Synthesis Of Vesicles Research Articles

Photophysiology of the haploid form of the cryptophyte Teleaulax amphioxeia.

Cryptophytes are abundant and ubiquitous microalgae that constitute a major plastid source for kleptoplastidic ciliates and dinoflagellates. Despite their ecological significance, the understanding of their light preferences and photophysiology remains limited. Here, we provide a comprehensive study of the response of the haploid strain Teleaulax amphioxeia (Cr10EHU) to varying light irradiance. This strain is capable of growing under a wide range of irradiance levels, notably by finely tuning the different pigments bound to the membrane light-harvesting proteins. Analysis of the luminal phycoerythrin content revealed remarkable flexibility, with phycoerythrin emerging as a pivotal protein facilitating acclimation to varying light levels. Detailed ultrastructure examinations unveiled that this adaptability was supported by the synthesis of large thylakoidal vesicles, likely enhancing the capture of green photons efficiently under low light, a phenomenon previously undocumented. Teleaulax amphioxeia Cr10EHU effectively regulated light utilization by using a cryptophyte state transition-like process, with a larger amplitude observed under high growth irradiance. Furthermore, our results revealed the establishment of growth irradiance-dependent non-photochemical quenching of fluorescence, likely inducing the dissipation of excess light. This study underscores the particularities and the significant photoadaptability of the plastid of the haploid form of T. amphioxeia. It constitutes a comprehensive photophysiological characterization of the Cr10EHU strain that paves the way for future studies of the kleptoplastidy process.

Vesicles Formed by Transition Metal Complexes: Synthesis, Morphology and Applications

AbstractVesicles are one of the most important self‐assembled manifestations because of their multidimensional applications in biology, catalysis and adsorption. Several synthetic vesicles have been prepared from small peptides, amphiphilic fatty acids, amino acids and organic molecules etc. Transition metal complexes derived from different types of ligands are found to be self assembled to form solid state networks and also supramolecular aggregates in solution. In this review article we have discussed about synthesis, morphology and utility of vesicles formed by 14 transition metal complexes of some amphiphilic and non‐amphiphilic ligands. Interestingly, vesicles derived from transition metal complexes with non‐amphiphilic ligands are mostly soluble in organic solvents, whereas those derived from amphiphilic ligands are soluble in aqueous medium. As a result, their potential applications can be explored in complementary solvents accordingly.

Robust Electrostatic-Templated Polymerization for Controllable Synthesis of Stable and Permeable Polyelectrolyte Vesicles.

Polymer vesicles are of profound interest for designing delivery vehicles and nanoreactors toward a variety of biomedical and catalytic applications, yet robust synthesis of stable and permeable vesicles remains challenging. Here, we propose an electrostatic-templated polymerization that enables fabrication of polyelectrolyte vesicles with simultaneously regulated stability and permeability. In our design, cationic monomers were copolymerized with cross-linkers in the presence of a polyanionic-neutral diblock copolymer as a template. By properly choosing the block length ratio of the template, we fabricated a type of polyion complex vesicle consisting of a cross-linked cationic membrane, electrostatically assembled with the template copolymer which can be removed by sequential dissociation and separation under concentrated salt. We finally obtained stable polyelectrolyte vesicles of regulated size, membrane permeability, and response properties by tuning the synthesis factors including ionic strength, cross-linker type, and fraction as well as different monomers and concentrations. As a proof-of-concept, lipase was loaded in the designed cationic vesicles, which exhibited enhanced enzyme stability and activity. Our study has developed a novel and robust strategy for controllable synthesis of a new class of stable and permeable polymer (polyelectrolyte) vesicles that feature great potential applications as functional delivery carriers and nanoreactors.

Assessment of spermatogenesis in camels via seminiferous tubule staging and testosterone profiling

Objective: To investigate the successive morphological stages of spermatogenesis, hormonal regulation, and testosterone profile in dromedary camel reproduction. Methods: Testicular tissue samples were obtained from 12 dromedary bull camels aged 7 to 8 at a local abattoir. The histological assessment involved tissue processing, hematoxylin and eosin (H & E) staining, and examination under a microscope. Stereological analysis, germ cell identification, and assessment of seminiferous tubules and maturation were conducted. Testosterone assay was performed by radioimmunoassay using blood samples collected at regular intervals. Results: The study revealed 12 phases of the dromedary camel's seminiferous epithelium cycle, highlighting distinct morphological characteristics and cellular processes. Acrosomal migration, maturation, cap formation, and the Golgi-mediated synthesis of proacrosomal vesicles were also explained in dimension, as were the steps of acrosome biogenesis. Spermatids and mature sperm cells were present when spermatogenesis phases were examined. An analysis of the dimensions of seminiferous tubules revealed specific measures for diameter, area, and epithelial height about luminal characteristics. Moreover, there were noticeable variations in the serum testosterone concentrations during the study period, indicating temporal dynamics. Conclusions: This study outlines the spermatogenesis process in dromedary camels across 12 stages, emphasizing cellular dynamics and acrosomal biogenesis. It also provides seminiferous tubule measurements and observes seasonal testosterone fluctuations, offering insights into reproductive regulation and potential strategies for camel breeding conservation.

Control of giant vesicle assemblies by stimuli-responsive lipids.

We describe a bottom-up synthesis of giant vesicles (GVs) utilizing an artificial stimuli-responsive diazobenzene lipid building block. Controlled by light, the GVs can exhibit dynamic behaviors, including reversible formation, the generation of highly multilamellar assemblies, and vesicle capturing and releasing events.

Synthesis and self-assembly of the amphiphilic homopolymers poly(4-hydroxystyrene) and poly(4-(4-bromophenyloxy)styrene)

Amphiphilic homopolymer synthesis and formation of micelles and vesicles in selective solvents.

Bottom-Up Synthesis of Crystalline Covalent Organic Framework Nanosheets, Nanotubes, and Kippah Vesicles: An Odd-Even Effect Induction.

Few-layer organic nanosheets are becoming increasingly attractive as two-dimensional (2D) materials due to their precise atomic connectivity and tailor-made pores. However, most strategies for synthesizing nanosheets rely on surface-assisted methods or top-down exfoliation of stacked materials. A bottom-up approach with well-designed building blocks would be the convenient pathway to achieve the bulk-scale synthesis of 2D nanosheets with uniform size and crystallinity. Herein, we have synthesized crystalline covalent organic framework nanosheets (CONs) by reacting tetratopic thianthrene tetraaldehyde (THT) and aliphatic diamines. The bent geometry of thianthrene in THT retards the out-of-plane stacking, while the flexible diamines introduce dynamic characteristics into the framework, facilitating nanosheet formation. Successful isoreticulation with five diamines with two to six carbon chain lengths generalizes the design strategy. Microscopic imaging reveals that the odd and even diamine-based CONs transmute to different nanostructures, such as nanotubes and hollow spheres. The single-crystal X-ray diffraction structure of repeating units indicates that the odd-even linker units of diamines introduce irregular-regular curvature in the backbone, aiding such dimensionality conversion. Theoretical calculations shed more light on nanosheet stacking and rolling behavior with respect to the odd-even effects.

Atomic insights of an up and down conformation of the Acinetobacter baumannii F1 -ATPase subunit ε and deciphering the residues critical for ATP hydrolysis inhibition and ATP synthesis.

The Acinetobacter baumannii F1 FO -ATP synthase (α3 :β3 :γ:δ:ε:a:b2 :c10 ), which is essential for this strictly respiratory opportunistic human pathogen, is incapable of ATP-driven proton translocation due to its latent ATPase activity. Here, we generated and purified the first recombinant A. baumannii F1 -ATPase (AbF1 -ATPase) composed of subunits α3 :β3 :γ:ε, showing latent ATP hydrolysis. A 3.0 Å cryo-electron microscopy structure visualizes the architecture and regulatory element of this enzyme, in which the C-terminal domain of subunit ε (Abε) is present in an extended position. An ε-free AbF1 -ɑβγ complex generated showed a 21.5-fold ATP hydrolysis increase, demonstrating that Abε is the major regulator of AbF1 -ATPase's latent ATP hydrolysis. The recombinant system enabled mutational studies of single amino acid substitutions within Abε or its interacting subunits β and γ, respectively, as well as C-terminal truncated mutants of Abε, providing a detailed picture of Abε's main element for the self-inhibition mechanism of ATP hydrolysis. Using a heterologous expression system, the importance of Abε's C-terminus in ATP synthesis of inverted membrane vesicles, including AbF1 FO -ATP synthases, has been explored. In addition, we are presenting the first NMR solution structure of the compact form of Abε, revealing interaction of its N-terminal β-barrel and C-terminal ɑ-hairpin domain. A double mutant of Abε highlights critical residues for Abε's domain-domain formation which is important also for AbF1 -ATPase's stability. Abε does not bind MgATP, which is described to regulate the up and down movements in other bacterial counterparts. The data are compared to regulatory elements of F1 -ATPases in bacteria, chloroplasts, and mitochondria to prevent wasting of ATP.

Biomimetic synthesis and optimization of extracellular vesicles for bone regeneration.

Critical-size bone defect repair is in high demand but is difficult to treat. Modern therapies, such as autograft and cell-based treatments, face limitations, including potential immunological rejection and tumorigenesis. Therefore, extracellular vesicle (EV)-based strategies have been proposed as a novel approach for tissue regeneration owing to EVs' complex composition of lipids, proteins, and nucleic acids, as well as their low immunogenicity and congenital cell-targeting features. Despite these remarkable features of EVs, biomimetic synthesis and optimization of natural EVs can lead to enhanced bioactivity, increased cellular uptake, and specific cell targeting, aiming to achieve optimal therapeutic efficacy. To maximize their function, these nanoparticles can be integrated into bone graft biomaterials for superior bone regeneration. Herein, we summarize the role of naturally occurring EVs from distinct cell types in bone regeneration, the current strategies for optimizing biomimetic synthetic EVs in bone regeneration, and discuss the recent advances in applying bone graft biomaterials for the delivery of EVs to bone defect repair. We focused on distinct strategies for optimizing EVs with different functions and the most recent research on achieving time-controlled release of nanoparticles from EV-loaded biomaterials. Furthermore, we thoroughly discuss several current challenges and proposed solutions, aiming to provide insight into current progress, inspiration for future development directions, and incentives for clinical application in this field.

Electrohydrodynamic Techniques for the Manufacture and/or Immobilization of Vesicles.

The development of accurate drug delivery systems is one of the main challenges in the biomedical field. A huge variety of structures, such as vesicles, nanoparticles, and nanofibers, have been proposed as carriers for bioactive agents, aiming for precision in administration and dosage, safety, and bioavailability. This review covers the use of electrohydrodynamic techniques both for the immobilization and for the synthesis of vesicles in a non-conventional way. The state of the art discusses the most recent advances in this field as well as the advantages and limitations of electrospun and electrosprayed amphiphilic structures as precursor templates for the in situ vesicle self-assembly. Finally, the perspectives and challenges of combined strategies for the development of advanced structures for the delivery of bioactive agents are analyzed.

Synthesis of protein vesicles for extending time window of ischemic stroke treatment through microcirculatory thrombolysis

Microcirculatory disturbances are a major barrier to extend the time window of ischemic stroke treatment. Herein, we developed a dynamic template method for the synthesis of protein vesicles, by which urokinase-type plasminogen activator (uPA) was crosslinked by poly(ethylene glycol) via pH sensitive host–guest interaction to form a protein gel-shell to encapsulate superoxide dismutase (SOD) for getting cascade biofunctions of microvessel thrombolysis and reperfusion injury inhibition. On a middle cerebral artery occlusion mouse model, the uPA@SOD vesicles administrated beyond intravenous thrombolysis time window (>4.5 h) could reopen the collateral circulation of brain, thus benefiting the followed interventional recanalization of large vessel in the late window (>6 h). These led to rapid reversion of hypoxia condition and more efficient rescue of injured neurons in the ischemic tissue. The synthesis of protein vesicles is highly beneficial for stroke treatment, meanwhile enabling the facile creation of multi-protein therapeutic systems.

Sec14-like-Lipid-Binding-4 Enables Hemoglobin Clearance By Liver Sinusoidal Endothelial Cells

Hemolysis of banked red blood cells (RBC storage lesion) contributes to lack of therapeutic benefit and higher mortality following transfusion therapy in patients. However, the molecular mechanism underlying storage lesion remains poorly understood. Using a multiple-ancestry genome-wide association analysis among blood donors, our group has recently identified Sec14-like-lipid-binding-4 (Sec14L4) protein to be associated with increased rate of hemolysis following blood storage. Although Sec14L4 is highly similar to yeast phosphatidylinositol transfer protein Sec14, which is known to be involved in the synthesis of golgi-derived transport vesicles, the exact function of Sec14L4 and how it promotes RBC storage lesions remains unknown. Our initial studies using fluorescence microscopy in our newly generated Sec14L4 null mice revealed significant enrichment of Sec14L4 in the live sinusoidal endothelial cells (LSECs), which positively correlated with elevated levels of iron in the liver. Scanning and transmission electron micrographs revealed impaired actin cytoskeleton organization in LSECs of Sec14L4 null mice, which was associated with upregulation of major GTPases RhoA, RhoB and Rac1 activity. The impairment of actin cytoskeletal organization in Sec14L4 null mice lead to impaired vesicle trafficking in LSECs and higher retention of RBCs in the liver sinusoids compared to control mice. These data are the first to demonstrate a previously unrecognized role for Sec14L4 protein in LSEC mediated endocytic trafficking by promoting actin polymerization. These findings also suggest that Sec14L4 may regulate RBC storage lesion through impaired hemoglobin clearance by LSECs secondary to cytoskeletal impairment.

The synthesis of extracellular vesicles by the protistan parasite Blastocystis.

Blastocystis is a genus of single-celled protist belonging to the stramenopile group. Prior studies have shown that isolates of Blastocystis subtype 7 (ST7) induced higher levels of intestinal epithelial cell damage and gut microbiota dysbiosis in comparison to other subtypes in in vivo and in vitro settings. Prior research has shown a link between gut dysbiosis and exposure to extracellular vesicles (EVs) produced by pathogenic microorganisms. This study demonstrates a protocol for the isolation of EVs from Blastocystis ST7 via ultracentrifugation. Nanoparticle tracking analysis and transmission electron microscopy were used to assess EV size and morphology. The protein content of isolated EVs was assessed by mass spectrophotometry and the presence of EV markers were evaluated by Western blotting. Finally, the EVs were cocultured with prominent human gut microbiome species to observe their effect on prokaryote growth. Our data shows that Blastocystis ST7 secretes EVs that are similar in morphology to previously characterized EVs from other organisms and that these EVs contain a limited yet unique protein cargo with functions in host-parasite intercellular communication and cell viability. This cargo may be involved in mediating the effects of Blastocystis on its surrounding environment.

Synchronous Synthesis of Polymeric Vesicles with Controllable Size and Low‐Polydispersity by Polymerization‐Induced Self‐Assembly

Comprehensive SummaryThe size and size distribution of polymeric nanoparticles have great impact on their physicochemical and biological properties. Polymerization‐induced self‐assembly (PISA) has been demonstrated to be an efficient method to fabricate various polymeric nanoparticles, among which polymeric vesicles have attracted great interest due to their unique hollow structure. However, polymeric vesicles with relatively broad size distributions and random size are normally formed, which is problematic for many potential applications. Herein, we report the synthesis of polymeric vesicles with low‐polydispersity and controllable size by polymerization‐induced self‐assembly. A mixed macro RAFT agent of three different poly(ethylene glycol) (PEGm‐CPADB) was used to mediate the RAFT dispersion copolymerization of 7‐(2‐methacryloyloxyethoxy)‐4‐methylcoumarin (CMA) and benzyl methacrylate (BzMA), which generated vesicles with low‐polydispersity (< 0.1) and controllable size in the scope of sub‐100 nm. In comparison, RAFT dispersion copolymerization of CMA and BzMA using a single PEG‐CPADB as the macro RAFT agent generated vesicles with adjustable but obviously larger size and broader size distributions (> 0.1). The results demonstrate that the ternary stabilizers play a crucial role in the formation of small and low‐polydispersity vesicles.

One-Pot Synthesis of Oxidation-Sensitive Supramolecular Gels and Vesicles.

Polypeptide-based nanoparticles offer unique advantages from a nanomedicine perspective such as biocompatibility, biodegradability, and stimuli-responsive properties to (patho)physiological conditions. Conventionally, self-assembled polypeptide nanostructures are prepared by first synthesizing their constituent amphiphilic polypeptides followed by postpolymerization self-assembly. Herein, we describe the one-pot synthesis of oxidation-sensitive supramolecular micelles and vesicles. This was achieved by polymerization-induced self-assembly (PISA) of the N-carboxyanhydride (NCA) precursor of methionine using poly(ethylene oxide) as a stabilizing and hydrophilic block in dimethyl sulfoxide (DMSO). By adjusting the hydrophobic block length and concentration, we obtained a range of morphologies from spherical to wormlike micelles, to vesicles. Remarkably, the secondary structure of polypeptides greatly influenced the final morphology of the assemblies. Surprisingly, wormlike micellar morphologies were obtained for a wide range of methionine block lengths and solid contents, with spherical micelles restricted to very short hydrophobic lengths. Wormlike micelles further assembled into oxidation-sensitive, self-standing gels in the reaction pot. Both vesicles and wormlike micelles obtained using this method demonstrated to degrade under controlled oxidant conditions, which would expand their biomedical applications such as in sustained drug release or as cellular scaffolds in tissue engineering.

Design and Manufacture of a Low-Cost Microfluidic System for the Synthesis of Giant Liposomes for the Encapsulation of Yeast Homologues: Applications in the Screening of Membrane-Active Peptide Libraries

The discovery of new membrane-active peptides (MAPs) is an area of considerable interest in modern biotechnology considering their ample applicability in several fields ranging from the development of novel delivery vehicles (via cell-penetrating peptides) to responding to the latent threat of antibiotic resistance (via antimicrobial peptides). Different strategies have been devised for such discovery process, however, most of them involve costly, tedious, and low-efficiency methods. We have recently proposed an alternative route based on constructing a non-rationally designed library recombinantly expressed on the yeasts’ surfaces. However, a major challenge is to conduct a robust and high-throughput screening of possible candidates with membrane activity. Here, we addressed this issue by putting forward low-cost microfluidic platforms for both the synthesis of Giant Unilamellar Vesicles (GUVs) as mimicking entities of cell membranes and for providing intimate contact between GUVs and homologues of yeasts expressing MAPs. The homologues were chitosan microparticles functionalized with the membrane translocating peptide Buforin II, while intimate contact was through passive micromixers with different channel geometries. Both microfluidic platforms were evaluated both in silico (via Multiphysics simulations) and in vitro with a high agreement between the two approaches. Large and stable GUVs (5–100 µm) were synthesized effectively, and the mixing processes were comprehensively studied leading to finding the best operating parameters. A serpentine micromixer equipped with circular features showed the highest average encapsulation efficiencies, which was explained by the unique mixing patterns achieved within the device. The microfluidic devices developed here demonstrate high potential as platforms for the discovery of novel MAPs as well as for other applications in the biomedical field such as the encapsulation and controlled delivery of bioactive compounds.

Inhibition of Sphingosine-1-Phosphate Receptor 2 by JTE013 Promoted Osteogenesis by Increasing Vesicle Trafficking, Wnt/Ca2+, and BMP/Smad Signaling.

Sphingosine-1-phosphate receptor 2 (S1PR2) is a G protein-coupled receptor that regulates various immune responses. Herein, we determine the effects of a S1PR2 antagonist (JTE013) or a S1PR2 shRNA on osteogenesis by culturing murine bone marrow stromal cells (BMSCs) in osteogenic media with JTE013, dimethylsulfoxide (DMSO), a S1PR2 shRNA, or a control shRNA. Treatment with JTE013 or the S1PR2 shRNA increased alkaline phosphatase and alizarin red s staining, and enhanced alkaline phosphatase, RUNX2, osteocalcin, and osterix mRNA levels in BMSCs compared with the controls. Protein analysis revealed that a high dose of JTE013 (4 or 8 μM) increased vesicle trafficking-associated proteins (F-actin, clathrin, Early Endosome Antigen 1 (EEA1), and syntaxin 6) and Wnt/Ca2+ signaling. On the other hand, a low dose of JTE013 (1 to 2 μM) increased BMP/Smad signaling. In contrast, the S1PR2 shRNA reduced vesicle trafficking-associated proteins and attenuated Wnts and BMP/Smad signaling, but enhanced p-CaMKII compared with the control, suggesting that the S1PR2 shRNA influenced osteogenesis via different signaling pathways. Moreover, inhibiting protein trafficking by brefeldin A in BMSCs suppressed Wnts and BMPRs expressions. These data supported that enhanced osteogenesis in JTE013-treated BMSCs is associated with increased vesicle trafficking, which promotes the synthesis and transport of osteogenic protein and matrix vesicles and enhances matrix mineralization.

Azobenzene-Based Cross-Linked Small-Molecule Vesicles for Precise Oxidative Damage Treatments Featuring Controlled and Prompt Molecular Release

Precise therapy has become prevalent in clinical practice owing to its accurate and efficient targeting treatment of diseases. Such treatments involving polymersomes as carriers have great potential to lesion sites without damage to normal tissues. However, due to the inherent thick hydrophobic layer of polymersomes, an instantaneous release response to external stimuli remains a challenge. To tackle this challenge, here, we report on the synthesis and applications of azobenzene-containing photochromic vesicles as delivery vectors. These vesicles are assembled from small-molecule amphiphiles that have been developed to provide a fast response and promote instantaneous release due to molecular size reduction compared with macromolecular polymersomes. After cross-linking, the stability of vesicles under a physiological environment is notably enhanced. By varying UV and visible light irradiation, the “gate” of vesicles can be opened and closed reversibly for the controlled release of capsuled cargoes. In vitro experiments display that the vesicles can be applied to load cysteamine for eliminating excess reactive oxygen. The synthesized vesicles here show high performance in controlled and instantaneous release in cells both in time and space. By our approach, oxidative damage to cellular biomolecules can be substantially reduced.

Expression of Fatty Acyl-CoA Ligase Drives One-Pot De Novo Synthesis of Membrane-Bound Vesicles in a Cell-Free Transcription-Translation System.

Despite the central importance of lipid membranes in cellular organization, it is challenging to reconstitute their formation de novo from minimal chemical and biological elements. Here, we describe a chemoenzymatic route to membrane-forming noncanonical phospholipids in which cysteine-modified lysolipids undergo spontaneous coupling with fatty acyl-CoA thioesters generated enzymatically by a fatty acyl-CoA ligase. Due to the high efficiency of the reaction, we were able to optimize phospholipid formation in a cell-free transcription-translation (TX-TL) system. Combining DNA encoding the fatty acyl-CoA ligase with suitable lipid precursors enabled one-pot de novo synthesis of membrane-bound vesicles. Noncanonical sphingolipid synthesis was also possible by using a cysteine-modified lysosphingomyelin as a precursor. When the sphingomyelin-interacting protein lysenin was coexpressed alongside the acyl-CoA ligase, the in situ assembled membranes were spontaneously decorated with protein. Our strategy of coupling gene expression with membrane lipid synthesis in a one-pot fashion could facilitate the generation of proteoliposomes and brings us closer to the bottom-up generation of synthetic cells using recombinant synthetic biology platforms.

Effects of different fluid fields on the formation of cyanobacterial blooms

Cyanobacterial blooms have been attracting more and more attention, and the mechanism is widely studied. However, the effects of fluid fields on the bloom formation were rarely reported. In this study, the effects of fluid fields formed under different external conditions were investigated. The results indicated that low wind speed (3 m/s) was conducive to the formation of cyanobacterial blooms, while high wind speed (6 m/s) was adverse. For low wind speed, an upward fluid field was detected by particle image velocimetry. This fluid field accelerated the algal growth by 58.6%, and improved the buoyancy by up-regulating the genes involved in the synthesis of gas vesicles and extracellular polymeric substances. In addition, the boundary shear stress induced the colony formation of cyanobacteria and improved the aggregation proportion significantly (p < 0.05), which was beneficial to bloom formation. As a result, cyanobacterial blooms are more likely to form on the lake shore under moderate breeze. When wind speed increased to 6 m/s, a downward fluid field was formed, causing algal cells to gather at the bottom and hindering the bloom formation. These results provided a theoretical basis for field researches related to the formation of cyanobacterial blooms and the treatment of cyanobacteria.