Poly(Glutamic Acid-Lysine) Hydrogels with Alternating Sequence Resist the Foreign Body Response in Rodents and Non-Human Primates.

The foreign body response (FBR) to implanted biomaterials and biomedical devices can severely impede their functionality and even lead to failure. The discovery of effective anti-FBR materials remains a formidable challenge. Inspire by the enrichment of glutamic acid (E) and lysine (K) residues on human protein surfaces, a class of zwitterionic polypeptide (ZIP) hydrogels with alternating E and K sequences to mitigate the FBR is prepared. When subcutaneously implanted, the ZIP hydrogels caused minimal inflammation after 2 weeks and no obvious collagen capsulation after 6 months in mice. Importantly, these hydrogels effectively resisted the FBR in non-human primate models for at least 2 months. In addition, the enzymatic degradability of the gel can be controlled by adjusting the crosslinking degree or the optical isomerism of amino acid monomers. The long-term FBR resistance and controlled degradability of ZIP hydrogels open up new possibilities for a broad range of biomedical applications.

Facile Synthesis of High Molecular Weight Poly(ethylene glycol)-b-poly(amino acid)s by Relay Polymerization.

Poly(amino acid)s (PAAs) are one kind of favorable biopolymer that can be used as a drug or gene carrier. However, conventional ring-opening polymerization of PAAs is slow and needs a strict anhydrous environment with an anhydrous reagent as well as the product without enough high molecular weight (Mn), which limits the expanding of PAAs' application. Herein, we took BLG-NCA as the monomer to quickly synthesize one kind of high Mn amphiphilic copolymer, poly(ethylene glycol)-b-poly(γ-benzyl-l-glutamic acid) (PEG-PBLG), by relay polymerization with a simple one-pot method within 3 h in mild conditions (open air, moisture insensitive). In the polymerization process, ring-opening polymerization-induced self-assembly in sodium bicarbonate aqueous solution first occurred to obtain low Mn PEG-PBLG seeds without purification. Then γ-benzyl-l-glutamate N-carboxyanhydride (BLG-NCA) dichloromethane solution was added into PEG-PBLG seeds directly and stirred vigorously to form am emulsion; during this process, the amphiphilic PEG-PBLG seeds will anchor on the interface of DCM and water to ensure the concentration of α-helix rigid PBLG in DCM to maintain the following relay polymerization. Then, high Mn PEG-PBLG was obtained in mild conditions in one pot. We found that the α-helix rigid structure was essential for relay polymerization by studying the synthetic speed of amphiphilic copolymer with different secondary structures. MOE simulation results showed that PBLG and BLG-NCA tended to form a double hydrogen bond, which was beneficial to relay polymerization because of higher local concentrations that can produce more double hydrogen bonds. Our strategy can quickly obtain high Mn PEG-PBLG (224.9 KDa) within 3 h from PEG-NH2 and BLG-NCA in one pot and did not need an extra initiator. After deprotection, the poly(ethylene glycol)-b-poly(l-glutamate acid) (PEG-PGA) with high Mn as a second product can be used as an excellent antitumor drug carrier. The high Mn PEG-PGA can achieve an encapsulation rate of 86.7% and a drug loading rate of 47.3%, which is twice that of the low Mn PEG-PGA. As a result, the synthesis of PEG-PBLG by relay polymerization simplified the process of PEG-PAA polymerization and increased the Mn. In addition, this method opened a way to obtain other kinds of high Mn PEG-PBLG values in the future.

Peptide Epimerase Responsible for d-Amino Acid Introduction in Poly-γ-glutamic Acid Biosynthesis.

Poly-γ-glutamic acid (PGA) is a natural polymer of d- and/or l-glutamic acid (Glu) linked by isopeptide bonds. We recently showed that PGA synthetase, an enzyme complex composed of PgsB, PgsC, and PgsA, uses only l-Glu for polymerization, and d-Glu residues are introduced by peptide epimerization. However, it remains unclear which of the three enzymes is responsible for epimerization because in vitro functional characterization of the membrane-associated PgsBCA complex has never been successful. Here, we performed gene exchange experiments and showed that PgsA is responsible for the epimerization. Additionally, we identified a region in PgsA that modulates epimerization activity based on homology modeling from the recently solved structure of MslH, which showed 53% identity to PgsA. Our results suggested that d/l-ratios of the PGA product can be altered by introducing amino acid substitutions in this region, which will be useful for the production of PGA with controlled d/l-ratios.

Coupling fermentation of glutamic acid and γ-polyglutamic acid and preparation of poly(amino acid) superabsorbent polymers.

γ-polyglutamic acid (γ-PGA) is a biomarker that can be directly obtained by microbial fermentation. Poly(amino acid) superabsorbent polymers (SAPs) were prepared with purified γ-PGA as raw material and ethylene glycol diglycidyl ether (EGDGE) as a cross-linking agent. However, γ-PGA fermentation broth has a high viscosity, requires complex extraction and separation processes, and entails high energy consumption, resulting in the high cost of poly (amino acid) SAPs. Therefore, the coupling fermentation processes of glutamate polyglutamic acid, the process of using glutamate fermentation broth instead of pure glutamate powder for fermentation, and the process of treating the fermentation broth under conditions of centrifugation, UV irradiation, and high temperature, were studied. The results showed that the yield of γ-PGA after centrifugation decreased by 5%, but it did not affect the synthesis of hydrogels, and the addition of γ-PGA fermentation broth had a significant effect on the performance of γ-PGA-co-PASP SAPs. The proposed method not only helps avoid the separation of complex γ-PGA fermentation broth and reduces the cost, but it also helps improve the performance of the super-absorbent resin, which has great application potential.

Physicochemical Characterization of Chitosan/Poly-γ-Glutamic Acid Glass-like Materials.

This paper sets up a new route for producing non-covalently crosslinked bio-composites by blending poly-γ-glutamic acid (γ-PGA) of microbial origin and chitosan (CH) through poly-electrolyte complexation under specific experimental conditions. CH and two different molecular weight γ-PGA fractions have been blended at different mass ratios (1/9, 2/8 and 3/7) under acidic pH. The developed materials seemed to behave like moldable hydrogels with a soft rubbery consistency. However, after dehydration, they became exceedingly hard, glass-like materials completely insoluble in water and organic solvents. The native biopolymers and their blends underwent comprehensive structural, physicochemical, and thermal analyses. The study confirmed strong physical interactions between polysaccharide and polyamide chains, facilitated by electrostatic attraction and hydrogen bonding. The materials exhibited both crystalline and amorphous structures and demonstrated good thermal stability and degradability. Described as thermoplastic and saloplastic, these bio-composites offer vast opportunities in the realm of polyelectrolyte complexes (PECs). This unique combination of properties allowed the bio-composites to function as glass-like materials, making them highly versatile for potential applications in various fields. They hold potential for use in regenerative medicine, biomedical devices, food packaging, and 3D printing. Their environmentally friendly properties make them attractive candidates for sustainable material development in various industries.

The distinct initiation sites and processing activities of TTLL4 and TTLL7 in glutamylation of brain tubulin.

Mammalian brain tubulins undergo a reversible posttranslational modification-polyglutamylation-which attaches a secondary polyglutamate chain to the primary sequence of proteins. Loss of its erasers can disrupt polyglutamylation homeostasis and cause neurodegeneration. Tubulin tyrosine ligase like 4 (TTLL4) and TTLL7 were known to modify tubulins, both with preference for the ß-isoform, but differently contribute to neurodegeneration. However, differences in their biochemical properties and functions remain largely unknown. Here, using an antibody-based method, we characterized the properties of a purified recombinant TTLL4 and confirmed its sole role as an initiator, unlike TTLL7, which both initiates and elongates the side chains. Unexpectedly, TTLL4 produced stronger glutamylation immunosignals for α-isoform than ß-isoform in brain tubulins. Contrarily, the recombinant TTLL7 raised comparable glutamylation immunoreactivity for two isoforms. Given the site selectivity of the glutamylation antibody, we analyzed modification sites of two enzymes. Tandem mass spectrometry analysis revealed their incompatible site selectivity on synthetic peptides mimicking carboxyl termini of α1- and ß2-tubulins and a recombinant tubulin. Particularly, in the recombinant α1A-tubulin, a novel region was found glutamylated by TTLL4 and TTLL7, that again at distinct sites. These results pinpoint different site specificities between two enzymes. Moreover, TTLL7 exhibits less efficiency to elongate microtubules premodified by TTLL4, suggesting possible regulation of TTLL7 elongation activity by TTLL4-initiated sites. Finally, we showed that kinesin behaves differentially on microtubules modified by two enzymes. This study underpins the different reactivity, site selectivity, and function of TTLL4 and TTLL7 on brain tubulins and sheds light on their distinct role in vivo.

Ameliorative effect of calcium poly(aspartic acid) (PASP-Ca) and calcium poly-γ-glutamic acid (γ-PGA-Ca) on soil acidity in different horizons.

Soil acidification is a worldwide eco-environmental problem detrimental to plant growth and threatening food security. In this study, calcium poly(aspartic acid) (PASP-Ca) and calcium poly-γ-glutamic acid (γ-PGA-Ca) were obtained through cation exchange and used to mitigate soil acidity owing to high solubility and complexing capability. Three rates at 6.7, 13.4, and 20.1 g kg-1, denoted as PASP-Ca1, PASP-Ca2, and PASP-Ca3, and γ-PGA-Ca (7.4 g kg-1) were surface-applied and compared with conventional lime (CaCO3, 2.5 g kg-1) along with control in two soil layers (top soil 0-10 cm, subsoil 10-20 cm). After leaching, various soil properties and aluminum fractions were measured to assess their ameliorative performance and mechanisms. Although lime achieved the highest soil pH (6.91) in the topsoil followed by PASP-Ca and γ-PGA-Ca (pH: 5.57-6.33), it had less effect on subsoil increase (5.3) vs. PASP-Ca and γ-PGA-Ca (pH: 5.44-5.74). Surface-applied PASP-Ca demonstrated efficiency in elevating soil pH and reducing exchangeable acidity, mainly as exchangeable Al3+, whereas γ-PGA-Ca addition superiorly improved soil pH buffering capacity (pHBC). Moreover, PASP-Ca and γ-PGA-Ca addition improved organic carbon by 34.4-44.9%, available P by 4.80-20.71%, and cation exchange capacity (CEC) by 6.19-29.2%, thus greatly enhanced soil fertility. Ca2+ from polyAA-Ca promoted the displacement of exchangeable Al3+ or H+ from soil colloid, which were subsequently complexed or protonated and facilitated leaching. Additionally, the transformation into stable organo-aluminum fractions via complexation inhibited further hydrolysis. Under PASP-Ca or γ-PGA-Ca addition, the saturation of aluminum in cation exchange complex was reduced 2.91-7.81% compared to the control without addition amendments. Thus, PASP-Ca and γ-PGA-Ca can serve as potent ameliorants to alleviate soil acidity and aluminum toxicity for sustainable agricultural development.

pH Responsive Poly(Amino Acid) Nanoparticles as Potent Carrier Adjuvants for Enhancing Cellular Immunity.

Adjuvants are widely used in vaccine to improve the protection or treatment efficacy. However, so far they inevitably produce side effects and are hard to induce cellular immunity in practical application. Herein, two kinds of amphiphilic poly(glutamic acid) nanoparticles (α-PGA-F and γ-PGA-F NPs) as nanocarrier adjuvants are fabricated to induce an effective cellular immune response. Amphiphilic PGA are synthesized by grafting phenylalanine ethyl ester to form biodegradable self-assembly nanoadjuvants in a water solution. The model antigen, chicken ovalbumin (OVA), can be loaded into PGA-F NPs (OVA@PGA-F NPs) with the high loading ratio >12%. Moreover, compared with γ-PGA-F NPs, the acidic environment can induce the α-helical secondary structure of α-PGA NPs, promoting membrane fusion and more fast antigen lysosomal escape. Hence, the antigen presenting cells treated with OVA@α-PGA-F NPs show higher secretion of inflammatory cytokines, and higher expression of major biological histocompatibility complex class I and CD80 than those of OVA@γ-PGA-F NPs. Overall, this work indicates that pH responsive α-PGA-F NPs as a carrier adjuvant can effectively improve the ability of cellular immune responses, leading to it being a potent candidate for vaccine applications.

Antimicrobial activity of gamma-poly (glutamic acid), a preservative coating for cherries.

We investigated the minimum inhibitory concentration (MIC), antibacterial activity, and preservation ability of four molar masses of γ-polyglutamic acid (PGA) against Escherichia coli, Bacillus subtilis, and yeast. The antibacterial mechanism was determined based on the cell structure, membrane permeability, and microscopic morphology of the microorganisms. We then measured the weight loss, decay rate, total acid, catalase activity, peroxidase activity, and malondialdehyde content toward the possible use of PGA as a preservative coating for cherries. When the molar mass was greater than 700 kDa, the MIC for Escherichia coli and Bacillus subtilis was less than 2.5 mg/mL. The mechanism of action of the four molar masses of PGA was different with respect to the three microbial species, but a higher molar mass of PGA corresponded to stronger inhibition against the microbes. PGA of 2000 kDa molar mass damaged the microbial cellular structure, resulting in excretion of alkaline phosphatase, but PGA of 1.5 kDa molar mass affected the membrane permeability and the amount of soluble sugar. Scanning electron microscopy indicated the inhibitory effect of PGA. The antibacterial mechanism of PGA was related to the molar mass of PGA and the microbial membrane structure. Compared with the control, a PGA coating effectively inhibit the spoilage rate, delay the ripening, and prolong the shelf life of cherries.

ß-Alanine enhancing the crosslink of chitosan/poly-(γ-glutamic acid) hydrogel for a potential alkaline-adapted wound dressing.

Tiny crosslink in chitosan (CS)/poly-(γ-glutamic acid) (γ-PGA) hydrogel leads to some disadvantages including low mechanical strength and high swelling. To enhance the crosslink of CS/γ-PGA hydrogel, amino acid (AA) was introduced to remove the drawbacks. The results indicated that AA can dramatically increase the crosslink and mechanical properties of CS/γ-PGA hydrogel, and AA chain length and concentration have a drastic effect on them. Particularly, 0.5 % ß-Alanine (ß-Ala) decreased the hydrogel by 70 % in porosity, 52 % in water solubility, and 30 % in swelling, but increased by 2.2-fold in elastic modulus, 2.08-fold in stress, and 1.53-fold in water retention. The porosity of the hydrogel correlates positively with the elastic modulus but negatively with the crosslinking degree. The effect of pH on CS/ß-Ala/γ-PGA hydrogel was investigated in the load and release of benzalkonium chlorides (BAC). ß-Ala strengthened pH response of the hydrogel in BAC load and release. The loading capacity increased with pH value, and 0.5 % ß-Ala increased the hydrogel by 1.25-fold in the release capacity in alkaline environment, suggesting a good buffering effect of ß-Ala on pH variation to accelerate the transportation of BAC. CS/ß-Ala/γ-PGA hydrogel will be competently applied as a potential material for wound dressing in alkaline environment.

Hydrocarbon Degradation and Microbial Survival Improvement in Response to γ-Polyglutamic Acid Application.

To improve the environmental sustainability of cleanup activities of contaminated sites there is a need to develop technologies that minimize soil and habitat disturbances. Cleanup technologies, such as bioremediation, are based on biological products and processes, and they are important for the future of our planet. We studied the potential of γ-poly glutamic acid (PGA) as a natural component of biofilm produced by Bacillus sp. to be used for the decomposition of petroleum products, such as heavy naphtha (N), lubricating oil (O), and grease (G). The study aimed to assess the impact of the use of different concentrations of PGA on the degradation process of various fractions of petroleum hydrocarbons (PH) and its effect on bacterial population growth in harsh conditions of PH contamination. In laboratory conditions, four treatments of PGA with each of the petroleum products (N, O, and G) were tested: PGA0 (reference), PGA1 (1% PGA), PGA1B (1% PGA with Bacillus licheniformis), and PGA10 (10% PGA). After 7, 28, 56, and 112 days of the experiment, the percentage yield extraction, hydrocarbon mass loss, geochemical ratios, pH, electrical conductivity, and microorganisms survival were determined. We observed an increase in PH removal, reflected as a higher amount of extraction yield (growing with time and reaching about 11% in G) and loss of hydrocarbon mass (about 4% in O and G) in all treatments of the PGA compared to the reference. The positive degradation impact was intensive until around day 60. The PH removal stimulation by PGA was also reflected by changes in the values of geochemical ratios, which indicated that the highest rate of degradation was at the initial stage of the process. In general, for the stimulation of PH removal, using a lower (1%) concentration of PGA resulted in better performance than a higher concentration (10%). The PH removal facilitated by PGA is related to the anionic homopoliamid structure of the molecule and its action as a surfactant, which leads to the formation of micelles and the gradual release of PH absorbed in the zeolite carrier. Moreover, the protective properties of PGA against the extinction of bacteria under high concentrations of PH were identified. Generally, the γ-PGA biopolymer helps to degrade the hydrocarbon pollutants and stabilize the environment suitable for microbial degraders development.

Structural and genetic insights into a poly-γ-glutamic acid with in vitro antioxidant activity of Bacillus velezensis VCN56.

Poly-γ­glutamic acid (γ­PGA) produced by Bacillus species is a natural biopolymer, which is widely used in various fields including food, pharmaceuticals, and cosmetics. In this study, the screening of 19 Bacillus isolates derived from traditionally fermented foods revealed that Bacillus velezensis VCN56 was the most potent γ­PGA producer. The maximum concentration of crude γ­PGA was 32.9 ± 1.5 g/L in the PGA-3 medium containing glycerol, citric acid, sodium glutamate, NH4Cl, and starch. The resulting γ-PGA was purified and then characterized by HPLC, FTIR, and 1H-NMR analyses. Molecular weight of purified γ­PGA was estimated to be 98 kDa with a polydisperse index of 2.04. Notably, the pure γ­PGA showed significant in vitro antioxidant scavenging activities against 1,1-diphenyl-2-picrylhydrazyl (72.0 ± 1.5%), hydroxyl (81.0 ± 0.6%), and superoxide (43.9 ± 0.8%) radicals at the concentration of 4 mg/mL. Using whole-genome sequencing, the genetic organization of pgs operon responsible for γ­PGA biosynthesis in B. velezensis VCN56 differs from those in other Bacillus genomes. Further genome analysis revealed metabolic pathways for γ-PGA production and degradation. For the first time, the present study provides a better understanding of γ-PGA with a promising antioxidant activity produced by B. velezensis at the phenotypic, biochemical, and genomic levels, which hold potential applications in the foods, cosmetics, and pharmaceutical industries.

Atropisomerism in a polyglutamate-based thermoresponsive alignment medium.

Structure elucidation via residual dipolar couplings (RDCs) relies on alignment media. We report on lyotropic liquid crystals (LLCs) of poly-γ-p-biphenyl(2'-methoxy-2-methyl)methyl-L-glutamate (PBPM3LG). Temperature dependent atropisomerism within the biphenyl group enables the acquisition of multiple RDC-datasets within one sample.

The evaluation of Bacillus-secreted polyglutamic acid as anti-scaling treatment for circulating cooling water.

Polyaspartic acid is considered a green agent for the treatment of circulating cooling water. However, its chemosynthetic process is not green, as it requires significant amounts of energy and causes water pollution. In this work, we identified an analog of polyaspartic acid, namely polyglutamic acid (γ-PGA), which could be directly produced by Bacillus spp., and we explored its performance and scale inhibition mechanism as a scale inhibitor. We found that γ-PGA secreted by B. megaterium with a molecular weight of ~ 70 kDa showed poor scale inhibition, while the γ-PGA secreted by B. licheniformis with a molecular weight of ~ 15 kDa had a 26.87% higher efficiency compared to commercially available polyaspartic acid. The scale inhibition mechanism was explored using the γ-PGA material secreted by B. licheniformis. Fourier transform spectrometer, field emission scanning electron microscopy, and X-ray photoelectron spectroscopy analysis demonstrated that the scale inhibition performance of γ-PGA was due to the combination of its functional groups and Ca2+, which affected the growth process of CaCO3 and inhibited the formation of CaCO3. This study provided deeper insight into scale inhibition performance related to the scale inhibition mechanism.

The effect of hydration and dehydration on the conformation, assembling behavior and photoluminescence of PBLG.

Hydration and dehydration play crucial roles in hydrophobic effects (HEs) and are yet to be understood. Poly(γ-benzyl-L-glutamate) (PBLG) homopolymers in THF/water with various water contents were investigated. We discovered that PBLG was hydrated at low water contents and adopted a helical conformation. The chain became dehydrated with increasing water content, which converted the PBLG100 helix to a PPII-helix. The variation in the conformation resulted in an alteration of the self-assembled morphologies from fibers to particles. For PBLG12 with a shorter chain, the chain underwent an α-to-ß transition in the conformation due to dehydration as the water content increased, and correspondingly the morphologies varied from tapes to helical ribbons, and eventually to toroids at a higher water content. We also observed that this α-to-ß transition is accompanied by an increase in intensity of the fluorescence, which is attributed to the through-space-conjugation of tightly packed phenyl groups within the ß-sheet. The discovered effect of hydration and dehydration on the PBLG chain conformation, self-assembling behavior and optical function is essential for the innovation of polypeptide materials and understanding of water-mediated biological systems.

Construction of an exosome-functionalized graphene oxide based composite bionic smart drug delivery system and its anticancer activity.

Graphene oxide has covalently modified by chito oligosaccharides andγ-polyglutamic acid to form GO-CO-γ-PGA, which exhibits excellent performance as a drug delivery carrier, but this carrier did not have the ability to actively target. In this study, the targeting property of breast cancer tumor cell exosomes was exploited to give GO-CO-γ-PGA the ability to target breast tumor cells (MDA-MB-231), and the drug mitoxantrone (MIT) was loaded to finally form EXO-GO-CO-γ-PGA-MIT with an encapsulation efficiency of 73.02%. The pH response of EXO-GO-CO-γ-PGA showed a maximum cumulative release rate of 56.59% (pH 5.0, 120 h) and 6.73% (pH 7.4, 120 h) for MIT at different pH conditions.In vitrocellular assays showed that EXO-GO-CO-γ-PGA-MIT was more potent in killing MDA-MB-231 cells due to its targeting ability and had a significantly higher pro-apoptotic capacity compared to GO-CO-γ-PGA-MIT. The results showed that this bionic nano-intelligent drug delivery system has good drug slow release function and it can increase the local drug concentration of tumor and enhance the pro-apoptotic ability of MIT, so this newly synthesized bionic drug delivery carriers (EXO-GO-CO-γ-PGA-MIT) has potential application in breast cancer treatment.

Dynamic regulable sodium alginate/poly(γ-glutamic acid) hybrid hydrogels promoted chondrogenic differentiation of stem cells.

Traditional hydrogels often fail to match the dynamic interactions between mechanical and cellular behaviors exhibited by the natural cartilage extracellular matrix. In this research, we constructed a novel hybrid hydrogels system based on sodium alginate and polyglutamic acid. By controlling the grafting rate and concentration of polymer, the gelation time and mechanical strength can be adjusted between range of 8-28 s and 60-144 kPa. By adding microcrystalline cellulose into the system, so that the degradation time was prolonged (125%) and the swelling rate was reduced (470%). Additionally, the presence of hydrazone bonds gives the system some dynamic response characteristics, and the hydrogel exhibits excellent self healing and injectable ability. It was found that the system had positive cytocompatibility (80%), which accelerated regulatory gene expression in cartilage tissue. In conclusion, this injectable hydrogel with self-healing and customizable mechanical strength will have broad application prospects in future biomedical engineering.

A novel low-molecular-weight chitosan/gamma-polyglutamic acid polyplexes for nucleic acid delivery into zebrafish larvae.

Many challenges, such as virus infection, extreme weather and long cultivation periods, during the development of fish larvae have been observed, especially in aquaculture. Gene delivery is a useful method to express functional genes to defend against these challengers. However, the methods for fish larvae are insufficient. In our earlier report, low-molecular-weight chitosan (LMWCS) showed a strong positive charge and may be useful for polyplex formulation. Herein, we present a simple self-assembly of LMWCS polyplexes (LMWCSrNPs) for gene delivery into zebrafish larvae. Different weight ratios of LMWCS/gamma-polyglutamic acid (γ-PGA)/plasmid DNA were analyzed by gel mobility assay. Delivery efficiency determined by green fluorescent protein (GFP) expression in zebrafish liver (ZFL) cells showed that delivery efficiency at a weight ratio of 20:8:1 was higher than others. Zeta potential and transmission electron microscopy (TEM) analysis showed that the round shape of the particle size varied. In our earlier reports, IRF9S2C could induce interferon-stimulated gene (ISG) expression to induce innate immunity in zebrafish and pufferfish. Further delivery of pcDNA3-IRF9S2C-HA plasmid DNA into ZFL cells and zebrafish larvae by LMWCSrNP successfully induced ISG expression. Collectively, LMWCSrNP could be a novel gene delivery system for zebrafish larvae and might be used to improve applications in aquaculture.

Optimization of fermentation conditions, purification and rheological properties of poly (γ-glutamic acid) produced by Bacillus subtilis 1006-3.

This study aimed to investigate the optimal fermentation condition, purification and rheological properties of extracellular polymers produced by Bacillus subtilis 1006-3. An optimum temperature of 30.2 °C, inoculation amount of 6.1%, and pH of 8.2 were determined via Response Surface Methodology. The result of amino acid analysis and gel electrophoresis indicated that the obtained polymer contained only glutamic acid, with a wide molecular weight range. This polymer was finally determined as γ-PGA by infrared spectroscopy. The γ-PGA solution displayed a behavior of pseudoplastic non-Newtonian fluid with shear thinning properties, which can be described by the Ostward-de Waele power law model. The apparent viscosity of γ-PGA solution increased with the increase in its concentration from 1% to 10%. The deviation in pH from neutral value, and the addition of NaCl or MgCl2 can reduce the apparent viscosity of γ-PGA solution, and it was more sensitive to Mg2+ than to Na+ addition. At the concentration of 4, 6, and 8%, γ-PGA solution showed predominantly viscous response in the range of 0.1-100 rad/s angular frequency (G″>G'). These results indicated the potential application of the γ-PGA as a thickening agent.

Therapeutic melanoma inhibition by local micelle-mediated cyclic nucleotide repression.

The acidic tumor microenvironment in melanoma drives immune evasion by up-regulating cyclic adenosine monophosphate (cAMP) in tumor-infiltrating monocytes. Here we show that the release of non-toxic concentrations of an adenylate cyclase (AC) inhibitor from poly(sarcosine)-block-poly(L-glutamic acid γ-benzyl ester) (polypept(o)id) copolymer micelles restores antitumor immunity. In combination with selective, non-therapeutic regulatory T cell depletion, AC inhibitor micelles achieve a complete remission of established B16-F10-OVA tumors. Single-cell sequencing of melanoma-infiltrating immune cells shows that AC inhibitor micelles reduce the number of anti-inflammatory myeloid cells and checkpoint receptor expression on T cells. AC inhibitor micelles thus represent an immunotherapeutic measure to counteract melanoma immune escape.