Spontaneous Transition of Spherical Coacervate to Vesicle-Like Compartment.

Numerous biological systems contain vesicle-like biomolecular compartments without membranes, which contribute to diverse functions including gene regulation, stress response, signaling, and skin barrier formation. Coacervation, as a form of liquid-liquid phase separation (LLPS), is recognized as a representative precursor to the formation and assembly of membrane-less vesicle-like structures, although their formation mechanism remains unclear. In this study, a coacervation-driven membrane-less vesicle-like structure is constructed using two proteins, GG1234 (an anionic intrinsically disordered protein) and bhBMP-2 (a bioengineered human bone morphogenetic protein 2). GG1234 formed both simple coacervates by itself and complex coacervates with the relatively cationic bhBMP-2 under acidic conditions. Upon addition of dissolved bhBMP-2 to the simple coacervates of GG1234, a phase transition from spherical simple coacervates to vesicular condensates occurred via the interactions between GG1234 and bhBMP-2 on the surface of the highly viscoelastic GG1234 simple coacervates. Furthermore, the shell structure in the outer region of the GG1234/bhBMP-2 vesicular condensates exhibited gel-like properties, leading to the formation of multiphasic vesicle-like compartments. A potential mechanism is proposed for the formation of the membrane-less GG1234/bhBMP-2 vesicle-like compartments. This study provides a dynamic process underlying the formation of biomolecular multiphasic condensates, thereby enhancing the understanding of these biomolecular structures.

Influence of the backbone chemistry and ionic functional groups of five pairs of oppositely charged polyelectrolytes on complex coacervation.

Complex coacervation plays an important role in various fields. Here, the influences of the backbone chemistry and ionic functional groups of five pairs of oppositely charged polyelectrolytes on complex coacervation were investigated. These pairs include synthetic polymers with aliphatic hydrocarbon backbones, peptides with amide bonds, and carbohydrates with glycosidic linkages. Despite sharing identical charged groups, specific pairs displayed distinct liquid/liquid and liquid/solid phase separations depending on the polyelectrolyte mixing ratio, buffer, and ionic strength. The coacervate phase boundary broadened in the orders: glycosidic linkages > amide backbone > aliphatic hydrocarbon backbone, and Tris-phosphate > Tris-acetate > Tris-chloride buffers. Coacervates prepared from polyelectrolytes with lower solubilities in water resisted disassembly at high salt concentrations, and their merge rate was slow. These observations suggest that the hydrophobic segments in polyelectrolytes interfere with the formation of complex coacervates; however, following coacervate formation, the hydrophobic segments render the coacervates stable and elastic.

Hydrophobicity of arginine leads to reentrant liquid-liquid phase separation behaviors of arginine-rich proteins.

Intrinsically disordered proteins rich in cationic amino acid groups can undergo Liquid-Liquid Phase Separation (LLPS) in the presence of charge-balancing anionic counterparts. Arginine and Lysine are the two most prevalent cationic amino acids in proteins that undergo LLPS, with arginine-rich proteins observed to undergo LLPS more readily than lysine-rich proteins, a feature commonly attributed to arginine's ability to form stronger cation-π interactions with aromatic groups. Here, we show that arginine's ability to promote LLPS is independent of the presence of aromatic partners, and that arginine-rich peptides, but not lysine-rich peptides, display re-entrant phase behavior at high salt concentrations. We further demonstrate that the hydrophobicity of arginine is the determining factor giving rise to the reentrant phase behavior and tunable viscoelastic properties of the dense LLPS phase. Controlling arginine-induced reentrant LLPS behavior using temperature and salt concentration opens avenues for the bioengineering of stress-triggered biological phenomena and drug delivery systems.

Ultrasound stimulation improves inflammatory resolution, neuroprotection, and functional recovery after spinal cord injury.

Spinal cord injury (SCI) is associated with limited functional recovery. Despite advances in neuroscience, realistic therapeutic treatments for SCI remain unavailable. In this study, the effects of non-invasive ultrasound (US) treatment on behavior and inflammatory responses were evaluated in a rat model of SCI. Adult female Sprague-Dawley rats were subjected to spinal cord contusion injury. Two different US parameters (SCIU5: 5% and SCIU40: 40% duty cycle) were applied, and their effects on behavioral recovery after SCI were quantified. Tissue and neuronal responses were detected. Immunofluorescence was used to detect inflammatory markers. In the rat model of SCI, motor function was more effectively restored, and the lesion cavity area was smaller in the SCIU5 group. Furthermore, the SCIU5 protocol elicited an anti-inflammatory response at the injury site by reducing degenerative FJC-labeled neurons, macrophage/microglia activation, and infiltration. Thus, the lesion area decreased, and tissue density increased. Meanwhile, the SCIU40 protocol did not improve motor function or induce an anti-inflammatory response at the injury site. The SCIU5 protocol effectively accelerated the rate of improved exercise performance in the rat model while reducing inflammation. Accordingly, appropriate US stimulation may represent a promising treatment modality for SCI with beneficial anti-inflammatory effects.

Methylene Blue Delivery Mediated by Focused Ultrasound-Induced Blood-Brain Barrier Disruption Reduces Neural Damage and Amyloid-Beta Plaques by AQP-4 Upregulation.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease worldwide, causing progressive cognitive decline, memory impairment, and neurological deficits. Methylene blue (MB), an antioxidant, has emerged as a potential drug for the treatment of AD owing to its cognitive improvement and neuroprotective functions. Despite the small molecular size of MB, which can cross the BBB, the therapeutic effective dosage using a BBB-permeable delivery system in a specific brain localization remains unclear. In this study, we presented magnetic resonance-guided focused ultrasound (MRgFUS) as a delivery system to enhance BBB permeability for the effective treatment of AD. MRgFUS using two ultrasound intensities (0.25 and 0.32 MPa) was used to intravenously deliver MB to the hippocampal region. Compared with treatment with 0.25 MPa FUS, treatment with 0.32 MPa FUS significantly enhanced MB brain accumulation. Deposition of amyloid-ß (Aß) plaques and neural cell damage was significantly reduced in 0.32 MPa FUS/MB-treated APP/PS1 mice. Furthermore, aquaporin-4 expression increased significantly in the 0.32 MPa FUS and 0.32 MPa FUS/MB groups without glial fibrillary acidic protein activation. The results from this study demonstrate that FUS improved MB delivery to the brain, and FUS/MB combination treatment reduced the number of Aß plaques. This study revealed the potential of FUS-BBBD as an effective strategy to enhance the efficacy of therapeutic drugs for AD.

Catalytically defective receptor protein tyrosine kinase PTK7 enhances invasive phenotype by inducing MMP-9 through activation of AP-1 and NF-κB in esophageal squamous cell carcinoma cells.

Protein tyrosine kinase 7 (PTK7), a member of the catalytically defective receptor protein tyrosine kinase family, is upregulated in various cancers including esophageal squamous cell carcinoma (ESCC). Here, we have explored the molecular mechanism of PTK7-dependent invasiveness in ESCC cells. PTK7 knockdown reduced gelatin degradation and MMP-9 secretion in cultures of ESCC TE-10 cells, and showed reduced levels of MMP9 mRNA using real-time RT-PCR and luciferase reporter assays. PTK7 knockdown decreased not only phosphorylation of NF-κB, IκB, ERK, and JNK, but also nuclear localization of NF-κB and AP-1 consisting of c-Fos and c-Jun. Activation of AP-1 and NF-κB requires PTK7-mediated activation of tyrosine kinases, including Src. In addition, NF-κB activation by PTK7 involves the PI3K/Akt signaling pathway. PTK7-mediated upregulation of MMP9 was also observed in other ESCC cell lines and in three-dimensional cultures of TE-10 cells. Moreover, MMP-9 expression positively correlated with PTK7 expression in ESCC tumor tissue. These findings demonstrate that PTK7 upregulates MMP9 through activation of AP-1 and NF-κB and, thus increases invasive properties of ESCC cells.