Chitosan/dextran-based organohydrogel delivers EZH2 inhibitor to epigenetically reprogram chemo/immuno-resistance in unresectable metastatic melanoma.

Melanoma either intrinsically possesses resistance or rapidly acquires resistance to anti-tumor therapy, which often leads to local recurrence or distant metastasis after resection. In this study, we found histone 3 lysine 27 (H3K27) demethylated by an inhibitor of histone methyltransferase EZH2 could epigenetically reverse the resistance to chemo-drug paclitaxel (PTX), or enhance the efficacy of immune checkpoint inhibitor anti-TIGIT via downregulating TIGIT ligand CD155. Next, to address the complexity in the combination of multiple bioactive molecules with distinct therapeutic properties, we developed a polysaccharides-based organohydrogel (OHG) configured with a heterogenous network. Therein, hydroxypropyl chitosan (HPC)-stabilized emulsions for hydrophobic drug entrapment were crosslinked with oxidized dextran (Odex) to form a hydrophilic gel matrix to facilitate antibody accommodation, which demonstrated a tunable sustained release profile by optimizing emulsion/gel volume ratios. As results, local injection of OHG loaded with EZH2 inhibitor UNC1999, PTX and anti-TIGIT did not only synergistically enhance the cytotoxicity of PTX, but also reprogrammed the immune resistance via bi-directionally blocking TIGIT/CD155 axis, leading to the recruitment of cytotoxic effector cells into tumor and conferring a systemic immune memory to prevent lung metastasis. Hence, this polysaccharides-based OHG represents a potential in-situ epigenetic-, chemo- and immunotherapy platform to treat unresectable metastatic melanoma.

Triple-crosslinked double-network alginate/dextran/dendrimer hydrogel with tunable mechanical and adhesive properties: A potential candidate for sutureless keratoplasty.

An ideal adhesive hydrogel must possess high adhesion to the native tissue, biocompatibility, eligible biodegradability, and good mechanical compliance with the substrate tissues. We constructed an interpenetrating double-network hydrogel containing polysaccharides (alginate and dextran) and nanosized spherical dendrimer by both physical and chemical crosslinking, thus endowing the hydrogel with a broad range of mechanical properties, adhesive properties, and biological functions. The double-network hydrogel has moderate pore sizes and swelling properties. The chelation of calcium ions significantly enhances the tensile and compressive properties. The incorporation of dendrimer improves both the mechanical and adhesive properties. This multicomponent interpenetrating network hydrogel has excellent biocompatibility, tunable mechanical and adhesive properties, and satisfied multi-functions to meet the complex requirements of wound healing and tissue engineering. The hydrogel exhibits promising corneal adhesion capabilities in vitro, potentially supplanting the need for sutures in corneal stromal surgery and mitigating the risks associated with donor corneal damage and graft rejection during corneal transplantation. This novel polysaccharide and dendrimer hydrogel also shows good results in sutureless keratoplasty, with high efficiency and reliability. Based on the clinical requirements for tissue bonding and wound closure, the hydrogel provides insight into solving the mechanical properties and adhesive strength of tissue adhesives.

Mechanistic Insights into the c-MYC G-Quadruplex and Berberine Binding inside an Aqueous Two-Phase System Mimicking Biomolecular Condensates.

We investigated the binding between the c-MYC G-quadruplex (GQ) and berberine chloride (BCl) in an aqueous two-phase system (ATPS) with 12.3 wt % polyethylene glycol and 5.6 wt % dextran, mimicking the highly crowded intracellular biomolecular condensates formed via liquid-liquid phase separation. We found that in the ATPS, complex formation is significantly altered, leading to an increase in affinity and a change in the stoichiometry of the complex with respect to neat buffer conditions. Thermodynamic studies reveal that binding becomes more thermodynamically favorable in the ATPS due to entropic effects, as the strong excluded volume effect inside ATPS droplets reduces the entropic penalty associated with binding. Finally, the binding affinity of BCl for the c-MYC GQ is higher than those for other DNA structures, indicating potential specific interactions. Overall, these findings will be helpful in the design of potential drugs targeting the c-MYC GQ structures in cancer-related biocondensates.

Thermo-responsive aqueous two-phase system for two-level compartmentalization.

Hierarchical compartmentalization responding to changes in intracellular and extracellular environments is ubiquitous in living eukaryotic cells but remains a formidable task in synthetic systems. Here we report a two-level compartmentalization approach based on a thermo-responsive aqueous two-phase system (TR-ATPS) comprising poly(N-isopropylacrylamide) (PNIPAM) and dextran (DEX). Liquid membraneless compartments enriched in PNIPAM are phase-separated from the continuous DEX solution via liquid-liquid phase separation at 25 °C and shrink dramatically with small second-level compartments generated at the interface, resembling the structure of colloidosome, by increasing the temperature to 35 °C. The TR-ATPS can store biomolecules, program the spatial distribution of enzymes, and accelerate the overall biochemical reaction efficiency by nearly 7-fold. The TR-ATPS inspires on-demand, stimulus-triggered spatiotemporal enrichment of biomolecules via two-level compartmentalization, creating opportunities in synthetic biology and biochemical engineering.

VMT/ACP/Dextran composite nanosheets against dental caries through promoting mineralization of dentin tubules, pH buffering, and antibacterial.

Dental caries is a worldwide public healthcare concern, and is closely related to the acidic environment that caused by bacterial decomposition of food. In this study, a two-step ion exchange liquid-phase stripping method was applied to strip out vermiculite (VMT) nanosheets, then amorphous calcium phosphate (ACP) and dextran were inserted between the VMT nanosheets interlayer to obtain a composite two-dimension nanosheets (VMT/ACP/Dextran). VMT/ACP/Dextran composite nanosheets exhibited excellent biocompatibility and could provide exogenous Ca2+and PO43- from ACP, provide SiO44-, Mg2+, Fe2+ and obtain buffering pH and antibacterial properties from VMT, as well as improve suspension stability and targeting Streptococcus mutans through glucan. The in vitro study showed that the composite materials could promote the mineralization and sealing of dentin tubules by releasing active ions, buffer pH 4.5 (a value close to the pH in the dental plaque environment) to pH 6.6-7.1 (values close to the pH in human saliva) through ion exchange, and exert antibacterial effects by targeting Streptococcus mutans and exerting oxidase like and peroxidase like activities to produce reactive oxygen species (ROS). The in vivo animal study showed that daily cleaning teeth using VMT/ACP/Dextran composite nanosheets could effectively reduce the incidence rate and severity of dental caries in rats. Taking together, the developed VMT/ACP/Dextran composite nanosheets, which integrated the excellent properties of VMT, ACP and dextran, can effectively prevent dental caries through a combination of factors such as buffering acids, antibacterial properties, and promoting calcification, and may be used as an active ingredient for daily oral hygiene or filling materials to prevent and treat dental caries.

Ultrahigh-Resolution Visualization of Vascular Heterogeneity in Brain Tumors via Magnetic Nanoparticles-Enhanced Susceptibility-Weighted Imaging.

The precise assessment of vascular heterogeneity in brain tumors is vital for diagnosing, grading, predicting progression, and guiding treatment decisions. However, currently, there is a significant shortage of high-resolution imaging approaches. Herein, we propose a contrast-enhanced susceptibility-weighted imaging (CE-SWI) utilizing the minimalist dextran-modified Fe3O4 nanoparticles (Dextran@Fe3O4 NPs) for ultrahigh-resolution mapping of vasculature in brain tumors. The Dextran@Fe3O4 NPs are prepared via a facile coprecipitation method under room temperature, and exhibit small hydrodynamic size (28 nm), good solubility, excellent biocompatibility, and high transverse relaxivity (r2*, 159.7 mM-1 s-1) under 9.4 T magnetic field. The Dextran@Fe3O4 NPs-enhanced SWI can increase the contrast-to-noise ratio (CNR) of cerebral vessels to 2.5 times that before injection and achieves ultrahigh-spatial-resolution visualization of microvessels as small as 0.1 mm in diameter. This advanced imaging capability not only allows for the detailed mapping of both enlarged peritumoral drainage vessels and the intratumoral microvessels, but also facilitates the sensitive imaging detection of vascular permeability deterioration in a C6 cells-bearing rat glioblastoma model. Our proposed Dextran@Fe3O4 NPs-enhanced SWI provides a powerful imaging technique with great clinical translation potential for the precise theranostics of brain tumors.

Targeting Tumor-Associated Macrophages with the Immune-Activating Nanomedicine for Achieving Strong Antitumor Activity with Rapid Clearance from the Body.

Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) crucial for the detection of infections and activation of downstream signaling pathways that lead to the production of pro-inflammatory cytokines and interferons. The TLR pathway is an attractive actively studied target pathway. Because of their strong immunostimulatory activity, TLRs are thought to be a "double-edged sword" for systemic treatment, even in the cancer field. To solve this, we have developed dextran-based TAM targeting activating conjugate (D-TAC) technology, which successfully uses tumor-associated macrophages (TAMs) to deliver the TLR7 agonist DSP-0509. We used low molecular weight dextran to target CD206 high M2-type macrophages, activate them, and induce a change in phenotype to antitumor M1-type macrophages with rapid clearance from the body and astonishing antitumor activity. We also demonstrated that the antitumor effect of our best drug candidate 5DEX-0509R is dependent on the abundance of TAMs, which is consistent with their mechanism of action. We believe that 5DEX-0509R generated by D-TAC technology can be a clinically applicable immunotherapy targeting the TLR signaling pathway.

Slowly hydrolyzable property of microbial dextrans at the small intestinal α-glucosidase levels leads to the modulated glycemic responses in the mouse model.

Dextran-type α-glucans have been known as non-digestible ingredients that can be considered prebiotics to promote colon health. However, recent studies have revealed that various α-linked glucosyl units are hydrolyzed to glucose by small intestinal α-glucosidases. This study analyzed the structural characteristics of exopolysaccharides (EPSs) from Weissella species, and the hydrolysis properties at both in vitro/in vivo levels were investigated. Compared with a previous in vitro digestion model using fungal α-hydrolytic enzymes, dextrans, which mainly consist of α-1,6 linkages with small amounts of α-1,3 linked glucose units, were slowly hydrolyzed to glucose by mammalian mucosal α-glucosidases, resulting in attenuation of the initial glycemic response following administration of EPS samples to mice via oral gavage. The results of this study demonstrate the concept of dextran-type α-glucans as glycemic carbohydrates rather than dietary fibers or prebiotics. Slowly digestible dextrans can be applied as a functional ingredient to regulate postprandial glucose delivery throughout the gastrointestinal tract.

Facile roll-to-roll production of nanoporous fiber coatings for advanced wound care sutures.

Theranostic sutures are derived from innovative ideas to enhance wound healing results by adding wound diagnostics and therapeutics to typical sutures by functionalizing them with additional materials. Here, we present a new direct electrospinning method for the fast, continuous, inexpensive, and high-throughput production of versatile nanofibrous-coated suture threads, with precise control over various essential microstructural and physical characteristics. The thickness of the coating layer and the alignment of nanofibers with the thread's direction can be adjusted by the user by varying the spooling speed and the displacement between the spinneret needle and thread. To show the flexibility of our method for a range of different materials selected, gelatin, polycaprolactone, silk fibroin, and PEDOT:PSS (poly(3,4-ethylene dioxythiophene):poly(styrene sulfonate)) were the resultant nanofibers characterized by scanning electron microscopy (SEM) imaging and conductivity tests. In a series of in vitro and ex vivo tests (pig skin), sutures were successfully tested for their flexibility and mechanical properties when used as weaving and knotting sutures, and their biocompatibility with a keratinocyte cell line. For temperature-based drug-releasing tests, two fluorescent molecules as drug models with high and low molecular weight, namely fluorescein isothiocyanate-dextran (20 kDa) and rhodamine B (470 Da), were used, and their steady release with incremental increase of temperature to 37 °C over 120 min was seen, which is appropriate for bacterial treatment drugs. Given the advantages of the presented technique, it seems to have promising potential to be used in future medical applications for wound closure and bacterial infection treatment via a temperature-triggered drug release strategy.

Ultrasound-assisted glycosylation of ovalbumin and dextran conjugate carrier for anthocyanins and their stability evaluation.

Anthocyanins (AC) are vulnerable to degradation when affected by external factors. The present study employed ultrasound-assisted glycosylation of ovalbumin (OVA) and dextran (Dex) to generate conjugate carrier for AC to improve its stability. The results showed that sonication significantly improved the progression of Maillard reaction to OVA. Compared to traditional glycosylation, ultrasound treatment showed a higher degree of grafting, a lower number of free-SH, and smaller particle size and uniform distribution. The SDS-PAGE results indicated covalent interaction. Intrinsic fluorescence (INF), Fourier transform infrared spectroscopy (FTIR), and Circular dichroism (CD) analysis results suggested that ultrasound-assisted glycosylation altered the OVA structure. The scanning electron microscope (SEM) and X-ray diffractometer (XRD) observed that the ultrasound-assisted complex had a more compact and smoother structure and protein unfolding were better. The protein solubility increased significantly after glycosylation. Thermal gravimetric analysis (TGA) and Differential scanning calorimetry (DSC) indicated that the glycosylated conjugates can significantly improve the thermal stability of AC In addition, the AC showed an improved processing and storage stability when conjugated with glycosylated carrier. The glycosylated protein-anthocyanins complex may help provide new ideas and scientific basis for the development of naturally sourced anthocyanins-relevant products in pharmaceutical and food industry applications.

Tailoring the specificity of ficin versus large hemoglobin and small casein by co-immobilizing inert proteins on the immobilized enzyme layer and further modification with aldehyde dextran.

Ficin has been immobilized at full loading on glyoxyl agarose beads. Then, ficin was blocked with 2,2'-dipyridyldisulfide. To be effective, the modification must be performed in the presence of 0.5 M urea, as the enzyme was not inhibited under standard conditions, very likely because the catalytic Cys was not fully exposed to the medium. Activity could be fully recovered by incubation with 1 M mercaptoethanol. This biocatalyst could hydrolyze hemoglobin and casein. The objective of this paper was to increase the enzyme specificity versus small proteins by generating steric hindrances to the access of large proteins. The step by step blocking via ionic exchange of the biocatalyst with aminated bovine serum albumin (BSA), aldehyde dextran and a second layer of aminated BSA produced a biocatalyst that maintained its activity versus small synthetic substrates, increased the biocatalyst stability, while reduced its activity to over 50 % versus casein. Interestingly, this treatment almost fully annulled the activity versus hemoglobin, more effectively at 37 °C than at 55 °C. The biocatalyst could be reused 5 times without changes in activity. The changes could be caused by steric hindrances, but it cannot be discarded some changes in enzyme sequence specificity caused by the modifications.

Unrevealing the potentialities in food formulations of a low-branched dextran from Leuconostoc mesenteroides.

The search for novel exopolysaccharides (EPS) with targeted functionalities is currently a topic of great interest. This study aimed to investigate the chemical characteristics and technological properties of a novel EPS (named EPS_O) from Leuconostoc mesenteroides. EPS_O was a high-molecular-weight dextran (>6.68 × 105 g/mol) characterized by high water-holding capacity (785 ± 73%) and high water solubility index (about 99%). EPS_O in water (<30 mg/mL) formed viscous solutions, whereas at concentrations >30 mg/mL, it formed weak gels. Notably, lower concentrations (4-5 mg/mL) exhibited antimicrobial activity against various foodborne pathogens, antibiofilm activity against Listeria monocytogenes, and radical-scavenging activity. These properties are significant for maintaining food quality and promoting health. Based on these findings, EPS_O presents itself as a promising food ingredient that could elevate food quality and confer health benefits to consumers.

Defining the Spatial Resolution of Analyte Recovery during Microperfusion-Based Sampling of Brain Parenchyma.

The unique architecture of the brain and the blood-brain barrier imposes challenges for the measurement of parenchyma-derived biomarkers that prevent sufficient understanding of transient neuropathogenic processes. One solution to this challenge is direct sampling of brain interstitial fluid via implanted microperfusion probes. Seeking to understand spatial limitations to microperfusion in the brain, we employed computational fluid dynamics modeling and empirical recovery of fluorescently labeled dextrans in an animal model. We found that dextrans were successfully recovered via microperfusion over a 6 h sampling period, especially at probes implanted 2 mm from the dextran infusion point relative to probes implanted 5 mm from the injection site. Experimental recovery was consistently around 1% of simulated, suggesting that this parameter can be used to set practical limits on the maximal tissue concentration of proteins measured in microperfusates and on the spatial domain sampled by our multimodal microperfusion probe.

Support Enzyme Loading Influences the Effect of Aldehyde Dextran Modification on the Specificity of Immobilized Ficin for Large Proteins.

It has been reported that the modification of immobilized glyoxyl-ficin with aldehyde dextran can promote steric hindrances that greatly reduce the activity of the immobilized protease against hemoglobin, while the protease still maintained a reasonable level of activity against casein. In this paper, we studied if this effect may be different depending on the amount of ficin loaded on the support. For this purpose, both the moderately loaded and the overloaded glyoxyl-ficin biocatalysts were prepared and modified with aldehyde dextran. While the moderately loaded biocatalyst had a significantly reduced activity, mainly against hemoglobin, the activity of the overloaded biocatalyst was almost maintained. This suggests that aldehyde dextran was able to modify areas of the moderately loaded enzyme that were not available when the enzyme was overloaded. This modification promoted a significant increase in biocatalyst stability for both biocatalysts, but the stability was higher for the overloaded biocatalyst (perhaps due to a combination of inter- and intramolecular crosslinking).

Effects of enzymatic hydrolysis combined with glycation on the emulsification characteristics and emulsion stability of peanut protein isolate.

Peanut protein isolate (PPI) has high nutritional value, but its poor function limits its application in the food industry. In this study, peanut protein isolate was modified by enzymatic hydrolysis combined with glycation. The structure, emulsification and interface properties of peanut protein isolate hydrolysate (HPPI) and dextran (Dex) conjugate (HPPI-Dex) were studied. In addition, the physicochemical properties, rheological properties, and stability of the emulsion were also investigated. The results showed that the graft degree increased with the increase of Dex ratio. Fourier transform infrared spectroscopy (FTIR) confirmed that the glycation of HPPI and Dex occurred. The microstructure showed that the structure of HPPI-Dex was expanded, and the molecular flexibility was enhanced. When the ratio of HPPI to Dex was 1:3, the emulsifying activity and the interface pressure of glycated HPPI reached the highest value, and the emulsifying activity (61.08 m2/g) of HPPI-Dex was 5.28 times that of PPI. The HPPI-Dex stabilized emulsions had good physicochemical properties and rheological properties. In addition, HPPI-Dex stabilized emulsions had high stability under heat treatment, salt ion treatment and freeze-thaw cycle. According to confocal laser scanning microscopy (CLSM), the dispersion of HPPI-Dex stabilized emulsions was better after 28 days of storage. This study provides a theoretical basis for developing peanut protein emulsifier and further expanding the application of peanut protein in food industry.

Marangoni Droplets of Dextran in PEG Solution and Its Motile Change Due to Coil-Globule Transition of Coexisting DNA.

Motile droplets using Marangoni convection are attracting attention for their potential as cell-mimicking small robots. However, the motion of droplets relative to the internal and external environments that generate Marangoni convection has not been quantitatively described. In this study, we used an aqueous two-phase system [poly(ethylene glycol) (PEG) and dextran] in an elongated chamber to generate motile dextran droplets in a constant PEG concentration gradient. We demonstrated that dextran droplets move by Marangoni convection, resulting from the PEG concentration gradient and the active transport of PEG and dextran into and out of the motile dextran droplet. Furthermore, by spontaneously incorporating long DNA into the dextran droplets, we achieved cell-like motility changes controlled by coexisting environment-sensing molecules. The DNA changes its position within the droplet and motile speed in response to external conditions. In the presence of Mg2+, the coil-globule transition of DNA inside the droplet accelerates the motile speed due to the decrease in the droplet's dynamic viscosity. Globule DNA condenses at the rear part of the droplet along the convection, while coil DNA moves away from the droplet's central axis, separating the dipole convections. These results provide a blueprint for designing autonomous small robots using phase-separated droplets, which change the mobility and molecular distribution within the droplet in reaction with the environment. It will also open unexplored areas of self-assembly mechanisms through phase separation under convections, such as intracellular phase separation.

Solanum melongena L. Extract Promotes Intestinal Tight Junction Re-Assembly via SIRT-1-Dependent Mechanisms.

Tight junction disruption can lead to pathogenesis of various diseases without therapeutic strategy to recover intestinal barrier integrity. The main objective of this study is to demonstrate the effect of Solanum melongena L. extract (SMLE) on intestinal tight junction recovery and its underlying mechanism. Intestinal barrier function is attenuated by Ca2+ depletion. SMLE treatment increased TER value across T84 cell monolayers. Permeability assay reveals that Ca2+ depletion promotes 4-kDa FITC-dextran permeability, but not 70-kDa FITC-dextran. SMLE suppresses the rate of 4-kDa FITC-dextran permeability, indicating that SMLE inhibits paracellular leak pathway permeability. SMLE-mediated TER increase and leak pathway suppression are abolished by neither calcium/calmodulin-dependent protein kinase kinase ß (CaMKKß) inhibitor nor AMP-activated protein kinase (AMPK) inhibitor. Furthermore, mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK) inhibitors have no effects on SMLE-mediated TER increase and leak pathway suppression. Interestingly, SMLE is unable to enhance TER value and diminish leak pathway permeability in T84 cell monolayers pre-treated with sirtuin-1 (SIRT-1) inhibitor. Immunofluorescence staining reveals that SMLE enhances re-assembly of tight junction proteins, including occludin and ZO-1 to intercellular space but this effect is abolished by SIRT-1 inhibitor. These data suggest that SMLE promotes intestinal tight junction re-assembly via SIRT-1-dependent manner.

The Discovery, Molecular Cloning, and Characterization of Dextransucrase LmDexA and Its Active Truncated Mutant from Leuconostoc mesenteroides NN710.

Dextransucrases play a crucial role in the production of dextran from economical sucrose; therefore, there is a pressing demand to explore novel dextransucrases with better performance. This study characterized a dextransucrase enzyme, LmDexA, which was identified from the Leuconostoc mesenteroides NN710. This bacterium was isolated from the soil of growing dragon fruit in Guangxi province, China. We successfully constructed six different N-terminal truncated variants through sequential analysis. Additionally, a truncated variant, ΔN190LmDexA, was constructed by removing the 190 amino acids fragment from the N-terminal. This truncated variant was then successfully expressed heterologously in Escherichia coli and purified. The purified ΔN190LmDexA demonstrated optimal hydrolysis activity at a pH of 5.6 and a temperature of 30 °C. Its maximum specific activity was measured to be 126.13 U/mg, with a Km of 13.7 mM. Results demonstrated a significant improvement in the heterologous expression level and total enzyme activity of ΔN190LmDexA. ΔN190LmDexA exhibited both hydrolytic and transsaccharolytic enzymatic activities. When sucrose was used as the substrate, it primarily produced high-molecular-weight dextran (>400 kDa). However, upon the addition of maltose as a receptor, it resulted in the production of a significant amount of oligosaccharides. Our results can provide valuable information for enhancing the characteristics of recombinant dextransucrase and potentially converting sucrose into high-value-added dextran and oligosaccharides.

Perivascular spaces around arteries exceed perivenous spaces in the mouse brain.

The perivascular space (PVS) surrounds cerebral blood vessels and plays an important role in clearing waste products from the brain. Their anatomy and function have been described for arteries, but PVS around veins remain poorly characterized. Using in vivo 2-photon imaging in mice, we determined the size of the PVS around arteries and veins, and their connection with the subarachnoid space. After infusion of 70 kD FITC-dextran into the cerebrospinal fluid via the cisterna magna, labeled PVS were evident around arteries, but veins showed less frequent labeling of the PVS. The size of the PVS correlated with blood vessel size for both pial arteries and veins, but not for penetrating vessels. The PVS around pial arteries and veins was separated from the subarachnoid space by a thin meningeal layer, which did not form a barrier for the tracer. In vivo, FITC-dextran signal was observed adjacent to the vessel wall, but minimally within the wall itself. Post-mortem, there was a significant shift in the tracer's location within the arterial wall, extending into the smooth muscle layer. Taken together, these findings suggest that the PVS around veins has a limited role in the exchange of solutes between CSF and brain parenchyma.

Towards Self-regeneration: Exploring the Limits of Protein Synthesis in the Protein Synthesis Using Recombinant Elements (PURE) Cell-free Transcription-Translation System.

Self-regeneration is a key function of living systems that needs to be recapitulated in vitro to create a living synthetic cell. A major limiting factor for protein self-regeneration in the PURE cell-free transcription-translation system is its high protein concentration, which far exceeds the system's protein synthesis rate. Here, we were able to drastically reduce the nonribosomal PURE protein concentration up to 97.3% while increasing protein synthesis efficiency. Although crowding agents were not effective in the original PURE formulation, we found that in highly dilute PURE formulations, addition of 6% dextran considerably increased protein synthesis rate and total protein yield. These new PURE formulations will be useful for many cell-free synthetic biology applications, and we estimate that PURE can now support the complete self-regeneration of all 36 nonribosomal proteins, which is a critical step toward the development of a universal biochemical constructor and living synthetic cell.