Driven progressive evolution of genome sequence complexity in Cyanobacteria

Progressive evolution, or the tendency towards increasing complexity, is a controversial issue in biology, which resolution entails a proper measurement of complexity. Genomes are the best entities to address this challenge, as they encode the historical information of a species’ biotic and environmental interactions. As a case study, we have measured genome sequence complexity in the ancient phylum Cyanobacteria. To arrive at an appropriate measure of genome sequence complexity, we have chosen metrics that do not decipher biological functionality but that show strong phylogenetic signal. Using a ridge regression of those metrics against root-to-tip distance, we detected positive trends towards higher complexity in three of them. Lastly, we applied three standard tests to detect if progressive evolution is passive or driven—the minimum, ancestor– descendant, and sub-clade tests. These results provide evidence for driven progressive evolution at the genome-level in the phylum Cyanobacteria.Generalitat Valenciana Prometeo/2018/A/133European Union (EU)Fulbright fellowship (Spanish Minister of Science, Innovation and Universities)SAF2015-65878-RAGL2017-88702-C2-2-RPGC2018-099344-B-I0

Progressive genome evolution in Cyanobacteria

Resumen del trabajo presentado a la VII Biennial Congress of Sociedad Española de Biología Evolutiva (SEBE), celebrada en Sevilla (España) del 5 al 7 de febrero de 2020.Progressive evolution, the tendency towards increasing complexity, is a controversial issue in Biology, whose resolution requires the proper measurement of complexity. To address this challenge, we consider that genomes are the best entities to measure complexity because they record the history and information gain of organisms in their ongoing biotic and environmental interactions. By recurring to six metrics that measure genome complexity, which are not primarily associated to functionality, we report the existence of progressive evolution towards higher genome complexity in the evolution of the Cyanobacteria phylum. We show that these complexity metrics plus three additional genome parameters present statistically significant phylogenetic signal in Cyanobacteria. Moreover, a ridge regression of genome complexity metrics against evolutionary age shows that three out of six present a positively driven evolutionary trend towards higher complexity. These findings support the existence of progressive genome evolution in this ancient and diverse group of organisms.Peer reviewe

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

Espectrómetro multicanal por conversión en tiempo a doble pendiente

Universidad Granada, Facultad de Ciencias. Leída el 27-06-197

Desarrollo de procesos de producción de proteínas bioterapéuticas: Aumento de la productividad especifica en células HEK293

La tesis doctoral se basa en la producción de proteinas recombinantes bioterapéuticas mediante el cultivo de células de mamífero. El trabajo realizado ha consistido en la obtención de células de riñón embrionario humano (HEK293) modificadas genéticamente para producir dos proteinas bioterapéuticas candidatas, el interferón gamma y el anticuerpo monoclonal trastuzumab. El objetivo principal del trabajo ha sido la mejora de las líneas celulares obtenidas, con el fin de aumentar la productividad especifica de las mismas. Durante el desarrollo del trabajo se han usado diversas aproximaciones para de conseguir este objetivo. Durante el capítulo 3 se ha realizado la construcción de las células productoras, comprobando que expresan los productos de interés y poniendo a punto las diversas técnicas necesarias para su cultivo. En el capítulo 4 se han evaluado diferentes elementos genéticos que regulan la expresión y la secreción de las proteinas recombinantes (Promotores y péptidos señal), encontrándose como elementos óptimos el promotor CMV y el péptido señal del interferón alfa-2. Con estos elementos se ha conseguido multiplicar por 5 la producción específica del anticuerpo monoclonal trastuzumab. En el capítulo 5, se ha explorado un elemento genético de gran importancia para la obtención de líneas celulares recombinantes, el marcador de selección basado en genes metabólicos. Estos marcadores son ampliamente usados en la industria biofarmacéutica, para seleccionar las células más productoras dentro de una población determinada sin necesidad de usar antibióticos para ello. Sin embargo, su aplicación en células HEK293 se ve dificultada por la expresión de enzimas endógenas que interfieren con los marcadores comercialmente disponibles. Los resultados obtenidos durante este capítulo han permitido la definición de dos marcadores de selección: La enzima fenilalanina hidroxilasa, aplicable a las células HEK293 no modificadas y la glutamina sintetasa, aplicada a células HEK293 mutantes obtenidas mediante CRISPR/Cas9. El uso de este último marcador, en combinación con un inhibidor, ha permitido una mejora de entre 3 y 5 veces en la producción de interferón gamma y de trastuzumab, en comparación con el marcador basado en antibióticos. En el capítulo 6 se ha explorado y desarrollado un método para integrar en el genoma de la célula HEK293, de forma dirigida, el vector que contienen el gen que codifica para las proteinas de interés. Para ello, se ha construido y caracterizado un sitio genómico de alta transcripción mediante el uso de la proteína verde fluorescente (eGFP), para, posteriormente, integrar en dicho lugar el transgen usando el sistema CRISPR/Cas9. Los resultados obtenidos demuestran que la técnica ensayada permite la integración dirigida en el punto deseado del ADN genómico de la célula, habiéndose logrado duplicar la producción especifica de interferón gamma y aumentando la producción de trastuzumab en un 30%. Finalmente, durante el capítulo 7 se ha llevado a cabo un estudio de escalado en la producción de interferón gamma usando biorreactores como paso previo a una potencial aplicación industrial. Se ha realizado un análisis de las condiciones fisiológicas de cultivo en biorreactor, habiendo sido necesario optimizar la concentración de CO2 y el pH con el fin de conseguir un proceso escalable desde el cultivo en matraz hasta el biorreactor y demostrando que las células HEK293 cultivadas en biorreactor necesitan una mínima cantidad de CO2 para conseguir un metabolismo optimo y mantener la productividad especifica. El proceso resultante ha sido escalado usando un reactor preindustrial de un solo uso de 50L. Se puede concluir, a tenor de los resultados obtenidos y, dada la variedad de modelos ensayados, que las técnicas desarrolladas resultan interesantes de cara a una posible aplicación en la producción de diferentes proteinas recombinantes de interés bioterapéutico, sector de gran importancia económica e industrial.The thesis is focused on the production of recombinant proteins of biotherapeutic interest using mammalian cell culture. During the work, several human embryonic kidney-based (HEK293) cells lines has been developed, applying genetic engineering techniques, to produce two candidate biotherapeutics: the cytokine Interferon gamma and the monoclonal antibody Trastuzumab. The main goal of the work has been the genetic improvement of the cell lines obtained, in order to increase the specific productivity of the candidates. Throughout the work, diverse approaches have been used to achieve this goal. Firstly, during the third chapter, development of productive cells was carried out, checking correct expression of products of interest and good practice of cell culture techniques. Afterwards, in chapter 4, different genetic elements regulating the expression and secretion of recombinant proteins (Promoters and signal peptides) were evaluated, finding CMV promoter and interferon alpha-2 signal peptide being the optimal elements. By using these elements, a 5-fold increase in specific productivity of trastuzumab expressing cells has been achieved. In Chapter 5, the metabolic selectable marker, a key genetic element in cell line development, has been explored. These markers are widely used in the biotherapeutics manufacturing industry, in order to select the most productive cells within a given population avoiding the use of antibiotics. However, its application in HEK293 cells is hampered by the expression of endogenous genes that interfere with commercially available markers. The results obtained during this chapter have allowed the definition of 2 selectable markers, one based on the enzyme phenylalanine hydroxylase, applicable to conventional HEK293 cells and another based on glutamine synthetase, applied to mutant HEK293 cells obtained by gene editing using CRISP/Cas9 technology. The use of glutamine synthetase, in combination with an inhibitor, has elicited a 3 to 5-fold increase in trastuzumab and interferon gamma production, in comparison with antibiotic resistance marker During Chapter 6, a targeted- integration method has been explored and developed to insert the vector containing the gene of interest into a particular location into HEK293 genome. To this purpose, a high transcription genomic site has been characterized using green fluorescent protein (eGFP), in order to integrate the construct of interest via CRISPR/Cas9. The technique developed in this chapter allows targeted-integration of transgenes into the expected site of genomic DNA of the host cell, doubling the specific production of interferon gamma and increasing the production of trastuzumab by a 30%. Finally, during chapter 7, we carried out a scale-up study on interferon gamma production using bioreactors, as previous step towards a potential industrial application. A complete analysis of the physiological conditions of culture in bioreactor has been conducted, being necessary an optimization of CO2 feeding and pH levels in order to achieve a scalable process from the shake flask to the bioreactor. These findings prove that HEK293 cells cultured in bioreactor need a minimum amount of CO2 to achieve an optimal metabolism, while maintaining specific productivity. The productive process has been successfully tested at pilot scale using a 50L single use bioreactor. It can be stated that, based on the results obtained and given the variety of models tested, the methods developed are potentially applicable to the production of different recombinant proteins of biotherapeutic interest, a sector of great economic and industrial significance

Compositional segmentation and long-range fractal correlations in DNA sequences. Phys Rev

A segmentation algorithm based on the Jensen-Shannon entropic divergence is used to decompose longrange correlated DNA sequences into statistically significant, compositionally homogeneous patches. By adequately setting the significance level for segmenting the sequence, the underlying power-law distribution of patch lengths can be revealed. Some of the identified DNA domains were uncorrelated, but most of them continued to display long-range correlations even after several steps of recursive segmentation, thus indicating a complex multi-length-scaled structure for the sequence. On the other hand, by separately shuffling each segment, or by randomly rearranging the order in which the different segments occur in the sequence, shuffled sequences preserving the original statistical distribution of patch lengths were generated. Both types of random sequences displayed the same correlation scaling exponents as the original DNA sequence, thus demonstrating that neither the internal structure of patches nor the order in which these are arranged in the sequence is critical; therefore, long-range correlations in nucleotide sequences seem to rely only on the power-law distribution of patch lengths

Isochore chromosome maps of the human genome

The human genome is a mosaic of isochores, which are long DNA segments (z.Gt;300 kbp) relatively homogeneous in G+C. Human isochores were first identified by density-gradient ultracentrifugation of bulk DNA, and differ in important features, e.g. genes are found predominantly in the GC-richest isochores. Here, we use a reliable segmentation method to partition the longest contigs in the human genome draft sequence into long homogeneous genome regions (LHGRs), thereby revealing the isochore structure of the human genome. The advantages of the isochore maps presented here are: (1) sequence heterogeneities at different scales are shown in the same plot; (2) pair-wise compositional differences between adjacent regions are all statistically significant; (3) isochore boundaries are accurately defined to single base pair resolution; and (4) both gradual and abrupt isochore boundaries are simultaneously revealed. Taking advantage of the wide sample of genome sequence analyzed, we investigate the correspondence between LHGRs and true human isochores revealed through DNA centrifugation. LHGRs show many of the typical isochore features, mainly size distribution, G+C range, and proportions of the isochore classes. The relative density of genes, Alu and long interspersed nuclear element repeats and the different types of single nucleotide polymorphisms on LHGRs also coincide with expectations in true isochores. Potential applications of isochore maps range from the improvement of gene-finding algorithms to the prediction of linkage disequilibrium levels in association studies between marker genes and complex traits. The coordinates for the LHGRs identified in all the contigs longer than 2 Mb in the human genome sequence are available at the online resource on isochore mapping: http://bioinfo2.ugr.es/isochores.This work was supported by grant BIO99-0651-CO2-01 from the Spanish Government

Desarrollo de procesos de producción de proteínas bioterapéuticas : aumento de la productividad especifica en células HEK293 /

La tesis doctoral se basa en la producción de proteinas recombinantes bioterapéuticas mediante el cultivo de células de mamífero. El trabajo realizado ha consistido en la obtención de células de riñón embrionario humano (HEK293) modificadas genéticamente para producir dos proteinas bioterapéuticas candidatas, el interferón gamma y el anticuerpo monoclonal trastuzumab. El objetivo principal del trabajo ha sido la mejora de las líneas celulares obtenidas, con el fin de aumentar la productividad especifica de las mismas. Durante el desarrollo del trabajo se han usado diversas aproximaciones para de conseguir este objetivo. Durante el capítulo 3 se ha realizado la construcción de las células productoras, comprobando que expresan los productos de interés y poniendo a punto las diversas técnicas necesarias para su cultivo. En el capítulo 4 se han evaluado diferentes elementos genéticos que regulan la expresión y la secreción de las proteinas recombinantes (Promotores y péptidos señal), encontrándose como elementos óptimos el promotor CMV y el péptido señal del interferón alfa-2. Con estos elementos se ha conseguido multiplicar por 5 la producción específica del anticuerpo monoclonal trastuzumab. En el capítulo 5, se ha explorado un elemento genético de gran importancia para la obtención de líneas celulares recombinantes, el marcador de selección basado en genes metabólicos. Estos marcadores son ampliamente usados en la industria biofarmacéutica, para seleccionar las células más productoras dentro de una población determinada sin necesidad de usar antibióticos para ello. Sin embargo, su aplicación en células HEK293 se ve dificultada por la expresión de enzimas endógenas que interfieren con los marcadores comercialmente disponibles. Los resultados obtenidos durante este capítulo han permitido la definición de dos marcadores de selección: La enzima fenilalanina hidroxilasa, aplicable a las células HEK293 no modificadas y la glutamina sintetasa, aplicada a células HEK293 mutantes obtenidas mediante CRISPR/Cas9. El uso de este último marcador, en combinación con un inhibidor, ha permitido una mejora de entre 3 y 5 veces en la producción de interferón gamma y de trastuzumab, en comparación con el marcador basado en antibióticos. En el capítulo 6 se ha explorado y desarrollado un método para integrar en el genoma de la célula HEK293, de forma dirigida, el vector que contienen el gen que codifica para las proteinas de interés. Para ello, se ha construido y caracterizado un sitio genómico de alta transcripción mediante el uso de la proteína verde fluorescente (eGFP), para, posteriormente, integrar en dicho lugar el transgen usando el sistema CRISPR/Cas9. Los resultados obtenidos demuestran que la técnica ensayada permite la integración dirigida en el punto deseado del ADN genómico de la célula, habiéndose logrado duplicar la producción especifica de interferón gamma y aumentando la producción de trastuzumab en un 30%. Finalmente, durante el capítulo 7 se ha llevado a cabo un estudio de escalado en la producción de interferón gamma usando biorreactores como paso previo a una potencial aplicación industrial. Se ha realizado un análisis de las condiciones fisiológicas de cultivo en biorreactor, habiendo sido necesario optimizar la concentración de CO2 y el pH con el fin de conseguir un proceso escalable desde el cultivo en matraz hasta el biorreactor y demostrando que las células HEK293 cultivadas en biorreactor necesitan una mínima cantidad de CO2 para conseguir un metabolismo optimo y mantener la productividad especifica. El proceso resultante ha sido escalado usando un reactor preindustrial de un solo uso de 50L. Se puede concluir, a tenor de los resultados obtenidos y, dada la variedad de modelos ensayados, que las técnicas desarrolladas resultan interesantes de cara a una posible aplicación en la producción de diferentes proteinas recombinantes de interés bioterapéutico, sector de gran importancia económica e industrial.The thesis is focused on the production of recombinant proteins of biotherapeutic interest using mammalian cell culture. During the work, several human embryonic kidney-based (HEK293) cells lines has been developed, applying genetic engineering techniques, to produce two candidate biotherapeutics: the cytokine Interferon gamma and the monoclonal antibody Trastuzumab. The main goal of the work has been the genetic improvement of the cell lines obtained, in order to increase the specific productivity of the candidates. Throughout the work, diverse approaches have been used to achieve this goal. Firstly, during the third chapter, development of productive cells was carried out, checking correct expression of products of interest and good practice of cell culture techniques. Afterwards, in chapter 4, different genetic elements regulating the expression and secretion of recombinant proteins (Promoters and signal peptides) were evaluated, finding CMV promoter and interferon alpha-2 signal peptide being the optimal elements. By using these elements, a 5-fold increase in specific productivity of trastuzumab expressing cells has been achieved. In Chapter 5, the metabolic selectable marker, a key genetic element in cell line development, has been explored. These markers are widely used in the biotherapeutics manufacturing industry, in order to select the most productive cells within a given population avoiding the use of antibiotics. However, its application in HEK293 cells is hampered by the expression of endogenous genes that interfere with commercially available markers. The results obtained during this chapter have allowed the definition of 2 selectable markers, one based on the enzyme phenylalanine hydroxylase, applicable to conventional HEK293 cells and another based on glutamine synthetase, applied to mutant HEK293 cells obtained by gene editing using CRISP/Cas9 technology. The use of glutamine synthetase, in combination with an inhibitor, has elicited a 3 to 5-fold increase in trastuzumab and interferon gamma production, in comparison with antibiotic resistance marker During Chapter 6, a targeted- integration method has been explored and developed to insert the vector containing the gene of interest into a particular location into HEK293 genome. To this purpose, a high transcription genomic site has been characterized using green fluorescent protein (eGFP), in order to integrate the construct of interest via CRISPR/Cas9. The technique developed in this chapter allows targeted-integration of transgenes into the expected site of genomic DNA of the host cell, doubling the specific production of interferon gamma and increasing the production of trastuzumab by a 30%. Finally, during chapter 7, we carried out a scale-up study on interferon gamma production using bioreactors, as previous step towards a potential industrial application. A complete analysis of the physiological conditions of culture in bioreactor has been conducted, being necessary an optimization of CO2 feeding and pH levels in order to achieve a scalable process from the shake flask to the bioreactor. These findings prove that HEK293 cells cultured in bioreactor need a minimum amount of CO2 to achieve an optimal metabolism, while maintaining specific productivity. The productive process has been successfully tested at pilot scale using a 50L single use bioreactor. It can be stated that, based on the results obtained and given the variety of models tested, the methods developed are potentially applicable to the production of different recombinant proteins of biotherapeutic interest, a sector of great economic and industrial significance

Analysis of symbolic sequences using the Jensen-Shannon divergence

We study statistical properties of the Jensen-Shannon divergence D, which quantifies the difference between probability distributions, and which has been widely applied to analyses of symbolic sequences. We present three interpretations of D in the framework of statistical physics, information theory, and mathematical statistics, and obtain approximations of the mean, the variance, and the probability distribution of D in random, uncorrelated sequences. We present a segmentation method based on D that is able to segment a nonstationary symbolic sequence into stationary subsequences, and apply this method to DNA sequences, which are known to be nonstationary on a wide range of different length scales