Native Liquid Chromatography and Mass Spectrometry to Structurally and Functionally Characterize Endo-Xylanase Proteoforms

Xylanases are of great value in various industries, including paper, food, and biorefinery. Due to their biotechnological production, these enzymes can contain a variety of post-translational modifications, which may have a profound effect on protein function. Understanding the structure–function relationship can guide the development of products with optimal performance. We have developed a workflow for the structural and functional characterization of an endo-1,4-β-xylanase (ENDO-I) produced by Aspergillus niger with and without applying thermal stress. This workflow relies on orthogonal native separation techniques to resolve proteoforms. Mass spectrometry and activity assays of separated proteoforms permitted the establishment of structure–function relationships. The separation conditions were focus on balancing efficient separation and protein functionality. We employed size exclusion chromatography (SEC) to separate ENDO-I from other co-expressed proteins. Charge variants were investigated with ion exchange chromatography (IEX) and revealed the presence of low abundant glycated variants in the temperature-stressed material. To obtain better insights into the effect on glycation on function, we enriched for these species using boronate affinity chromatography (BAC). The activity measurements showed lower activity of glycated species compared to the non-modified enzyme. Altogether, this workflow allowed in-depth structural and functional characterization of ENDO-I proteoforms.     

Land use regression model for ultrafine particles in Amsterdam

There are currently no epidemiological studies on health effects of long-term exposure to ultrafine particles (UFP), largely because data on spatial exposure contrasts for UFP is lacking. The objective of this study was to develop a land use regression (LUR) model for UFP in the city of Amsterdam. Total particle number concentrations (PNC), PM10, PM2.5, and its soot content were measured directly outside 50 homes spread over the city of Amsterdam. Each home was measured during one week. Continuous measurements at a central urban background site were used to adjust the average concentration for temporal variation. Predictor variables (traffic, address density, land use) were obtained using geographic information systems. A model including the product of traffic intensity and the inverse distance to the nearest road squared, address density, and location near the port explained 67% of the variability in measured PNC. LUR models for PM2.5, soot, and coarse particles (PM10, PM2.5) explained 57%, 76%, and 37% of the variability in measured concentrations. Predictions from the PNC model correlated highly with predictions from LUR models for PM2.5, soot, and coarse particles. A LUR model for PNC has been developed, with similar validity as previous models for more commonly measured pollutants.     

An Artificial Nocturnal Flower via Humidity‐Gated Photoactuation in Liquid Crystal Networks

Beyond their colorful appearances and versatile geometries, flowers can self‐shape‐morph by adapting to environmental changes. Such responses are often regulated by a delicate interplay between different stimuli such as temperature, light, and humidity, giving rise to the beauty and complexity of the plant kingdom. Nature inspires scientists to realize artificial systems that mimic their natural counterparts in function, flexibility, and adaptation. Yet, many of the artificial systems demonstrated to date fail to mimic the adaptive functions, due to the lack of multi‐responsivity and sophisticated control over deformation directionality. Herein, a new class of liquid‐crystal‐network (LCN) photoactuators whose response is controlled by delicate interplay between light and humidity is presented. Using a novel deformation mechanism in LCNs, humidity‐gated photoactuation, an artificial nocturnal flower is devised that is closed under daylight conditions when the humidity level is low and/or the light level is high, while it opens in the dark when the humidity level is high. The humidity‐gated photoactuators can be fueled with lower light intensities than conventional photothermal LCN actuators. This, combined with facile control over the speed, geometry, and directionality of movements, renders the “nocturnal actuator” promising for smart and adaptive bioinspired microrobotics.     

Correlation of Immunological and Histopathological Features with Gene Expression-Based Classifiers in Colon Cancer Patients

The purpose of this study was to evaluate the association between four distinct histopathological features: (1) tumor infiltrating lymphocytes, (2) mucinous differentiation, (3) tumor-stroma ratio, plus (4) tumor budding and two gene expression-based classifiers—(1) consensus molecular subtypes (CMS) plus (2) colorectal cancer intrinsic subtypes (CRIS). All four histopathological features were retrospectively scored on hematoxylin and eosin sections of the most invasive part of the primary tumor in 218 stage II and III colon cancer patients from two independent cohorts (AMC-AJCC-90 and AC-ICAM). RNA-based CMS and CRIS assignments were independently obtained for all patients. Contingency tables were constructed and a χ2 test was used to test for statistical significance. Odds ratios with 95% confidence intervals were calculated. The presence of tumor infiltrating lymphocytes and a mucinous phenotype (>50% mucinous surface area) were strongly correlated with CMS1 (p < 0.001 and p = 0.008) and CRIS-A (p = 0.006 and p < 0.001). The presence of mucus (≥ 10%) was associated with CMS3: mucus was present in 64.1% of all CMS3 tumors (p < 0.001). Although a clear association between tumor-stroma ratio and CMS4 was established in this study (p = 0.006), still 32 out of 61 (52.5%) CMS4 tumors were scored as stroma-low, indicating that CMS4 tumors cannot be identified solely based on stromal content. Higher budding counts were seen in CMS4 and CRIS-B tumors (p = 0.045 and p = 0.046). No other associations of the measured parameters were seen for any of the other CRIS subtypes. Our analysis revealed clear associations between histopathologic features and CMS or CRIS subtypes. However, identification of distinct molecular subtypes solely based on histopathology proved to be infeasible. Combining both molecular and morphologic features could potentially improve patient stratification.     

Introduction of a stabilizing 10 residue {beta}-hairpin in Bacillus subtilis neutral protease

A 10 residue ß-hairpin, which is characteristic ofthermostable Bacillus neutral proteases, was engineered intothe thermolabile neutral protease of Bacillus subtilis. Therecipient enzyme remained fully active after introduction ofthe loop. However, the mutant protein exhibited autocatalyticnicking and a 0.4°C decrease in thermostability. Two additionalpoint mutations designed to improve the interactions betweenthe enzyme surface and the introduced ß-hairpin resultedin reduced nicking and increased thermostability. After theintroduction of both additional mutations in the loopcontainingmutant, nicking was largely prevented and an increase in thermostabilityof 1.1°C was achieved.     

The effect of cavity-filling mutations on the thermostability of Bacillus stearothermophilus neutral protease

Cavities in the hydrophobic core of the neutral protease ofBacillus stearothermophilus were analyzed using a threedimensionalmodel that was inferred from the crystal structure of thermolysin,the highly homologous neutral protease of B.thermoproteolyticus(85% sequence identity). Site–directed mutagenesis wasused to fill some of these cavities, thereby improving hydrophobicpacking in the protein interior. The mutations had small effectson the thermostability, even after drastic changes, such asLeu284Trp and Met168Trp. The effects on T50, the temperatureat which 50% of the enzyme is irreversibly inactivated in 30min, ranged from 0.0 to +0.4°C. These results can be explainedby assuming that the mutations have positive and negative structuraleffects of approximately the same magnitude. Alternatively,it could be envisaged that the local unfolding steps, whichrender the enzyme susceptible towards autolysis and which arerate limiting in the process of thermal inactivation, are onlyslightly affected by alterations in the hydrophobic core.     

Uncertainties in the fate of nitrogen I: An overview of sources of uncertainty illustrated with a Dutch case study

This study focuses on the uncertainties in the fate of nitrogen (N) in the Netherlands. Nitrogen inputs into the Netherlands in products, by rivers, and by atmospheric deposition, and microbial and industrial fixation of atmospheric N₂ amount to about 4450 Gg N y^–1. About 60% of this N is transported out of the Netherlands in products. The fate of the remaining 40%, however, is less clear. We discuss uncertainties in losses to the atmosphere (as ammonia or through denitrification), by leaching and runoff, and in N accumulation in biomass and soils. These processes may account for the fate of about 40% of the N in the Netherlands, and for the fate of about 60% of the N in Dutch agricultural soils. Reducing uncertainties in the estimates of these fluxes is necessary for reducing the impact of excess N in the environment. In particular, monitoring the environmental effects of ammonia emissions and nitrate leaching to groundwater and aquatic systems requires an increased understanding of the fate of N. Uncertainties arise because (1) some N fluxes cannot be measured directly and are usually quantified indirectly as the balance in N budgets, (2) direct measurements of N fluxes have inevitable inaccuracies, (3) lack of experimental data and other information (e.g. statistics) needed for upscaling, (4) large spatial and temporal variability of fluxes, and (5) poor understanding of the processes involved. These uncertainties can be reduced by additional experimental studies and by further development of process-based models and N budget studies. We prioritize these future research needs according to a range of different criteria.     

Achieving flue gas desulphurization with membrane gas absorption

Membrane gas absorption (MGA) is an efficient and flexible way to carry out gas-liquid (G-L) contacting operations with hollow fibre membranes. The main advantages of membrane gas absorption over conventional G-L contactors is the compactness and low weight of the installation, the standard modular construction elements and the flexible operation. Gas and liquid flow can be controlled independently in the MGA process, which makes operation over a wide range of liquid and gas flow rates possible. In a MGA installation no entrainment, flooding, channelling or foaming occurs. Rob Klaassen and Albert E Jansen, TNO Environment, Energy and Process Innovation, discuss the principle of this technology, the benefits of newly developed membrane modules and their potential applications.     

Discovery of an Acyclic Nucleoside Phosphonate that Inhibits Mycobacterium tuberculosis ThyX Based on the Binding Mode of a 5‐Alkynyl Substrate Analogue

The urgent need for new antibiotics poses a challenge to target un(der)exploited vital cellular processes. Thymidylate biosynthesis is one such process due to its crucial role in DNA replication and repair. Thymidylate synthases (TS) catalyze a crucial step in the biosynthesis of thymidine 5‐triphosphate (TTP), an elementary building block required for DNA synthesis and repair. To date, TS inhibitors have only been successfully applied in anticancer therapy due to their lack of specificity for antimicrobial versus human enzymes. However, the discovery of a new family of TS enzymes (ThyX) in a range of pathogenic bacteria that is structurally and biochemically different from the “classic” TS (ThyA) has opened the possibility to develop selective ThyX inhibitors as potent antimicrobial drugs. Here, the interaction of the known inhibitor 5‐(3‐octanamidoprop‐1yn‐1yl)‐2′‐deoxyuridine‐5′‐monophosphate ( 1 ) with Mycobacterium tuberculosis ThyX enzyme is explored using molecular modeling starting from published crystal structures, with further confirmation through NMR experiments. While the deoxyuridylate (dUMP) moiety of compound 1 occupies the cavity of the natural substrate in ThyX, the rest of the ligand (the “5‐alkynyl tail”) extends to the outside of the enzyme between two of its four subunits. The hydrophobic pocket that accommodates the alkyl part of the tail is formed by displacement of Tyr 44.C, Tyr 108.A and Lys 165.A. Changes to the resonance of the Lys 165 NH₃ group upon ligand binding were monitored in a titration experiment by 2D HISQC NMR. Guided by the results of the modeling and NMR studies, and inspired by the success of acyclic antiviral nucleosides, compounds where a 5‐alkynyl uracyl moiety is coupled to an acyclic nucleoside phosphonate (ANP) were synthesized and evaluated. Of the compounds evaluated, sodium (6‐(5‐(3‐octanamidoprop‐1‐yn‐1‐yl)‐2,4‐dioxo‐3,4‐dihydropyrimidin‐1(2H)‐yl)hexyl)phosphonate ( 3 e ) exhibited 43 % of inhibitory effect on ThyX at 50 μM . While only modest activity was achieved, this is the first example of an ANP inhibiting ThyX, and these results can be used to further guide structural modifications to this class to develop more potent compounds with potential application as antibacterial agents acting through a novel mechanism of action.