A salt/salt aqueous two-phase system based on pH-switchable deep eutectic solvent for the extraction and separation of mulberry polysaccharides.

With the aim of expanding the potential application scope of mulberries, eleven pH-switchable deep eutectic solvents were screened for the ultrasonic-assisted extraction of mulberry polysaccharides, and a salt/salt aqueous two-phase system was constructed for the efficient separation of mulberry polysaccharides by regulating the system pH. DES-9 (tetraethylammonium chloride: octanoic acid molar ratio = 1: 2) with a critical response pH value of approximately 6.1 was concluded to be the best extraction solvent for extracting mulberry polysaccharides. A maximum polysaccharide extraction yield of 270.71 mg/g was obtained under the optimal conditions. The maximum polysaccharide extraction efficiency was 78.09 % for the pH-driven tetraethylammonium chloride/K2HPO4 aqueous two-phase system. An acidic ß-pyran mulberry polysaccharide with a low-molecular weight of 9.26 kDa and a confirmed monosaccharide composition were obtained. This efficient and environmentally friendly polysaccharide separation method offers a new approach for the efficient extraction and utilization of other plant polysaccharides.

A brief review of preparation and applications of monolithic aerogels in atmospheric environmental purification.

Monolithic aerogels are promising candidates for use in atmospheric environmental purification due to their structural advantages, such as fine building block size together with high specific surface area, abundant pore structure, etc. Additionally, monolithic aerogels possess a unique monolithic macrostructure that sets them apart from aerogel powders and nanoparticles in practical environmental clean-up applications. This review delves into the available synthesis strategies and atmospheric environmental applications of monolithic aerogels, covering types of monolithic aerogels including SiO2, graphene, metal oxides and their combinations, along with their preparation methods. In particular, recent developments for VOC adsorption, CO2 capture, catalytic oxidation of VOCs and catalytic reduction of CO2 are highlighted. Finally, challenges and future opportunities for monolithic aerogels in the atmospheric environmental purification field are proposed. This review provides valuable insights for designing and utilizing monolithic aerogel-based functional materials.

Phagosome Isolation with Magnetic Beads and Purification of Toll-Like Receptor (TLR) Complexes in Phagosomes.

Phagocytosis is a central process by which macrophage cells internalize and eliminate microbes as well as apoptotic cells. The nascent phagosome undergoes a complex maturation process involving sequential fusion with endosomal compartments. The endosomal TLRs, including TLR3, -7, -8, and -9, play a critical role in innate immunity by sensing bacterial or viral nucleic acids and are preferentially transported to the phagosomal membrane of innate immune cells upon activation. Therefore, phagosome isolation is helpful for studies on pathogenic invasion and the functions of phagosome proteins, including endosomal TLRs.

Chromatographic Purification and Polishing of AAV Particles.

The production of Adeno-associated virus (AAV) vectors in the lab setting has typically involved expression in adherent cells followed by purification through ultracentrifugation in density gradients. This production method is, however, not easily scalable, presents high levels of cellular impurities that co-purify with the virus, and results in a mixture of empty and full capsids. Here we describe a detailed AAV production protocol that overcomes these limitations through AAV expression in suspension cells followed by AAV affinity purification and AAV polishing to separate empty and full capsids, resulting in high yields of ultra-pure AAV that is highly enriched in full capsids.

Generating ESC-Derived RGCs for Cell Replacement Therapy.

In several ocular diseases, degeneration of retinal neurons can lead to permanent blindness. Transplantation of stem cell (SC)-derived RGCs has been proposed as a potential therapy for RGC loss. Although there are reports of successful cases of SC-derived RGC transplantation, achieving long-distance regeneration and functional connectivity remains a challenge. To address these hurdles, retinal organoids are being used to study the regulatory mechanism of stem cell transplantation. Here we present a modified protocol for differentiating human embryonic stem cells (ESCs) into retinal organoids and transplanting organoid-derived RGCs into the murine eyes.

Development of an effective method for purifying trypsin using a recombinant inhibitor.

A trypsin affinity material was prepared by covalently immobilizing buckwheat trypsin inhibitor (BTI) on epichlorohydrin-activated cross-linked agarose gel (Selfinose CL 6 B). The optimal conditions for activating Selfinose CL 6 B were 15 % epichlorohydrin and 0.8 M NaOH at 40 °C for 2 h. The optimal pH for immobilizing BTI was 9.5. BTI-Sefinose CL 6 B showed a maximum adsorption capacity of 2.25 mg trypsin/(g support). The material also displayed good reusability, retaining over 90 % of its initial adsorption capacity after 30 cycles. High-purity trypsin was obtained from locust homogenate using BTI-Selfinose CL 6 B through one-step affinity chromatography. The molecular mass and Km value of locust trypsin were determined as 27 kDa and 0.241 mM using N-benzoyl-DL-arginine-nitroanilide as substrate. The optimal temperature and pH of trypsin activity were 55 °C and 9.0, respectively. The enzyme exhibited good stability in the temperature range of 30-50 °C and pH range of 4.0-10.0. BTI-Selfinose CL 6 B demonstrates potential application in the preparation of high-purity trypsin and the discovery of more novel trypsin from various species.

A simple procedure for preparing biotinylated immunoglobulin M from hybridoma culture medium.

We previously reported a chromatography system for purifying immunoglobulin M (IgM) using N,N,N',N'-ethylenediaminetetrakis(methylenephosphonic acid)-modified zirconia particles that selectively absorb immunoglobulins. Here, we report a simple procedure for preparing biotinylated IgM from hybridoma culture medium using this zirconia-based chromatography system. The culture medium of an IgM-producing hybridoma cell line was used as the starting sample solution, and the IgM in the medium was concentrated and partially purified by zirconia chromatography. Next, 9-(biotinamido)-4,7-dioxanonanoic acid N-succinimidyl ester was added to react with the proteins in the sample. Subsequently, only the biotinylated IgM was isolated by Capto Core 400 polishing column chromatography. The entire process was easy to perform, could be completed within 2 h, and provided highly pure biotin-labeled IgM. This procedure is expected to be applicable to the labeling of IgM with various compounds and drugs.

Simplifying Recombinant Protein Production: Combining Golden Gate Cloning with a Standardized Protein Purification Scheme.

Recombinant protein production is pivotal in molecular biology, enabling profound insights into cellular processes through biophysical, biochemical, and structural analyses of the purified samples. The demand for substantial biomolecule quantities often presents challenges, particularly for eukaryotic proteins. Escherichia coli expression systems have evolved to address these issues, offering advanced features such as solubility tags, posttranslational modification capabilities, and modular plasmid libraries. Nevertheless, existing tools are often complex, which limits their accessibility and necessitate streamlined systems for rapid screening under standardized conditions. Based on the Golden Gate cloning method, we have developed a simple "one-pot" approach for the generation of expression constructs using strategically chosen protein purification tags like hexahistidine, SUMO, MBP, GST, and GB1 to enhance solubility and expression. The system allows visual candidate screening through mScarlet fluorescence and solubility tags are removable via TEV protease cleavage. We provide a comprehensive protocol encompassing oligonucleotide design, cloning, expression, His-tag affinity chromatography, and size-exclusion chromatography. This method, therefore, streamlines prokaryotic and eukaryotic protein production, rendering it accessible to standard molecular biology laboratories with basic protein biochemical equipment.

Optimization, purification and characterization of laccase from a new endophytic Trichoderma harzianum AUMC14897 isolated from Opuntia ficus-indica and its applications in dye decolorization and wastewater treatment.

BACKGROUND: Hazardous synthetic dye wastes have become a growing threat to the environment and public health. Fungal enzymes are eco-friendly, compatible and cost-effective approach for diversity of applications. Therefore, this study aimed to screen, optimize fermentation conditions, and characterize laccase from fungal endophyte with elucidating its ability to decolorize several wastewater dyes. RESULTS: A new fungal endophyte capable of laccase-producing was firstly isolated from cladodes of Opuntia ficus-indica and identified as T. harzianum AUMC14897 using ITS-rRNA sequencing analysis. Furthermore, the response surface methodology (RSM) was utilized to optimize several fermentation parameters that increase laccase production. The isolated laccase was purified to 13.79-fold. GFC, SDS-PAGE revealed laccase molecular weight at 72 kDa and zymogram analysis elucidated a single band without any isozymes. The peak activity of the pure laccase was detected at 50 °C, pH 4.5, with thermal stability up to 50 °C and half life span for 4 h even after 24 h retained 30% of its activity. The Km and Vmax values were 0.1 mM, 22.22 µmol/min and activation energy (Ea) equal to 5.71 kcal/mol. Furthermore, the purified laccase effectively decolorized various synthetic and real wastewater dyes. CONCLUSION: Subsequently, the new endophytic strain produces high laccase activity that possesses a unique characteristic, it could be an appealing candidate for both environmental and industrial applications.

Replication-Competent Oropouche Virus in Semen of Traveler Returning to Italy from Cuba, 2024.

A febrile man in Italy who had traveled to Cuba in July 2024 was diagnosed with Oropouche fever. Reverse transcription PCR detected prolonged shedding of Oropouche virus RNA in whole blood, serum, urine, and semen. Sixteen days after symptom onset, replication-competent virus was detected in semen, suggesting risk for sexual transmission.

Production of Norovirus VLPs of the Nine Representative Genotypes Widely Distributed in Japan using the Silkworm-Baculovirus Expression Vector System.

Norovirus (NoV) is one of the major causes of acute viral gastroenteritis in humans. Genetic variation is abundant, and prevalent genotypes vary from year to year and region to region. Since NoVs are difficult to amplify in cultured cells, genome RNA-free virus-like particles (VLPs) that mimic the capsid structure of the virus are promising vaccine candidates for the prevention of NoVs infection, and the development of multivalent VLP vaccines is required to prevent NoV infection in a wide range of genotypes. In this study, we attempted to produce NoV VLPs of the top nine genotypes that have a history of epidemics in Japan using the silkworm-baculovirus expression vector system (silkworm-BEVS), which has a proven track record in the mass production of recombinant proteins. In silkworm pupae infected with recombinant baculoviruses constructed to express VP1s, the major protein that forms VLP, the NoV VP1 protein was expressed in large amounts. Most genotypes of VP1 accumulated in the cytoplasm as soluble proteins, but solubility was reduced for that of two genotypes. VP1s of five genotypes could be purified in large quantities (>0.9mg per pupa) by a two-step purification process, and gel filtration chromatography analysis confirmed the formation of VLPs. This study demonstrates the utility of silkworm-BEVS in producing NoV VLPs of multiple genotypes and provides the basis for the development of a multivalent vaccine against genetically diverse NoV infections.

Case report: Manual carbon hemoperfusion for the treatment of meloxicam toxicity in a cat and suspected ibuprofen toxicity in a dog.

Extracorporeal blood purification (ECBP) has become a popular treatment option for non-steroidal anti-inflammatory drug (NSAID) toxicity in small animals. However, challenges arise when using ECBP for small dogs and cats because the priming volume required by most machine-based ECBP platforms might be excessive, leading to cardiovascular instability if a blood prime is not used. This report describes the successful use of manual carbon hemoperfusion (MCHP) to reduce plasma meloxicam levels in a cat following an inadvertent overdose and its use in a dog following suspected ibuprofen ingestion. In both animals, MCHP reduced the circuit volume needed for ECBP from 125 mL with a machine-based therapeutic plasma exchange or 104 mL with an in-series carbon hemoperfusion on an intermittent hemodialysis platform to just 40-50 mL. In the cat, MCHP reduced plasma meloxicam levels by 44%, and in both animals, the use of MCHP in these pets was well-tolerated and safe. Due to pre-existing anemia, the cat required a blood transfusion but the dog did not. MCHP is technically simple and can be performed at any hospital with access to carbon filters and blood bank resources. This technique may represent a reasonable alternative to treat NSAID toxicities in animals that are too small for conventional extracorporeal decontamination methods using either machine-based platforms without using a blood prime or in locations where these machines are unavailable.

Flow-electrode capacitive separation of organic acid products and recovery of alkali cations after acidic CO2 electrolysis.

Acidic CO2 electrolysis, enhanced by the introduction of alkali cations, presents a strategic approach for improving carbon efficiency compared to processes conducted in neutral and alkaline environments. However, a significant challenge arises from the dissolution of both organic acids and alkali cations in a strongly acidic feed stream, resulting in a considerable energy penalty for downstream separation. In this study, we investigate the feasibility of using flow-electrode capacitive deionization (FCDI) technology to separate organic acids and recover alkali cations from a strongly acidic feed stream (pH ~ 1). We show that organic acids, such as formic acid and acetic acid, are retained in molecular form in the separation chamber, achieving a rejection rate of over 90% under all conditions. Alkali cations, such as K+ and Cs+, migrate to the cathode chamber in ionic form, with their removal and recovery significantly influenced by their concentration and the pH of the feed stream, but responding differently to the types and concentrations of organic acids. The energy consumption for the removal and recovery of K+ is 4 to 8 times higher than for Cs+, and the charge efficiency is significantly influenced by the types of organic acid products and alkali cations. We conduct a series of electrochemical measurements and analyze the impedance spectroscopy, identifying that hindered mass transfer governed the electrode process. Our findings underscore the potential of FCDI as an advanced downstream separation technology for acidic electrocatalysis processes.

An unconventional strategy for purifying recombinant SARS-CoV-2 spike protein.

The soluble domain of the trimeric SARS-CoV-2 spike protein is a promising candidate for a COVID-19 vaccine. Purification of this protein from mammalian cell culture supernatant using conventional resin-based chromatography is challenging as its large size (∼550 kDa) restricts its access and mobility within the pores of the resin particles. This reduces binding capacity and process robustness very significantly as extremely low flow rates need to be used during purification. Convection-based ion-exchange membrane chromatography has been found to be suitable in this respect. However, the high ionic strength of mammalian cell culture supernatant makes it difficult to bind this protein on charged membranes without dilution with a suitable buffer. An unconventional strategy involving size-exclusion chromatography as the first step, followed by cation exchange membrane chromatography as the second step is proposed in this paper. In the size exclusion chromatography step, the spike protein is excluded from the pores and can therefore be isolated in the void volume fraction. This step removes small molecule impurities and also serves as a desalting and buffer exchange step, making the partially purified material suitable for the cation exchange membrane chromatography step. The proposed process is variant-independent, fast and scalable and addresses some of the challenges associated with the currently used purification methods.

Purification Resistance Index: A new water quality assessment method toward drinking water production.

Water quality assessment plays a significant role in ensuring the availability of clean and safe water. The Water Quality Index (WQI) model method has been developed to provide a basis for assessing water quality by integrating various water quality parameters. However, existing WQIs do not "actively" consider the difficulty of water treatment from raw water to specific water use scenarios. This study proposes a novel model framework, named as Purification Resistance Index (PRI), quantitatively evaluating not only the exceedance of pollutants but also how difficult they can be removed in the water treatment process. The framework is built based on the conventional drinking water treatment processes, with sub-indices for coagulation-sedimentation (rc), filtration (rf), disinfection (rd), and advanced treatment (ra). The model considers appropriate weights assigned to each sub-index to calculate the purification resistance, resulting in a comprehensive index for water quality evaluation. Case studies on nationwide and citywide water source reservoirs demonstrated the applicability of PRI approach. PRI breakthrough the traditional water quality risk assessment paradigm and extents to engineering region and provide useful tools for water source supervision, drinking water treatment plant planning and updating, operation control, and other purposes. Water authority, water utility and municipal design institute will all benefit. It is open for more localized practices validation and discussion.

MOF Nanosheet-Functionalized Poly(lactic acid) Meta-membranes for Long-Term Air Purification and Intelligent Monitoring.

The wide use of conventional polymeric air filters is causing a dramatically increasing accumulation of plastic and microplastic pollution. The development of poly(lactic acid) (PLA) fibrous membranes for efficient air purification is of important significance but frequently challenged by the rapid decay of filtration performance due to the intrinsically poor electret properties of PLA. Here, we propose an electroactivity promotion methodology, involving the one-step synthesis and homogeneous incorporation of high-dielectric ZIF-8 nanosheets (ZIFNSs), to facilitate interfacial polarization and fiber refinement during electrospinning of PLA nanofibers. The preparative electrospun PLA/ZIFNS meta-membranes exhibited an unusual combination of significantly reduced nanofiber diameter (∼462 nm), enhanced surface potential (approaching 10 kV), and increased surface activity and facilitated the formation of electroactive phases. With well-controlled morphological features, the highly electroactive PLA/ZIFNS meta-membranes exhibited exceptional filtration efficiencies for PM2.5 and PM0.3 (99.2 and 96.0%, respectively) even at the highest airflow rate of 85 L/min, in clear contrast to that of its pure PLA counterpart (only 79.3 and 74.6%). Arising from the increased electroactivity and active contact sites, remarkable triboelectric performance and self-charging mechanisms were demonstrated for the PLA/ZIFNS meta-membranes, contributing to long-term efficient PM0.3 filtration (97.5% for over 360 min). Moreover, as triggered by physiological activities like respiration and speaking, the electroactive PLA/ZIFNS meta-membranes enabled real-time monitoring with high sensitivity and specificity. The proposed strategy affords significant promotion of electroactivity and triboelectric performance for PLA nanofibers, which may motivate the development of ecofriendly protective membranes for respiratory healthcare and real-time monitoring.

Evaluation, optimization study, and life cycle assessment of novel eco-friendly PVA-based nanocomposite hydrogel adsorbents for methylene blue and paracetamol removal.

In this study, an eco-friendly and novel hydrogel based on a crosslinked polyvinyl alcohol (PVA), iota carrageenan (IC) and polyvinylpyrrolidone (PVP) scaffold, containing a large amount (10-50 wt%) of nanoscale palm fronds (NPF) as additives, for water purification was demonstrated. A life cycle assessment (LCA) findings on NPF as biomass waste incorporated into PVA_PVP_IC polymer matrix was presented, and the results highlight the necessity of focused actions to reduce environmental impact and support the palm waste utilization in a sustainable manner. The multicomponent nanocomposite hydrogels were examined as adsorbents in a system work in batches for methylene blue (MB) and paracetamol (PCT) removal. The results show that, the presence of NPF, which dispersed in the hydrogel PVA_PVP_IC scaffolds containing both covalent and non-covalent cross-linking bonds, greatly enhanced the MB and PCT adsorption efficiency. A response surface methodology (RSM) model was used to find the best operating parameters of contaminant adsorption, including time, adsorbent dose, and starting concentration of pollutants. By using this statistical model, it was found that the optimal conditions for the adsorption reaction to achieve the complete removal of MB are 66.7 h adsorption time duration, 98.5 mg L-1 starting concentration, and an adsorbent dose of 5.9 mg, while for the complete removal of PCT, it is 57.6 h adsorption time duration, 80 mg L-1 starting concentration, and an adsorbent dose of 6 mg. The reusability of the nanocomposite hydrogels were tested for 5 cycles, all showed high adsorption capacity, indicating the potential for practical application of this nanocomposite hydrogel system. This study indicates that the prepared nanocomposite hydrogel raises the standard used for treatment of wastewater and also gives a solution to protect the environment and mitigate global warming.

Biogas slurry treatment and biogas upgrading by microalgae-based systems under the induction of different phytohormones.

The low grade of biogas and the difficulty of treating biogas slurry are the two major bottlenecks limiting the sustainable development of the fermentation engineering. This study investigates the potential role of microalgae-microbial symbiosis and phytohormones in solving this challenge. Chlorella microalgae were combined with endophytic bacteria (S395-2) and Clonostachys fungus to construct symbiotic systems. Growth, photosynthetic activity, and carbon dioxide and pollutant removal out of biogas slurry and biogas were analyzed under treatment with three different phytohormones (cytokinin, synthetic strigolactones (GR24), natural strigolactones). The Chlorella-S395-2-Clonostachys symbiont achieved the highest purification efficiency under GR24 induction, with removal efficiency exceeding 86% for chemical oxygen demand, total phosphorous, and total nitrogen, as well as over 76% for CO2. Economic efficiency can be increased by about 150%. The positive correlation between treatment effectiveness and co-culture performance suggests a promising avenue for developing symbiotic systems for biogas slurry treatment and biogas upgrading.

Optimizing roof-harvested rainwater storage: Impact of dissolved oxygen regime on self-purification and quality dynamics.

Roof-harvested rainwater presents a promising, unconventional, and sustainable water resource for both potable and non-potable uses. However, there is a significant gap in understanding the quality evolution of stored rainwater under varying dissolved oxygen conditions and its suitability for various applications. This study investigated the evolution of rainwater quality under three distinct storage conditions: aerated, open, and sealed. Additionally, the microbial community and metabolic functions were analyzed to systematically evaluate the self-purification performance over long-term storage durations. The results indicate that aerated storage enhances microbial carbon metabolism, leading to a degradation rate of 54.4 %. Sealed and open storage conditions exhibited primary organic matter degradation during the early and late stages, respectively. Roof-rainwater harvesting (RRWH) systems showed limited denitrification activity across all three dissolved oxygen conditions. The maximum accumulation of NO3-N during the storage period reached 5.23 mg/L. In contrast, sealed storage demonstrated robust self-purification performance, evidenced by a comprehensive coefficient of 15.83 calculated by Streeter-Phelps model. These findings provide valuable insights into the mechanisms governing rainwater quality changes under various storage conditions, emphasizing the necessity for developing effective management strategies for the storage and utilization of roof-harvested rainwater.

Activated carbon co-modified by chitosan and SDS for targeted removal of aflatoxins from fragrant peanut oil.

The pore size structure and surface structure of activated carbon (AC) may be ameliorated using the hydrophobic groups and negative surface charge of sodium dodecyl sulfate (SDS), along with the abundant hydroxyl and amine groups of chitosan (CS). In this study, a composite adsorbent (CS/SDS/AC) created through co-modification of AC with CS and SDS was employed to remove aflatoxins from fragrant peanut oil. CS/SDS/AC exhibited a superior adsorption capacity (89.9 %) for adsorbing aflatoxins. More significantly, CS/SDS/AC improved the retention of sterols (64-99.3 %), tocopherols (61.4-82.7 %), and volatile flavor compounds (68.4-82.3 %) in the treated oil. CS/SDS/AC composite adsorbent emerged as a promising candidate characterized by high detoxification efficiency and low preparation costs but also helped retain nutrients and flavors in the fragrant oil. This development could present a novel strategy for oil industry to address aflatoxins.