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.

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.

Large-Scale Transient Transfection of Suspension-Adapted Chinese Hamster Ovary Cells for the Production of the Trimeric SARS-CoV-2 Spike Protein.

The production and purification of the secreted ectodomain of SARS-CoV-2 spike protein (S protein) were performed by transiently transfecting suspension-adapted Chinese hamster ovary cells (ExpiCHO). The method involved the separate addition of plasmid DNA expressing the S protein and polyethyleneimine to a suspension culture at a density of 5 × 106 cells/mL; and the subsequent addition of dimethyl sulfoxide at 2% (v/v). The transfected ExpiCHO cells were cultivated at 31 °C with agitation by orbital shaking under 5% CO2. On day six post-transfection, the culture was centrifuged, and the supernatant was filtered to remove cells and cell debris. Finally, the secreted recombinant S protein was recovered from the supernatant by a single step of affinity chromatography to the Twin-Strep-Tag of the recombinant S protein.

A system for efficient and sustainable cogeneration of water and electricity: Temperature difference induced by photothermal conversion and evaporative cooling.

Solar energy, with its sustainable properties, has garnered considerable attention for its potential to produce green electricity and clean water. This paper proposes a multistage energy transfer co-generation system (MWCNTs-covered thermoelectric module with aerogel and cooler, AC-CTEM) combining power generation and evaporative cooling. On the light-absorbing surface, the hot side of a thermoelectric module is covered with a hydrophobic coating made of PDMS and MWCNT. The cold side transfers heat to the evaporation zone using a heat sink. Aerogel evaporators are cross-linked with chitosan and polyurethane, which reduces the enthalpy of evaporation and facilitates efficient interfacial evaporation to remove heat and return it to refrigeration. Additionally, with the addition of Fresnel lenses and wind energy to the enhancement device, the system achieved an evaporation rate of 3.445 kg m-2 h-1 and an open-circuit voltage of 201.12 mV under 1 kW m-2 solar irradiation. The AC-CTEM system also demonstrated long-term stability and effectiveness in treating various types of non-potable water. Furthermore, we demonstrated the practical utility of the system by successfully cultivating grass seeds and powering electronic equipment. The AC-CTEM system exemplifies a practical energy-saving approach for the development of highly efficient co-generation systems.

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.

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.

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.

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.

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.

A sustainable approach for efficient ammonium recovery, and simultaneous TOC removal from aqueous solutions by vesicle-like iron phosphate-based carbon nanomaterials.

NH4+ is an ion with versatile potential; however, the release of wastewater containing this component, regardless of its high or low concentration, causes severe eutrophication in aquatic systems and contaminates numerous manufacturing processes. Thus, this study developed a sustainable method that can simultaneously remove, recover NH4+, polish water, oxidize organic matter, and yet release a material that can still be used as fertilizer. Regarding NH4+ removal, FeP400 rapidly exhibited an exceptional NH4+ uptake capacity (343.5 mg g-1) within 8 min, even in dairy processing wastewater with high NH4+ concentrations and diverse co-existing components. FeP400 could oxidize organic compounds spontaneously to remove TOC, indirectly enhancing its NH4+ uptake up to 33.5 % through charge balance mechanisms. The adsorption process involved both chemical (i.e., double-salt precipitation) and physical mechanisms (i.e., H-bonding and electrostatic interaction), as confirmed by thermodynamics, FT-IR, and XPS analyses. Regarding recovery, FeP400 can be reused for over 10 cycles with high removal (81 %) and NH4+ recovery (88 %), a significant improvement over conventional options. FeP400 also performed efficiently under flowing conditions using low-range NH4+ and TOC samples over 10 cycles, polishing not only 34.1 L of water with undetected NH4+, neutral pH, and extremely low TOC but also effectively recovering the NH4+ uptake at an economical cost. Lastly, its environmentally friendly nature, which contains essential nutrients for plant growth, further enhances its recyclability after release. Thus, FeP400 is believed to offer a transformative, sustainable, and highly efficacious solution to the NH4+contamination and critical ultrapure water issues that industries urgently address.

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.

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.

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.

Interactors and neighbors of ULK1 complex members.

The ULK1 kinase complex plays a crucial role in autophagosome biogenesis. To identify interactors or regulators of ULK1 complex assembly influencing autophagosome biogenesis, we performed an interaction proteomics screen. Employing both affinity purification and proximity labeling of N- and C-terminal tagged fusion proteins coupled to quantitative mass spectrometry, we identified 317 high-confidence interactors or neighbors of the four ULK1 complex members, including both member-specific and common interactors. Interactions with selective macroautophagy/autophagy receptors indicate the activation of selective autophagy pathways by 90 min of nutrient starvation. Focusing on the ULK1 effector protein BAG2, a common interactor identified by both approaches, we highlight that ULK1 phosphorylates BAG2, supporting the localization of the scaffold and autophagy inducer AMBRA1 to the ER, thereby positively regulating autophagy initiation.Abbreviation: AMBRA1: autophagy and beclin 1 regulator 1; ATG: autophagy related; ER: endoplasmic reticulum; HA: hemagglutinin; KD: knockdown; KO: knockout; MS: mass spectrometry; PTM: posttranslational modification; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1; WIPI2: WD repeat domain, phosphoinositide interacting 2.

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.

Sustainable Water Management with Photocatalytic Janus Mesh: Efficient Fog Harvesting, Water Purification, and Microbial Disinfection.

Water scarcity is a critical global challenge, especially in arid and semiarid regions. Fog harvesting has emerged as a promising solution; however, concerns about air pollution and bacterial growth in humid environments have raised doubts about the safety and sustainability of such systems. This study introduces a Janus mesh with asymmetric wettability on its two faces, fabricated through a simple and scalable method. The unique design of the Janus mesh enables the transport of water droplets from the superhydrophobic side to the hydrophilic side in a unidirectional manner, enhancing its fog harvesting efficiency. The mesh's photocatalytic properties not only elevate the fog harvesting rate to 4.7 kg·m-2h-1 but also effectively purify the harvested water by removing organic contaminants (94%) and microbial impurities (99.98%). Additionally, its inherent bactericidal activity prevents biofouling, ensuring sustained efficiency in water collection. The mesh's self-cleaning abilities through photocatalysis maintain its surface integrity, promising long-term stability for fog harvesting applications. This technological advancement in fog harvesting offers a sustainable and economical solution to water scarcity concerns, addressing safety and sustainability issues associated with existing systems. By potentially transforming the livelihoods of communities struggling with water scarcity, this innovation paves the way for a more sustainable future.

Protocol for validating liquid-liquid phase separation as a driver of membraneless organelle assembly in vitro and in human cells.

Liquid-liquid phase separation (LLPS) of scaffold proteins has often been proposed to drive the biogenesis of membraneless cellular compartments. Here, we present a protocol to link in vitro LLPS propensity to localization in vivo. We describe steps for examining LLPS in vitro in the presence of crowding agents or cytomimetic media. We complement our in vitro studies with recombinant proteins with experiments of protein electroporation into mitotic HeLa cells. In addition, we discuss steps to assess protein localization and delivery levels. For complete details on the use and execution of this protocol, please refer to Hedtfeld et al.1.

Dihydrotetrazine-Functionalized Zirconium-Based Metal-Organic Frameworks for High-Capacity Oil Denitrogenation.

High structural stability, dual organic-inorganic nature, and tunability in chemical functionality are promising characteristics of zirconium-based metal-organic frameworks (Zr-MOFs). These properties assist Zr-MOFs in extending their applications in various fields, especially adsorptive removal of pollutants. In this work, two well-known Zr-MOFs (UiO-66(Zr) and MIL-140(Zr) with the formula Zr6O4(OH)4(BDC)6, H2BDC is benzene 1,4-dicarboxylic acid) were synthesized and decorated with a dihydrotetrazine functional group through postsynthesis linker exchange (PSLE). Two dihydrotetrazine (DHTZ)-functionalized frameworks, UiO-66(Zr)-DHTZ and MIL-140(Zr)-DHTZ, were applied for the removal of quinoline (Qui) and indole (Ind) from the model oil. The results of adsorption experiments at room temperature display that these functionalized Zr-MOFs have significantly improved removal capacities for Qui (875% for UiO-66(Zr)-DHTZ and 303% for MIL-140(Zr)-DHTZ) and Ind (722% for UiO-66(Zr)-DHTZ and 257% for MIL-140(Zr)-DHTZ). Mechanistic studies based on X-ray photoelectron (XPS) and Fourier-transform infrared (FT-IR) spectroscopies reveal that there is a specific kind of host-guest interaction between dihydrotetrazine and nitrogen-containing compounds (NCCs). UiO-66(Zr)-DHTZ adsorbs 1426 mg·g-1 Qui and 1176 mg·g-1 Ind, while MIL-140(Zr)-DHTZ adsorbs 619 mg·g-1 Qui and 511 mg·g-1 Ind. The lower adsorption capacities of MIL-140(Zr)-DHTZ compared to UiO-66(Zr)-DHTZ are related to its lower surface area (783 m2·g-1 versus 330 m2·g-1). The recyclability of the frameworks goes up to five cycles without any significant decrease in the removal capacity. These results indicate that dihydrotetrazine-functionalized Zr-MOFs are highly stable platforms with superior adsorption capacity compared to basic and neutral NCCs.

Separation and purification of antimicrobial substances from Paenibacillus polymyxa KH-19 and analysis of its physicochemical characterization.

Soft rot is one of the top ten most dangerous plant pathogens in agricultural production, storage, and transport, and the use of microorganisms and their metabolites to control soft rot is a current research hotspot. In this study, we identified the antimicrobial substance in the metabolite of Paenibacillus polymyxa KH-19, and determined that the antimicrobial substance of this strain was an active protein. The protein was completely precipitated at 40-60% ammonium sulphate saturation and showed good inhibitory effects against seven pathogenic bacteria including Pectobacterium carotovorum BC2 and seven pathogenic fungi including Pyricularia oryzae. The MIC of the protein was 51.563 µg/mL, temperature acid-base UV and light stability insensitive to protease, with high-temperature resistance. The antimicrobial protein was isolated and purified by DEAE-anion exchange column and Sephadex G-75 gel filtration chromatography, and the LC-MS/MS assay identified the protein as lysophosphatidyl esterase with a molecular weight of 25.255 kDa. The purified antimicrobial protein increased the inhibitory effect against P. carotovorum BC2, with the diameter of the circle of inhibition being 26.50 ± 0.915 mm. Bioinformatics analysis showed that the protein has the molecular formula of C1117H1732N316O338S5, encodes 224 amino acids, has an aliphatic index of 88.39, and belongs to the category of hydrophilic unstable proteins. The present study is the first report of an active protein with extreme thermoplastic and resistance to P. carotovorum BC2, which provides a reference for the preparation and application of the antimicrobial substances of P. polymyxa KH-19, as well as a theoretical basis for the study of the function of lysophosphodiesterase protein and its use as a microbial preparation.