Fitness cost associated with cell phenotypic switching drives population diversification dynamics and controllability.

Isogenic cell populations can cope with stress conditions by switching to alternative phenotypes. Even if it can lead to increased fitness in a natural context, this feature is typically unwanted for a range of applications (e.g., bioproduction, synthetic biology, and biomedicine) where it tends to make cellular response unpredictable. However, little is known about the diversification profiles that can be adopted by a cell population. Here, we characterize the diversification dynamics for various systems (bacteria and yeast) and for different phenotypes (utilization of alternative carbon sources, general stress response and more complex development patterns). Our results suggest that the diversification dynamics and the fitness cost associated with cell switching are coupled. To quantify the contribution of the switching cost on population dynamics, we design a stochastic model that let us reproduce the dynamics observed experimentally and identify three diversification regimes, i.e., constrained (at low switching cost), dispersed (at medium and high switching cost), and bursty (for very high switching cost). Furthermore, we use a cell-machine interface called Segregostat to demonstrate that different levels of control can be applied to these diversification regimes, enabling applications involving more precise cellular responses.

Is heterogeneity in large-scale bioreactors a real problem in recombinant protein synthesis by Pichia pastoris?

Culture medium heterogeneity is inherent in industrial bioreactors. The loss of mixing efficiency in a large-scale bioreactor yields to the formation of concentration gradients. Consequently, cells face oscillatory culture conditions that may deeply affect their metabolism. Herein, cell response to transient perturbations, namely high methanol concentration combined with hypoxia, has been investigated using a two stirred-tank reactor compartiments (STR-STR) scale-down system and a Pichia pastoris strain expressing the gene encoding enhanced green fluorescent protein (eGFP) under the control of the alcohol oxidase 1 (AOX1) promoter. Cell residence times under transient stressing conditions were calculated based on the typical hydraulic circulation times of bioreactors of tens and hundreds cubic metres. A significant increase in methanol and oxygen uptake rates was observed as the cell residence time was increased. Stressful culture conditions impaired biomass formation and triggered cell flocculation. More importantly, both expression levels of genes under the control of pAOX1 promoter and eGFP specific fluorescence were higher in those oscillatory culture conditions, suggesting that those a priori unfavourable culture conditions in fact benefit to recombinant protein productivity. Flocculent cells were also identified as the most productive as compared to ovoid cells. KEY POINTS: • Transient hypoxia and high methanol trigger high level of recombinant protein synthesis • In Pichia pastoris, pAOX1 induction is higher in flocculent cells • Medium heterogeneity leads to morphological diversification.

Avoiding the All-or-None Response in Gene Expression During E. coli Continuous Cultivation Based on the On-Line Monitoring of Cell Phenotypic Switching Dynamics.

Different expression vectors are available for the effective production of recombinant proteins by bacterial populations. However, the productivity of such systems is limited by the inherent noise of the gene circuits used for the synthesis of recombinant products. An extreme case of cell-to-cell heterogeneity that has been previously reported for the ara- and lac-based expression systems in E. coli is the all-or-none response. According to this mode of response, two subpopulations of cells are generated, i.e., a "low-" subpopulation exhibiting a shallow expression level and a "high-" subpopulation exhibiting a high-expression level. The "low-" subpopulation can be considered as a cluster of non-producing cells contributing to the loss of productivity. Here we describe the setup, design, and operation of a continuous culture where inducer addition is operated based on microbial population dynamics. The determination of population dynamics is done based on an automated flow cytometry (FC) procedure previously denoted as segregostat. We illustrate how this setup can be used to control the activation of an ara-based expression system and avoid phenotypic diversification leading to an all-or-none response. Upon the determination of the natural frequency of the gene circuit used as an expression system, our current protocol can be set up without the requirement of a feedback controller.

Related factors to non-adherence to antiretroviral therapy in HIV/AIDS patients.

OBJECTIVE: To identify sociodemographic, clinical, and pharmacological factors  associated with nonadherence to antiretroviral treatment in patients with  human immunodeficiency virus/acquired immunodeficiency syndrome treated  between 2017 and 2020 in four cities in Colombia. METHOD: An observational, cross-sectional, retrospective study was conducted of a population of patients with human immunodeficiency virus/acquired immunodeficiency syndrome treated between 2017 and 2020. The Morisky-Green scale, the simplified medication adherence  questionnaire, and the simplified scale to detect adherence problems to  antiretroviral treatment were applied to determine patient adherence. A  binomial multiple logistic regression was performed to evaluate the factors that  best explain nonadherence. RESULTS: A total of 9,835 patients were evaluated, of whom 74.4% were men,  71.1% were aged between 18 and 44 years, 76.0% had attended at most  secondary school, 78.1% were single, and 97.6% resided in an urban area.  After applying three different scales to each patient, 10% of the study  population were identified as nonadherent to treatment. The risk of  nonadherence was significantly higher in patients who presented any drug- related problem or had an adverse reaction to antiretroviral drugs. CONCLUSIONS: The variables most strongly associated with nonadherence to  antiretroviral treatment were drug-related problems, adverse drug reactions, a  history of nonadherence to treatment, and psychoactive substance use.

OBJETIVO: Identificar los factores sociodemográficos, clínicos y farmacológicos asociados a la no adherencia al tratamiento antirretroviral en pacientes con infección por virus de la inmunodeficiencia humana/sida atendidos entre 2017 y 2020 en diferentes ciudades de Colombia.Método: Se realizó un estudio observacional, de corte transversal y retrospectivo, con una población de pacientes con infección por virus de la  inmunodeficiencia humana/sida atendidos entre 2017 a 2020. Se aplicaron las  escalas Morisky-Green, el cuestionario simplificado de adherencia a la  medicación y la escala simplificada para detectar problemas de adherencia al  tratamiento antirretroviral, para determinar la adherencia de los pacientes. Se  realizó una regresión logística múltiple para evaluar los factores que mejor  explican la no adherencia. RESULTADOS: Se evaluaron 9.835 pacientes, de los cuales el 74,4% eran hombres, el 71,1% tenían una edad entre 18 a 44 años, el 76,0% cursó como máximo hasta secundaria, el 78,1% eran solteros y el 97,6%  residían en zona urbana. Se encontró una proporción de no adherencia al  tratamiento del 10% después de aplicar tres escalas diferentes a cada paciente. Las personas que presentaron algún problema relacionado con los medicamentos tuvieron un riesgo significativamente mayor de no ser adherentes, al igual que aquellos que tuvieron alguna reacción adversa a los medicamentos antirretrovirales. CONCLUSIONES: Los problemas relacionados con el uso de medicamentos, las  reacciones adversas a medicamentos, los antecedentes de no adherencia al  tratamiento y el consumo de sustancias psicoactivas fueron las variables que  más se asociaron con la no adherencia al tratamiento antirretroviral.

Factores relacionados con la no adherencia a la terapia antirretroviral en pacientes con VIH/sida / Related factors to non-adherence to antiretroviral therapy in HIV/AIDS patients

Objetivo: Identificar los factores sociodemográficos, clínicos y farmacológicos asociados a la no adherencia al tratamiento antirretroviral enpacientes con infección por virus de la inmunodeficiencia humana/sidaatendidos entre 2017 y 2020 en diferentes ciudades de Colombia.Método: Se realizó un estudio observacional, de corte transversal yretrospectivo, con una población de pacientes con infección por virusde la inmunodeficiencia humana/sida atendidos entre 2017 a 2020.Se aplicaron las escalas Morisky-Green, el cuestionario simplificado deadherencia a la medicación y la escala simplificada para detectar problemas de adherencia al tratamiento antirretroviral, para determinar laadherencia de los pacientes. Se realizó una regresión logística múltiplepara evaluar los factores que mejor explican la no adherencia.Resultados: Se evaluaron 9.835 pacientes, de los cuales el 74,4% eranhombres, el 71,1% tenían una edad entre 18 a 44 años, el 76,0% cursócomo máximo hasta secundaria, el 78,1% eran solteros y el 97,6% residían en zona urbana. Se encontró una proporción de no adherencia altratamiento del 10% después de aplicar tres escalas diferentes a cadapaciente. Las personas que presentaron algún problema relacionado conlos medicamentos tuvieron un riesgo significativamente mayor de no seradherentes, al igual que aquellos que tuvieron alguna reacción adversa alos medicamentos antirretrovirales. (AU)

Objective: To identify sociodemographic, clinical, and pharmacological factors associated with nonadherence to antiretroviral treatment inpatients with human immunodeficiency virus/acquired immunodeficiencysyndrome treated between 2017 and 2020 in four cities in Colombia.Method: An observational, cross-sectional, retrospective study was conducted of a population of patients with human immunodeficiency virus/acquired immunodeficiency syndrome treated between 2017 and 2020.The Morisky-Green scale, the simplified medication adherence questionnaire, and the simplified scale to detect adherence problems to antiretroviral treatment were applied to determine patient adherence. A binomialmultiple logistic regression was performed to evaluate the factors that bestexplain nonadherence.Results: A total of 9,835 patients were evaluated, of whom 74.4%were men, 71.1% were aged between 18 and 44 years, 76.0% hadattended at most secondary school, 78.1% were single, and 97.6% resided in an urban area. After applying three different scales to eachpatient, 10% of the study population were identified as nonadherent totreatment. The risk of nonadherence was significantly higher in patientswho presented any drug-related problem or had an adverse reaction toantiretroviral drugs. (AU)

Overexpression of the mitochondrial pyruvate carrier reduces lactate production and increases recombinant protein productivity in CHO cells.

Chinese hamster ovary (CHO) cells are characterized by a low glucose catabolic efficiency, resulting in undesirable lactate production. Here, it is hypothesized that such low efficiency is determined by the transport of pyruvate into the mitochondria. The mitochondrial pyruvate carrier (MPC), responsible for introducing pyruvate into the mitochondria, is formed by two subunits, MPC1 and MPC2. Stable CHO cell lines, overexpressing the genes of both subunits, were constructed to facilitate the entry of pyruvate into the mitochondria and its incorporation into oxidative pathways. Significant overexpression of both genes, compared to the basal level of the control cells, was verified, and subcellular localization of both subunits in the mitochondria was confirmed. Kinetic evaluation of the best MPC overexpressing CHO cells showed a reduction of up to 50% in the overall yield of lactate production with respect to the control. An increase in specific growth rate and maximum viable cell concentration, as well as an increase of up to 40% on the maximum concentration of two recombinant model proteins transiently expressed (alkaline phosphatase or a monoclonal antibody), was also observed. Hybrid cybernetic modeling, that considered 89 reactions, 25 extracellular metabolites, and a network of 62 intracellular metabolites, explained that the best MPC overexpression case resulted in an increased metabolic flux across the mitochondrial membrane, activated a more balanced growth, and reduced the Warburg effect without compromising glucose consumption rate and maximum cell concentration. Overall, this study showed that transport of pyruvate into the mitochondria limits the efficiency of glucose oxidation, which can be overcome by a cell engineering approach.

Shikimic Acid Production in Escherichia coli: From Classical Metabolic Engineering Strategies to Omics Applied to Improve Its Production.

Shikimic acid (SA) is an intermediate of the SA pathway that is present in bacteria and plants. SA has gained great interest because it is a precursor in the synthesis of the drug oseltamivir phosphate (OSF), an efficient inhibitor of the neuraminidase enzyme of diverse seasonal influenza viruses, the avian influenza virus H5N1, and the human influenza virus H1N1. For the purposes of OSF production, SA is extracted from the pods of Chinese star anise plants (Illicium spp.), yielding up to 17% of SA (dry basis content). The high demand for OSF necessary to manage a major influenza outbreak is not adequately met by industrial production using SA from plants sources. As the SA pathway is present in the model bacteria Escherichia coli, several "intuitive" metabolically engineered strains have been applied for its successful overproduction by biotechnological processes, resulting in strains producing up to 71 g/L of SA, with high conversion yields of up to 0.42 (mol SA/mol Glc), in both batch and fed-batch cultures using complex fermentation broths, including glucose as a carbon source and yeast extract. Global transcriptomic analyses have been performed in SA-producing strains, resulting in the identification of possible key target genes for the design of a rational strain improvement strategy. Because possible target genes are involved in the transport, catabolism, and interconversion of different carbon sources and metabolic intermediates outside the central carbon metabolism and SA pathways, as genes involved in diverse cellular stress responses, the development of rational cellular strain improvement strategies based on omics data constitutes a challenging task to improve SA production in currently overproducing engineered strains. In this review, we discuss the main metabolic engineering strategies that have been applied for the development of efficient SA-producing strains, as the perspective of omics analysis has focused on further strain improvement for the production of this valuable aromatic intermediate.