Amorphous Solubility Advantage: Theoretical Considerations, Experimental Methods, and Contemporary Relevance.

Twenty-five years ago, Hancock and Parks asked a provocative question: "what is the true solubility advantage for amorphous pharmaceuticals?" Difficulties in determining the amorphous solubility have since been overcome due to significant advances in theoretical understanding and experimental methods. The amorphous solubility is now understood to be the concentration after the drug undergoes liquid-liquid or liquid-glass phase separation, forming a water-saturated drug-rich phase in metastable equilibrium with an aqueous phase containing molecularly dissolved drug. While crystalline solubility is an essential parameter impacting the absorption of crystalline drug formulations, amorphous solubility is a vital factor for considering absorption from supersaturating formulations. However, the amorphous solubility of drugs is complex, especially in the presence of formulation additives and gastrointestinal components, and concentration-based measurements may not indicate the maximum drug thermodynamic activity. This review discusses the concept of the amorphous solubility advantage, including a historical perspective, theoretical considerations, experimental methods for amorphous solubility measurement, and the contribution of supersaturation and amorphous solubility to drug absorption. Leveraging amorphous solubility and understanding the associated physicochemical principles can lead to more effective development strategies for poorly water-soluble drugs, ultimately benefiting therapeutic outcomes.

The role of m6A-associated membraneless organelles in the RNA metabolism processes and human diseases.

N6-methyladenosine (m6A) is the most abundant post-transcriptional dynamic RNA modification process in eukaryotes, extensively implicated in cellular growth, embryonic development and immune homeostasis. One of the most profound biological functions of m6A is to regulate RNA metabolism, thereby determining the fate of RNA. Notably, the regulation of m6A-mediated organized RNA metabolism critically relies on the assembly of membraneless organelles (MLOs) in both the nucleus and cytoplasm, such as nuclear speckles, stress granules and processing bodies. In addition, m6A-associated MLOs exert a pivotal role in governing diverse RNA metabolic processes encompassing transcription, splicing, transport, decay and translation. However, emerging evidence suggests that dysregulated m6A levels contribute to the formation of pathological condensates in a range of human diseases, including tumorigenesis, reproductive diseases, neurological diseases and respiratory diseases. To date, the molecular mechanism by which m6A regulates the aggregation of biomolecular condensates associated with RNA metabolism is unclear. In this review, we comprehensively summarize the updated biochemical processes of m6A-associated MLOs, particularly focusing on their impact on RNA metabolism and their pivotal role in disease development and related biological mechanisms. Furthermore, we propose that m6A-associated MLOs could serve as predictive markers for disease progression and potential drug targets in the future.

Liquid Phase Electron Microscopy of Bacterial Ultrastructure.

Recent advances in liquid phase scanning transmission electron microscopy (LP-STEM) have enabled the study of dynamic biological processes at nanometer resolutions, paving the way for live-cell imaging using electron microscopy. However, this technique is often hampered by the inherent thickness of whole cell samples and damage from electron beam irradiation. These restrictions degrade image quality and resolution, impeding biological interpretation. Using graphene encapsulation, scanning transmission electron microscopy (STEM), and energy-dispersive X-ray (EDX) spectroscopy to mitigate these issues provides unprecedented levels of intracellular detail in aqueous specimens. This study demonstrates the potential of LP-STEM to examine and identify internal cellular structures in thick biological samples. Specifically, it highlights the use of LP-STEM to investigate the radiation resistant, gram-positive bacterium, Deinococcus radiodurans using various imaging techniques.

Absence of ATG9A and synaptophysin demixing on Rab5 mutation-induced giant endosomes.

ATG9A is the only integral membrane protein among core autophagy-related (ATG) proteins. We previously found that ATG9A does not co-assemble into synaptophysin-positive vesicles, but rather, localizes to a distinct pool of vesicles within synapsin condensates in both fibroblasts and nerve terminals. The endocytic origin of these vesicles further suggests the existence of different intracellular sorting or segregation mechanisms for ATG9A and synaptophysin in cells. However, the precise underlying mechanism remains largely unknown. In this follow-up study, we investigated the endosomal localization of these two proteins by exploiting the advantages of a Rab5 mutant that induces the formation of enlarged endosomes. Notably, ATG9A and synaptophysin intermix perfectly and do not segregate on giant endosomes, indicating that the separation of these two proteins is not solely caused by the inherent properties of the proteins, but possibly by other unknown factors.

Emerging roles of liquid-liquid phase separation in liver innate immunity.

Biomolecular condensates formed by liquid-liquid phase separation (LLPS) have become an extensive mechanism of macromolecular metabolism and biochemical reactions in cells. Large molecules like proteins and nucleic acids will spontaneously aggregate and assemble into droplet-like structures driven by LLPS when the physical and chemical properties of cells are altered. LLPS provides a mature molecular platform for innate immune response, which tightly regulates key signaling in liver immune response spatially and physically, including DNA and RNA sensing pathways, inflammasome activation, and autophagy. Take this, LLPS plays a promoting or protecting role in a range of liver diseases, such as viral hepatitis, non-alcoholic fatty liver disease, liver fibrosis, hepatic ischemia-reperfusion injury, autoimmune liver disease, and liver cancer. This review systematically describes the whole landscape of LLPS in liver innate immunity. It will help us to guide a better-personalized approach to LLPS-targeted immunotherapy for liver diseases.

Method to Purify Phase-Separated p62 Bodies Using Fluorescence-Activated Particle Sorting.

p62 bodies are ubiquitin-positive cytoplasmic condensates formed by liquid-liquid phase separation. They are targeted by selective autophagy and play important roles in intracellular quality control and stress responses. However, little is known about their constituents. In this chapter, we describe a method for purifying p62 bodies using fluorescence-activated particle sorting. This method contributes to the identification of novel components of p62 bodies under various physiological and stress conditions.

Sequence programmable nucleic acid coacervates.

Nature uses bottom-up self-assembly to build structures with remarkable complexity and functionality. Understanding how molecular-scale interactions translate to macroscopic properties remains a major challenge and requires systems that effectively bridge these two scales. Here, we generate DNA and RNA liquids with exquisite programmability in their material properties. Nucleic acids are negatively charged, and in the presence of polycations, they may condense to a liquid-like state. Within these liquids, DNA and RNA retain sequence-specific hybridization abilities. We show that intermolecular hybridization in the condensed phase cross-links molecules and slows down chain dynamics. This reduced chain mobility is mirrored in the macroscopic properties of the condensates. Molecular diffusivity and material viscosity scale with the intermolecular hybridization energy, enabling precise sequence-based modulation of condensate properties over orders of magnitude. Our work offers a robust platform to create self-assembling programmable fluids and may help advance our understanding of liquid-like compartments in cells.

Liquid-liquid phase separation of alpha-synuclein increases the structural variability of fibrils formed during amyloid aggregation.

Protein liquid-liquid phase separation (LLPS) is a rapidly emerging field of study on biomolecular condensate formation. In recent years, this phenomenon has been implicated in the process of amyloid fibril formation, serving as an intermediate step between the native protein transition into their aggregated state. The formation of fibrils via LLPS has been demonstrated for a number of proteins related to neurodegenerative disorders, as well as other amyloidoses. Despite the surge in amyloid-related LLPS studies, the influence of protein condensate formation on the end-point fibril characteristics is still far from fully understood. In this work, we compare alpha-synuclein aggregation under different conditions, which promote or negate its LLPS and examine the differences between the formed aggregates. We show that alpha-synuclein phase separation generates a wide variety of assemblies with distinct secondary structures and morphologies. The LLPS-induced structures also possess higher levels of toxicity to cells, indicating that biomolecular condensate formation may be a critical step in the appearance of disease-related fibril variants.

Liquid-Phase Exfoliation of 3D Metal-Organic Frameworks into Nanosheets.

Traditionally, the acquisition of 2D materials involved the exfoliation of layered crystals. However, the anisotropic bonding arrangements within 3D crystals indicate they are mechanically reminiscent of 2D counterparts and could also be exfoliated into nanosheets. This report delineates the preparation of 2D nanosheets from six representative 3D metal-organic frameworks (MOFs) through liquid-phase exfoliation. Notably, the cleavage planes of exfoliated nanosheets align perpendicular to the direction of the minimum elastic modulus (Emin) within the pristine 3D frameworks. The findings suggest that the in-plane and out-of-plane bonding forces of the exfoliated nanosheets can be correlated with the maximum elastic modulus (Emax) and Emin of the 3D frameworks, respectively. Emax influences the ease of cleaving adjacent layers, while Emin governs the ability to resist cracking of layers. Hence, a combination of large Emax and small Emin indicates an efficient exfoliation process, and vice versa. The ratio of Emax/Emin, denoted as Amax/min, is adopted as a universal index to quantify the ease of mechanical exfoliation for 3D MOFs. This ratio, readily accessible through mechanical experiments and computation, serves as a valuable metric for selecting appropriate exfoliation methods to produce surfactant-free 2D nanosheets from various 3D materials.

[Rapid determination of nine preservatives in tobacco flavor by three phase-hollow fiber-liquid phase microextraction-high performance liquid chromatography].

Tobacco flavors are extensively utilized in traditional tobacco products, electronic nicotine, heated tobacco products, and snuff. To inhibit fungal growth arising from high moisture content, preservatives such as benzoic acid (BA), sorbic acid (SA), and parabens are often incorporated into tobacco flavors. Nonetheless, consuming preservatives beyond safety thresholds may pose health risks. Therefore, analytical determination of these preservatives is crucial for both quality assurance and consumer protection. For example, BA and SA can induce adverse reactions in susceptible individuals, including asthma, urticaria, metabolic acidosis, and convulsions. Parabens, because of their endocrine activity, are classified as endocrine-disrupting chemicals. Despite extensive research, the concurrent quantification of trace-level hydrophilic (BA and SA) and hydrophobic (methylparaben, ethylparaben, isopropylparaben, propylparaben, butylparaben, isobutylparaben, and benzylparaben) preservatives in tobacco flavors remains challenging. Traditional liquid phase extraction coupled with high performance liquid chromatography (HPLC) often results in high false positive rates and inadequate sensitivity. In contrast, tandem mass spectrometry offers high sensitivity and specificity; however, its widespread application is limited by laborious sample preparation and significant operational costs. Therefore, it is crucial to establish a fast and sensitive sample pretreatment and analysis method for the nine preservatives in tobacco flavors. In this study, a method for the simultaneous determination of the nine preservatives (SA, BA and seven parabens) in tobacco flavor was established based on three phase-hollow fiber-liquid phase microextraction (3P-HF-LPME) technology combined with HPLC. To obtain the optimal pretreatment conditions, extraction solvent type, sample phase pH, acceptor phase pH, sample phase volume, extraction time, and mass fraction of sodium chloride, were examined. Additionally, the HPLC parameters, including UV detection wavelength and mobile phase composition, were refined. The optimal extraction conditions were as follows: dihexyl ether was used as extraction solvent, 15 mL sample solution (pH 4) was used as sample phase, sodium hydroxide aqueous solution (pH 12) was used as acceptor phase, and the extraction was carried out at 800 r/min for 30 min. Chromatographic separation was accomplished using an Agilent Poroshell 120 EC-C18 column (100 mm×3 mm, 2.7 µm) and a mobile phase comprising methanol, 0.02 mol/L ammonium acetate aqueous solution (containing 0.5% acetic acid), and acetonitrile for gradient elution. Under the optimized conditions, the nine target analytes showed good linear relationships in their respective linear ranges, the correlation coefficients (r) were ≥0.9967, limits of detection (LODs) and quantification (LOQs) were 0.02-0.07 mg/kg and 0.08-0.24 mg/kg, respectively. Under two spiked levels, the enrichment factors (EFs) and extraction recoveries (ERs) of the nine target analytes were 30.6-91.1 and 6.1%-18.2%, respectively. The recoveries of the nine target analytes ranged from 82.2% to 115.7% and the relative standard deviations (RSDs) (n=5) were less than 14.5% at low, medium and high levels. The developed method is straightforward, precise, sensitive, and well-suited for the rapid screening of preservatives in tobacco flavor samples.

The OsSRO1c-OsDREB2B complex undergoes protein phase transition to enhance cold tolerance in rice.

Cold stress is one of the major abiotic stress factors affecting rice growth and development, leading to significant yield loss in the context of global climate change. Exploring natural variants that confer cold resistance and the underlying molecular mechanism responsible for this is the major strategy to breed cold tolerant rice varieties. Here, we show that the natural variations of a SIMILAR to RCD ONE (SRO) gene, OsSRO1c, confer cold tolerance in rice at both seedling and booting stages. OsSRO1c possesses intrinsic liquid-liquid phase separation ability in vivo and in vitro and recruits an AP2/ERF transcription factor and positive cold stress regulator, OsDREB2B, into its biomolecular condensates in the nucleus, resulting in elevated transcriptional activity of OsDREB2B. The OsSRO1c-OsDREB2B complex directly responds to low temperature through dynamic phase transitions and regulates key cold response genes, including COLD1. Furthermore, introgression of an elite haplotype of OsSRO1c into a cold susceptible indica rice significantly increases its cold resistance. Collectively, our work reveals a novel cold tolerance regulatory module in rice and provides promising genetic targets for molecular breeding of cold-tolerant rice varieties.

Growth mechanisms of amorphous nanoparticles in solution and during heat drying.

The purpose of this study was twofold: to identify the growth mechanisms of amorphous nanoparticles in solution and during the drying process at high temperatures, and to guide the process condition and stabilizer selection for amorphous nanoparticle formulations. In contrast to nanocrystals that are mostly mechanically robust, amorphous nanoparticles tend to undergo deformation under stress. As a result, development of a stable formulation and evaluation of the drying process for re-dispersible amorphous nanoparticles presents considerable challenges. Although amorphous nanoparticles have stability issues, they have several pros in terms of production, high monodispersity, and diverse applications in drug delivery. In this study, amorphous nanoparticles were prepared via liquid-liquid phase separation, and their growth mechanisms were investigated both in solution and during the drying process. In solution, particles were found to be susceptible to flocculation, crystallization, coalescence, and Ostwald ripening, with coalescence being a preliminary step providing the driving force for Ostwald ripening. However, during the heat drying process, coalescence and crystallization were found to be the primary mechanisms for particle growth, with Ostwald ripening being negligible due to reduced molecular mobility. The glass transition temperature (Tg) of these amorphous nanoparticles was found to be a crucial factor both in solution and during the drying process. At temperatures < Tg, particles remained in a rigid, glassy state thereby inhibiting coalescence, whereas at or above Tg, particles transition from glassy to rubbery state, making them more susceptible to deformation and coalescence. The mechanistic understanding of particle growth from this study can also be extended to the stabilization of other types of soft nanoparticles.

Exploring Intrinsic Disorder in Human Synucleins and Associated Proteins.

In this work, we explored the intrinsic disorder status of the three members of the synuclein family of proteins-α-, ß-, and γ-synucleins-and showed that although all three human synucleins are highly disordered, the highest levels of disorder are observed in γ-synuclein. Our analysis of the peculiarities of the amino acid sequences and modeled 3D structures of the human synuclein family members revealed that the pathological mutations A30P, E46K, H50Q, A53T, and A53E associated with the early onset of Parkinson's disease caused some increase in the local disorder propensity of human α-synuclein. A comparative sequence-based analysis of the synuclein proteins from various evolutionary distant species and evaluation of their levels of intrinsic disorder using a set of commonly used bioinformatics tools revealed that, irrespective of their origin, all members of the synuclein family analyzed in this study were predicted to be highly disordered proteins, indicating that their intrinsically disordered nature represents an evolutionary conserved and therefore functionally important feature. A detailed functional disorder analysis of the proteins in the interactomes of the human synuclein family members utilizing a set of commonly used disorder analysis tools showed that the human α-synuclein interactome has relatively higher levels of intrinsic disorder as compared with the interactomes of human ß- and γ- synucleins and revealed that, relative to the ß- and γ-synuclein interactomes, α-synuclein interactors are involved in a much broader spectrum of highly diversified functional pathways. Although proteins interacting with three human synucleins were characterized by highly diversified functionalities, this analysis also revealed that the interactors of three human synucleins were involved in three common functional pathways, such as the synaptic vesicle cycle, serotonergic synapse, and retrograde endocannabinoid signaling. Taken together, these observations highlight the critical importance of the intrinsic disorder of human synucleins and their interactors in various neuronal processes.

Observation of the Liquid-Liquid Phase Separation of FUS-LC Using Vacuum-Ultraviolet Circular Dichroism Spectroscopy.

To reveal the structural mechanism by which the low-complexity domain of the fused in sarcoma protein (FUS-LC) mediates liquid-liquid phase separation (LLPS), we conducted a vacuum-ultraviolet circular dichroism (VUV-CD) spectroscopic study, a technique to analyze the secondary structures of proteins. The VUV-CD measurements were performed at the BL12 VUV-CD station at the Hiroshima Synchrotron Radiation Center (HiSOR) in Japan. CD spectra were measured between 180 and 260 nm while controlling the temperature of samples from 37°C to 5°C to obtain the LLPS of FUS-LC. The CD spectrum obtained at 37°C exhibited a large negative peak at 195 nm and a small negative shoulder near 220 nm. The peak intensity around 195 nm decreased as the sample temperature decreased. The spectral changes originated from the LLPS formation.

Multiscale Tunable Nanorings Based on Bi-Component Micellar-Configuration-Transformation Induced by Hydrophobicity.

Ringy nanostructures are amazing materials, displaying unique optical, magnetic, and electronic properties highly related to their dimensions. A strategy capable of continuously tailoring the diameter of nanorings is the key to elucidating their structure-function relationship. Herein, a method of bi-component micellar-configuration-transformation induced by hydrophobicity for the synthesis of nanorings with diameters ranging from submicron (≈143 nm) to micron (≈4.8 µm) and their carbonaceous analogs is established. Remarkably, the nanorings fabricated with this liquid phase strategy achieve the record for the largest diameter span. Through varying the molecular lengths of fatty alcohols and copolymers, shortening the molecular length of fatty alcohol can swell the primary micelles, improve the exposure of hydrophobic component and boost the assembly kinetics for ultra-large nanorings is shown here. On the other hand, shortening the molecular length of the copolymer will give rise to ultra-small nanorings by reducing the size of primary micelles and shortening the assembly time. When assembling the nanorings into monolayer arrays and then depositing Au, such substrate displays enhanced surface-enhanced Raman scattering (SERS) performance. This research develops a facile method for the controllable synthesis of ringy materials with multiscale tunable diameters and may inspire more interesting applications in physics, optical, and sensors.

Combining Protein Phase Separation and Bio-orthogonal Linking to Coimmobilize Enzymes for Cascade Biocatalysis.

The designed and ordered co-immobilization of multiple enzymes for vectorial biocatalysis is challenging. Here, a combination of protein phase separation and bioorthogonal linking is used to generate a zeolitic imidazole framework (ZIF-8) containing co-immobilized enzymes. Zn2+ ions induce the clustering of minimal protein modules, such as 6-His tag, proline-rich motif (PRM) and SRC homology 3 (SH3) domains, and allow for phase separation of the coupled aldoketoreductase (AKR) and alcohol dehydrogenase (ADH) at low concentrations. This is achieved by fusing SpyCatcher and PRM-SH3-6His peptide fragments to the C and N termini of AKR, respectively, and the SpyTag to ADH. Addition of 2-methylimidazole results in droplet formation and enables in situ spatial embedding the recombinant AKR and ADH to generate the cascade biocalysis system encapsulated in ZIF-8 (AAE@ZIF). In synthesizing (S)-1-(2-chlorophenyl) ethanol, ater 6 cycles, the yield can still reach 91%, with 99.99% enantiomeric excess (ee) value for each cycle. However, the yield could only reach 72.9% when traditionally encapsulated AKR and ADH in ZIF-8 are used. Thus, this work demonstrates that a combination of protein phase separation and bio-orthogonal linking enables the in situ creation of a stable and spatially organized bi-enzyme system with enhanced channeling effects in ZIF-8.

Sustainable approaches to analyzing phenolic compounds: a green chemistry perspective.

Innovative and eco-friendly methodologies for the determination of phenolic compounds, showing a paradigm shift in analytical chemistry toward sustainability. Phenolic compounds, valued for their diverse health benefits, have historically been analyzed using methods that often involve hazardous solvents and energy-intensive processes. This review focuses on green analytical chemistry principles, emphasizing sustainability, reduced environmental impact, and analytical efficiency. The use of DES, specifically Ch: Chl-based DES, emerges as a prominent green alternative for extracting phenolic compounds from various sources. The integration of UAE with DES enhances extraction efficiency, contributing to a more sustainable analytical approach. Furthermore, the review highlights the significance of DLLME and SPME in reducing solvent consumption and simplifying extraction procedures. These techniques exemplify the commitment to making phenolic compound analysis environmentally friendly. The incorporation of portable measurement tools, such as smartphones, into analytical methodologies is a notable aspect discussed in the review. Techniques like UA-DLLME leverage portable devices, making phenolic compound determination more accessible and versatile. Anticipating the future, the review foresees ongoing advancements in sustainable analytical approaches, driven by collaborative efforts across diverse disciplines. Novel solvents, extraction techniques, and portable measurement methods are expected to play pivotal roles in the continuous evolution of green analytical methodologies for the analysis of phenolic compounds. The review encapsulates a transformative journey toward environmentally responsible and efficient analytical practices, paving the way for further research and application in diverse analytical settings.

PRMT1 and TDRD3 promote stress granule assembly by rebuilding the protein-RNA interaction network.

Stress granules (SGs) are membrane-less organelles (MLOs) or cytosolic compartments formed upon exposure to environmental cell stress-inducing stimuli. SGs are based on ribonucleoprotein complexes from a set of cytoplasmic proteins and mRNAs, blocked in translation due to stress cell-induced polysome disassembly. Post-translational modifications (PTMs) such as methylation, are involved in SG assembly, with the methylation writer PRMT1 and its reader TDRD3 colocalizing to SGs. However, the role of this writer-reader system in SG assembly remains unclear. Here, we found that PRMT1 methylates SG constituent RNA-binding proteins (RBPs) on their RGG motifs. Besides, we report that TDRD3, as a reader of asymmetric dimethylarginines, enhances RNA binding to recruit additional RNAs and RBPs, lowering the percolation threshold and promoting SG assembly. Our study enriches our understanding of the molecular mechanism of SG formation by elucidating the functions of PRMT1 and TDRD3. We anticipate that our study will provide a new perspective for comprehensively understanding the functions of PTMs in liquid-liquid phase separation driven condensate assembly.

Important yet Overlooked HONO Source from Aqueous-phase Photochemical Oxidation of Nitrophenols.

Nitrites (NO2-/HONO), as the primary source of hydroxyl radicals (•OH) in the atmosphere, play a key role in atmospheric chemistry. However, the current understanding of the source of NO2-/HONO is insufficient and therefore hinders the accurate quantification of atmospheric oxidation capacity. Herein, we highlighted an overlooked HONO source by the reaction between nitrophenols (NPs) and •OH in the aqueous phase and provided kinetic data to better evaluate the contribution of this process to atmospheric HONO. Three typical NPs, including 4-nitrophenol (4NP), 2-nitrophenol (2NP), and 4-nitrocatechol (4NC), underwent a denitration process to form aqueous NO2- and gaseous HONO through the •OH oxidation, with the yield of NO2-/HONO varied from 15.0 to 33.5%. According to chemical composition and structure analysis, the reaction pathway, where the ipso addition of •OH to the NO2 group on 4NP generated hydroquinone, can contribute to more than 61.9% of the NO2-/HONO formation. The aqueous photooxidation of NPs may account for HONO in the atmosphere, depending on the specific conditions. The results clearly suggest that the photooxidation of NPs should be considered in the field observation and calculation to better evaluate the HONO budget in the atmosphere.

Biomolecular condensates with liquid properties formed during viral infections.

During a viral infection, several membraneless compartments with liquid properties are formed. They can be of viral origin concentrating viral proteins and nucleic acids, and harboring essential stages of the viral cycle, or of cellular origin containing components involved in innate immunity. This is a paradigm shift in our understanding of viral replication and the interaction between viruses and innate cellular immunity.