Search

Bacillus licheniformis WX-02 is a well-studied strain to produce poly-γ-glutamic acid (γ-PGA) with numerous applications. This study is to improve WX-02 strain's capability of assimilating glycerol, a major byproduct of biofuels industries, through metabolic manipulation. Through gene knockout, the GlpK pathway was identified as the sole functional glycerol catabolism pathway, while the DhaK pathway was inactive for this strain under either aerobic or anaerobic conditions. The enhancement of glycerol utilization was attempted by substituting the native glpFK promoter with the constitutive promoter (P43), ytzE promoter (PytzE), and bacABC operon promoter (PbacA), respectively. The glycerol consumptions of the corresponding mutant strains WX02-P43glpFK, WX02-PytzEglpFK, and WX02-PbacAglpFK were 30.9, 26.42, and 18.8% higher than that of the WX-02 strain, respectively. The γ-PGA concentrations produced by the three mutant strains were 33.71, 23.39, and 30.05% higher than that of WX-02 strain, respectively. When biodiesel-derived crude glycerol was used as the carbon source, the mutant WX02-P43glpFK produced 16.63 g L of γ-PGA, with a productivity of 0.35 g L h. Collectively, this study demonstrated that glycerol can be used as an effective substrate for producing γ-PGA by metabolic engineering B. licheniformis strains. [ABSTRACT FROM AUTHOR]

The most important drawbacks of the thermo sensitive hydrogels based on N -isopropylacrylamide (NIPAAm) are the lack of other responsive groups and these hydrogels are not biodegradable; moreover, chemical cross-linking agents were commonly used in hydrogels formation process. In order to overcome these inconvenience, poly (NIPAAm- co - poly (γ-glutamic acid) - Allyl glycidyl ether) (poly (NIPAAm- co -γ-PGA-AGE)) hydrogels were obtained by cross-linking polymerization of the corresponding monomers. Poly (γ-glutamic acid) (γ-PGA) of biocompatible and biodegradable was functionalized with an appropriate amount of vinyl groups, thus acting both as a co-monomer and as a bio-degradable cross-linker. The morphology, thermal-behavior, swelling/deswelling kinetics, pH sensitivity and biodegradability of poly (NIPAAm- co -γ-PGA-AGE) hydrogels were studied. The results indicated that the hydrogels not only had good thermal response sensitivities, but also behaved with pH response sensitivities and showed degradation behavior. The hydrogels were loaded with the model drug naproxen and the release studies were performed under simulated physiological conditions. The results showed that drug was efficiently loaded and released in a controlled manner from these hydrogels. In addition, the more amount of γ-PGA-AGE added to the poly (NIPAAm- co -γ-PGA-AGE) hydrogels, the better cytocompatibility of the hydrogels. These hydrogels have more attractive application prospects in the biomedical field. [ABSTRACT FROM AUTHOR]

Poly-γ-glutamic acid (PGA) was modified with phosphorylating agents such as sodium metaphosphate and potassium metaphosphate in the culture medium of Bacillus subtilis ( natto ). The highly phosphorylated PGA derivatives were prepared and investigated for their chemical and physicochemical properties. The PGA derivatives had approximately 7% (W/W) inorganic phosphorus and characteristic absorbance PO 2 − bands at 1082 cm −1 and 1260 cm −1 by Fourier Transform Infrared Spectroscopy. The derivative modified by sodium metaphosphate (J-5) was easily hydrated in water and had extremely low viscosity. The shear rate-induced transition leading to the decrease of viscosity was not observed in J-5 whereas the derivative modified by potassium metaphosphate (J-6) as well as unmodified PGA (J-1) showed the typical decrease of viscosity. In circular dichroism (CD) measurement of J-5, there was a significant loss of the negative chirality CD signal, implying that protein aggregation occured at decreasing pH from 6.2 to 4.4. The thioflavin T fluorescence intensity of the aqueous solution in the J-5 was extremely high despite the absence of heat-treatment. The results indicate that the J-5 is the likeliest type of aggregation by β-sheet cross-linking which is relevant to protein diseases like Alzheimer’s disease. [ABSTRACT FROM AUTHOR]

Background: Polysaccharides and poly-γ-glutamic acid (γ-PGA) are biomacromolecules that have been reported as bioflocculants, and they exhibit high flocculating activity in many industrial applications. Bacillus licheniformis CGMCC 2876 can produce polysaccharide and γ-PGA bioflocculants under different culture conditions. Several key genes are involved in the metabolic pathway of polysaccharides in B. licheniformis, but the impacts of the regulation of these genes on the production of polysaccharide bioflocculants have not been illustrated completely. To increase the bioflocculant production and identify the correlation between the synthesis of polysaccharides and γ-PGA in B. licheniformis, a few key genes were investigated to explore their influence on the synthesis of the bioflocculants. Results: Overexpressing epsB from the eps gene cluster not only improved the bioflocculant crude yield by 13.98% but also enhanced the flocculating activity by 117.92%. The composition of the bioflocculant from the epsB recombinant strain was 28.95% total sugar, 3.464% protein and 44.03% γ-PGA, while in the original strain, these components represented 53.67%, 3.246% and 34.13%, respectively. In combination with an analysis of the transcriptional levels of several key genes involved in γ-PGA synthesis in B. licheniformis, we inferred that epsB played a key role in the synthesis of both polysaccharide and γ-PGA. The bioflocculant production of the epsB recombinant strain was further evaluated during batch fermentation in a 2 L fermenter; the flocculating activity reached 9612.75 U/mL, and the bioflocculant yield reached 10.26 g/L after 72 h, representing increases of 224% and 36.62%, respectively, compared with the original strain. Moreover, we found that the tandem expression of phosphoglucomutase (pgcA) and UTP-glucose- 1-phosphate uridylyltransferase (gtaB1) could enhance the crude yield of the bioflocculant by 20.77% and that the overexpression of epsA could enhance the bioflocculant yield by 23.70% compared with the original strain. Conclusions: This study provides a new method to greatly increase the bioflocculant production in B. licheniformis, and it demonstrates the correlation between the biosynthesis of polysaccharide and γ-PGA during EPS fermentation by regulating the expression of EpsB. [ABSTRACT FROM AUTHOR]

BACKGROUND: Nowadays complex bone defects have become a great challenge to orthopedists. A synergistic contribution of various growth factors and a crosstalk between their signaling pathways have been suggested as determinatives for the overall osteogenic outcome. OBJECTIVE: To develop calcium phosphate cement (CPC) incorporated with γ-polyglutamic acid/carboxymethyl chitosan (PGA/CMCS), and to evaluate its physical and chemical properties and sustained-release function. METHODS: The γ-PGA/CMCS polymer composites were prepared by graft copolymerization and spray freeze drying methods, and then loaded with recombinant human bone morphogenetic protein 2 (rhBMP-2) growth factor. CPC served as control group, and γ-PGA/CMCS-CPC containing different contents of rhBMP-2 as experimental groups. A γ-PGA/CMCS-CPC scaffold with regular blade-like crystalline structure was fabricated by injection compression molding. Before mixed with the liquid phase, the solid additives were properly mixed by wet method of CPC solid and the γ-PGA/CMCS carrier, then the pre-blended mix was freeze-dried. The setting time and compressive strength of bone cement in each group were detected, and the microstructure of the material surface was observed under scanning electron microscopy. In vitro release of rhBMP-2 was investigated. The effect of bone cement extracts on cell proliferation was determined through MTS assay. RESULTS AND CONCLUSION: γ-PGA/CMCS-CPC had the same physicochemical properties to the CPC. Initial and final setting time, compressive strength of bone cement had no significant differences among groups. The scanning electron microscope results showed that the γ-PGA/CMCS-CPC scaffold was covered by regular blade-like crystalline structure and the γ-PGA/CMCS particles were uniformly dispersed in the CPC crystals. A sustained release of rhBMP-2 was observed from the γ-PGA/CMCS-CPC. The cell experiments exhibited that the samples with regular blade-like crystalline structure had better cell response compared to CPC control groups with irregular crystalline structure. These findings indicate that γ-PGA/CMCS-CPC can maintain good physicochemical properties, and release growth factor or drug to promote bone formation. [ABSTRACT FROM AUTHOR]

Poly-γ-glutamic acid (γ-PGA) is a microbe-secreted isopeptide shown to promote growth and enhance crop stress tolerance. However, its downstream signaling pathways are unknown. Here, we studied γ-PGA-induced tolerance to salt and cold stresses. Pretreatment with γ-PGA contributed to enhance stress tolerance of canola seedlings by promoting proline accumulation and total antioxidant capacity (T-AOC) improvement. Further, Ca 2+ , H 2 O 2 , brassinolide, and jasmonic acid were found to be involved in the γ-PGA-induced process. First, using signal blockers, we concluded that γ-PGA activated Ca 2+ fluctuations in canola seedling leaves. Second, the activated Ca 2+ further elicited H 2 O 2 production by Ca 2+ -binding proteins CBL9, CPK4, and CPK5. Third, the H 2 O 2 signal promoted brassinolide and jasmonic acid biosynthesis by upregulating key genes ( DWF4 and LOX2 , respectively) for synthesizing these compounds. Lastly, brassinolide and jasmonic acid increased H 2 O 2 which promoted proline accumulation and T-AOC improvement and further enhanced Ca 2+ -binding proteins including CaM, CBL10, and CPK9. [ABSTRACT FROM AUTHOR]

This study aimed to develop non-food fermentation for the cost-effective production of poly-(γ-glutamic acid) (γ-PGA) using a novel strain of Bacillus amyloliquefaciens NX-2S. The new isolate assimilated inulin more efficiently than other carbohydrates from Jerusalem artichoke, without hydrolytic treatment. To investigate the effect of inulin on γ-PGA production, the transcript levels of γ-PGA synthetase genes ( pgsB , pgsC , pgsA ), regulatory genes ( comA , degQ , degS ), and the glutamic acid biosynthesis gene ( glnA ) were analyzed; inulin addition upregulated these key genes. Without exogenous glutamate, strain NX-2S could produce 6.85 ± 0.22 g/L of γ-PGA during fermentation. Exogenous glutamate greatly enhances the γ-PGA yield (39.4 ± 0.38 g/L) and productivity (0.43 ± 0.05 g/L/h) in batch fermentation. Our study revealed a potential method of non-food fermentation to produce high-value products. [ABSTRACT FROM AUTHOR]

Mild crosslinking methods, which have a strong influence on biomedical hydrogels and scaffolds, have attracted wide attention in recent years. In this project, native chemical ligation (NCL) was utilized to prepare biocompatible and biodegradable hydrogels using naturally derived poly(glutamic acid) (PGA) with no additives or by-products. Firstly, thiolactone-grafted poly(glutamic acid) (PGA-HC) and cysteine-grafted poly(glutamic acid) (PGA-C) precursors were synthesized. Their structure was confirmed by nuclear magnetic resonance (NMR). Then, hydrogels crosslinked by NCL were formed by blending buffered solutions of PGA-HC and PGA-C with no additives under physiological conditions. After that, the equilibrium water content, morphology, degradation rate and mechanical properties of the hydrogels were characterized in detail. The data showed that the PGA hydrogels had gelation times, water contents and mechanical properties that were tunable by adjusting the precursor composition. Furthermore, the biocompatibility of the hydrogels was confirmed by an MTT assay. These characteristics provide a potential opportunity for the NCL hydrogels as wound dressings, skin fillings, drug delivery vehicles and tissue regeneration matrices. [ABSTRACT FROM AUTHOR]

Nowadays, combination therapy became a standard in oncology. In this study, we compare the activity of two polymeric carriers bearing a combination of the anticancer drugs paclitaxel (PTX) and doxorubicin (DOX), which differ mainly in their architecture and supramolecular assembly. Drugs were covalently bound to a linear polymer, polyglutamic acid (PGA) or to a dendritic scaffold, polyglycerol (PG) decorated with poly(ethylene glycol) (PEG), forming PGA-PTX-DOX and PG-PTX-bz-DOX-PEG, respectively. We explored the relationship between the polymeric architectures and their performance with the aim to augment the pharmacological benefits of releasing both drugs simultaneously at the tumor site at a synergistic ratio. We recently designed and characterized a PGA-PTX-DOX conjugate. Here, we describe the synthesis and characterization of PG dendritic scaffold bearing the combination of PTX and DOX. The performance of both conjugates was evaluated in a murine model of mammary adenocarcinoma in immunocompetent mice, to investigate whether the activity of the treatments is affected by the immune system. Drug conjugation to a nano-sized polymer enabled preferred tumor accumulation by extravasation-dependent targeting, making use of the enhanced permeability and retention (EPR) effect. Both PGA-PTX-DOX and PG-PTX-bz-DOX-PEG nano-sized conjugates exhibited superior anti-tumor efficacy and safety compared to the combination of the free drugs, at equivalent concentrations. However, while PGA-PTX-DOX was more efficient than a mixture of each drug conjugated to a separate PGA chain, as was previously shown, PG-PTX-bz-DOX-PEG had similar activity to the mixture of the PG-PTX-bz-PEG and PG-DOX-PEG conjugates. Our results show that both conjugates are potential candidates as precision combination nanomedicines for the treatment of breast cancer. [ABSTRACT FROM AUTHOR]

Lyotropic liquid crystalline (LLC) phases of polyglutamic acid derivatives, such as poly-γ-benzyl- l-glutamate, are suitable alignment media for organic structure elucidation by NMR spectroscopy. Their helical structure is responsible for enantiodiscrimination. As part of our ongoing investigations concerning the alignment mechanism(s) of these systems, we considered whether an additional chiral center in the side chain could improve enantiodiscrimination relative to the helical polymer with an achiral side chain. Therefore, the diastereoisomers poly-γ- S-2-methylbutyl- l-glutamate (PSMBLG) and poly-γ- S-2-methylbutyl- d-glutamate (PSMBDG) were synthesized. These two polymers were tested for their liquid crystallinity and suitability as alignment media. The spatial structure of the solutes (−)-curcumol and isopinocampheol (IPC) were validated by the residual dipolar coupling data obtained. Additionally, enantiodiscrimination of IPC was observed in the new alignment media and compared with the enantiodiscrimination of IPC in other homopolypeptides. [ABSTRACT FROM AUTHOR]

Background: Sucrose is an naturally abundant and easily fermentable feedstock for various biochemical production processes. By now, several sucrose utilization pathways have been identified and characterized. Among them, the pathway consists of sucrose permease and sucrose phosphorylase is an energy-conserving sucrose utilization pathway because it consumes less ATP when comparing to other known pathways. Bacillus amyloliquefaciens NK-1 strain can use sucrose as the feedstock to produce poly-γ-glutamic acid (γ-PGA), a highly valuable biopolymer. The native sucrose utilization pathway in NK-1 strain consists of phosphoenolpyruvate-dependent phosphotransferase system and sucrose-6-P hydrolase and consumes more ATP than the energy-conserving sucrose utilization pathway. Results: In this study, the native sucrose utilization pathway in NK-1 was firstly deleted and generated the B. amyloliquefaciens 3A strain. Then four combination of heterologous energy-conserving sucrose utilization pathways were constructed and introduced into the 3Δ strain. Results demonstrated that the combination of cscB (encodes sucrose permease) from Escherichia coli and suc P (encodes sucrose phosphorylase) from Bifidobacterium adolescentis showed the highest sucrose metabolic efficiency. The corresponding mutant consumed 49.4% more sucrose and produced 38.5% more γ-PGA than the NK-1 strain under the same fermentation conditions. Conclusions: To our best knowledge, this is the first report concerning the enhancement of the target product production by introducing the heterologous energy-conserving sucrose utilization pathways. Such a strategy can be easily extended to other microorganism hosts for reinforced biochemical production using sucrose as substrate. [ABSTRACT FROM AUTHOR]

In the management of bladder cancer, surgical resection of the tumour is usually followed by intravesical instillation of immunomodulatives and/or chemotherapeutics. The purpose of this local intravesical therapy is to eliminate residual malignant cells after surgical intervention. The main limitation of a localised adjuvant therapy is the insufficient concentration of the active pharmaceutical ingredient (API) in malignant cells due to the unique structure of the human urothelium making it an exclusively hard to overcome barrier in the human body. Different strategies such as electromotive drug administration or local hyperthermia are employed to ameliorate intravesical drug uptake. Previous studies on biorecognitive targeting showed promising results for lectin-, especially wheat germ agglutinin (WGA), mediated drug delivery. Here, we present a targeted conjugate that provides enough binding sites for a possible API as well as high cytoadhesive and cytoinvasive potential. The conjugate should comprise the following components: First WGA, as the targeting moiety, second poly- l -glutamic acid (PGA) as a polymeric backbone providing more than 300 possible binding sites for an API and third fluorescein cadaverine (Fc), a fluorescent dye we coupled to PGA rendering the conjugate traceable. After purification via size exclusion chromatography (SEC) the WGA containing and therefore binding conjugate was isolated from the reaction mix. In flow-cytometric and fluorimetric experiments with single cells and cell monolayers, respectively, binding and internalisation of the conjugate representing a high molecular weight (>100 kDa) could be demonstrated. Fluorescent PGA without the WGA component showed neither binding nor internalisation potential. Microscopic colocalization studies with cell monolayers and single cells confirmed the cytoadhesive and cytoinvasive potential of the WGA containing conjugate. In accordance with the results of specificity studies the interaction between the conjugate and the cell surface depended solely on the WGA component of the conjugate. With the help of this targeted drug delivery system limiting factors of intravesical adjuvant therapy could eventually be overcome and thereby treatment efficacy of instillative chemotherapy could be improved. In addition, traceable drug conjugates might bear interesting advantages for theranostic purposes in the treatment of bladder cancer. [ABSTRACT FROM AUTHOR]

Purpose: To devise an efficient strategy for the separation and recovery of high-quality γ-PGA by investigation of the physical properties, pigment properties and microfiltration mode of high-viscosity fermentation broth. Methods: The bacterial strain, Bacillus subtilis 115, was used in this study. The viscosity of the fermentation broth was determined by digital viscometer with spindle SP-2 at 25 °C. The concentrations of glucose and L-glutamate were analyzed with a biosensor equipped with both glucose oxidase and Lglutamate oxidase electrodes. The pigment in the fermentation liquid was scanned with a UV spectrophotometer at wavelength range of 200 - 500 nm and was removed using activated carbon. Measurement of IR spectrum was performed using an IR spectrophotometer with KBr pellet. Results: The results showed that the γ-PGA yield was 35 g/L. The viscosity of the fermentation broth was 1600 mPa.s at the end of the batch fermentation. After 3-fold dilution, the viscosity was reduced to one-fortieth of the original value at 65 °C for 30 min., which allowed effective removal of Bacillus subtilis 115 from the broth. Maximum UV absorption of the pigment was occurred at 260 nm. The pigment was removed by shaking with 0.6% activated carbon powder at 50 rpm for 20 min, resulting in 88% decolorization. Concentration with hollow-fiber membrane (MWCO 500,000) resulted in complete removal of residual glucose and glutamic acid from the aqueous solution of γ-PGA. The molecular weight of the γ-PGA was 1095 kDa, and its UV scanning spectrum showed an absorption peak at 216 nm. The decomposition temperature (Td) of the γ-PGA was 312.92 oC. Its IR spectrum was consistent with the presence of carboxyl, hydroxyl, carbonyl and amide groups. Conclusion: An efficient method for the extraction and purification of high-quality γ-PGA from highviscosity fermentation broth. [ABSTRACT FROM AUTHOR]

A series of ionic complexes with a comb-like architecture and a nearly stoichiometric composition were prepared from bacterial poly(γ, dl -glutamic acid) (PGGA) and alkyltrimethyl phosphonium bromides ( n ATMP·Br) bearing long linear alkyl chains with even numbers of carbon atoms ( n ) ranging from 12 to 22. The n ATMP·PGGA complexes were non-water soluble but readily soluble in organic solvents, and they displayed a high thermal stability. Combined DSC and XRD studies revealed that these complexes adopted an amphiphilic layered structure with the polypeptide chain and the alkyl chain separated in two differentiated phases with a nanoperiodicity that increased steadily with the length of the alkyl chain. The paraffinic phase was found to be partially crystallized in an extent that decreased with n , so that complexes with n = 12 and 14 did not show any sign of crystallinity whereas those with n from 16 to 22 showed crystalline melting in the ∼30–70 °C range. The structural transitions taking place by temperature effects were characterized by simultaneous SAXS/WAXS using synchrotron radiation at real time. In all cases, a shortening of the layer periodicity occurred upon heating with recovering of the initial structure after cooling. n ATMP·PGGA with n ≤ 16 showed strong antimicrobial activity against both E. coli and S. aureus, a property that could be related to the weak dissociation of the complexes happening upon incubation in water. The structure and properties of these complexes where comparatively discussed taking as reference their analog complexes made from PGGA and alkyltrimethylammonium bromides already studied by us. [ABSTRACT FROM AUTHOR]

Due to the various needs in the current applications, multifunctional composite materials with high strength and high toughness were highly desired now. Many scholars dedicated their time to find a simple, green and fast method for the preparation of multi-functional materials. In this study, inspired by the hierarchical 'brick-and-mortar' structure and excellent mechanical performance of nacre, a fast green vacuum-filtration method was used to fabricate strong and multifunctional polyglutamic acid/layered double hydroxide (PGA/LDH) films mimicking nacre. The experimental results confirm that the artificial nacre has hierarchical 'brick-and-mortar' structure. It exhibits excellent strength (93.5 MPa) and flexibility (easily bendable fold), combining with outstanding properties of UV-blocking and translucence properties. This work provides a way of fabricating multifunctional organic-inorganic hybrid films, which has potential applications in the areas of optical, transportation and construction fields. [ABSTRACT FROM AUTHOR]

This study found that high ferric ion (Fe 3+ ) concentrations lead to high poly-γ-glutamic acid (γ-PGA) production and low-molecular-weight γ-PGA. In cultures containing 7.4 mmol/L FeCl 3 , the average molecular weight of γ-PGA reached 3.18 ± 0.3 × 10 5 Da, which is a 76% decrease from that in the culture without FeCl 3 (1.31 ± 0.14 × 10 6 Da). Cultures containing increasing concentrations of FeCl 3 (0–7.4 mmol/L) showed a corresponding increase in γ-PGA yield from 26.57 ± 1.64 to 34.93 ± 1.05 g/L. Transcription level analysis indicated that high FeCl 3 concentrations could enhance the expression of γ-PGA synthetase genes ( pgsA , pgsB, and pgsC ), which may contribute to the improved production of γ-PGA. In addition, γ-PGA degradation genes ggt (encoding γ-glutamyltransferase) and cwlO were found to contribute to a lower γ-PGA molecular weight in cultures containing 7.4 mmol/L FeCl 3 . The result of γ-glutamyltransferase activity was in agreement with the results of the transcription level analysis. Overall, this study would provide a novel method for the regulation of synthesis of γ-PGA with low molecular weight. [ABSTRACT FROM AUTHOR]

In this study, we investigated chmemoenzymatic synthesis of amylose-grafted poly(γ-glutamic acid) (PGA) as a new artificial saccharide-peptide conjugate composed of two biological macromolecules. Maltooligosaccharide as a primer of enzymatic polymerization by phosphorylase catalysis was first introduced on the PGA main chain by the condensation reaction using the condensing agent in NaOH aq. Thermostable phosphorylase-catalyzed enzymatic polymerization of α- d -glucose 1-phosphate (G-1-P) as a monomer was then performed from the primer chain ends of the product to obtain amylose-grafted PGAs, which formed hydrogels in reaction media depending on the G-1-P/primer feed ratios. The powder X-ray diffraction patterns of lyophilized samples (cryogels) from the hydrogels suggested that the amylose graft chains formed double helixes, which acted as cross-inking points for self-assembling hydrogelation. The scanning electron microscopic images of the cryogels showed regularly controlled porous morphologies. Moreover, pore sizes of the cryogels increased with increasing the G-1-P/primer feed ratios, whereas the degrees of substitution of primer on the PGA main chain did not obviously affect pore sizes. [ABSTRACT FROM AUTHOR]

Mammals is a powerful tool to study pathophysiologic process of cardiovascular disease, but for a long time it is just limited to large animals, like pigs, rabbits, and big rats. The mice with reproduction fast cycle, lower costing, and close to human genes, can be showed about some phenomenon quickly, also more easy to form model of atherosclerosis. Thus, a mouse model of vascular injury is vitally important for researching the pathophysiological mechanisms to restenosis after percutaneous transluminal coronary angioplasty (PTCA) as well as translational approaches. In this Experiment, we established a unique murine model of femoral artery injury through a home-made wire. The mouse model of vascular injury may be used to explore the molecular mechanism of post-angioplasty restenosis with lowest costing. [ABSTRACT FROM AUTHOR]

Neural cell adhesion molecule (NCAM) expression is known to be associated with an aggressive biological behavior, increased metastatic capacity and expression of stem-cell markers in several tumor types. NCAM was also found to be expressed on tumor endothelial cells while forming new capillary-like tubes, but not on normal endothelial cells. An NCAM-targeted polymer-drug conjugate can be used both to target tumors expressing high levels of NCAM as well as the angiogenic vessels and cancer stem cells populations characterized by NCAM expression within tumors. Here, we describe the design, synthesis, physico-chemical characterization and the biological evaluation of an NCAM-targeted conjugate of polyglutamic acid with paclitaxel that was developed and evaluated on neuroblastoma, a high NCAM-expressing tumor. This conjugate inhibited tumor growth to a higher extent compared to the control conjugates and was less toxic than free paclitaxel. The dose of the conjugate could be increased at least twice than the maximum tolerated dose of paclitaxel to achieve better activity without aggravating toxicity. This work presents evidence that NCAM targeting can highly increase the efficacy of nanomedicines in the appropriate tumor models. [ABSTRACT FROM AUTHOR]

Recently we reported the development of 100 nm polyglutamic acid (PGA)-based nanocapsules, which were intended to carry anticancer drugs to the lymphatic system (Abellan-Pose et al., 2016). In this work, the objective was to further assess the potential “lympho-targeting” properties of radiolabeled 111 In-PGA and 111 In-PGA-PEG, following intravenous or subcutaneous administration. The results indicate that, following intravenous administration, both types of nanocapsules exhibit a modest accumulation in the lymph nodes (⩽2.3% ID/g). On the contrary, following subcutaneous administration, and irrespective of the presence of PEG on their surface, the nanocapsules were found to form a reservoir at the injection site, from which they drained slowly into the popliteal and the iliac lymph nodes. The significant accumulation of the radiolabeled nanocapsules in the lymph nodes was attained at 24 and 48 h post-injection, reaching values comprised between 70% and 187% ID/g in the popliteal lymph nodes. Altogether, the results led us to validate our hypothesis about the ability of the PGA and PGA-PEG nanocapsules to reach the lymphatic system, especially following subcutaneous administration. [ABSTRACT FROM AUTHOR]

To investigate the often reported disagreement in food folate quantitation between the microbiological assay and high-performance liquid chromatography methods, different foods were analyzed both by a microbiological assay and by a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. For the LC-MS/MS analysis we emphasize the need for complete deconjugation of polyglutamic folate forms. Moreover, our results revealed no need for an additional enzyme treatment except in the deconjugation step. To check the efficiency of deconjugation without additional sample preparations, the amount of diglutamates was quantified and samples were screened for additional polyglutamates. A thorough investigation of a substance with a polyglutamate chain deconjugated like the folates revealed that it was an oxidation product of 5-methyltetrahydrofolate, a pyrazino- s-triazine called MeFox in previous reports. The latter is not microbiologically active and, therefore, does not contribute to the amount of total folates. But we found it is commonly present in foods, especially in those low in ascorbic acid. The microbiological assay showed different responses to the single vitamers. Therefore, it was necessary to perform calibration with the folate that had the highest portion in the folate distribution. The investigations showed that both methods can provide similar results when they both include a deconjugation step. This is particularly important for LC-MS/MS but probably also for the microbiological assay. Additionally, consideration of the folate distribution was found to be crucial for the accurate calibration of the microbiological assay. [ABSTRACT FROM AUTHOR]

The growth regulator gibberellic acid (GA 3 ) has several uses in the field, improving germination, plant development, productivity, and the quality of food. This work describes the development of a nanocarrier system for GA 3 , based on the poly(γ-glutamic acid) (γ-PGA) and chitosan (CS) polymers, Nanoparticles without GA 3 (nano-γPGA/CS-GA 3 ) showed colloidal characteristics, with an average size of 117 ± 9 nm, PDI of 0.43 ± 0.07, and zeta potential of −29 ± 0.5 mV. The encapsulated nanoparticles (nano-γPGA/CS-GA 3 ) presented an average size of 134 ± 9 nm, PDI of 0.35 ± 0.05, zeta potential of 27.9 ± 0.5 mV, and 61% encapsulation. The images of nanoparticles observed by Transmission and scanning electron microscopy (TEM and SEM) showed a spherical shape of the nanoparticles. The system showed sustained release, with 58% release after 48 h. Evaluation of thermal properties using DSC and TGA analyses indicated that there was an interaction between the CS and γ-PGA polymers. In tests using Phaseolus vulgaris seeds, nano-γPGA/CS-GA 3 showed high biological activity, enhancing the rate of germination in the first day (50–70%) when compared with free GA 3 (10–16%). Encapsulated GA 3 was also more efficient than the free hormone in the increase of leaf area and the induction of root development (including the formation of lateral roots). These effects were not observed when seeds were treated with nano-γPGA/CS without GA 3 . The results demonstrated the considerable potential of nano-γPGA/CS-GA 3 for use in agriculture. [ABSTRACT FROM AUTHOR]

Background: Poly-gamma-glutamic acid (γ-PGA) is a promising macromolecule with potential as a replacement for chemosynthetic polymers. γ-PGA can be produced by many microorganisms, including Bacillus species. Bacillus licheniformis CGMCC2876 secretes γ-PGA when using glycerol and trisodium citrate as its optimal carbon sources and secretes polysaccharides when using glucose as the sole carbon source. To better understand the metabolic mechanism underlying the secretion of polymeric substances, SWATH was applied to investigate the effect of glucose on the production of polysaccharides and γ-PGA at the proteome level. Results: The addition of glucose at 5 or 10 g/L of glucose decreased the γ-PGA concentration by 31.54 or 61.62%, respectively, whereas the polysaccharide concentration increased from 5.2 to 43.47%. Several proteins playing related roles in γ-PGA and polysaccharide synthesis were identified using the SWATH acquisition LC-MS/MS method. CcpA and CcpN co-enhanced glycolysis and suppressed carbon flux into the TCA cycle, consequently slowing glutamic acid synthesis. On the other hand, CcpN cut off the carbon flux from glycerol metabolism and further reduced γ-PGA production. CcpA activated a series of operons (glm and epsA-O) to reallocate the carbon flux to polysaccharide synthesis when glucose was present. The production of γ-PGA was influenced by NrgB, which converted the major nitrogen metabolic flux between NH4+ and glutamate. Conclusion: The mechanism by which B. licheniformis regulates two macromolecules was proposed for the first time in this paper. This genetic information will facilitate the engineering of bacteria for practicable strategies for the fermentation of γ-PGA and polysaccharides for diverse applications. [ABSTRACT FROM AUTHOR]

Nucleoplasmin (Npm) is the most abundant protein in the nucleus of oocytes which can promote in vitro nucleosome formation. In this study, to improve the efficiency of SCNT in Banna Mini-pig Inbred Line (BMI), we obtained the recombinant nucleoplasmin (Npm) by using a prokaryotic expression system and compared the difference on the developmental effects between exogenous Npm and its structural analog, polyglutamic acid (PGA). We chose the pMD18-T vector for Npm expression and transformed E. coli BL21 (DE3) cells to construct a prokaryotic expression system. The results showed that the recombinant Npm protein of 32 kDa could be obtained. Recombinant Npm or PGA was injected into porcine oocytes at different concentrations. The results showed that the blastocyst rates in injection 560 ng/μL Npm and 1000 ng/μL PGA groups were higher than those of other groups. Moreover, the blastocyst rate was highest in injection 560 ng/μL Npm group and was lowest in injection 100 ng/μL PGA group. These data suggested that addition of recombinant Npm obtained by prokaryotic expression or PGA could improve the development of SCNT embryos in the BMI. [ABSTRACT FROM AUTHOR]

Vaccination is an innovative strategy for cancer treatment by leveraging various components of the patients' immunity to boost an anti-tumor immune response. Rationally designed nanoparticles are well suited to maximize cancer vaccination by the inclusion of immune stimulatory adjuvants. Also, nanoparticles might control the pharmacokinetics and destination of the immune potentiating compounds. Poly-γ-glutamic acid (γ-PGA) based nanoparticles (NPs), which have a natural origin, can be easily taken up by dendritic cells (DCs), which leads to the secretion of cytokines which ameliorates the stimulation capacity of T cells. The intrinsic adjuvant properties and antigen carrier properties of γ-PGA NPs have been the focus of recent investigations as they can modulate the tumor microenvironment, can contribute to systemic anti-tumor immunity and subsequently inhibit tumor growth. This review provides a comprehensive overview on the potential of γ-PGA NPs as antigen carriers and/or adjuvants for anti-cancer vaccination., Competing Interests: Declaration of Competing Interest The authors would like to acknowledge the Nanotechnology Research Center, Mashhad University of Medical Sciences (MUMS)., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Following the global COVID-19 pandemic, the incidence of tracheal epithelial injury is increasing. However, the repair of tracheal epithelial injury remains a challenge due to the slow renewal rate of tracheal epithelial cells (TECs). In traditional nebulized inhalation treatments, drugs are enriched in the lungs or absorbed into the blood, reducing drug concentration at the tracheal injury site. In this study, we prepared an epidermal growth factor (EGF)-loaded gamma-polyglutamic acid (γ-PGA)/epsilon-poly-L-lysine (ε-PLL) (PP) hydrogel (EGF@PP) to promote the repair of tracheal epithelial injury. Epidermal growth factor promotes the proliferation of TECs and enhances vascularization, thereby accelerating injury repair. The PP hydrogel exhibits outstanding wet adhesion, slow drug release, and antibacterial and anti-inflammatory properties, making it suitable for application in the airways and creating an environment conducive to epithelial repair. Here, we established a rabbit model of tracheal injury using a laser to destroy the tracheal epithelium and delivered EGF@PP powder to the injury site under fiberoptic bronchoscopy guidance. Our findings revealed that this was an effective therapeutic strategy for accelerating the repair of tracheal epithelial injury.