Velvet family of proteins whose molecular function is unknown. Here we provide the first evidence that these WOPR and Velvet proteins interact, and that Velvet proteins associate with DNA to drive gene expression. Using genome-wide chromatin immunoprecipitation studies, we determine that Ryp1, Ryp2, and Ryp3 associate with a large common set of genomic loci that includes known virulence genes, indicating that the Ryp factors directly control genes required for pathogenicity in addition to their role in regulating cell morphology. We further dissect the Ryp regulatory circuit by determining that a fourth transcription factor, which we name Ryp4, is required for yeast-phase growth and gene expression, associates with DNA, and displays interdependent regulation with Ryp1, Ryp2, and Ryp3. Finally, we define cis-acting motifs that recruit the Ryp factors to their interwoven network of temperature-responsive target genes. Taken together, our results reveal a positive feedback circuit that directs a broad transcriptional switch between environmental and pathogenic states in response to temperature.

First detection and complete genome sequence of Deformed wing virus in Chilean honeybees.

Deformed wing virus (DWV) is one of the most common viruses affecting honey bee specimens. Although the presence of DWV has been reported in many countries, there is no data of the current situation in Chile. In this report, we detected the presence of DWV in apiaries from two different locations in central Chile. Furthermore, the genome of a Chilean DWV isolate was completely sequenced. This is the first report of the presence of a honey bee virus in Chile.

Analysis of manganese-regulated gene expression in the ligninolytic basidiomycete Ceriporiopsis subvermispora.

In this work, we explore the use of the unbiased cDNA-AFLP strategy to identify genes involved in Mn(2+) homeostasis in Ceriporiopsis subvermispora. In this ligninolytic white-rot fungus, whose genome has not yet been sequenced, three Mn peroxidase genes responding to Mn(2+) have been characterized. Using cDNA-AFLP to identify transcript-derived fragments (TDFs), a total of 37 differentially expressed cDNA fragments were identified by comparing band intensities among cDNA-AFLP patterns obtained from mycelia from cultures supplemented with different concentrations of Mn(2+). Of 21 differentially expressed TDFs, nine were classified as upregulated, five as downregulated and seven as unregulated. Of these, six upregulated and two downregulated TDFs were selected for further characterization. The expected TDFs for the known Mn peroxidases were not isolated, but several genes encoding proteins related to protein sorting, storage and excretion of excess Mn(2+) were identified. Transcripts induced under Mn(2+) supplementation exhibited homologies to the elongation factor eEF3, a HDEL sequence binding protein and the ARD1 subunit of the N-acetyltransferase complex, among others. Overall, the results obtained in this study suggest a complex picture of Mn(2+) homeostasis and provide the possibility to search for common regulatory elements in the promoters of the novel putatively identified genes.

Seudotrombocitopenia dependiente de EDTA / EDTA-dependent pseudothrombocytopenia

Introducción. La seudotrombocitopenia dependiente de etilendiaminotetracetato (EDTA) es un fenómeno conocido consistente en la formación de agregados de plaquetas in vitro en muestras de sangre anticoaguladas con EDTA y que puede conducir a diagnósticos erróneos de trombocitopenia. Es poco frecuente entre la población pediátrica y carece de significación patológica. Su correcta identificación puede evitar estudios innecesarios y tratamientos potencialmente peligrosos. Caso clínico. Niña de 10 años que ingresó en nuestro centro para estudio de trombopenia sin episodios previos ni clínica actual sugestiva de diátesis hemorrágica. El recuento de plaquetas fue de 31 x 109/L. En el examen microscópico de la sangre se observaron frecuentes agregados plaquetarios. Al realizar un hemograma de una muestra anticoagulada con heparina se evidenció un recuento de plaquetas normal, emitiéndose el diagnóstico de seudotrombocitopenia dependiente de EDTA. Discusión. La mayoría de casos de seudotrombopenia dependiente de EDTA obedecen a aglutininas de tipo IgM o IgG con máxima actividad entre 4 y 20 °C. Se desconoce el mecanismo íntimo de interacción con las plaquetas, pero se supone que en presencia de EDTA ocurre un cambio conformacional de la superficie plaquetaria que induce la exposición de un neoantíngeno al que se unirían las aglutininas (AU)