Search Results (62)

Porcine Rotavirus (PoRV), a predominant causative agent of neonatal diarrhea in piglets, shares substantial genetic homology with human rotavirus and represents a considerable threat to both public health and the global swine industry in the absence of specific antiviral interventions. The VP6 protein, an internal capsid component, is characterized by exceptional sequence conservation and robust immunogenicity, rendering it an ideal candidate for viral genotyping and vaccine development. In the present study, the recombinant plasmid pET28a(+)-VP6 was engineered to facilitate the high-yield expression and purification of the VP6 antigen. BALB/c mice were immunized to generate monoclonal antibodies (mAbs) through hybridoma technology, and the antigenic specificity of the resulting mAbs was stringently validated. Subsequently, a panel of truncated protein constructs was designed to precisely map linear B-cell epitopes, followed by comparative conservation analysis across diverse PoRV strains. Functional validation demonstrated that all three mAbs exhibited high-affinity binding to VP6, with a peak detection titer of 1:3,000,000 and exclusive specificity toward PoRVA. These antibodies effectively recognized representative genotypes such as G3 and X1, while exhibiting no cross-reactivity with unrelated viral pathogens; however, their reactivity against other PoRV serogroups (e.g., types B and C) remains to be further elucidated. Epitope mapping identified two novel linear B-cell epitopes, ¹²⁸YIKNWNLQNR¹³⁷ and ¹³⁸RQRTGFVFHK¹⁴⁷, both displaying strong sequence conservation among circulating PoRV strains. Collectively, these findings provide a rigorous experimental framework for the functional dissection of VP6 and reinforce its potential as a valuable diagnostic and immunoprophylactic target in PoRV control strategies. Full article

The rabbit is a widely used experimental model for human translational research and stem cell therapy. Many studies have focused on rabbit mesenchymal stem cells from different biological sources for their possible application in regenerative medicine. However, a minimal number of studies have been published aimed at rabbit hematopoietic stem/progenitor cells, mainly due to the lack of specific anti-rabbit CD34 antibodies. In general, CD34 antigen is commonly used to identify and isolate hematopoietic stem/progenitor cells in humans and other animal species. The aim of this study was to develop novel monoclonal antibodies highly specific to rabbit CD34 antigen. We used hybridoma technology, two synthetic peptides derived from predicted rabbit CD34 protein, and a recombinant rabbit CD34 protein as immunogens to produce monoclonal antibodies (mAbs) specific to rabbit CD34. The produced antibodies were screened for their binding activity and specificity using ELISA, flow cytometry, and Western blot analysis. Finally, four mAbs (58/47/26, 58/47/34, 182/7/80, and 575/36/8) were selected for the final purification process. The purified mAbs recognized up to 2–3% of total rabbit bone marrow cells, while about 2% of those cells exhibited CD45 expression, which are likely rabbit primitive hematopoietic stem cells and their hematopoietic progenitors, respectively. The newly generated and purified mAbs specifically recognize CD34 antigen in rabbit bone marrow or peripheral blood and can be therefore used for further immunological applications, to study rabbit hematopoiesis or to establish a new animal model for hematopoietic stem cell transplantation studies. Full article

Ag/AgCl-decorated layered lanthanum oxide (La₂O₃) and niobium pentoxide (Nb₂O₅) plasmonic photocatalysts are fabricated through an ionic liquid-mediated co-precipitation method. Scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), powder X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), diffuse reflectance spectroscopy (DRS), and photoluminescence (PL) techniques were used to illustrate the physicochemical properties of the materials. The photoactivity was evaluated for the degradation of Acid Blue 92 (AB92) azo dye, a typical organic contaminant from the textile industry, and U251 cancer cell inactivation. According to the results, Nb₂O₅–Ag/AgCl was able to remove >99% of AB92 solution in 35 min with the rate constant of 0.12 min⁻¹, 2.4 times higher than that of La₂O₃–Ag/AgCl. A pH of 3 and a catalyst dosage of 0.02 g were determined as the optimized factors to reach the highest degradation efficiency under solar energy at noon, which was opted to have the highest sunlight intensity over the reactor. Also, 0.02 mg/mL of Nb₂O₅–Ag/AgCl was determined to be of great potential to reduce cancer cell viability by more than 50%, revealed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and mitochondrial membrane potential (MMP) examinations. The mechanism of degradation was also discussed, considering the key role of Ag⁰ nanoparticles in inducing a plasmonic effect and improving the charge separation. This work provides helpful insights to opt for an efficient rare metal oxide with good biocompatibility as support for the plasmonic photocatalysts with the goal of environmental purification under sunlight. Full article

In this study, an orthogonal array design was employed to optimize total flavonoid extraction conditions. The results showed that the optimal conditions were an ethanol concentration of 70%, an ultrasonic power of 250 W, a solid–liquid ratio of 1:30 g/mL, and an ultrasonic time of 25 min. Under these optimal extraction conditions, the total flavonoid yield was 169.3 mg/g plant material. The purification effects of LX-38, LX-60, LS-46, LS-306, XDA-8, AB-8, and D101 macroporous resins on the total flavonoids of Eucommia ulmoides leaves were also investigated. The parameters of the process using XDA-8 macroporous resin for the purification of the crude extract of total flavonoids from Eucommia ulmoides leaves were investigated. The adsorption conditions of the XDA-8 resin consisted of an initial sample concentration of 2.0 mg/mL, a sample pH value of 5.0, an adsorption flow rate of 1.5 mL/min, and a temperature of 25 °C. The desorption conditions of the XDA-8 resin consisted of 60% ethanol used as a desorption solution and a 2.0 mL/min desorption flow rate of the eluent. The total flavonoids from the Eucommia ulmoides leaves were purified under these conditions, and, afterward, the flavonoid content was 51.5%. The main components of the purified flavonoids from the Eucommia ulmoides leaves were isolated using high-performance liquid chromatography (HPLC), and they included chlorogenic acid, rutin, isoquercetin, kaempferol-3-O-rutinoside, quercetin 3-rhamnoside, hyperoside, and quercetin. The antioxidant activities were measured, and those of the purified total flavonoids from the Eucommia ulmoides leaves were higher than those of dibutylhydroxytoluene (BHT) and lower than those of ascorbic acid (Vc). Additionally, the purified total flavonoids from the Eucommia ulmoides leaves exhibited significant antioxidant activities. Full article

Aglycone-type soy isoflavones, recognized for their bioactive phytoestrogen properties, face industrial limitations due to their low natural abundance and inefficient conversion. This study optimized a multi-enzyme synergistic catalysis system using soybean sprout powder, achieving high conversion rates and purity through response surface methodology. The optimal enzyme system comprised β-glucosidase (25 U/mL), cellulase (200 U/mL), hemicellulase (400 U/mL), and β-galactosidase (900 U/mL) at pH 5.0, 50 °C, and 3.2 h. This system yielded an aglycone conversion rate of 92% and glycoside hydrolysis rate of 97%, outperforming single-enzyme approaches. Upon post-purification with AB-8 macroporous resin, the product reached a purity of 58.1 ± 0.54% and exhibited strong antioxidant activity, with DPPH and ABTS radical scavenging rates of 81.01 ± 0.78% and 71.37 ± 1.01%, respectively. In a zebrafish central nervous system injury model induced by mycophenolate mofetil, the 500 μg/mL sample group significantly reduced neural apoptosis fluorescence intensity compared to controls (p < 0.05), achieving a neuroprotective rate of 76.58%, which was similar to the effect of L-reducing glutathione. This study offers an efficient, cost-effective enzymatic strategy for producing aglycone soy isoflavones, highlighting their potential in functional foods and neuroprotective applications. Full article

The growing demand for therapeutic monoclonal antibodies (mAbs) has heightened the need for efficient and scalable purification strategies. A major challenge in downstream processing is the removal of antibody aggregates, which can compromise drug safety, efficacy, and regulatory compliance. This review explores the use of multimodal chromatography for aggregate separation, providing an in-depth analysis of commercially available resins and emerging adsorbent prototypes. It also examines the mechanisms of aggregate formation during bioprocessing. A comparative evaluation of conventional single-mode chromatography techniques—affinity, ion exchange, and hydrophobic interaction—is presented alongside multimodal chromatography, which integrates ion-exchange, hydrophobic, and other non-covalent interactions for enhanced aggregate clearance and process flexibility. The review primarily assesses commercial multimodal resins in terms of aggregate removal efficiency, binding capacity, and scalability. Additionally, advancements in prototype resins and multimodal membranes are discussed. Finally, the advantages, limitations, and future directions of multimodal chromatography in mAb aggregate removal are outlined. As purification demands continue to evolve, multimodal chromatography is poised to play an increasingly critical role in achieving the high purity standards required for therapeutic antibodies. Full article

Background/Objectives: Pharmaceutical companies are aware of the ongoing effort to satisfy the increasing global demand for therapeutic-grade monoclonal antibodies (mAbs), an especially difficult challenge for poor and developing countries. We present a simple, economical, single-step purification approach at neutral pH for polyclonal human IgG (hIgG), which does not require any expensive ligands, chromatography columns, polymers, or membranes. Methods/Results: Instead, porous precipitates of commercial, recyclable aromatic [bathophenanthroline:cation] complexes were found to efficiently capture impurity proteins from CHO cells or E. coli lysate while maintaining the majority of the highly concentrated hIgG (5–15 mg/mL) in the supernatant. [(Batho)₃:Zn²⁺] complexes were the most promising, resulting in hIgG with a purity of ≈95%, by SDS-PAGE. This purified hIgG is monomeric (by dynamic light scattering, DLS) and preserves the native secondary structure (by far UV circular dichroism spectroscopy, CD). The process yield is >90% (by densitometry) and is maintained after a 100-fold increase in the reaction volume, which required only proportional increases in reagents. Conclusions: Although Protein A chromatographic columns, the industry gold standard, have a limited binding capacity, are costly, and require familiarity with column maintenance, we are attempting, by our efforts, to help to produce a more efficient, simple, and economical purification platform. Full article

Sea buckthorn pomace (SBP) is a rich source of flavonoid compounds with potential healthy properties. This study optimized ultrasonic-enzymatic-assisted extraction (UEAE) of flavonoids from SBP and investigated its chemical composition and biological activities. Under the optimal conditions (pectinase addition of 1500 U/g, ultrasonic power of 300 W, ethanol concentration of 48%, liquid–solid ratio of 34:1, extract temperature of 50 °C, and extraction time of 28 min), the yield of SBP flavonoid extracts (SBFEs) was 21.57 ± 0.45 mg/g, well-matched with the predicted value (21.72 mg/g). The chemical composition was detected by ultrahigh-performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS^E) and mainly including isorhamnetin, kaempferol, and quercetin’s derivatives. After purification with AB-8 macroporous resin, the purified product (PSBFE) exhibited a significantly enhanced scavenging capability for 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (947.17 ± 3.85 and 427.33 ± 0.67 μmol Trolox/g, respectively) and ferric reducing antioxidant power (2.68 ± 0.01 mmol FeSO₄·7H₂O/g). Moreover, PSBFE possessed a pronounced inhibitory rate on α-glucosidase and α-amylase, with the IC₅₀ at 52.89 ± 0.09 and 97.81 ± 0.42 μg/mL, respectively. These findings indicate that it is a reliable, optimal extraction method to obtain potential antioxidant and hypoglycemic flavonoids from SBP for comprehensive development in functional food. Full article

To explore anthocyanins in black bean peel, the conditions of ultrasound-assisted deep eutectic solvents (DESs) were screened and optimized using the method of response surface optimization. After that, the purification of the anthocyanins was performed before investigating their antioxidant activity and stability. The results showed that the choline chloride–citric acid system was more suitable for the extraction of anthocyanins from black bean peel, and the maximum amount of 61.00 ± 2.73 mg C3GE/100 g DW anthocyanins was obtained with the following optimized conditions: extraction time, 40 min; ultrasonic power, 60 KHz; material–liquid ratio, 1:20 g/mL; and ultrasonic temperature, 50 °C. The purity of the anthocyanins increased to 193.62 mg C3GE/100 g after purification with AB-8 resin, which also significantly improved the ability to screen DPPH and ABTS radicals. The anthocyanins from black bean peel were sensitive to light, temperature, pH, and additives. Full article

Avian infectious bronchitis is caused by the avian infectious bronchitis virus (IBV), which poses a significant threat to the poultry industry and public health. The S1 protein of IBV plays a crucial role in the process of the virus invading host cells. To investigate the significant antigenic targets within the S1 protein, in this study, the truncated S1 sequence of the IBV M41 strain was cloned with approximately 660 bp and expressed. After purification and renaturation, the recombinant S1 protein was immunized into BALB/c mice. Then, following fusion with lymphocytes and SP2/0 cells, the indirect ELISA and Western blotting techniques were employed to screen hybridoma cell lines secreting monoclonal antibodies (mAbs) targeting the S1 protein. Antigenic epitopes of the mAbs were identified using truncated S1 fragments and peptide scanning. The results indicated that three hybridoma cell lines stably secreting S1 protein-specific mAbs (2A10, 4E9, and 5E12) were screened. The heavy chains of the three mAbs were IgG1, and all three mAbs contained kappa light chains. The identified minimal B-cell epitopes were ¹³²RVSAMK¹³⁷ and ¹⁴²FYNLTV¹⁴⁷. Homology analysis showed these both epitopes were conserved across IBV subtypes and located on the S1 protein surface. The conserved β-sheet epitope ¹³²RVSAMK¹³⁷ and the surface-exposed, flexible loop epitope ¹⁴²FYNLTV¹⁴⁷ serve as ideal targets for broad-spectrum diagnostics and early infection detection, respectively. These epitopes provide unique structural advantages for antibody binding, enabling the design of multivalent epitope vaccines or the development of immunomodulatory drugs. They offer novel biomaterials and targets for antibody-based drug development and rapid detection methods for avian infectious bronchitis virus (IBV), holding significant potential for the prevention and control of IBV. Full article

Keratan sulfate (KS) is a negatively charged carbohydrate linked to proteins. Several KS-bearing structural glycosaminoglycans participate to maintain the homeostasis of a functional extracellular matrix. Dysfunction of its biochemical composition and structure might therefore lead to pathological situations. For this reason, imaging of KS in tissues is an important diagnostic tool. Here, we describe the identification of the KS paratope derived from the ancestral anti-KS IgG mAb MZ15, as well as the engineering, functional recombinant expression in E. coli, and purification of an anti-KS single-chain variable fragment (ScFv). The ScFv enabled in vitro imaging of KS in cryosections of rat cornea by immunofluorescence microscopy comparable to the ancestral IgG MZ15. Full article

The synthesis of indole phytoalexin-like analogs related to alkyl (((1-(4-substitutedphenyl)-3-oxo-3-phenylpropyl)thio)carbonothioyl)-ʟ-tryptophanate 1a–d and the evaluation of their antifungal activity against the phytopathogen Fusarium oxysporum is reported. The target compounds were synthesized in the following two stages: (1) the initial esterification of ʟ-tryptophan, which reacted with trimethyl silane chloride and simple aliphatic alcohols (R = Me, Et) under microwave irradiation (MWI) at 100 °C to obtain the respective alkyl ester 2a–b; (2) the resulting mixture of ʟ-tryptophanates 2a–b with carbon disulfide and (E)-chalcone 3a–b under MWI at 50 °C during 60 min, followed by purification through classical column chromatography (55–76% yields). The products were obtained as mixtures of (S,R) and (S,S) diastereoisomers. An LC-DAD-MS analysis allowed us to establish the ratio of these diastereoisomers, and subsequent DFT/B3LYP-based computational calculations of the NMR ¹H chemical shifts suggested that the major diastereoisomer involved an (S,R) absolute configuration, comprising more than 60% of the mixture. The compounds 1a–d were subjected to an antifungal activity test against the phytopathogen F. oxysporum using an amended medium-based assay. Compound series 1 showed inhibition percentages of 80% at the first concentration and IC₅₀ values between 0.33 and 5.71 mM, demonstrating greater potential as antifungal agents compared to other ʟ-tryptophan derivatives like alkyl (2S)-3-(1H-indol-3-yl)-2-{[(1Z)-3-oxobut-1-en-1-yl]amino}propanoate, which presented lower inhibition percentages. In summary, phytoalexin analogs derived from ʟ-tryptophan and (E)-chalcones significantly inhibited the mycelial growth of Fusarium oxysporum, indicating their potential as effective antifungal agents. Full article

Novel multicomponent Nb–Ni–Ti–Zr–Co alloys with 20–55 at.% Nb were synthesized from metal powders by arc melting. The resulting alloys consist primarily of Nb-rich and eutectic body-centered (BCC) phases. The content of the eutectic BCC phase is highest for an equimolar composition, while the content of the Nb-rich BCC phase increases with Nb content in the alloy. The content of secondary phases is the highest for the alloy with 32 at.% Nb. According to ab initio calculations, hydrogen occupies tetrahedral interstitial sites in the Nb-rich phase and octahedral sites in the eutectic BCC phase. For different Nb concentrations, hydrogen-binding energies were calculated. An increase in the Nb-rich phase leads to softening of multicomponent alloys. The alloys with 20 and 32 at.% Nb demonstrate high hydrogen permeability (1.05 and 0.96 × 10⁻⁸ molH₂m⁻¹s⁻¹Pa^−0.5, respectively) at 400 °C, making them promising for hydrogen purification membrane application. Multicomponent alloys with a high Nb content (55 at.%) have low resistance to hydrogen embrittlement. Full article

Background: Schwann cells (SCs) and their plasticity contribute to the peripheral nervous system’s capacity for nerve regeneration after injury. The Egr2/Krox20 promoter antisense RNA (Egr2-AS) recruits chromatin remodeling complexes to inhibit Egr2 transcription following peripheral nerve injury. Methods: RNA-seq and ATAC-seq were performed on control cells, Lenti-GFP-transduced cells, and cells overexpressing Egr2-AS (Lenti-AS). Egr2 AS-RNA was cloned into the pLVX-DsRed-Express2-N1 lentiviral expression vector (Clontech, Mountain View, CA, USA), and the levels of AS-RNA expression were determined. Ezh2 and Wdr5 were immunoprecipitated from rat SCs and RT-qPCR was performed against AS-Egr2 RNA. ChIP followed by DNA purification columns was used to perform qPCR for relevant promoters. Hi-C, HiC-DC+, R, Bioconductor, and TOBIAS were used for significant and differential loop analysis, identifications of COREs and CORE-promotor loops, comparisons of TF activity at promoter sites, and identification of site-specific TF footprints. OnTAD was used to detect TADs, and Juicer was used to identify A/B compartments. Results: Here we show that a Neuregulin-ErbB2/3 signaling axis mediates binding of the Egr2-AS to YY1^Ser184 and regulates its expression. Egr2-AS modulates the chromatin accessibility of Schwann cells and interacts with two distinct histone modification complexes. It binds to EZH2 and WDR5 and enables targeting of H3K27me3 and H3K4me3 to promoters of Egr2 and C-JUN, respectively. Expression of the Egr2-AS results in reorganization of the global chromatin landscape and quantitative changes in the loop formation and contact frequency at domain boundaries exhibiting enrichment for AP-1 genes. In addition, the Egr2-AS induces changes in the hierarchical TADs and increases transcription factor binding scores on an inter-TAD loop between a super-enhancer regulatory hub and the promoter of mTOR. Conclusions: Our results show that Neuregulin-ErbB2/3-YY1 regulates the expression of Egr2-AS, which mediates remodeling of the chromatin landscape in Schwann cells. Full article

Host cell proteins (HCPs) are one of the process-related impurities that need to be well characterized and controlled throughout biomanufacturing processes to assure the quality, safety, and efficacy of monoclonal antibodies (mAbs) and other protein-based biopharmaceuticals. Although ELISA remains the gold standard method for quantification of total HCPs, it lacks the specificity and coverage to identify and quantify individual HCPs. As a complementary method to ELISA, the LC-MS/MS method has emerged as a powerful tool to identify and profile individual HCPs during the downstream purification process. In this study, we developed a sensitive, robust, and reproducible analytical flow ultra-high-pressure LC (UHPLC)-high-resolution accurate mass (HRAM) data-dependent MS/MS method for HCP identification and monitoring using an Orbitrap Ascend BioPharma Tribrid mass spectrometer. As a case study, the developed method was applied to an in-house trastuzumab product to assess HCP clearance efficiency of the newly introduced POROS™ Caprylate Mixed-Mode Cation Exchange Chromatography resin (POROS Caprylate mixed-mode resin) by monitoring individual HCP changes between the trastuzumab sample collected from the Protein A pool (purified by Protein A chromatography) and polish pool (purified by Protein A first and then further purified by POROS Caprylate mixed-mode resin). The new method successfully identified the total number of individual HCPs in both samples and quantified the abundance changes in the remaining HCPs in the polish purification sample. Full article