Quantitative characterization of metabolism and metabolic shifts during growth of the new human cell line AGE1.HN using time resolved metabolic flux analysis

For the improved production of vaccines and therapeutic proteins, a detailed understanding of the metabolic dynamics during batch or fed-batch production is requested. To study the new human cell line AGE1.HN, a flexible metabolic flux analysis method was developed that is considering dynamic changes in growth and metabolism during cultivation. This method comprises analysis of formation of cellular components as well as conversion of major substrates and products, spline fitting of dynamic data and flux estimation using metabolite balancing. During batch cultivation of AGE1.HN three distinct phases were observed, an initial one with consumption of pyruvate and high glycolytic activity, a second characterized by a highly efficient metabolism with very little energy spilling waste production and a third with glutamine limitation and decreasing viability. Main events triggering changes in cellular metabolism were depletion of pyruvate and glutamine. Potential targets for the improvement identified from the analysis are (i) reduction of overflow metabolism in the beginning of cultivation, e.g. accomplished by reduction of pyruvate content in the medium and (ii) prolongation of phase 2 with its highly efficient energy metabolism applying e.g. specific feeding strategies. The method presented allows fast and reliable metabolic flux analysis during the development of producer cells and production processes from microtiter plate to large scale reactors with moderate analytical and computational effort. It seems well suited to guide media optimization and genetic engineering of producing cell lines

Kostenki 17 (Spitsynskaya): new data on the stratigraphy, chronology and conditions of the occurrence of the cultural layers

This paper describes the results of 2017—2020 fieldwork at the Upper Palaeolithic site of Kostenki 17 (Spitsynskaya). This work established the presence of five new horizons of finds, in addition to the two cultural layers known since the 1950s. Given the thickness of the Upper Humic Bed, it is clear that the horizons of finds are separated by sterile layers, and correspond to the existence of different sites on this part of the promontory. Despite the small number of finds, the contents of each horizon differ in both the composition of faunal remains and lithic artefacts, as well as raw materials, state of preservation and degree of weathering. The investigated area of the lower (second) cultural layer demonstrates a complex nature of the post-deposition processes that influenced its preservation. A sharp slope of the Lower Humic Bed and a significant difference in the depth of the finds indicate a partial displacement along the slope of some sections of the cultural layer, which was not recorded in previous excavations. The present publication includes a stratigraphic characterization of deposits, a description of the occurrence of the cultural layers and horizons of finds, preliminary results of a technical and typological study of new collections of stone tools and ornaments from cultural layer II, and the results of radiocarbon dating

Validation and invalidation of chemical probes for the human N-myristoyltransferases

On-target, cell-active chemical probes are of fundamental importance in both chemical and cell biology, whereas the application of poorly-characterised probes often leads to invalid conclusions.Human N-myristoyltransferase (NMT) has attracted increasing interest as a target in cancer and infectious diseases; here we report an in-depth comparison of five compounds widely applied as human NMT inhibitors, using a combination of quantitative whole-proteome N-myristoylation profiling, biochemical enzyme assays, cytotoxicity, in-cell protein synthesis and cell cycle assays. We find that N-myristoylation is unaffected by 2-hydroxymyristic acid (100 μM), D-NMAPPD (30 μM) or Tris-DBA palladium (10 μM), with the latter compounds causing cytotoxicity through mechanisms unrelated to NMT. In contrast, drug-like inhibitors IMP-366 (DDD85646) and IMP-1088 delivered complete and specific inhibition of N-myristoylation in a range of cell lines at 1 μM and 100 nM, respectively. This study enables the selection of appropriate on-target probes for future studies and suggests the need for reassessment of previous studies which used off-target compounds