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PurposeFor biochemical evaluation of soft tissues of the knee, T1rho magnetic resonance imaging (MRI) has been proposed. Purpose of this study was to compare three T1rho sequences based on fast advanced spin echo (FASE), ultrashort echo time (UTE), and magnetization-prepared angle-modulated partitioned k-space spoiled gradient echo snapshots (MAPSS) acquisitions for the knee evaluation.Materials and methodsWe developed two T1rho sequences using 3D FASE or 3D radial UTE acquisitions. 3D MAPSS T1rho was provided by the manufacturer. Agarose phantoms with varying concentrations were imaged. Additionally, bilateral knees of asymptomatic subjects were imaged sagittally. T1rho values of the phantoms and 4 regions of interest (ROI) of the knees (i.e., anterior and posterior meniscus, femoral and tibial cartilage) were determined.ResultsIn phantoms, all T1rho values monotonically decreased with increasing agarose concentration. 3D MAPSS T1rho values of 51, 34, and 38 ms were found for 2, 3, and 4% agarose, respectively, similar to published values on another platform. In the knee, the raw images were detailed with good contrast. Cartilage and meniscus T1rho values varied with the pulse sequence, being the lowest in the 3D UTE T1rho sequence. Comparing different ROIs, menisci generally had lower T1rho values compared to cartilage, as expected in healthy knees.ConclusionWe have successfully developed and implemented the new T1rho sequences and validated them using agarose phantoms and volunteer knees. All sequences were optimized to be clinically feasible (~ 5 min or less) and yielded satisfactory image quality and T1rho values consistent with the literature.

The Geobacillus zalihae strain T1 produces a thermostable T1 lipase that could be used for industrial purposes. Previously, the GST-T1 lipase was purified through two chromatographic steps: affinity and ion exchange (IEX) but the recovery yield was only 33%. To improve the recovery yield to over 80%, the GST tag from the pGEX system was replaced with a poly-histidine at the N-terminal of the T1 lipase sequence. The novel construct of pGEX/His-T1 lipase was developed by site-directed mutagenesis, where the XbaI restriction site was introduced upstream of the GST tag, allowing the removal of tag via double digestion using XbaI and EcoRI (existing cutting site in the pGEX system). Fragment of 6 × His-T1 lipase fusion was synthesized, cloned into the pGEX4T1 system, and expressed in Escherichia coli BL21 (DE3) pLysS, resulting in lipase-specific activity at 236 U/mg. The single purification step of His-T1 lipase was successfully achieved using nickel Sepharose 6FF with an optimized concentration of 5 mM imidazole for binding, yielding the recovery of 98%, 1,353 U/mg lipase activity, and a 5.7-fold increase in purification fold. His-T1 lipase was characterized and was found to be stable at pH 5–9, active at 70 °C, and optimal at pH 9. [ABSTRACT FROM AUTHOR]

Electrically conductive biomaterials direct cell behavior by capitalizing on the effect of bioelectricity in tissue homeostasis and healing. Many studies have leveraged conductive biomaterials to influence cells and improve tissue healing, even in the absence of external stimulation. However, most studies using electroactive materials neglect characterizing how the inclusion of conductive additives affects the material's mechanical properties, and the interplay between substrate electrical and mechanical properties on cell behavior is poorly understood. Furthermore, mechanisms dictating how electrically conductive materials affect cell behavior in the absence of external stimulation are not explicit. In this study, we developed a mechanically and electrically tunable conductive hydrogel using agarose and the conductive polymer PEDOT:PSS. Under certain conditions, we observed that the hydrogel physical and electrical properties were decoupled. We then seeded human mesenchymal stromal cells (MSCs) onto the hydrogels and observed enhanced adhesion and spreading of MSCs on conductive substrates, regardless of the hydrogel mechanical properties, and despite the gels having no cell-binding sites. To explain this observation, we measured protein interaction with the gels and found that charged proteins adsorbed significantly more to conductive hydrogels. These data demonstrate that conductivity promotes cell adhesion, likely by facilitating increased adsorption of proteins associated with cell binding, providing a better understanding of the mechanism of action of electrically conductive materials.

Enzyme-assisted ultrasonic extraction (EAUE) was utilized and optimized for extracting polysaccharides from Schizochytrium limacinum meal (SLMPs) via the response surface methodology. The optimal EAUE conditions were determined as follows: enzyme concentration at 5.18%, ultrasonic temperature at 53 °C, ultrasonic duration of 40 min, ultrasonic power at 60 W, and a liquid-to-material ratio of 34 mL/g, achieving a polysaccharide extraction yield of 11.86 ± 0.61%. The purified polysaccharide component, SLMP1-1, isolated using DEAE Sepharose Fast Flow and Sephadex G-100 columns, exhibited potent antioxidant activity. SLMP1-1, with a molecular weight of 25.5 kDa, comprises glucose, mannose, arabinose, and galactose in a molar ratio of 16.39:14.75:1:693.03. 1H NMR analysis revealed the α configuration of SLMP1-1. Antioxidant assessments, including DPPH, ABTS, and ferric ion reduction assays, were detected with inhibitory values at 21.82–82.98%, 38.21–98.46%, and 3.30–20.30% at 0.2–1.0 mg/mL. This confirmed the effective antioxidant capacity of SLMP1-1, which is notably enhanced post oral and gastric digestion. The findings suggest that polysaccharides extracted from Schizochytrium limacinum meal hold significant promise as natural antioxidants. [ABSTRACT FROM AUTHOR]

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a useful technique for mapping the spatial distribution of molecules across biological samples. Sample preparation is crucial for MALDI-IMS; samples must be flat, dry, and cocrystallized with a matrix prior to analysis. Agarose-based samples can be difficult to consistently prepare as they are susceptible to environmental changes, which can lead to inconsistent drying and wrinkling on the sample surface. Small height differences may cause low ionization of target analytes or introduce artifacts in imaging data depending on the instrument used for analysis. To overcome the variations, a home-built robotic spinner was constructed and applied to agarose-based samples. This robotic spinner is inexpensive and easy to assemble, and when it was applied to agarose-based samples, accelerated the drying process and reduced wrinkles, improving the overall quality of the resulting IMS data.

A water‐soluble polysaccharide (GFP) was isolated from Grateloupia filicina and fractionated using a DEAE Sepharose Fast Flow column to evaluate immunostimulatory activity. Carbohydrates (62.0%–68.4%) and sulfates (29.3%–34.3%) were the major components of GFP and its fractions (GFP‐1 and GFP‐2), with relatively lower levels of proteins (4.5%–15.4%) and uronic acid (1.4%–3.9%). The average molecular weight (Mw) for GFP and its fractions was calculated between 98.2%–243.7 kDa. The polysaccharides were composed of galactose (62.1%–87.2%), glucose (4.5%–33.2%), xylose (3.1%–5.3%), mannose (1.4%–2.2%), rhamnose (1.2%–2.0%), and arabinose (0.9%–1.7%) units connected through →3)‐Galp‐(1→, →4)‐Galp‐(1→, →2)‐Galp‐(1→, →6)‐Galp‐(1→, →3,4)‐Galp ‐(1→, →3,6)‐Galp‐(1→, →4,6)‐Galp‐(1→, →3,4,6)‐Galp‐(1→, →2,3)‐Galp‐(1→, →2,4)‐Galp‐(1→, →4)‐Glcp‐(1→, →6)‐Glcp‐(1→ and →4,6)‐Glcp‐(1→residues. The isolated polysaccharides effectively induced RAW264.7 murine macrophages by releasing nitric oxide (NO) and various cytokines via nuclear factor kappa light chain enhancer of activated B cells (NF‐κB) and mitogen‐activated protein kinase (MAPK) signaling pathways. Further, the expression of toll‐like receptor‐2 (TLR‐2) and TLR‐4 in RAW264.7 cells indicated their activation through TLR‐2 and TLR‐4 binding receptors. Among the polysaccharides, GFP‐1 highly stimulated the activation of RAW264.7 cells, which was mainly constituted of (→1) terminal‐D‐galactopyranosyl, (1→3)‐linked‐ᴅ‐galactopyranosyl, (1→4)‐linked‐ᴅ‐galactopyranosyl and (1→3,4) ‐linked‐ᴅ‐galactopyranosyl residues. These findings demonstrate that GFP‐1 from G. filicina are effective at stimulating the immune system and this warrants further investigation to determine potential biomedical applications. [ABSTRACT FROM AUTHOR]

Copyright of Modern Food Science & Technology is the property of Editorial Office of Modern Food Science & Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

To investigate the possible degradation of the intramuscular connective tissue (IMCT) with cathepsin L, isolated IMCTs were incubated with purified cathepsin L in vitro. Here, we prepared purified cathepsin L from bovine pancreas by using DEAE Sephacel, Sephacryl S-100 HR, SP Sepharose FF, and con A-Sepharose affinity chromatography in sequence. An SDS-PAGE analysis of CNBr-digested peptides showed that the degradation of collagen in IMCT could take place on terminal non-helical peptides rather than the triple helix region. Decorin (DCN) was clearly degraded at a pH of 5.0. The TP and TO of intramuscular connective tissue decreased to 41.41 °C and 43.79 °C, respectively. In the cathepsin L treatment of pH 5.0, the decreases in the TP and TO of IMCT were more sensitive than they were at pH 5.5~6.5. [ABSTRACT FROM AUTHOR]

In this research, for the first time, A. flavus uricase gene was cloned in pPink-UOX plasmid under strong alcohol oxidase promoter of Pichia pink expression system after codon optimization. After selecting the best uricase producing clone with an activity of 0.7 U/ml at the Flask level, a 5-L fermenter was used to increase the expression of the enzyme. Within 60 h, the fermentation process produced 1500 g of biomass from 4 L of semi defined culture media and expressed 2.5 g/L of the enzyme. The purity of recombinant uricase production using three consecutive DEAE Sepharose, CM Sepharose and Phenyl Sepharose columns was above 99%, which was confirmed by SDS-PAGE and RP-HPLC analyses. Size exclusion chromatography analysis showed that the purified enzyme has comparable heterogeneity to the Rasburicase. The yield of recombinant uricase production in this study was 63% and its specific activity was 24 U/mg. The high expression of recombinant uricase in the Pichia pink strain and the increased enzyme activity compared to the standard sample indicate the potential of therapeutic and diagnostic applications of recombinant uricase in the present study. [ABSTRACT FROM AUTHOR]

The isolation and quantitative characterization of circulating tumor cells (CTCs) are of great importance in cancer diagnosis and prognosis. However, isolating and detecting CTCs in whole blood presents a significant challenge due to the low numbers of CTCs (often ranging from one to five) in samples containing billions of erythrocytes. Recently, point-of-care devices that use antibody trapping coupled with remote immunofluorescence analyses have been described to identify the number and type of CTCs in blood. In this study, we propose a novel method for trapping and quantifying CTCs using Sepharose 6B beads of 45–160 μm size that are engineered with capture antibodies. Specifically, we employed CD44 antibody conjugates (bearing a maleimide group) that are specific to the CTCs of breast cancer to thiol-Sepharose beads 6B. These beads, when mixed with MDAMB231 and Jurkat cells and filtered through a 40 μm filter, can capture ~80% of MDAMB231 cells. Furthermore, the antibody-modified Sepharose 6B can be stored at four degrees Celsius for a period exceeding six months. [ABSTRACT FROM AUTHOR]

Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection. The fast and accurate diagnosis of sepsis by procalcitonin (PCT) has emerged as an essential tool in clinical medicine. Although in use in the clinical laboratory for a long time, PCT quantification has not yet been standardized. The International Federation of Clinical Chemistry working group on the standardization of PCT (IFCC-WG PCT) aims to provide an LC-MS/MS-based reference method as well as the highest metrological order reference material to address this diagnostic need. Here, we present the systematic evaluation of the efficiency of an immuno-enrichment method, based on functionalized Sepharose, magnetic-core, or polystyrene (latex) nano-particles, to quantitatively precipitate PCT from different human sample materials. This method may be utilized for both mass spectrometric and proteomic purposes. In summary, only magnetic-core nano-particles functionalized by polyclonal PCT antibodies can fulfil the necessary requirements of the international standardization of PCT. An optimized method proved significant benefits in quantitative and specific precipitation as well as in the subsequent LC-MS/MS detection of PCT in human serum samples or HeLa cell extract. Based on this finding, further attempts of the PCT standardization process will utilize a magnetic core-derived immuno-enrichment step, combined with subsequent quantitative LC-MS/MS detection. [ABSTRACT FROM AUTHOR]

Carbonic anhydrases (CAs) are a metalloenzyme that contains zinc ions in their active sites, and they reversibly catalyze the reactions of hydration of CO2 and dehydration of HCO3 - in the living. In this study, human carbonic anhydrase II (hCAII) isoenzyme was purified from human erythrocytes by affinity column (Sepharose 4B-L-tyrosine-sulfanilamide). The inhibitory effect of artificial tear drops on hCAII isoenzyme was investigated. IC50 values were calculated as 1.01 µL for sodium hyaluronate, 140.06 µL for polyvinyl alcohol+povidone, and 49.51 for polyethylene glycol+propylene glycol. The Ki value for sodium hyaluronate was found as 0.339±0.121 mM. According to these results, sodium hyaluronate was the most effective inhibitor in terms of application volume (microliter) among the three drugs examined. [ABSTRACT FROM AUTHOR]

Diverse classes of silencing small (s)RNAs operate via ARGONAUTE-family proteins within RNA-induced-silencing-complexes (RISCs). Here, we have streamlined various embodiments of a Q-sepharose-based RISC-purification method that relies on conserved biochemical properties of all ARGONAUTEs. We show, in multiple benchmarking assays, that the resulting 15-min benchtop extraction procedure allows simultaneous purification of all known classes of RISC-associated sRNAs without prior knowledge of the samples-intrinsic ARGONAUTE repertoires. Optimized under a user-friendly format, the method - coined 'TraPR' for Trans-kingdom, rapid, affordable Purification of RISCs - operates irrespectively of the organism, tissue, cell type or bio-fluid of interest, and scales to minute amounts of input material. The method is highly suited for direct profiling of silencing sRNAs, with TraPR-generated sequencing libraries outperforming those obtained via gold-standard procedures that require immunoprecipitations and/or lengthy polyacrylamide gel-selection. TraPR considerably improves the quality and consistency of silencing sRNA sample preparation including from notoriously difficult-to-handle tissues/bio-fluids such as starchy storage roots or mammalian plasma, and regardless of RNA contaminants or RNA degradation status of samples.

Posttranslational modifications play a critical and diverse role in regulating cellular activities. Despite their fundamentally important role in cellular function, there has been no report to date of an effective generalized approach to the targeting, extraction, and characterization of the critical c-terminal regions of natively prenylated proteins. Various chemical modification and metabolic labeling strategies in cell culture have been reported. However, their applicability is limited to cell culture systems and does not allow for analysis of tissue samples. The chemical characteristics (hydrophobicity, low abundance, highly basic charge) of many of the c-terminal regions of prenylated proteins have impaired the use of standard proteomic workflows. In this context, we sought a direct approach to the problem in order to examine these proteins in tissue without the use of labeling. Here we demonstrate that prenylated proteins can be captured on chromatographic resins functionalized with mixed disulfide functions. Protease treatment of resin-bound proteins using chymotryptic digestion revealed peptides from many known prenylated proteins. Exposure of the protease-treated resin to reducing agents and hydro organic mixtures released c-terminal peptides with intact prenyl groups along with other enzymatic modifications expected in this protein family. Database and search parameters were selected to allow for c-terminal modifications unique to these molecules such as CAAX box processing and c-terminal methylation. In summary, we present a direct approach to enrich and obtain information at a molecular level of detail about prenylation of proteins from tissue and cell extracts using high-performance LC-MS without the need for metabolic labeling and derivatization.

Salmon nasal cartilage proteoglycan (PG) was orally administered to mice. The PG digest was recovered from the small intestine, and its sugar chain size and unsaturated disaccharide content were examined. The elution position of the PG digest following Sepharose CL-4B chromatography was consistent with that of actinase-digested PG prior to administration. The PG digest was incubated with chondroitinase ABC, which resulted in the elution pattern of the unsaturated disaccharides being identical to that of the degraded product of actinase-digested PG. The core protein of PG was digested in the mouse small intestine, but chondroitin sulfate, which is the sugar chain of PG, was not degraded at all. Then, the effects of chondroitin 4- and 6-sulfates on human colon cancer cells were examined. These chondroitin sulfates were found to suppress the expression of interleukin-6 induced by TNF-α. Overall, the chondroitin sulfate chain may act on the intestinal epithelium and suppress inflammation of the intestinal tract. • Chondroitin sulfate is not digested or degraded at all in the mouse small intestine. • Chondroitin sulfate suppresses IL6 expression in a small intestinal epithelial model. • Oral administration of proteoglycans may help ameliorate inflammatory bowel disease. [ABSTRACT FROM AUTHOR]

Integrin αMβ2 (Mac-1, CD11b/CD18, CR3) is an important adhesion receptor expressed on monocytes. Mac-1 is responsible for mediating cell migration, phagocytosis, degranulation as well as cell-cell fusion. It is also the most promiscuous integrin in terms of ligand specificity with over 100 ligands, most of which use the αMI-domain as their binding site. Despite the importance of αMI-domain in defining ligand interactions of Mac-1, structural studies of αMI-domain's interactions with ligands are lacking. In particular, solution NMR studies of αMI-domain's interaction with ligands have not been possible because the most commonly used active αMI-domain mutants (I316G and ΔK315) are not sufficiently stable and soluble to be used in solution NMR. The goal of this study is to identify an αMI-domain active mutant that's amenable to NMR characterization. By screening known activating mutations of αMI-domain, we determined that the Q163C/Q309C mutant, which converts the αMI-domain into its active form through the formation of an intramolecular disulfide bond, can be produced with a high yield and is more stable than other active mutants. In addition, the Q163C/Q309C mutant has better NMR spectral quality than other active mutants and its affinity for ligands is comparable to other active mutants. Analysis of the Co2+-induced pseudocontact shifts in the Q163C/Q309C mutant showed the structure of the mutant is consistent with the active conformation. Finally, we show that the minor fraction of the Q163C/Q309C mutant without the disulfide bond can be removed through the use of carboxymethyl sepharose chromatography. We think the availability of this mutant for NMR study will significantly enhance structural characterizations of αMI-domain-ligand interactions. [ABSTRACT FROM AUTHOR]

Lipoprotein lipase (LPL), the enzyme that hydrolyzes triglycerides in plasma lipoproteins, is assumed to be active only as a homodimer. In support of this idea, several groups have reported that the size of LPL, as measured by density gradient ultracentrifugation, is ∼110 kDa, twice the size of LPL monomers (∼55 kDa). Of note, however, in those studies the LPL had been incubated with heparin, a polyanionic substance that binds and stabilizes LPL. Here we revisited the assumption that LPL is active only as a homodimer. When freshly secreted human LPL (or purified preparations of LPL) was subjected to density gradient ultracentrifugation (in the absence of heparin), LPL mass and activity peaks exhibited the size expected of monomers (near the 66-kDa albumin standard). GPIHBP1-bound LPL also exhibited the size expected for a monomer. In the presence of heparin, LPL size increased, overlapping with a 97.2-kDa standard. We also used density gradient ultracentrifugation to characterize the LPL within the high-salt and low-salt peaks from a heparin-Sepharose column. The catalytically active LPL within the high-salt peak exhibited the size of monomers, whereas most of the inactive LPL in the low-salt peak was at the bottom of the tube (in aggregates). Consistent with those findings, the LPL in the low-salt peak, but not that in the high-salt peak, was easily detectable with single mAb sandwich ELISAs, in which LPL is captured and detected with the same antibody. We conclude that catalytically active LPL can exist in a monomeric state.

Copyright of Food Research & Development is the property of Food Research & Development Editorial Department and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

This article introduces the aptamer affinity column (AAC) with nucleic acid aptamer as an affinity ligand for the extraction of four aminoglycoside antibiotics (AGs). The AAC was prepared by loading the aptamer functionalized Sepharose into an extraction column, which was conjugated by covalent binding between NHS-activated Sepharose and amino-modified aptamers with a coupling time of 2 h. After the sample solution flowed through the AAC, the AGs were retained because of the affinity between the AGs and aptamer, then AGs were eluted and analyzed by UPLC-MS/MS. Under the optimized conditions, the maximum adsorption of AGs on the AAC could reach 8.0 μg. Moreover, the proposed AAC could be reused more than 20 times. The resultant AAC that conjugated with the aptamer was successfully applied in the enrichment and purification of four AGs in a milk sample and good recovery results in the range of 83.3–98.8% were obtained (with RSD in the range of 0.6–5.8%). The proposed AAC for recognition of multi-target AGs exhibited good enrichment and purification effects, showing great application potential for targets with their related aptamers. [ABSTRACT FROM AUTHOR]

The hA5G18 peptide (DDFVFYVGGYPS) identified from the human laminin α5 chain G domain promotes cell attachment and spreading when directly coated on a plastic plate, but does not show activity when it is conjugated on a chitosan matrix. Here, we focused on the structural requirement of hA5G18 for activity. hA5G18 was stained with Congo red and formed amyloid-like fibrils. A deletion analysis of hA5G18 revealed that FVFYV was a minimum active sequence for the formation of amyloid-like fibrils, but FVFYV did not promote cell attachment. Next, we designed functional fibrils using FVFYV as a template for amyloid-like fibrils. When we conjugated an integrin binding sequence Arg-Gly-Asp (RGD) to the FVFYV peptide with Gly-Gly (GG) as a spacer, FVFYVGGRGD promoted cell attachment in a plate coat assay, but a negative control sequence RGE conjugated peptide, FVFYVGGRGE, also showed activity. However, when the peptides were conjugated to Sepharose beads, the FVFYVGGRGD beads showed cell attachment activity, but the FVFYVGGRGE beads did not. These results suggest that RGD and RGE similarly contribute to cell attachment activity in amyloid-like fibrils, but only RGD contributes the activity on the Sepharose beads. Further, we conjugated a basic amino acid (Arg, Lys, and His) to the FVFYV peptide. Arg or Lys-conjugated FVFYV peptides, FVFYVGGR and FVFYVGGK, showed cell attachment activity when they were coated on a plate, but a His-conjugated FVFYV peptide FVFYVGGH did not show activity. None of the basic amino acid-conjugated peptides showed cell attachment in a Sepharose bead assay. The cell attachment and spreading on FVFYVGGR and FVFYVGGK were inhibited by an anti-integrin β1 antibody. These results suggest that the Arg and Lys residues play critical roles in the interaction with integrins in amyloid-like fibrils. FVFYV is useful to use as a template for amyloid-like fibrils and to develop multi-functional biomaterials. [ABSTRACT FROM AUTHOR]

To investigate anti-inflammatory effects and molecular mechanisms of polysaccharides derived from Apocynum venetum L. roots on lipopolysaccharide (LPS)-induced mouse mononuclear macrophage leukemia (RAW 264.7) cells based on their structural characterization. Water soluble polysaccharides (ALRP) purified from Apocynum venetum L. roots were processing by diethylaminoethyl (DEAE)-cellulose, Sepharose CL-6B or Sephadex G100, and semi-preparative high performance gel permeation chromatography (HPGPC), successively. After that, two neutral homogeneous heteropolysaccharides (ALRPN-1 and ALRPN-2) were obtained. Of which, ALRPN-1 was composed of glucose, galactose and arabinose with weight-average molecular (Mw) of 1.542 × 104 Da. While, ALRPN-2 was composed of glucose, galactose and mannose with Mw of 5.105 × 103 Da. Structural characterization suggested that ALRPN-1 and ALRPN-2 had similar linear backbone with different branches. Meanwhile, the senior structure and micromorphology of ALRPN-1 and ALRPN-2 were demonstrated different. Furthermore, they exerted potent anti-inflammatory activities by weakening the phagocytosis to a certain extent, significantly reducing the level of nitric oxide (NO), tumor necrosis factor (TNF-α) and interleukin (IL-6, IL-1β), as well as down-regulating the phosphorylation level of extracellular signal-related kinase (ERK1/2) of mitogen-activated protein kinases (MAPKs) signal pathway in LPS-induced RAW 264.7 cells. This study systematically analyzed the two new neutral polysaccharides purified from Apocynum venetum L. roots and demonstrated their potent anti-inflammatory activities. Our study would provide theoretical support for potential applications of Apocynum venetum L. during traditional use and the health care fields. [Display omitted] • Two polysaccharides (ALRPN-1, ALRPN-2) were purified from Apocynum venetum L. roots. • The primary and senior structure of two polysaccharides were fully characterized. • ALRPN-1/2 have potent anti-inflammatory activity on LPS-induced RAW 264.7 cells. • Anti-inflammatory activity is partly explained from structure-function relationship. [ABSTRACT FROM AUTHOR]

PurposeTo compare the apparent diffusion coefficient (ADC) of hyperpolarized (HP) [13 C,15 N]urea to the ADC of endogenous water in healthy and fibrotic mouse liver.Materials and methodsADC measurements for water and [13 C]urea were made in agarose phantoms at 14.1T. Next, the ADC of water and injected HP [13 C,15 N]urea were measured in eight CD1 mouse livers before and after induction of liver fibrosis using CCl4 . Liver fibrosis was quantified pathologically using the modified Brunt fibrosis score and compared to the measured ADC of water and urea.ResultsIn cell-free phantoms with 12.5% agarose, water ADC was nearly twice the ADC of urea (1.93 × 10-3 mm2 /s vs. 1.00 × 10-3 mm2 /s). The mean ADC values of water and [13 C,15 N]urea in healthy mouse liver (±SD) were nearly identical [(0.75 ± 0.11) × 10-3 mm2 /s and (0.75 ± 0.22) × 10-3 mm2 /s, respectively]. Mean water and [13 C,15 N]urea ADC values in fibrotic liver (±SD) were (0.84 ± 0.22) × 10-3 mm2 /s and (0.75 ± 0.15) × 10-3 mm2 /s, respectively. Neither water nor urea ADCs were statistically different in the fibrotic livers compared to baseline (P = 0.14 and P = 0.99, respectively). Water and urea ADCs were positively correlated at baseline (R2  = 0.52 and P = 0.045) but not in fibrotic livers (R2  = 0.23 and P = 0.23).ConclusionADC of injected hyperpolarized urea in healthy liver reflects a smaller change as compared to free solution than ADC of water. This may reflect differences in cellular compartmentalization of the two compounds. No significant change in ADC of either water or urea were observed in relatively mild stages of liver fibrosis.Level of evidence1 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2018;47:141-151.

C-reactive protein (CRP) is a circulating marker of inflammation yet with ill-defined biological functions. This is partly due to the uncharacterized activities of endogenous CRP in mice, the major animal model used to define protein function. The hurdles for purification and characterization of mouse CRP are its low circulating levels and the lack of specific antibodies. To clear these hurdles, here we developed an efficient expression system by constructing recombinant Pichia pastoris cells for secretion of native conformation mouse CRP. The recombinant expression of mouse CRP in Escherichia coli failed to yield sufficient amount of native protein, reflecting the importance of post-translational modification of glycosylation in aiding proper folding. By contrast, sufficient amount of native mouse CRP was successfully purified from P. pastoris. Preliminary purification was performed by Nickel Chelating Sepharose Fast-Flow affinity chromatography with 6 × His tags attached to the protein. Subsequently, p -Aminophenyl Phosphoryl Choline Agarose resin affinity chromatography was used for tandem purification. The purified mouse CRP showed native pentamer and capabilities of PC binding. Moreover, the 6 × His tag provides a convenient tool for detecting the interactions of mouse CRP with ligands. [ABSTRACT FROM AUTHOR]

A microwave-assisted extraction procedure for the crude Salvia miltiorrhiza polysaccharides (SMPs) obtained from Salvia miltiorrhiza Bunge was optimized. Four independent variables were studied: microwave power, extraction time, solvent-to-solid ratio, and concentration of ethanol, with optimal settings of 1200 W, 12 min, 38, and 86 %, respectively. The SMPs were successively purified by DEAE Sepharose Fast Flow and Sephadex G-100 chromatography to produce a novel polysaccharide termed SMP1. The SMP1 was composed of glucose, galactose, and fructose in a molar ratio of 1:1.67:1.12 with an average molecular weight of 6087 Da. Pharmacological studies showed that SMP1 protected from OGD/R-induced ferroptosis and lipid peroxidation by activating Nrf2/HO-1 pathway in PC12 cells. Our research systematically indicated that polysaccharide could inhibit ferroptosis to alleviate oxidative stress injury, which laid the foundation for the future clinical application of Salvia miltiorrhiza polysaccharide. [Display omitted] • The extraction condition of crude Salvia miltiorrhiza polysaccharides was optimized. • The primary structure of a novel polysaccharide named SMP1 was analyzed. • SMP1 activated Nrf2/HO-1pathway to inhibit ferroptosis in PC12 cells. [ABSTRACT FROM AUTHOR]

Polysaccharides from Pimpinella anisum were isolated using water at elevated temperature and DEAE Sepharose FF chromatography to examine their chemical structure and activation capacity on immune cells. P. anisum fractions (PAF1, PAF2 and PAF3) were mainly composed of neutral sugars (84.0–98.2%) and uronic acids (2.1–11.8%) with weight average molecular weight (M w) ranging from 186.6 to 5474.5 × 103 g/mol. Polysaccharides induced a significant inflammatory response in RAW264.7 murine macrophage cells releasing nitric oxide and expressing TNF-α, IL-1β, IL-6 and IL-10 cytokines. The induction of NK-92 natural killer cells resulted in TNF-α and IFN-γ production and activation of GrB/perforin-, NKG2D- and FasL-mediated cytotoxicity. Polysaccharides triggered the phosphorylation of NF-κB, ERK, JNK and p38 proteins in RAW264.7 and NK-92 cells indicating the involvement of NF-κB and MAPKs signaling pathways. The most active polysaccharide was a galactoarabinan with complex structure. • A galactoarabinan (PAF2) was isolated from Pimpinella anisum. • Galactoarabinan stimulated RAW264.7 and NK cells to produce proinflammatory mediators. • RAW264.7 and NK cells activated through NF-κB and MAPKs pathways. • Optimum M w and conformation was essential for cell activation capacity of galactoarabinan. [ABSTRACT FROM AUTHOR]

Advances in methods that determine cell mechanical phenotype, or mechanotype, have demonstrated the utility of biophysical markers in clinical and research applications ranging from cancer diagnosis to stem cell enrichment. Here, we introduce quantitative deformability cytometry (q-DC), a method for rapid, calibrated, single-cell mechanotyping. We track changes in cell shape as cells deform into microfluidic constrictions, and we calibrate the mechanical stresses using gel beads. We observe that time-dependent strain follows power-law rheology, enabling single-cell measurements of apparent elastic modulus, Ea, and power-law exponent, β. To validate our method, we mechanotype human promyelocytic leukemia (HL-60) cells and thereby confirm q-DC measurements of Ea = 0.53 ± 0.04 kPa. We also demonstrate that q-DC is sensitive to pharmacological perturbations of the cytoskeleton as well as differences in the mechanotype of human breast cancer cell lines (Ea = 2.1 ± 0.1 and 0.80 ± 0.19 kPa for MCF-7 and MDA-MB-231 cells). To establish an operational framework for q-DC, we investigate the effects of applied stress and cell/pore-size ratio on mechanotype measurements. We show that Ea increases with applied stress, which is consistent with stress stiffening behavior of cells. We also find that Ea increases for larger cell/pore-size ratios, even when the same applied stress is maintained; these results indicate strain stiffening and/or dependence of mechanotype on deformation depth. Taken together, the calibrated measurements enabled by q-DC should advance applications of cell mechanotype in basic research and clinical settings.

Aims: Staphylococcus aureus is an opportunistic pathogen whose treatment is further complicated by its ability to form biofilms. In this study, we examine the impact of growing S. aureus biofilms on different polymerizing surfaces, specifically agar and agarose, on the pathogen's tolerance to fluoroquinolones., Methods and Results: Biofilms of two methicillin-resistant strains of S. aureus were grown on agar or agarose in the presence of the same added nutrients, and their antibiotic susceptibility to two fluoroquinolones, moxifloxacin (MXF) and delafloxacin (DLX), were measured. We also compared the metabolism and extracellular polymeric substances (EPS) production of biofilms that were grown on agar and agarose., Conclusions: Biofilms that were grown on agarose were consistently more susceptible to antibiotics than those grown on agar. We found that in biofilms that were grown on agar, extracellular protein composition was higher, and adding EPS to agarose-grown biofilms increased their tolerance to DLX to levels that were comparable to agar-grown biofilms., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Bacteria are often found in environments where space is limited, and they attach themselves to surfaces. One common form of movement on these surfaces is bacterial twitching motility, which is powered by the extension and retraction of type IV pili. Although twitching motility in unrestricted conditions has been extensively studied, the effects of spatial confinement on this behaviour are not well understood. In this study, we explored the diffusive properties of individual twitching Pseudomonas aeruginosa cells in spatially confined conditions. We achieved this by placing the bacteria between layers of agarose and glass, and then tracking the long-term twitching motility of individual cells. Interestingly, we found that while confinement reduced the immediate speed of twitching, it paradoxically increased diffusion. Through a combination of mechanical and geometrical analysis, as well as numerical simulations, we showed that this increase in diffusion could be attributed to mechanical factors. The constraint imposed by the agarose altered the diffusion pattern of the bacteria from normal to superdiffusion. These findings provide valuable insights into the motile behaviour of bacteria in confined environments., (© 2024 John Wiley & Sons Ltd.)

Two polysaccharide fractions from Pogostemon cablin (Blanco) Benth. (P. cablin) (designated as PCB-1 and PCB2-1) were isolated by water extraction and purified by Sepharose chromatography. The chemical properties of the polysaccharides were characterised, and their antioxidant activities were evaluated. The sugar content of the crude polysaccharide (PCB), PCB-1, and PCB2-1 was 58.74, 90.23 and 88.61%, respectively. The molecular weights of PCB-1 and PCB2-1 were determined to be 97.8 and 12.8 kDa, respectively. Monosaccharide composition analysis showed that all the three polysaccharides consisted of mannose, rhamnose, galacturonic acid, galactose, glucose, and arabinose, but with varying molar ratios. The polysaccharides exhibited significantly high antioxidant activities in vitro based on the scavenging activity against hydroxyl radicals, metal ion-chelating and ferric-reducing abilities. In vivo experiments in an oxidatively damaged mice model showed that PCB-1 increased the levels of antioxidant enzymes, including superoxide dismutase, catalase, and glutathione peroxidase, and inhibited malondialdehyde formation in the serum and liver. These findings suggest that PCB-1 has significant potential as an antioxidant in functional foods. [ABSTRACT FROM AUTHOR]

The article presents the results of the comparative development of different variants of immunoaffinity sorbents for the purification of human IL-7, namely: Sepharose 6B and sol-gel materials based on tetraethoxysilane (TEOS). The optimal conditions for the synthesis of a chromatographic sorbent based on TEOS, which provide acceptable characteristics of the immobilization of a biological substance, were experimentally established. The sorbent based on TEOS and Sepharose 6B provide identical characteristics for the sorption of antibodies after 12 hours of the experiment. To establish rational conditions for immunoaffinity chromatography, various combinations of sorbents, anti-IL-7 monoclonal antibodies, and elution solutions were studied. It was shown that a variant of immunoaffinity chromatography based on TEOS sorbent using 325D2 anti-IL-7 monoclonal antibodies and 4M magnesium chloride solution as eluate, is more effective. Immunochromatographic columns synthesized on the basis of Sepharose 6B and TEOS are characterized by high recovery rates (at least 95%) in a wide range of human IL-7 concentrations (0.05...20 µg/ml), however, the column based on Sepharose 6B is less than stable with repeated use (more than 12 cycles without regeneration). The developed method makes it possible to obtain recombinant human IL-7 with a purity of 98%. [ABSTRACT FROM AUTHOR]

This study aimed to purify human chorionic gonadotropin (HCG) from the urine of pregnant women with high biological activity (10811 IU/mg) and purity (98.2%), by simple capturing of HCG using DEAE Sepharose FF and polishing using Sephacryl S200 HR. The HCG obtained was characterized by SDS-PAGE and dissociated into alpha and beta subunits using the urea treatment method. The βHCG subunits were injected into rabbits for the production of highly specific polyclonal anti-βHCG antisera. The polyclonal anti-βHCG was locally produced in rabbits and assessed for binding titer (1/10000), displacement (84.8%), and specificity (98.8%). Purified HCG along with locally prepared polyclonal anti-βHCG antisera were used as basic components of the in-house Radioimmunoassay system for quantitative estimation of HCG in human serum. [ABSTRACT FROM AUTHOR]

Purpose: Sulfated polysaccharide from Codium species has been reported for its antiinflammatory activities. However, the effect of sulfated polysaccharide from Codium edule on allergic responses has not been studied. The study was conducted to determine the effect of sulfated polysaccharide (F1) from C. edule on allergic contact dermatitis (ACD) induced by 2,4-dinitrofluorobenzene (DNFB) in female BALB/c mice. Methods: F1 was isolated using DEAE sepharose gel chromatography and chemically identified by LC-MS analyses. The effects of F1 on changes in ear thickness, allergic responses, and histology were evaluated. The effects of F1 on the production of inflammatory cytokines interferon gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) in serum were also quantified and compared with standard prednisolone therapy. Results: F1 was identified as a heteropolysaccharide with β-D-galactans and β-L-arabinans units. F1 was non-toxic at 2000 mg/kg. Administration of F1 in DNFB-challenged mice significantly suppressed the increase in ear thickness, erythema, desquamation, and proliferation of inflammatory cells. F1 significantly decreased the production of inflammatory markers, IFN-γ and TNF-a in a dose-dependent manner when compared to the untreated group (P<0.05). Conclusion: Results suggest that F1 from C. edule is a bioactive sulfated heteropolysaccharide with anti-inflammatory activity and might be a valuable candidate molecule for the treatment of allergic diseases such as ACD. [ABSTRACT FROM AUTHOR]

Extracellular vesicles (EVs) are a versatile group of cell-secreted membranous nanoparticles present in body fluids. They have an exceptional diagnostic potential due to their molecular content matching the originating cells and accessibility from body fluids. However, methods for EV isolation are still in development, with size exclusion chromatography (SEC) emerging as a preferred method. Here we compared four types of SEC to isolate EVs from the CSF of patients with severe traumatic brain injury. A pool of nine CSF samples was separated by SEC columns packed with Sepharose CL-6B, Sephacryl S-400 or Superose 6PG and a ready-to-use qEV10/70 nm column. A total of 46 fractions were collected and analysed by slot-blot followed by Ponceau staining. Immunodetection was performed for albumin, EV markers CD9, CD81, and lipoprotein markers ApoE and ApoAI. The size and concentration of nanoparticles in fractions were determined by tunable resistive pulse sensing and EVs were visualised by transmission electron microscopy. We show that all four SEC techniques enabled separation of CSF into nanoparticle- and free protein-enriched fractions. Sepharose CL-6B resulted in a significantly higher number of separated EVs while lipoproteins were eluted together with free proteins. Our data indicate that Sepharose CL-6B is suitable for isolation of EVs from CSF and their separation from lipoproteins. [ABSTRACT FROM AUTHOR]

[Display omitted] • Developed a proof-of-concept scFv-based immunochromatographic (IAC) assay to detect a widely misused substance, salbutamol. • The IAC assay demonstrated the application of moderate-affinity antibodies for high-sensitivity detection platforms. Widespread misuse of salbutamol as an athletic performance enhancer and livestock growth promoter necessitates its detection in biological matrices. Antibodies remain important immunodiagnostic reagents in the detection of salbutamol. Previously, a novel anti-salbutamol chicken scFv (clone OP2B1C4) was generated from a SAL-KLH-immunized chicken antibody phage display library. This scFv (K D = ∼0.592 μM) was used to develop an indirect competitive (ic) ELISA capable of detecting salbutamol at the high ng/mL to low μg/mL levels for human doping urinalysis but was incapable of low ng/mL detection. In this study, the same scFv OP2B1C4 was used to develop a prototype immunoaffinity chromatographic (IAC) adsorbent for higher sensitivity applications. The scFv was immobilized on CNBr-activated Sepharose 4B and the scFv-IAC was profiled for its binding capacity and recovery. From three consecutive runs, estimated binding capacity was > 7 μg with elution recoveries of 59.43 %, 69.34 % and 73.8 %. When used as a clean-up SPE prior to LC-MS/MS analysis, the scFv-IAC could isolate salbutamol at a concentration of 1 ng/mL (∼100-fold lower than ic-ELISA LOD) in salbutamol-spiked porcine urines with relative recoveries ranging from 76.96 % − 99.85 %. The results highlight the potential of often-neglected moderate-affinity antibodies for high-sensitivity applications through extension onto IAC-based immunodiagnostic platforms. [ABSTRACT FROM AUTHOR]

3D printing of biological architectures that mimic the structural and functional features of in vivo tissues is of great interest in tissue engineering and the development of transplantable organ constructs. Printable bio-inks that are compatible with cellular activities play critical roles in the process of 3D bio-printing. Although a variety of hydrogels have been used as bio-inks for 3D bio-printing, they inherit poor mechanical properties and/or the lack of essential protein components that compromise their performance. Here, a hybrid Matrigel-agarose hydrogel system has been demonstrated that possesses both desired rheological properties for bio-printing and biocompatibility for long-term (11 days) cell culture. The agarose component in the hybrid hydrogel system enables the maintenance of 3D-printed structures, whereas Matrigel provides essential microenvironments for cell growth. When human intestinal epithelial HCT116 cells are encapsulated in the printed Matrigel-agarose constructs, high cell viability and proper cell spreading morphology are observed. Given that Matrigel is used extensively for 3D cell culturing, the developed 3D-printable Matrigel-agarose system will open a new way to construct Matrigel-based 3D constructs for cell culture and tissue engineering.

Comprehensive Summary: A convenient and highly efficient strategy was developed for the simplification of enzymatic synthesis and purification of oligosaccharides. Glycosyl acceptors terminated with a thiol tag were extended by enzymatic modular assembly (EMA) in the aqueous phase, and the desired products were captured during solid‐phase workup (SPW) using commercially available thiopropyl Sepharose 6B resin through a reversible disulfide linkage. Finally, the products were released from solid‐phase resin in the presence of dithiothreitol (DTT) as reducing agent and the resin could be recovered and reused. This strategy overcomes the uncertain influence of polymer‐ or ionic‐supports commonly used in enzymatic glycosylation, and showed perfect compatibility with all 10 enzyme modules for the rapid assembly of a series of complex oligosaccharides. [ABSTRACT FROM AUTHOR]

Scyphozoan Nemopilema nomurai envenomation is an unresolved threat to human health in Asian waters. Nemopilema nomurai venom metalloproteinases show important toxicities in skin damage and inflammation, but there is still no purified protein for further studies. In this study, high proteinase activity fractions in tentacle autolysis were isolated by ammonium sulfate precipitation, DEAE Sepharose Fast Flow, and Superdex 75 chromatography successively. Purification was guided by azocasein hydrolysis activity and SDS-PAGE. The final products were analyzed by LC-MS/MS. Four elution peaks purified by Superdex 75 chromatography had multiple protein bands but did not show proteinase activity. These fractions would recover proteinase activity after mixing again. Regulation mechanisms were speculated as binding metalloproteinase regulator or disaggregating metalloproteinase inhibitor by LC-MS/MS analysis. For the first time, a synergistic effect in N. nomurai proteinase activity was found in the purification process. [ABSTRACT FROM AUTHOR]

Aim. To develop a method for isolation and purification of recombinant human interleukin-7 from bacterial inclusion bodies. Methods. Protein synthesis via fermentation, densitometry, refolding method of gel filtration, two-stage chromatographic purification of the protein. Results. The inclusion bodies obtained via fermentation in E.coli cells contained recombinant human interleukin-7, which was solubilized at 22ºС for 1 hour in the following buffer solution: 7M of guanidine hydrochloride, 100mM of Tris-НСl, 0.1 % Tween-20 and 50mM of dithiothreitol. Renaturation was conducted by replacing the buffer on column XK26 / 40 (GE Healthcare) with Sephadex G-25 fine equilibrated Clark-Labs solution containing L-arginine hydrochloride and tween-20. Subsequent purification of rIL-7 on the gel filtration column was performed in two stages: at the first stage, purification of rIL-7 was performed on a column packed with 20 ml of Q sepharose, at the second stage, the rIL-7 fraction purified on Q sepharose was purified on SP sepharose. Elution of rIL-7 was performed using double-stage gradient NaCL (0.2 and 0.7M) in a buffer 0.05 M of KH2PO4/NaOH with pH 6.0; 0.1 М of L-arginine; 0.1 % Tween-80. Conclusions. The amount of the target protein after all the proposed stages of purification was 10 % with the purity ratio about 95 %, which is sufficient for further development of the various pharmaceutical forms. [ABSTRACT FROM AUTHOR]

Hirsutella sinensis , the anamorph of Ophiocordyceps sinensis (syn. Cordyceps sinensis), is a great substitute for precious and rare wild Cordyceps sinensis to effectively treat a variety of lung and kidney diseases. In this study, an α-glucan (named as HSWP-2a) was obtained by hot water extraction, DEAE-cellulose separation, and Sepharose CL-6B purification from H. sinensis mycelia. Different from known α-glucans, HSWP-2a is an α-(1 → 4)-D-glucan that branched at O -6, O -3, or O -2 with a terminal 1-linked α-D-Glc p as side chain, with an average molecular weight of 870.70 kDa. Immunological tests showed that HSWP-2a could remarkably enhance the phagocytosis of macrophages and increase the production of NO, IL-1β, IL-6, and TNF-α, via activating the p38, JNK, and NF-κB signaling pathways. Moreover, HSWP-2a could significantly promote splenic lymphocyte proliferation. Taken together, HSWP-2a may be potentially utilized as a natural immunomodulatory agent. • A water-soluble polysaccharide (HSWP-2a) was isolated from Hirsutella sinensis mycelia. • HSWP-2a was an α-(1 → 4)-D-glucan that branched at O -6, O -3, or O -2 with a terminal 1-linked α-D-Glc p as side chain. • HSWP-2a exhibited remarkable immune-enhancing activities on RAW264.7 cells through the p38, JNK, and NF-κB signaling pathways. • HSWP-2a could significantly promote the proliferation of T and B lymphocytes. [ABSTRACT FROM AUTHOR]

Sulfated polysaccharides presenting in the cell wall of seaweeds and aquatic plants exhibit a diverse range of biological functions mainly due to their particular structural and molecular properties. The aim of the current study was to evaluate the inhibition of oxidation reactions and activities of digestive enzymes including α-amylase and αglucosidase by sulfated polysaccharides from aquatic plant Myriophyllum spicatum. After the removal of pigments and low molecular weight compounds, polysaccharides were extracted by distilled water at 60°C for 2 hours. Polysaccharide purification using DEAE Sepharose FF led to production of two fractions with different electric charges including F1 and F2. Obtained polysaccharides were considerably capable of scavenging DPPH free radicals (20.62-64.81%) and reducing Fe3+ ions (0.24-0.50 Abs) with F1 fraction exhibiting the greatest activity. Polysaccharides inhibitions against α-amylase activity were greatly different (22.50- 63.20%) with the lowest IC50 being 1.31 mg/mL for F2 fraction. Compared with control group, α-glucosidase activity was reduced to various levels (1.30-89.5%) and the lowest IC50 of 10.0 mg/mL was observed for crude polysaccharide. Overall, these findings indicated that sulfated polysaccharides from M. spicatum L. possess promising antioxidant, α-amylase and α-glucosidase inhibition potentials depending on their chemical and molecular structure and thus could be considered as antioxidant and anti-diabetic agents. [ABSTRACT FROM AUTHOR]

The research includes the partial purification of the angiotensin-converting enzyme (ACE) from the serum of normal human blood and determine the effect of two kind of inhibitor having a nitrile group on the ACE activity. The enzyme was purified in three steps, precipitation by ammonium sulfate (70%), dialysis and gel filtration by sepharose 6-B. Also, molecular docking studies were applied by using AutoDock 4.2, not only to determine the binding mode of the inhibitors, but also to determine the correlation between the docking score values. The results confirmed a marked increase in angiotensin-converting enzyme activity and specific activity and a decrease in total activity as the purification stages progressed. The descending of the purer enzyme with the highest efficacy from the separation column after the passage of samples in the purification stages was identical to the descending of the higher efficacy. The results showed inhibition of enzyme activity with inhibitors 1-(3- mercapto-2-methylpropanoyl) pyrrolidine-2-carbonitrile (1) and 5N-(acetonitrile)-1,1-dimethylbiguanide) (2) and the inhibition percentage was directly proportional to inhibitory concentrations for experimental part. Molecular docking calculations showed that inhibtor 1 and 2 as ligands having binding affinity with ACE as target. The effect of nitrile derivatives has been studied on the ability to inhibit purified ACE, as this type of derivative was chosen because of the presence of the active nitrile group, which has many drug applications and inhibiting activities of enzymes. Besides experimental data, docking studies revealed that inhibitor 2 complex is more effective and has different interaction and conformation trends than inhibitor 1 complex against the related target protein, ACE. [ABSTRACT FROM AUTHOR]

The small GTPase Rat sarcoma virus proteins (RAS) are key regulators of cell growth and involved in 20-30% of cancers. RAS switches between its active state and inactive state via exchange of GTP (active) and GDP (inactive). Therefore, to study active protein, it needs to undergo nucleotide exchange to a non-hydrolysable GTP analog. Calf intestine alkaline phosphatase bound to agarose beads (CIP-agarose) is regularly used in a nucleotide exchange protocol to replace GDP with a non-hydrolysable analog. Due to pandemic supply problems and product shortages, we found the need for an alternative to this commercially available product. Here we describe how we generated a bacterial alkaline phosphatase (BAP) with an affinity tag bound to an agarose bead. This BAP completely exchanges the nucleotide in our samples, thereby demonstrating an alternative to the commercially available product using generally available laboratory equipment., Competing Interests: Declaration of Competing interest The authors declare they have no conflicts of interest with the contents of this article., (Copyright © 2024 Elsevier Inc. All rights reserved.)

There is growing interest in understanding the biological implications of single cell heterogeneity and heteroplasmy of mitochondrial DNA (mtDNA), but current methodologies for single-cell mtDNA analysis limit the scale of analysis to small cell populations. Although droplet microfluidics have increased the throughput of single-cell genomic, RNA, and protein analysis, their application to sub-cellular organelle analysis has remained a largely unsolved challenge. Here, we introduce an agarose-based droplet microfluidic approach for single-cell, single-mtDNA analysis, which allows simultaneous processing of hundreds of individual mtDNA molecules within >10,000 individual cells. Our microfluidic chip encapsulates individual cells in agarose beads, designed to have a sufficiently dense hydrogel network to retain mtDNA after lysis and provide a robust scaffold for subsequent multi-step processing and analysis. To mitigate the impact of the high viscosity of agarose required for mtDNA retention on the throughput of microfluidics, we developed a parallelized device, successfully achieving ~95 % mtDNA retention from single cells within our microbeads at >700,000 drops/minute. To demonstrate utility, we analyzed specific regions of the single-mtDNA using a multiplexed rolling circle amplification (RCA) assay. We demonstrated compatibility with both microscopy, for digital counting of individual RCA products, and flow cytometry for higher throughput analysis., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Background: Highly toxic organophosphorus nerve agents often exist in the form of gas in the environment and can damage human neuroregulatory system by inhibiting the activity of acetylcholinesterase (AChE). However, fluorescent probes based on small organic molecules bring a secondary burden to environment, and their sensitivity and specificity for sarin simulant diethyl chlorophosphate (DCP) detection are unsatisfactory. Nanozyme cascade systems with signal amplification can be used for highly sensitive identification of analytes, but are rarely used in ratiometric analysis of DCP. Combination of enzyme cascades and ratiometric fluorescence ensures the accuracy and sensitivity of the output signal., Results: We prepared a self-assembled nanohybrid (Ag-AuNCs@UiO-66-NH 2 ) by metal-organic framework material and gold nanoclusters. On the one hand, UiO-66-NH 2 with enzyme-like activity was used to hydrolyze DCP into diethyl phosphate (DEP) and chloridion (Cl - ). Cl - hindered aggregation-induced enhanced emission (AIEE) of AuNCs by binding with Ag + and decreased the fluorescence of AuNCs. On the other hand, ligand metal charge transfer effect (LMCT) of UiO-66-NH 2 was blocked by DCP to enhance the fluorescence of UiO-66-NH 2 . Combining ratiometric analysis and nanozyme cascade reaction, an ultra-sensitive fluorescence sensor for detecting DCP was constructed, and ensured the accuracy of experimental results. In addition, Ag-AuNCs@UiO-66-NH 2 was embedded into the agarose hydrogel substrate, the resulting agarose hydrogel film allowed quantitative assessment of DCP vapor and high sensitivity was demonstrated (detection limit as low as 1.02 ppb)., Significance: A strategy combining enzyme cascade with ratiometric fluorescence was proposed, which improved the accuracy and sensitivity of the analysis results. The soft-solid platform based on agarose hydrogel film was constructed to realize the quantitative monitoring of sarin simulant gas. The LOD value obtained in this work is much lower than the immediately life-threatening or health threatening concentration of sarin., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Rapid and accurate detection of the zoonotic nematode Anisakis is poised to control its epidemic. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-associated assay shows great potential in the detection of pathogenic microorganisms. The one-tube method integrated the CRISPR system with the recombinase polymerase amplification (RPA) system to avoid the risk of aerosol pollution; however, it suffers from low sensitivity due to the incompatibility of the two systems and additional manual operations. Therefore, in the present study, the agarose hydrogel boosted one-tube RPA-CRISPR/Cas12a assay was constructed by adding the CRISPR system to the agarose hydrogel, which avoided the initially low amplification efficiency of RPA caused by the cleavage of Cas12a and achieved reaction continuity. The sensitivity was 10-fold higher than that of the one-tube RPA-CRISPR/Cas12a system. This method was used for Anisakis detection within 80 min from the sample to result, achieving point-of-care testing (POCT) through a smartphone and a portable device. This study provided a novel toolbox for POCT with significant application value in preventing Anisakis infection.

Proximity-dependent biotinylation (PDB) techniques provide information about the molecular neighborhood of a protein of interest, yielding insights into its function and localization. Here, we assessed how different labeling enzymes and streptavidin resins influence PDB results. We compared the high-confidence interactors of the DNA/RNA-binding protein transactive response DNA-binding protein 43 kDa (TDP-43) identified using either miniTurbo (biotin ligase) or APEX2 (peroxidase) enzymes. We also evaluated two commercial affinity resins for purification of biotinylated proteins: conventional streptavidin sepharose versus a new trypsin-resistant streptavidin conjugated to magnetic resin, which significantly reduces the level of contamination by streptavidin peptides following on-bead trypsin digestion. Downstream analyses involved liquid chromatography coupled to mass spectrometry in data-dependent acquisition mode, database searching, and statistical analysis of high-confidence interactors using SAINTexpress. The APEX2-TDP-43 experiment identified more interactors than miniTurbo-TDP-43, although miniTurbo provided greater overlap with previously documented TDP-43 interactors. Purifications on sepharose resin yielded more interactors than magnetic resin in small-scale experiments using a range of magnetic resin volumes. We suggest that resin-specific background protein binding profiles and different lysate-to-resin ratios cumulatively affect the distributions of prey protein abundance in experimental and control samples, which impact statistical confidence scores. Overall, we highlight key experimental variables to consider for the empirical optimization of PDB experiments.

Biomaterials capable of delivering therapeutic proteins are relevant in biomedicine, yet their manufacturing relies on centralized manufacturing chains that pose challenges to their remote implementation at the point of care. This study explores the viability of confined cell-free protein synthesis within porous hydrogels as biomaterials that dynamically produce and deliver proteins to in vitro and in vivo biological microenvironments. These functional biomaterials have the potential to be assembled as implants at the point of care. To this aim, we first entrap cell-free extracts (CFEs) from Escherichia coli containing the transcription-translation machinery, together with plasmid DNA encoding the super folded green fluorescence protein (sGFP) as a model protein, into hydrogels using various preparation methods. Agarose hydrogels result in the most suitable biomaterials to confine the protein synthesis system, demonstrating efficient sGFP production and diffusion from the core to the surface of the hydrogel. Freeze-drying (FD) of agarose hydrogels still allows for the synthesis and diffusion of sGFP, yielding a more attractive biomaterial for its reconstitution and implementation at the point of care. FD-agarose hydrogels are biocompatible in vitro, allowing for the colonization of cell microenvironments along with cell proliferation. Implantation assays of this biomaterial in a preclinical mouse model proved the feasibility of this protein synthesis approach in an in vivo context and indicated that the physical properties of the biomaterials influence their immune responses. This work introduces a promising avenue for biomaterial fabrication, enabling the in vivo synthesis and targeted delivery of proteins and opening new paths for advanced protein therapeutic approaches based on biocompatible biomaterials.

In this study, we conducted Ferguson plot analyses using both agarose and polyacrylamide gels in native electrophoresis and SDS-PAGE. The results revealed intriguing differences in the behavior of bovine serum albumin (BSA) and other model proteins. Specifically, BSA exhibited Ferguson plot slopes that were dependent on the oligomer size in agarose native gel electrophoresis, while such size-dependent behavior was not observed in native-PAGE or SDS-PAGE. These findings suggest that Ferguson plot analysis is a suitable approach when using agarose gel under the electrophoretic conditions employed in this study. Furthermore, our investigation extended to model proteins with acidic isoelectric points and larger molecular weights, namely Ferritin and caseinolytic peptidase B (ClpB). Notably, these proteins displayed distinct Ferguson plot slopes when subjected to agarose gel electrophoresis. Intriguingly, when polyacrylamide gel was employed, ClpB exhibited multiple bands, each with its unique Ferguson plot slope, deviating from the expected behavior based on molecular size. This divergence in Ferguson plot characteristics between agarose and polyacrylamide gels points to an interesting and complex interplay between protein properties and gel electrophoresis conditions., Competing Interests: Declaration of competing interest This work was supported by Kyokuto Pharmaceutical Industrial Co., Ltd. Y.T., and T.A. (Teruo Akuta) are employees of the for-profit company Kyokuto Pharmaceuticals. M.T. is emeritus professor of the Kagoshima university and has no conflict of interest. T.A. (Tsutomu Arakawa) used to belong to the for-profit company Alliance Protein Laboratories but currently has no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A new type of core-shell microsphere was prepared by a pre-crosslinking method, consisting of cross-linked agarose microspheres as the core and agarose-dextran as the shell. After optimizing the preparation process, the microspheres with a uniform particle size were obtained and characterized using cryo-scanning electron microscopy to determine their surface and cross-sectional morphology. Results from flow rate-pressure and chromatographic performance tests showed that the core-shell agarose microspheres were supported by the core microspheres and composed of composite polysaccharides, forming an interpenetrating polymer network structure as a hard shell. The core-shell agarose microspheres showed a 300.5 % increase in linear flow rate compared to composite polysaccharide microspheres prepared from shell materials and a 141.5 % increase compared to 6 % agarose microspheres. Additionally, the large pore structure of the shell combined with the fine pore structure of the core improved the material separation efficiency in the range of 0.1-2000 kDa. These findings suggest that core-shell natural polysaccharide microspheres have great potential as a separation chromatographic medium., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Postoperative abdominal adhesions often occur after abdominal surgery; barrier membranes which mimic peritoneal tissue can be constructed to prevent abdominal adhesions. To this end, silk fibroin (SF) sheets were coated with polyvinyl alcohol (PVA) and agarose (AGA) at PVA:AGA ratios of 100:0, 70:30, 50:50, 30:70, and 0:100 to create a composite anti-adhesive barrier and allow us to identify a suitable coating ratio. The membranes were characterized in terms of their molecular organization, structure, and morphology using Fourier transform Infrared spectrometer (FT-IR), differential scanning calorimeter (DSC), and scanning electron microscope (SEM), respectively. The physical and mechanical properties of the membranes and their biological performance (i.e., fibroblast proliferation and invasion) were tested in vitro. Each membrane showed both smooth and rough surface characteristics. Membranes coated with PVA:AGA at ratios of 100:0, 70:30, 50:50, and 30:70 exhibited more -OH and amide III moieties than those coated with 0:100 PVA:AGA, which consequently affected structural organization, degradation, and fibroblast viability. The 0:100 PVA:AGA-coated degraded the fastest. Barrier membranes coated with 100:0 and 70:30 PVA: AGA demonstrated reduced fibroblast proliferation and attachment. The membrane coated with 70:30 PVA:AGA exhibited a stable appearance, and did not curl under wet conditions. Therefore, SF sheets coated with 70:30 PVA:AGA show promise as anti-adhesive barrier membranes for further development., Competing Interests: Declaration of competing interest On behave of the corresponding and co-authors, I would like to declare that we have no conflict of interest on this manuscript., (Copyright © 2024 Elsevier Ltd. All rights reserved.)