Electrocardiographic alterations in persons with a positive serology for toxoplasmosis]

To each of 11,161 randomly taken people from urban and peri-urban localities of the first seven regions of Chile (geographic area of distribution of Chagas' disease in the country), with negative serology for Chagas' disease, an indirect hemagglutination test (IHAT) for toxoplasmosis and an EKG were practiced. The IHAT for toxoplasmosis resulted positive in 3,519 individuals (31.5%). The EKG showed alterations in 10.9% of the IHAT positive individuals and in 7.9% of the IHAT negative ones. This difference between the proportion of altered EKG in IHAT positive people and altered EKG in those with negative IHAT is statistically significative with p < 0.001. These results suggest the convenience of considering toxoplasmosis as a cause of chronic myocardiopathy in epidemiological studies on Chagas' disease, since not discard the presence of Toxoplasma gondii infection should overvaluete the magnitude of the impact of Trypanosoma cruzi in the genesis of such a myocardiopathy.     

Preventing pyruvate kinase muscle expression in Chinese hamster ovary cells curbs lactogenic behavior by altering glycolysis,gating pyruvate generation,and increasing pyruvate flux into the TCA cycle

Deletion of the pyruvate kinase muscle (PKM) gene, which is involved in conversion of phosphoenolpyruvate to pyruvate, has been shown to curb lactogenic behavior in Chinese hamster ovary (CHO) cells. This study describes the generation of pyruvate kinase muscle isoforms 1 and 2 knockout (PKM-KO) and pyruvate kinase muscle isoform-1 knockout (PKM1-KO) CHO host cells to understand metabolic shifts that reduce lactate secretion in these cells. Glucose and amino acids uptake levels in wild-type (WT), PKM-KO, and PKM1-KO stable cell lines, expressing two different antibodies, were analyzed in 14-day fed-batch production assays using different vessels. PKM-KO and PKM1-KO cells consumed more glucose per cell, altered amino acids metabolism, had higher flux of pyruvate into the tricarboxylic acid (TCA) cycle, and as previously shown reduced lactate secretion levels compared with the WT cells. Additionally, both PKM-KO and PKM1-KO cells had higher specific productivity and lower cell growth rates compared with the WT cells. Our findings suggest that rewiring the flux of pyruvate to the TCA cycle by deletion of PKM or PKM1 reduced cell growth and increased specific productivity in CHO cells. Overall, PKM1-KO cells had similar product quality and comparable or better titers relative to the WT cells, hence, targeted deletion of this isoform for curbing lactogenic behavior in CHO cells is suggested.     

Purification and characterization of a new mammalian serum protein with the ability to inhibit actin polymerization and promote depolymerization of actin filaments

A protein with capacity to bind G-actin and the ability to inhibit polymerization and promote depolymerization of actin filaments has been isolated from the serum of rabbit. The protein, SAIP (for serum actin inhibitory protein), has been purified by affinity chromatography of serum over actin-Sepharose followed by protein fractionation with ammonium sulfate and chromatography over DEAE-cellulose. Five milligrams of purified SAIP is obtained from 100 mL of serum. Rabbit SAIP is resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into two closely related polypeptides of 60000 and 56000 daltons, respectively (ratio 5.1:1). Each of these polypeptides consists of two isoelectric variants. SAIP binds to monomeric actin with a stoichiometry of 1:1 and a Kd of 0.12 microM. The SAIP-actin complex binds to DNase I. Actin polymerization is completely inhibited by incubation of actin with an equal concentration of SAIP. At equimolar concentrations to F-actin, SAIP induces complete depolymerization of the actin filaments. SAIP is also present in calf serum.     

Endothelial intercellular cell adhesion molecule 1 contributes to cell aggregate formation in CHO cells cultured in serum-free media

Chinese hamster ovary (CHO) cells have been adapted to grow in serum-free media and in suspension culture to facilitate manufacturing needs. Some CHO cell lines, however, tend to form cell aggregates while being cultured in suspension. This can result in reduced viability and capacity for single cell cloning (SCC) via limiting dilution, and process steps to mitigate cell aggregate formation, for example, addition of anti-cell-aggregation agents. In this study, we have identified endothelial intercellular cell adhesion molecule 1 (ICAM-1) as a key protein promoting cell aggregate formation in a production competent CHO cell line, which is prone to cell aggregate formation. Knocking out (KO) the ICAM-1 gene significantly decreased cell aggregate formation in the culture media without anti-cell-aggregation reagent. This trait can simplify the process of transfection, selection, automated clone isolation, and so on. Evaluation in standard cell line development of ICAM-1 KO and wild-type CHO hosts did not reveal any noticeable impacts on titer or product quality. Furthermore, analysis of a derived nonaggregating cell line showed significant reductions in expression of cell adhesion proteins. Overall, our data suggest that deletion of ICAM-1 and perhaps other cell adhesion proteins can reduce cell aggregate formation and improve clonality assurance during SCC.