The degradation of environmental conditions, such as nutrient depletion and accumulation of toxic waste products over time, often lead to premature apoptotic cell death in mammalian cell cultures and suboptimal protein yield. Although apoptosis has been extensively researched, the changes in the whole cell proteome during prolonged cultivation, where apoptosis is a major mode of cell death, have not been examined. To our knowledge, the work presented here is the first whole cell proteome analysis of non-induced apoptosis in mammalian cells. Flow cytometry analyses of various activated caspases demonstrated the onset of apoptosis in Chinese hamster ovary cells during prolonged cultivation was primarily through the intrinsic pathway. Differential in gel electrophoresis proteomic study comparing protein samples collected during cultivation resulted in the identification of 40 differentially expressed proteins, including four cytoskeletal proteins, ten chaperone and folding proteins, seven metabolic enzymes and seven other proteins of varied functions. The induction of seven ER chaperones and foldases is a solid indication of the onset of the unfolded protein response, which is triggered by cellular and ER stresses, many of which occur during prolonged batch cultures. In addition, the upregulation of six glycolytic enzymes and another metabolic protein emphasizes that a change in the energy metabolism likely occurred as culture conditions degraded and apoptosis advanced. By identifying the intracellular changes during cultivation, this study provides a foundation for optimizing cell line-specific cultivation processes, prolonging longevity and maximizing protein production.

Functional assessment of proteins with significantly altered expression levels revealed several mechanisms thought to be involved in bacterial heavy metal detoxification, including general stress adaptation, anti-oxidative stress and heavy metal efflux proteins. This information may contribute to the development of plant growth-promoting bacteria mediated phytoremediation processes.

The Asian citrus psyllid, Diaphorina citri Kuwayama, is among the most important pests of citrus. It is the main vector of the Huanglongbing (HLB) pathogen Candidatus Liberibacter asiaticus (CLas), which causes severe losses in citrus crops. Control of D. citri is therefore of paramount importance to reduce the spread of HLB. In this regard, using RNA interference (RNAi) to silence target genes is a useful strategy to control psyllids. In this study, using RNAi, we examined the biological functions of the V‐ATPase subunit E (V‐ATP‐E) gene of D. citri, including its effect on acquisition of CLas. The amino acid sequence of V‐ATP‐E from D. citri had high homology with proteins from other insects. V‐ATP‐E was expressed at all D. citri life stages analyzed, and the expression level in mature adults was higher than that of teneral adults. Silencing of V‐ATP‐E resulted in a significant increase in mortality, reduced body weight, and induced cell apoptosis of the D. citri midgut. The reduced expression of V‐ATP‐E was indicated to inhibit CLas passing through the midgut and into the hemolymph, leading to a majority of CLas being confined to the midgut. In addition, double‐stranded RNA of D. citri V‐ATP‐E was safe to non‐target parasitic wasps. These results suggest that V‐ATP‐E is an effective RNAi target that can be used in D. citri control to block CLas infection.

No abstract