An overview of mechanistic modeling of liquid chromatography

Abstract

Liquid chromatography is considered to be the bottleneck for purification of therapeutic proteins. Development and optimization of chromatography process is a cumbersome activity due to the increasing complexities in the types and content of impurities present in the high product titer cell culture harvest obtained from the upstream processing. Further, regulatory expectations are continuously rising with the recent initiatives of quality by design and process analytical technology expecting the manufacturer to have a deeper understanding of the process and the product. Mechanistic modeling is one approach to gain this deeper understanding of a process step. It involves modeling of the underlying physicochemical processes. A well calibrated model with acceptable predictability can be very effective in both process optimization and process characterization activities. In this paper we provide an overview of mechanistic modeling of liquid chromatography. We discuss the various components that such a model entails and also presents the status quo of this area.     

Lysine Succinylation of VBS Contributes to Sclerotia Development and Aflatoxin Biosynthesis in Aspergillus flavus

Aspergillus flavus is a common saprophytic and pathogenic fungus, and its secondary metabolic pathways are one of the most highly characterized owing to its aflatoxin (AF) metabolite affecting global economic crops and human health. Different natural environments can cause significant variations in AF synthesis. Succinylation was recently identified as one of the most critical regulatory post-translational modifications affecting metabolic pathways. It is primarily reported in human cells and bacteria with few studies on fungi. Proteomic quantification of lysine succinylation (Ksuc) exploring its potential involvement in secondary metabolism regulation (including AF production) has not been performed under natural conditions in A. flavus. In this study, a quantification method was performed based on tandem mass tag labeling and antibody-based affinity enrichment of succinylated peptides via high accuracy nano-liquid chromatography with tandem mass spectrometry to explore the succinylation mechanism affecting the pathogenicity of naturally isolated A. flavus strains with varying toxin production. Altogether, 1240 Ksuc sites in 768 proteins were identified with 1103 sites in 685 proteins quantified. Comparing succinylated protein levels between high and low AF-producing A. flavus strains, bioinformatics analysis indicated that most succinylated proteins located in the AF biosynthetic pathway were downregulated, which directly affected AF synthesis. Versicolorin B synthase is a key catalytic enzyme for heterochrome B synthesis during AF synthesis. Site-directed mutagenesis and biochemical studies revealed that versicolorin B synthase succinylation is an important regulatory mechanism affecting sclerotia development and AF biosynthesis in A. flavus. In summary, our quantitative study of the lysine succinylome in high/low AF-producing strains revealed the role of Ksuc in regulating AF biosynthesis. We revealed novel insights into the metabolism of AF biosynthesis using naturally isolated A. flavus strains and identified a rich source of metabolism-related enzymes regulated by succinylation.     

Uptake,Transport and Storage of Cardenolides in Foxglove. Cardenolide Sinks and Occurrence of Cardenolides in the Sieve Tubes of Digitalis lanata1

Cardiac glycoside transport was investigated on the organ and whole plant level. Uptake experiments were carried out with shoot and root cultures of Digitalis lanata. In both systems primary cardenolides, i.e., those with a terminal glucose in their oligosaccharide side chain, were taken up against their concentration gradient, whereas the glucose-free secondary cardenolides were not. Active uptake of primary cardenolides was further evidenced by KCN inhibition of uptake. Using plantlets grown in vitro the long-distance transport of primary cardenolides from the leaves to the roots was demonstrated. Cardenolides were also detected in etiolated leaves, induced on plants with green leaves, which are supposed to be unable to synthezise cardenolides de novo, providing further evidence for long-distance transport. Several primary cardenolides were detected in the honeydew excreted by aphids fed on Digitalis lanata leaves, indicating that the phloem is a transporting tissue for cardenolides. On the other hand, the xylem sap obtained by applying the pressure-chamber technique was cardenolide-free. It was concluded that in Digitalis primary cardenolides serve as both the transport and the storage form of cardenolides. After their synthesis they are either stored in the vacuoles of the source tissue or loaded into the sieve tubes, from which they are unloaded at other sites where they are trapped in the vacuoles of the respective sink tissue.     

Time of day effect on balance performance,functional capacities and risk of fall in women with rheumatoid arthritis

ABSTRACT

Objective: This study explored the time of day effect of balance performance, functional capacities and risk of fall in three different times in patients with rheumatoid arthritis (RA) and the association between these variations and those of RA symptoms.

Methods: A “discontinual” protocol, composed of three test sessions, carried out at 6 am, 2 pm and 10 pm was set up, in order to investigate the time of day effect of balance performance, functional capacities, risk of fall, stiffness, range of motion, swollen and painful joints in women with RA.

Results: Time Up and Go Test (TUGT), Functional Reach Test (FRT) and tinetti test scores were significantly higher (p < .01) at 6 am and at 10 pm compared to 2 pm. Stiffness, range of motion, swollen and painful joints values were significantly higher (p < .01) at 6 am and at 10 pm compared to 2 pm. A significant difference was observed on the stiffness, range of motion and swollen joints values between 6 am and 10 pm that were higher at 6 am (p < .05).

Using Pearson’s coefficient, correlations were found between RA symptom values; and TUGT, FRT and Tinetti test scores.

Conclusion: Results showed a time of day effect of balance performance, functional capacities and risk of falls in women with RA. This variation indicates an alteration of performance at 6 am and 10 pm. Fluctuations of stiffness, limited range of motion, swollen and painful joints noted are concomitant to those of balance performance, functional capacities, and risk of fall.

Abbreviations: RA: rheumatoid arthritis; H&O questionnaire: Horne and Ostberg questionnaire; PSQI: Pittsburgh sleep quality index; HAQ: health assessment questionnaire; SF-36: the short form-36; WOMAC: Western Ontario and McMaster Universities Osteoarthritis Index; TUGT: Time Up and Go Test; FRT: Functional Reach Test     

Anterograde Axonal Transport of Peptidylglycine α-Amidating Monooxygenase in Rat Sciatic Nerves

Axonal transport of peptidylglycine alpha-amidating monooxygenase (PAM) activity was studied in rat sciatic nerves from 12 to 120 h after double ligations. The anterograde axonal transport increased and reached a plateau between 48 and 72 h and then decreased. The flow rate was 100 mm/day, and the molecular mass of the active entity was 70 kDa, which was determined by gel filtration. In contrast, there was no evidence for significant retrograde axonal transport. Anterograde axonal transport of immunoreactive cholecystokinin, a carboxy-terminal-amidated putative neuropeptide, was also found. These results suggest that PAM is transported by a rapid axonal flow and may play a role as a processing enzyme during transport or in the terminals of rat sciatic nerves.     

Structural and Functional Impact of SRP54 Mutations Causing Severe Congenital Neutropenia

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Turnover of cell surface-bound capsular polysaccharide in Staphylococcus aureus

The capsular polysaccharide (CPS) of Staphylococcus aureus strain Smith was labelled by growth of bacteria in the presence of radioactive N-acetylglucosamine and was separated from labelled cell wall components by affinity chromatography on wheat germ agglutinin following dissolution of the cells by lysostaphin. The products were partially characterised chemically and immunochemically. Similar labelled components were found in the culture fluid during growth. In a pulse-chase experiment, cell-bound CPS was released continuously into the culture fluid at the same rate as cell wall turnover and there was no evidence of direct excretion of CPS.     

Planktonic indices in the evaluation of the ecological status and the trophic state of the longest lake in Poland

Due to the intensive mixing polymictic lakes should be homogenous. However, morphometric diversity and high water dynamics contribute to the differentiation of many parameters in various areas of the lakes. This study analyzes both phytoplankton and zooplankton to assess differences in water quality along the north–south axis of the longest lake in Poland. New phytoplankton indicators were applied for determining the lake's ecological status: the Q index based on functional groups and the PMPL (Phytoplankton Metric for Polish Lakes) index based on phytoplankton biomass. TSI_ROT index (Rotifer Trophic State Index), which comprises the percentage of species indicating a high trophic state in the indicatory group and the percentage of bacteriovorus in the Rotifera population, was used for zooplankton analysis.TP content was different at different sites – we observed its gradual increase from the south to the north. Spatial variation of phosphorus did not considerably affect plankton diversity. The phytoplankton was dominated by Oscillatoriales, typical of shallow, well-mixed eutrophic lakes. The ecological status of the lake based on the EQR (Ecological Quality Ratio) was poor or moderate. The zooplankton was dominated by rotifers (at almost all sites), which indicates a eutrophic state of the lake. The values of phytoplankton indices at the studied sites did not differ considerably; the differences resulted more from local conditions such as the contaminant inflow and the macrophyte development than water dynamics.We have demonstrated that in the lake dominated by filamentous Cyanobacteria the ecological status should be determined according to the PMPL index or other indices dependent on the dominant Cyanobacteria species. Since the Q index does not include the functional group S1, the results can lead to the false conclusion that water quality improves with an increased amount of phytoplankton. The high abundance of Cyanobacteria in the lake may have contributed to the poor growth of rotifers.     

Occurrence of two structural types of mercury reductases among Gram-positive bacteria

Structural variants of mercury reductase containing the N-terminal domain, which is easily cleaved by trypsin, have been found in Gram-positive bacteria with a low genomic G + C content (Bacillus, Staphylococcus and, possibly, some other genera). Mercury reductases without the N-terminal domain and relatively resistant to limited proteolysis are typical for Gram-positive bacteria with a high genomic G + C content (Arthrobacter, Citreobacterium, Micrococcus, Mycobacterium, Rhodococcus). Both types of mercury reductase genes may be located on plasmids.