Anion-exchange Chromatography Research Articles

Non-affinity platform for processing knob-into-hole bispecific antibody

Bispecific antibodies hold significant potential as next-generation biotherapeutics owing to their ability to simultaneously bind to two targets. However, the development of bispecific antibodies as biotherapeutics has been hindered by the high levels of byproducts produced, including both high molecular weight and low molecular weight variants. In addition, the inevitable expression of homodimers in host cells presents further obstacles to the commercial development of bispecific antibodies as therapeutics. These byproducts, which share similar physicochemical properties with the target, pose several challenges for downstream purification processes. In this study, we present a non-protein A purification platform that employ a one-step polishing chromatography to purify bispecific antibodies. Mixed-mode Capto adhere resin was used to capture the target protein at pH 7.90 ± 0.10, followed by anion exchange chromatography as a polishing step. Overall, the results of this two-step chromatography purification method demonstrated at final product purity of 98% as assessed by size-exclusion high-performance liquid chromatography (SEC-HPLC) and 98% by reversed-phase-high-performance liquid chromatography (RP-HPLC), with residual host cell proteins controlled at 10 ppm and an excellent recovery rate of approximately 60%. This study presents a non-protein A capture platform, offering a simplified, streamlined, and competitive alternative to conventional affinity chromatography.Graphical

Biochemical Characterization of Mannanase from Newly Isolated Acinetobacter sp. KUB-ST1-1 and its Hydrolysate Containing Mannooligosaccharides: Potential as Applied Prebiotic for Pet Food

The β-mannanase enzyme derived from isolated Acinetobacter sp. KUB-ST1-1 was studied for its potential for mannooligosaccharide production. Extracellular mannanase from Acinetobacter sp. KUB-ST1-1, cultured in nutrient broth with 0.5% (w/v) locust bean gum, was purified using ultrafiltration, anion exchange chromatography, and cation exchange chromatography. The enzyme, with a molecular weight of 57 kDa, had high activity in a 50 mM phosphate buffer at pH 6.0 and was stable from pH 4.0 to 7.0. It had high-temperature stability at 40 °C and 50 °C for up to 18 h. This enzyme was highly active toward konjac and galactomannan, especially with locust bean gum and copra meal. The hydrolysis products consisted mainly of the 2 and 3 units of mannooligosaccharide. The mannooligosaccharide exhibited prebiotic properties by promoting increased growth of some beneficial lactic acid bacteria. Furthermore, the defatted copra meal hydrolysate had considerable resistance to pepsin, trypsin, and bile salts under simulated gastrointestinal conditions for dogs. These characteristics highlighted the enzyme's excellent properties and suggested its potential as a promising candidate for applications in the food and bio-industries, particularly in pet food production.

Mixed-mode separation of antisense oligonucleotides using a single column with complementary anion-exchange and hydrophobic interaction chromatography approaches

The current study investigates the use of mixed-mode chromatography as a combination of anion-exchange (AEX) and hydrophobic interaction chromatography (HIC) for the analysis and purification of single-stranded antisense oligonucleotides with stereo-controlled phosphorothioate inter- nucleotide linkages.Initially a Scherzo-SS-C18 trimodal stationary phase with reversed-phase/AEX/ cation-exchange (CEX) functionalities is systematically evaluated to reveal the presence of U-shaped retention composed of two retention modes namely AEX and HIC, where the latter was also observed on related trimodal Scherzo SM and SW analogues. For the first time, retention and separation of deprotected oligonucleotides was described on a single mixed-mode column using a combination of AEX and HIC. This methodology was later applied to an alternative reversed-phase/AEX support, Newcrom BH, displaying similar retention trends under dual salt / organic modifier gradients. The merit of the method was assessed on the basis of separations between a phosphodiester (PO) impurity and phosphorothioate (PS) target for an assortment of selected 2’-O-methoxyethyl 18- to 20-mer single-stranded antisense oligonucleotides. Various parameters were evaluated mostly under HIC conditions including organic modifier percentage, type of salt, temperature, pH and type of buffer in the mobile phase. Retention of the oligonucleotides was significantly affected by the acetonitrile composition and the type of salt ((NH4)2SO4, NaBr, NaCl) where the latter NaCl also afforded resolution between the PS target and closely eluting PO impurities. Small changes in pH between 6.5 and 7 using MES and TRIS respectively demonstrated notable differences in retention and resolution. The optimized methods were compared against a range of traditional supports and applied to various mixed-mode analogues, possessing embedded amino acid, complex forming weak cation-exchange and terminal strong cation-exchange functionalities. The mixed-mode supports displayed HIC retention and better resolution between PS target and PO impurity was evident with a more focused 5% acetonitrile gradient over its 10% counterpart. Overall, throughout the study AEX and HIC demonstrated comparable resolution trends.Finally, the optimized HIC method was applied to a selected 18-mer antisense oligonucleotide at semi-preparative scale using the Newcrom BH column, affording purities of 60-80% and recoveries of 52-76%. Although HIC does not provide better separations between PS and PO than AEX, it opens possibilities to operate under non-denaturing conditions and allows purification of samples containing high salt content, as a standalone method or post AEX without a prior desalting step, which can result in 20-30% sample loss.

Structure and characterization of an extracellular polysaccharide from Paenibacillus polymyxa 88A.

Levan-type polysaccharides, produced by various organisms, are nontoxic, biocompatible, and biodegradable polymers with a wide range of biological activities. They have high potential for use in medicine, cosmetology, and industry. A large amount of levan (41.1 g L-1) was recovered by ethanol precipitation from a liquid nutrient medium of Paenibacillus polymyxa 88A that contained 15 % w/v sucrose as a carbon source. The levan was fractionated by gel-permeation and anion-exchange chromatography and was analyzed by DRIFT and NMR spectroscopy. It was found that levan was represented by slightly branched chains composed of β-(2→6)-Fruf residues. The average molecular mass of the levan was about 1.9 MDa. When shear stress was applied at different temperatures, aqueous levan solutions showed pseudoplastic behavior. As found by SEM, a freeze-dried powdered levan sample had a microporous structure. The levan had excellent emulsifying activity toward sunflower oil, forming an emulsion with long-term stability. Analysis of the antioxidant activity of the levan showed a higher, dose-dependent activity toward ABTS, as compared with that toward DPPH. Finally, the bioactivity of the levan was examined by MTT assay by using human cervical carcinoma (HeLa) cells.

Antimicrobial and antioxidant activities of peptide derived from turmeric plant (Curcuma longa L).

The overuse and inappropriate use of antibiotics have led to the emergence of several antibiotic resistant bacteria. As a result, there is growing interest in exploring alternative agents as antimicrobial peptides (AMPs), which operate through unique mechanisms to effectively counteract bacterial resistance. In this study, peptides smaller than 3 kDa were isolated by cation exchange chromatography, anion exchange chromatography and reverse-phase chromatography. Subsequently, 12 candidate peptides were selected and chemically synthesized for a comparative study of growth inhibition in pathogenic bacteria. They demonstrated potent antibacterial activity toward A. baumannii, S. epidermidis, S. aureus, and S. enterica. Exposure to the Cur-1 peptide induced changes in bacterial proteins associated with metabolite interconversion and translation. In addition, all peptides derived from turmeric peptic hydrolysate exhibited antioxidant activity as assessed by ABTS, DPPH and FRAP assays. Cur-1 peptide displayed both high antibacterial and antioxidant potential, positioning it as a promising natural option for antibacterial management and applications within food industry.

Fine structure of starch biomacromolecules and digestibility: The regulative role of amylose and amylopectin in the digestive hydrolysis of starch in rice

The digestibility of starch in staple foods has rarely been examined at the bio-macromolecular level. This study addresses this by investigating the fine structures of amylose and amylopectin to understand their roles in starch digestibility in cooked white rice. Using the static INFOGEST protocol and oral processing by human volunteers, we assessed the starch digestion characteristics of 13 rice varieties, with amylose and amylopectin chain length distribution being analyzed using size-exclusion chromatography and high-performance anion exchange chromatography, respectively. Kinetic modelling revealed that chewed white rice follows a typical parallel digestion pattern, with rapidly (SF) and slowly digestible starch (SS) being digested simultaneously at distinctly different rates. Amylose content (AC) and amylose weight were significantly and positively correlated with the digestion rate and extent of SS, whereas the digestion rate and extent of SF were closely linked to amylopectin, particularly its short and intermediate chains (degree of polymerization 13–36). Compared to low-amylose rice (AC < 25 %), high-amylose rice exhibited significantly higher SS but with a lower digestion rate, attributed to its higher AC with shorter chains and fewer short to intermediate Ap branches. These findings provide insights into starch structure-digestibility relationships, aiding the development of rice varieties with slower digestion rates.

Detecting the Major Degradation Products of Phosphorothioate Oligonucleotides by Chemical Derivatization and Weak Anion Exchange Chromatography.

Novel polar cysteine analogues have been synthesized for the derivatization of oligonucleotide depurination impurities that may be formed under acidic conditions. Depurination impurities belong to a group that includes deamination and phosphate diester impurities, which are similar in chemical structure to each other and the parent oligonucleotide, and thus coelute by most chromatographic separation methods. The polar cysteine analogues react with depurination impurities and enable their complete separation from the parent oligonucleotide by weak anion exchange (WAX) chromatography. Optimized conditions for the derivatization reaction and the WAX analysis are presented. The ability of the WAX method to chromatographically separate deamination and phosphate diester impurities is also demonstrated, and therefore, the combination of chemical derivatization and WAX chromatography permits detection and quantification of the three major degradation products of phosphorothioate (PS) oligonucleotides.

Development of a Dual-Stage CIM® CDI Reactor with Immobilized Glucuronan Lyases and Laccases for Sustainable Synthesis of Antioxidant Phenolized Oligoglucuronan

Immobilized enzyme reactors (IMERs) are critical tools for developing novel oligosaccharides based on the enzymatic catalysis of polysaccharides. In this paper, a novel glucuronan lyase from Peteryoungia rosettiformans was produced, purified, and then immobilized on a CIM® CDI disk for cleaving glucuronan. The results showed that around 63.6% of glycuronan lyases (800.9 μg) were immobilized on the disk. The Vmax values of immobilized glucuronan lyases did not significantly change (56.9 ± 4.7 μM∙min−1), while the Km values (0.310 ± 0.075 g∙L−1) increased by 2.5 times. It is worth noting that immobilized glucuronan lyases overcame the catalytic inhibition of free enzymes observed under high glucuronan concentrations (0.5–2 g∙L−1). circumscribed central composite design (CCCD) and response surface methodology (RSM) showed that glucuronan concentration, flow rate, and reaction time significantly affected the yield of oligoglucuronans. The degree of polymerization (DP) of degraded glucuronan ranged from DP 2–8 according to the results obtained by high performance anion exchange chromatography coupled with a pulsed amperometric detector (HPAEC-PAD). The IMER retained 50.9% activity after running 2373 column volumes of glucuronan. Finally, this glucuronan lyase reactor was tentatively connected to an immobilized laccase reactor to depolymerize, and gallic acid (GA) was added to glucuronan. Approximately 8.5 mg of GA was added onto 1 g of initial glucuronan, and the GA–oligoglucuronan conjugates showed notable antioxidant activity.

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.

The diversity of glycan chains in jellyfish mucin of three Cubozoan species: the contrast in molecular evolution rates of the peptide chain and Glycans.

The O-glycan composition of jellyfish (JF) mucin (qniumucin: Q-mucin) extracted from three Cubozoan species was studied after the optimization of the purification protocol. Application of a stepwise gradient of ionic strength to anion exchange chromatography (AEXC) was effective for isolating Q-mucin from coexisting impurities. In the three species, the amino acid sequence of the tandem repeat (TR) region in Q-mucin in all three Cubozoans seemed to remain the same as that in all Scyphozoans, although their glycan chains seemed to exhibit clear diversity. In particular, the amounts of acidic moieties on the glycan chains of Q-mucin from the Cubozoans markedly varied even in these genetically close species. In two of the three Cubozoan species, the fraction of disaccharides was large, showing a sharp contrast to that of the glycans of Q-mucin in Scyphozoans. This study also indicates that the simple sequence of TR commonly inherited in all Cubozoan and Scyphozoan JF species after the long term of evolution over 500Myears. According to this research, the glycans and the TR of mucin-type glycoproteins (MTGPs), forming a hierarchical structure, appear to complement each other in the evolutionary changes because the time required for their hereditary conversion is considerably different. The cooperation of these mechanisms is a strategy to achieve the contradictory functions of biosystems, namely species conservation and diversity acquisition.

Potential of Indonesian cocoa pod husk for production of ligninolytic enzymes laccase and manganese peroxidase from Trametes hirsuta

Lignocellulosic biomass waste is a great potential source of cellulose and cellulose derivative bioproducts. Biomass pretreatment which involves ligninolytic enzymes is an environmentally friendly process that minimizes the use of harsh chemicals and energy input. This study evaluated white rot fungi and inducers (corncob, pineapple leaf, sorghum, and cocoa pod husk) to produce laccase (Lac) and manganese peroxidase (MnP). Enzyme production was optimized by using Plackett Burman and response surface methodology. Purification was undertaken by ammonium sulfate precipitation, dialysis, and anion exchange chromatography. Trametes hirsuta and cocoa pod husk as producer and inducer, respectively, resulted in the highest enzyme activities. The activities of Lac and MnP were optimum at 762.25 U/L and 596.92 U/L, respectively. The optimum pH and temperature for Lac and MnP were pH 3 (room temperature) and 60 °C (pH 5). The expected Lac and MnP were observed at ~65 kDa and 35–50 kDa protein band, respectively.

Limitation of anion exchange chromatography and potential application of hydrophobic interaction chromatography for monitoring AAV9 capsid degradation upon thermal stress

Adeno-Associated Virus (AAV) is often selected as the vector of choice for gene therapy due to its superior clinical performance compared to other gene delivery systems. Currently the characterization of AAV degradation, especially the chemical degradation of capsid, has been limited due to lack of suitable methods. Our study using AAV9 as a model molecule shows that anion exchange chromatography (AEX) as a charge-based separation method has limitations in monitoring the chemical degradation of AAV9 capsid due to a confounding effect from DNA cargo ejection. We developed a hydrophobic interaction chromatography (HIC) method, free from DNA interference, that could serve as a quick and reliable alternative to resource-demanding peptide mapping method for monitoring AAV capsid chemical degradation. Compared with brief thermal stress at 75 °C, AAV9 capsid exhibited much higher levels of chemical degradation but slower capsid titer loss upon extended exposure for 4 weeks at 40 °C.

Efficient Cd separation protocols for high-precision cadmium isotope analyses of diverse samples by double spike MC-ICP-MS

Cadmium isotope analyses are applied for research in planetary, Earth, environmental and life sciences. However, there is still a lack of efficient methods for the separation of the trace element Cd from the different types of samples that are of interest for isotopic analyses. This study presents new and improved Cd separation and purification techniques for meteorite, diverse terrestrial and seawater samples prior to Cd isotope measurements by multiple collector ICP-MS using the double spike approach for mass bias correction. The first separation stage for meteorites and terrestrial samples employs an existing anion exchange chromatography method that uses AG MP-1M resin, whilst AG 1-X8 and Nobias Chelate PA-1 resins were applied for the initial isolation of Cd from small and large seawater samples, respectively. Teflon microcolumns and AG MP-1M anion exchange resin was then employed for further Cd purification of all sample types. The methods consistently isolate Cd from matrix elements more efficiently than previous protocols, whilst achieving consistently high Cd recoveries, of >95% for meteorite and terrestrial samples and >80% for seawater samples, combined with low procedural blanks of <0.08 ng. The performance of the new protocols is demonstrated by repeated Cd isotope analyses of well-characterized geological and biological reference materials, which produced data that are in excellent agreement with results that were obtained with previous, more laborious separation methods. Furthermore, precise Cd isotope compositions (δ114Cd) are presented for the Murchison meteorite and, for the first time, the ordinary chondrite GRO 95504. Finally, analyses of seven seawater samples, with Cd concentrations between 0.25 and 0.91 nmol kg-1 using sample volumes of up to 2 L, generated results consistent with those of previous studies. Overall, the new methods thus enable unbiased δ114Cd measurements for diverse sample types with a 2SD precision of better than ±0.07‰, even for samples with 10 ng or less of natural Cd.

Comparison of Two Analytical Techniques of Biomass Burning Markers in PM1 and PM2.5 From Zabrze, Southern Poland.

With increasing attention to alternative energy sources due to environmental concerns, biomass emerges as a promising solution. However, biomass combustion emits particulate matter and harmful substances, impacting air quality and human health. The study compares gas chromatography-mass spectrometry (GC-MS) and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) methods for biomass combustion marker determination. GC-MS involves derivatization and subsequent analysis, while HPAEC-PAD utilizes water extraction, bypassing derivatization. Both methods' analytical performance characteristics were compared, showing HPAEC-PAD to have slightly lower detection limits and simplified sample preparation. The comparison revealed similar concentrations of levoglucosan and mannosan between the methods. However, galactosan (GA) concentrations showed discrepancies, possibly due to method sensitivity and recovery. HPAEC-PAD exhibited advantages in GA detection due to its direct analysis and better recovery. The study highlights the suitability of HPAEC-PAD for biomass combustion marker analysis, especially for GA determination, owing to its simplicity. However, GC-MS offers advantages in complex matrix identification. The findings provide insights for future research on atmospheric aerosol analysis and comparative studies of analytical techniques for other monosaccharides.

Optimization, structural characterization, and biological applications of exopolysaccharide produced by Enterococcus faecium KT990028

The aim of this study was to select the best exopolysaccharide (EPS) producer among the Enterococcus strains to optimize, characterize, and evaluate its biological properties. Among the eleven strains, Enterococcus faecium KT990028 was selected, and the production conditions were optimized: 16.3 % (w/v) sucrose, 0.70 % (w/v) yeast extract, 8.3 % (w/v) reconstituted skimmed milk, at 38 °C in 15 h of incubation, producing 2.880 g/L of EPS. High performance anion exchange chromatography (HPAEC) analysis revealed that the molecular weight was 166.98 kDa. HPAEC, spectroscopy (FTIR), and nuclear magnetic resonance (1H NMR) analyses revealed that the EPS was a heteropolysaccharide composed of galactose (37.74 %), rhamnose (19.79 %), arabinose (17.71 %), glucose (9.50 %), fucose (7.93 %), and mannose (7.33 %). Scanning electron microscopy showed a three-dimensional microstructure in the form of decompressed plates, with wrinkles, and pores. By means of dynamic light scattering (DLS), the EPS showed an average size varying from 135.25 ± 10.56 nm and 410.60 ± 45.20 nm, as the concentration was increased from 0.5 mg/mL to 2.0 mg/mL, respectively. X-ray diffraction revealed that the EPS has an amorphous and crystalline nature, while thermogravimetric analysis indicated stability up to 400 °C. The antioxidant effect (5 mg/mL) against DPPH, ABTS, OH, and O2 was 64.50 ± 0.71 %, 47.50 ± 0.10 %, 68.36 ± 0.59 %, and 44.83 ± 0.86 %, respectively. It was also able to inhibit and biofilm disruption of Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 6057 and had an antimicrobial effect from 50 mg/mL for the strains of against Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Listeria monocytogenes ATCC 19117, Staphylococcus aureus ATCC 6538, and Enterococcus faecalis ATCC 6057. Cell cytotoxicity carried out using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that the EPS was safe and promoted the proliferation of Vero cells. Thus, the results indicated that the EPS from E. faecium KT990028 is a promising functional biopolymer for possible applications in the food and pharmaceutical fields.

Laccase purification and azo dye decolorization potential of Sparassis latifolia

Sparassis latifolia often referred to as Cauliflower mushroom possess both medicinal and edibility values. In this research work, first time laccase purification and dye colourisation efficacy of Sparassis latifolia’s purified laccase were assessed. Optimal laccase potential was noted after 12th day of incubation with 4 pH of medium at 45 °C. As supplementary nutritional source, sucrose and ammonium sulphate were unveiled as most effective synthetic carbon and nitrogen sources while wheat straw found as proficient lignocellulosic biomass for enhanced laccase production. Following optimisation of laccase production parameters, laccase was purified to 14.2 fold by ammonium sulphate precipitation, dialysis and anion exchange chromatography. Laccase molecular weight determined through SDS PAGE was found to be of 65 KDa. This purified laccase was tested for its ability to decolourise the Congo red at definite pH and temperature. It was found that this fungus has capability to decolourise up to 98.6% of Congo-red dye.

Structural, molecular, and physicochemical properties of starch in high-amylose durum wheat lines

There is growing interest in the development of high-amylose cereals such as wheat due to their functional properties and nutritional value in foods. The increase in amylose content is associated with significant changes in the physicochemical, molecular, and structural characteristics of wheat starch which could affect its processing quality. The present study aimed to analyze the physicochemical, molecular, and structural characteristics of high-amylose starch from three durum wheat near isogenic lines (NILs), each obtained using three different recurrent parents. Morphology, granule size distribution, pasting and thermal properties, X-ray diffraction, and structural features were determined. The NILs showed the highest amylose content (65.3 and 69.8%), granules with an elongated shape, size between the wild-type and the mutant, restricted swelling in the pasting profile, the highest average and final gelatinization temperature, and B-polymorphism. The starches from the NILs showed the highest molar mass of amylopectin (AP) (7.0–7.6 × 107 Da) and the lowest amylose (AM) (1.1–1.8 × 106 Da), an issue associated with the biosynthesis of both components. The study on the debranched sample by high-performance size-exclusion chromatography showed that the high-amylose starch had the highest content of long B2 and B3 chains of AP (23.9–26.6%), which was corroborated with the high-performance anion-exchange chromatography analysis where the chains with degree of polymerization >37 had the highest content (18.3–19.1%). The PC1 is highly associated with the MwAM and the AM fraction in the debranched starch analyzed by HPSEC. Characterizing high-amylose durum wheat starch provides information to determine the structure-function relationship and design strategies to produce crosses with structural characteristics of AM and AP for specific applications.

Electronegative ApoCIII-Rich HDL promotes cellular damage and vascular aging

Abstract Background Experimental studies attributing HDL a cardioprotective effect are challenged by well-designed randomized controlled trials showing largely neutral results, which might be related to more recent observations that certain HDL particles exert detrimental effects. This study explores the effects of electronegative HDL on cellular damage and vascular aging. Using anion-exchange chromatography, HDL was divided into five subclasses (H1 to H5) based on increasing electronegativity. Compared to the health-promoting H1, the most electronegative subclass, H5 contains significantly higher levels of ApoCIII and triglycerides (TG). Purpose Herein, we aim to explore the cellular and molecular mechanisms underpinning H5-induced pathology in human umbilical vein vascular endothelial cells (HUVECs), which may accelerate vascular aging preceding the development of atherosclerotic cardiovascular disease (ASCVD). Methods H1 and H5 were isolated from patients with metabolic syndrome (MetS). To gauge the impact of H5 on vascular integrity, HUVECs were treated with solvent, H1, or H5 at concentrations of 25, 50, or 100 µg/mL. Cell viability and apoptosis were evaluated using the MTT assay and PI/Calcein fluorescent staining, respectively. Immunofluorescence staining and immunoblotting, focusing on 8-oxodG, P53, and β-gal, proxies of DNA damage and cellular senescence, were performed. Senescence-associated secretory phenotype (SASP) analysis was done to elucidate the senescent mechanism. By harnessing Mitosox imaging reactive oxygen species (ROS) synthesis was monitored. Finally, unbiased lipid- and transcriptomics were used to identify lipid components potentially involved in H5 signaling. Results As compared to controls, MetS patients exhibited more electronegative HDL particles, as indicated by increased H5 (1.604±0.410% vs 4.598±4.712%, P=0.0016). Remarkable elevations were noted in individuals with severe clinical complications, as shown in the representative patient with left ventricular hypertrophy, chronic coronary syndrome, and heart failure (Fig. 1). In HUVECs, H5 but not H1 reduced cell viability and increased apoptosis in a concentration-dependent manner. In parallel, H5 but not H1 induced cell aging, ROS production, and DNA damage, with SASP cytokines including IL-1β, IL-6, CCL2, TNF-α, and γH2AX (Fig. 1). Notably, compared to H1, H5 exhibited high contents of various TG species, impairing H5’s anti-inflammatory and antioxidant capacities, along with reductions in several phosphatidylcholines that may constitute essential cell membrane structural components, as evidenced by lipidomic studies (Fig. 2). Conclusion H5, the most electronegative and dysfunctional HDL subfraction, is capable of inducing DNA damage, oxidative stress, and senescence in vascular ECs. Its notable elevation in patients with MetS highlights a previously unrecognized etiology, shedding light on how electronegative HDL may contribute to and expedite ASCVD.Fig 1Fig 2

Mechanistic model of minute virus of mice elution behavior in anion exchange chromatography purification.

This study aimed to propose a methodology for developing a mechanistic model for viral clearance of the minute virus of mice (MVM) on flow-through anion exchange (AEX) chromatography. Protein surface analysis was applied to investigate the possibility of molecular interaction between the recombinant biotherapeutic and MVM. The protein product-free Tris buffers were spiked with MVM, and the MVM elution profile from AEX chromatography was quantitatively analyzed using quantitative polymerase chain reaction (qPCR) for pooled fractions. GoSilico™ Chromatography Modeling Software was applied to develop the mechanistic models for MVM species. For evaluating the visual fit of the developed model, the R2 of intact MVM virions and uncoated capsids between the simulated and measured amount in each fraction are 0.880 and 0.948, respectively. Response surface plots of logarithmic reduction values (LRV) against pH and conductivity in loaded sample were generated to show the range for suitable loaded sample conditions for achieving a good LRV. To evaluate the applicability of the developed MVM elution model to a recombinant biotherapeutic, two demonstrations of AEX chromatography purification were performed with a loaded sample of a model monoclonal antibody. The peaks of the MVM species in the elution step of both runs were accurately simulated by the developed model. In addition, to assess the possibility of molecular interaction between the virus and the target protein significantly affecting the MVM elution behavior, the antibody's surface was evaluated in terms of hydrophobicity/hydrophilicity using surface analysis.

Exploring the catalytic potential of watermelon urease: Purification, biochemical characterization, and heavy metal precipitation

Bioactive urease from watermelon (Citrullus lanatus) seeds was purified using acetone fractionation, anion-exchange, and size-exclusion chromatography, achieving a 121-fold increase and specific activity of 3216 U/mg. The enzyme appeared as a single band on native and SDS-PAGE, with a molecular mass of 480 ± 10 kDa and subunit mass of 80 ± 10 kDa, indicating six identical subunits. Atomic absorption spectroscopy revealed 1.46 nickel ions per subunit. Watermelon urease exhibited serological similarities with jack bean and pigeon pea ureases, an optimal pH of 7.3, an activation energy of 3 kcal/mol, Vmax of 3571 μmol/min/mg, and Km of 0.16 mM. The enzyme displayed biphasic thermal and pH inactivation kinetics, a strong preference for urea, and a half-life of 70 days with 1 mM DTT. This study highlights watermelon urease's role in bioremediation by facilitating the precipitation of heavy metals as stable carbonates, promoting environmental sustainability.