Inclusion of maintenance energy improves the intracellular flux predictions of CHO

Chinese hamster ovary (CHO) cells are the leading platform for the production of biopharmaceuticals with human-like glycosylation. The standard practice for cell line generation relies on trial and error approaches such as adaptive evolution and high-throughput screening, which typically take several months. Metabolic modeling could aid in designing better producer cell lines and thus shorten development times. The genome-scale metabolic model (GSMM) of CHO can accurately predict growth rates. However, in order to predict rational engineering strategies it also needs to accurately predict intracellular fluxes. In this work we evaluated the agreement between the fluxes predicted by parsimonious flux balance analysis (pFBA) using the CHO GSMM and a wide range of ¹³C metabolic flux data from literature. While glycolytic fluxes were predicted relatively well, the fluxes of tricarboxylic acid (TCA) cycle were vastly underestimated due to too low energy demand. Inclusion of computationally estimated maintenance energy significantly improved the overall accuracy of intracellular flux predictions. Maintenance energy was therefore determined experimentally by running continuous cultures at different growth rates and evaluating their respective energy consumption. The experimentally and computationally determined maintenance energy were in good agreement. Additionally, we compared alternative objective functions (minimization of uptake rates of seven nonessential metabolites) to the biomass objective. While the predictions of the uptake rates were quite inaccurate for most objectives, the predictions of the intracellular fluxes were comparable to the biomass objective function.     

The SARS‐unique domain (SUD) of SARS‐CoV and SARS‐CoV‐2 interacts with human Paip1 to enhance viral RNA translation

The ongoing outbreak of severe acute respiratory syndrome (SARS) coronavirus 2 (SARS‐CoV‐2) demonstrates the continuous threat of emerging coronaviruses (CoVs) to public health. SARS‐CoV‐2 and SARS‐CoV share an otherwise non‐conserved part of non‐structural protein 3 (Nsp3), therefore named as “SARS‐unique domain” (SUD). We previously found a yeast‐2‐hybrid screen interaction of the SARS‐CoV SUD with human poly(A)‐binding protein (PABP)‐interacting protein 1 (Paip1), a stimulator of protein translation. Here, we validate SARS‐CoV SUD:Paip1 interaction by size‐exclusion chromatography, split‐yellow fluorescent protein, and co‐immunoprecipitation assays, and confirm such interaction also between the corresponding domain of SARS‐CoV‐2 and Paip1. The three‐dimensional structure of the N‐terminal domain of SARS‐CoV SUD (“macrodomain II”, Mac2) in complex with the middle domain of Paip1, determined by X‐ray crystallography and small‐angle X‐ray scattering, provides insights into the structural determinants of the complex formation. In cellulo, SUD enhances synthesis of viral but not host proteins via binding to Paip1 in pBAC‐SARS‐CoV replicon‐transfected cells. We propose a possible mechanism for stimulation of viral translation by the SUD of SARS‐CoV and SARS‐CoV‐2.     

Combination of hydrophobicity and codon usage bias determines sorting of model K+ channel protein to either mitochondria or endoplasmic reticulum

When the K⁺ channel-like protein Kesv from Ectocarpus siliculosus virus 1 is heterologously expressed in mammalian cells, it is sorted to the mitochondria. This targeting can be redirected to the endoplasmic reticulum (ER) by altering the codon usage in distinct regions of the gene or by inserting a triplet of hydrophobic amino acids (AAs) into the protein's C-terminal transmembrane domain (ct-TMD). Systematic variations in the flavor of the inserted AAs and/or its codon usage show that a positive charge in the inserted AA triplet alone serves as strong signal for mitochondria sorting. In cases of neutral AA triplets, mitochondria sorting are favored by a combination of hydrophilic AAs and rarely used codons; sorting to the ER exhibits the inverse dependency. This propensity for ER sorting is particularly high when a common codon follows a rarer one in the AA triplet; mitochondria sorting in contrast is supported by codon uniformity. Since parameters like positive charge, hydrophobic AAs, and common codons are known to facilitate elongation of nascent proteins in the ribosome the data suggest a mechanism in which local changes in elongation velocity and co-translational folding in the ct-TMD influence intracellular protein sorting.     

Quantitation of antibody uptake on A group erythrocytes using immunoautoradiography and monoclonal IgM anti-A

The number of antibody molecules on individual erythrocytes was counted in A1, A2, A3 B and A group individuals using immunoautoradiography (IAR) and monoclonal IgM anti-A1. Quantitation was also done for A group pregnant women. The number of antibody molecules on different red cells of an individual varied widely. Gross variations were also noted in cells of different individuals from one and the same group. The mean values of the uptake of the number of antibody molecules showed the following range A1 greater than A2 greater than Ax greater than A3B. When compared to the average for total A1 adults, red cells of pregnant women and newborn infants showed a 10.7% and 19.7% reduction respectively, in antibody uptake. The mean number of antibody molecules per A1 adult red cells was 5.6 +/- 3 X 10(4), while A2 had 0.85 +/- 0.35 X 10(4) molecules, thus showing a significant quantitative variation.     

Identification of the site of human mannan-binding lectin involved in the interaction with its partner serine proteases: the essential role of Lys55

Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys(55)Ala abolished interaction with the MASPs and MAp19 and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys(55)Gln and Lys(55)Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys(55)Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3. Mutation Arg(47)Glu inhibited interaction with MAp19 and decreased the ability of MBL to trigger the lectin pathway. Mutant Arg(47)Lys showed no interaction with the MASPs or MAp19, likely resulting from a defect in oligomerization. In contrast, mutation Arg(47)Ala had no impact on the interaction with the MASPs and MAp19, nor on the ability of MBL to trigger the lectin pathway. Mutation Pro(53)Ala only had a slight effect on the interaction with MASP-1 and -3, whereas mutations at residues Leu(49) and Leu(56) were ineffective. In conclusion, the MASP binding site of MBL involves a sequence stretch centered on residue Lys(55), which may form an ionic bond representing the major component of the MBL-MASP interaction. The binding sites for MASP-2/MAp19 and MASP-1/3 have common features but are not strictly identical.     

QM/MM studies of enzymes

Combined quantum-mechanics/molecular-mechanics (QM/MM) methods are making rapid progress both methodologically and with respect to their range of application. Mechanistic studies on enzymes, including contributions towards the understanding of enzyme catalysis, continue to be a major target. They are joined by calculations of pK(a) values, redox properties, ground- and excited-state spectroscopic parameters, and excited-state dynamics. Methodological advances include improved QM/MM schemes, in particular new approaches for an effective treatment of the QM-MM electrostatic interactions, and the incorporation of new efficient and accurate QM methods in QM/MM schemes.     

The role of frogs in the epidemiology of Weil's disease

Summary 1. A high percentage of frog sera (ofRana esculenta andRana fusca) reacts positively with respect to many pathogenic and apathogenic leptospira-strains; this reaction is highest (100%) with respect to the incomplete biotype ofL. icterohaemorrhagiae.2. Under natural conditions frogs do not excrete leptospirae with the urine and neither is this the case, when a single leptospira has somehow succeeded in penetrating into them. This penetration does not easily take place.3. By the inoculation of many virulent leptospirae it is possible to break the congenital immunity. Up till 3 days after the inoculation leptospirae can be detected in the blood, up till 7 days in the liver and kidneys. The frogs did not appear to fall ill.4. Frogs do not play a part in the epidemiology of Weil's disease.5. In normal frog sera precipitation of the agglutinins by means of ammonium sulphate and acetone alcohol could not be observed so definitely as in immune sera. Normal frog serum is thermostabile at 56–57°C., immune serum thermolabile at the same temperature. These differences are explained, in accordance withBordet, by the lesser strength of the antibodies in normal sera.