Guidelines for analytical method development and validation of biotechnological synthesis of drugs. Production of a hydroxyprogesterone as model

In connection with biotechnological synthesis of pharmaceutical drugs, validated methods for quantification of both product and substrate at different time intervals are essential for proper calculation of rate coefficients. In this field, there still exist no guidelines for analytical validation, unlike the situation in the bioanalytical field. Therefore, in this study the detailed guidelines by FDA for bioanalytical method validation were applied to a typical biotechnological process; the enzymatic synthesis of 9alpha-hydroxyprogesterone in E. coli using progesterone as substrate. The process liquid was extracted and analyzed using an HPLC-DAD system. The quality control (QC) samples of the product demonstrated excellent precision (C.V.<1.5%) and accuracy between 99.3 and 107%. The study showed that the recommendations and the validation terms for bioanalytical methods can be used also for biotechnological production, but with some important exceptions. The tolerances (C.V. values) of the validation terms should be much narrower; the internal standard (I.S.) must be present in the process liquid before the start of the process and must be much different in structure from the substrate (so as not to participate in the biotechnological process). In addition, the selectivity must be checked very frequently during the process due to the changes in the blank process liquid with time.     

Comprehensive screening of Arabidopsis mutants suggests the lysine histidine transporter 1 to be involved in plant uptake of amino acids

Plant nitrogen (N) uptake is a key process in the global N cycle and is usually considered a "bottleneck" for biomass production in land ecosystems. Earlier, mineral N was considered the only form available to plants. Recent studies have questioned this dogma and shown that plants may access organic N sources such as amino acids. The actual mechanism enabling plants to access amino acid N is still unknown. However, a recent study suggested the Lysine Histidine Transporter 1 (LHT1) to be involved in root amino acid uptake. In this study, we isolated mutants defective in root amino acid uptake by screening Arabidopsis (Arabidopsis thaliana) seeds from ethyl methanesulfonate-treated plants and seeds from amino acid transporter T-DNA knockout mutants for resistance against the toxic D-enantiomer of alanine (Ala). Both ethyl methanesulfonate and T-DNA knockout plants identified as D-Ala resistant were found to be mutated in the LHT1 gene. LHT1 mutants displayed impaired capacity for uptake of a range of amino acids from solutions, displayed impaired growth when N was supplied in organic forms, and acquired substantially lower amounts of amino acids than wild-type plants from solid growth media. LHT1 mutants grown on mineral N did not display a phenotype until at the stage of flowering, when premature senescence of old leaf pairs occurred, suggesting that LHT1 may fulfill an important function at this developmental stage. Based on the broad and unbiased screening of mutants resistant to D-Ala, we suggest that LHT1 is an important mediator of root uptake of amino acids. This provides a molecular background for plant acquisition of organic N from the soil.     

Age at onset determines severity and choice of treatment in early rheumatoid arthritis: a prospective study

Introduction

Disease activity, severity and comorbidity contribute to increased mortality in patients with rheumatoid arthritis (RA). We evaluated the impact of age at disease onset on prognostic risk factors and treatment in patients with early disease.

Methods

In this study, 950 RA patients were followed regularly from the time of inclusion (<12 months from symptom onset) for disease activity (erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), tender and/or swollen joints, Visual Analogue Scale pain and global scores, and Disease Activity Score in 28 joints (DAS28)) and function (Health Assessment Questionnaire (HAQ)). Disease severity, measured on the basis of radiographs of the hands and feet (erosions based on Larsen score), extraarticular disease, nodules, and comorbidities and treatment (disease-modifying antirheumatic drugs (DMARDs), corticosteroids, biologics and nonsteroidal anti-inflammatory drugs) were recorded at the time of inclusion and at 5 years. Autoantibodies (rheumatoid factor, antinuclear antibodies and antibodies against cyclic citrullinated peptides (ACPAs)) and genetic markers (human leucocyte antibody (HLA) shared epitope and protein tyrosine phosphatase nonreceptor type 22 (PTPN22)) were analysed at the time of inclusion. Data were stratified as young-onset RA (YORA) and late-onset RA (LORA), which were defined as being below or above the median age at the time of onset of RA (58 years).

Results

LORA was associated with lower frequency of ACPA (P < 0.05) and carriage of PTPN22-T variant (P < 0.01), but with greater disease activity at the time of inclusion measured on the basis of ESR (P < 0.001), CRP (P < 0.01) and accumulated disease activity (area under the curve for DAS28 score) at 6 months (P < 0.01), 12 months (P < 0.01) and 24 months (P < 0.05), as well as a higher HAQ score (P < 0.01) compared with YORA patients. At baseline and 24 months, LORA was more often associated with erosions (P < 0.01 for both) and higher Larsen scores (P < 0.001 for both). LORA was more often treated with corticosteroids (P < 0.01) and less often with methotrexate (P < 0.001) and biologics (P < 0.001). YORA was more often associated with early DMARD treatment (P < 0.001). The results of multiple regression analyses supported our findings regarding the impact of age on chosen treatment.

Conclusion

YORA patients were more frequently ACPA-positive than LORA patients. LORA was more often associated with erosions, higher Larsen scores, higher disease activity and higher HAQ scores at baseline. Nevertheless, YORA was treated earlier with DMARDs, whilst LORA was more often treated with corticosteroids and less often with DMARDs in early-stage disease. These findings could have implications for the development of comorbidities.     

Thermodynamic and kinetic approaches for evaluation of monoclonal antibody - Lipoprotein interactions

Two complementary instrumental techniques were used, and the data generated was processed with advanced numerical tools to investigate the interactions between anti-human apoB-100 monoclonal antibody (anti-apoB-100 Mab) and apoB-100 containing lipoproteins. Partial Filling Affinity Capillary Electrophoresis (PF-ACE) combined with Adsorption Energy Distribution (AED) calculations provided information on the heterogeneity of the interactions without any a priori model assumptions. The AED calculations evidenced a homogenous binding site distribution for the interactions. Quartz Crystal Microbalance (QCM) studies were used to evaluate thermodynamics and kinetics of the Low-Density Lipoprotein (LDL) and anti-apoB-100 Mab interactions. High affinity and selectivity were observed, and the emerging data sets were analysed with so called Interaction Maps. In thermodynamic studies, the interaction between LDL and anti-apoB-100 Mab was found to be predominantly enthalpy driven. Both techniques were also used to study antibody interactions with Intermediate-Density (IDL) and Very Low-Density (VLDL) Lipoproteins. By screening affinity constants for IDL-VLDL sample in a single injection we were able to distinguish affinity constants for both subpopulations using the numerical Interaction Map tool.     

Symbiosis constraints: Strong mycobiont control limits nutrient response in lichens

Symbioses such as lichens are potentially threatened by drastic environmental changes. We used the lichen Peltigera aphthosa—a symbiosis between a fungus (mycobiont), a green alga (Coccomyxa sp.), and N₂‐fixing cyanobacteria (Nostoc sp.)—as a model organism to assess the effects of environmental perturbations in nitrogen (N) or phosphorus (P). Growth, carbon (C) and N stable isotopes, CNP concentrations, and specific markers were analyzed in whole thalli and the partners after 4 months of daily nutrient additions in the field. Thallus N was 40% higher in N‐fertilized thalli, amino acid concentrations were twice as high, while fungal chitin but not ergosterol was lower. Nitrogen also resulted in a thicker algal layer and density, and a higher δ¹³C abundance in all three partners. Photosynthesis was not affected by either N or P. Thallus growth increased with light dose independent of fertilization regime. We conclude that faster algal growth compared to fungal lead to increased competition for light and CO₂ among the Coccomyxa cells, and for C between alga and fungus, resulting in neither photosynthesis nor thallus growth responded to N fertilization. This suggests that the symbiotic lifestyle of lichens may prevent them from utilizing nutrient abundance to increase C assimilation and growth.     

Food selectivity versus prey availability: a study using the marine fish Pomatoschistus microps

Summary The food selection of the common goby, Pomatoschistus microps Krøyer, was studied in the field and in laboratory experiments on the Swedish west coast. The three most important prey organisms for P. microps in the study area were Corophium volutator, chironomid larvae and Nereis spp. Corophium was consumed more than any other prey, even when it was not the most abundant prey species in the bottom. One reason may be the higher activity of Corophium above the sediment surface, which may increase its visibility and consequently its vulnerability to visual predators. When P. microps was offered Corophium and chironomid larvae with similar exposure in laboratory experiments, it showed no preference for either of the prey items. It always took the closest mobile prey, regardless of species and size.     

Continuous extraction of β-d-galactosidase from Escherichia coli in an aqueous two-phase system: effects of biomass concentration on partitioning and mass transfer

High concentrations of Escherichia coli disintegrate move the binodial of a poly(ethylene glycol) (PEG) 4000/potassium phosphate aqueous two-phase system towards lower concentrations. It has also been shown that the yield and purification factor of β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) in the PEG phase was gradually improved by moving the experimental system to a composition closer to the binodial. The mass transfer rates of cell debris, total protein, β-d-galactosidase and DNA have been studied and were found to be fast enough to reach equilibrium between the phases after 1.9 s of mixing in a static mixer with 24 mixing elements. A continuous extraction process for β-d-galactosidase from E. coli has been designed on the basis of these studies with a mean residence time of 6.3 min from the disintegrator inlet to the β-d-galactosidase containing PEG-phase outlet of the centrifuge. This PEG phase contained 83.5% of the total β-d-galactosidase with a purification factor of 13.6, and only 2.8% of the total protease activity of the disintegrate. All cell debris and almost all DNA were confined to the potassium phosphate phase.     

Phenol oxidation in soft cuticle and blood of crayfish compared with that in other arthropods and activation of the phenol oxidases by fungal and other cell walls

Phenol oxidase from blood as well as from soft cuticle of different crayfish was highly activated by cell walls of a number of fungi and other plants, but the enzymes from other crustaceans tested and from some insects were not. Attachment of the phenol oxidase on blood and soft cuticle to the cell wall surface of certain plants and subsequent heavy melanization of the walls with natural or added substrate may be a unique property of crayfish since it did not occur in any other arthropods tested. Interestingly, blood and cuticle of crayfish showed the same specificity in these processes and the mechanisms might therefore be similar, if not the same, in both. The activity of the nonadsorbed enzyme dispersed in the blood was higher in a crayfish species resistant to fungal attack than in susceptible ones, but the relative degree of activation by adding cell walls was greater in the latter.The natural substrate for the phenol-oxidase activity in crayfish blood was released or mobilized by the blood cells. The substrate for the activity in cuticle was not available in the intact cuticle itself. It appeared only after a “signal” had been transmitted through the cuticle to the cells inside. The possibility that these mechanisms are involved in the cuticular as well as the blood defense system against fungal and other microbial parasites is discussed.     

Water repellency,mat formation,and leaf-stimulated growth of some ectomycorrhizal fungi

Summary Ectomycorrhizal short roots, mycelia, rhizomorphs and mats from conifer soil were examined in relation to their hydrophobic properties. In some cases connected fruit bodies were included in the study. Mycorrhizal soils gathered from the forest and/or colonized in a laboratory rhizoscope were studied, as were mycelia in pure culture. Most forest-derived species were hydrophobic. The drought-resistant Cenococcum geophilum and the more ruderal and moisture-dependent Thelephora terrestris were both strongly hydrophilic. The hydrophobic mycelium seemed solely responsible for the water repellence properties, and adjacent soil and plant debris remained unaffected and hydrophilic. In hydrophobic fungi, mat formation was induced in the rhizoscope by hyphal contact with alder litter leaves. This stimulating effect was not found when the leaves were covered by water or when fresh, green alder leaves were used. Thelephora terrestris did not form such mats in vitro and spread sparsely in air pockets as well as in the adjacent water film. The possibility is discussed that many mycorrhizal fungi in the forest may partly control their soil environment via aeration created by their hydrophobia.