Gel Filtration Research Articles

Anionic Oligo(ethylene glycol)-Based Molecular Brushes: Thermo- and pH-Responsive Properties

Anionic thermo- and pH-responsive copolymers were synthesized by photoiniferter reversible addition–fragmentation chain transfer polymerization (PI-RAFT). The thermo-responsive properties were provided by oligo(ethylene glycol)-based macromonomer units containing hydrophilic and hydrophobic moieties. The pH-responsive properties were enabled by the addition of 5–20 mol% of strong (2-acrylamido-2-methylpropanesulfonic) and weak (methacrylic) acids. Upon initiation by visible light at 470 nm and in the absence of radical initiators, yields from the ternary copolymers reached 94% in 2.5 h when the process was carried out in continuous flow mode using 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid as a light-sensitive RAFT agent. The polymers were characterized using size exclusion chromatography, IR and NMR spectroscopy, and differential scanning calorimetry. The copolymers featured a sufficiently high molecular weight (93–146 kDa) consistent with theoretical values and satisfactory dispersities in the range of 1.18–1.45. The pH-responsive properties were studied in deionized water, saline, and buffer solutions. Dramatic differences in LCST behavior were observed in strong and weak acid-based polyelectrolytes. The introduction of sulfonic acid units, even in very small amounts, completely suppressed the LCST transition in deionized water while maintaining it in the saline and buffer solutions, with a negligible LCST dependence on the pH. In contrast, the incorporation of weak methacrylic acid demonstrated a pronounced pH dependence. The peculiarities of micelle formation in aqueous solutions were investigated and critical micelle concentrations and their ability to retain pyrene, a hydrophobic drug model, were determined. It was observed that anionic molecular brushes formed small micelles with aggregation numbers of 1–2 at concentrations in the order of 10−4 mg/mL. These micelles have a high ability to entrap pyrene, which makes them a promising tool for targeted drug delivery.

Plasma-assisted destruction of polystyrene nanoplastics.

This study addresses the critical need for the effective removal of nanoplastics (1 nm to 1000 nm), which pose a significant environmental challenge due to their ease of entry into biological systems and poorly understood health impacts. We report our investigation of a plasma-assisted methodology with a falling film plasma reactor to destroy and remove 200 nm polystyrene nanoplastic particles from their aqueous solution. Using the nanoparticle tracking analysis, size exclusion chromatography, and total organic carbon (TOC) analysis, we examined the degradation kinetics of the nanoplastics upon plasma-assisted treatment. A nanoplastic removal rate of 98.4% by particle count was achieved in one hour of treatment. This rate increased to 99.3% after three hours of treatment, along with a 27.4% reduction in the TOC of the solution. The chromatography results indicate that the observed elimination of nanoplastic contaminants was likely through the production of short polystyrene oligomers with molecular weights roughly equivalent to those of two styrene units. The superior efficacy of the plasma-assisted methodology over traditional ozonation to destroy nanoplastics was also illustrated. Our results not only elucidate a hypothesized polystyrene radical decay mechanism but also demonstrate a potential and complementary approach for mitigating nanoplastic pollution in water purification strategies.

Identification of an Intrinsically Disordered Region (IDR) in Arginyltransferase 1 (ATE1).

Arginyltransferase 1 (ATE1) catalyzes arginylation, an important posttranslational modification (PTM) in eukaryotes that plays a critical role in cellular homeostasis. The disruption of ATE1 function is implicated in mammalian neurodegenerative disorders and cardiovascular maldevelopment, while posttranslational arginylation has also been linked to the activities of several important human viruses such as SARS-CoV-2 and HIV. Despite the known significance of ATE1 in mammalian cellular function, past biophysical studies of this enzyme have mainly focused on yeast ATE1, leaving the mechanism of arginylation in mammalian cells unclear. In this study, we sought to structurally and biophysically characterize mouse (Mus musculus) ATE1. Using size-exclusion chromatography (SEC), small-angle X-ray scattering (SAXS), and hydrogen-deuterium exchange mass spectrometry (HDX-MS), assisted by AlphaFold modeling, we found that mouse ATE1 is structurally more complex than yeast ATE1. Importantly, our data indicate the existence of an intrinsically disordered region (IDR) in all mouse ATE1 splice variants. However, comparative HDX-MS analyses show that yeast ATE1 does not have such an IDR, consistent with prior X-ray, cryo-EM, and SAXS analyses. Furthermore, bioinformatics approaches reveal that mammalian ATE1 sequences, as well those as in a large majority of other eukaryotes, contain an IDR-like sequence positioned in proximity to the ATE1 GNAT active-site fold. Computational analysis suggests that the IDR facilitates the formation of a complex between ATE1 and tRNAArg, adding a new complexity to the ATE1 structure and providing new insights for future studies of ATE1 functions.

Amniotic fluid-derived stem cells: potential factories of natural and mimetic strategies for congenital malformations

Background:Mesenchymal stem cells (MSCs) derived from gestational tissues offer a promising avenue for prenatal intervention in congenital malformations although their application is hampered by concerns related to cellular plasticity and the need for invasive, high-risk surgical procedures. Here, we present naturally occurring exosomes (EXOs) isolated from amniotic fluid-derived MSCs (AF-MSCs) and their mimetic analogs (MIMs) as viable, reproducible, and stable alternatives. These nanovesicles present a minimally invasive therapeutic option, addressing the limitations of MSC-based treatments while retaining therapeutic efficacy.Methods:MIMs were generated from AF-MSCs by combining sequential filtration steps through filter membranes with different porosity and size exclusion chromatography columns. A physicochemical, structural, and molecular comparison was conducted with exosomes (EXOs) released from the same batch of cells. Additionally, their distribution patterns in female mice were evaluated following in vivo administration, along with an assessment of their safety profile throughout gestation in a mouse strain predisposed to neural tube defects (NTDs). The possibility to exploit both formulations as mRNA-therapeutics was explored by evaluating cell uptake in two different cell types(fibroblasts, and macrophages) and mRNA functionality overtime in an in vitro experimental setting as well as in an ex vivo, whole embryo culture using pregnant C57BL6 dams.Results:Molecular and physiochemical characterization showed no differences between EXOs and MIMs, with MIMs determining a threefold greater yield. Biodistribution patterns following intraperitoneal administration were comparable between the two particle types, with the uterus being among targeted organs. No toxic effects were observed in the dams during gestation, nor were there any malformations or significant differences in the number of viable versus dead fetuses detected. MIMs delivered a more intense and prolonged expression of mRNA encoding for green fluorescent protein in macrophages and fibroblasts. An ex-vivo whole embryo culture demonstrated that MIMs mainly accumulate at the level of the yolk sac, while EXOs reach the embryo.Conclusions:The present data confirms the potential application of EXOs and MIMs as suitable tools for prevention and treatment of NTDs and proposes MIMs as prospective vehicles to prevent congenital malformations caused by in utero exposure to drugs.

MICROCOMPOUNDING OF SMALL SAMPLES OF NATURAL RUBBER

ABSTRACT We describe a microcompounding method that can be used to characterize and compare cured small natural rubber samples. First, commercial Hevea and dried guayule (Parthenium argentatum) latex samples were microcompounded to validate the method. Latex was then extracted from the ground branches of six greenhouse shrubs of different ages and genotypes of wild type and transgenic guayule and coagulated into rubber samples. Size exclusion chromatography showed that the samples had different molecular weights and oligomer content. Little variation in physical properties was found between rubber extracted from shrubs of different age within a genotype, but a larger variation among genotypes was found. The new method allows testing of cured rubber samples from experimental variants and early screening out of genotypes to assess the impact of variations in cultivation practices or lab-scale processing conditions on rubber quality.

Glass Silicone Oil Free Pre-filled Syringe as Primary Container in Autoinjector.

Pre-filled syringes (PFSs) have become popular as a convenient and cost-effective container closure system for delivering biotherapeutics. However, standard siliconized PFSs may compromise the stability of therapeutic proteins due to their exposure to the silicone oil-water interface. To address this concern, silicone oil-free (SOF) glass syringes coupled with silicone-oil free plunger stoppers have been developed. This study aims to compare the impact of silicone oil-free (SOF) and siliconized syringes as primary container on protein stability and device functionality of the combination products. The stability of proteins with different modalities was assessed in SOF and siliconized 1mL glass syringes for up to 6months at 5℃, 25℃, and 40℃ with levels of subvisible particles and soluble aggregate determined by micro-flow imaging (MFI) and ultra performance size-exclusion chromatography (UP-SEC). The functionality of SOF glass syringes, including break loose force, extrusion force and delivery time in autoinjectors, was evaluated at different time points during the stability study. Additionally, SOF glass syringes were filled with viscosity surrogate ranging from 1 to 90 cP to understand the impact of solution viscosity on break loose force, extrusion force, and autoinjector delivery time. SOF demonstrates compatibility with proteins and exhibited significantly low particle counts compared to siliconized PFS. SOF syringes show significantly higher break-loose and extrusion forces. However, unlike siliconized syringes where silicone oil migration increases extrusion force, no significant change in functionality was observed in SOF glass syringe during stability testing. Overall, SOF glass syringes showed great potential as an alternative package for biologics with comparable performance on functionality as siliconized PFS. The combination of SOF glass and its PTFE coated stopper presents a new primary container closure system with both adequate protein stability and desired functionality features.

Faricimab maintains substance integrity and sterility after compounding and storage in two different polypropylene syringe types.

Compounding and storage of intravitreal anti-vascular endothelial growth factor (anti-VEGF) agents in syringes is commonly performed in an off-label manner. However, the preservation of compound integrity and microbiological safety must be guaranteed. The aim of this study was to compare the chemical and physical stability, sterility and binding affinity to vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) of faricimab, a novel bispecific anti-VEGF/Ang-2 biologic, after compounding and storage in two different polypropylene syringe types for up to 28 days. Faricimab was compounded into silicone oil-free and silicone oil-containing polypropylene syringes under controlled aseptic clean room conditions and stored under light protection at 2-8 °C for up to 28 days. Compound integrity was analysed by size exclusion chromatography, nano differential scanning fluorimetry, UV-vis and dynamic light scattering. The analysis of the simultaneous binding of VEGF and Ang-2 was performed by grating-coupled interferometry. Additionally, samples were tested for sterility and presence of bacterial endotoxins. One-way ANOVA test was used to analyse statistical significance (p ≤ 0.05). No significant differences in VEGF and ANG-2 binding affinity were found in faricimab samples stored in either syringe type after 28 days compared to control. Chemical and physical stability testing revealed no statistically significant variation. Furthermore, sterility and the absence of bacterial endotoxins could be maintained. Our findings confirm the pharmaceutical safety of compounded faricimab after storage for up to 28 days. This may facilitate a cost-effective off-label use of faricimab in clinical practice while maintaining safety in the treatment of patients.

How Do DNA Molecular Springs Modulate Protein-Protein Interactions: Experimental and Theoretical Results.

Deoxyribonucleic acid (DNA) nanomachines have been widely exploited in enzyme activity regulation, protein crystallization, protein assembly, and control of the protein-protein interaction (PPI). Yet, the fundamental biophysical framework of DNA nanomachines in the case of regulating protein-protein interactions remains elusive. Here, we established a DNA nanospring-mCherry model with mCherry homodimers of different Kd. Using size exclusion chromatography and fluorescence polarization, we profiled the DNA nanospring-mediated manipulation of PPI as an entropy-reducing process. The energy transfer efficiency was a function of the length of the complementary sequence and the geometry of the DNA nanospring construction. With basic force analysis and physical chemistry calculation, we proposed a unified model of the correlation between the dissociation constant, local concentration, construction of DNA nanospring, and kinetics of protein dimerization. Overall, we demonstrated that the DNA nanospring-mCherry conjugate was a simple and practical model to analyze DNA-controlled protein-protein interaction.

Purification, characterization and molecular docking studies of analogous alpha amylase inhibitor compounds

BackgroundA large number of protein inhibitors are found in higher plants, cereals and legumes. These inhibitors are helpful in the prevention as well as medical treatment of metabolic syndromes such as type 2 diabetes. Basically, the diabetes mellitus is a chronic disorder that occurs either due to inadequate insulin secretions or when body fails to utilize the produced insulin. The alpha amylase inhibitors are termed as starch blockers. They catalyze the hydrolysis of α-(1,4)-D-glycosidic linkages of starch and other glucose polymer. They play a significant role in inhibition of the activity of salivary and pancreatic amylase in vitro and in vivo. MethodThe present study was based on the purification, characterization and molecular docking studies of the alpha amylase inhibitor isolated from the kidney bean sample. The seed sample was collected from G.B. Pant Nagar University. The crude extract was prepared, the in-vitro studies and heat stability were determined. Following it, purification was carried out using ammonium sulphate precipitation and size exclusion chromatography was done. Later, characterization and molecular docking studies of the purified sample was done after obtaining GC–MS results. ResultThe in-vitro analysis was done and noted that the inhibitory activity was 96.5 ± 0.84 % post size exclusion chromatography. The molecular weight was about 54 kDa. The molecular docking studies revealed that there were interactions between the ligand molecules (the constituents that were selected from the GC–MS chromatogram peaks based on the height and area) and the human pancreatic alpha amylase (1HNY). The hydrogen bonding as affinity binding capacity was also been observed in each of the ligand–protein interaction. ConclusionFrom the tests, it has been elucidated that the alpha amylase inhibitor isolated from the sample has a great potential to serve as an anti-diabetic drug. However, in order to check the potential effects and to explore the optimization and development of the anti-diabetic drug the in-vivo studies can also be done further.

Evaluation of the analytic performance and macroprolactin sensitivity of a new prolactin immunoassay.

Prolactin measurement is essential in endocrine diagnostics. Challenges such as the hook effect and reactivity to macroprolactin, which varies according to the reagent, complicate accurate measurement. The present study evaluated a newly marketed reagent to detect prolactin, IDS Prolactin, comparing it to an established reagent, Roche Elecsys Prolactin, assessing its behavior toward macroprolactin and polyethylene glycol (PEG) treatment, and establishing reference intervals. The IDS Prolactin and Roche Elecsys Prolactin assays were compared using 44 samples containing macroprolactin confirmed on gel filtration chromatography (macroprolactin: BBPRL) and 104 samples for which the diagnosis of macroprolactin was excluded (monomeric prolactin: MNPRL). Analytic performance of the IDS Prolactin assay was also assessed. The new reagent showed satisfactory analytic performance, meeting EFLM standards for repeatability and intermediate imprecision. Comparison between the two methods found robust correlation for monomeric samples (y=1.060x-18.28; r2=0.993). Compared to the Roche assay, which is particularly low in its reaction to macroprolactin, the IDS assay displayed a higher level of detection. PEG precipitation effectively separated monomeric and macroprolactin samples when a cut-off of 65% recovery was used or at the threshold of 444 mIU/L (20.9 µg/L) for post-PEG monomeric prolactin upper limit of normal. Reference intervals were established for women, with ROC curve analysis demonstrating high sensitivity and specificity. The IDS Prolactin assay showed excellent analytic performance and satisfactory characteristics on macroprolactinemic samples.

The molecular structure of leaf starch from three cereal crops

Plants produce storage and transient starches in seeds and in leaves, respectively. Understanding molecular fine structure and synthesis of transient starch can help improve plant quality (e.g. by helping breeders produce slowly digested amylopectin, which is beneficial for human nutrition). In the present study, leaf starches from rice, wheat and barley were isolated with cesium chloride gradient centrifugation. Starch fine structure was measured using size-exclusion chromatography and flurophore-assisted carbohydrate electrophoresis. The chain-length distribution (CLD) of amylopectin leaf starch was trimodal in wheat and barley leaf starch. The global peak of leaf starch was at degree of polymerization (DP) 22, and leaf amylopectin containeds more long branches, which are generally considered to hinder starch digestion, suggesting that leaf-specific starch synthesis enzymes could be expressed in the endosperm by genetic modification to produce amylopectin with more long chains, which would be more slowly digested, with advantages to human health. HypothesisThe biosynthetic processes for leaf starch and storage starch in a given plant species will show significant differences.

Removal of pharmaceutical compounds from sewage effluent by the nanofiltration membrane

Membrane is a promising technology for water treatment, and its commercial production has emerged globally. However, commercial membranes' effectiveness is still uncertain due to the new types of contaminants, such as pharmaceutical compounds (PC). Thus, in the current work, the performance of two commercial membranes (NFX and GC) has been evaluated using feed water from the effluent of sewage treatment plants (STP) and hospital wastewater (HWW). The spiked feed water contains PC classes of analgesics/anti-inflammatory, anti-hypertensives, beta-blockers, and psychiatric/antidepressants. The percentage difference between feed and permeate was measured. While the fouling properties of the membranes are also evaluated using STP and HWW as feed, along with the pure water flux in a variable-pressure setting, the results showed that NFX exhibits excellent, consistent rejection for the targeted PCs (>80 %). GC showed a broader range of rejection (10 % to 90 %) and presented a better flux flow than NFX. The main rejection factor is due to the membrane's pore size variation and the layer construction, which refer to the absorption, size exclusion, and diffusion of the PCs instead of the type of feed water and the flux. Current work provides scientific data on the diverse fabrication and market-available types of membranes, which would pose various efficiencies towards the type of targeted pollution.

Expression, purification, and biophysical analysis of a part of the C-terminal domain of human hypoxia inducible factor-2α (HIF-2α)

Hypoxia inducible factor 2α (HIF-2α) is a member of the basic helix-loop-helix(bHLH)-Per-Arnt-Sim (PAS) family of transcription factors. It is overexpressed in several cancers, associated with poor prognosis of the patients and resistance to treatment. Here, we study the residues 366-704 of the C-terminal end of human HIF-2α, which contains the N-transcriptional activation domain (NTAD), the oxygen-dependent degradation domain (ODD), and a part of the inhibitory domain (IH). An efficient protocol was developed to produce the 366-704 domain of human HIF-2α protein. Subsequently, we analyzed its biophysical characteristics using circular dichroism spectroscopy and size exclusion chromatography showing that the protein forms an antiparallel beta sheet conformation, and a computational model of the HIF-2α structure was produced. Our data offer new structural information for the unique biological properties of HIF-2α.

Structure-function relationship of PE11 esterase of Mycobacterium tuberculosis with respect to its role in virulence

The lipolytic enzymes of Mycobacterium tuberculosis play a critical role in immunomodulation and virulence. Among these proteins, PE11 which also belongs to the PE/PPE family, is the smallest (∼10.8 kDa) and play a significant role in cell wall remodelling and virulence. PE11 is established to be an esterase, but its enzymatic and structural properties are not yet characterized. In this study, using homology modelling we deduced the putative structure which shows the presence of both α-helix and β-sheet structures which is in close agreement with that observed by CD spectra of the purified protein. PE11 was found to contain a Gx3Sx4G motif homologous to canonical ‘GxSxG’ motif present in many serin hydrolases. The catalytic triad appears to be located within this motif as substitution of Serine26 and Glycine31 residues abrogated its enzymatic activity. Gel-filtration chromatography data indicate that PE11 possibly exists as dimer and tetramer showing positive cooperativity for binding its substrates. In addition, PE11 esterase activity was found to be critical for cell wall remodelling, antibiotic resistance and conferring survival advantages to M. tuberculosis. Our data suggest that PE11 can be targeted for designing potential therapeutic strategies.

Production and compositional analysis of full-length influenza virus hemagglutinin in Nanodiscs: Insights from multi-angle light scattering.

The global threat of pandemics highlights the urgency of developing innovative vaccine strategies. Viral spike proteins are the primary antigens recognized by the immune system and serve as key targets for vaccine development. This study reports the production of full-length Influenza A virus surface glycoprotein, hemagglutinin (HA), and its incorporation into Nanodiscs (NDs). HA was expressed in insect cells and purified using detergents, maintaining its functional integrity. Characterization by size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) confirmed that HA could be incorporated into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) NDs as a single trimer. SEC-MALS was instrumental in analysing the composition of NDs, which included HA, membrane scaffold proteins, lipids, and glycans. These findings provide a robust framework for the production and reconstitution of glycoproteins in NDs, and offers valuable insights into the study of multi-component nanoparticles using MALS. Our work highlights the potential of NDs for studying viral glycoproteins and advances the development of well-defined recombinant ND-based vaccines.

Degradation of (1→3)(1→6)-α-D-dextran by ultrasound: Molecular weight, viscosity and kinetics

The (1→3)(1→6)-α-D-dextran (alternating dextran) produced by Leuconostoc citreum SK24.002 is a novel functional exopolysaccharide, and its low molecular weight derivatives have potential applications in the food, pharmaceutical, and chemical industries. In this work, we used sonication, a green polysaccharide disruption method, to study the degradation process of this dextran by changing the intensity and duration of the sonication treatment and the concentration of the dextran solution. The molecular weight and viscosity of the dextran products were measured with a high-performance size exclusion column chromatography–multi-angle laser light scattering–refractive index system and by rheometry, respectively. The degradation efficiency of dextran was directly affected by the duration and intensity of the ultrasonic treatment and the concentration of the dextran solution. The polydispersity index fluctuated as the duration of the sonication treatment increased. The combination of a high intensity (672 W/cm2) and long (120 min) sonication treatment and a low solution concentration (3 g dextran/100 mL) was most effective for reducing the apparent and complex viscosities of dextran. The storage modulus of dextran was always slightly larger than its loss modulus, indicating that it formed a gel-like structure. The second-order kinetic model (1/Mwt - 1/Mw0 = kt) was the best fit to explain the degradation dynamics of dextran by sonication at intensities of 168 W/cm2–834 W/cm2 and with dextran solution concentrations of 1 g/100 mL – 7 g/100 mL. Our findings show that sonication is an effective way to reduce the molecular weight of alternating dextran.

Identification of disease-specific extracellular vesicle-associated plasma protein biomarkers for Duchenne Muscular Dystrophy and Facioscapulohumeral Muscular Dystrophy.

Reliable, circulating biomarkers for Duchenne, Becker and facioscapulohumeral muscular dystrophies (DBMD and FSHD) remain unvalidated. Here, we investigated the plasma extracellular vesicle (EV) proteome to identify disease-specific biomarkers that could accelerate therapy approvals. We extracted EVs from the plasma of DBMD and FSHD patients and healthy controls using size-exclusion chromatography, conducted mass spectrometry on the extracted EV proteins, and performed comparative analysis to identify disease-specific biomarkers. We correlated the levels of these biomarkers with clinical outcome measures and confounding factors. The muscle-associated proteins PYGM, MYOM3, FLNC, MYH2 and TTN were exclusively present in DBMD EVs. PYGM, MYOM3, and TTN negatively correlated with age. PYGM and MYOM3 levels were elevated in patients without cardiomyopathy, and PYGM levels were specifically elevated in ambulatory DMD patients. On the other hand, female FSHD patients displayed significantly higher MBL2 and lower GPLD1 levels. However, male FSHD patients exhibited higher C9 and lower C4BPB levels. Additionally, desmosome proteins JUP and DSP were uniquely found in FSHD males. MBL2 positively correlated with age and C4BPB negatively correlated with FSHD severity in male patients. Our findings underscore the sensitivity of analyzing circulating EV content to identify disease-specific protein biomarkers for DBMD and FSHD. Our results also emphasize the potential of EV-based biomarker discovery as a promising approach to monitor disease progression as well as effectiveness of therapies in muscular dystrophy, potentially contributing to their approval. Further research with larger cohorts is needed to validate these biomarkers and explore their clinical implications.

Cyclooctyne End-Functionalized Poly(morpholine-2,5-dione)s.

The cyclooctyne-functionalized alcohol (1R,8S,9S)-bicyclo-[6.1.0]non-4-yn-9-ylmethanol (BCN-OH) is applied as initiator for the organo-catalyzed ring-opening polymerization (ROP) of morpholine-2,5-diones based on the l-amino acids valine, isoleucine, and phenylalanine. The ROP is catalyzed by a binary system of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and 1-(3,5-bis(trifluoromethyl)phenyl)-3-cyclohexylthiourea (TU) applying a feed ratio of [M]/[I]/[DBU]/[TU] of 100/1/1/10. Kinetic studies reveal that BCN-OH is capable to initiate the polymerization of morpholine-2,5-diones, which proceed in a controlled manner until monomer conversions of 80%. Characterization by means of 1HNMR spectroscopy, size exclusion chromatography (SEC), and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry confirm the covalent attachment of the cyclooctyne moiety as α-end group of the poly(morpholine-2,5-dione)s with maximum dispersities of 1.25. As a proof of concept, a vitaminA end-functionalized poly(2-ethyl-2-oxazoline) is coupled to a poly(ester amide) by strain-promoted azide-alkyne cycloaddition. Characterization of the block copolymer by SEC and DOSYNMR spectroscopy confirm the successful attachment of the two building blocks. The versatile cyclooctyne moiety shall facilitate a metal-free attachment of other polymer blocks, targeting ligands or dyes at the α-end group of well-defined poly(morpholine-2,5-dione)s. In consequence, the approach provides access to a new generation of functionalized poly(ester amide)s, which can be customized for specific needs.

Lung contusion complicated by pneumonia worsens lung injury via the inflammatory effect of alveolar small extracellular vesicles on macrophages and epithelial cells.

Lung contusion (LC) complicated by pneumonia is associated with a higher risk of acute lung injury (ALI) mediated by activation of immune cells and injury to the lung epithelium. Small extracellular vesicles (sEVs) are essential mediators of cellular crosstalk; however, their role in the development of postinjury ALI remains unclear. We hypothesized that LC complicated by pneumonia increases the pro-inflammatory effect of alveolar sEVs on macrophages and the cytotoxicity of alveolar sEVs to pulmonary epithelial cells, worsening the severity of ALI. Studies in C57BL/6 mice were designed with four groups: sham, LC, Pneumonia (Pneu), and LC + Pneu. Lung contusion was induced by a cortical controlled impactor, while pneumonia was conducted by intratracheal injection of 105 cfu Pseudomonas aeruginosa. Bronchoalveolar lavage fluid (BAL) was harvested 24 hours postinfection, and sEVs were purified by centrifugation and size exclusion chromatography. To evaluate the effect of alveolar sEV on cells, sEVs from each group were cocultured with macrophages (RAW 264.7) to assess cytokine release and lung epithelial cells (MLE 12) to assess epithelial cytotoxicity. The LC + Pneu group severely injured lungs histologically and increased the susceptibility to the bacteria. The LC + Pneu group showed higher concentrations of proteins, macrophage inflammatory protein 1-alpha (MIP1α), and intercellular adhesion molecule 1 (ICAM-1) in BAL. MIP1α and ICAM-1 expression in the macrophages increased after incubation with sEVs from the LC + Pneu group. Moreover, the sEVs demonstrated higher cytotoxicity to epithelial cells and increased apoptosis in epithelial cells after incubation with sEVs from the LC + Pneu group. Lung contusion complicated by pneumonia increased the pro-inflammatory effect of alveolar sEVs on macrophages and the cytotoxicity of alveolar sEVs to pulmonary epithelial cells, worsening the severity of ALI. These results demonstrate the potential importance of alveolar sEVs in lung inflammation following a bacterial infection after trauma.

Thermal treatment of sewage sludge: Impact of the sludge type and origin on the formation of recalcitrant compounds

In a municipal wastewater treatment plant, the thermal treatment of sludge can be an efficient way of increasing the final sludge cake dryness and boosting anaerobic digestion performances. However, such treatments generate refractory compounds which, once returned to headworks, can affect the quality compliance of effluent discharges, particularly concerning organic nitrogen. This study explores the effects of thermal hydrolysis (TH) and hydrothermal carbonization (HTC) of municipal sludge on the refractory organic compound production. A novel approach using ultra-performance liquid chromatography with size-exclusion chromatography and UV/fluorescence detection (UPLC-SEC-UV/Fluo) was employed to characterise recalcitrant dissolved organic matter (rDOM), which typically consists of Maillard reaction products (MRP). Specifically, UPLC-SEC-UV/Fluo was combined with principal component analysis (PCA) to enable a more thorough examination of the chemical diversity of MRPs produced. Greater temperatures during the thermal treatment step lead to increased production of rDOM and rDON. Protein-rich sludge with a great AS:PS ratio yields the greatest rDOM levels. MRP characteristics, such as molecular weight distribution and aromaticity, are primarily influenced by temperature and plant origin. UPLC-SEC-UV/Fluo provides information on the structures of MRPs useful to optimize the thermal treatment process and in understanding their fate in subsequent processes (chemical oxidation, biodegradation).These insights have practical implications for sludge treatment processes, including optimizing TH and HTC conditions to control rDOM production and adapt the sludge treatment line of a water resource recovery facility.