Site-directed mutagenesis and X-ray crystallography of two phospholipase A2 mutants: Y52F and Y73F

Tyr52 and Tyr73 are conserved amino acid residues throughoutall vertebrate phospholipases A₂. They are part of an extendedhydrogen bonding system that links the N-terminal -NH⁺₃ -groupto the catalytic residues His48 and Asp99. These tyrosines werereplaced by phenylalanines in a porcine pancreatic phospholipaseA₂ mutant, in which residues 62–66 had been deleted (62–66PLA₂).The mutations did not affect the catalytic properties of theenzyme, nor the folding kinetics. The stability against denaturatlonby guanidine hydrochloride was decreased, however. To analysehow the enzyme compensates for the loss of the tyrosine hydroxylgroup, the X-ray structures of the Y52F and AY73F mutants weredetermined. After crystallographic refinement the final crystallographicR-factors were 18.1% for the %Y52F mutant (data between 7 and2.3 Å resolution) and 19.1% for the Y73F mutant (databetween 7 and 2.4 Å resolution). No conformational changesoccurred in the mutants compared with the 62–66PLA₂, butan empty cavity formed at the site of the hydroxyl group ofthe former tyrosine. In both mutants the Asp99 side chain losesone of its hydrogen bonds and this might explain the observeddestabilization.     

The effect of engineering surface loops on the thermal stability of Bacillus subtilis neutral protease

Using genetic techniques the contribution of surface loops tothe thermal stability of Bacillus subtUis neutral protease (NPsub)wasstudied. Mutations were designed to make the surface of NP-submore similar to the surface of more thermostable neutral proteasessuch as thermolysin (TLN). The mutations included the replacementof an irregular loop by a shorter variant and the introductionof a ten–residue (3– hairpin. In general, thesedrastic mutations had little effect on the production and activityof NP–sub, indicating the feasibility of major structuralrearrangements at the surface of proteins. In the most stablemutant, exhibiting an increase in thermal stability of 1.1°C, 10% of the surface of NP–sub was modified. Several NP–subvariants carrying multiple mutations were constructed. Non–additiveeffects on thermal stability were observed, which were interpretedon the basis of a model for thermal inactivation, that emphasizesthe importance of local unfolding processes for thermal stability     

Media photo‐degradation in pharmaceutical biotechnology – impact of ambient light on media quality,cell physiology,and IgG production in CHO cultures

BACKGROUND

Many vital components in bioprocess media are prone to photo‐conversion or photo‐degradation upon exposure to ambient light, with severe negative consequences for biomass yield and overall productivity. However, there is only limited awareness of light irradiation as a potential risk factor when working in transparent glass bioreactors, storage vessels or disposable bag systems. The chemical complexity of most media renders a root‐cause analysis difficult. This study investigated in a novel, holistic approach how light‐induced changes in media composition relate to alterations in radical burden, cell physiology, morphology, and product formation in industrial Chinese hamster ovary (CHO) bioprocesses.

RESULTS

Two media formulations from proprietary and commercial sources were tested in a pre‐hoc light exposure scenario prior to cultivation. Using fluorescence excitation/emission (EEM) matrix spectroscopy, a photo‐sensitization of riboflavin was identified as a likely cause for drastically decreased IgG titers (up to ?80%) and specific growth rates (?50% to ?90%). Up to three‐fold higher radical levels were observed in photo‐degraded medium. On the biological side, this resulted in significant changes in cell morphology and aberrations in the normal IgG biosynthesis/secretion pathway.

CONCLUSION

These findings clearly illustrate the underrated impact of room light after only short periods of exposure, occurring accidentally or knowingly during bioprocess development and scale‐ up. The detrimental effects, which may share a common mechanistic cause at the molecular level, correlate well with changes in spectroscopic properties. This offers new perspectives for online monitoring concepts, and improved detectability of such effects in future. © 2018 The Authors. Journal of Chemical Technology & Biotechnology published by JohnWiley & Sons Ltd on behalf of Society of Chemical Industry.

     

Expert elicitation on ultrafine particles: likelihood of health effects and causal pathways

Background

Exposure to fine ambient particulate matter (PM) has consistently been associated with increased morbidity and mortality. The relationship between exposure to ultrafine particles (UFP) and health effects is less firmly established. If UFP cause health effects independently from coarser fractions, this could affect health impact assessment of air pollution, which would possibly lead to alternative policy options to be considered to reduce the disease burden of PM. Therefore, we organized an expert elicitation workshop to assess the evidence for a causal relationship between exposure to UFP and health endpoints.

Methods

An expert elicitation on the health effects of ambient ultrafine particle exposure was carried out, focusing on: 1) the likelihood of causal relationships with key health endpoints, and 2) the likelihood of potential causal pathways for cardiac events. Based on a systematic peer-nomination procedure, fourteen European experts (epidemiologists, toxicologists and clinicians) were selected, of whom twelve attended. They were provided with a briefing book containing key literature. After a group discussion, individual expert judgments in the form of ratings of the likelihood of causal relationships and pathways were obtained using a confidence scheme adapted from the one used by the Intergovernmental Panel on Climate Change.

Results

The likelihood of an independent causal relationship between increased short-term UFP exposure and increased all-cause mortality, hospital admissions for cardiovascular and respiratory diseases, aggravation of asthma symptoms and lung function decrements was rated medium to high by most experts. The likelihood for long-term UFP exposure to be causally related to all cause mortality, cardiovascular and respiratory morbidity and lung cancer was rated slightly lower, mostly medium. The experts rated the likelihood of each of the six identified possible causal pathways separately. Out of these six, the highest likelihood was rated for the pathway involving respiratory inflammation and subsequent thrombotic effects.

Conclusion

The overall medium to high likelihood rating of causality of health effects of UFP exposure and the high likelihood rating of at least one of the proposed causal mechanisms explaining associations between UFP and cardiac events, stresses the importance of considering UFP in future health impact assessments of (transport-related) air pollution, and the need for further research on UFP exposure and health effects.     

Convective and radiant (IR) curing of bulk and waterborne epoxy coatings as thin layers. Part II: Infrared curing

In the first part of this paper, a method for studying the curing of 100-μm-thick epoxy-based layers on steel substrates was described. In this second part, we extend our study to radiative (infrared) heating conditions. A simple waterborne epoxy reactive system based on a diglycidylether of bisphenol-A prepolymer and a polyether triamine was used. The chemical kinetics during infrared heating were studied with the reaction kinetics recorded during convective heating. A second order autocatalytic model described the data. The curing mode had no significant influence on the heating kinetics but the heating rate had a significant influence on the curing kinetics. Higher heating rates can be achieved by infrared heating conditions compared with convective heating. The gelation phenomenon was changed for infrared conditions. Microdielectrometry can be used to record in situ the extent of reaction of the epoxy reactive system for thin layers during radiant heating.     

A Structure-Activity Relationship Study of Bimodal BODIPY-Labeled PSMA-Targeting Bioconjugates

The aim of this study was to identify a high-affinity BODIPY peptidomimetic that targets the prostate-specific membrane antigen (PSMA) as a potential bimodal imaging probe for prostate cancer. For the structure-activity study, several BODIPY (difluoroboron dipyrromethene) derivatives with varying spacers between the BODIPY dye and the PSMA Glu-CO-Lys binding motif were prepared. Corresponding affinities were determined by competitive binding assays in PSMA-positive LNCaP cells. One compound was identified with comparable affinity (IC₅₀=21.5±0.1 nM) to Glu-CO-Lys-Ahx-HBED-CC (PSMA-11) (IC₅₀=18.4±0.2 nM). Radiolabeling was achieved by Lewis-acid-mediated ¹⁹F/¹⁸F exchange in moderate molar activities (∼0.7 MBq nmol⁻¹) and high radiochemical purities (>99 %) with mean radiochemical yields of 20–30 %. Cell internalization of the ¹⁸F-labeled high-affinity conjugate was demonstrated in LNCaP cells showing gradual increasing PSMA-mediated internalization over time. By fluorescence microscopy, localization of the high-affinity BODIPY-PSMA conjugate was found in the cell membrane at early time points and also in subcellular compartments at later time points. In summary, a high-affinity BODIPY-PSMA conjugate has been identified as a suitable candidate for the development of PSMA-specific dual-imaging agents.     

Molecular Properties of Human Guanylate Cyclase-Activating Protein 3 (GCAP3) and Its Possible Association with Retinitis Pigmentosa

The cone-specific guanylate cyclase-activating protein 3 (GCAP3), encoded by the GUCA1C gene, has been shown to regulate the enzymatic activity of membrane-bound guanylate cyclases (GCs) in bovine and teleost fish photoreceptors, to an extent comparable to that of the paralog protein GCAP1. To date, the molecular mechanisms underlying GCAP3 function remain largely unexplored. In this work, we report a thorough characterization of the biochemical and biophysical properties of human GCAP3, moreover, we identified an isolated case of retinitis pigmentosa, in which a patient carried the c.301G > C mutation in GUCA1C, resulting in the substitution of a highly conserved aspartate residue by a histidine (p.(D101H)). We found that myristoylated GCAP3 can activate GC1 with a similar Ca²⁺-dependent profile, but significantly less efficiently than GCAP1. The non-myristoylated form did not induce appreciable regulation of GC1, nor did the p.D101H variant. GCAP3 forms dimers under physiological conditions, but at odds with its paralogs, it tends to form temperature-dependent aggregates driven by hydrophobic interactions. The peculiar properties of GCAP3 were confirmed by 2 µs molecular dynamics simulations, which for the p.D101H variant highlighted a very high structural flexibility and a clear tendency to lose the binding of a Ca²⁺ ion to EF3. Overall, our data show that GCAP3 has unusual biochemical properties, which make the protein significantly different from GCAP1 and GCAP2. Moreover, the newly identified point mutation resulting in a substantially unfunctional protein could trigger retinitis pigmentosa through a currently unknown mechanism.     

计及时变系统完整非线性的振荡模式分析

提出评估非自治非线性因素对振荡行为影响程度的指标。其绝对值在定常的线性模型下严格为0，并随着时变性或非线性影响的增强而增加。据此，可量化地比较不同因素的影响。受扰轨迹完整地反映了时滞环节及离散控制等本质非线性因素和时变因素对系统动态行为的影响。采用小波脊方法估算受扰轨迹在适当宽度的时间窗口内的振荡模式，并随着窗口的滑动得到振荡模式的时间序列。用该序列可以量化非自治非线性振荡的特性，并指导对大振幅低频振荡的分析与控制。仿真发现，即使是3机9节点的小系统，其动态行为也可能不同于平衡点特征根的描述，而平衡点特征根甚至可能丢掉最危险的非线性模式。