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1.
Environ Geochem Health ; 46(8): 289, 2024 Jul 06.
Article in English | MEDLINE | ID: mdl-38970698

ABSTRACT

Low molecular weight organic acids (LMWOAs) are important soil components and play a key role in regulating the geochemical behavior of heavy metal(loid)s. Biochar (BC) is a commonly used amendment that could change LMWOAs in soil. Here, four LMWOAs of oxalic acid (OA), tartaric acid (TA), malic acid (MA), and citric acid (CA) were evaluated for their roles in changing Cd and SB desorption behavior in contaminated soil with (S1-BC) or without BC (S1) produced from Paulownia biowaste. The results showed that OA, TA, MA, and CA reduced soil pH with rising concentrations, and biochar partially offset the pH reduction by LMWOAs. The LMWOAs reduced Cd desorption from the soil at low concentrations but increased Cd desorption at high concentrations, and CA was the most powerful in this regard. The LMWOAs had a similar effect on Sb desorption, and CA was the most effective species of LMWOAs. Adding BC to the soil affects Cd and Sb dynamics by reducing the Cd desorption but increasing Sb desorption from the soil and increasing the distribution coefficient (Kd) values of Cd but lowering the Kd values of Sb. This study helped understand the effects of LMWOAs on the geochemical behavior of Cd and Sb in the presence of biochar, as well as the potential risks of biochar amendment in enhancing Sb desorption from contaminated soil.


Subject(s)
Charcoal , Metals, Heavy , Soil Pollutants , Soil , Charcoal/chemistry , Soil Pollutants/chemistry , Metals, Heavy/chemistry , Soil/chemistry , Molecular Weight , Hydrogen-Ion Concentration , Cadmium/chemistry , Tartrates/chemistry , Malates/chemistry , Citric Acid/chemistry , Environmental Restoration and Remediation/methods , Oxalic Acid/chemistry , Adsorption , Oryza/chemistry
2.
Pharmeur Bio Sci Notes ; 2024: 90-105, 2024.
Article in English | MEDLINE | ID: mdl-38967620

ABSTRACT

Human immunoglobulin products are used for the treatment of a number of diseases, such as primary or secondary immunodeficiencies and autoimmune conditions due to the complete absence of antibodies or the production of defective immunoglobulins. Quality control of human immunoglobulin products is essential to ensure therapeutic functionality and safety. This includes testing for Fc function and anticomplementary activity (ACA), as well as verification of appropriate molecular size distribution using size-exclusion chromatography as prescribed in the European Pharmacopoeia (Ph. Eur.) monographs 0338, 0918, 2788 and 1928. To this end, specific biological reference preparations (BRPs) must be used. Stocks of the Ph. Eur. Human immunoglobulin (molecular size) BRP were running low and therefore a collaborative study was run by the European Directorate for the Quality of Medicines & HealthCare (EDQM), under the aegis of the Biological Standardisation Programme, to calibrate replacement batches. Eighteen laboratories, including manufacturers and Official Medicines Control Laboratories, took part in the study. Three batches of candidate BRPs were assessed and compared to Ph. Eur. Human immunoglobulin (molecular size) BRP 3 to ensure continuity. Based on the study results, the candidate BRPs were adopted by the Ph. Eur. Commission as Ph. Eur. Human immunoglobulin (molecular size) BRP batch 4, 5 and 6.


Subject(s)
Immunoglobulins , Quality Control , Humans , Immunoglobulins/analysis , Reference Standards , Chromatography, Gel/standards , Molecular Weight , Europe
3.
Drug Deliv ; 31(1): 2372285, 2024 Dec.
Article in English | MEDLINE | ID: mdl-38952133

ABSTRACT

In this study, chitosan low molecular weight (LCH) and chitosan medium molecular weight (MCH) were employed to encapsulate a yarrow extract rich in chlorogenic acid and dicaffeoylquinic acids (DCQAs) that showed antiproliferative activity against colon adenocarcinoma cells. The design of CH micro/nanoparticles to increase the extract colon delivery was carried out by using two different techniques: ionic gelation and spray drying. Ionic gelation nanoparticles obtained were smaller and presented higher yields values than spray-drying microparticles, but spray-drying microparticles showed the best performance in terms of encapsulation efficiency (EE) (> 94%), also allowing the inclusion of a higher quantity of extract. Spray-drying microparticles designed using LCH with an LCH:extract ratio of 6:1 (1.25 mg/mL) showed a mean diameter of 1.31 ± 0.21 µm and EE values > 93%, for all phenolic compounds studied. The release profile of phenolic compounds included in this formulation, at gastrointestinal pHs (2 and 7.4), showed for most of them a small initial release, followed by an increase at 1 h, with a constant release up to 3 h. Chlorogenic acid presented the higher release values at 3 h (56.91% at pH 2; 44.45% at pH 7.4). DCQAs release at 3 h ranged between 9.01- 40.73%, being higher for 1,5- and 3,4-DCQAs. After gastrointestinal digestion, 67.65% of chlorogenic and most DCQAs remained encapsulated. Therefore, spray-drying microparticles can be proposed as a promising vehicle to increase the colon delivery of yarrow phenolics compounds (mainly chlorogenic acid and DCQAs) previously described as potential agents against colorectal cancer.


Subject(s)
Achillea , Cell Proliferation , Chitosan , Chlorogenic Acid , Colorectal Neoplasms , Nanoparticles , Particle Size , Plant Extracts , Chitosan/chemistry , Humans , Plant Extracts/pharmacology , Plant Extracts/administration & dosage , Plant Extracts/chemistry , Achillea/chemistry , Chlorogenic Acid/pharmacology , Chlorogenic Acid/administration & dosage , Chlorogenic Acid/chemistry , Nanoparticles/chemistry , Cell Proliferation/drug effects , Colorectal Neoplasms/drug therapy , Cell Line, Tumor , Quinic Acid/analogs & derivatives , Quinic Acid/pharmacology , Quinic Acid/chemistry , Quinic Acid/administration & dosage , Drug Liberation , Drug Delivery Systems/methods , Antineoplastic Agents, Phytogenic/pharmacology , Antineoplastic Agents, Phytogenic/administration & dosage , Antineoplastic Agents, Phytogenic/chemistry , Colon/drug effects , Colon/metabolism , Drug Carriers/chemistry , Molecular Weight
4.
Int J Mol Sci ; 25(13)2024 Jul 05.
Article in English | MEDLINE | ID: mdl-39000481

ABSTRACT

Pollen, in addition to allergens, comprise low molecular weight components (LMC) smaller than 3 kDa. Emerging evidence indicates the relevance of LMC in allergic immune responses. However, the interaction of birch pollen (BP)-derived LMC and epithelial cells has not been extensively studied. We investigated epithelial barrier modifications induced by exposure to BP LMC, using the human bronchial epithelial cell line 16HBE14o-. Epithelial cell monolayers were apically exposed to the major BP allergen Bet v 1, aqueous BP extract or BP-derived LMC. Barrier integrity after the treatments was monitored by measuring transepithelial electrical resistance at regular intervals and by using the xCELLigence Real-Time Cell Analysis system. The polarized release of cytokines 24 h following treatment was measured using a multiplex immunoassay. Epithelial barrier integrity was significantly enhanced upon exposure to BP LMC. Moreover, BP LMC induced the repair of papain-mediated epithelial barrier damage. The apical release of CCL5 and TNF-α was significantly reduced after exposure to BP LMC, while the basolateral release of IL-6 significantly increased. In conclusion, the results of our study demonstrate that BP-derived LMC modify the physical and immunological properties of bronchial epithelial cells and thus regulate airway epithelial barrier responses.


Subject(s)
Betula , Bronchi , Epithelial Cells , Molecular Weight , Pollen , Humans , Bronchi/metabolism , Bronchi/cytology , Bronchi/drug effects , Epithelial Cells/metabolism , Epithelial Cells/drug effects , Cell Line , Allergens , Cytokines/metabolism , Respiratory Mucosa/metabolism , Respiratory Mucosa/drug effects
5.
J Agric Food Chem ; 72(29): 16334-16346, 2024 Jul 24.
Article in English | MEDLINE | ID: mdl-38994810

ABSTRACT

Boletus aereus Fr. ex Bull. stands out as a delectable edible mushroom with high nutritional and medicinal values, featuring polysaccharides as its primary nutrient composition. In our continuous exploration of its beneficial substances, a novel polysaccharide (BAPN-1) with a molecular weight of 2279 kDa was prepared. It was identified as a glucan with a backbone composed of the residues →4)-α-Glcp-(1→ and →4,6)-α-Glcp-(1→ connected in a proportion of 5:1 and a ß-Glcp-(1→ side residue attached at C6 of the →4,6)-α-Glcp-(1→ residue. Biologically, BAPN-1 exhibited broad-spectrum antiproliferative activities against various NHL cells, including HuT-78, OCI-LY1, OCI-LY18, Jurkat, RL, and Karpas-299, with IC50 values of 0.73, 1.21, 3.18, 1.52, 3.34, and 4.25 mg/mL, respectively. Additionally, BAPN-1 significantly induced cell cycle arrest in the G2/M phase and caused apoptosis of NHL cells. Mechanistically, bulk RNA sequencing and Western blot analysis revealed that BAPN-1 could upregulate cyclin B1 and enhance cleaved caspase-9 expression through the inhibition of FGFR3 and RAF-MEK-ERK signaling pathways. This work supports the improved utilization of B. aereus in high-value health products.


Subject(s)
Antineoplastic Agents , Apoptosis , Cell Proliferation , Lymphoma, Non-Hodgkin , Polysaccharides , Humans , Cell Proliferation/drug effects , Cell Line, Tumor , Apoptosis/drug effects , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Lymphoma, Non-Hodgkin/drug therapy , Polysaccharides/chemistry , Polysaccharides/pharmacology , Polysaccharides/isolation & purification , Molecular Weight , Basidiomycota/chemistry
6.
BMC Microbiol ; 24(1): 259, 2024 Jul 13.
Article in English | MEDLINE | ID: mdl-38997685

ABSTRACT

BACKGROUND: Bacterial genomes often encode structures similar to phage capsids (encapsulins) and phage tails which can be induced spontaneously or using genotoxic compounds such as mitomycin C. These high molecular-weight (HMW) putative antibacterial proteins (ABPs) are used against the competitive strains under natural environment. Previously, it was unknown whether these HMW putative ABPs originating from the insect pathogenic Gram-positive, spore-forming bacterium Brevibacillus laterosporus (Bl) isolates (1821L, 1951) are spontaneously induced during the growth and pose a detrimental effect on their own survival. Furthermore, no prior work has been undertaken to determine their biochemical characteristics. RESULTS: Using a soft agar overlay method with polyethylene glycol precipitation, a narrow spectrum of bioactivity was found from the precipitated lysate of Bl 1951. Electron micrographs of mitomycin C- induced filtrates showed structures similar to phage capsids and contractile tails. Bioactivity assays of cell free supernatants (CFS) extracted during the growth of Bl 1821L and Bl 1951 suggested spontaneous induction of these HMW putative ABPs with an autocidal activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of spontaneously induced putative ABPs showed appearance of ~ 30 kDa and ~ 48 kDa bands of varying intensity across all the time intervals during the bacterial growth except in the initial hours. Statistically, spontaneously induced HMW putative ABPs of Bl 1951 exhibited a significant decrease in the number of viable cells of its producer strain after 18 h of growth in liquid. In addition, a significant change in pH and prominent bioactivity of the CFS of this particular time period was noted. Biochemically, the filtered supernatant derived from either Bl 1821L or Bl 1951 maintained bioactivity over a wide range of pH and temperature. CONCLUSION: This study reports the spontaneous induction of HMW putative ABPs (bacteriocins) of Bl 1821L and Bl 1951 isolates during the course of growth with potential autocidal activity which is critically important during production as a potential biopesticide. A narrow spectrum of putative antibacterial activity of Bl 1951 precipitate was found. The stability of HMW putative ABPs of Bl 1821L and Bl 1951 over a wide range of pH and temperature can be useful in expanding the potential of this useful bacterium beyond the insecticidal value.


Subject(s)
Anti-Bacterial Agents , Bacterial Proteins , Brevibacillus , Molecular Weight , Brevibacillus/metabolism , Brevibacillus/genetics , Brevibacillus/isolation & purification , Animals , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Mitomycin/pharmacology , Kinetics , Insecta/microbiology , Hydrogen-Ion Concentration , Electrophoresis, Polyacrylamide Gel
7.
Molecules ; 29(13)2024 Jul 01.
Article in English | MEDLINE | ID: mdl-38999090

ABSTRACT

Phaeodactylum tricornutum is identified by its capacity for rapid growth, reproduction, and in vitro cultivation, as well as the presence of a range of high-value active compounds, including proteins, with potential food applications. The objective of this study was to investigate the effects of pH shift treatments (pH of 3, 5, 7, 9, and 11) on the structural and functional properties of the Phaeodactylum tricornutum protein (PTP). The molecular weight of the PTP was predominantly distributed within the following ranges: below 5 kDa, 5-100 kDa, and above 100 kDa. Compared to the acidic environment, the PTP demonstrated higher solubility and greater free sulfhydryl group content in the alkaline environment. Additionally, PTP had a smaller particle size and higher thermal stability in alkaline environments. The PTP exhibited superior foaming ability (135%), emulsification activity index (3.72 m2/g), and emulsion stability index (137.71 min) in alkaline environments. The results of this investigation provide a foundation for the future development and application of the PTP in the food industry.


Subject(s)
Diatoms , Hydrogen-Ion Concentration , Diatoms/chemistry , Solubility , Molecular Weight , Particle Size
8.
Transl Psychiatry ; 14(1): 300, 2024 Jul 20.
Article in English | MEDLINE | ID: mdl-39033141

ABSTRACT

The Poly (I:C) (polyriboinosinic-polyribocytidilic acid) paradigm of maternal immune activation (MIA) is most widely used as experimental model for the evaluation of the effects of gestational infection on the brain and behavior of the progeny. We have previously reported significant batch-to-batch variability in the effects of Poly (I:C), purchased from the same supplier (Sigma-Aldrich), on maternal and fetal immune responses and found these differences to be dependent on the relative amount of synthetic double-stranded RNA fragments in the high versus low molecular weight (LMW) range contained in the compound. We here resorted to Poly (I:C) purified for LMW dsRNA fragments to establish a MIA paradigm with increased reproducibility and enhanced standardization in an effort to refine the MIA paradigm and characterize its effect on offspring behavior. We found that the parallel application of LMW Poly (I:C) in two different MIA-experienced laboratories (Vienna and Zurich) yielded differential outcomes in terms of maternal immune responses and behavioral phenotypes in the offspring generation. In both experimental sites, administration of LMW Poly (I:C) induced a significant sickness response and cytokine induction in the pregnant dam and fetal brains, while the expected deficit in sociability as one main behavioral outcome parameter in the MIA progeny, was only present in the Zurich, but not the Vienna cohort. We conclude that although using Poly (I:C) purified for a defined molecular weight range reduces batch-to-batch variability, it does not make the MIA model more reliable and robust. The differential response in behavioral phenotypes of the MIA offspring between the two laboratories illustrates the highly complex interaction between prenatal and postnatal milieus - including the laboratory environment - that determine offspring phenotypic outcomes after MIA. Consequently, establishing a new MIA protocol or implementing the MIA model firstly under new or changed environmental conditions must include the assessment of offspring behavior to ensure solid and reproducible experimental outcomes.


Subject(s)
Poly I-C , Prenatal Exposure Delayed Effects , Poly I-C/pharmacology , Female , Pregnancy , Animals , Prenatal Exposure Delayed Effects/immunology , Molecular Weight , Disease Models, Animal , Cytokines/immunology , Behavior, Animal/drug effects , Male
9.
Biomed Res Int ; 2024: 2222098, 2024.
Article in English | MEDLINE | ID: mdl-39015602

ABSTRACT

In this study, we aimed to isolate and purify catalase from human blood erythrocytes by using a newly synthesized affinity gel. The synthesized ω-amino hexyl agarose-1,2,3-triazole-5-carboxylic acid affinity gel was analyzed by FT-IR. Then, different buffer, pH, and ionic strength parameters were optimized to determine the equilibration, washing, and elution buffer conditions. The catalase was purified from human blood erythrocytes with a specific activity of 45.58 EU/mg, purification fold of 529.50, and a yield of 0.416% using the synthesized new affinity gel. The purity and molecular weight of the enzyme were analyzed by SDS-PAGE, and a single band at 60 kDa was observed for catalase. The optimum reaction temperature of the catalase was found to be 30°C, while the thermal stability temperature was 60°C. The Km and Vmax of the enzyme for hydrogen peroxide were calculated at 0.125 mM and 2500 U mL-1, respectively.


Subject(s)
Catalase , Chromatography, Affinity , Erythrocytes , Humans , Catalase/chemistry , Catalase/isolation & purification , Catalase/metabolism , Erythrocytes/enzymology , Chromatography, Affinity/methods , Hydrogen-Ion Concentration , Temperature , Enzyme Stability , Kinetics , Hydrogen Peroxide/chemistry , Spectroscopy, Fourier Transform Infrared/methods , Molecular Weight
10.
Anal Chem ; 96(28): 11326-11333, 2024 Jul 16.
Article in English | MEDLINE | ID: mdl-38953527

ABSTRACT

Herein, the aptamer-antibody sandwich module was first introduced to accurately recognize a low molecular weight compound (mycotoxin). Impressively, compared with the large steric hindrance of a traditional dual-antibody module, the aptamer-antibody sandwich with low Gibbs free energy and a low dissociation constant has high recognition efficiency; thus, it could reduce false positives and false negatives caused by a dual-antibody module. As a proof of concept, a sensitive electrochemiluminescence (ECL) biosensor was constructed for detecting mycotoxin zearalenone (ZEN) based on an aptamer-antibody sandwich as a biological recognition element and porous ZnO nanosheets (Zn NSs) supported Cu nanoclusters (Cu NCs) as the signal transduction element, in which the antibody was modified on the vertex of a tetrahedral DNA nanostructure (TDN) with a rigid structure to increase the kinetics of target recognition for promoting the detection sensitivity. Moreover, the Cu NCs/Zn NSs exhibited an excellent ECL response that was attributed to the aggregation-induced ECL enhancement through electrostatic interactions. The sensing platform achieved trace detection of ZEN with a low detection limit of 0.31 fg/mL, far beyond that of the enzyme-linked immunosorbent assay (ELISA, the current rapid detection method) and high-performance liquid chromatography (HPLC, the national standard detection method). The strategy has great application potential in food analysis, environmental monitoring, and clinical diagnosis.


Subject(s)
Aptamers, Nucleotide , Biosensing Techniques , Electrochemical Techniques , Zearalenone , Aptamers, Nucleotide/chemistry , Biosensing Techniques/methods , Zearalenone/analysis , Zearalenone/immunology , Electrochemical Techniques/methods , Copper/chemistry , Limit of Detection , Antibodies/chemistry , Antibodies/immunology , Luminescent Measurements/methods , Zinc Oxide/chemistry , Molecular Weight
11.
J Agric Food Chem ; 72(28): 15998-16009, 2024 Jul 17.
Article in English | MEDLINE | ID: mdl-38949246

ABSTRACT

Aflatoxin B1 is a notorious mycotoxin with mutagenicity and carcinogenicity, posing a serious hazard to human and animal health. In this study, an AFB1-degrading dipeptidyl-peptidase III mining from Aspergillus terreus HNGD-TM15 (ADPP III) with a molecular weight of 79 kDa was identified. ADPP III exhibited optimal activity toward AFB1 at 40 °C and pH 7.0, maintaining over 80% relative activity at 80 °C. The key amino acid residues that affected enzyme activity were identified as H450, E451, H455, and E509 via bioinformatic analysis and site-directed mutagenesis. The degradation product of ADPP III toward AFB1 was verified to be AFD1. The zebrafish hepatotoxicity assay verified the toxicity of the AFB1 degradation product was significantly weaker than that of AFB1. The result of this study proved that ADPP III presented a promising prospect for industrial application in food and feed detoxification.


Subject(s)
Aflatoxin B1 , Aspergillus , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases , Fungal Proteins , Zebrafish , Aflatoxin B1/metabolism , Aflatoxin B1/chemistry , Aspergillus/enzymology , Aspergillus/genetics , Aspergillus/chemistry , Aspergillus/metabolism , Animals , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/chemistry , Fungal Proteins/genetics , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Enzyme Stability , Kinetics , Molecular Weight , Hydrogen-Ion Concentration , Substrate Specificity
12.
J Agric Food Chem ; 72(28): 15811-15822, 2024 Jul 17.
Article in English | MEDLINE | ID: mdl-38975865

ABSTRACT

Colanic acid (CA) is exopolysaccharide that presents growing potential in the food and healthcare industry as a versatile polymer. Previously, we have constructed the Escherichia coli strain WWM16 which can efficiently produce CA. In this study, WWM16 has been further engineered to produce a higher yield of CA with low molecular mass and viscosity. The gene mcbR encoding a transcriptional factor, and the genes opgD, opgG, and opgH related to the biosynthesis of osmoregulated periplasmic glucans were deleted in E. coli WWM16, and the resulting strain WWM166 produced 18.1 g/L CA. The expression level of wcaD encoding the polymerase in WWM166 was downregulated using CRISPRi. As a result, the strain WWM166/pWpD1 could produce 49.9 g/L CA with lower molecular mass. CA products were purified from both WWM166 and WWM166/pWpD1, and their molecular mass, viscosity, fluidity, hygroscopicity, and antioxidant activity were determined and compared. These findings demonstrate the potential application of CA with different molecular masses to prolong life and protect skin in the food and cosmetic industries.


Subject(s)
Escherichia coli , Molecular Weight , Escherichia coli/genetics , Escherichia coli/metabolism , Viscosity , Metabolic Engineering , Polysaccharides/metabolism , Polysaccharides/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/chemistry
13.
J Med Virol ; 96(6): e29733, 2024 Jun.
Article in English | MEDLINE | ID: mdl-38874268

ABSTRACT

Viruses in human semen may be sexually transmitted via free and cell-mediated viral infection. The potential effects of semen on the infection and sexual transmission of most viruses in semen remain largely unclear. The present study elucidated the inhibitory effects of human seminal plasma (SP) on Jurkat cell (JC)-mediated mumps virus (MuV) infection. We demonstrated that MuV efficiently infected JCs and that the JCs infected by MuV (JC-MuV) mediated MuV infection of HeLa cells. Remarkably, SP was highly cytotoxic to JCs and inhibited JC-MuV infection of HeLa cells. The cytotoxic factor possessed a molecular weight of less than 3 kDa, whereas that of the viricidal factor was over 100 kDa. The cooperation of cytotoxic and viricidal factors was required for the SP inhibition of JC-MuV infection, and prostatic fluid (PF) was responsible for both the cytotoxic and viricidal effects of SP. The cytotoxic effects we observed were resistant to the treatment of PF with boiling water, proteinase K, RNase A, and DNase I. Our results provide novel insights into the antiviral properties of SP, which may limit cell-mediated sexual viral transmission.


Subject(s)
Mumps virus , Semen , Humans , Mumps virus/physiology , Semen/virology , Male , HeLa Cells , Lymphocytes/virology , Jurkat Cells , Cell Survival , Molecular Weight
14.
Int J Mol Sci ; 25(11)2024 Jun 06.
Article in English | MEDLINE | ID: mdl-38892442

ABSTRACT

Biopharmaceutical products, in particular messenger ribonucleic acid (mRNA), have the potential to dramatically improve the quality of life for patients suffering from respiratory and infectious diseases, rare genetic disorders, and cancer. However, the quality and safety of such products are particularly critical for patients and require close scrutiny. Key product-related impurities, such as fragments and aggregates, among others, can significantly reduce the efficacy of mRNA therapies. In the present work, the possibilities offered by size exclusion chromatography (SEC) for the characterization of mRNA samples were explored using state-of-the-art ultra-wide pore columns with average pore diameters of 1000 and 2500 Å. Our investigation shows that a column with 1000 Å pores proved to be optimal for the analysis of mRNA products, whatever the size between 500 and 5000 nucleotides (nt). We also studied the influence of mobile phase composition and found that the addition of 10 mM magnesium chloride (MgCl2) can be beneficial in improving the resolution and recovery of large size variants for some mRNA samples. We demonstrate that caution should be exercised when increasing column length or decreasing the flow rate. While these adjustments slightly improve resolution, they also lead to an apparent increase in the amount of low-molecular-weight species (LMWS) and monomer peak tailing, which can be attributed to the prolonged residence time inside the column. Finally, our optimal SEC method has been successfully applied to a wide range of mRNA products, ranging from 1000 to 4500 nt in length, as well as mRNA from different suppliers and stressed/unstressed samples.


Subject(s)
Chromatography, Gel , RNA, Messenger , RNA, Messenger/genetics , RNA, Messenger/chemistry , Chromatography, Gel/methods , Humans , Porosity , Molecular Weight , Magnesium Chloride/chemistry
15.
Anal Methods ; 16(26): 4310-4321, 2024 Jul 04.
Article in English | MEDLINE | ID: mdl-38888190

ABSTRACT

The main objective of this study was to design, build, and test a compact, multi-well, portable dry film FTIR system for industrial food and bioprocess applications. The system features dry film sampling on a circular rotating disc comprising 31 wells, a design that was chosen to simplify potential automation and robotic sample handling at a later stage. Calibration models for average molecular weight (AMW, 200 samples) and collagen content (68 samples) were developed from the measurements of industrially produced protein hydrolysate samples in a controlled laboratory environment. Similarly, calibration models for the prediction of lactate content in samples from cultivation media (59 samples) were also developed. The portable dry film FTIR system showed reliable model characteristics which were benchmarked with a benchtop FTIR system. Subsequently, the portable dry film FTIR system was deployed in a bioprocessing plant, and protein hydrolysate samples were measured at-line in an industrial environment. This industrial testing involved building a calibration model for predicting AMW using 60 protein hydrolysate samples measured at-line using the portable dry film FTIR system and subsequent model validation using a test set of 26 samples. The industrial calibration in terms of coefficient of determination (R2 = 0.94), root mean square of cross-validation (RMSECV = 194 g mol-1), and root mean square of prediction (RMSEP = 162 g mol-1) demonstrated low prediction errors as compared to benchtop FTIR measurements, with no statistical difference between the calibration models of the two FTIR systems. This is to the authors' knowledge the first study for developing and employing a portable dry film FTIR system in the enzymatic protein hydrolysis industry for successful at-line measurements of protein hydrolysate samples. The study therefore suggests that the portable dry film FTIR instrument has huge potential for in/at-line applications in the food and bioprocessing industries.


Subject(s)
Protein Hydrolysates , Spectroscopy, Fourier Transform Infrared/methods , Spectroscopy, Fourier Transform Infrared/instrumentation , Protein Hydrolysates/analysis , Protein Hydrolysates/chemistry , Calibration , Molecular Weight , Collagen/chemistry , Collagen/analysis
16.
FASEB J ; 38(13): e23747, 2024 Jul 15.
Article in English | MEDLINE | ID: mdl-38924451

ABSTRACT

In this study, fibrinolytic protease was isolated and purified from Perinereis aibuhitensis Grub, and the extraction process was optimized. The properties of the enzyme, such as the amino acid composition, thermal stability, optimal temperature, and pH, were investigated. After detoxification, proteins collected from fresh Clamworm (Perinereis aibuhitensis Grub) were concentrated via ammonium sulfate precipitation. The crude protease was purified using gel filtration resin (Sephadex G-100), anion exchange resin (DEAE-Sepharose FF), and hydrophobic resin (Phenyl Sepharose 6FF). The molecular weight of the protease was determined by polyacrylamide gel electrophoresis (SDS-PAGE). The optimum temperature and optimum pH of the protease were determined. The activity of crude protease in the 40-60% salt-out section was the highest, reaching 467.53 U/mg. The optimal process for purifying crude protein involved the application of DEAE-Sepharose FF and Phenyl Sepharose 6FF, which resulted in the isolation of a single protease known as Asp60-D1-P1 with the highest fibrinolytic activity; additionally, the enzyme activity was measured at 3367.76 U/mg. Analysis by Native-PAGE and SDS-PAGE revealed that the molecular weight of Asp60-D1-P1 was 44.5 kDa, which consisted of two subunits with molecular weights of 6.5 and 37.8 kDa, respectively. The optimum temperature for Asp60-D1-P1 was 40°C, and the optimal pH was 8.0.


Subject(s)
Fibrinolysin , Animals , Hydrogen-Ion Concentration , Fibrinolysin/metabolism , Fibrinolysin/isolation & purification , Polychaeta/enzymology , Temperature , Molecular Weight , Enzyme Stability , Metals/pharmacology , Electrophoresis, Polyacrylamide Gel , Fibrinolytic Agents/isolation & purification , Fibrinolytic Agents/chemistry , Fibrinolytic Agents/pharmacology , Fibrinolytic Agents/metabolism
17.
Int J Biol Macromol ; 273(Pt 2): 132807, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38825289

ABSTRACT

It is well known that Rosa roxburghii, as a homology of both medicine and food, is rich in polysaccharides. To discover bioactive macromolecules for combating cancer, the polysaccharides in R. roxburghii were investigated, leading to the purification of a polysaccharide (RRTP80-1). RRTP80-1 was measured to have an average molecular weight of 8.65 × 103 g/mol. Monosaccharide composition analysis revealed that RRTP80-1 was formed from three types of monosaccharides including arabinose, glucose, and galactose. Methylation and GC-MS analysis suggested that the backbone of RRTP80-1 consisted of →5)-α-l-Araf-(1→, →6)-α-d-Glcp-(1→, →2,5)-α-l-Araf-(1→, →4,6)-ß-d-Galp-(1→, and →3)-α-l-Araf-(1→, with branch chains composed of α-l-Araf-(1→. In vivo studies indicated that RRTP80-1 exhibited inhibitory activity against the growth and proliferation of neoplasms in the zebrafish tumor xenograft model by suppressing angiogenesis. Additionally, RRTP80-1 was found to upregulate reactive oxygen species (ROS) and nitric oxide (NO) production levels in zebrafish models. All these studies suggest that RRTP80-1 activates the immune system to inhibit tumors. The potential role of the newly discovered homogeneous polysaccharide RRTP80-1 in cancer treatment was preliminarily clarified in this study.


Subject(s)
Monosaccharides , Polysaccharides , Rosa , Zebrafish , Rosa/chemistry , Polysaccharides/pharmacology , Polysaccharides/chemistry , Polysaccharides/isolation & purification , Animals , Humans , Monosaccharides/analysis , Cell Line, Tumor , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Xenograft Model Antitumor Assays , Cell Proliferation/drug effects , Molecular Weight , Methylation , Nitric Oxide/metabolism
18.
Int J Biol Macromol ; 273(Pt 1): 132914, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38844290

ABSTRACT

Pinus taeda L. is a fast-growing softwood with significant commercial value. Understanding structural changes in hemicellulose during growth is essential to understanding the biosynthesis processes occurring in the cell walls of this tree. In this study, alkaline extraction is applied to isolate hemicellulose from Pinus taeda L. stem segments of different ages (1, 2, 3, and 4 years old). The results show that the extracted hemicellulose is mainly comprised of O-acetylgalactoglucomannan (GGM) and 4-O-methylglucuronoarabinoxylan (GAX), with the molecular weights and ratios (i.e., GGM:GAX) of GGM and GAX increasing alongside Pinus taeda L. age. Mature Pinus taeda L. hemicellulose is mainly composed of GGM, and the ratio of (mannose:glucose) in the GGM main chain gradually increases from 2.45 to 3.60 with growth, while the galactose substitution of GGM decreases gradually from 21.36% to 14.65%. The acetylation of GGM gradually increases from 0.33 to 0.45 with the acetyl groups mainly substituting into the O-3 position in the mannan. Furthermore, the contents of arabinose and glucuronic acid in GAX gradually decrease with growth. This study can provide useful information to the research in genetic breeding and high-value utilization of Pinus taeda L.


Subject(s)
Pinus taeda , Polysaccharides , Polysaccharides/metabolism , Polysaccharides/chemistry , Pinus taeda/metabolism , Pinus taeda/growth & development , Xylans/metabolism , Xylans/chemistry , Mannans/metabolism , Mannans/chemistry , Molecular Weight , Cell Wall/metabolism , Cell Wall/chemistry , Acetylation
19.
Int J Biol Macromol ; 273(Pt 1): 132999, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38866280

ABSTRACT

This study aimed to confirm macrophage-stimulatory component from Korean meadowsweet (Filipendula glaberrima; FG) and characterize its compositional and structural properties. FG-CWH, prepared via cool-water extraction and ethanol precipitation, induced the highest secretion of NO (6.0-8.0 µM), TNF-α (8.7-9.5 ng/mL), and IL-6 (1.0-5.7 ng/mL) compared to other samples at 0.4-10 µg/mL in RAW 264.7 cells. Analytical results revealed that FG-CWH is a high-molecular-weight component with an average molecular weight of 220 kDa, constituting a polysaccharide-protein mixture. Chemical and enzymatic treatment of FG-CWH indicated its primary composition as arabinogalactan protein (AGP)-rich glycoprotein, with activity likely associated with the chemical and structural characteristics of AGP. FG-CWH treatment resulted in significant and concentration-dependent increases in iNOS (20.0-29.6 folds), TNFα (10.6-18.6 folds) and IL6 (10.9-155.6 folds) gene expression, as well as the secretion of NO (5.3-6.3 µM), TNF-α (35.4-44.3 ng/mL), and IL-6 (4.1-8.4 ng/mL) secretion, even at a reduced concentration range of 125-500 ng/mL, compared to the negative control group. Immunoblotting analysis indicated FG-CWH-induced macrophage stimulation significantly associated with the activation of MAPK (ERK, JNK, and p38) and NF-κB (p65 and IκBα). These findings can serve as valuable groundwork for developing FG-derived AGP as novel functional ingredients to enhance human immunity.


Subject(s)
Macrophage Activation , Macrophages , Mucoproteins , Plant Proteins , Mice , Animals , RAW 264.7 Cells , Mucoproteins/chemistry , Mucoproteins/metabolism , Macrophage Activation/drug effects , Plant Proteins/chemistry , Plant Proteins/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Nitric Oxide/metabolism , Tumor Necrosis Factor-alpha/metabolism , Interleukin-6/metabolism , Molecular Weight , NF-kappa B/metabolism
20.
Int J Biol Macromol ; 273(Pt 1): 133070, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38866292

ABSTRACT

In recent years, researchers have put much attention on the improvements and upgrades of novel wet strength agent in the papermaking fields, especially in the usage of household paper. Herein, PEIM-KH560 by polyethyleneimine (PEI) and γ-glycidyl ether propyl trimethoxysilane (KH560) was synthesized with five molecular weights (Mw) of PEI at 600, 1800, 10,000, 70,000 and 750,000. Results showed that the molecular weight greatly influenced the physicochemical properties of PEI-KH560, such as the size and thermal stability. The intrinsic cationic charge of PEI-KH560 provided the bonding sites with the paper fibers, forming strengthened fiber-fiber joints. It was shown that the dry, wet strength and hydrophobicity of cellulosic paper sheets were obviously improved. When the m (PEI):m(KH560) is 1:2, the strength of papers after sizing by Mw of PEI at 600 and 1800 is the most obvious, with the dry strength increased by 227.9 % and 187.5 %, and the wet strength increased by 183.8 % and 207.8 %, respectively. The maximum hydrophobicity was found at the PEI1800-KH560 with the contact angle value of 130.6°. The resultant environmental-friendly agent (PEI-KH560) obtained in this work provides valuable significance for the preparation of household and food packaging paper.


Subject(s)
Hydrophobic and Hydrophilic Interactions , Molecular Weight , Paper , Polyethyleneimine , Polyethyleneimine/chemistry , Silanes/chemistry , Surface Properties
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