Gel Filtration Research Articles

Isolation and Characterization of Exosomes from Ocular Samples.

Exosomes are microsize vesicles secreted by nearly all cells to the extracellular space. The vesicles transport cell signaling and communicate with other cells. Ultracentrifugation is the standard method to isolate exosomes from culture media or body fluid. Without ultracentrifuge, exosomes can be precipitated by polyethylene glycol or separated by size exclusion chromatography. After isolation, nanoparticle tracking analysis can help to estimate the size and concentration of exosome samples. Transmission electron microscopy can directly show the size and morphology of exosomes. Moreover, the sample should be characterized by the expression of several exosome biomarker proteins. Exosomal contents such as proteins and miRNAs could be profiled using appropriate technologies.

Coupling Online Size Exclusion Chromatography with Charge Detection-Mass Spectrometry Using Hadamard Transform Multiplexing.

Charge detection mass spectrometry (CD-MS) is a powerful technique for the analysis of large, heterogeneous biomolecules. By directly measuring the charge states of individual ions, CD-MS can measure the masses from spectra where conventional deconvolution approaches fail due to the lack of isotopic resolution or distinguishable charge states. However, CD-MS is inherently slow because hundreds or thousands of spectra need to be collected to produce adequate ion statistics. The slower speed of CD-MS complicates efforts to couple it with online separation techniques, which limit the number of spectra that can be acquired during a chromatographic peak. Here, we present the application of Hadamard transform multiplexing to online size exclusion chromatography (SEC) coupled with Orbitrap CD-MS, with a goal of using SEC for separating complex mixtures prior to CD-MS analysis. We developed a microcontroller to deliver pulsed injections from a large sample loop onto a SEC for online CD-MS analysis. Data showed a series of peaks spaced according to the pseudorandom injection sequence, which were demultiplexed with a Hadamard transform algorithm. The demultiplexed data revealed improved CD-MS signals while preserving retention time information. This multiplexing approach provides a general solution to the inherent incompatibilities of online separations and CD-MS detection that will enable a range of applications.

Pancreatic β-cells package double C2-like domain beta protein into extracellular vesicles via tandem C2 domains

IntroductionDouble C2-like domain beta (DOC2B) is a vesicle priming protein critical for glucose-stimulated insulin secretion in β-cells. Individuals with type 1 diabetes (T1D) have lower levels of DOC2B in their residual functional β-cell mass and platelets, a phenotype also observed in a mouse model of T1D. Thus, DOC2B levels could provide important information on β-cell dys(function).ObjectiveOur objective was to evaluate the DOC2B secretome of β-cells. In addition to soluble extracellular protein, we assessed DOC2B localized within membrane-delimited nanoparticles – extracellular vesicles (EVs). Moreover, in rat clonal β-cells, we probed domains required for DOC2B sorting into EVs.MethodUsing Single Extracellular VEsicle Nanoscopy, we quantified EVs derived from clonal β-cells (human EndoC-βH1, rat INS-1 832/13, and mouse MIN6); two other cell types known to regulate glucose homeostasis and functionally utilize DOC2B (skeletal muscle rat myotube L6-GLUT4myc and human neuronal-like SH-SY5Y cells); and human islets sourced from individuals with no diabetes (ND). EVs derived from ND human plasma, ND human islets, and cell lines were isolated with either size exclusion chromatography or differential centrifugation. Isolated EVs were comprehensively characterized using dotblots, transmission electron microscopy, nanoparticle tracking analysis, and immunoblotting.ResultsDOC2B was present within EVs derived from ND human plasma, ND human islets, and INS-1 832/13 β-cells. Compared to neuronal-like SH-SY5Y cells and L6-GLUT4myc myotubes, clonal β-cells (EndoC-βH1, INS-1 832/13, and MIN6) produced significantly more EVs. DOC2B levels in EVs (over whole cell lysates) were higher in INS-1 832/13 β-cells compared to L6-GLUT4myc myotubes; SH-SY5Y neuronal-like cells did not release appreciable DOC2B. Mechanistically, we show that DOC2B was localized to the EV lumen; the tandem C2 domains were sufficient to confer sorting to INS-1 832/13 β-cell EVs.DiscussionClonal β-cells and ND human islets produce abundant EVs. In cell culture, appreciable DOC2B can be packaged into EVs, and a small fraction is excreted as a soluble protein. While DOC2B-laden EVs and soluble protein are present in ND plasma, further studies will be necessary to determine if DOC2B originating from β-cells significantly contributes to the plasma secretome.

Detection of renal tubular transporter proteins in canine urinary extracellular vesicles using liquid chromatography-tandem mass spectrometry.

Urinary extracellular vesicles (UEVs) are membranous particles that carry renal tubular transporter proteins. Here, we evaluate whether selected renal tubular transporter proteins can be detected in UEVs isolated from small volume (1-5 mL) canine urine samples of healthy dogs and canine patients with elevated circulating parathyroid hormone (PTH)/PTH-related peptide (PTHrp) concentrations, hypercortisolism, and primary hypoadrenocorticism using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The total creatinine content of each urine sample was calculated from urine volume and creatinine concentration. UEVs were isolated by size exclusion chromatography prior to quantification by nanoparticle tracking analysis and proteomic analysis by LC-MS/MS. Group comparisons were made using non-parametric statistics. Aquaporin-2 (AQP2) and the renal sodium/phosphate co-transporter (NPT2A) were detected in UEVs isolated from small volume samples of almost all healthy dogs but were not detected in most dogs with elevated circulating PTH/PTH related peptide (PTHrp) concentrations, hypercortisolism and primary hypoadrenocorticism. Total creatinine content of the urine sample was strongly positively correlated with the number of UEVs (rs = .84, P < .001); thus, total creatinine was used as a surrogate marker of UEV number. In healthy dogs, AQP2 and NPT2A were both detected in samples containing at least 1.7 × 109 UEVs or 24 μmol creatinine, however in non-healthy dogs, AQP2 and NPT2A were not detected in most samples containing up to 6.3 × 109 UEVs or 32 μmol creatinine.

Enhancing gene delivery efficiency with amphiphilic chitosan modified by myristic acid and tertiary amino groups

The aim of this study is to synthesize new amphiphilic chitosan containing myristic acid as the hydrophobic tail and tertiary amine groups as the hydrophilic head and to evaluate the gene delivery efficiency. In this context, the primary amine groups of chitosan were first modified with myristic acid (Chi-M), followed by the modification of the methylol groups with 3-dimethylamino-1-propyl chloride hydrochloride. The chemical characterization of this chitosan formulation (Chi-MA) was determined using nuclear magnetic resonance (NMR), Fourier-transform infrared spectroscopy (FTIR) analysis and gel permeation chromatography-size exclusion chromatography. Chi-MA nanoparticles were prepared via ionic gelation, and particle size, polydispersity and zeta potential were determined. The nanoparticles were evaluated for their proton buffering capacity and gene complexing capacity. Additionally, the cytotoxicity of Chi-MA on HEK293T cells was determined via MTT assay, and the transfection efficiency of Chi-MA was analyzed by a flow cytometer. The results indicate a significant increase in gene complexing capacity (8-fold) and nanoparticle formation ability of Chi-MA compared to other chitosan formulations. Chi-MA nanoparticles showed no toxicity against HEK293T cells and exhibited the highest transfection efficiency with significantly lower nanoparticle: gene ratios compared to previous studies. These findings demonstrate the effective use of amphiphilic Chi-MA as a gene carrier.

Evaluating the influence of the initial high molecular weight level on monoclonal antibody particle formation kinetics using a short-term chemical stress study

Protein formulations may form proteinaceous particles that vary in size from nanometers to millimeters. Monitoring the kinetics of protein particle formation, e.g., through accelerated degradation studies, is an attempt to understand and assess the rate and progression of particle populations. Little is known about whether the initial level of high molecular weight (HMW) species, or initial HMW level (IHL), of a protein solution influences the propagation of protein particle formation, and thus affects the storage stability of proteins.In this study, we have established a method to generate protein solutions of different IHLs by thermal stress. We have evaluated a 16-week thermal stability study at 40 °C of two monoclonal antibodies (mAb-A and mAb-B) at different IHLs using size exclusion chromatography (SEC) and sub-visible particle analysis. We have performed an isothermal stress study with guanidinium hydrochloride (GuaHCl) at room temperature for 300-min to evaluate the formation of HMWs analysed by SEC. The application of the Finke-Watzky (F-W) two-step nucleation model allowed us to mathematically describe the kinetics of HMW formation and to extract kinetic parameters of this process.For mAb-A, the IHLs had a marginal influence on the loss of monomer rate; instead, mAb-A exhibited fragmentation at 40 °C, which was independent of the IHL. Nevertheless, above a threshold of ≥ 7 % IHL, existing trimers/tetramers undergo conversion into higher-order oligomers at 40 °C, which is not observed at lower IHLs. In contrast, mAb-B exhibited an increased HMW formation rate above a threshold of ≥ 4 % IHL, which was reflected in the monomer decay rates at 40 °C and the F-W kinetic parameters of the chemical stress study.This case study shows that the initial level of HMWs exerts a differential influence on the progression of HMW formation. In one instance, there is a discernible acceleration in the formation of HMWs with rising IHLs. Conversely, in another example, the IHL exerts only a slight influence on HMW formation. Moreover, the results of our short-term chemical stress study are in accordance with those of a classical storage stability study conducted at 40 °C, which evaluated different IHLs. The analysis of HMW formation kinetics will enhance our understanding of the protein particle formation process and facilitate the formulation development of biotherapeutics.

Comprehensive chromatographic profiling and structural analysis of key anticoagulant components in enoxaparin

Heparin is the most widely used anticoagulant in clinical practice, with enoxaparin being one of the most important low molecular weight heparins (LMWHs). In this study, an antithrombin III (ATIII) affinity column was used. Enoxaparin and its oligosaccharides of varying sizes, prepared using preparative size exclusion chromatography (SEC), were fractionated through the ATIII affinity column. The different affinity fractions from each oligosaccharide size were profiled using strong anion exchange (SAX) chromatography. Each peak was automatically transferred to an SEC column for desalting prior to mass spectrometry (MS) analysis, which enabled structural identification using a multiple heart-cut (MHC) 2D LC-MS system (SAX-SEC-MS). The high-affinity fraction from enoxaparin was further analyzed using the MHC 2D LC system (SEC-SAX). SAX profiles of the high-affinity oligosaccharides, prepared by both size and affinity fractionation, were consistent with those obtained by direct SEC-SAX analysis. The possible sequences of several high-affinity hexasaccharides and the domain compositions of high-affinity octa- and decasaccharides in enoxaparin were further elucidated by disaccharide analysis after manual collection of the oligosaccharides. This work advances the understanding of enoxaparin's structural features and offers a potential approach to improve the quality of enoxaparin, as well as to identify key structural motifs in heparin/LMWHs that contribute to protein binding.

Insight into the Coextrusion Mechanism between Whey Protein Isolate and Cysteine.

The disulfide cross-linking sites of whey protein isolate (WPI) coextruded with dissolved cysteine (Cys) at concentrations of 0, 20, 40, 60, 80, and 100 mM were analyzed by liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) combined with pLink software, and the structure and gel water distribution of WPI during coextrusion (≤50 °C) were also investigated. LC/MS/MS demonstrated that α-La (6) and α-La (120) were the most active sites for intermolecular disulfide cross-linking of α-La. Meanwhile, the molecular weight of protein polymers in coextruded WPI-Cys was the largest at 100 mM Cys, and α-lactalbumin was the main reactant for polymerization from the result of SDS-PAGE and size exclusion chromatography. Additionally, the high concentration of Cys caused the secondary structure of WPI to gradually change from a highly ordered to a disordered structure during coextrusion. In addition, with an increasing concentration of Cys, the free sulfhydryl group of proteins and the binding force to immobilized water gradually increased. Therefore, this work revealed the disulfide cross-linking mechanism between WPI and Cys under low-temperature coextrusion at the molecular level, and the obtained coextruded cross-linked WPI could serve as a novel food ingredient with excellent water-holding capacity for the food industry.

Quantifying the effects of size exclusion on colloidal particle transport in porous media at a pore scale and column scale

Quantifying the effects of size exclusion on colloidal particle transport in porous media at a pore scale and column scale

Characterization of adeno-associated virus capsid proteins using denaturing size-exclusion chromatography coupled with mass spectrometry

Recombinant adeno-associated viruses (AAVs) are a highly effective platform for gene delivery for the treatment of many human diseases. Characterization of AAV viral protein attributes (VP), such as serotype identity, VP stoichiometry, and VP post-translational modifications, is essential to ensure product and process consistency. While size-exclusion chromatography (SEC) coupled with mass spectrometry (MS) is commonly used in the biopharmaceutical industry for analyzing protein therapeutics, its application to intact AAV VP components has not gained traction, presumably due to difficulties in achieving adequate resolution of VP(1−3) monomers. Herein, we describe the development of a denaturing SEC method and optimization of SEC parameters, including stationary phase pore size, column temperature, and mobile phase composition, to achieve effective chromatographic separation of VP(1−3). We demonstrate that an optimized dSEC-MS method featuring MS-compatible formic acid, can effectively separate VP(1−3) across AAV1, 2, 5, 6, 8, and 9 serotypes using a single column and mobile phase condition. A case study was included to showcase successful application of the dSEC-MS method in analyzing changes across different AAV production processes, yielding similar conclusions to an orthogonal approach, such as hydrophilic interaction chromatography (HILIC)- MS. Additionally, dSEC integrated with fluorescence (FLR) and ultraviolet (UV) detection can be used to semi-quantitatively identify both AAV DNA and VP components from empty and full AAV samples. Overall, this robust and MS-friendly methodological advancement could greatly streamline the development and analytical quality control processes for AAV-based gene therapies, providing a highly sensitive method for intact VP characterization.

A plant-based oligomeric CD2v extracellular domain antigen exhibits equivalent immunogenicity to the live attenuated vaccine ASFV-G-∆I177L.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a deadly, highly contagious disease in both domestic pigs and wild boar. With mortality up to 100%, the disease has been making a serious impact on the swine industry worldwide. Because no effective antiviral treatment has been observed, proactive prevention such as vaccination remains the key to controlling the outbreak. In the pursuit of expediting vaccine development, our current work has made the first report for heterologous production of the viral outer envelope glycoprotein CD2v extracellular domain (CD2v ED), a proposed promising vaccine antigen candidate in the "green" synthetic host Nicotiana benthamiana. Protein oligomerization strategies were implemented to increase the immunogenicity of the target antigen. Herein, the protein was expressed in oligomeric forms based on the C-terminally fused GCN4pII trimerization motif and GCN4pII_TP oligomerization motif. Quantitative western blot analysis showed significantly higher expression of trimeric CD2v ED_GCN4pII with a yield of about 12mg/100g of fresh weight, in comparison to oligomeric CD2v ED_GCN4pII_TP, revealing the former is the better choice for further studies. The results of purification and size determination by size exclusion chromatography (SEC) illustrated that CD2v ED_GCN4pII was successfully produced in stable oligomeric forms throughout the extraction, purification, and analysis process. Most importantly, purified CD2v ED_GCN4pII was demonstrated to induce both humoral and cellular immunity responses in mice to extents equivalent to those of the live attenuated vaccine ASFV-G-∆I177L, suggesting it as the potential subunit vaccine candidate for preventing ASFV.

Folds from fold: Exploring topological isoforms of a single-domain protein

Expanding the protein fold space beyond linear chains is of fundamental significance, yet remains largely unexplored. Herein, we report the creation of seven topological isoforms (i.e., linear, cyclic, knot, lasso, pseudorotaxane, and catenane) from a single protein fold precursor by rewiring the connectivity of secondary structure elements of the SpyTag-SpyCatcher complex and mutating the reactive residue on SpyTag to abolish the isopeptide bonding. These topological isoforms can be directly expressed in cells. Their topologies were confirmed by combined techniques of proteolytic digestion, fluorescence correlation spectroscopy (FCS), size-exclusion chromatography (SEC), and topological transformation. To study the effects of topology on their structures and properties, their biophysical properties were characterized by differential scanning calorimetry (DSC), heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy (HSQC-NMR), and circular dichroism (CD) spectroscopy. Molecular dynamics (MD) simulations were further performed to reveal the atomic details of structural changes upon unfolding. Both experimental and simulation results suggest that they share a similar, well-folded hydrophobic core but exhibit distinct folding/unfolding dynamic behaviors. These results shed light onto the folding landscape of topological isoforms derived from the same protein fold. As a model system, this work improves our understanding of protein structure and dynamics beyond linear chains and suggests that protein folds are highly amenable to topological variation.

A Rare Case of Homozygous Hemoglobin G-Philadelphia &amp; Alpha Thalassemia

Abstract Introduction/Objective Hemoglobin (Hb) G-Philadelphia (GPh) is an alpha chain gene variant caused by a missense mutation resulting in a change from Glu to Asp at position 68. This change leads to alterations in the Hb structure and oxygen affinity. The mutation is inherited in an autosomal codominant pattern allowing for an asymptomatic carrier state. While the most common alpha chain variant, GPh heterozygosity is still infrequent, estimated at 1:5000 in African Americans. In African Americans, GPh is frequently linked to an alpha thalassemia deletion. On its own it is considered a benign variant that does not result in significant health issues. Homozygous HbGPh frequency is estimated at 1 in 25 million. Methods/Case Report A postpartum woman in her 4th decade of life was found to have a Hb of 10.8 and a mean corpuscular volume of 74. Initial work up included Hb electrophoresis that quantified HbA at 0%, Hb F at 0%, HbA2 at 0%, and 2 Hb variants representing 96.1% and 3.8% of the total globin. The major Hb variant migrated in the D or G position by alkaline and acid gel Hb electrophoresis. The minor variant was consistent with HbG2. Due to this abnormal Hb electrophoresis, a sample was sent out for a Hb evaluation reflexive cascade (HERC). HERC analysis by high-performance liquid chromatography and capillary gel electrophoresis found 99.9% of Hb consistent with the alpha chain variant HbG. Analysis of the alpha globin gene region revealed 2 copies of an alpha globin gene deletion that was interpreted as alpha-thal trait (-a/-a). This result is consistent with homozygous alpha-thal-2 and homozygous HbGPh (alpha^G/thal + alpha^G/thal). Results (if a Case Study enter NA) NA Conclusion Alpha^G/thal + alpha^G/thal presents a unique and complex challenge for diagnosis due to its rarity and because HbG2 is not always visualized by alkaline gel electrophoresis. The presence of a Hb G or D variant, a HbG2 variant, and the lack of both HbA and HbA2 are key electrophoretic findings. Clinically, alpha^G/thal + alpha^G/thal behaves essentially like a homozygous alpha-thal-2 trait with persistent microcytosis and erythrocytosis. Thus, co- inheritance of the alpha-thal trait will be suggested by a thalassemic complete blood count pattern.

Proteomic and metabolomic profiles of plasma-derived Extracellular Vesicles differentiate melanoma patients from healthy controls

BackgroundPlasma-derived Extracellular Vesicles (EVs) have been suggested as novel biomarkers in melanoma, due to their ability to reflect the cell of origin and ease of collection. This study aimed to identify novel EV biomarkers that can discriminate between disease stages. This was achieved by characterising the plasma-derived EVs of patients with melanoma, and comparing their proteomic and metabolomic profile to those from healthy controls. MethodsEVs were isolated from the plasma of 36 patients with melanoma and 13 healthy controls using Size Exclusion Chromatography. Proteomic and Metabolomic Analyses were performed, and machine learning algorithms were used to identify potential proteins and metabolites to differentiate the plasma-derived EVs from melanoma patients of different disease stages. ResultsThe concentration and size of the EV population isolated was similar between groups. Proteins (APOC4, PRG4, PLG, TNC, VWF and SERPIND1) and metabolites (lyso PC a C18:2, PC ae C44:3) previously associated with melanoma pathogenesis were identified as relevant in differentiating between disease stages. ConclusionThe results further support the continued investigation of circulating plasma-derived EVs as biomarkers in melanoma. Furthermore, the potential of combined proteo-metabolomic signatures for differentiation between disease stages may provide valuable insights into early detection, prognosis, and personalised treatment strategies.

Isolation of small extracellular vesicles from regenerating muscle tissue using tangential flow filtration and size exclusion chromatography

We have recently made the strikingly discovery that upon a muscle injury, Wnt7a is upregulated and secreted from new regenerating myofibers on the surface of exosomes to elicit its myogenerative response distally. Despite recent advances in extracellular vesicle (EVs) isolation from diverse tissues, there is still a lack of specific methodology to purify EVs from muscle tissue. To eliminate contamination with non-EV secreted proteins and cytoplasmic fragments, which are typically found when using classical methodology, such as ultracentrifugation, we adapted a protocol combining Tangential Flow Filtration (TFF) and Size Exclusion Chromatography (SEC). We found that this approach allows simultaneous purification of Wnt7a, bound to EVs (retentate fraction) and free non-EV Wnt7a (permeate fraction). Here we described this optimized protocol designed to specifically isolate EVs from hind limb muscle explants, without cross-contamination with other sources of non-EV bounded proteins. The first step of the protocol is to remove large EVs with sequential centrifugation. Extracellular vesicles are then concentrated and washed in exchange buffer by TFF. Lastly, SEC is performed to remove any soluble protein traces remaining after TFF. Overall, this procedure can be used to isolate EVs from conditioned media or biofluid that contains EVs derived from any cell type or tissue, improving reproducibility, efficiency, and purity of EVs preparations. Our purification protocol results in high purity EVs that maintain structural integrity and thus fully compatible with in vitro and in vivo bioactivity and analytic assays.

Structure and Antigenicity of the Porcine Astrovirus 4 Capsid Spike

Porcine astrovirus 4 (PoAstV4) has been recently associated with respiratory disease in pigs. In order to understand the scope of PoAstV4 infections and to support the development of a vaccine to combat PoAstV4 disease in pigs, we designed and produced a recombinant PoAstV4 capsid spike protein for use as an antigen in serological assays and for potential future use as a vaccine antigen. Structural prediction of the full-length PoAstV4 capsid protein guided the design of the recombinant PoAstV4 capsid spike domain expression plasmid. The recombinant PoAstV4 capsid spike was expressed in Escherichia coli, purified by affinity and size-exclusion chromatography, and its crystal structure was determined at 1.85 Å resolution, enabling structural comparisons to other animal and human astrovirus capsid spike structures. The recombinant PoAstV4 capsid spike protein was also used as an antigen for the successful development of a serological assay to detect PoAstV4 antibodies, demonstrating that the recombinant PoAstV4 capsid spike retains antigenic epitopes found on the native PoAstV4 capsid. These studies lay a foundation for seroprevalence studies and the development of a PoAstV4 vaccine for swine.

Otimização do conjugado Anti-IGG canino no kit diagnóstico da Leishmaniose Visceral Canina (LVC)

Introduction: The diagnosis of canine visceral leishmaniasis (CVL) is of great importance in the control of visceral leishmaniasis, a disease that is neglected worldwide and represents a significant public health problem. Enzyme-linked immunosorbent assay (ELISA) test is widely used because of the possibility of large-scale diagnosis and low cost. Objective: The objective of this study was to optimize the stoichiometry of the conjugate used in the EIE/CVL kit; for this purpose, some conjugation and monitoring parameters were modified through molecular exclusion chromatography to improve the final product. Method: The conjugate produced by Bio-Manguinhos was optimized by purification using SEC gel filtration chromatography and evaluated using the EIE-leishmaniasis-visceral-canine-Bio-Manguinhos kit (EIE-LVC). The conjugates were produced with different stoichiometries (IgG X peroxidase): current conjugate, optimized conjugate and optimized and purified conjugate. Results: The results obtained were validated using the manual Canine Visceral Leishmaniasis test. The homogeneity of the conjugate means in the ELISA test was evaluated using the chi-square test, thereby demonstrating the normality and reliability of the data. Based on statistical data, positive samples obtained by the ELISA test showed homogeneity at a significance level of 0.05, i.e. a data reliability level of 95%. The optimization of the production of the canine anti-IgG conjugate, an extremely important component used in the composition of the Bio-Manguinhos EIE LVC KIT, contributed to ensuring the quality of the kit and improving the diagnosis of canine visceral leishmaniasis disease, making it more sensitive and inexpensive for public health. Conclusions: This work demonstrates that it is possible to optimize the anti-IgG canine peroxidase (HRP) conjugate used in the EIE CVL kit to make it more specific and ultimately more economical.

Novel Antioxidant Peptides from Fermented Whey Protein by Lactobacillus rhamnosus B2-1: Separation and Identification by in Vitro and in Silico Approaches.

Whey is a byproduct of the dairy industry and is rich in protein. To enhance the significance of such byproducts and find efficacious antioxidants for combating oxidative stress, this study reported on the preparation, purification, and identification of novel peptides with antioxidant activities from whey protein metabolites following fermentation by Lactobacillus rhamnosus B2-1. The isolation and identification processes involved macroporous adsorption resin column chromatography, gel filtration column chromatography, and liquid chromatography-tandem mass spectrometry. Therein, three novel antioxidant peptides (PKYPVEPF, LEASPEVI, and YPFPGPIHNS) were selected to be synthesized, and they demonstrated remarkable antioxidant activities in vitro chemical assays. PKYPVEPF, LEASPEVI, and YPFPGPIHNS (100 μg/mL) displayed a notable cytoprotective impact on HepG2 cells under oxidative stress induced by H2O2, increasing the cell viability from 49.02 ± 3.05% to 88.59 ± 10.49%, 82.38 ± 19.16%, and 85.15 ± 7.19%, respectively. Moreover, the peptides boosted the activities of catalase and superoxide dismutase in damaged cells and reduced reactive oxygen species levels. The molecular docking studies highlighted that these antioxidant peptides efficiently bound to key amino acids in the Kelch domain of Keap1, thereby preventing the interaction between Keap1 and Nrf2. In conclusion, PKYPVEPF, LEASPEVI, and YPFPGPIHNS demonstrated substantial antioxidant activity, suggesting their potential for widespread application as functional food additives and ingredients.

Bacterial Degradation of Low-Density Polyethylene Preferentially Targets the Amorphous Regions of the Polymer.

Low-density polyethylene (LDPE) is among the most abundant synthetic plastics in the world, contributing significantly to the plastic waste accumulation problem. A variety of microorganisms, such as Cupriavidus necator H16, Pseudomonas putida LS46, and Pseudomonas chlororaphis PA2361, can form biofilms on the surface of LDPE polymers and cause damage to the exterior structure. However, the damage is not extensive and complete degradation has not been achieved. The changes in polymer structure were analyzed using Time-domain Nuclear Magnetic Resonance (TD-NMR), High-Temperature Size-Exclusion Chromatography (HT-SEC), Differential Scanning Calorimetry (DSC), and Gas Chromatography with a Flame Ionization Detector (GC-FID). Limited degradation of the LDPE powder was seen in the first 30 days of incubation with the bacteria. Degradation can be seen in the LDPE weight loss percentage, LDPE degradation products in the supernatant, and the decrease in the percentage of amorphous regions (from >47% to 40%). The changes in weight-average molar mass (Mw), number-average molar mass (Mn), and the dispersity ratio (Đ) indicate that the low-molar mass fractions of the LDPE were preferentially degraded. The results here confirmed that LDPE degradation is heavily dependent on the presence of amorphous content and that only the amorphous content was degraded via bacterial enzymatic action.

Improving poly(lactic acid) fire performances via blending with benzoxazine

Improving the mechanical performance of PLA via reactive blending with thermosets is an interesting approach, however, not well explored for its fire performance improvement. With our approach, the fire performances of PLA were improved by blending PLA with a commercially available bisphenol-F-based benzoxazine. To establish the proof of concept, the benzoxazine was initially pre-cured at either 100 °C or 150 °C for a pre-selected time i.e. 60′, 80′, or 120′. The benzoxazine samples were then blended with PLA matrix via an extrusion process at 10 wt.% or 20 wt.% loadings. Detailed thermal and chemical investigations via Differential Scanning Calorimetry (DSC) and Size Exclusion Chromatography (SEC), and the evaluation of the mechanical properties, confirmed that the addition of benzoxazine does not influence the intrinsic properties of PLA. The fire performance was tested by Mass Loss Cone (MLC) calorimetry. PLA formulation with 20 wt.% of the benzoxazine cured 80′ at 150 °C, lead to a 43 % reduction of the peak of heat release rate compared to neat PLA. This was attributed to increased char formation during the combustion process. Also, the char formation permits a significant delay in the temperature increase of the sample.