Polyphosphate Polymers Research Articles

Effect of NIR-II on cancer nanocomposite vaccines for lymph node targeted delivery and enhanced immunotherapy.

e20506 Background: Cancervaccines have become one of the hot areas of research in recent years. However, different types of tumors and the tumor tissue itself are heterogeneous. Some cancervaccines contain a single epitope and have weak immunogenicity. The antigen-presenting cells cannot adequately absorb and present the antigen peptide contained in the vaccine. As a result, the body fails to elicit an effective immune response, which greatly limits the therapeutic effect of the cancervaccine. In situ cancervaccines composed of antigens and adjuvants are a promising cancer treatment modality; however, convenient manufacturing of vaccines in vivo and their efficient delivery to lymph nodes (LNs) remain a significant challenge. In this study, we outline a facile approach to simultaneously achieve in situ programming of vaccines via two synergistic nanomedicines to generate robust immune responses. Methods: We first prepared two synergistic nanocomposite systems: Gold nanorods, amphiphilic polymer polyphosphate mPEG- b-PHEP encapsulating the immune adjuvant R848, and nanoparticles doped with cationic liposomes DOTAP. Two nanomedicines were characterized and the efficacy of the nanovaccine was verified at the Cellular Experiments. At the animal level, the lymph node targeting of the nanoparticles was demonstrated through IVIS. We also constructed distant model and lung metastasis model to verify the nanovaccine's effect on enhancing systemic immunotherapy. Results: We used amphiphilic polymer polyphosphate mPEG- b-PHEP to encapsulate TLR7/8 agonist R848 to obtain VNPR848 nanoparticles. Gold nanorods (~ 100nm) can produce a strong photothermal effect mediated by NIR-II, killing tumor cells and releasing abundant antigens at the same time. The small size of VNPR848 (~ 30nm) is beneficial to the enrichment of lymph nodes. After capturing the antigens produced by photothermal, nanoparticles will be targeted and delivered to the tumor draining lymph nodes together with R848, and will be effectively absorbed by DC cells. This in situ tumor vaccine with the ability of targeted delivery of lymph nodes can trigger a strong anti-tumor immune response. This therapy can completely inhibit distant tumors in some mice and produce a long-term immune memory effect on NSCLC metastasis. Conclusions: We propose a simple personalized in situ cancervaccine strategy, which uses nanotechnology to remove tumors in situ and release antigens. Antigens and immune adjuvants are simultaneously delivered to the draining lymph nodes to initiate the antigen presentation function of DC cells, resulting in an anti-tumor immune response to inhibit tumor recurrence and metastasis. The nano-vaccine based on powerful therapeutic effect provides an attractive technology for in situ programming of personalized cancervaccine and an effective strategy for the development of cancervaccine.

Transforming the Stability, Encapsulation, and Sustained Release Properties of Calcium Alginate Beads through Gel-Confined Coacervation.

Calcium alginate (Ca2+/alginate) gel beads find use in diverse applications, ranging from drug delivery and tissue engineering to bioprocessing, food formulation, and agriculture. Unless modified, however, these gels have limited stability in alkaline media (including phosphate buffers), and their high solute permeability limits their ability to efficiently encapsulate and slowly release water-soluble small molecules. Here, we show how these limitations can be addressed by mixing the alginate solutions used in the bead preparation with the nontoxic anionic polymer polyphosphate (PP). Upon complexing Ca2+ ions, PP undergoes complex coacervation (i.e., liquid/liquid phase separation into a Ca2+/PP-rich coacervate phase and a dilute supernatant phase). At lower PP concentrations, the Ca2+/PP coacervate appears to simply remain dispersed within the beads. Though its presence makes the beads more stable in alkaline media (phosphate-buffered saline and seawater), it has little impact on the bead stiffness, morphology, and (at least in the absence of substantial payload/coacervate association) encapsulation and release properties. When the PP concentrations exceed a critical value, however, Ca2+/PP coacervation within the gelling Ca2+/alginate beads collapses the resulting beads into more compact, interpenetrating polymer networks. Besides their enhanced stability to alkaline environments, these hybrid beads exhibit irregular morphologies with wrinkled and dimpled surface structures and macroscopic (closed) internal pores, and their collapse into these polymer-rich networks also makes them significantly stiffer than their PP-free counterparts. Crucially, these beads also exhibit a much lower solute permeability, which enables highly efficient encapsulation and multiday release of water-soluble small molecules (with the beads encapsulating >90% of the added model payload and sustaining its release over 3-5 d). Collectively, these findings provide a mild and simple (single-step) pathway to generating ionically cross-linked alginate beads with significantly enhanced stability, encapsulation efficiency, and sustained release.

Ppx1 putative exopolyphosphatase is essential for polyphosphate accumulation in Lacticaseibacillus paracasei.

The linear polymer polyphosphate (poly-P) is present across all three domains of life and serves diverse physiological functions. The enzyme polyphosphate kinase (Ppk) is responsible for poly-P synthesis, whereas poly-P degradation is carried out by the enzyme exopolyphosphatase (Ppx). In many Lactobacillaceae, the Ppk-encoding gene (ppk) is found clustered together with two genes encoding putative exopolyphosphatases (ppx1 and ppx2) each having different domain compositions, with the gene order ppx1-ppk-ppx2. However, the specific function of these ppx genes remains unexplored. An in-frame deletion of ppx1 in Lacticaseibacillus paracasei BL23 resulted in bacteria unable to accumulate poly-P, whereas the disruption of ppx2 did not affect poly-P synthesis. The expression of ppk was not altered in the Δppx1 strain, and poly-P synthesis in this strain was only restored by expressing ppx1 in trans. Moreover, no poly-P synthesis was observed when ppk was expressed from a plasmid in the Δppx1 strain. Purified Ppx2 exhibited in vitro exopolyphosphatase activity, whereas no in vitro enzymatic activity could be demonstrated for Ppx1. This observation corresponds with the absence in Ppx1 of conserved motifs essential for catalysis found in characterized exopolyphosphatases. Furthermore, assays with purified Ppk and Ppx1 evidenced that Ppx1 enhanced Ppk activity. These results demonstrate that Ppx1 is essential for poly-P synthesis in Lc. paracasei and have unveiled, for the first time, an unexpected role of Ppx1 exopolyphosphatase in poly-P synthesis.IMPORTANCEPoly-P is a pivotal molecular player in bacteria, participating in a diverse array of processes ranging from stress resilience to pathogenesis while also serving as a functional component in probiotic bacteria. The synthesis of poly-P is tightly regulated, but the underlying mechanisms remain incompletely elucidated. Our study sheds light on the distinctive role played by the two exopolyphosphatases (Ppx) found in the Lactobacillaceae bacterial group, of relevance in food and health. This particular group is noteworthy for possessing two Ppx enzymes, supposedly involved in poly-P degradation. Remarkably, our investigation uncovers an unprecedented function of Ppx1 in Lacticaseibacillus paracasei, where its absence leads to the total cessation of poly-P synthesis, paralleling the impact observed upon eliminating the poly-P forming enzyme, poly-P kinase. Unlike the anticipated role as a conventional exopolyphosphatase, Ppx1 demonstrates an unexpected function. Our results added a layer of complexity to our understanding of poly-P dynamics in bacteria.

Polyphosphate-loaded silk fibroin membrane as hemostatic agent in oral surgery: a pilot study

PurposePost-interventional hemorrhage can result in serious complications, especially in patients with hemostatic disorders. Identification of safe and efficient local hemostatic agents is important, particularly in the context of an ageing society and the emergence of new oral anticoagulants. The aim of this in vitro study was to investigate the potential of silk fibroin membranes coated with the inorganic polymer polyphosphate (polyP) as a novel hemostatic device in oral surgery.MethodsCocoons of the silkworm Bombyx mori were degummed and dissolved. Varying amounts of long-chain polyP (2–2000 µg/mm2) were adsorbed to the surface of silk fibroin membranes. Analysis of the procoagulant effect of polyP-coated silk membranes was performed using real-time thrombin generation assays in human plasma. Increasing concentrations of polyP (0.15–500 µg/ml) served as a positive control, while uncoated silk fibroin membranes were used as negative control.ResultsPolyP-coated silk fibroin membranes triggered coagulation when compared to plasma samples and pure silk fibroin membranes. A polyP-dose-dependent effect of thrombin generation could be found with a maximum (ETP = 1525.7 nM⋅min, peak thrombin = 310.1 nM, time to peak = 9.8 min, lag time = 7.6 min.) at 200 µg/mm2 of polymer loading on the silk fibroin membrane surface.ConclusionsIn this study, it was demonstrated that silk fibroin membranes coated with polyP have the potential to act as a promising novel hemostatic device.Graphical

Hexametaphosphate, a Common Food Additive, Aggregated the Hen Egg White Lysozyme.

Polyphosphate polymers are chains of phosphate monomers chemically bonded together via phosphoanhydride bonds. They are found in all prokaryotic and eukaryotic organisms and are among the earliest, most anionic, and most mysterious molecules known. They are everywhere, from small cellular components to additives in our food. There is a strong association between hyperphosphatemia and mortality. That is why it is crucial to assess how polyphosphates, as food additives, affect the quality of edible proteins. This study investigated the effect of inexpensive and widely used food additives (hexametaphosphate labeled as E452) on bakery items, meat products, fish, and soft drinks. Using various spectroscopic and microscopic techniques, we examined how hexametaphosphate affected the aggregation propensity, structure, and stability of a commonly used food protein: hen egg white lysozyme (HEWL). The solubility of HEWL is affected in a bimodal fashion by the concentration of hexametaphosphate. The bimodal concentration-dependent effect was also observed in the tertiary and secondary structural changes. Hexametaphosphate-induced HEWL aggregates were amorphous, as evidenced by ThT fluorescence, far-UV CD, and TEM imaging. This study showed that the food additive (hexametaphosphate) may denature and aggregate proteins and may lead to undesirable health issues.

The physiological polyphosphate as a healing biomaterial for chronic wounds: Crucial roles of its antibacterial and unique metabolic energy supplying properties

• Wound healing, especially the healing of chronic wounds, requires an increased demand for metabolic energy (ATP). • The physiological polymer polyphosphate (polyP) is an efficient source for the generation of ATP during wound regeneration. • In addition, polyP undergoes coacervation, a process during which bacteria are killed. • These two pillars are the basis for the successful regeneration of chronic wounds in humans. Insufficient metabolic energy, in the form of adenosine triphosphate (ATP), and bacterial infections are among the main causes for the development of chronic wounds. Previously we showed that the physiological inorganic polymer polyphosphate (polyP) massively accelerates wound healing both in animals (diabetic mice) and, when incorporated into mats, in patients with chronic wounds. Here, we focused on a hydrogel-based gel formulation, supplemented with both soluble sodium polyP (Na-polyP) and amorphous calcium polyP nanoparticles (Ca-polyP-NP). Exposure of human epidermal keratinocytes to the gel caused a significant increase in extracellular ATP level, an effect that was even enhanced when Na-polyP was combined with Ca-polyP-NP. Furthermore, it is shown that the added polyP in the gel is converted into a coacervate, leading to encapsulation and killing of bacteria. The data on human chronic wounds showed that the administration of hydrogel leads to the complete closure of these wounds. Histological analysis of biopsies showed an increased granulation of the wounds and an enhanced microvessel formation. The results indicate that the polyP hydrogel, due to its properties to entrap bacteria and generate metabolic energy, is a very promising formulation for a new therapy for chronic wounds.

Molecular and biochemical approach for understanding the transition of amorphous to crystalline calcium phosphate deposits in human teeth.

Calcium phosphate (CaP) deposition during bone mineralization starts with the aggregation of Posner's clusters Ca9(PO4)6 into amorphous Ca-phosphate (ACP), which then transforms into crystalline CaP and finally maturates to hydroxyapatite (HA). Using dentin/enamel of human teeth as a model system, we show that the physiological inorganic polymer polyphosphate (polyP), a phosphate donor in mineralization, prevents the transition from amorphous to crystalline CaP at concentrations>15wt%. Stabilization of the amorphous phase of CaP by polyP is reversed by hydrolysis of the polymer by alkaline phosphatase (ALP), an enzyme that releases phosphate for mineralization. It is still present in calcified enamel and dentin, as shown here by immunostaining and enzyme activity measurements. The phase transfer into crystalline CaP can be prevented by the ALP inhibitor levamisole. Besides TEM and SEM, the modulating effects of polyP and ALP on the kinetics of the phase transition from amorphous to crystalline CaP are demonstrated and confirmed by XRD and FTIR analyses. Molecular modeling studies show that the polyP chains, due to their dimensions, are able to penetrate into the channels between the Posner molecules, preventing cluster association to ACP and impairing HA crystal formation.

Patterning of the Autotrophic, Mixotrophic, and Heterotrophic Proteomes of Oxygen-Evolving Cyanobacterium Synechocystis sp. PCC 6803.

Proteomes of an oxygenic photosynthetic cyanobacterium, Synechocystis sp. PCC 6803, were analyzed under photoautotrophic (low and high CO2, assigned as ATLC and ATHC), photomixotrophic (MT), and light-activated heterotrophic (LAH) conditions. Allocation of proteome mass fraction to seven sub-proteomes and differential expression of individual proteins were analyzed, paying particular attention to photosynthesis and carbon metabolism–centered sub-proteomes affected by the quality and quantity of the carbon source and light regime upon growth. A distinct common feature of the ATHC, MT, and LAH cultures was low abundance of inducible carbon-concentrating mechanisms and photorespiration-related enzymes, independent of the inorganic or organic carbon source. On the other hand, these cells accumulated a respiratory NAD(P)H dehydrogenase I (NDH-11) complex in the thylakoid membrane (TM). Additionally, in glucose-supplemented cultures, a distinct NDH-2 protein, NdbA, accumulated in the TM, while the plasma membrane-localized NdbC and terminal oxidase decreased in abundance in comparison to both AT conditions. Photosynthetic complexes were uniquely depleted under the LAH condition but accumulated under the ATHC condition. The MT proteome displayed several heterotrophic features typical of the LAH proteome, particularly including the high abundance of ribosome as well as amino acid and protein biosynthesis machinery-related components. It is also noteworthy that the two equally light-exposed ATHC and MT cultures allocated similar mass fractions of the total proteome to the seven distinct sub-proteomes. Unique trophic condition-specific expression patterns were likewise observed among individual proteins, including the accumulation of phosphate transporters and polyphosphate polymers storing energy surplus in highly energetic bonds under the MT condition and accumulation under the LAH condition of an enzyme catalyzing cyanophycin biosynthesis. It is concluded that the rigor of cell growth in the MT condition results, to a great extent, by combining photosynthetic activity with high intracellular inorganic carbon conditions created upon glucose breakdown and release of CO2, besides the direct utilization of glucose-derived carbon skeletons for growth. This combination provides the MT cultures with excellent conditions for growth that often exceeds that of mere ATHC.

Effects of Polyphosphate on Leukocyte Function.

Leukocytes are immune cells derived from hematopoietic stem cells of the bone marrow which play essential roles in inflammatory and immune responses. In contrast to anucleate platelets and erythrocytes, leukocytes are differentiated from other blood cells by the presence of a nucleus, and consist of monocytes, neutrophils, lymphocytes, basophils, and eosinophils. Factors released from platelets mediate immune responses in part by recruitment and regulation of leukocyte activity. Platelet dense granules contain the highly anionic polymer polyphosphate (polyP) with monomer chain lengths of approximately 60-100 phosphates long, which are released into the microenvironment upon platelet activation. Recent studies suggest that polyP released from platelets plays roles in leukocyte migration, recruitment, accumulation, differentiation, and activation. Furthermore, bacterial-derived polyphosphate, generally consisting of phosphate monomer lengths in the hundreds to thousands, appear to play a role in pathogenic evasion of the host immune response. This review will discuss the effects of host and pathogenic-derived polyphosphate on leukocyte function.

Regulation of inorganic polyphosphate is required for proper vacuolar proteolysis in fission yeast

Regulation of cellular proliferation and quiescence is a central issue in biology that has been studied using model unicellular eukaryotes, such as the fission yeast Schizosaccharomyces pombe. We previously reported that the ubiquitin/proteasome pathway and autophagy are essential to maintain quiescence induced by nitrogen deprivation in S. pombe; however, specific ubiquitin ligases that maintain quiescence are not fully understood. Here we investigated the SPX-RING-type ubiquitin ligase Pqr1, identified as required for quiescence in a genetic screen. Pqr1 is found to be crucial for vacuolar proteolysis, the final step of autophagy, through proper regulation of phosphate and its polymer polyphosphate. Pqr1 restricts phosphate uptake into the cell through ubiquitination and subsequent degradation of phosphate transporters on plasma membranes. We hypothesized that Pqr1 may act as the central regulator for phosphate control in S. pombe, through the function of the SPX domain involved in phosphate sensing. Deletion of pqr1+ resulted in hyperaccumulation of intracellular phosphate and polyphosphate and in improper autophagy-dependent proteolysis under conditions of nitrogen starvation. Polyphosphate hyperaccumulation in pqr1+-deficient cells was mediated by the polyphosphate synthase VTC complex in vacuoles. Simultaneous deletion of VTC complex subunits rescued Pqr1 mutant phenotypes, including defects in proteolysis and loss of viability during quiescence. We conclude that excess polyphosphate may interfere with proteolysis in vacuoles by mechanisms that as yet remain unknown. The present results demonstrate a connection between polyphosphate metabolism and vacuolar functions for proper autophagy-dependent proteolysis, and we propose that polyphosphate homeostasis contributes to maintenance of cellular viability during quiescence.

Dynamic Polyphosphate Metabolism Coordinating with Manganese Ions Defends against Oxidative Stress in the Extreme Bacterium Deinococcus radiodurans.

Deinococcus radiodurans is an extreme bacterium with unparalleled resistance to oxidative stresses. Accumulation of intracellular Mn2+ complexing with small metabolites is the key contributor to the tolerance of D. radiodurans against oxidative stress. However, the intracellular reservoir of Mn ions and homeostatic regulation of the Mn complex in D. radiodurans remain unclear. We identified an evolutionarily ancient and negatively charged phosphate polymer (polyphosphate [PolyP]) in D. radiodurans We investigated PolyP metabolism in the response of D. radiodurans to oxidative stress. The genes dr1939, encoding polyphosphatase kinase (PPKDr; the subscript "Dr" refers to D. radiodurans), and dra0185, encoding exopolyphosphatase (PPXDr), were identified. PPXDr is a novel exopolyphosphatase with a cofactor preference to Mn2+, which enhances the dimerization and activity of PPXDr to allow the effective cleavage of PolyP-Mn. PPKDr and PPXDr exhibited different dynamic expression profiles under oxidative stress. First, ppkDr was upregulated leading to the accumulation of PolyP, which chelated large amounts of intracellular Mn ions. Subsequently, the expression level of ppkDr decreased while ppxDr was substantially upregulated and effectively hydrolyzed inactive PolyP-Mn to release phosphate (Pi) and Mn2+, which could form into Mn-Pi complexes to scavenge O2- and protect proteins from oxidative damage. Hence, dynamic cellular PolyP metabolites complexed with free Mn ions highlight a defense strategy of D. radiodurans in response to oxidative stress.IMPORTANCE The Mn-phosphate complex (Mn-Pi) plays a key role in the cellular resistance of radioresistant bacteria. The evolutionarily ancient polyphosphate polymers (polyphosphate [PolyP]) could effectively chelate Mn2+ and donate phosphates. However, the intracellular reservoir of Mn ions and homeostatic regulation of the Mn-Pi complex remain unclear. Here, we investigated the relationship of PolyP metabolites and Mn2+ homeostasis and how they function to defend against oxidative stress in the radioresistant bacterium Deinococcus radiodurans We found that PPXDr (the subscript "Dr" refers to D. radiodurans) is a novel exopolyphosphatase with a cofactor preference for Mn2+, mediating PolyP-Mn degradation into Pi and Mn ions. The formed Mn-Pi complexes effectively protect proteins. The dynamic PolyP metabolism coordinating with Mn ions is a defense strategy of D. radiodurans in response to oxidative stress. The findings not only provide new insights into the resistance mechanism of the extreme bacterium D. radiodurans but also broaden our understanding of the functions of PolyP metabolism in organisms.

Nanoparticle-directed and ionically forced polyphosphate coacervation: a versatile and reversible core\u2013shell system for drug delivery

A drug encapsulation/delivery system using a novel principle is described that is based on an intra-particle migration of calcium ions between a central Ca2+-enriched nanoparticle core and the surrounding shell compartment. The supply of Ca2+ is needed for the formation of a coacervate shell around the nanoparticles, acting as the core of drug-loadable core–shell particles, using the physiological inorganic polymer polyphosphate (polyP). This polyanion has the unique property to form, at an alkaline pH and in the presence of a stoichiometric surplus of calcium ions, water-insoluble and stabile amorphous nanoparticles. At neutral pH a coacervate, the biologically active form of the polymer, is obtained that is composed of polyP and Ca2+. The drug-loaded core–shell particles, built from the Ca–polyP core and the surrounding Ca–polyP shell, were fabricated in two successive steps. First, the formation of the nanoparticle core at pH 10 and a superstoichiometric 2:1 molar ratio between CaCl2 and Na–polyP into which dexamethasone, as a phosphate derivative, was incorporated. Second, the preparation of the coacervate shell, loaded with ascorbic acid, by exposure of the Ca–polyP core to soluble Na–polyP and L-ascorbate (calcium salt). EDX analysis revealed that during this step the Ca2+ ions required for coacervate formation migrate from the Ca–polyP core (with a high Ca:P ratio) to the shell. Electron microscopy of the particles show an electron-dense 150–200 nm sized core surrounded by a less sharply delimited electron-sparse shell. The core–shell particles exhibited strong osteogenic activity in vitro, based on the combined action of polyP and of dexamethasone and ascorbic acid, which reversibly bind to the anionic polyP via ionic Ca2+ bonds. Drug release from the particles occurs after contact with a peptide/protein-containing serum, a process which is almost complete after 10 days and accompanied by the conversion of the nanoparticles into a coacervate. Human osteosarcoma SaOS-2 cells cultivated onto or within an alginate hydrogel matrix showed increased growth/viability and mineralization when the hybrid particles containing dexamethasone and ascorbic acid were embedded in the matrix. The polyP-based core–shell particles have the potential to become a suitable, pH-responsive drug encapsulation/release system, especially for bone, cartilage and wound healing.

The biomaterial polyphosphate blocks stoichiometric binding of the SARS-CoV-2 S-protein to the cellular ACE2 receptor.

The effect of the polyanionic polymer of inorganic polyphosphate (polyP) involved in innate immunity on the binding of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein to the cellular ACE2 receptor was studied. The RBD surface comprises a basic amino acid stretch of four arginine residues which interact with the physiological polyP (polyP40) and polyP3. Subsequently, the interaction of RBD with ACE2 is sensitively inhibited. After the chemical modification of arginine, an increased inhibition by polyP, at a 1 : 1 molar ratio (polyP : RBP), is measured already at 0.1 μg mL-1. Heparin was ineffective. The results suggest a potential therapeutic benefit of polyP against SARS-CoV-2 infection.

Polyphosphate, Zn2+ and high molecular weight kininogen modulate individual reactions of the contact pathway of blood clotting

BackgroundInorganic polyphosphate modulates the contact pathway of blood clotting, which is implicated in thrombosis and inflammation. Polyphosphate polymer lengths are highly variable, with shorter polymers (approximately 60‐100 phosphates) secreted from human platelets, and longer polymers (up to thousands of phosphates) in microbes. We previously reported that optimal triggering of clotting via the contact pathway requires very long polyphosphates, although the impact of shorter polyphosphate polymers on individual proteolytic reactions of the contact pathway was not interrogated. Objectives and methodsWe conducted in vitro measurements of enzyme kinetics to investigate the ability of varying polyphosphate sizes, together with high molecular weight kininogen and Zn2+, to mediate four individual proteolytic reactions of the contact pathway: factor XII autoactivation, factor XII activation by kallikrein, prekallikrein activation by factor XIIa, and prekallikrein autoactivation. ResultsThe individual contact pathway reactions were differentially dependent on polyphosphate length. Very long‐chain polyphosphate was required to support factor XII autoactivation, whereas platelet‐size polyphosphate significantly accelerated the activation of factor XII by kallikrein, and the activation of prekallikrein by factor XIIa. Intriguingly, polyphosphate did not support prekallikrein autoactivation. We also report that high molecular weight kininogen was required only when kallikrein was the enzyme (ie, FXII activation by kallikrein), whereas Zn2+ was required only when FXII was the substrate (ie, FXII activation by either kallikrein or FXIIa). Activation of prekallikrein by FXIIa required neither Zn2+ nor high molecular weight kininogen. ConclusionsPlatelet polyphosphate and Zn2+ can promote subsets of the reactions of the contact pathway, with implications for a variety of disease states.

A fully degradable and photocrosslinked polysaccharide-polyphosphate hydrogel for tissue engineering

Extracellular matrix degradability meditates cell behaviors and gains increasing importance in the development of implantation materials for tissue engineering. Here, we developed a fully biodegradable hydrogel combining the unique features of synthetic polyphosphate polymer and natural polysaccharide polymer. Polyphosphate copolymer poly(butynyl phospholane)-random-poly(ethylethylene phosphate) (PBYP-r-PEEP) bearing pendent alkynes was synthesized through a facile one-pot reaction. Subsequently, thiol-yne “click” reaction was employed to fabricate the fully degradable and photocrosslinked hydrogel by mixing PBYP-r-PEEP with thiolated biodegradable hyaluronic acid (HA-SH). The generated HA/PPE hydrogels show viscoelastic properties and enzymatic biodegradability, supporting the growth of human mesenchymal stem cells (hMSCs). HA/PPE hydrogel is permissive to the covalent conjugation of cell-adhesive peptide RGD, which can enhance the cell-cell interactions. This HA/PPE hydrogel system provides a fully biodegradable platform that can support hMSCs growth and facilitate the formation of cell clustering, expanding the range of fully degradable materials for tissue engineering and regenerative medicine.

Determination of individual chain length and chain-length distribution of polyphosphates in microalgae by 31P-DOSY-NMR

Due to its ecological and biotechnological relevance, polyphosphate in microalgae is currently the focus of intense research. Numerous biological functions are performed by or dependent on polyphosphate, and they depend, among other factors, on its chain length. Chain length determination is important for understanding polyphosphate metabolism and for maximizing intracellular polyphosphate abundance per unit weight of biomass. 31P-DOSY NMR virtually separates various polyphosphate polymers in a mixture based on different translational diffusion coefficients. The diffusion coefficient of a polyphosphate molecule correlates with its molecular weight, enabling determination of individual chain lengths. Moreover, the polydispersity index can also be uniquely determined by DOSY as a measure of the overall chain-length distribution of polyphosphates. By contrast, conventional 31P NMR is only able to estimate the average chain length of the entire polyphosphate pool. Therefore, DOSY provides the opportunity to deepen our insight into polyphosphate metabolism and dynamics in algal biomass.

Synthesis of Polyphosphate Polymer Employing the Bisphenol (BHBF) and the Dichloride of Phenylphosphonic (PPDC): Evaluation of the Thermal Characteristics

This work presents studies about the synthesis and thermal characterization of a new polymer synthetized from the chloride phenylphosphonic molecule containing. The interfacial polycondensation method was utilized for the synthesis at low temperature.

Transformation of Construction Cement to a Self-Healing Hybrid Binder.

A new biomimetic strategy to im prove the self-healing properties of Portland cement is presented that is based on the application of the biogenic inorganic polymer polyphosphate (polyP), which is used as a cement admixture. The data show that synthetic linear polyp, with an average chain length of 40, as well as natural long-chain polyP isolated from soil bacteria, has the ability to support self-healing of this construction material. Furthermore, polyP, used as a water-soluble Na-salt, is subject to Na+/Ca2+ exchange by the Ca2+ from the cement, resulting in the formation of a water-rich coacervate when added to the cement surface, especially to the surface of bacteria-containing cement/concrete samples. The addition of polyP in low concentrations (<1% on weight basis for the solids) not only accelerated the hardening of cement/concrete but also the healing of microcracks present in the material. The results suggest that long-chain polyP is a promising additive that increases the self-healing capacity of cement by mimicking a bacteria-mediated natural mechanism.

Polyphosphate in Lactobacillus and Its Link to Stress Tolerance and Probiotic Properties.

The synthesis of the inorganic polymer polyphosphate (poly-P) in bacteria has been linked to stress survival and to the capacity of some strains to sequester heavy metals. In addition, synthesis of poly-P by certain strains of probiotic lactobacilli has been evidenced as a probiotic mechanism due to the homeostatic properties of this compound at the intestinal epithelium. We analyzed the link between poly-P synthesis, stress response, and mercury toxicity/accumulation by comparing wild-type strains of Lactobacillus and their corresponding mutants devoid of poly-P synthesis capacity (defective in the poly-P kinase, ppk, gene). Results showed that resistance to salt (NaCl) and acidic (pH 4) stresses upon ppk mutation was affected in Lactobacillus casei, while no effect was observed in two different Lactobacillus plantarum strains. Inorganic [Hg(II)] and organic (CH3Hg) mercury toxicity was generally increased upon ppk mutation, but no influence was seen on the capacity to retain both mercurial forms by the bacteria. Notwithstanding, the culture supernatants of ppk-defective L. plantarum strains possessed a diminished capacity to induce HSP27 expression, a marker for cell protection, in cultured Caco-2 cells compared to wild-type strains. In summary, our results illustrate that the role of poly-P in stress tolerance can vary between strains and they reinforce the idea of probiotic-derived poly-P as a molecule that modulates host-signaling pathways. They also question the relevance of this polymer to the capacity to retain mercury of probiotics.

Silencing of RpATG6 impaired the yolk accumulation and the biogenesis of the yolk organelles in the insect vector R. prolixus.

In oviparous animals, the egg yolk is synthesized by the mother in a major metabolic challenge, where the different yolk components are secreted to the hemolymph and delivered to the oocytes mostly by endocytosis. The yolk macromolecules are then stored in a wide range of endocytic-originated vesicles which are collectively referred to as yolk organelles and occupy most of the mature oocytes cytoplasm. After fertilization, the contents of these organelles are degraded in a regulated manner to supply the embryo cells with fundamental molecules for de novo synthesis. Yolk accumulation and its regulated degradation are therefore crucial for successful development, however, most of the molecular mechanisms involved in the biogenesis, sorting and degradation of targeted yolk organelles are still poorly understood. ATG6 is part of two PI3P-kinase complexes that can regulate the recruitment of the endocytic or the autophagy machineries. Here, we investigate the role of RpATG6 in the endocytosis of the yolk macromolecules and in the biogenesis of the yolk organelles in the insect vector Rhodnius prolixus. We found that vitellogenic females express high levels of RpATG6 in the ovaries, when compared to the levels detected in the midgut and fat body. RNAi silencing of RpATG6 resulted in yolk proteins accumulated in the vitellogenic hemolymph, as a consequence of poor uptake by the oocytes. Accordingly, the silenced oocytes are unviable, white (contrasting to the control pink oocytes), smaller (62% of the control oocyte volume) and accumulate only 40% of the yolk proteins, 80% of the TAG and 50% of the polymer polyphosphate quantified in control oocytes. The cortex of silenced oocytes present atypical smaller vesicles indicating that the yolk organelles were not properly formed and/or sorted, which was supported by the lack of endocytic vesicles near the plasma membrane of silenced oocytes as seen by TEM. Altogether, we found that RpATG6 is central for the mechanisms of yolk accumulation, emerging as an important target for further investigations on oogenesis and, therefore, reproduction of this vector.