Comprehensive stable-isotope tracing of glucose and amino acids identifies metabolic by-products and their sources in CHO cell culture.

Mammalian cell culture processes are widely utilized for biotherapeutics production, disease diagnostics, and biosensors, and hence, should be optimized to support robust cell growth and viability. However, toxic by-products accumulate in cultures due to inefficiencies in metabolic activities and nutrient utilization. In this study, we applied comprehensive C stable-isotope tracing of amino acids and glucose to two Immunoglobulin G (IgG) producing Chinese Hamster Ovary (CHO) cell lines to identify secreted by-products and trace their origins.

Chemical inhibitors of hexokinase-2 enzyme reduce lactate accumulation, alter glycosylation processing, and produce altered glycoforms in CHO cell cultures.

Chinese hamster ovary (CHO) cells, predominant hosts for recombinant biotherapeutics production, generate lactate as a major glycolysis by-product. High lactate levels adversely impact cell growth and productivity. The goal of this study was to reduce lactate in CHO cell cultures by adding chemical inhibitors to hexokinase-2 (HK2), the enzyme catalyzing the conversion of glucose to glucose 6-phosphate, and examine their impact on lactate accumulation, cell growth, protein titers, and N-glycosylation.

Pathogenic mechanisms contributing to thrombocytopenia in patients with systemic lupus erythematosus.

SummarySystemic lupus erythematosus (SLE) is an autoimmune condition developing thrombocytopenia in about 10-15% of cases, however, mechanisms leading to low platelet count were not deeply investigated in this illness. Here we studied possible causes of thrombocytopenia, including different mechanisms of platelet clearance and impairment in platelet production. Twenty-five SLE patients with and without thrombocytopenia were included.

Functional polymorphisms of DNA repair genes in Latin America reinforces the heterogeneity of Myelodysplastic Syndrome.

Nucleotide excision repair pathway (NER) is an essential mechanism for single-strand breaks (SSB) repair while xeroderma pigmentosum family (XPA to XPG) is the most important system to NER. Myelodysplastic syndrome (MDS) is a heterogeneous hematological cancer characterized by cytopenias and risk of acute myeloid leukemia (AML) transformation. MDS pathogenesis has been associated with problems of DNA repair system.

Prognostic assessment for chronic myelomonocytic leukemia in the context of the World Health Organization 2016 proposal: a multicenter study of 280 patients.

Knowledge on chronic myelomonocytic leukemia (CMML) patients from Argentina and Brazil is limited. Our series of 280 patients depicted an older age at diagnosis (median 72 years old), 26% of aberrant karyotypes, and a prevalence of myelodysplastic (60%) and CMML-0 subtypes (56%). The median overall survival (OS) was 48.

Improving the Methanol Tolerance of an Methylotroph via Adaptive Laboratory Evolution Enhances Synthetic Methanol Utilization.

There is great interest in developing synthetic methylotrophs that harbor methane and methanol utilization pathways in heterologous hosts such as for industrial bioconversion of one-carbon compounds. While there are recent reports that describe the successful engineering of synthetic methylotrophs, additional efforts are required to achieve the robust methylotrophic phenotypes required for industrial realization. Here, we address an important issue of synthetic methylotrophy in : methanol toxicity.

Collagen microarchitecture mechanically controls myofibroblast differentiation.

Altered microarchitecture of collagen type I is a hallmark of wound healing and cancer that is commonly attributed to myofibroblasts. However, it remains unknown which effect collagen microarchitecture has on myofibroblast differentiation. Here, we combined experimental and computational approaches to investigate the hypothesis that the microarchitecture of fibrillar collagen networks mechanically regulates myofibroblast differentiation of adipose stromal cells (ASCs) independent of bulk stiffness.

[Nosocomial urinary tract infection: an analysis beyond urinary catheterization].

Introduction: Nosocomially acquired urinary tract infections (NAUTI) represent an important public health issue, but its characteristics when they are not catheter associated (CA-UTI) or when they take place outside intensive care units (ICU) are poorly understood.

Objectives: To determine the patients' characteristics, etiology and antimicrobial susceptibility of NAUTI, both CA-UTI and no CA-UTI, in general ward and ICU.

Methods: We conducted a retrospective analytic cross-sectional study, between 2009 and 2013, in a third level universitary hospital.

Enzyme Replacement Therapy in Pregnant Women with Fabry Disease: A Case Series.

Fabry disease is a rare inherited lysosomal storage disorder caused by the deficiency of the enzyme alpha-galactosidase A. There is uncertainty regarding the safety of enzyme replacement therapy during pregnancy. We describe the course and outcome of seven pregnancies in six patients with Fabry disease who continued or reinitiated enzyme replacement therapy during pregnancy.

Benefit of an electronic medical record-based alarm in the optimization of stress ulcer prophylaxis.

Background: The use of stress ulcer prophylaxis (SUP) has risen in recent years, even in patients without a clear indication for therapy.

Aim: To evaluate the efficacy of an electronic medical record (EMR)-based alarm to improve appropriate SUP use in hospitalized patients.

Methods: We conducted an uncontrolled before-after study comparing SUP prescription in intensive care unit (ICU) patients and non-ICU patients, before and after the implementation of an EMR-based alarm that provided the correct indications for SUP.

Dissecting the genetic and metabolic mechanisms of adaptation to the knockout of a major metabolic enzyme in .

Unraveling the mechanisms of microbial adaptive evolution following genetic or environmental challenges is of fundamental interest in biological science and engineering. When the challenge is the loss of a metabolic enzyme, adaptive responses can also shed significant insight into metabolic robustness, regulation, and areas of kinetic limitation. In this study, whole-genome sequencing and high-resolution C-metabolic flux analysis were performed on 10 adaptively evolved knockouts of catalyzes the first reaction in glycolysis, and its loss results in major physiological and carbon catabolism pathway changes, including an 80% reduction in growth rate.

Expression of heterologous non-oxidative pentose phosphate pathway from Bacillus methanolicus and phosphoglucose isomerase deletion improves methanol assimilation and metabolite production by a synthetic Escherichia coli methylotroph.

Synthetic methylotrophy aims to develop non-native methylotrophic microorganisms to utilize methane or methanol to produce chemicals and biofuels. We report two complimentary strategies to further engineer a previously engineered methylotrophic E. coli strain for improved methanol utilization.

Methanol assimilation in Escherichia coli is improved by co-utilization of threonine and deletion of leucine-responsive regulatory protein.

Methane, the main component of natural gas, can be used to produce methanol which can be further converted to other valuable products. There is increasing interest in using biological systems for the production of fuels and chemicals from methanol, termed methylotrophy. In this work, we have examined methanol assimilation metabolism in a synthetic methylotrophic E.

Metabolism of the fast-growing bacterium Vibrio natriegens elucidated by C metabolic flux analysis.

Vibrio natriegens is a fast-growing, non-pathogenic bacterium that is being considered as the next-generation workhorse for the biotechnology industry. However, little is known about the metabolism of this organism which is limiting our ability to apply rational metabolic engineering strategies. To address this critical gap in current knowledge, here we have performed a comprehensive analysis of V.

Fast growth phenotype of E. coli K-12 from adaptive laboratory evolution does not require intracellular flux rewiring.

Adaptive laboratory evolution (ALE) is a widely-used method for improving the fitness of microorganisms in selected environmental conditions. It has been applied previously to Escherichia coli K-12 MG1655 during aerobic exponential growth on glucose minimal media, a frequently used model organism and growth condition, to probe the limits of E. coli growth rate and gain insights into fast growth phenotypes.

Influence of TNF and IL6 gene polymorphisms on the severity of cytopenias in Argentine patients with myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) represent a heterogeneous group of hematologic disorders characterized by cytopenia(s) and predisposition to leukemic progression. An immune dysregulation and an aberrant bone marrow microenvironment seem to be key elements in the physiopathological process of MDS. In order to evaluate a possible association between susceptibility and clinic-pathologic features, we genotyped 153 MDS patients for functional cytokine polymorphisms: TNF (-308 G/A), IFNG (+874 A/T and +875 CAn), IL6 (-174 G/C), and TGFB1 (+869 C/T and +915 G/C).

Comprehensive analysis of glucose and xylose metabolism in Escherichia coli under aerobic and anaerobic conditions by C metabolic flux analysis.

Glucose and xylose are the two most abundant sugars derived from the breakdown of lignocellulosic biomass. While aerobic glucose metabolism is relatively well understood in E. coli, until now there have been only a handful of studies focused on anaerobic glucose metabolism and no C-flux studies on xylose metabolism.

Engineering the biological conversion of methanol to specialty chemicals in Escherichia coli.

Methanol is an attractive substrate for biological production of chemicals and fuels. Engineering methylotrophic Escherichia coli as a platform organism for converting methanol to metabolites is desirable. Prior efforts to engineer methylotrophic E.

C metabolic flux analysis of microbial and mammalian systems is enhanced with GC-MS measurements of glycogen and RNA labeling.

C metabolic flux analysis (C-MFA) is a widely used tool for quantitative analysis of microbial and mammalian metabolism. Until now, C-MFA was based mainly on measurements of isotopic labeling of amino acids derived from hydrolyzed biomass proteins and isotopic labeling of extracted intracellular metabolites. Here, we demonstrate that isotopic labeling of glycogen and RNA, measured with gas chromatography-mass spectrometry (GC-MS), provides valuable additional information for C-MFA.

Tracing metabolism from lignocellulosic biomass and gaseous substrates to products with stable-isotopes.

Engineered microbes offer a practical and sustainable alternative to traditional industrial approaches. To increase the economic feasibility of biological processes, microbial isolates are engineered to take up inexpensive feedstocks (including lignocellulosic biomass, syngas, methane, and carbon dioxide), and convert them into substrates of central metabolism and further into value-added products. To trace the metabolism of these feedstocks into products, isotopic tracers are applied together with isotopomer analysis techniques such as C-metabolic flux analysis to provide a detailed picture of pathway utilization.

Characterization of physiological responses to 22 gene knockouts in Escherichia coli central carbon metabolism.

Understanding the impact of gene knockouts on cellular physiology, and metabolism in particular, is centrally important to quantitative systems biology and metabolic engineering. Here, we present a comprehensive physiological characterization of wild-type Escherichia coli and 22 knockouts of enzymes in the upper part of central carbon metabolism, including the PTS system, glycolysis, pentose phosphate pathway and Entner-Doudoroff pathway. Our results reveal significant metabolic changes that are affected by specific gene knockouts.