A production-adapted, multi-auxotrophic, CHO cell line for reducing monoclonal and bispecific antibody cell line development timelines

Please click Additional Files below to see the full abstract

Mercury isotopes in a forested ecosystem: Implications for air‐surface exchange dynamics and the global mercury cycle

Forests mediate the biogeochemical cycling of mercury (Hg) between the atmosphere and terrestrial ecosystems; however, there remain many gaps in our understanding of these processes. Our objectives in this study were to characterize Hg isotopic composition within forests, and use natural abundance stable Hg isotopes to track sources and reveal mechanisms underlying the cycling of Hg. We quantified the stable Hg isotopic composition of foliage, forest floor, mineral soil, precipitation, and total gaseous mercury (THg (g) ) in the atmosphere and in evasion from soil, in 10‐year‐old aspen forests at the Rhinelander FACE experiment in northeastern Wisconsin, USA. The effect of increased atmospheric CO 2 and O 3 concentrations on Hg isotopic composition was small relative to differences among forest ecosystem components. Precipitation samples had δ 202 Hg values of −0.74 to 0.06‰ and ∆ 199 Hg values of 0.16 to 0.82‰. Atmospheric THg (g) had δ 202 Hg values of 0.48 to 0.93‰ and ∆ 199 Hg values of −0.21 to −0.15‰. Uptake of THg (g) by foliage resulted in a large (−2.89‰) shift in δ 202 Hg values; foliage displayed δ 202 Hg values of −2.53 to −1.89‰ and ∆ 199 Hg values of −0.37 to −0.23‰. Forest floor samples had δ 202 Hg values of −1.88 to −1.22‰ and ∆ 199 Hg values of −0.22 to −0.14‰. Mercury isotopes distinguished geogenic sources of Hg and atmospheric derived sources of Hg in soil, and showed that precipitation Hg only accounted for ~16% of atmospheric Hg inputs. The isotopic composition of Hg evasion from the forest floor was similar to atmospheric THg (g) ; however, there were systematic differences in δ 202 Hg values and MIF of even isotopes (∆ 200 Hg and ∆ 204 Hg). Mercury evasion from the forest floor may have arisen from air‐surface exchange of atmospheric THg (g) , but was not the emission of legacy Hg from soils, nor re‐emission of wet‐deposition. This implies that there was net atmospheric THg (g) deposition to the forest soils. Furthermore, MDF of Hg isotopes during foliar uptake and air‐surface exchange of atmospheric THg (g) resulted in the release of Hg with very positive δ 202 Hg values to the atmosphere, which is key information for modeling the isotopic balance of the global mercury cycle, and may indicate a shorter residence time than previously recognized for the atmospheric mercury pool. Key points Atmospheric Hg was fractionated during uptake by foliage (‐2.89 permil δ202Hg) Hg evading from soil was from atmospheric Hg interaction with soil environment Air‐surface exchange of Hg releases Hg with positive δ202Hg to global reservoirPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/97463/1/2011GB004202RRts04.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/97463/2/2011GB004202RRts05.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/97463/3/2011GB004202RRts01.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/97463/4/gbc20021.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/97463/5/2011GB004202RRts06.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/97463/6/2011GB004202RRts02.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/97463/7/2011GB004202RRts07.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/97463/8/2011GB004202RRts03.pd

Chemical and Genetic Modulation of Complex I of the Electron Transport Chain Enhances the Biotherapeutic Protein Production Capacity of CHO Cells

Chinese hamster ovary (CHO) cells are the cell line of choice for producing recombinant therapeutic proteins. Despite improvements in production processes, reducing manufacturing costs remains a key driver in the search for more productive clones. To identify media additives capable of increasing protein production, CHOZN® GS−/− cell lines were screened with 1280 small molecules, and two were identified, forskolin and BrdU, which increased productivity by ≥40%. While it is possible to incorporate these small molecules into a commercial-scale process, doing so may not be financially feasible or could raise regulatory concerns related to the purity of the final drug substance. To circumvent these issues, RNA-Seq was performed to identify transcripts which were up- or downregulated upon BrdU treatment. Subsequent Reactome pathway analysis identified the electron transport chain as an affected pathway. CRISPR/Cas9 was utilized to create missense mutations in two independent components of the electron transport chain and the resultant clones partially recapitulated the phenotypes observed upon BrdU treatment, including the productivity of recombinant therapeutic proteins. Together, this work suggests that BrdU can enhance the productivity of CHO cells by modulating cellular energetics and provides a blueprint for translating data from small molecule chemical screens into genetic engineering targets to improve the performance of CHO cells. This could ultimately lead to more productive host cell lines and a more cost-effective method of supplying medication to patients

Discovery of Novel Secreted Virulence Factors from Salmonella enterica Serovar Typhimurium by Proteomic Analysis of Culture Supernatants ▿ #

Salmonella enterica serovar Typhimurium is a leading cause of acute gastroenteritis throughout the world. This pathogen has two type III secretion systems (TTSS) encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) that deliver virulence factors (effectors) to the host cell cytoplasm and are required for virulence. While many effectors have been identified and at least partially characterized, the full repertoire of effectors has not been catalogued. In this proteomic study, we identified effector proteins secreted into defined minimal medium designed to induce expression of the SPI-2 TTSS and its effectors. We compared the secretomes of the parent strain to those of strains missing essential (ssaK::cat) or regulatory (ΔssaL) components of the SPI-2 TTSS. We identified 20 known SPI-2 effectors. Excluding the translocon components SseBCD, all SPI-2 effectors were biased for identification in the ΔssaL mutant, substantiating the regulatory role of SsaL in TTS. To identify novel effector proteins, we coupled our secretome data with a machine learning algorithm (SIEVE, SVM-based identification and evaluation of virulence effectors) and selected 12 candidate proteins for further characterization. Using CyaA′ reporter fusions, we identified six novel type III effectors and two additional proteins that were secreted into J774 macrophages independently of a TTSS. To assess their roles in virulence, we constructed nonpolar deletions and performed a competitive index analysis from intraperitoneally infected 129/SvJ mice. Six mutants were significantly attenuated for spleen colonization. Our results also suggest that non-type III secretion mechanisms are required for full Salmonella virulence