Catechins prevent monoclonal antibody fragmentation during production via fed-batch culture of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are widely used for the industrial production of therapeutic monoclonal antibodies (mAbs). To meet the increasing market demands, high productivity, and quality are required in cell culture. One of the critical attributes of mAbs, from a safety perspective, is mAb fragmentation. However, methods for preventing mAbs fragmentation in CHO cell culture are limited. In this study, we observed that the antibody fragment content increased with increasing titers in fed-batch cultures for all three cell lines expressing recombinant antibodies. Adding copper sulfate to the culture medium further increased the fragment content, suggesting the involvement of reactive oxygen species (ROS) in the fragmentation process. Though antioxidants may be helpful to scavenge ROS, several antioxidants are reported to decrease the productivity of CHO cells. Among the antioxidants examined, we observed that the addition of catechin or (-)-epigallocatechin gallate to the culture medium prevented fragmentation content by about 20% and increased viable cell density and titer by 30% and 10%, respectively. Thus, the addition of catechins or compounds of equivalent function would be beneficial for manufacturing therapeutic mAbs with a balance between high titers and good quality.

Endothelial specific deletion of FOXO1 alters pericyte coverage in the developing retina.

Pericytes are mural cells that cover small blood vessels. While defects in pericyte coverage are known to be involved in various vessel related pathologies, including diabetic retinopathy, the molecular mechanisms underlying pericyte coverage are not fully understood. In this study, we investigated the contribution of the forkhead transcription factor FOXO1 in endothelial cells to pericyte coverage in the developing retina. We observed retinal pericytes in tamoxifen-inducible endothelium-specific Foxo1 deletion mice. Tamoxifen was injected at postnatal day 1-3 and the retinas were harvested at P21. Our results demonstrated that Foxo1 deletion in the endothelium affected arteriole pericyte morphology without altering pericyte number, proliferation, and apoptosis. We hypothesized that abnormal pericyte morphogenesis in the knockout retina was caused by impaired pericyte differentiation. FOXO1 silencing by siRNA in the primary artery endothelium further revealed that THBS1 (thrombospondin 1), which promotes pericyte differentiation via TGFß activation, was reduced in the FOXO1-deficient endothelium. Immunohistochemistry of FOXO1 knockout mice showed reduced numbers of phospho-Smad3+ arteriole pericytes compared with wild-type mice. In addition, endothelium-pericyte co-culture analysis revealed that pericytes cultured with FOXO1-deficient endothelial cells failed to differentiate sufficiently; this failure was partially rescued by the addition of recombinant THBS1 to the supernatant. The findings suggest that endothelial FOXO1 contributes to pericyte differentiation via regulation of THBS1 expression. This study provides new insights into the molecular mechanism of pericyte coverage in the context of endothelium-derived regulation and highlights a new therapeutic target for pericyte-related pathology.

Sleep deprivation impairs spontaneous object-place but not novel-object recognition in rats.

Effects of sleep deprivation (SD) on one-trial recognition memory were investigated in rats using either a spontaneous novel-object or object-place recognition test. Rats were allowed to explore a field in which two identical objects were presented. After a delay period, they were placed again in the same field in which either: (1) one of the two objects was replaced by another object (novel-object recognition); or (2) one of the sample objects was moved to a different place (object-place recognition), and their exploration behavior to these objects was analyzed. Four hours SD immediately after the sample phase (early SD group) disrupted object-place recognition but not novel-object recognition, while SD 4-8h after the sample phase (delayed SD group) did not affect either paradigm. The results suggest that sleep selectively promotes the consolidation of hippocampal dependent memory, and that this effect is limited to within 4h after learning.

Idiopathic severe constriction of the fetal ductus arteriosus: a possible underestimated pathophysiology.

Idiopathic intrauterine constriction/closure of the ductus arteriosus, which is distinct from that secondary to maternal exposure to non-steroidal anti-inflammatory drugs, such as indomethacin, or structural cardiac defect, is an uncommon event that often results in severe fetal-neonatal morbidity and mortality. We reported a case of idiopathic fetal ductal constriction, in which the diagnosis was confirmed by documentation of an abnormal four-chamber view of the fetal heart at 38 weeks of gestation on obstetric ultrasound examination. A female infant weighing 2,816 g was born by Cesarean section, and her postnatal course was mild; transient tachypnea requiring only several days of supplemental oxygen with spontaneous regression of the abnormal echocardiographic findings by 3 months of age. The incidence of idiopathic constriction/closure of the fetal ductus arteriosus may be underestimated, particularly with a negative history of maternal drug exposure and mild postnatal clinical presentation.

Dual regulatory pathways integrating the RcsC-RcsD-RcsB signalling system control enterohaemorrhagic Escherichia coli pathogenicity.

Bacterial pathogenesis is strictly regulated in response to changes in environmental conditions. A His-Asp phosphorelay system consisting of a sensor kinase and response regulator is used by Gram-positive and Gram-negative bacteria to control gene expression in response to environmental stimuli. We screened His-Asp phosphorelay systems for their effect on virulence expression in enterohaemorrhagic Escherichia coli (EHEC), and found rcsD or rcsB overexpression enhanced locus for enterocyte effacement (LEE) gene transcription and adherence to Caco-2 cells through transcriptional activation of the ler regulatory gene. An EHEC-specific regulator GrvA, encoded by ECs1274, was required for ler transcription activation by RcsB. Furthermore, GrvA activated ler transcription in E. coli K12. Stimulation of the RcsDCB regulatory system by RcsF overexpression slightly increased EspB expression in the wild type but not the ECs1274 mutant. However, EspB expression in an rcsB deletion mutant increased compared with wild type, suggesting that RcsB negatively regulates LEE gene expression and that active RcsB protein is present under normal growth conditions. Deletion of pchA, which encodes a positive regulator for ler, abolished the effect of the rcsB deletion, suggesting that pchA mediated the negative RcsB effect. pchA transcript levels decreased when RcsB expression increased. Thus, LEE gene transcription may be regulated by RcsB through two oppositely regulated O157-specific regulators, PchA and GrvA.