Monitoring Intracellular IP6 with a Genetically Encoded Fluorescence Biosensor.

Inositol hexakisphosphate (IP6), a naturally occurring metabolite of inositol with specific functions in different organelles or tissues, participates in numerous physiological processes and plays a key role in mammalian metabolic regulation. However, current IP6 detection methods, i.e., high-performance liquid chromatography and gel electrophoresis, require sample destruction and lack spatiotemporal resolution. Here, we construct and characterize a genetically encoded fluorescence biosensor named HIPSer that enables ratiometric quantitative IP6 detection in HEK293T cells and subcellular compartments. We demonstrate that HIPSer has a high sensitivity and relative selectivity for IP6 in vitro. We also provide proof-of-concept evidence that HIPSer can monitor IP6 levels in real time in HEK293T cells and can be targeted for IP6 detection in the nucleus of HEK293T cells. Moreover, HIPSer could also detect changes in IP6 content induced by chemical inhibition of IP6-metabolizing enzymes in HEK293T cells. Thus, HIPSer achieves spatiotemporally precise detection of fluctuations in endogenous IP6 in live cells and provides a versatile tool for mechanistic investigations of inositol phosphate functions in metabolism and signaling.

Purification and biochemical characterization of phytase from Bacillus cereus isolated from gastrointestinal tract of African giant snail (Achatina fulica) / Purificación y caracterización bioquímica de fitasa de Bacillus cereus aislada del tracto gastrointestinal de caracol gigante africano (Achatina fulica)

Phytases are specialized enzymes meant for phytic acid degradation. They possess ability to prevent phytic acid indigestion, including its attendant environmental pollution. This study was aimed at investigating biochemical properties of purified phytase of B. cereus isolated from Achatina fulica. Phytase produced from Bacillus cereus that exhibited optimal phytate degrading-ability of all the bacteria isolated was purified in a three-step purification. The biochemical properties of the purified enzyme were also determined. The phytase homogeny of approximately 45 kDa exhibited 12.8-purification fold and 1.6% yield with optima phytate degrading efficiency and maximum stability at pH 7 and 50 °C. Remaining activity of 52 and 47% obtained between 60 and 70 °C after 2 h further established thermostability of the purified phytase. Mg2+ and Zn2+ enhanced phytate hydrolysis by the enzyme, while Na+ showed mild inhibition but Hg2+ severely inhibited the enzymatic activity. Km and Vmax were estimated to be 0.11 mM and 55.6 μmol/min/mL, displaying enzyme-high substrate affinity and catalytic efficiency, respectively. Phytase purified from Bacillus cereus, isolated from African giant snails, has shown excellent characteristics suitable for phytic acid hydrolysis and could be employed in industrial and biotechnological applications.(AU)

Purification and biochemical characterization of phytase from Bacillus cereus isolated from gastrointestinal tract of African giant snail (Achatina fulica).

Phytases are specialized enzymes meant for phytic acid degradation. They possess ability to prevent phytic acid indigestion, including its attendant environmental pollution. This study was aimed at investigating biochemical properties of purified phytase of B. cereus isolated from Achatina fulica. Phytase produced from Bacillus cereus that exhibited optimal phytate degrading-ability of all the bacteria isolated was purified in a three-step purification. The biochemical properties of the purified enzyme were also determined. The phytase homogeny of approximately 45 kDa exhibited 12.8-purification fold and 1.6% yield with optima phytate degrading efficiency and maximum stability at pH 7 and 50 °C. Remaining activity of 52 and 47% obtained between 60 and 70 °C after 2 h further established thermostability of the purified phytase. Mg2+ and Zn2+ enhanced phytate hydrolysis by the enzyme, while Na+ showed mild inhibition but Hg2+ severely inhibited the enzymatic activity. Km and Vmax were estimated to be 0.11 mM and 55.6 µmol/min/mL, displaying enzyme-high substrate affinity and catalytic efficiency, respectively. Phytase purified from Bacillus cereus, isolated from African giant snails, has shown excellent characteristics suitable for phytic acid hydrolysis and could be employed in industrial and biotechnological applications.

Construction of hydrophobic fire retardant coating on cotton fabric using a layer-by-layer spray coating method.

Multifunctional cotton fabric was prepared through a two-step layer-by-layer spray coating method, where the first layer of the coating comprising chitosan and ammonium phytate provided fire retardancy, and the second one with PDMS-ZnO composite imparted hydrophobicity to the fabric. A molecular dynamics (MD) simulation study was carried out to calculate interfacial adhesion of different components of the coating, based on which the sequencing of the coating layers was determined and used to prepare coated samples. The coated fabric demonstrated a significant improvement in fire retardancy through an increase in LOI from 18 % in control to 30 %, a reduction in char length from 30 cm to 7 cm, and a decrease in peak and total heat release rate values by 75 % and 33 %, respectively. The hydrophobicity of coated fabric was tested via water drop test where coated sample maintained a contact angle of 148° for up to 120 s, while the control sample showed 0°.

Adsorption mechanisms of inositol hexakisphosphate in the presence of phosphate at the amorphous aluminum oxyhydroxide-water interface.

Myo-inositol hexakisphosphate (myo-IHP) is one of the most common soil organic phosphorus (P) species in soil. Its retention in soil is often competed by phosphate, making bioavailability of P species difficult. In this study, the adsorption mechanism of myo-IHP at the amorphous aluminum (oxyhydr)oxide (AAH)-water interface was investigated at pH 6.5 in the presence of phosphate using batch adsorption experiments and solution 31P NMR spectroscopy. The ratio of [myo-IHP]i/[phosphate]i (Ri) was kept 0.33-3 while ligand addition was varied. In the absence of phosphate, myo-IHP forms inner-sphere surface complexes in AAH via P1,3, P2, P4,6, and P5 functional group coordination. When two ligands were simultaneously added, fewer P functional groups of myo-IHP coordinated to AAH and the surface complexes were altered with the coordination of mainly P1,3 and P2 functional groups. When phosphate was pre-adsorbed, myo-IHP adsorption decreased by 8.0-44% compared to the respective simultaneous addition system. P2 or P5 functional group was predominantly coordinated to the AAH surfaces at Ri = 0.33. Myo-IHP pre-adsorption resulted in an increase in the final myo-IHP adsorption compared to that in the simultaneous addition system under the respective Ri values (0.33-3). In this system, P1,3, P2, P4,6, and P5 functional groups were coordinated to form inner-sphere surface complexes regardless of Ri. The study revealed that the functional group specific adsorption mechanism of myo-IHP at the AAH-water interface was affected by addition sequence and Ri of two ligands. The competitive adsorption between organic P and phosphate plays an important role in the fate of P in soils.

Phytate-mediated phosphorylation of starch by dry heating with rice bran extract.

Rice and corn starches were subjected to dry heating with rice bran extract or sodium trimetaphosphate (STMP)/sodium tripolyphosphate (STPP) for starch phosphorylation. Phytate in rice bran extract or STMP/STPP increased the concentration of phosphorus in rice and maize starches. The highest concentrations of phosphorus were induced in rice starch with rice bran extract and in corn starch with STMP/STPP. 31P NMR analysis indicated that the rice bran extract and STMP/STPP produced monostarch monophosphate under the same reaction conditions. Rice and corn starches phosphorylated with rice bran extract or STMP/STPP demonstrated great peak viscosity and low pasting temperatures. Although starch phosphorylated with either rice bran extract or STMP/STPP showed higher paste clarity, solubility, and swelling power than native starch, these parameters were optimal in rice starch phosphorylated with rice bran extract. Therefore, dry heating with rice bran extract induced phytate-mediated phosphorylation with the typical physicochemical properties of starch phosphates.

Improving the color and functional properties of seabuckthorn seed protein with phytase treatment combined with alkaline solubilization and isoelectric precipitation.

BACKGROUND: Reducing anti-nutritional factors like phytates in seed protein products requires an ongoing effort. This study was the first to investigate the phytic acid content in seabuckthorn seed protein (SSP) and its reduction by an exogenous phytase during protein isolation from seabuckthorn seed meal through the common alkaline solubilization-isoelectric precipitation process. RESULTS: The additional phytase treatment could reduce the content of phytic acid from 22.46 to 13.27 g kg-1 , leading to SSP products with lighter color (lower ΔE* ), higher protein solubility, higher in vitro digestibility, but lower phenolic antioxidant content (including flavonoids and procyanidins) and some beneficial ions like Ca, Fe, Mg, and Zn. The Fourier transform infrared (FTIR) results indicated that the secondary structure of protein changed under the treatment with phytase. Correlation analysis showed that L* was significantly negatively correlated with TP, TPC and TF (P < 0.001), while a* and b* were significantly positively correlated with them (P < 0.001). CONCLUSIONS: There may be a trade-off between protein functionalities and other health-promoting components when a phytase treatment is included in SSP isolation. © 2021 Society of Chemical Industry.

Improving the flame retardancy and accelerating the degradation of poly (lactic acid) in soil by introducing fully bio-based additives.

In this study, a novel bio-based flame retardant LC-PA is prepared by the Mannich reaction between phytic acid (PA) and L-citrulline (LC). LC-PA is combined with tannic acid (TA) and introduced into PLA to improve fire performance and accelerate biodegradability. Compared with control PLA, the PLA composite containing 10% LC-PA/TA increases the LOI value to 26.9%, reaches a V-0 rating in the UL-94 test, and reduces the peak heat release rate and total heat release by 24.5% and 21.1%, respectively. More importantly, the introduction of LC-PA/TA accelerates the degradation rate of PLA in soil, which is of significance for biodegradable materials. The addition of LC-PA/TA can attract water and provide a suitable energy source for microbial proliferation, accelerating the hydrolysis and microbial degradation of PLA. This work provides a practical approach for high flame retardancy and rapid biodegradability in the soil to the bio-based polymer.

Secondary-structure switch regulates the substrate binding of a YopJ family acetyltransferase.

The Yersinia outer protein J (YopJ) family effectors are widely deployed through the type III secretion system by both plant and animal pathogens. As non-canonical acetyltransferases, the enzymatic activities of YopJ family effectors are allosterically activated by the eukaryote-specific ligand inositol hexaphosphate (InsP6). However, the underpinning molecular mechanism remains undefined. Here we present the crystal structure of apo-PopP2, a YopJ family member secreted by the plant pathogen Ralstonia solanacearum. Structural comparison of apo-PopP2 with the InsP6-bound PopP2 reveals a substantial conformational readjustment centered in the substrate-binding site. Combining biochemical and computational analyses, we further identify a mechanism by which the association of InsP6 with PopP2 induces an α-helix-to-ß-strand transition in the catalytic core, resulting in stabilization of the substrate recognition helix in the target protein binding site. Together, our study uncovers the molecular basis governing InsP6-mediated allosteric regulation of YopJ family acetyltransferases and further expands the paradigm of fold-switching proteins.

Using Biotinylated myo-Inositol Hexakisphosphate to Investigate Inositol Pyrophosphate-Protein Interactions with Surface-Based Biosensors.

Inositol pyrophosphates (PP-InsPs) are highly phosphorylated molecules that have emerged as central nutrient messengers in eukaryotic organisms. They can bind to structurally diverse target proteins to regulate biological functions, such as protein-protein interactions. PP-InsPs are strongly negatively charged and interact with highly basic surface patches in proteins, making their quantitative biochemical analysis challenging. Here, we present the synthesis of biotinylated myo-inositol hexakisphosphates and their application in surface plasmon resonance and grating-coupled interferometry assays, to enable the rapid identification, validation, and kinetic characterization of InsP- and PP-InsP-protein interactions.

A promising potential candidate for vascular replacement materials with anti-inflammatory action, good hemocompatibility and endotheliocyte-cytocompatibility: phytic acid-fixed amniotic membrane.

Due to its excellent biocompatibility and anti-inflammatory activity, amniotic membrane (AM) has attracted much attention from scholars. However, its clinical application in vascular reconstruction was limited for poor processability, rapid biodegradation, and insufficient hemocompatibility. A naturally extracted substance with good cytocompatibility, phytic acid (PA), which can quickly form strong and stable hydrogen bonds on the tissue surface, was used to crosslink decellularized AM (DAM) to prepare a novel vascular replacement material. The results showed that PA-fixed AM had excellent mechanical strength and resistance to enzymatic degradation as well as appropriate surface hydrophilicity. Among all samples, 2% PA-fixed specimen showed excellent human umbilical vein endothelial cells (HUVECs)-cytocompatibility and hemocompatibility. It could also stimulate the secretion of vascular endothelial growth factor and endothelin-1 from seeded HUVECs, indicating that PA might promote neovascularization after implantation of PA-fixed specimens. Also, 2% PA-fixed specimen could inhibit the secretion of tumor necrosis factor-αfrom co-cultured macrophages, thus might reduce the inflammatory response after sample implantation. Finally, the results ofex vivoblood test andin vivoexperiments confirmed our deduction that PA might promote neovascularization after implantation. All the results indicated that prepared PA-fixed DAM could be considered as a promising small-diameter vascular replacement material.

Flame Retardant Functionalization of Microcrystalline Cellulose by Phosphorylation Reaction with Phytic Acid.

The functionalization of microcrystalline cellulose (MCC) is an important strategy for broadening its application fields. In the present work, MCC was functionalized by phosphorylation reaction with phytic acid (PA) for enhanced flame retardancy. The conditions of phosphorylation reaction including PA concentration, MCC/PA weight ratio and temperature were discussed, and the thermal degradation, heat release and char-forming properties of the resulting PA modified MCC were studied by thermogravimetric analysis and pyrolysis combustion flow calorimetry. The PA modified MCC, which was prepared at 90 °C, 50%PA and 1:3 weight ratio of MCC to PA, exhibited early thermal dehydration with rapid char formation as well as low heat release capability. This work suggests a novel strategy for the phosphorylation of cellulose using PA and reveals that the PA phosphorylated MCC can act as a promising flame retardant material.

Pharmacokinetic properties of a novel formulation of S-adenosyl-L-methionine phytate.

S-adenosyl-L-methionine (SAM), the main endogenous methyl donor, is the adenosyl derivative of the amino acid methionine, which displays many important roles in cellular metabolism. It is widely used as a food supplement and in some countries is also marketed as a drug. Its interesting nutraceutical and pharmacological properties prompted us to evaluate the pharmacokinetics of a new form of SAM, the phytate salt. The product was administered orally to rats and pharmacokinetic parameters were evaluated by comparing the results with that obtained by administering the SAM tosylated form (SAM PTS). It was found that phytate anion protects SAM from degradation, probably because of steric hindrance exerted by the counterion, and that the SAM phytate displayed significant better pharmacokinetic parameters compared to SAM PTS. These results open to the perspective of the use of new salts of SAM endowed with better pharmacokinetic properties.

Alginate enriched with phytic acid for hydrogels preparation. Therapeutic applications.

In the last decade, numerous innovative strategies have been used to obtain highly efficient synthetic or semi-synthetic biomaterials. Between these innovative biomaterials, hydrogels occupy a distinct place due to their superior biological and physico-chemical characteristics. Alginate is a natural linear polysaccharide with important physico-chemical and biological properties. Recently, we obtained a new hydrogel based on alginate and phytic acid with improved physico-chemical properties. In the present study, the hydrogels previously obtained were tested in terms of their biological properties and possibilities of use in the biomedical field. For this purpose, the hydrogels were loaded with norfloxacin (NRF), an antibacterial compound utilised in the treatment against Gram-negative and Gram-positive organisms. Unfortunately, NRF has low solubility and permeability. In order to provide protection against loss, but also for enhanced bioavailability, and controlled-release of norfloxacin, a drug inclusion complex with cyclodextrin was realized. The effect of complexation on the release profile was highlighted. The addition of NRF to the hydrogel matrices greatly improved the antibacterial activity of the tested compounds. The presence of CD did not affect the homogeneity of the drug distribution. Changes in the polymeric matrix structure were registered after the incorporation of the drug, which were attributed to the relaxation of the network subsequently to the penetration and diffusion of the drug solution simultaneously with the swelling process. The release of NRF from Alg_PA polymeric network has been successfully modulated by the use of CD as a host molecule.

Zinc delivery system constructed from food-borne nanoparticles derived from Undaria pinnatifida.

Food-borne nanoparticles from Undaria pinnatifida (UPFNs) were prepared and successfully applied as nanocarriers for microelement zinc delivery. UPFNs were spherical nanoparticles with average sizes of about 4.07 ± 1.09 nm, which chelated with zinc ions through amino nitrogen and carboxyl oxygen atoms as characterized by X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, and 1H nuclear magnetic resonance spectroscopy. Thermodynamic analysis revealed that the overall chelation process between UPFNs and zinc ions was a spontaneous enthalpy-driven endothermic reaction. Compared to zinc sulfate, UPFN-Zn2+ showed higher solubility both in phytic acid solution and the process of gastrointestinal digestion. Meanwhile, no obvious cytotoxicity was found in UPFNs and UPFN-Zn2+. Specifically, UPFN-Zn2+ could successfully rescue cell viability, DNA replication activity and restore cell proliferation ability in zinc-deficient cells induced by a specific zinc chelator TPEN. Overall, UPFNs might serve as efficient, stable, and safe nanocarriers for zinc delivery.

Preparation of a superhydrophilic SiO2 nanoparticles coated chitosan-sodium phytate film by a simple ethanol soaking process.

The development of environmentally friendly and transparent superhydrophilic food packaging materials is essential in our daily lives. The objective of this study was to develop a simple method of preparing a superhydrophilic, transparent, and biodegradable composite film. The composite film was obtained by soaking a chitosan-sodium phytate film in an ethanol solution of SiO2 nanoparticles. The results showed that when the chitosan-sodium phytate film was coated with SiO2 nanoparticles that were dissolved in 75% ethanol, its water contact angle (WCA) was reduced from 100° to 3°, and the film surface was changed from a hydrophobic to a superhydrophilic. Furthermore, the oxygen transmission rate (OTR) was significantly reduced, and the mechanical properties of the film were improved. The method is easy to carry out and can be used for the potential production of superhydrophilic materials.

Role of metal complexation on the solubility and enzymatic hydrolysis of phytate.

Phytate is a dominant form of organic phosphorus (P) in the environment. Complexation and precipitation with polyvalent metal ions can stabilize phytate, thereby significantly hinder the hydrolysis by enzymes. Here, we studied the stability and hydrolyzability of environmentally relevant metal phytate complexes (Na, Ca, Mg, Cu, Zn, Al, Fe, Al/Fe, Mn, and Cd) under different pHs, presence of metal chelators, and thermal conditions. Our results show that the order of solubility of metal phytate complexes is as follows: i) for metal species: Na, Ca, Mg > Cu, Zn, Mn, Cd > Al, Fe, ii) under different pHs: pH 5.0 > pH 7.5), and iii) in the presence of chelators: EDTA> citric acid. Phytate-metal complexes are mostly resistant towards acid hydrolysis (except Al-phytate), and dry complexes are generally stable at high pressure and temperature under autoclave conditions (except Ca phytate). Inhibition of metal complex towards enzymatic hydrolysis by Aspergillus niger phytase was variable but found to be highest in Fe phytate complex. Strong chelating agents such as EDTA are insufficient for releasing metals from the complexes unless the reduction of metals (such as Fe) occurs first. The insights gained from this research are expected to contribute to the current understanding of the fate of phytate in the presence of various metals that are commonly present in agricultural soils.

Role of ß-glucan content, molecular weight and phytate in the bile acid binding of oat ß-glucan.

There is controversy about the role of viscosity and co-migrating molecules on the bile acid binding of beta-glucan. Thus, this study aimed to investigate the impact of ß-glucan molecular weight and the content of both ß-glucan and phytate on the mobility of bile acids by modelling intestinal conditions in vitro. Two approaches were used to evaluate factors underlying this binding effect. The first studied bile acid binding capacity of soluble ß-glucan using purified compounds. Viscosity of the ß-glucan solution governed mainly the mobility of bile acid since both a decrease in ß-glucan concentration and degradation of ß-glucan by enzyme hydrolysis resulted in decreased binding. The second approach investigated the trapping of bile acids in the oat bran matrix. Results suggested trapping of bile acids by the ß-glucan gel network. Additionally, hydrolysis of phytate was shown to increase bile acid binding, probably due to better extractability of ß-glucan in this sample.

Molecular mechanism of N-terminal acetylation by the ternary NatC complex.

Protein N-terminal acetylation is predominantly a ribosome-associated modification, with NatA-E serving as the major enzymes. NatC is the most unusual of these enzymes, containing one Naa30 catalytic subunit and two auxiliary subunits, Naa35 and Naa38; and substrate selectivity profile that overlaps with NatE. Here, we report the cryoelectron microscopy structure of S. pombe NatC with a NatE/C-type bisubstrate analog and inositol hexaphosphate (IP6), and associated biochemistry studies. We find that the presence of three subunits is a prerequisite for normal NatC acetylation activity in yeast and that IP6 binds tightly to NatC to stabilize the complex. We also describe the molecular basis for IP6-mediated NatC complex stabilization and the overlapping yet distinct substrate profiles of NatC and NatE.

Alginate enriched with phytic acid for hydrogels preparation.

Alginate hydrogels are extremely versatile and flexible biomaterials, with an enormous potential for bio-applications use. Their similarity with extracellular matrix is a key factor in their performance for cell and tissue regeneration. In this study superabsorbent high porous hydrogels based on sodium alginate physical crosslinked with a natural crosslinker compound namely phytic acid were prepared and evaluated from the viewpoint of their specific properties. The resulting hydrogels obtained with different ratios between alginate and phytic acid were characterized by Fourier transform infrared spectroscopy technique, scanning electron microscopy, XRD measurements, swelling tests in physiological environment, and thermal analysis by using a simultaneous TG/FT-IR/MS system. There are put into evidence the differences in physico-chemical properties of the hydrogels in relation with their composition, which endows them tunable properties and versatility.