Gel Filtration Research Articles

Online coupling of size exclusion chromatography to capillary-enhanced Raman spectroscopy for the identification of protein classes in hemolyzed blood serum

Online coupling of size exclusion chromatography to capillary-enhanced Raman spectroscopy for the identification of protein classes in hemolyzed blood serum

Enrichment protocols for human conjunctival extracellular vesicles and their characterization.

The understanding of the role played by extracellular vesicles (EVs) in different tissues has improved significantly in the last years, but remains limited concerning the conjunctiva, a complex eye tissue whose role is pivotal for corneal protection. Here, we conducted a comparative study to isolate and characterize EVs from human conjunctival epithelial (IM-HConEpiC) and human conjunctival mesenchymal stromal cell (Conj-MSCs) secretomes using different isolation methods: differential ultracentrifugation (UC), and a combination of ultrafiltration (UF) with precipitation or size exclusion chromatography (SEC). EVs were characterized by total protein content, size, morphology, and expression of protein markers. EV functional effect was tested in an in vitro oxidative stress model. We successfully recovered EVs with the three methods, although significantly higher yields were obtained with UF-precipitation. Dynamic light scattering analysis confirmed the presence of nano-sized particles, being UC-isolated EVs larger than those isolated by UF-precipitation and UF-SEC. Atomic Force Microscopy showed EVs with a slightly ellipsoidal morphology. EVs enriched with UF-precipitation method were further analyzed, confirming the expression of Alix, CD63, TSG101, and Syntenin-1 by Western blotting and showing that Conj-MSC-derived EVs significantly reduced oxidative stress on IM-HConEpiC. Therefore, we conclude that UF-precipitation is the most efficient method for conjunctival EV enrichment.

The potent PHL4 transcription factor effector domain contains significant disorder.

The phosphate-starvation response transcription-factor protein family is essential to plant response to low-levels of phosphate. Proteins in this transcription factor (TF) family act by altering various gene expression levels, such as increasing levels of the acid phosphatase proteins which catalyze the conversion of inorganic phosphates to bio-available compounds. There are few structural characterizations of proteins in this TF family, none of which address the potent TF activation domains. The phosphate-starvation response-like protein-4 (PHL4) protein from this family has garnered interest due to the unusually high TF activation activity of the N-terminal domain. Here, we demonstrate using solution nuclear magnetic resonance (NMR) measurements that the PHL4 N-terminal activating TF effector domain is mainly an intrinsically disordered domain of over 200 residues, and that the C-terminal region of PHL4 is also disordered. Additionally, we present evidence from size-exclusion chromatography, diffusion NMR measurements, and a cross-linking assay suggesting full-length PHL4 forms a trimeric or tetrameric assembly. Together, the data indicate the N- and C-terminal disordered domains in PHL4 flank a central folded region that likely forms the ordered oligomer of PHL4. This work provides a foundation for future studies detailing how the conformations and molecular motions of PHL4 change as it acts as a potent activator of gene expression in phosphate metabolism. Such a detailed mechanistic understanding of TF function will benefit genetic engineering efforts that take advantage of this activity to boost transcriptional activation of genes across different organisms.

Purification and Characterization of γ-Glutamyl Transpeptidase from Toona sinensisand Its Function on Formation of Sulfur-Containing Volatiles.

γ-Glutamyl transpeptidase (GGT) in Toona sinensis shoot (TS) was extracted and purified through salt precipitation, ion-exchange column, and gel filtration chromatography methods. The GGT had two peptides with M.W. 35 and 50 kDa. Its function in the formation of TS sulfur-containing volatiles (SCVs) was evaluated. The highest GGT hydrolytic activity was at pH 7.0 and 50 °C. Its activity was enhanced by Ca2+, Mg2+, K+, Na+, and Ba2+ and reduced by Cu2+, Fe3+, Mn2+, and Zn2+. The SCVs reached the highest level at GGT to TS sulfur-containing precursors extract 1:20. The aroma produced by GGT from the extract was similar to that of raw TS, especially for the cooked onion-like/TS-like flavor. These results revealed the characteristics and function of GGT in TS on sulfurous aroma formation. It could be applied to retrieve the production of sulfur-containing aroma from thermally preserved TS or similar vegetables, which lost GGT activity irreversibly during heat drying.

Efficient Neutralizing Antibodies Induction by Human Parvovirus B19 Epitope-Presenting Protein Nanoparticles.

Human parvovirus B19 (B19V) causes fetal hydrops in pregnant women. Despite the significant impact of this virus, effective vaccines remain unclear. In this study, we successfully engineered B19V protein nanoparticles by fusing the N-terminal receptor-binding domain corresponding to 5-80 amino acids of VP1 with two distinct types of self-assembling protein nanoparticles. Gel filtration and electron microscopic analysis confirmed the spherical assembly of the antigen-fused nanoparticles. The purified nanoparticles are efficiently bound to the surface of UT7/Epo-S1 cells, which are semi-permissive hosts for B19V infection. Immunization of BALB/c mice with VP1u 5-80 nanoparticles elicited a robust production of B19V-specific IgG antibodies compared to single VP1u 5-80 peptides. Moreover, a neutralization assay using B19V derived from a blood donor sample revealed that antibodies from mice immunized with VP1u 5-80 nanoparticles exhibited stronger infection-neutralizing activity. These findings suggest that nanoparticle formation plays a crucial role in enhancing the immunogenicity of the B19V VP1u 5-80 amino acid peptide and that these nanoparticles could serve as promising vaccine candidates, effectively inducing immunity against B19V.

Chondroitin Sulfate-Coated Heteroduplex-Molecular Spherical Nucleic Acids.

Molecular Spherical Nucleic Acids (MSNAs) are atomically uniform dendritic nanostructures and potential delivery vehicles for oligonucleotides. The radial formulation combined with covalent conjugation may hide the oligonucleotide content and simultaneously enhance the role of appropriate conjugate groups on the outer sphere. The conjugate halo may be modulated to affect the delivery properties of the MSNAs. In the present study, [60]fullerene-based molecular spherical nucleic acids, consisting of a 2'-deoxyribonucleotide and a ribonucleotide sequence, were used as hybridization-mediated carriers (''DNA and RNA-carriers'') for an antisense oligonucleotide, suppressing Tau protein, (i.e. Tau-ASO) and its conjugates with chondroitin sulfate tetrasaccharides (CS) with different sulfation patterns. The impact of the MSNA carriers, CS-moieties on the conjugates and the CS-decorations on the MSNAs on cellular uptake and - activity (Tau-suppression) of the Tau-ASO was studied with hippocampal neurons in vitro. The formation and stability of these heteroduplex ASO-MSNAs were evaluated by UV melting profile analysis, polyacrylamide gel electrophoresis (PAGE), dynamic light scattering (DLS) and size exclusion chromatography equipped with a multi angle light scattering detector (SEC-MALS). The cellular uptake and - activity were studied by confocal microscopy and Western blot analysis, respectively.

Side-Chain Engineering of Boron β-Dialdiminate Homopolymer for Solvent-Dependent Emission Properties.

Luminescent materials that respond to changes in microscopic chemical conditions are essential for visualizing and evaluating molecular environments. Boron complexes are often employed as robust scaffolds for constructing such responsive systems because of their inherent stimuli-responsive properties. However, in the case of nonresponsive luminophores, the absence of these intrinsic features limits their range of applications. In this study, environment-responsive luminescent homopolymers based on the boron β-dialdiminate complex are developed, which is intrinsically less responsive, by introducing optimized side chains. As a key finding, the triethylene glycol-decorated polymer exhibits more intense luminescence in chloroform but weaker luminescence in N,N-dimethylformamide. Structural analyses using NMR and size-exclusion chromatography suggest that this polymer forms larger aggregates in polar solvents because of the solvophobicity of its main chain, while the polar side chains assist in maintaining adequate dispersibility of these aggregates. Photophysical measurements indicate that interchromophore interactions within the aggregates should be responsible for the reduced luminescence in polar solvents. These findings suggest that side-chain engineering should be an effective strategy for creating stimuli-responsive polymers from otherwise nonresponsive luminophores.

Freeze-Thaw Cycle Events Enable the Deep Disintegration of Biochar: Release of Dissolved Black Carbon and Its Structural-Dependent Carbon Sequestration Capacity.

Biochar is widely regarded as a recalcitrant carbon pool. However, the impact of freeze-thaw cycle events on its storage capacity, particularly on the release of dissolved black carbon (DBC), has remained poorly investigated. This study investigated the release behavior of DBC from biochar pyrolyzed at 300-700 °C during freeze-thaw cycles and their retention capacity in soil. Freeze-thaw cycles dramatically promoted DBC release (33.08-230.74 mg C L-1), exhibiting an order of magnitude higher than those without freeze-thaw process. The release kinetics of freeze-thaw-induced DBC varied depending on the pyrolysis temperature of biochar due to the different disintegration mechanisms. Interestingly, the retention capacity of freeze-thaw-induced DBC in soil showed a reduction ranging from 7.7 to 29.5% compared to DBC without the freeze-thaw process. This reduction can be attributed to numerous hydrophilic low-molecular-weight compounds (16.97-75.31%) in freeze-thaw-induced DBC, as evidenced by the results of size exclusion chromatography, fluorescence excitation/emission matrix, Fourier transform infrared spectroscopy, and nuclear magnetic resonance. These compounds tend to concentrate in the aqueous phase rather than being retained in the soil, potentially exacerbating the outflow of dissolved organic carbon. These findings clarify the release behavior of DBC during freeze-thaw cycles and reveal their contribution to the attenuation of carbon pools in cold regions.

Abstract PR013: Highly accurate detection of early-stage colorectal cancer using tumor and immune extracellular vesicles biomarkers

Abstract Introduction: Colorectal cancer (CRC) is one of the most common cancers worldwide, and early detection is critical for successful treatment and improved survival rates. However, precancerous and early-stage CRC present significant diagnostic challenges due to the small size of lesions and the very low expression of tumor-specific biomarkers in the bloodstream. Current genomics-based diagnostic methods struggle to detect these lesions, making it imperative to develop more sensitive and specific approaches. Circulating extracellular vesicles (EVs) are emerging as a promising solution for early-stage CRC detection, since EVs are produced by tumor cells as well as the tumor microenviroment and host immune cells. Thus, EVs may offer the means to detect and monitor small early-stage tumors through both direct detection of tumor- associated biomarkers as well through tumor specific host response and tumor microenvironment biomarkers. Methods: To identify novel early-stage CRC specific biomarkers, we performed proteomics analysis on EVs purified from patient plasma using size exclusion chromatography and a proprietary buffer system that enhances EV and corona protein recovery. TrueDiscovery™ Data-independent acquisition (DIA) mass spectrometry (MS) analysis was conducted on 24 pre- cancer/stage 0 and 25 stage 1 CRC patients and 75 normal patient plasma samples. An in-house developed machine learning pipeline was used to identify differentially expressed proteins and model candidate multiplexes to identify those with extremely high diagnostic accuracy (> 0.99). Results: An average of ∼2,500 proteins were identified per sample and included in bioinformatics analysis. Comparative analysis between control patient EVs and either pre- cancerous/Stage 0, or Stage 1 colon cancer EVs using an in-house Machine Learning pipeline identified 336 and 493 differentially expressed proteins for each group (FDR adjusted p-value < 0.001). Of these, the best 17 pre/stage 0 proteins and 53 stage 1 proteins were trained and tested using Support Vector Machine to assess all potential 3-plexes in order to identify those with mean diagnostic accuracy > 99%. This yielded 5 3-plexes in precancer/stage 0 and 34 3-plexes in stage 1 with near perfect diagnostic accuracy. These plexes are currently being assessed in single analyte and multiplex immunoassays with the goal of translating candidate 3-plexes to the clinical. Conclusions: Key biomarkers from CRC patient EVs indicate the potential to detect and diagnose early-stage and low tumor burden cancers using our methods. Interestingly, the most accurate 3-plexes consist of proteins from immune, inflammatory and metabolic processes, suggesting that for the detection of early stage cancer it may be critical to include both tumor and host specific biomarkers. Citation Format: Poorva Mudgal, Cheryl Bandoski, Zachary Opheim, Martin Mehnert, Valesca Anschau, Issa Isaac, Alan Ezrin, Todd Hembrough. Highly accurate detection of early-stage colorectal cancer using tumor and immune extracellular vesicles biomarkers [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr PR013.

Abstract A011: Identifying surface biomarkers from colorectal cancer-derived extracellular vesicles using a bead-based approach

Abstract Colorectal cancer (CRC) accounts for the second most common cause of cancer-related mortality globally. The search for prognostic biomarkers subserving to detected cancer at an early stage, to improve tumor classification and to stratify therapeutic patient management groups is actively ongoing. Surgical resections and chemotherapeutics remain a mainstay in the cure and control of solid tumors. Despite their usefulness in the clinical scenario, shortcomings are well recognized, leading researchers to investigate for a circulating source of tumor-derived materials in biofluids, including mostly circulating tumor DNA (ctDNA), circulating tumor cells (CTCs) and extracellular vesicles (EVs). Characterization of EVs surface markers and assessment of their molecular cargo, can lead to the identification of early metastatic colorectal cancer signatures. Cell surface markers of three CRC cell lines (HCT116, DLD1 and LOVO) were characterized using flow cytometry. EVs were isolated from 48-hour serum-deprived cell lines using the polymer pull down method (20% Polyethelene glycol (PEG)). The concentration and particle size distribution of isolated EVs was checked by the NanoSight NS300. Avidin-coated Polystyrene beads were conjugated with CD9 biotin antibodies; and were used to immunoprecipitated (IP) CD9+ EVs. Following a 2-hour incubation and a series of washing steps, the PE-labelled antibodies of interest (CD9, CD63, EpCAM, Vim, CD44, CD133 and CD29) were added and read using xMAP technology. To increase the number of surface markers that can be read in tandem, IP was multiplexed using EpCAM, CD9 and CD63. This was followed by the isolation of EVs from plasma of metastatic CRC patients (N=3) using size exclusion chromatography (Izon qEV1 columns-35nm in conjunction with the automatic fraction collector) and polymer pull down. The concentration and size were assessed by Nanosight NS300 and surface markers of tumor-derived EVs were assessed using the immunoprecipitation technique on the Luminex system. Comparison of signals between EVs isolated from cell lines and matched originator cells showed that cell surface signatures in EVs match closely with their originator cells. The mean fluorescence intensities quantified cell surface marker expression in the different exosome-bead complexes. Interestingly, one of the metastatic cancer patients with invasive colorectal adenocarcinoma and lung metastasis showed EpCAM and Vimentin positivity suggesting epithelial-mesenchymal plasticity. These results show the importance of multiplexing both at the level of immunoprecipitation and at end-measurement, given the dynamics of cell surface marker changes during disease progression. The use of the mesenchymal marker; Vimentin in a panel for cell surface markers stems from studies on biotinylated cell surface markers on various cancer cell lines and identification of Vimentin in such models. Interestingly we find Vimentin on EVs surface in this study. Citation Format: Jessica Debattista, Laura Grech, Christian Scerri, Malcolm Buhagiar, Godfrey Grech. Identifying surface biomarkers from colorectal cancer-derived extracellular vesicles using a bead-based approach [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A011.

Abstract B017: MicroRNA and proteomic analyses of plasma extracellular vesicles detect diffuse large B-cell lymphoma

Abstract Background: Liquid biopsy offers a non-invasive method for detection of non-Hodgkin lymphoma (NHL). Tissue biopsy, the standard method of NHL detection, causes patient discomfort and increases risk of infection. Extracellular vesicles (EVs) are secreted by cells as particles with cargos of nucleic acids and proteins enclosed in a lipid bilayer. The most common NHL subtype is diffuse large B-cell lymphoma (DLBCL). We analyzed plasma-derived EVs from DLBCL patients and matching healthy controls for microRNA (miRNA) and protein to identify potential liquid biopsy biomarkers. Approach: Plasma samples (1 mL) from DLBCL patients and healthy controls were eluted through Izon size-exclusion chromatography (SEC) to obtain EVs. The size distribution of EVs was characterized with nanoparticle tracking analysis. For miRNA analysis, SEC EVs from three DLBCL patients and three matching healthy controls underwent further selection by immunocapture and release according to NHL antigens CD19, CD20, CD22, and C37, with an EV microfluidic affinity purification (EV-MAP) chip. Western blot confirmed the presence of these NHL antigens in NHL cell lysate and NHL cell culture SEC EVs, with flow cytometry verifying expression on NHL cells. MiRNA from these immunocaptured EVs were then isolated and sequenced with Qiagen kits. MiRNA levels were assessed for fold change (FC) expression between cases and controls. Upregulated miRNAs from DLBCL patients were compiled with the miRNA Enrichment Analysis and Annotation Tool (miEAA). For proteomic analysis, SEC EVs from ten DLBCL patients and ten matching healthy controls were measured with label-free mass spectrometry and a custom peptide library. Protein levels were assessed for FC expression and compiled with the Mann-Whitney U test. Results: The size of EVs ranged from 50–300 nm. Exclusively different upregulated miRNAs (FC>2, p<0.05, FDR p<0.05) were found by selection with different antigens: None unique with CD19, one with CD20, eight with CD22, and one with CD37. MiR-320b was upregulated with CD20 (FC=52, p=0.0002, FDR p=0.02), and miR-3168 with CD37 (FC=906, p=0.0003, FDR p=0.02). With CD22, miR-190b-5p, let-7c-5p, miR-1-3p, miR-125b-5p, miR-133a-3p, miR-153-3p, miR- 184, and miR-206 were upregulated in DLBCL patients (FC>17, p<0.003, FDR p<0.05). The miEAA recognized all the upregulated CD22 miRNAs as overrepresented in B-cell lymphoma, except for miR-190b-5p. Proteomic analysis of EVs found 42 different exo-proteins with AUC>0.90 (p<0.05) and 12 different exo-proteins with AUC>0.95 (p<0.009) upregulated in DLBCL patients. Two upregulated exo-proteins (FC>2.81, p<0.0002, adjusted p<0.004), ADAM metallopeptidase with thrombospondin type 1 motif 13 (ADAMTS13) and von Willebrand Factor (vWF), achieved a perfect AUC=1 (p=0.00001) for all DLBCL patients and healthy controls. Conclusions: These results support the hypothesis that liquid biopsy of plasma EVs detect DLBCL. Upregulated miRNA and proteomic signatures via EVs indicate the presence of DLBCL for non-invasive NHL detection. Citation Format: Arthur A. Lee, Sagar Rayamajhi, Mengjia Hu, Harsh Pathak, Rajni V. Puri, Kamilla Isakova, Mei Han, Claire S. Berquist, Samuel G. Mackintosh, Stephanie D. Byrum, Dennis W. Province, Mohammod M. Rahman, Foyez Ahmmed, Leonidas E. Bantis, Malgorzata A. Witek, Steven A. Soper, Andrew K. Godwin, Haitham Abdelhakim. MicroRNA and proteomic analyses of plasma extracellular vesicles detect diffuse large B-cell lymphoma [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr B017.

Reassessing the Photochemical Upcycling of Polystyrene Using Acridinium Salts.

Approaches aimed at deconstructing PS by photocatalytic means struggle to generate high-energy species capable of cleaving the C-H and C-C bonds of PS. We show herein that 9-mesityl-10-methylacridinium perchlorate (MA) is capable of upcycling various grades of PS susbstrates into benzoic acid (BAc) up to 40%, with small proportions of acetophenone and formic acid, under visible light (456 nm) or through solar radiation. Time-resolved emission and absorption spectroscopy evidence that a reaction with oxygen is the primary photochemical step in oxygen-saturated solutions, accounting for 77% of the photons absorbed vs. 1% for the direct reaction with PS. These results suggest a mechanism in which MA-mediated photocycling of PS to BAc occurs through the abstraction of benzylic H atoms by reactive oxygen species generated by energy or electron transfer from the excited state of MA. Addition of triplet O2 to these radicals, followed by intra- or inter-molecular hydrogen atom transfer (HAT), generates C- or O-centered radicals then undergoing β-scission or hydroperoxide fragmentation. The formation of intermediate oligomers functionalized by terminal carbonyl groups is demonstrated by both infrared analysis and mass spectrometry. These oligomers undergo further photoinduced conversion in the absence of MA, as evidenced by size exclusion chromatography analysis.

Reassessing the Photochemical Upcycling of Polystyrene Using Acridinium Salts

Approaches aimed at deconstructing PS by photocatalytic means struggle to generate high‐energy species capable of cleaving the C‐H and C‐C bonds of PS. We show herein that 9‐mesityl‐10‐methylacridinium perchlorate (MA) is capable of upcycling various grades of PS susbstrates into benzoic acid (BAc) up to 40%, with small proportions of acetophenone and formic acid, under visible light (456 nm) or through solar radiation. Time‐resolved emission and absorption spectroscopy evidence that a reaction with oxygen is the primary photochemical step in oxygen‐saturated solutions, accounting for 77% of the photons absorbed vs. 1% for the direct reaction with PS. These results suggest a mechanism in which MA‐mediated photocycling of PS to BAc occurs through the abstraction of benzylic H atoms by reactive oxygen species generated by energy or electron transfer from the excited state of MA. Addition of triplet O2 to these radicals, followed by intra‐ or inter‐molecular hydrogen atom transfer (HAT), generates C‐ or O‐centered radicals then undergoing β‐scission or hydroperoxide fragmentation. The formation of intermediate oligomers functionalized by terminal carbonyl groups is demonstrated by both infrared analysis and mass spectrometry. These oligomers undergo further photoinduced conversion in the absence of MA, as evidenced by size exclusion chromatography analysis.

H-NS is a Transcriptional Repressor of the CRISPR-Cas System in Acinetobacter baumannii ATCC 19606.

Acinetobacter baumannii is a multidrug-resistant opportunistic pathogen primarily associated with hospital-acquired infections. The bacterium can gain multidrug resistance through several mechanisms, including horizontal gene transfer. A CRISPR-Cas system including several Cas genes could restrict the horizontal gene transfer. However, the molecular mechanism of CRISPR- Cas transcriptional regulation remains unclear. We identified a type I-F CRISPR-Cas system in A. baumannii ATCC 19606T standard strain based on sequence analysis. We focused on the transcriptional regulation of Cas3, a key protein of the CRISPR-Cas system. We performed a DNA affinity chromatography-pulldown assay to identify transcriptional regulators of the Cas3 promoter. We identified several putative transcriptional factors, such as H-NS, integration host factor, and HU, that can bind to the promoter region of Cas3. We characterized AbH-NS using size exclusion chromatography and cross-linking experiments and demonstrated that the Cas3 promoter can be regulated by AbH-NS in a concentration-dependent manner via an in vitro transcription assay. CRISPR-Cas expression levels in wild-type and hns mutant strains in the early stationary phase were examined by qPCR and β-galactosidase assay. We found that H-NS can act as a repressor of Cas3. Our transformation efficiency results indicated that the hns mutation decreased the transformation efficiency, while the Cas3 mutation increased it. We report the existence and characterization of the CRISPR-Cas system in A. baumannii 19606T and demonstrate that AbH-NS is a transcriptional repressor of CRISPR-Cas-related genes in A. baumannii.

Dual-Responsive Nanoparticles for Smart Drug Delivery: A NIR Light-Sensitive and Redox-Reactive PEG-PCL-Based System.

Stimuli-responsive polymeric nanoparticles (NPs) can serve as smart drug delivery systems (DDSs) by triggering drug release upon external or internal stimuli. A dual-responsive DDS made of a triblock poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCL-SS-PEG-SS-PCL) copolymer, bearing disulfide bonds between PCL and PEG, was synthesized. The copolymer was functionalized with coumarin and sensitive to near-infrared (NIR) light irradiation, while the S-S bonds could be cleaved by GSH (10 mM). Characterization was achieved by nuclear magnetic resonance, size exclusion chromatography, and Fourier transform infrared analyses. Nile Red (NR)-loaded NPs were prepared through self-assembly of the copolymer in water and analyzed by dynamic light scattering and field-emission scanning electron microscopy. The NR release upon ultraviolet (UV)/NIR light irradiation as well as by GSH concentrations was monitored by using fluorescence spectroscopy, while simultaneous exposure to UV/NIR light and intracellular GSH concentration led to faster NR release. AlamarBlue assay showed satisfactory cell viability of the NR-loaded NPs, while their cellular uptake in human dermal fibroblast cells was investigated by fluorescence microscopy and fluorescence emission measurements.

Film Properties of Heparin Cross-Linked with Epichlorohydrin in Absence or Presence of Imidazole

We cross-linked unfractionated heparin (H) using epichlorohydrin (E), in the absence or presence of imidazole (I), using various ratios of H, E, and I substances. The objectives and goals were to use the reaction for the preparation of medical materials suitable for blood sample applications. Nuclear magnetic resonance indicated the involvement of an H-end sequence [H-(1→4)-β-D-GlcA-(1→3)-β-D-Gal-(1→3)-β-D-Gal-(1→4)-β-D-Xyl-α-Ser] in the linkage with the 2-hydroxypropyl bridge. The yields of the individual experiments were found to increase in the following ratios: 1H/1E/3I (24%) < 1H/1E/2I (32%) < 1H/3E (42%) < 1H/1E/1I (46%) < 1H/2E (64%) < 1H/1E (77%). According to size-exclusion chromatography with multiple-angle light scattering (SEC-MALS) analysis, the mass at the peak increased in the following order: H (9292 g/mol) < 1H/1E (9294 g/mol) < 1H/2E (9326 g/mol) < 1H/3E (9708 g/mol) < 1H/1E/2I (11,212 g/mol) < 1H/1E/3I (12,301 g/mol) < 1H/1E/1I (13,800 g/mol) and in the reverse order with the increase in amount of epichlorohydrin and imidazole, i.e., 1H/1E > 1H/2E > 1H/3E and 1H/1E/1I > 1H/1E/2I > 1H/1E/3I. X-ray diffraction revealed that all prepared films were amorphous. An evaluation of the surface morphology using atomic force microscopy (AFM) confirmed a relatively low films roughness (~0.9–3.6 nm). The surface reduced elastic modulus, determined by the PeakForce quantitative nanomechanical mapping (PF-QNM) technique, was found to increase by up to ~63% for films cross-linked with E in the absence of I when compared with the results for the H substrate. A negligible change in modulus was, however, observed for films cross-linked in the presence of I, or was even reduced by ~15% (1H/1E/3I) compared to that for the H substrate. This could be explained by the parallel cross-linking of H only with E within its serine end unit and in competition with only one nitrogen of I. According to the highest yield (77%) of 1H/1E, the preferred product is the following: H-(1→4)-β-D-GlcA-(1→3)-β-D-Gal-(1→3)-β-D-Gal-(1→4)-β-D-Xyl-α-Ser-CH2-CH(OH)-CH2-OH. For the 1H/1E/1I (46% yield), 1H/1E/2I (32%), and 1H/1E/3I (24%) products, the cross-linked motif was the same, and the difference represented the surplus amount of the imidazolium cation ionically bound to the heparin anionic groups.

Extraction, characterization, and evaluation of galactomannan from Barbatimão Verdadeiro as a potential additive for enhanced oil recovery fluids

Polymers play a crucial role in enhanced oil recovery (EOR) by increasing the viscosity of injection fluids and enhancing oil displacement. The widely used polymer, partially hydrolyzed polyacrylamide (HPAM), faces performance degradation in high-salinity and high-temperature environments. This study explores galactomannan from Stryphnodendron polyphyllum seeds as an alternative viscosifying agent for EOR fluids. An extraction process for galactomannan from Barbatimão Verdadeiro seeds was developed, yielding 23.3% m/m. The biopolymer was characterized using Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), thermogravimetric analysis (TGA), and size exclusion chromatography (SEC). Rheological behavior was tested in brines of different salinities, and injectivity/filterability tests were performed following API RP 63:1990 standards. The galactomannan obtained from Stryphnodendron polyphyllum exhibited a mannose-to-galactose ratio of 1.32. Rheological analysis showed pseudoplastic behavior, with viscosities of 38 cP in brine solutions at 7.37 s−1 and 60 °C. Filterability tests demonstrated satisfactory injectivity with factors of 1.09 (8 µm) and 0.67 (1.2 µm). Compared to HPAM, galactomannan exhibited superior viscosity retention in high-salinity conditions, where HPAM dropped to 6 cP. The galactomannan extracted from Stryphnodendron polyphyllum seeds is a promising biopolymer for EOR applications, offering superior performance under harsh conditions compared to traditional HPAM. Its resilience to high salinity and temperature makes it a viable alternative for pre-salt and offshore fields, contributing to more sustainable and efficient oil recovery.

Double Hydrophilic Hyperbranched Copolymer-Based Lipomer Nanoparticles: Copolymer Synthesis and Co-Assembly Studies

Double hydrophilic, random, hyperbranched copolymers were synthesized via reversible addition–fragmentation chain transfer (RAFT) polymerization of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) and 2-(dimethylamino)ethyl methacrylate (DMAEMA) utilizing ethylene glycol dimethacrylate (EGDMA) as the branching agent. The resulting copolymers were characterized in terms of their molecular weight and dispersity using size exclusion chromatography (SEC), and their chemical structure was confirmed using FT-IR and 1H-NMR spectroscopy techniques. The choice of the two hydrophilic blocks and the design of the macromolecular structure allowed the formation of self-assembled nanoparticles, partially due to the pH-responsive character of the DMAEMA segments and their interaction with -COOH end groups remaining from the chain transfer agent. The copolymers showed pH-responsive properties, mainly due to the protonation–deprotonation equilibria of the DMAEMA segments. Subsequently, a nanoscopic polymer–lipid (lipomer) mixed system was formulated by complexing the synthesized copolymers with cosmetic amphiphilic emulsifiers, specifically glyceryl stearate (GS) and glyceryl stearate citrate (GSC). This study aims to show that developing lipid–polymer hybrid nanoparticles can effectively address the limitations of both liposomes and polymeric nanoparticles. The effects of varying the ionic strength and pH on stimuli-sensitive polymeric and mixed polymer–lipid nanostructures were thoroughly investigated. To achieve this, the structural properties of the hybrid nanoparticles were comprehensively characterized using physicochemical techniques providing insights into their size distribution and stability.

Water-Soluble Intracellular Polysaccharides (IPSW-2 to 4) from Phellinus igniarius Mycelia: Fractionation, Structural Elucidation, and Antioxidant Activity

Phellinus igniarius is a medicinal fungus. Nonetheless, research on its water-soluble intracellular polysaccharides (IPSW-2 to 4) fractionation, structural elucidation, and antioxidant activity is limited. In this study, water-soluble intracellular polysaccharides (IPSW-2 to 4) were extracted and fractionated from P. igniarius mycelia, and their antioxidant and structural properties were assessed using GC-FID, GC-MS, FTIR, and NMR spectroscopy (1H and 13C). In the water-eluted P. igniarius polysaccharide fractions (IPS30W, IPS60W, and IPS80W) of anion-exchange chromatography, the polysaccharide content was 79.05%, 68.25%, and 62.06%, with higher yields of 25.07%, 21.38%, and 20.34%, respectively. In contrast, the salt (NaCl) elution fractions (IPS30S1, IPS60S1, IPS60S2, and IPS80S1) of anion-exchange chromatography had lower polysaccharide content and yield. Hence, water elution fractions (IPS30W, IPS60W, and IPS80W) were selected for further purification. After repeated purification using size-exclusion chromatography, IPSW-2 to 4 were obtained with a yield of 8% to 15.83%. The IPSW-2 to IPSW-4 structures were elucidated, and they showed no triple helical conformation. Based on periodate oxidation, Smith degradation, methylation analysis, and 1H and 13C NMR spectroscopy, the primary structures of IPSW-2, IPSW-3, and IPSW-4 were all glucan, with the main chain consisting of (1→6)-α-D-Glcp, (1→3,4)-α-D-Glcp, and (1→3, 6)-α-D-Glcp, with α-D-Glcp as a side chain. Finally, antioxidant analysis showed that IPS30W, IPS60W, and IPS80W were all more capable of scavenging superoxide anions than the polysaccharides of Phyllostachys (13.8%) and floribunda (15.1%) at the same concentration (0.40 mg/mL). This will serve as a guide for the development of functional foods.

Evaluation of Antibacterial Activity of Milk Peptides Released during the Growth of Lactobacilli in Milk

The present study is carried out to evaluate the antibacterial peptides released during the growth of lactobacilli in milk. Among 8 isolates of lactobacilli and yoghurt cultures used for study, the yoghurt culture released the maximum quantity of peptides (90 ± 1.25 µg/ml). When tested for antibacterial activity only yoghurt culture filtrate showed the antibacterial potential. Further, yoghurt culture lysate was prepared, which was used to treat the milk to bring in proteolysis. The supernatant obtained after centrifugation of this treated milk was used to partially purify peptides by gel filtration using Sephadex G-50. One of the three fractions (F2) showed antibacterial activity against test organisms, E. coli, Salmonella sp. and Shigella dysentriae. The peptide fraction was incorporated into curd samples inoculated with different lactic cultures and spiked with E. coli to determine antibacterial activity. It was found that peptide showed a bacteriostatic effect on E. coli, and the viable count of it was almost reduced by two log cycles when compared with the control.