Purification Cycles Research Articles

Organometallic Compound Stabilizes All-Inorganic Tin-Based Perovskite Nanocrystals Against Antisolvent Post-Treatment.

The isolation and purification of all-inorganic Sn-based perovskite nanocrystals (PNCs) remain troublesome, as common antisolvents accelerate the collapse of the optically active perovskite structure. Here, we mitigate such instabilities and endow strong resistance to antisolvent by incorporating the organometallic compound zinc diethyldithiocarbamate, Zn(DDTC)2, during the solution-based synthesis of all-inorganic CsSnI3 nanocrystals. Thiourea is shown to form through the thermal-driven conversion of Zn(DDTC)2 during synthesis, which binds to un-passivated Sn sites on the crystal surface and shields it from irreversible oxidation reactions. The CsSnI3 PNCs capped with thiourea show great stability after two purification cycles using methyl acetate, with negligible change in morphology, phase, and optical properties. Moreover, the modified PNCs are resistant to other commonly used antisolvents, like ethyl acetate, 1-pentanol, and isopropanol, offering a platform to explore all-inorganic Sn-based nanocrystalline thin films and optoelectronics.

GC–MS-based metabolite fingerprinting reveals the presence of novel anticancer compounds in the microcolonial fungus Aureobasidium sp. TD-062 from the under-explored Thar Desert

Aureobasidium sp., strain TD-062, a micro colonial black yeast, was obtained as part of a screening program from the Thar Desert of India, which has been inadequately explored for novel microorganisms/bioactive metabolites. The anticancer activity of aqueous and organic solvent extracts of culture supernatant of TD-062 was investigated against A-375 myeloma and MCF-7 breast cancer cell lines. Following column chromatography, bioactive fractions were subjected to GC–MS analysis in two cycles. The GC–MS metabolite fingerprinting revealed 20 compounds belonging to various chemical groups. Further, based on observed and calculated mass ion spectra, retention time, and retention indices, many compounds could be considered novel. The second purification cycle resulted in three compounds- ATD-1, ATD-2 and ATD-3 with different calculated retention indices from that of the nearest matching compounds, squalene and tocopherol. In silico prediction study of their ADMET profiles, suggests that these new compounds have a suitable contour for use as safe anticancer drugs, without the toxicity normally associated with anticancer compounds.

Serotype-agnostic affinity purification of adeno-associated virus (AAV) via peptide-functionalized chromatographic resins

Adeno-associated viruses (AAVs) have emerged as a prominent family of vectors for gene delivery, providing therapeutic options to diseases once deemed incurable. At the same time, they necessitate efficient and affordable purification methods that can be platformed to serve all AAV serotypes. Current chromatographic tools, while affording high product purity, fail to bind certain serotypes, provide limited yield and lifetime, and impose harsh elution conditions that can compromise the vector's activity and safety. Addressing these challenges, this work demonstrates the application of new peptide ligands as the first serotype-agnostic technology for AAV purification by affinity chromatography. Our study reveals a pH-dependent affinity interaction: AAV2, AAV3, AAV6, AAV9, and AAVrh.10 are effectively captured at neutral pH, while binding AAV1, AAV5, AAV7, and AAV8 is stronger in a slightly acidic environment. The elution of bound AAVs was achieved using magnesium chloride at neutral pH for all serotypes, consistently affording capsid yields above 50% and genome yields above 80%, together with a >100-fold reduction in host cell proteins and nucleic acids. In particular, peptide ligand A10 exhibited remarkable binding capacity (> 1014 vp per mL of resin) and purification performance for all AAV serotypes, demonstrating broad applicability for gene therapy manufacturing. Finally, this work introduces novel alkaline-stable variants of A10 and demonstrates their use as the first affinity ligands capable of performing multiple cycles of AAV2, AAV8, and AAV9 purification with intermediate caustic cleaning without loss of capacity or product quality. Collectively, these results demonstrate the promise of this technology to further the impact and affordability of gene therapy.

Oriented immobilization of nanobodies using SpyCatcher/SpyTag significantly enhances the capacity of affinity chromatography

The use of nanobodies (Nbs) in affinity chromatography for biomacromolecule purification is gaining popularity. However, high-performance Nb-based affinity resins are not readily available, mainly due to the lack of suitable immobilization methods. In this study, we explored an autocatalytic coupling strategy based on the SpyCatcher/SpyTag chemistry to achieve oriented immobilization of Nb ligands. To facilitate this approach, a variant cSpyCatcher003 (cSC003) was coupled onto agarose microspheres, providing a specific attachment site for SpyTagged nanobody ligands. The cSC003 easily purified from Escherichia coli through a two-step procedure, exhibits exceptional alkali resistance and structural recovery capability, highlighting its robustness as a linker in the coupling strategy. To validate the effectiveness of cSC003-derivatized support, we employed VHSA, a nanobody against human serum albumin (HSA), as the model ligand. Notably, the immobilization of SpyTagged VHSA onto the cSC003-derivatized support was achieved with a coupling efficiency of 90 %, significantly higher than that of traditional thiol-based coupling method. This improvement directly correlated to the preservation of the native conformation of nanobodies during the coupling process. In addition, the Spy-immobilized resin demonstrated better performance in the binding capacity, with a 3-fold improvement in capture efficiency, underscoring the advantages of the Spy immobilization strategy for oriented immobilization of VHSA ligands. Moreover, online purification and immobilization of SpyTagged VHSA from crude bacterial lysate was achieved using the cSC003-derivatized support. The resulting resin exhibited high binding specificity towards HSA, yielding a purity above 95 % directly from human serum, and maintained good stability throughout multiple purification cycles. These findings highlight the potential of the Spy immobilization strategy for developing Nb-based affinity chromatographic materials, with significant implications for biopharmaceutical downstream processes.

Exploring the Potential of Endophytic Microorganisms and Nanoparticles for Enhanced Water Remediation.

Endophytic microorganisms contribute significantly to water bioremediation by enhancing pollutant degradation and supporting aquatic plant health and resilience by releasing bioactive compounds and enzymes. These microorganisms inhabit plant tissues without causing disease or any noticeable symptoms. Endophytes effectively aid in eliminating contaminants from water systems. Nanoparticles serve as potent enhancers in bioremediation processes, augmenting the efficiency of pollutant degradation by increasing surface area and bioavailability, thereby improving the efficacy and rate of remediation. Their controlled nutrient release and ability to stabilize endophytic colonization further contribute to the enhanced and sustainable elimination of contaminated environments. The synergistic effect of endophytes and nanoparticles in water remediation has been widely explored in recent studies, revealing compelling outcomes. Water pollution poses significant threats to human health, ecosystems, and economies; hence, the sixth global goal of the Sustainable Development Agenda 2030 of the United Nations aims to ensure the availability and sustainable management of water resources, recognizing their crucial importance for current and future generations. Conventional methods for addressing water pollution exhibit several limitations, including high costs, energy-intensive processes, the production of hazardous by-products, and insufficient effectiveness in mitigating emerging pollutants such as pharmaceuticals and microplastics. Noticeably, there is an inability to effectively remove various types of pollutants, thus resulting in incomplete purification cycles. Nanoparticle-enhanced water bioremediation offers an innovative, eco-friendly alternative for degrading contaminants. A growing body of research has shown that integrating endophytic microorganisms with nanoparticles for water bioremediation is a potent and viable alternative. This review examines the potential of using endophytic microorganisms and nanoparticles to enhance water remediation, exploring their combined effects and applications in water purification. The paper also provides an overview of synthetic methods for producing endophyte-nanoparticle composites to optimize their remediation capabilities in aqueous environments. The final section of the review highlights the constraints related to integrating endophytes with nanoparticles.

Organic A‐Site Cations Improve the Resilience of Inorganic Lead‐Halide Perovskite Nanocrystals to Surface Defect Formation

AbstractLead halide perovskite nanocrystals (LHP NCs) are generally prone to surface defect formation during the purification process, resulting in a reduced photoluminescence (PL) quantum yield. The purification of LHP NCs using antisolvents leads to the detachment of surface atoms and ligandsin the form of alkylammonium‐halides or A‐carboxylates (A: Cs, methylammonium, MA, or formamidinium, FA. Currently, intense research is being carried out to improve the surface stability of LHP NCs using various long‐chain organic ligands that strongly bind to the surface of NCs. Herein, the findings on the higher surface stability of hybrid (MAPbBr3 and FAPbBr3) LHP NCs compared to that of inorganic CsPbBr3 NCs against purification with polar antisolvents are reported. It is discovered that the CsPbBr3 NC surface stability can be enhanced (reaching that of hybrid perovskite NCs) by the incorporation of a small amount of MA or FA A‐site organic cations into their lattice, corroborated by the evidence that the PL quantum yield of mixed A‐cation CsxFA1‐xPbBr3 and CsxMA1‐xPbBr3 NCs remains unaffected after several purification cycles. It is hypothesized that this is attributed to hydrogen bonding between the organic A‐site cations and the neighboring halides on the surface. These findings are not only fundamentally important but are also expected to have wide implications in the field of metal‐halide perovskite optoelectronics.

Isolation and monoculture of functional primary astrocytes from the adult mouse spinal cord.

Astrocytes are a widely heterogenic cell population that play major roles in central nervous system (CNS) homeostasis and neurotransmission, as well as in various neuropathologies, including spinal cord injury (SCI), traumatic brain injury, and neurodegenerative diseases, such as amyotrophic lateral sclerosis. Spinal cord astrocytes have distinct differences from those in the brain and accurate modeling of disease states is necessary for understanding disease progression and developing therapeutic interventions. Several limitations to modeling spinal cord astrocytes in vitro exist, including lack of commercially available adult-derived cells, lack of purchasable astrocytes with different genotypes, as well as time-consuming and costly in-house primary cell isolations that often result in low yield due to small tissue volume. To address these issues, we developed an efficient adult mouse spinal cord astrocyte isolation method that utilizes enzymatic digestion, debris filtration, and multiple ACSA-2 magnetic microbead purification cycles to achieve an astrocyte monoculture purity of ≅93-98%, based on all markers assessed. Importantly, the isolated cells contain active mitochondria and express key astrocyte markers including ACSA-1, ACSA-2, EAAT2, and GFAP. Furthermore, this isolation method can be applied to the spinal cord of male and female mice, mice subjected to SCI, and genetically modified mice. We present a primary adult mouse spinal cord astrocyte isolation protocol focused on purity, viability, and length of isolation that can be applied to a multitude of models and aid in targeted research on spinal-cord related CNS processes and pathologies.

Valorization of Acid Leaching Post-Consumer Gypsum Purification Wastewater

Industries are required to utilize treatment technologies to reduce contaminants in wastewater prior to discharge and to valorize by-products to increase sustainability and competitiveness. Most acid leaching gypsum purification studies have obviated the treatment of the highly acidic wastewater produced. In this work, acidic wastewater from acid leaching purification of post-consumer gypsum was treated to recover a valuable solid product and reusable water. The main aims of this work were to determine the impact of recirculating acidic and treated wastewaters on the efficiency of the acid leaching purification process and to valorize the impurities in the wastewater. Samples were characterized through X-ray fluorescence and X-ray diffraction. SimaPro 9.5 and the ReCiPe 2016 midpoint method were used for the life cycle assessment of three sustainable wastewater management approaches. The reuse of the acidic wastewater did not improve the chemical purity of gypsum. Soluble impurities were precipitated at pH 10.5 as a magnesium-rich gypsum that could be commercialized as fertilizer or soil ameliorant. The alkaline-treated water was reused for six acid leaching purification cycles without impacting the efficiency of the purification process. An acid leaching–neutralization–filtration–precipitation approach demonstrated superior overall environmental performance. Barriers and enabling measures for the implementation of an in-house wastewater treatment were identified.

Biobased biodegradable hydrogel containing modified cellulosic nanofiber-ZnO nanohybrid as efficient metal ions removers with recyclable capacity

In the past decades, immense attention has been paid to the development of biobased hydrogel nanocomposite (HNC) to remove metal ions from wastewater owing to their high adsorption capacity, biodegradability, and reusability. In this work, we have demonstrated the fabrication of a nanohybrid using modified cellulose nanofiber and zinc oxide nanoparticles using an eco-friendly and non-toxic approach and subsequently incorporated it into a starch/itaconic acid/acrylic acid-based hydrogel matrix. The structure and morphology of the prepared nanohybrid and HNC were characterized by different spectroscopic and microscopic techniques. The resulting HNCs were employed for the removal of metal ions (Cu(II) and Fe(II)) from aqueous solution and manifested noteworthy adsorption ability. With the incorporation of 0.25 wt% nanohybrid, the HNC showed the highest water swelling capacity of 580 g/g. Besides, the incorporation of the nanohybrid enhanced the metal ion adsorption ability of the HNC, and the removal capacity of 122 and 70 mg/g was obtained for Cu(II) and Fe(II) ions, respectively. The adsorption kinetics followed the pseudo-second-order model and isotherms were found to be fitted well with the Freundlich model. Additionally, the prepared nano-structural adsorbent can be removed easily and could be used for 3 repeated purification cycles without significant loss of efficiency. Thus, this work leads to the development of nanohybrid-containing biobased HNC as a promising candidate for the removal of toxic metal ions with remarkable reusability as well as adsorption capacity.

A specific nanobody-based affinity chromatography resin as a platform for small ubiquitin-related modifier fusion protein purification

As an excellent fusion tag for expressing heterologous proteins, yeast SUMO (small ubiquitin-related modifier) has unique advantages such as improving solubility, promoting stability, and reducing degradation, but it lacks a simple and rapid purification method. Camelid single-domain antibodies (VHHs or nanobodies) show great promise as an efficient tool in analytical application. In this study, VHHs against SUMO protein were isolated for the first time using biopanning of an immune camelid nanobody library. Among these nanobodies, VS2 demonstrated a high expression level (1.12 g L − 1), and a high affinity for SUMO (2.26 nM). Meanwhile, VHHs were coupled to agarose resins by cysteine at the C-terminal to form affinity chromatography resins. The VS2 resin showed excellent specificity and a dynamic binding capacity for SUMO, SUMO-DsbA (disulfide oxidoreductase) and SUMO-SAM (S-adenosylmethionine synthetase) were 2.41 mg/mL resin, 7.57 mg/mL resin and 16.23 mg/mL resin, respectively. Furthermore, the VS2 resin enabled one-step purification of SUMO-fusions [SUMO-Fc (human IgG1-Fc fragment), SUMO-IGF1 (human insulin-like growth factor 1), SUMO-FGF21 (human fibroblast growth factor 21), SUMO-G-CSF (human Granulocyte colony-stimulating factor), SUMO-PDGF (human platelet-derived growth factor) and SUMO-PAS200 (conformationally disordered polypeptide chains with expanded hydrodynamic volume comprising the small residues Pro, Ala-and Ser)], and maintained binding capacity and selectivity over 25 purification cycles, each including 15 min of cleaning-in-place with 0.1 M NaOH. This study demonstrated that the VS2 resin was a useful tool at the laboratory scale for one-step purification of various SUMO fusions from complex mixtures.

Double buffer of MAPbBr3 QDs using a bulk crystal source by the LARP method and MABr modifier for CsPbBr3 perovskite light-emitting diodes

In this work, we employed MAPbBr3 quantum dots (QDs) and MABr salt as buffer layers to boost the optoelectronic characteristics of CsPbBr3 perovskite light-emitting diodes (PeLEDs). The MAPbBr3 QDs were prepared by ligand-assisted reprecipitation method and using MAPbBr3 bulk single crystals as source material, but traditional powder source. The MAPbBr3 bulk single crystals were obtained by inverse temperature crystallization method. The purification cycles of bulk-crystal source MAPbBr3 quantum dots (bc-MAPbBr3 QDs) were studied. The bc-MAPbBr3 QDs and MABr salt were applied into the PeLED structures. The best brightness reached 7555 cd m−2, and the external quantum efficiency reached 0.318%.

Investigation on nonequilibrium crystallization of highly undercooled Cu–Ni–Co alloys

Deep undercooling rapid solidification experiments of Cu–Ni–Co alloys were carried out using the molten glass purification cycle superheat method. 130 K, 177 K and 277 K undercooling degrees were obtained and analysed for Cu55Ni43Co2 alloy. The crystallographic information obtained by EBSD and the dynamics of the solidification front taken by high speed photography were analysed and it was concluded that both the formation of relatively rounded equiaxed grains during the first refinement and the subsequent formation of finer subcrystals were due to the release of a large amount of solidification energy during the solidification process. The re-glow stage releases a large amount of latent heat of solidification resulting in the remelting of the dendrites. TEM tests were carried out on Cu55Ni43Co2 alloy with a maximum undercooling of 277 K. It was found that part of the substructure showed a high density dislocation network. By analysing the effect of increasing undercooling on the microstructure evolution, combined with the EBSD and TEM characterisation data, it was found that the stress-induced recrystallization mechanism was the dominant factor in the grain refinement mechanism at large undercooling.

Protein engineering of a nanoCLAMP antibody mimetic scaffold as a platform for producing bioprocess-compatible affinity capture ligands

Protein A affinity chromatography is widely used for the large-scale purification of antibodies because of its high yield, selectivity, and compatibility with NaOH sanitation. A general platform to produce robust affinity capture ligands for proteins beyond antibodies would improve bioprocessing efficiency. We previously developed nanoCLAMPs (nano Clostridial Antibody Mimetic Proteins), a class of antibody mimetic proteins useful as lab-scale affinity capture reagents. This work describes a protein engineering campaign to develop a more robust nanoCLAMP scaffold compatible with harsh bioprocessing conditions. The campaign generated an improved scaffold with dramatically improved resistance to heat, proteases, and NaOH. To isolate additional nanoCLAMPs based on this scaffold, we constructed a randomized library of 1× 1010 clones and isolated binders to several targets. We then performed an in-depth characterization of nanoCLAMPs recognizing yeast SUMO, a fusion partner used for the purification of recombinant proteins. These second-generation nanoCLAMPs typically had a Kd of <80nM, a Tm of >70 °C, and a t1/2 in 0.1mg/ml trypsin of >20h. Affinity chromatography resins bearing these next-generation nanoCLAMPs enabled single-step purifications of SUMO fusions. Bound target proteins could be eluted at neutral or acidic pH. These affinity resins maintained binding capacity and selectivity over 20 purification cycles, each including 10min of cleaning-in-place with 0.1M NaOH, and remained functional after exposure to 100% DMF and autoclaving. The improved nanoCLAMP scaffold will enable the development of robust, high-performance affinity chromatography resins against a wide range of protein targets.

Sustainable soy protein microsponges for efficient removal of lead (II) from aqueous environments

Protein-based materials recently emerged as good candidates for water cleaning applications, due to the large availability of the constituent material, their biocompatibility and the ease of preparation. In this work, new adsorbent biomaterials were created from Soy Protein Isolate (SPI) in aqueous solution using a simple environmentally friendly procedure. Protein microsponge-like structures were produced and characterized by means of spectroscopy and fluorescence microscopy methods. The efficiency of these structures in removing Pb2+ ions from aqueous solutions was evaluated by investigating the adsorption mechanisms. The molecular structure and, consequently, the physico-chemical properties of these aggregates can be readily tuned by selecting the pH of the solution during production. In particular, the presence of β-structures typical of amyloids as well as an environment characterized by a lower dielectric constant seem to enhance metal binding affinity revealing that hydrophobicity and water accessibility of the material are key features affecting the adsorption efficiency. Presented results provide new knowledge on how raw plant proteins can be valorised for the production of new biomaterials. This may offer extraordinary opportunities towards the design and production of new tailorable biosorbents which can also be exploited for several cycles of purification with minimal reduction in performance. SynopsisInnovative, sustainable plant-protein biomaterials with tunable properties are presented as green solution for water purification from lead(II) and the structure-function relationship is discussed.

A refined protocol for the isolation and monoculture of primary mouse renal peritubular endothelial cells.

During an episode of acute kidney injury (AKI), a sudden and rapid decline in renal function is often accompanied by a persistent reduction in mitochondrial function, microvasculature dysfunction/rarefaction, and tubular epithelial injury/necrosis. Additionally, patients who have experienced an AKI are at an elevated risk of developing other progressive renal, cardiovascular, and cardiorenal related diseases. While restoration of the microvasculature is imperative for oxygen and nutrient delivery/transport during proper renal repair processes, the mechanism(s) by which neovascularization and/or inhibition of microvascular dysfunction improves renal recovery remain understudied. Interestingly, pharmacological stimulation of mitochondrial biogenesis (MB) post-AKI has been shown to restore mitochondrial and renal function in mice. Thus, targeting MB pathways in microvasculature endothelial cell (MV-EC) may provide a novel strategy to improve renal vascular function and repair processes post-AKI. However, limitations to studying such mechanisms include a lack of commercially available primary renal peritubular MV-ECs, the variability in both purity and outgrowth of primary renal MV-EC in monoculture, the tendency of primary renal MV-ECs to undergo phenotypic loss in primary monoculture, and a limited quantity of published protocols to obtain primary renal peritubular MV-ECs. Thus, we focused on refining the isolation and phenotypic retention of mouse renal peritubular endothelial cells (MRPEC) for future physiological and pharmacological based studies. Here, we present a refined isolation method that augments the purity, outgrowth, and phenotypic retention of primary MRPEC monocultures by utilizing a collagenase type I enzymatic digestion, CD326+ (EPCAM) magnetic microbead epithelial cell depletion, and two CD146+ (MCAM) magnetic microbead purification cycles to achieve a monoculture MRPEC purity of ≅ 91-99% by all markers evaluated.

Design and creation of local mobile plants for stock watering and sheep wool washing units in Kalmykia

The purpose of the study The article deals with the issues of theoretical and experimental study of water purification processes for mobile watering points based on local underground water supply sources and the creation of a mobile installation with a closed cycle of water purification of wool washing, taking into account the regional characteristics of farms of the Republic of Kalmykia. Groundwater abstraction in the area of animal pens is justified; water purification up to the regulatory requirements for watering. The issues of purification production with the use of chocolate clays as a reagent, deposits of which are available in the northern part of Kalmykia, in the Priergeninsky district; determination of the total income from the practice of increasing the efficiency of farming; mobile purification technologies with the reuse of purified water have been developed.

Large-scale heterogeneous synthesis of monodisperse high performance colloidal CsPbBr3 nanocrystals

Colloidal lead halide perovskite nanocrystals (LHP NCs) are promising semiconductor materials for optoelectronic devices, but the high ionicity of LHP NCs makes their crystallization control and post-treatment difficult. Here, phosphonic acids (PAs) are employed as ligands to design a solid-liquid heterogeneous reaction system to regulate the LHP NC crystallization and achieve the desired focusing growth. During the heterogeneous synthesis, the precursors in the liquid phase are responsible for the burst nucleation and initial growth of NCs. Afterwards, the focusing growth of NCs is supported by the precursors released from the solid phase. In addition, the strong binding ability of PAs enables effective passivation of LHP NCs. Without post-treatment, gram-scale monodisperse CsPbBr3 NCs having photoluminescence with a full width at half-maximum of 18 nm and a quantum yield of near-unity are obtained. The CsPbBr3 NCs covered by a compact ligand layer keep initial quantum yield even after 18 cycles of purification, exhibiting excellent stability against polar solvents, ultraviolet irradiation and heat treatment. As scintillators, the prepared CsPbBr3 NCs show strong radioluminescence emission and high-resolution X-ray imaging.

Alkaline treatment enhances mass transfer in Protein A affinity chromatography

Cycle stability is important for preparative chromatography resins. Up to 200 cycles have been reported for Protein A affinity resins when used under optimized operating conditions. Through engineered ligands, alkaline resistant Protein A resins are available that can withstand repeated cleaning-in-place cycles with even 1 M NaOH. This enables an increase of purification cycles through the reduction of fouling while maintaining high binding capacities. Previously, non-intuitive changes in dynamic binding capacity after alkaline treatment have been observed for these novel Protein A resins, where sharper breakthrough curves and increased capacities were reported. In this work, we have systematically investigated resins with both low and high alkaline stability and studied the changes in static and dynamic binding capacities and elution behavior. We propose that the observed mass transfer increases of up to 40% are due to a switch in diffusion mechanism, as shown by confocal laser scanning microscopy. Based on our results, only a small window of alkaline treatment conditions exists, where dynamic binding capacity can be increased. Our findings may help to explain previous findings and observations of others.

Margaret of York as the Two Marys: Flexible Iconography in the Frontispiece of Le Dyalogue de la duchesse de Bourgogne à Jésus Christ

THE ILLUMINATED FRONTISPIECE OF Margaret of York’s Le Dyalogue de la duchesse de Bourgogne à Jésus Christ (The Dialogue of the Duchess of Burgundy with Jesus Christ) has often been used as a visual example of late medieval lay devotion. As such, the miniature has frequently been compared with such scenes as the noli me tangere, Christ’s apocryphal appearance to the Virgin Mary after his resurrection, and—although less often—to the Annunciation. This article examines the iconography of the bed’s curtain-sack, which hangs between Christ and Margaret, to confirm that the frontispiece draws on Annunciation imagery specifically. It then offers a new reading of the illumination as iconographically flexible, combining the three aforementioned iconographies into a single image. By viewing the frontispiece through the interpretive lens of the biblical Song of Songs, it demonstrates how the miniature was intended to lead Margaret through the cycle of spiritual purification and virginity, pregnancy, and generation.

Optimization of Recombinant GPCR Proteins for Biophysical and Structural Studies Using Virus-like Particles.

Overexpression of biologically functional GPCRs and homogeneous purified protein solutions are required to enable structural studies and protein-based biophysical assay development. Iterative and time-consuming optimization cycles of protein engineering, expression, and purification are often needed to achieve the desired protein quantity and quality. Here, we describe the reconstitution of GPCRs in virus-like particles (VLPs) and their use in biophysical assays to characterize protein yield, stability, and small molecule ligand binding. This approach prevents the need for time-consuming detergent solubilization and protein purification during recombinant GPCR protein optimization.