Size Exclusion Chromatography Analysis Research Articles

Gel‐forming properties of Bambara groundnut globulin and protein subfractions: structural and rheological characterisation

SummaryThe gel‐forming ability of the pulse proteins influences the formation of food products with desirable texture, structure, and stability. In this study, Bambara globulin was fractionated into legumin and vicilin for structural and physicochemical analyses, and heat‐induced gel rheological characterisation. Bambara globulin showed major vicilin (7S, Mw: 120 kDa) and minor legumin (11S, Mw: 410 kDa) components by size exclusion chromatography and gel electrophoresis analyses. Legumin had the lowest amount of all the charged amino acids. One basic subunit (22 kDa) was identified in the legumin fraction with predominant helical structures. The sol–gel transition temperatures increased in the order of globulin (40 °C) < legumin (50 °C) < vicilin (80 °C). The G′ and G″ of globulin showed relatively low dependency on heating time beyond the gel point compared to legumin and vicilin subfractions, suggesting a more rapid establishment of its gel network during gelation. Vicilin gel consisted of a microporous structure with a small lath sheet‐like structure compared to others. Bambara vicilin could be a prospective ingredient in the development of new products with defined textural characteristics.

Structure–function characterization of two enzymes from novel subfamilies of manganese peroxidases secreted by the lignocellulose-degrading Agaricales fungi Agrocybe pediades and Cyathus striatus

BackgroundManganese peroxidases (MnPs) are, together with lignin peroxidases and versatile peroxidases, key elements of the enzymatic machineries secreted by white-rot fungi to degrade lignin, thus providing access to cellulose and hemicellulose in plant cell walls. A recent genomic analysis of 52 Agaricomycetes species revealed the existence of novel MnP subfamilies differing in the amino-acid residues that constitute the manganese oxidation site. Following this in silico analysis, a comprehensive structure–function study is needed to understand how these enzymes work and contribute to transform the lignin macromolecule.ResultsTwo MnPs belonging to the subfamilies recently classified as MnP-DGD and MnP-ESD—referred to as Ape-MnP1 and Cst-MnP1, respectively—were identified as the primary peroxidases secreted by the Agaricales species Agrocybe pediades and Cyathus striatus when growing on lignocellulosic substrates. Following heterologous expression and in vitro activation, their biochemical characterization confirmed that these enzymes are active MnPs. However, crystal structure and mutagenesis studies revealed manganese coordination spheres different from those expected after their initial classification. Specifically, a glutamine residue (Gln333) in the C-terminal tail of Ape-MnP1 was found to be involved in manganese binding, along with Asp35 and Asp177, while Cst-MnP1 counts only two amino acids (Glu36 and Asp176), instead of three, to function as a MnP. These findings led to the renaming of these subfamilies as MnP-DDQ and MnP-ED and to re-evaluate their evolutionary origin. Both enzymes were also able to directly oxidize lignin-derived phenolic compounds, as seen for other short MnPs. Importantly, size-exclusion chromatography analyses showed that both enzymes cause changes in polymeric lignin in the presence of manganese, suggesting their relevance in lignocellulose transformation.ConclusionsUnderstanding the mechanisms used by basidiomycetes to degrade lignin is of particular relevance to comprehend carbon cycle in nature and to design biotechnological tools for the industrial use of plant biomass. Here, we provide the first structure–function characterization of two novel MnP subfamilies present in Agaricales mushrooms, elucidating the main residues involved in catalysis and demonstrating their ability to modify the lignin macromolecule.

Analytical evaluation of the performance of pyrolysis-gasification (Py-Gs) combination within hybrid thermochemical-biological biorefinery

Thermochemical treatments like pyrolysis and gasification, were proposed to circumvent hydrolysis bottleneck of conventional 2nd generation biorefineries. Within this big-picture, the target of this article was to establish the type and amount of bioavailable matter that can be obtained through intermediate pyrolysis of biomass followed by gasification of biochar. To establish the amount of “chemical energy” partitioned among different products’ chemical oxygen demand (COD), which is proportional to higher heating value (HHV) and often utilized for biological systems, was applied for the evaluation of thermochemical conversion of a lignocellulosic feedstock (fir wood). The most abundant product of intermediate pyrolysis was biochar, which retains from 33 % to 40 % of feedstock COD (with temperatures starting from 450 °C). The yield of water-soluble pyrolysis products (WS) slightly increases from 24 % at 450 °C to 27 % at 650 °C, whereas the yield of WI increases considerably with the same temperature. Pyrolysis temperature showed a minor effect on the composition of WS as revealed by GC-MS analysis of main compounds and size exclusion chromatography. Preliminary gasification experiments, performed at 850 °C under CO2 atmosphere, provided gasification rates for different biochars, which was equal to 0.004, 0.005, 0.006 min−1 for biochars obtained at 650, 550, and 450 °C respectively. Syngas obtained from gasification of 450 °C and 550 °C biochar was almost tar-free, whereas gasification of 650 °C biochar yielded a detectable amount of tars (0.4 %). Increasing the gasification temperature to 950 °C sharply increases the gasification rate of biochar obtained at 450 °C, allowing to obtain 55% conversion yield. Within the scope of hybrid thermochemical-biological (HTB) processes, the obtained results show that intermediate pyrolysis, when coupled with subsequent 950 °C CO2 gasification of biochar, can deliver 64% of chemical energy (by COD basis) of the lignocellulosic feedstock as bioavailable constituents, which are defined to syngas and WS based on recent biological studies. Whereas downstream fermentation can process syngas and WS materials in an effective way, such yields could surpass the holocellulose-targeted methods based on hydrolysis.

Understanding the Effects of Site-Specific Light Chain Conjugation on Antibody Structure Using Hydrogen Exchange-Mass Spectrometry (HX-MS)

Antibody drug conjugates (ADCs) represent one of the fastest growing classes of cancer therapeutics. Drug incorporation through site-specific conjugation in ADCs leads to uniform drug load and distribution. These site-specific modifications may have an impact on ADC quality attributes including protein higher order structure (HOS), which might impact safety and efficacy. In this study, we conducted a side-by-side comparison between the conjugated and unconjugated mAb. In the ADC, the linker-pyrrolobenzodiazepine was site specifically conjugated to an engineered unpaired C215 residue within the Fab domain of the light chain. Differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF) indicated a decrease in thermal stability for the CH2 transition of the ADC. Size exclusion chromatography (SEC) analysis showed that conjugation of the mAb resulted in earlier aggregation onset and increased aggregation propensity after 4 weeks at 40 °C. Differential hydrogen-exchange mass spectrometry (HX-MS) indicated that upon conjugation, light chain residues 150-155 and 197-204, close to the conjugation site, showed significantly faster HX kinetics, suggesting an increase in backbone flexibility within this region, while heavy chain residues 32-44 exhibited significantly slower kinetics, suggesting distal stabilization of the mAb backbone.

LC-MS Characterization and Stability Assessment Elucidate Correlation Between Charge Variant Composition and Degradation of Monoclonal Antibody Therapeutics.

Aggregation stability of monoclonal antibody (mAb) therapeutics is influenced by many critical quality attributes (CQA) such as charge and hydrophobic variants in addition to environmental factors. In this study, correlation between charge heterogeneity and stability of mAbs for bevacizumab and trastuzumab has been investigated under a variety of stresses including thermal stress at 40°C, thermal stress at 55°C, shaking (mechanical), and low pH. Size- and charge-based heterogeneities were monitored using analytical size exclusion chromatography(SEC) and cation exchange chromatography(CEX), respectively, while dynamic light scattering was used to assess changes in hydrodynamic size. CEX analysis revealed an increase in cumulative acidic content for all variants of both mAbs post-stress treatment attributed to increased deamidation. Higher charge heterogeneity was observed in variants eluting close to the main peak than the ones eluting further away (25-fold and 42-fold increase in acidic content for main and B1 of bevacizumab and 19-fold for main of trastuzumab, respectively, under thermal stress; 50-fold increase in acidic for main and B1 of bevacizumab and 10% rise in basic content of main of trastuzumab under pH stress). Conversely, variants eluting far away from main exhibit greater aggregation as compared to close-eluting ones. Aggregation kinetics of variants followed different order for the different stresses for both mAbs (2nd order for thermal and pH stresses and 0th order for shaking stress). Half-life of terminal charge variants of both mAbs was 2- to 8-fold less than main indicating increased degradation propensity.

Investigating the Impact of Drone Transport on the Stability of Monoclonal Antibodies for Inter-Hospital Transportation

In the field of healthcare logistics, the reliance on conventional transport methods such as cars for the delivery of monoclonal antibodies (mAbs) is susceptible to challenges posed by traffic and infrastructure, leading to increased and unpredictable transport times. Recognizing the potential role of drones in mitigating these challenges, we aimed to investigate the impact of medical drone transport on the stability of mAbs. Compromised stability could lead to aggregation and immunogenicity, thereby jeopardizing the efficacy and safety of mAbs. We studied the transportation of vials as well as ready-to-administer infusion bags with blinatumomab, tocilizumab, and daratumumab. The methodology involved the measurement of both temperature and mechanical shock during drone transport. Moreover, the analytical techniques High Performance Size-Exclusion Chromatography (HP-SEC), Dynamic Light Scattering (DLS), Light Obscuration (LO), Micro-Flow Imaging (MFI), and Nanoparticle Tracking Analysis (NTA) were employed to comprehensively assess the presence of aggregates and particle formation. The key findings revealed no significant differences between car and drone transport, indicating that the stability of mAbs in both vials and infusion bags was adequately maintained during drone transport. This suggests that medical drones are a viable and reliable means for the inter-hospital transport of mAbs, paving the way for more efficient and predictable logistics in healthcare delivery.

Biodegradation behavior of poly (lactic acid) samples obtained by three‐dimensional printing: Influence of temperature and pigment presence

AbstractIn this study, the biodegradation behavior in the soil of natural and pigmented poly (lactic acid) (PLA) plates obtained by three‐dimensional (3D) printing or by compression molding was evaluated by the respirometry method at three different temperatures: 21, 28, and 35°C. PLA samples before and after aerobic biodegradation tests were characterized by Fourier transform infrared spectroscopy, differential scanning calorimetry (DSC), thermogravimetric analysis (TG), and size exclusion chromatography analysis. The increase in test temperature enhanced the biodegradation process. At a test temperature of 35°C, the maximum biodegradation rates of natural PLA and pigmented PLA were 65% and 68% higher, respectively, compared to the maximum biodegradation rate observed at 21°C. DSC and TG analysis indicate that pigmented PLA samples were more susceptible to degradation by hydrolysis. However, the rate of biodegradation efficiency was lower in pigmented PLA samples, with a decrease of 16%, 24%, and 14% at 21, 28, and 35°C, respectively, compared to natural PLA. This behavior indicated that pigment hindered the hydrolysis by‐products absorption by microorganisms. Additionally, the presence of micro voids in PLA samples obtained through 3D printing accelerated the rate of biodegradation efficiency by 32% at 28°C compared to pressed samples.Highlights Poly (lactic acid) (PLA) plate biodegradation in soil was analyzed using respirometry method. Temperature, pigment presence, and sample preparation method were considered. Higher temperatures enhanced both natural and pigmented PLA biodegradation. Pigments appeared to act as antimicrobial agents, slowing down the process. Water presence in the micro voids accelerated hydrolysis and biodegradation.

Synthesis of Self‐assembled Star/Linear Block Copolymer Blends via Aqueous RAFT Dispersion Polymerization

Comprehensive SummaryReversible addition‐fragmentation chain transfer (RAFT)‐mediated polymerization‐induced self‐assembly (PISA) of star block copolymer and linear block copolymer using a binary mixture of a star‐like macro‐RAFT agent and a linear macro‐RAFT agent is reported. With this formulation, star block copolymer and diblock copolymer were formed simultaneously to generate colloidally stable star/linear block copolymer assemblies. Size exclusion chromatography (SEC) analysis confirmed the presence of two types of polymers in the final samples. The molar ratio of the star‐like macro‐RAFT agent and the linear macro‐RAFT agent has a significant impact on the morphology of polymer assemblies. It was found that increasing the amount of star‐like macro‐RAFT agent facilitated the formation of higher‐order morphologies. Additionally, effects of other reaction parameters including the length/number of the arm of the star‐like macro‐RAFT agent, degree of polymer (DP), monomer concentration on the morphology of star/linear block copolymer assemblies were also investigated. We expect that this work will offer new possibilities for the scalable preparation of polymer assemblies with unique structures and functions.

From Pulp to Aromatic Products-Reaction Pathways of Lignin Depolymerization.

This study investigated the depolymerization of lignin into aromatic monomer compounds under hydrothermal conditions. A reaction scheme highlighting secondary alkylation reactions as well as the molecular weight shift was developed based on the experimental data. Lignin is produced in large quantities in paper production and dissolved in what is known as black liquor (BL). To avoid lignin recovery as an additional process step, BL is used directly as feedstock in the hydrothermal liquefaction (HTL) in this work. We performed various batch experiments in micro autoclaves with BL and model substances at different reaction temperatures (TR = 250-400 °C) and a holdingtime of tR = 20 min, as well as continuous experiments (TR = 325-375 °C, tR = 20 min). We were able to show that different derivatives of catechols are the main products among the monomers in our process. With the help of the model substance experiments, we were able to work out three main reactions: demethoxylation, demethylation, and alkylation. This behavior could be observed in the case of BL from hardwood as well as from softwood. 31P nuclear magnetic resonance (NMR) spectroscopy analysis has shown that these reactions take place on aromatic monomers as well as on larger aromatic oligomer structures. At higher temperatures, a large fraction of the carbon ends up in the solid product, while the yields of the monomers decrease sharply. 13C NMR spectroscopy of the solid material shows that the monomers are probably incorporated into the solid phase by repolymerization. We were also able to see this effect using size exclusion chromatography analysis based on the relative molecular weight. From all of the analytical results of the products, a reaction scheme was developed that describes the reaction pathways of the lignin during HTL. Based on this, a reaction kinetic model can be developed in the next step.

Investigation of the Reaction between a Homemade PEEK Oligomer and an Epoxy Prepolymer: Optimisation of Critical Parameters Using Physico-Chemical Methods.

Several researchers have examined the interest in using a thermoplastic to increase thermoset polymers' shock resistance. However, fewer studies have examined the nature of the mechanisms involved between both kinds of polymers. This was the objective of our work, which was carried out using a gradual approach. First, we describe the synthesis of a poly(ether ether ketone) oligomer (oPEEK) with hydroxyl terminations from the reaction of hydroquinone and 4,4'-difluorobenzophenone in N-methyl-2-pyrrolidone. Then, the main physicochemical properties of this oligomer were determined using different thermal analyses (i.e., differential scanning calorimetry (DSC), thermogravimetric (ATG), and thermomechanical analyses) to isolate its response alone. The chemical characterisation of this compound using conventional analytical chemistry techniques was more complex due to its insolubility. To this end, it was sulfonated, according to a well-known process, to make it soluble and enable nuclear magnetic resonance (NMR) and size exclusion chromatography (SEC) experiments. Additional information about the structural and chemical characteristics of the oligomer and its average molecular weight could thus be obtained. The synthesis of an oligoPEEK with α,ω-hydroxyl end-groups and a molecular weight of around 5070 g/mol was thus confirmed by NMR. This value was in accordance with that determined by SEC analysis. Next, the reaction of oPEEK with an epoxy prepolymer was demonstrated using DSC and dynamic rheometry. To this end, uncured mixtures of epoxy prepolymer (DGEBA) with different proportions of oPEEK (3, 5, 10 and 25%) were prepared and characterised by both techniques. Ultimately, the epoxy-oPEEK mixture was cured with isophorone diamine. Finally, topological analyses were performed by atomic force microscopy (AFM) in tapping mode to investigate the interface quality between the epoxy matrix and the oPEEK particles indirectly. No defects, such as decohesion areas, microvoids, or cracks, were observed between both systems.

Coumarin-DPPO a new bio-based phosphorus additive for poly(lactic acid): Processing and flame retardant application

This study addresses very important aspects of the sustainable development of new flame retardant materials: (i) the preparation of a novel flame retardant (FR) additive from bio-resource, (ii) its use as FR additive in poly(lactic acid) (PLA) based composites and, (iii) the in vitro toxicity assessment of the FR. The synthesis of this bio-based FR additive was achieved through a phospha-Michael addition of diphenylphosphine oxide (DPPO) and the naturally occurring compound coumarin, yielding 4-(diphenylphosphoryl)chroman-2-one (CU-DPPO). The flame-retarded CU-DPPO PLA composites were prepared either via conventional melt blending of commercially available PLA or through reactive extrusion (REX) of L-lactide. Characterization of the composites was performed through nuclear magnetic resonance (NMR), size exclusion chromatography (SEC) and thermogravimetric analysis (TGA). TGA analysis of the FR-PLA composites showed an increase in thermal stability, confirming the beneficial effect of the CU-DPPO additive. Direct insertion probe mass spectrometry (DIP-MS) analysis was used to gain insight on the mechanism of action of the FR additive, suggesting gas-phase activity. Rheological measurements exhibited the thermo-oxidative stabilizing effect in the modified PLA. The flame retardancy of the melt-blended PLA composites with CU-DPPO was investigated by limited oxygen index (LOI), cone calorimetry and vertical burning tests (UL 94 and BKZ-VB). Samples containing 10 wt.% of CU-DPPO showed improved fire performance with a limiting oxygen index (LOI) of 29%, passing Swiss vertical burning test (BKZ-VB) and obtaining a V-0 rating in the UL 94 test. Moreover, a preliminary toxicity assessment of the CU-DPPO, carried out using an established in vitro platform, showing no adverse effects on cell viability.

The insightful water quality analysis and predictive model establishment via machine learning in dual-source drinking water distribution system

Dual-source drinking water distribution systems (DWDS) over single-source water supply systems are becoming more practical in providing water for megacities. However, the more complex water supply problems are also generated, especially at the hydraulic junction. Herein, we have sampled for a one-year and analyzed the water quality at the hydraulic junction of a dual-source DWDS. The results show that visible changes in drinking water quality, including turbidity, pH, UV254, DOC, residual chlorine, and trihalomethanes (TMHs), are observed at the sample point between 10 and 12 km to one drinking water plant. The average concentration of residual chlorine decreases from 0.74 ± 0.05 mg/L to 0.31 ± 0.11 mg/L during the water supplied from 0 to 10 km and then increases to 0.75 ± 0.05 mg/L at the end of 22 km. Whereas the THMs shows an opposite trend, the concentration reaches to a peak level at hydraulic junction area (10–12 km). According to parallel factor (PARAFAC) and high-performance size-exclusion chromatography (HPSEC) analysis, organic matters vary significantly during water distribution, and tryptophan-like substances and amino acids are closely related to the level of THMs. The hydraulic junction area is confirmed to be located at 10–12 km based on the water quality variation. Furthermore, data-driven models are established by machine learning (ML) with test R2 higher than 0.8 for THMs prediction. And the SHAP analysis explains the model results and identifies the positive (water temperature and water supply distance) and negative (residual chlorine and pH) key factors influencing the THMs formation. This study conducts a deep understanding of water quality at the hydraulic junction areas and establishes predictive models for THMs formation in dual-sources DWDS.

Proteomics of serum-derived extracellular vesicles are associated with the severity and different clinical profiles of patients with COVID-19: An exploratory secondary analysis

Coronavirus disease 2019 (COVID-19) is characterized by a broad spectrum of clinical manifestations with the potential to progress to multiple organ dysfunction in severe cases. Extracellular vesicles (EVs) carry a range of biological cargoes, which may be used as biomarkers of disease state. An exploratory secondary analysis of the SARITA-2 and SARITA-1 datasets (randomized clinical trials on patients with mild and moderate/severe COVID-19) was performed. Serum-derived EVs were used for proteomic analysis to identify enriched biological processes and key proteins, thus providing insights into differences in disease severity. Serum-derived EVs were separated from patients with COVID-19 by size exclusion chromatography and nanoparticle tracking analysis was used to determine particle concentration and diameter. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was performed to identify and quantify protein signatures. Bioinformatics and multivariate statistical analysis were applied to distinguish candidate proteins associated with disease severity (mild versus moderate/severe COVID-19). No differences were observed in terms of the concentration and diameter of enriched EVs between mild (n = 14) and moderate/severe (n = 30) COVID-19. A total of 414 proteins were found to be present in EVs, of which 360 were shared while 48 were uniquely present in severe/moderate compared to mild COVID-19. The main biological signatures in moderate/severe COVID-19 were associated with platelet degranulation, exocytosis, complement activation, immune effector activation, and humoral immune response. Von Willebrand factor, serum amyloid A-2 protein, histone H4 and H2A type 2-C, and fibrinogen β-chain were the most differentially expressed proteins between severity groups. Exploratory proteomic analysis of serum-derived EVs from patients with COVID-19 detected key proteins related to immune response and activation of coagulation and complement pathways, which are associated with disease severity. Our data suggest that EV proteins may be relevant biomarkers of disease state and prognosis.

Biophysical characterization and insights into the oligomeric nature of CD2-associated protein.

Glomerular podocytes are specialized epithelial cells localized to the blood-urine interface of the kidney. Podocyte slit-diaphragm (SD), a size-and-charge-selective junction, is instrumental in blood ultrafiltration and the formation of protein-free urine. The SD consists of macromolecular complexes of several proteins, such as nephrin, podocin, and CD2-associated protein (CD2AP). CD2AP is an adapter protein and is considered to be crucial for the integrity of SD. Mutations in the SD proteins cause nephrotic syndrome (NS), characterized by proteinuria. SD proteins' structural features must be elucidated to understand the mechanism of proteinuria in NS. In this study, we expressed, purified, and biophysically characterized heterologously expressed human CD2AP. Codon-optimized human CD2AP was expressed in E. coli Rosetta cells. The recombinant protein was induced with 1 mM IPTG and purified by Ni-NTA affinity chromatography. Analytical size-exclusion chromatography, blue native-PAGE, circular dichroism, and fluorescence spectroscopy were performed to decipher the oligomeric nature, secondary structural content, and tertiary packing of CD2AP. Our analysis revealed that CD2AP adopts a predominantly disordered secondary structure despite exhibiting moderate tertiary packing, characterized by low helical and β-sheet content. CD2AP readily assembles into homo-oligomers, with octamers and tetramers constituting the primary population. Interestingly, the inherent flexibility of CD2AP's secondary structural elements appears resistant to thermal denaturation. Frameshift mutation (p.K579Efs*7) that leads to loss of the coiled-coil domain promotes aberrant oligomerization of CD2AP through SH3 domains. We successfully expressed full-length human CD2AP in a heterologous system, wherein the secondary structure of CD2AP is predominantly disordered. CD2AP can form higher-order oligomers, and the significance of these oligomers and the impact of mutations in the context of size-selective permeability of SD needs further investigation.

Characterization of water-soluble brown carbon chromophores from wildfire plumes in the western USA using size-exclusion chromatography

Abstract. Wildfires are an important source of carbonaceous aerosol in the atmosphere. Organic aerosol that absorbs light in the ultraviolet to visible spectral range is referred to as brown carbon (BrC), and its impact on Earth's radiative budget has not been well characterized. We collected water-soluble brown carbon using a particle-into-liquid sampler (PILS) on board a Twin Otter aircraft during the Fire Influence on Regional to Global Environments and Air Quality (FIREX-AQ) campaign. Samples were collected downwind of wildfires in the western United States from August to September 2019. We applied size-exclusion chromatography (SEC) with ultraviolet-visible spectroscopy to characterize the molecular size distribution of BrC chromophores. The wildfire plumes had transport ages of 0 to 5 h, and the absorption was dominated by chromophores with molecular weights <500 Da. With BrC normalized to a conserved biomass burning tracer, carbon monoxide, a consistent decrease in BrC absorption with plume age was not observed during FIREX-AQ. These findings are consistent with the variable trends in BrC absorption with plume age reported in recent studies. While BrC absorption trends were broadly consistent between the offline SEC analysis and the online PILS measurements, the absolute values of absorption and their spectral dependence differed. We investigate plausible explanations for the discrepancies observed between the online and offline analyses. This included solvent effects, pH, and sample storage. We suspect that sample storage impacted the absorption intensity of the offline measurements without impacting the molecular weight distribution of BrC chromophores.

Molecular-level transformations of biomass burning-derived water-soluble organic carbon during dark aqueous OH oxidation: Insights from absorption, fluorescence, high-performance size exclusion chromatography and high-resolution mass spectrometry analysis

Biomass burning (BB) releases large amounts of water-soluble organic carbon (WSOC), which would undergo heterogenous oxidation processes that induce transformations in both molecular structures and compositions within BB WSOC. This study designed an aqueous oxidation initiated by OH radicals in the absence of light for WSOC extracted from smoke particles generated by burning of corn straw and fir wood. The BB WSOC was comprehensively characterized using a combination of UV–visible spectra, excitation-emission matrix fluorescence in conjunction with parallel factor analysis (EEM-PARAFAC), high-performance size exclusion chromatography (HPSEC), and high-resolution mass spectrometry (HRMS) analyses. Over the course of oxidation, both chromophores and fluorophores exhibited gradual decreases. Moreover, EEM-PARAFAC revealed a preferential degradation of larger-sized protein-like/phenol-like organic matters, accompanied by the accumulation and/or formation of humic-like substances in aged BB WSOC. HPSEC analysis showed notable changes in molecular weight (MW) distributions for both types of BB WSOC during oxidation. Specifically, high MW species (>1 kDa) displayed a tendency to form along with oxidation, possibly attributed to the formation of assemblies via intermolecular weak forces. After oxidation, evidence of CHO compound degradation and enrichment/formation of CHON compounds was observed for both types of BB WSOC. Remarkably, the resistant, degraded and produced molecules for BB WSOC were dominated by CHO (38–73 %) and lignin-like molecules (41–47 %), suggesting diverse responses to oxidation within these two groups. Furthermore, polyphenols experienced selective degradation, while CHON, aliphatic and poly-aromatic molecules tended to form during the oxidation process for both types of BB WSOC. In summary, this study provides a comprehensive understanding of the molecular-level transformations undergone by BB WSOC during dark aqueous OH oxidation. The findings significantly contribute to our insights into atmospheric evolution of BB WSOC, thereby playing a crucial role in accurately assessing their effects within climate models and informing policy decisions.

Combined continuous nanoparticle synthesis with chromatographic size classification

In this paper, we report a combination of the continuous flow synthesis of gold nanoparticles (AuNPs) with subsequent purification and narrowing of the particle size distribution (PSD) by size-exclusion chromatography (SEC) by adapting the flow rates of synthesis and classification. First, we show scalability of chromatographic classification with respect to column dimension and the absence of irreversible nanoparticle adhesion on the column material. Two different syntheses lead to a large and widely distributed and a small and narrowly distributed AuNP dispersion, which are classified by a semipreparative column. The PSDs of individual fractions are characterized by analytical SEC. The broadly distributed AuNP dispersion was classified into three fractions with distinct PSDs. For the narrowly distributed AuNPs, the separation is almost independent of the mobile phase flow rate: coarse and fine fractions with almost identical PSDs and separation efficiency curves are observed irrespective of the flow rate. Even NP samples with narrow PSDs can be classified into multiple fractions with tailored PSDs while simultaneously removing dissolved impurities from the dispersion. With our study, we demonstrate the potential of a direct combination of continuous NP synthesis with chromatographic classification for the optimization of final PSDs and the simultaneous purification of nanoparticulate dispersions.

Effects of Dimerization on the Deacylase Activities of Human SIRT2.

Human sirtuin isoform 2 (SIRT2) is an NAD+-dependent enzyme that functions as a lysine deacetylase and defatty-acylase. Here, we report that SIRT2 readily dimerizes in solution and in cells and that dimerization affects its ability to remove different acyl modifications from substrates. Dimerization of recombinant SIRT2 was revealed with analytical size exclusion chromatography and chemical cross-linking. Dimerized SIRT2 dissociates into monomers upon binding long fatty acylated substrates (decanoyl-, dodecanoyl-, and myristoyl-lysine). However, we did not observe dissociation of dimeric SIRT2 in the presence of acetyl-lysine. Analysis of X-ray crystal structures led us to discover a SIRT2 double mutant (Q142A/E340A) that is impaired in its ability to dimerize, which was confirmed with chemical cross-linking and in cells with a split-GFP approach. In enzyme assays, the SIRT2(Q142A/E340A) mutant had normal defatty-acylase activity and impaired deacetylase activity compared with the wild-type protein. These results indicate that dimerization is essential for optimal SIRT2 function as a deacetylase. Moreover, we show that SIRT2 dimers can be dissociated by a deacetylase and defatty-acylase inhibitor, ascorbyl palmitate. Our finding that its oligomeric state can affect the acyl substrate selectivity of SIRT2 is a novel mode of activity regulation by the enzyme that can be altered genetically or pharmacologically.

Fractionation of Arctic Brown Algae (Fucus vesiculosus) Biomass Using 1-Butyl-3-methylimidazolium-Based Ionic Liquids.

Arctic brown algae are considered a promising industrial-scale source of bioactive sub-stances as polysaccharides, polyphenols, and low-molecular secondary metabolites. Conventional technologies for their processing are focused mainly on the isolation of polysaccharides and involve the use of hazardous solvents. In the present study a "green" approach to the fractionation of brown algae biomass based on the dissolution in ionic liquids (ILs) with 1-butil-3-methylimidazolium (bmim) cation with further sequential precipitation of polysaccharides and polyphenols with acetone and water, respectively, is proposed. The effects of IL cation nature, temperature, and treatment duration on the dissolution of bladderwrack (Fucus vesiculosus), yields of the fractions, and their chemical composition were studied involving FTIR and NMR spectroscopy, as well as size-exclusion chromatography and monosaccharide analysis. It was shown that the use of bmim acetate ensures almost complete dissolution of plant material after 24 h treatment at 150 °C and separate isolation of the polysaccharide mixture (alginates, cellulose, and fucoidan) and polyphenols (phlorotannins) with the yields of ~40 and ~10%, respectively. The near-quantitative extraction of polyphenolic fraction with the weight-average molecular mass of 10-20 kDa can be achieved even under mild conditions (80-100 °C). Efficient isolation of polysaccharides requires harsh conditions. Higher temperatures contribute to an increase in fucoidan content in the polysaccharide fraction.

Protein composition and nutritional aspects of pea protein fractions obtained by a modified isoelectric precipitation method using fermentation.

Pea albumins are promising for their nutritional, biological, and techno-functional properties. However, this fraction is usually discarded in the industry due to its low protein content compared to globulin fraction and the presence of some anti-nutritional compounds. In the present study, we used an alternative method of pea protein extraction based on alkaline solubilization/isoelectric precipitation in which the reduction of pH was achieved by lactic acid fermentation using specific starters instead of mineral acids. Hence, the main objective of this study was to examine the protein profile and the content of anti-nutritional and nutritional active compounds in pea albumin-rich fractions obtained by the isoelectric extraction method without (control) or with fermentation with different lactic acid bacteria (Streptococcus thermophilus, Lactiplantibacillus plantarum, and their co-culture). Different pea cultivars (Cartouche, Ascension, and Assas) were used here for their differences in protein profile. The results revealed a higher total nitrogen content in albumin-rich fraction for fermented samples and, in particular, for co-culture. The majority of total nitrogen was determined as non-protein (~50%), suggesting the degradation of proteins by LAB to small peptides and amino acids, which were solubilized in the soluble fraction (albumin) as confirmed by size exclusion chromatography (SEC-HPLC) analysis. Moreover, the higher antioxidant activity of fermented albumin samples was attributed to the production of small peptides during extraction. Lactic acid fermentation also resulted in a significant reduction of trypsin inhibitor activity, α-galactoside, and phytic acid content of this fraction compared to control.