Gel Filtration Research Articles

Separation and purification of antimicrobial substances from Paenibacillus polymyxa KH-19 and analysis of its physicochemical characterization.

Soft rot is one of the top ten most dangerous plant pathogens in agricultural production, storage, and transport, and the use of microorganisms and their metabolites to control soft rot is a current research hotspot. In this study, we identified the antimicrobial substance in the metabolite of Paenibacillus polymyxa KH-19, and determined that the antimicrobial substance of this strain was an active protein. The protein was completely precipitated at 40-60% ammonium sulphate saturation and showed good inhibitory effects against seven pathogenic bacteria including Pectobacterium carotovorum BC2 and seven pathogenic fungi including Pyricularia oryzae. The MIC of the protein was 51.563µg/mL, temperature acid-base UV and light stability insensitive to protease, with high-temperature resistance. The antimicrobial protein was isolated and purified by DEAE-anion exchange column and Sephadex G-75 gel filtration chromatography, and the LC-MS/MS assay identified the protein as lysophosphatidyl esterase with a molecular weight of 25.255kDa. The purified antimicrobial protein increased the inhibitory effect against P. carotovorum BC2, with the diameter of the circle of inhibition being 26.50 ± 0.915mm. Bioinformatics analysis showed that the protein has the molecular formula of C1117H1732N316O338S5, encodes 224 amino acids, has an aliphatic index of 88.39, and belongs to the category of hydrophilic unstable proteins. The present study is the first report of an active protein with extreme thermoplastic and resistance to P. carotovorum BC2, which provides a reference for the preparation and application of the antimicrobial substances of P. polymyxa KH-19, as well as a theoretical basis for the study of the function of lysophosphodiesterase protein and its use as a microbial preparation.

Unbiased complexome profiling and global proteomics analysis reveals mitochondrial impairment and potential changes at the intercalated disk in presymptomatic R14Δ/+ mice hearts.

Phospholamban (PLN) is a sarco-endoplasmic reticulum (SER) membrane protein that regulates cardiac contraction/relaxation by reversibly inhibiting the SERCA2a Ca2+-reuptake pump. The R14Δ-PLN mutation causes severe cardiomyopathy that is resistant to conventional treatment. Protein complexes and higher-order supercomplexes such as intercalated disk components and Ca+2-cycling domains underlie many critical cardiac functions, a subset of which may be disrupted by R14Δ-PLN. Complexome profiling (CP) is a proteomics workflow for systematic analysis of high molecular weight (MW) protein complexes and supercomplexes. We hypothesize that R14Δ-PLN may alter a subset of these assemblies, and apply CP workflows to explore these changes in presymptomatic R14Δ/+ mice hearts. Ventricular tissues from presymptomatic 28wk-old WT and R14Δ/+ mice were homogenized under non-denaturing conditions, fractionated by size-exclusion chromatography (SEC) with a linear MW-range exceeding 5 MDa, and subjected to quantitative data-independent acquisition mass spectrometry (DIA-MS) analysis. Unfortunately, current workflows for the systematic analysis of CP data proved ill-suited for use in cardiac samples. Most rely upon curated protein complex databases to provide ground-truth for analysis; however, these are derived primarily from cancerous or immortalized cell lines and, consequently, cell-type specific complexes (including cardiac-specific machinery potentially affected in R14Δ-PLN hearts) are poorly covered. We thus developed PERCOM: a novel CP data-analysis strategy that does not rely upon these databases and can, furthermore, be implemented on widely available spreadsheet software. Applying PERCOM to our CP dataset resulted in the identification of 296 proteins with disrupted elution profiles. Hits were significantly enriched for mitochondrial and intercalated disk (ICD) supercomplex components. Changes to mitochondrial supercomplexes were associated with reduced expression of mitochondrial proteins and maximal oxygen consumption rate. The observed alterations to mitochondrial and ICD supercomplexes were replicated in a second cohort of "juvenile" 9wk-old mice. These early-stage changes to key cardiac machinery may contribute to R14Δ-PLN pathogenesis.

Purification and characterization of α-galactosidases from Penicillium griseoroseum for efficient soymilk hydrolysis

Soybean utilization is limited by the presence of raffinose oligosaccharides (RFO), which are not digested by humans and cause gastrointestinal discomfort. This study explores the potential of α-galactosidases from Penicillium griseoroseum for RFO hydrolysis in soymilk. Two distinct α-galactosidase enzymes, designated α-Gal1 and α-Gal2, were purified using a combination of ion-exchange chromatography and native polyacrylamide gel electrophoresis. Both enzymes exhibited characteristics of multimeric proteins and displayed similar biochemical properties. Optimal activity was observed at a pH range of 4.5–5.0 and a temperature range of 40–45 °C. Notably, α-Gal1 demonstrated high thermostability with a half-life of 16 h at 40 °C. The α-galactosidases displayed different substrate affinitiesfor the substrates ρ-NP-αGal, o-NP-αGal, rD-raffinose, d-stachyose, and mD-melibiose. The Michaelis-Menten constant (Km) values for α-Gal1 were 1.06, 1.31, 28.74, 19.88, and 4.77 mmol/L, respectively, while those for α-Gal2 were 0.8, 1.26, 30.46, 21.74 and 5.01 mmol/L, respectively. Both α-Gal1 and α-Gal2 were strongly inhibited by metal ions (Ag⁺, Cu2⁺, Fe2⁺, and Hg2⁺) and moderately inhibited by d-melibiose. Importantly, both enzymes efficiently hydrolyzed RFOs, achieving complete d-stachyose elimination from soymilk after a 6-h incubation. These findings propose the promising application of these α-galactosidases in industrial soymilk production, potentially enhancing its nutritional value and alleviating gastrointestinal issues in consumers.

Facile Synthesis of Valsartan and Ribose Prodrug and Studying its Influence on the Pancreatic Lipase

Valsartan is a widely used drug that belongs to the type of angiotensin receptor blockers generally specified to control high blood pressure (hypertension) and heart failure. In the present work, a new ester prodrug utilizes ribose sugar as a carrier for the valsartan through an esterification reaction. The synthesized compound was characterized by the FT-IR and 1H NMR spectroscopic. The investigation furthermore involved measuring the activity of the lipase enzyme in the blood serum of individuals with high blood pressure and healthy individuals and purifying the lipase enzyme utilizing gel filtration technology. Besides, the investigation studied the kinetics of the lipase enzyme, the impact of various substrate concentrations, the impact of pH and optimal temperature on the enzyme's activity, and the impact of reaction time on the enzyme's effectiveness. It was found that the effectiveness of the lipase enzyme reduced with raising the concentration of the synthesized compound, and the sort of inhibition was determined as competitive.

Identification of Structure-Linked Activity on Bioactive Peptides from Sea Cucumber (Stichopus japonicus): A Compressive In Silico/In Vitro Study.

A sea cucumber (Stichopus japonicus) is an invertebrate rich in high-quality protein peptides that inhabits the coastal seas around East Asian countries. Such bioactive peptides can be utilized in targeted disease therapies and practical applications in the nutraceutical industry. Bioactive peptides were isolated from Stichopus japonicus through ultrafiltration and Sephadex G-10 size exclusion chromatography. The low-molecular-weight fraction (ACSH-III) showed the highest hydroxyl radical scavenging and angiotensin-converting enzyme (ACE) inhibitory activities. Subsequent purification of ACSH-III resulted in four fractions, of which ACSH-III-F3 and ACSH-III-F4 exhibited significant bioactivity. Peptides identified in these fractions, including Phenylalanine-Proline-Threonine-Tyrosine (FPTY) and Tyrosine-Proline-Serine-Tyrosine-Proline-Serine (YPSYPS), were characterized using high-performance liquid chromatography (HPLC) and quadrupole time-of-flight mass spectrometry (QTOF-MS). FPTY demonstrated the most potent antioxidant and antihypertensive activities among these peptides, with IC50 values of 0.11 ± 0.01 mg/mL for hydroxyl radicals and 0.03 ± 0.01 mg/mL for ACE inhibition. Docking simulations revealed strong binding affinities of these peptides to the active site of the ACE, with FPTY displaying interactions similar to those of the synthetic inhibitor lisinopril. These findings suggest that the identified peptides, particularly FPTY, have potential applications as natural antioxidants and functional foods.

Preservative Properties of Antimicrobial Peptides of Germinated Seeds of Pisum sativum (Garden Pea) On Roselle Flowers (Hibiscus sabdarifa L.) Drink

Study’s Excerpt This research presents the use of antimicrobial peptides derived from Pisum sativum (pea) seeds as natural preservatives for extending the shelf life of local African beverages. It demonstrated that peptides, particularly from germinated peas on day four, exhibits strong antimicrobial activity against bacteria. Plant-derived peptides could replace synthetic preservatives in the food and beverage industry. Full Abstract The demand for natural and effective food preservatives has increased due to the growing concern over the safety and quality of processed beverages. Antimicrobial peptides have gained significant attention as natural alternatives to synthetic preservatives, offering potential benefits such as broad-spectrum activity and lower toxicity. Germinating seeds of Pisum sativum were harvested on days 2, 4, 6 and 8 and peptides were extracted and purified using Ammonium Sulphate Precipitation at 70% saturation, Dialysis, Gel filtration and the peaks were obtained. The molecular weight of peptides was determined using SDS-PAGE while Six (6) Bacteria and Four (4) Fungal species were isolated and characterized using standard procedures. The highest protein concentration was obtained with germinated peas day four (P4) with a value of 1.7866mg/mL, while the highest zone diameter of inhibition was recorded with peptide of germinated pea seeds day six fraction three (P6:3) with a value of 17.000±0.577mm against B. cereus. The least MIC value, expressed in protein concentration (mg/mL), was obtained as 0.0253mg/mL with 100% concentration of the peptide of germinated peas on day four (P4). All four (4) fungal species recorded no activity/zone diameter of inhibition. Sanitizing activity of the peptides on the ‘Zobo’ drink adjusted to pH 2.3, at 0hr, 24hr, 48hr and 72hrs was carried out, and all the purified peptides extended the shelf life of the drink except for peptide of germinated peas on day two. Sensory evaluation equally indicated that the drink was preserved with 10% (P4:4) at pH 2.3, being the most preferred treatment by the judges with a percentage likeness of 72.22% after 72hrs of storage. Findings from this study highlight the potential of these peptides in developing innovative and safe preservation techniques for extending the shelf life of local African beverages. This study further offers promising insights into the application of Pisum sativum peptides in preserving traditional drinks like 'Zobo.'

Does Fungal Chitosan Leave Noticeable Traces in Treated Wines?

Background (1): The use of fungal chitosan as an antiseptic in wine appears as a promising alternative to sulfur dioxide for the elimination of Brettanomyces bruxellensis sensitive strains. Nevertheless, its utilization raises the question, "how are the treated wines different from the untreated ones?" Methods (2): Chitosan treatment residues were sought in the oligosaccharide and polysaccharide fractions and among 224 low MW ions (<1800 g·mol-1) in several wines by using liquid chromatography (size exclusion HPLC or LC-MS) and GC-MS. Standard oenological parameters were also examined as well as possible sensory modifications by a panel of tasters composed of experts and non-experts. Results (3): None of these methods enabled the reproducible and reliable identification of a treated wine without comparing it to its untreated control. The fingerprints of treatment are not reliably detectable by the analytical methods used in this study. However, the treated wines seem permanently protected against the development of chitosan-sensitive strains of B. bruxellensis. Conclusions (4): If chitosan treatment modifies the wine, the associated changes were not identified by the liquid chromatography method mentioned above and they were not perceived by most people in our taster panel. However, the expected antimicrobial action of chitosan was observed on B. bruxellensis sensitive strains and persisted at least one year. Tolerant strains were less affected by these persistent effects.

Comparative Efficiency of Polyethylene Glycol, Ammonium Sulphate, Methanol Precipitation, and Ultrafiltration Techniques for the Down Streaming of Viral Antigen

Background: Foot and mouth disease (FMD) is ubiquitous worldwide but endemic in many countries of Africa, Asia, South America, and the Middle East. Many reasons contribute to the incidence of viral diseases even in vaccinated animals. These reasons include low antigenic payload, low PD50, improper formulation, unstable vaccine containing antigen, and genetically different from field strain. Among these, the most important one is the low antigenic load per dose of the vaccine. Vaccine failure is mainly due to the direct use of virus suspension in the vaccine without the concentration of viral antigen. Another reason to concentrate the antigen is small volume storage in the vaccine bank. These issues are mostly concerned with developing countries like Pakistan which lack antigen concentration technology. The concentration of the virus is a major milestone to be achieved for the production of an effective vaccine as well as for the diagnostic tool.Methods: Different techniques including precipitation with polyethylene glycol, ammonium sulfate, methanol, and filtration through an ultra-filter membrane were used for the concentration of viral suspension. Antigen quantification in terms of µg/ml was determined through size exclusion chromatography by using Sephacryl S-300 as a stationary phase.Results: Percentage recovery of FMDV calculated through analysis of chromatograms found 77.80%, 59.75%, 32.50%, and 13.83% for polyethylene glycol, ammonium sulfate, ultra-filtration, and methanol treated samples respectively.Conclusion: Classical polyethylene glycol precipitation showed a maximum percentage recovery of foot and mouth disease virus as compared to other concentration methods.Keywords: Foot and mouth disease virus (FMDV); Concentration Methods; Polyethylene Glycol (PEG); Size Exclusion Chromatography (SEC); Sephacryl S-300

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.

Double 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). Our 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. Using 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. DOC2B 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. Clonal 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.

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

AbstractColloid‐facilitated transport has been widely acknowledged as a significant contributor to surface water and groundwater pollution. Compared to water‐dissolved solutes, colloids are subject to a size exclusion effect (SEE) due to their inherent size when transported through porous media. However, only a few studies have quantitatively assessed SEE on colloid transport in porous media. In this study, we conducted dynamic observation experiments (in a quartz sand micro‐model) using a high‐speed camera and directly observed the size exclusion phenomenon of colloid transport in pores. Moreover, we further performed column experiments to quantitatively characterize the breakthrough curves of silica colloids with diameters ranging from 0.1 to 5 µm through different‐size sands. Silica colloids displayed a distinct SEE, as demonstrated by their earlier breakthroughs compared to the NaNO3 tracer. This observation served as a quantitative indicator of the SEE. The breakthrough curves were fitted using the advection‐dispersion equation to quantify this phenomenon, allowing for calculating the water saturation that is inaccessible to mobile colloids (γ). It was determined that γ exhibited a significant negative linear correlation (p &lt; 0.01) with the average grain size (d50) of porous media. Additionally, power–law correlations were identified between γ and the colloid diameter (dp) and dp/d50. When the grain size of porous media remained below 1031 µm, an increase in the colloid diameter (from 0.1 to 5 µm) resulted in a more noticeable SEE, leading to the proportion of inaccessible pore regions to all pores increasing from 0.017 to 0.098, with this change following a power–law trend. These findings provide valuable insights into quantifying the SEE on silica colloids during transport in porous media. Furthermore, they provide theoretical support for future quantitative research on the mechanisms underlying colloid‐facilitated transport.

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.

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.

Eco-friendly production and probiotic purification of agarose degradation products: Oligosaccharides and 3,6-anhydro-L-galactose

Algal biomass offers a solution to global resource scarcity, with agarose, a key component of Gelidium amansii, containing valuable products like oligosaccharides and 3,6-anhydro-L-galactose. However, current purification methods limit their commercial viability. In this study, we utilized gel filtration chromatography to purify agaro-oligosaccharides and neoagaro-oligosaccharides with varying degrees of polymerization, achieving a novel purification of odd-numbered neoagaro-oligosaccharides. Additionally, by fermenting a mixture of 3,6-anhydro L-galactose and D-galactose with six probiotics, our results demonstrate that five probiotics—Lactobacillus plantarum, Bifidobacterium adolescentis, Streptococcus thermophilus, Lactobacillus acidophilus, and Lactobacillus rhamnosus effectively utilize D-galactose in mixed carbon sources while retaining 3,6-anhydro L-galactose. This approach enables efficient, low-cost, and eco-friendly purification of 3,6-anhydro L-galactose, opening avenues for its widespread utilization.