Extraction of Modified Adeno-Associated Virus-Like Particles Displaying Peptides on Their Surface.

Virus-like particles (VLPs) of the adeno-associated virus (AAV) can be produced using the baculovirus expression vector system. Insertion of small peptides on the surface of the AAV or AAV VLPs has been used to redirect the AAV to different target tissues and for vaccine development. Usually, the VLPs self-assemble intracellularly, and an extraction step must be performed before purification. Here, we describe the method we have used to extract AAV VLPs from insect cells successfully with peptide insertions on their surface.

Purification Process for a Secreted Protein Produced in Insect Cells.

The Baculovirus Expression Vector System (BEVS) is used with cultured insect cells to produce a wide variety of heterologous proteins, which can be secreted into the culture medium during the transient infection process (Smith et al. Mol Cell Biol 12:2156-2165, 1983). When the infection process is complete, centrifugation is often used to separate the desired protein from the spent insect cells. The desired product in the harvested supernatant is contaminated with baculovirus, amino acids, lipids, detergents, oils, lysed cells from the infection process, genomic DNA from the insect cells, and proteases due to the lytic nature of the baculovirus infection process and many other contaminants (Ikonomou et al. Appl Microbiol Biotechnol 62:1-20, 2003). All these contaminants that are present in the centrifuged supernatant with the desired secreted protein make the initial chromatographic capture step critical for effective purification of the desired protein. A purification scheme will be outlined for a slightly acidic secreted protein using cation exchange chromatography (Lundanes et al. Chromatography: basic principles, sample preparations and related methods, 1st edn. Wiley, 2013).

Recombinant AAV Purification.

Purification of rAAV is a crucial unit operation of the AAV production process. It enables the capture of AAV and removal of contaminants such as host cell proteins, host cell DNA, and other cell culture-related impurities. Here we describe the purification of rAAV produced in insect cells Sf9/rBEV by immuno-affinity capture chromatography. The method is fully scale-amenable unlike other traditional purification methods based on ultracentrifugation. The method reported herein has two main steps: (1) the clarification of cell lysate by depth filtration and (2) the selective capture and single-step purification of AAV via immune-affinity chromatography. This purification method has been successfully implemented to purify the majority of wild-type AAV serotypes.

One-Step Purification Strategy for Cowpea Chlorotic Mottle Virus-Like Particles Produced by the IC-BEVS.

Virus-like particles (VLP) of the cowpea chlorotic mottle virus (CCMV), a plant virus, have been shown to be safe and noncytotoxic vehicles for delivering various cargos, including nucleic acids and peptides, and as scaffolds for presenting epitopes. Thus, CCMV-VLP have acquired increasing attention to be used in fields such as gene therapy, drug delivery, and vaccine development. Regardless of their production method, most reports purify CCMV-VLP through a series of ultracentrifugation steps using sucrose density gradient ultracentrifugation, which is a complex and time-consuming process. Here, the use of anion exchange chromatography is described as a one-step protocol for purification of CCMV-VLP produced by the insect cell-baculovirus expression vector system (IC-BEVS).

Protocol for phospho-SrcKD: rPTPεD1 complex preparation and BLI binding assays to demonstrate their exosite interface.

Here, we present a protocol for the in vitro phosphorylation of Src kinase domain (SrcKD), preparation of phospho-SrcKD in complex with the D1 domain of rPTP epsilon (rPTPεD1), and binding assays using biolayer interferometry (BLI). We describe steps for the in vitro phosphorylation of SrcKD and preparation of the phospho-SrcKD: rPTPεD1 complex for small-angle X-ray scattering (SAXS) experiments. We then detail instructions for the BLI binding assay to determine the binding affinity between phospho-SrcKD and rPTPεD1. For complete details on the use and execution of this protocol, please refer to EswarKumar et al.1.

Optimization of thorium solvent extraction process from feed solution with Cyanex 272 by response surface methodology (RSM).

Due to the limited reserves of uranium, the abundance of thorium compared to it and other advantages, the development of the thorium fuel cycle is of interest in different countries. The optimization of thorium extraction from a feed solution produced by Saghand ore with bis(2,4,4-trimethylpentyl) phosphinic acid (Cyanex 272) on a laboratory scale was evaluated by response surface method. The operating variables include Cyanex 272 concentration of 0.001 to 0.2 mol/L, pH of 0 to 2, equilibrium time of 5 to 60 min and aqueous to organic phase ratio of 0.5 to 2.5 were conducted. The value of R2 = 0.9695 and an error of less than 4% indicate the validity of the model. Therefore, the model is in good agreement with the experimental results. It can be said that there are significant interactions between operational parameters, which vindicate different feedbacks of the system in different operational conditions. The results showed that the 4 mol/L sulfuric acid was a suitable agent for recovering thorium ions from the loaded organic phase. In optimum conditions, the thorium purity percentage and thorium stripping efficiency were obtained 98.99 and 94.12%, respectively.

Protocol to detect and quantify interactions between proteins expressed in Drosophila S2 cells.

Here, we present a protocol to quantify interactions among difficult-to-express proteins from Drosophila cells using the select western blot-free tagged-protein interaction (SWFTI) assay. We describe steps for plasmid design, cell plating, protein expression, and immunoprecipitation preparation. We then detail procedures for protein labeling, gel purification, and protein quantification. This protocol offers a fluorescence-based technique for rapid quantification of ectopically expressed proteins that are fused to SNAP and CLIP tags without the need for membrane transfer. For complete details on the use and execution of this protocol, please refer to Lin et al.1.

Polydopamine coating for enhanced electrostatic adsorption of methylene blue by multiwalled carbon nanotubes in alkaline environments.

The removal of dye molecules in alkaline environments is an issue that should receive increased attention. In this study, the interaction mechanism between polydopamine-modified multiwalled carbon nanotubes (P-MWCNTs) and multiwalled carbon nanotubes (MWCNTs) with the cationic dye methylene blue (MB) in alkaline environments was explained in depth by adsorption, spectroscopy, and density functional theory (DFT). The mechanism of action and dominant forces between the adsorbent and adsorbate were analyzed graphically by introducing energy decomposition analysis (EDA) and an independent gradient model (IGM) into the DFT calculations. In addition, the force distribution was investigated through an isosurface. Moreover, batch adsorption studies were conducted to evaluate the performance of MWCNTs and P-MWCNTs for MB removal in alkaline environments. The maximum MB adsorption capacities of the MWCNTs and P-MWCNTs in solution were 113.3 mg‧g-1 and 230.4 mg‧g-1, respectively, at pH 9. The IGM and EDA showed that the better adsorption capacity of the P-MWCNTs originated from the enhancement of the electrostatic effect by the proton dissociation of polydopamine. Moreover, the adsorption of MB by MWCNTs and P-MWCNTs in alkaline environments was governed by dispersion and electrostatic effects, respectively. Through this study, it is hoped that progress will be made in the use of DFT to explore the mechanism of adsorbent-adsorbate interactions.

Fractionated plasma separation and adsorption integrated with continuous veno-venous hemofiltration in patients with acute bipyridine herbicide poisoning.

OBJECTIVE: To evaluate the clinical efficacy and safety of fractionated plasma separation and adsorption combined with continuous veno-venous hemofiltration (FPSA-CVVH) treatment in patients with acute bipyridine herbicide poisoning. METHODS: A retrospective analysis of 18 patients with acute bipyridine herbicide poisoning was conducted, of which 9 patients were poisoned by diquat and 9 patients by paraquat. All patients underwent FPSA-CVVH treatment. The serum cytokine levels in pesticide-poisoned patients were assessed. The efficacy of FPSA-CVVH in eliminating cytokines, the 90-d survival rate of poisoned patients, and adverse reactions to the treatment were observed. RESULTS: Fourteen patients (77.8%) had acute kidney injuries and 10 (55.6%) had acute liver injuries. The serum cytokine levels of high mobility group protein B-1 (HMGB-1), interleukin-6 (IL-6), IL-8, interferon-inducible protein-10 (IP-10), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1ß (MIP-1ß) were significantly elevated. A total of 41 FPSA-CVVH treatment sessions were administered. After a single 8-h FPSA-CVVH treatment, the decreases in HMGB-1, IL-6, IL-8, IP-10, MCP-1, and MIP-1ß were 66.0%, 63.5%, 73.3%, 63.7%, 53.9%, and 54.1%, respectively. During FPSA-CVVH treatment, one patient required a filter change due to coagulation in the plasma component separator, and one experienced a bleeding adverse reaction. The 90-d patient survival rate was 50%, with 4 patients with diquat poisoning and 5 patients with paraquat poisoning, and both liver and kidney functions were restored to normal. CONCLUSION: Cytokine storms may play a significant role in the progression of multiorgan dysfunction in patients with acute bipyridine herbicide poisoning. FPSA-CVVH can effectively reduce cytokine levels, increase the survival rate of patients with acute bipyridine herbicide poisoning, and decrease the incidence of adverse events.

Biotechnological lactic acid production from low-cost renewable sources via anaerobic microbial processes.

Lactic acid (LA) production from microbial fermentation using low-cost renewable sources has emerged as an attractive alternative to the use of petroleum-based products. This approach not only offers sustainable solutions for waste management but also enables the production of value-added products in an eco-friendly manner. However, to make this approach economically viable, optimizing the production process for high yield, productivity, and purity while minimizing costs is crucial. To address these challenges, various approaches have been proposed, including the use of neutralizing agents, high cell density cultures, co-cultures, fed-batch fermentation, and product removal strategies. Overall, this review underscores the potential of microbial fermentation for LA production as a sustainable and cost-effective solution to meet the growing demand for eco-friendly products. Further optimization of fermentation processes and the development of new microbial strains and fermentation techniques are key to advancing this approach. The production of LA through microbial fermentation presents a sustainable and eco-friendly solution to the increasing demand for eco-friendly products. With continued innovation, we can expect to see a significant reduction in the environmental impact of industrial processes, coupled with a more cost-effective and high-purity source of lactic acid for various industries.

Blood purification therapy in chronic renal failure and its impact on renal index, serological index, and inflammatory factors.

Introduction: This study aimed to explore the clinical effects of blood purification therapy in patients with chronic renal disease, measured by renal function index and inflammation. Methodology: Data were collected from a tertiary care hospital in Pakistan between June 2022 and September 2023. Eighty-four patients undergoing maintenance hemodialysis for chronic renal failure were retrospectively included in this cohort. Results: Age, sex, BMI, course of disease, primary disease, and educational level were not related to the response to blood purification treatment. Blood purification therapy positively affected renal function, serological indices, and inflammatory factors (P<0.05). Conclusion: Blood purification therapy can improve toxin clearance and renal function and reduce inflammation. Therefore, the authors can conclude that this is an effective therapy for our population.

Detection of the GH analog somatrogon in sports drug testing: Immunological approaches and LC-HRMS/MS.

Due to the presumed lipolytic and anabolic properties, the misuse of human growth hormone (hGH) and its synthetic analogs in sports is prohibited both in- and out-of-competition. Within this research project, the detectability of somatrogon, a recombinant fusion glycoprotein of 22 kDa hGH and the C-terminal peptide (CTP) of the human chorionic gonadotropin (hCG) ß-subunit, with current WADA-approved doping control assays for hGH and hCG was investigated. For that purpose, cross-reactivity tests and a somatrogon administration study were conducted, and only "Kit 2" of the GH isoform differential immunoassays proved applicable to the detection of somatrogon administration in serum. In urine, the immunoassay specific for total hCG yielded presumptively positive findings for several post-administration samples, which can probably be attributed to the presence of an immunoreactive fragment of the hCG ß-subunit. As the detectability of somatrogon with these approaches was found to be limited, a highly specific detection assay (LOD: 10 ng/mL) for the drug in serum samples was developed by using affinity purification with GH receptor (GHR)-conjugated magnetic beads, proteolytic digestion, and liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Following optimization, the approach was comprehensively characterized, and authentic post-administration serum samples were successfully analyzed as proof-of-concept, indicating a detection window of at least 96 h. Consequently, the presented method can be employed to confirm the presence of somatrogon in serum samples, where only "Kit 2" of the currently used immunoassay kits yielded an abnormally high Rec/Pit ratio.

Digital design of an integrated purification system for continuous pharmaceutical manufacturing.

In this work dynamic models of the continuous crystallization, filtration, deliquoring, washing, and drying steps are introduced, which are developed in the open-source pharmaceutical modeling tool PharmaPy. These models enable the simulation and digital design of an integrated continuous two-stage crystallization and filtration-drying carousel system. The carousel offers an intensified process that can manufacture products with tailored properties through optimal design and control. Results show that improved crystallization design enhances overall process efficiency by improving critical material attributes of the crystal slurry for downstream filtration and drying operations. The digital design of the integrated process achieves enhanced productivity while satisfying multiple design and product quality constraints. Additionally, the impact of model uncertainty on the optimal operating conditions is investigated. The findings demonstrate the systematic process development potential of PharmaPy, providing improved process understanding, design space identification, and optimized robust operation.

Enhancing environmental sustainability: Butea monosperma leaves as a key component in WO3-based composites for water purification and therapeutic applications.

In this research, a novel nano-biocomposite material, namely, tungsten trioxide-Butea monosperma leaf powder (WO3@BLP), is an effective and eco-friendly adsorbent used for the mitigation of congo red (CR) and crystal violet (CV) dyes from its aqueous phase. The as-prepared WO3@BLP was characterized using Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), field emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), DLS analysis, and TGA. Many factors such as solution pH, WO3@BLP dose, temperature, contact time, and initial CR/CV dye concentrations were exploited to monitor the adsorption efficiency of WO3@BLP composites. The biosorption of both CR and CV dyes followed the Langmuir isotherm, with maximum adsorption capacities (qmax) reaching 84.91 mg g-1 for CR at pH 2.3 and 162.75 mg g-1 for CV at pH 8, fitting of kinetics data to the PSO model with closed values of qeexp (mg g-1) and qecal (mg g-1), i.e., 25.69 to 25.38 mg g-1 for CR dye and 29.06 to 29.08 mg g-1 for CV dye. The interaction mechanism behind the adsorption of CR and CV dyes onto the WO3@BLP bionanocomposite includes electrostatic interaction and surface complexation. The synthesized materials were tested for antifungal activity against three different Candida cells, i.e., C. albicans ATCC 90028, C. glabrata ATCC 90030, and C. tropicalis ATCC 750, by using broth dilution method on the minimum inhibiting concentration (MIC). Furthermore, the cytotoxicity of nano-formulated WO3@BLP was studied by in vitro hemolytic assay on a human host. Overall, this research presents a pioneering nano-biocomposite, WO3@BLP, as a sustainable adsorbent for CR and CV dye removal, adhering to Langmuir isotherm and pseudo-second-order kinetics. Its multifaceted approach includes elucidating interaction mechanisms, demonstrating antifungal activity, and assessing cytotoxicity, marking a significant advancement in environmental remediation.

Photocatalytically self-cleaning graphene oxide nanofiltration membranes reinforced with bismuth oxybromide for high-performance water purification.

Graphene oxide (GO) membranes have emerged as promising candidates for water purification applications, owing to their unique physicochemical attributes. Nevertheless, the trade-off between permeability and selectivity, coupled with their vulnerability to membrane fouling, poses significant challenges to their widespread industrial deployment. In this study, we introduce an innovative in-situ growth and layer-by-layer assembly technique for fabricating multilayer GO membranes reinforced with bismuth oxybromide (BiOBr) on commonly employed Nylon substrates. This method allows for the creation of two-dimensional lamellar membranes capable of photocatalytic self-cleaning and tunable nanochannel dimensions. The synthesized GO/BiOBr composite membranes exhibit remarkable water permeance rates (approximately 493.9 LMH/bar) and high molecular rejection efficiency (>99 % for Victoria Blue B and Congo Red dyes). Notably, these membranes showcase an enhanced photocatalytic self-cleaning performance upon exposure to visible light. Our work provides a viable route for the fabrication of functionalized GO-based nanofiltration membranes with BiOBr inclusions, offering a synergistic combination of high water permeability, modifiable nanochannels, and effective self-cleaning capabilities through photocatalysis.

In vitro inhibitor effect and molecular docking of thiamine (vitamin B1), riboflavin (vitamin B2), and reference inhibitor captopril on angiotensin-converting enzyme purified from sheep plasma.

OBJECTIVE: Angiotensin-converting enzyme (ACE, EC 3.4.15.1) is a very important factor in the regulation of blood pressure. Also, the inhibition of ACE with natural compounds has been a very important research area in the treatment of high blood pressure. ACE was purified and characterized from sheep plasma. Molecular docking studies and the inhibition effect of thiamine, riboflavin, and captopril on ACE were investigated. METHODS: Herein, ACE was purified from sheep plasma by affinity chromatography. The effect of thiamine and riboflavin on ACE was researched. Molecular docking studies were performed to understand the molecular interactions between thiamine, riboflavin, and captopril with ACE. RESULTS: The purification coefficient was found to be 8636 fold. The binding energy of thiamine, riboflavin, and captopril was found to be -6.7 kcal/mol, -8.1 kcal/mol, and -5.5 kcal/mol, respectively. Thiamine conformed to three conventional hydrogen bonds with ASP:415, HIS:513, and LYS:454. Riboflavin formed four conventional hydrogen bonds with GLN:281, GLU:376, THR:282, and TYR:520. Captopril formed two conventional hydrogen bonds with ARG:124, one conventional hydrogen bond with TYR:62 and ASN:85, and one carbon-hydrogen bond with ASN:66. Molecular docking results showed that thiamine, riboflavin, and captopril interacted with ACE through hydrogen bonding and hydrophobic interactions. Thiamine and riboflavin indicated significant inhibition effects on ACE. The IC50 values of thiamine, riboflavin, and captopril were found as 960.56 µM, 11.02 µM, and 1.60 nM, respectively. Ki values for thiamine, riboflavin, and captopril were determined as 1352.04 µM, 12.30 µM, and 1.06 nM, respectively. CONCLUSION: In this work, it was concluded that thiamine and riboflavin may have preventive and therapeutical impacts against high blood pressure with their ACE inhibitor effect. Thiamine and riboflavin showed a lower inhibitory effect with a higher IC50 than captopril. However, when the inhibitory effect of thiamine and riboflavin vitamins is compared to captopril, it is concluded that they may be natural inhibitors with fewer side effects.

Exploring the versatility of biodegradable biomass aerogels: In-depth evaluation of Firmiana simplex bark microfibers depolymerized by deep eutectic solvent.

In this study, we investigated the use of deep eutectic solvents (DESs) at different molar ratios and temperatures as a green and efficient approach for microfibers (MFs) extraction. Our approach entailed the utilization of Firmiana simplex bark (FSB) fibers, enabling the production of different dimensions of FSB microfibers (FSBMFs) by combining DES pretreatment and mechanical disintegration technique. The proposed practice demonstrates the simplicity and effectiveness of the method. The morphology of the prepared microfibers was studied using the Scanning electron microscopic (SEM) technique. Additionally, the results revealed that the chemical and mechanical treatments did not significantly alter the well-preserved cellulose structure of microfibers, and a crystallinity index of 56.6 % for FSB fibers and 63.8 % for FSBMFs was observed by X-ray diffraction (XRD) analysis. Furthermore, using the freeze-drying technique, FSBMFs in water solutions produced effective aerogels for air purification application. In comparison to commercial mask (CM), FSBMF aerogels' superior hierarchical cellular architectures allowed them to attain excellent filtration efficiencies of 94.48 % (PM10) and 91.51 % (PM2.5) as well as excellent degradation properties were analyzed. The findings show that FSBMFs can be extracted from Firmiana simplex bark, a natural cellulose-rich material, using DES for environmentally friendly aerogel preparation and applications.

Heterogenous Expression and Purification of Lipid II Flippase from Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is a common pathogen with strains that are resistant to existing antibiotics. MurJ from S. aureus (SaMurJ), an integral membrane protein functioning as Lipid II flippase, is a potential target for developing new antibacterial agents against this pathogen. Successful expression and purification of this protein shall be useful in the development of drugs against this target. OBJECTIVE: In this study, we demonstrated the optimized expression and purification procedures of SaMurJ, identified suitable detergent for extracting and solubilizing the protein, and examined the peptidisc system to generate a detergent-free environment. METHODS: SaMurJ fused with N-terminal ten-His tag was expressed without induction. Six detergents were selected for screening the most efficient candidate for extraction and solubilization of the protein. The thermostability of the detergent-solubilized protein was assessed by evaluated temperature incubation. Different ratios of peptidisc bi-helical peptide (NSPr) to SaMurJ were mixed and the on-bead peptidisc assembly method was applied. RESULTS: SaMurJ expressed in BL21(DE3) was confirmed by peptide fingerprinting, with a yield of 1 mg SaMurJ per liter culture. DDM was identified as the optimum detergent for solubilization and the nickel affinity column enabled SaMurJ purification with a purity of ~88%. However, NSPr could not stabilize SaMurJ. CONCLUSION: The expression and purification of SaMurJ were successful, with high purity and good yield. SaMurJ can be solubilized and stabilized by a DDM-containing buffer.

Performance Evaluation of Sinusoidal Power Supplies for Ozone Generation in Water Purification Applications.

Ozone generation is a water disinfection method, superior to chlorine in terms of fewer byproducts and no residual taste. However, its high production cost limits its widespread adoption. This paper designs an ozone generation sinusoidal power supply for water treatment. Ozone generation requires a high-frequency and high-voltage power supply to produce ozone from oxygen molecules. The study evaluates two power supply topologies, one with a parallel LC filter and the other with an LCL filter, assessing their feasibility, effectiveness, and reliability. Theoretically, the LCL filter achieves higher gains than the parallel LC filter. The larger inductance in the parallel LC filter reduces gain, while the larger inductance in the LCL filter increases gain. Simulation and practical results validate these findings, achieving gains of 40 for the parallel LC filter and 150 for the LCL filter.

All-Organic Piezo-Photocatalytic Film with Highly Efficient Catalysis, Weak-Force Excitation, and Recyclability.

Polyaniline (PANI), a typical organic photocatalyst, has an adjustable structure and good stability, can be easily synthesized on a large scale, and is economical. PANI is doped with ions to regulate its internal structure and improve its photocatalytic performance. However, its photocatalytic performance is limited by the doping concentration and its intrinsic properties, hindering its further application. Herein, PANI films with a piezo-photocatalytic function are fabricated to improve photocatalytic performance and explore their self-powered environmental purification property. PANI/poly(vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP) sandwich films, with PVDF-HFP as the interlayer, are prepared by introducing a piezoelectric field into PANI photocatalysts, thereby achieving excellent piezo-photocatalytic performance. The as-fabricated piezo-photocatalyst degrades methyl orange at a rate of 91.2% after 60 min under magnetic stirring. Owing to the low Young's modulus of the all-organic catalyst, self-powered purification is realized using the PANI/PVDF-HFP film. Leaf surfaces are functionalized by loading the film in them for removing pollutants under sunlight and water flow. Thus, this study proposes a common strategy, wherein a piezoelectric interlayer is introduced to load the organic photocatalyst for preparing an all-organic piezo-photocatalyst. This piezo-photocatalyst can be easily recycled and responds to weak forces, realizing its application for self-powered environmental purification.