No abstract

Cell type-specific modifications of conventional endosomal trafficking pathways lead to the formation of lysosome-related organelles (LROs). C. elegans gut granules are intestinally restricted LROs that coexist with conventional degradative lysosomes. The formation of gut granules requires the Rab32 family member GLO-1. We show that the loss of glo-1 leads to the mistrafficking of gut granule proteins but does not significantly alter conventional endolysosome biogenesis. GLO-3 directly binds to CCZ-1 and they both function to promote the gut granule association of GLO-1, strongly suggesting that together, GLO-3 and CCZ-1 activate GLO-1. We found that a point mutation in GLO-1 predicted to spontaneously activate, and function independently of it guanine nucleotide exchange factor (GEF), localizes to gut granules and partially restores gut granule protein localization in ccz-1(-) and glo-3(-) mutants. CCZ-1 forms a heterodimeric complex with SAND-1(MON1), which does not function in gut granule formation, to activate RAB-7 in trafficking pathways to conventional lysosomes. Therefore, our data suggest a model whereby the function of a Rab GEF can be altered by subunit exchange. glo-3(-) mutants, which retain low levels of GLO-3 activity, generate gut granules that lack GLO-1 and improperly accumulate RAB-7 in a SAND-1 dependent process. We show that GLO-1 and GLO-3 restrict the distribution of RAB-7 to conventional endolysosomes, providing insights into the segregation of pathways leading to conventional lysosomes and LROs.

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This investigation employed a viral screening method detect endogenous bovine virus contaminants in commercially supplied fetal bovine serum. Fifty-one lots of fetal bovine serum from 14 suppliers were examined. Over 30% of the lots tested were found to contain bovine viruses; they included bovine virus diarrhea virus, parainfluenza type3-like virus, bovine herpesvirus-1, bovine enterovirus type 4, and an unidentified cytopathogenic agent. Of the 51 lots, 20 had been pretested by the suppliers and were considered to be free of known viral contaminants. Our viral screening methods revealed that five of these pretested lots, or 25%, contained endogenous bovine viruses.

Recent advances in metabolite quantification and identification have enabled new research into the detection and control of titer inhibitors and promoters. This paper presents a bigdata analytics study to identify both inhibitors and promoters using multivariate data analysis of metabolomics data. By applying multi-way partial least squares (PLS) model to metabolite data from four fed-batch bioreactor conditions where feed formulation and selection agent concentrations varied, metabolites which exhibited the most significant impact on titer during cultivation were ranked from highest to lowest. The model outputs were then constrained to reduce the number of statistically relevant inhibitors or promoters to the top ten, which were used to conduct metabolic pathway analysis. Furthermore, a method is presented for identifying amino acids that prevent the accumulation of the inhibitors and/or enhance the formation of promoters during production. Finally, the metabolomics and pathway analysis results were integrated and validated with transcriptomics data to characterize metabolic changes occurring among different growth conditions. From these results, new feeding strategies were implemented which resulted in increased fed-batch production titer. Methodology from this work could be applied to future process optimization strategies for biotherapeutic production.