Synthesis, characterization of chitosan/tripolyphosphate nanoparticles loaded with 4-chloro-2-methylphenoxyacetate sodium salt and its herbicidal activity against Bidens pilosa L.

Herbicides are widely used to control weeds in agriculture filed, however, the excessive use of the conventional formulation causes harmful side effects on the environment. To relieve this problem, natural polymer nanoparticles as herbicide carrier were rapidly developed and applied in recent years. In the present study, chitosan/tripolyphosphate (CS/TPP) nanoparticles were synthesized as nanocarrier to load herbicide 4-chloro-2-methylphenoxyacetate sodium salt (MCPA-Na). The encapsulation efficiency (EE) of 51.32% was obtained through measuring indirectly by high performance liquid chromatography (HPLC). The free and MCPA-Na-loaded CS/TPP nanoparticles were characterized by using dynamic light scattering (DLS), zeta potential, Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). The encapsulation of MCPA-Na in CS/TPP nanoparticles resulted in the change of MCPA-Na release profile in different pH media and displayed effective sustained-release under neutral condition. The evaluation of herbicidal activity against Bidens pilosa L. showed that the efficacy enhancement of MCPA-Na was realized after encapsulation in CS/TPP nanoparticles. The proposed herbicide nanoformulation presented a good potential as a sustainable alternative for weed control in agriculture.

Diquafosol Improves Corneal Wound Healing by Inducing NGF Expression in an Experimental Dry Eye Model.

Dry eye disease (DED) is caused by inflammation and damage to the corneal surface due to tear film instability and hyperosmolarity. Various eye drops are used to treat this condition. Each eye drop has different properties and mechanisms of action, so the appropriate drug should be used according to clinical phenotypes. This study aims to compare the therapeutic mechanisms of cyclosporine A (CsA) and diquafosol tetrasodium (DQS). An experimental in vivo/in vitro model of DED using hyperosmolarity showed decreased cell viability, inhibited wound healing, and corneal damage compared to controls. Treatment with cyclosporine or diquafosol restored cell viability and wound healing and reduced corneal damage by hyperosmolarity. The expression of the inflammation-related genes il-1ß, il-1α, and il-6 was reduced by cyclosporine and diquafosol, and the expression of Tnf-α, c1q, and il-17a was reduced by cyclosporine. Increased apoptosis in the DED model was confirmed by increased Bax and decreased Bcl-2 and Bcl-xl expression, but treatment with cyclosporine or diquafosol resulted in decreased apoptosis. Diquafosol increased NGF expression and translocation into the extracellular space. DED has different damage patterns depending on the progression of the lesion. Thus, depending on the type of lesion, eye drops should be selected according to the therapeutic target, focusing on repairing cellular damage when cellular repair is needed or reducing inflammation when inflammation is high and cellular damage is severe.

Polyphosphate Plays a Significant Role in the Maturation of Spores in Myxococcus xanthus.

Myxococcus xanthus synthesizes polyphosphates (polyPs) with polyphosphate kinase 1 (Ppk1) and degrades short- and long-chain polyPs with the exopolyphosphatases, Ppx1 and Ppx2, respectively. M. xanthus polyP:AMP phosphotransferase (Pap) generates ADP from AMP and polyPs. Pap expression is induced by an elevation in intracellular polyP concentration. M. xanthus synthesized polyPs during the stationary phase; the ppk1 mutant died earlier than the wild-type strain after the stationary phase. In addition, M. xanthus cells cultured in phosphate-starved medium, H2O2-supplemented medium, or amino acid-deficient medium increased the intracellular polyP levels by six- to ninefold after 6 h of incubation. However, the growth of ppk1 and ppx2 mutants in phosphate-starved medium and H2O2-supplemented medium was not significantly different from that of wild-type strain, nor was there a significant difference in fruiting body formation and sporulation in starvation condition. During development, no difference was observed in the adenylate energy charge (AEC) values in the wild-type, ppk1 mutant, and pap mutant strains until the second day of development. However, after day 3, the ppk1 and pap mutants had a lower ADP ratio and a higher AMP ratio compared to wild-type strain, and as a result, the AEC values of these mutants were lower than those of the wild-type strain. Spores of ppk1 and pap mutants in the nutrient medium germinated later than those of the wild-type strain. These results suggested that polyPs produced during development may play an important role in cellular energy homeostasis of the spores by being used to convert AMP to ADP via Pap.

Multifunctional Cerium Oxide Nanozyme for Synergistic Dry Eye Disease Therapy.

Dry eye disease (DED) is a chronic multifactorial ocular surface disease mainly caused by the instability of tear film, characterized by a series of ocular discomforts and even visual disorders. Oxidative stress has been recognized as an upstream factor in DED development. Diquafosol sodium (DQS) is an agonist of the P2Y2 receptor to restore the integrity/stability of the tear film. With the ability to alternate between Ce3+ and Ce4+, cerium oxide nanozymes could scavenge overexpressed reactive oxygen species (ROS). Hence, a DQS-loaded cerium oxide nanozyme was designed to boost the synergistic treatment of DED. Cerium oxide with branched polyethylenimine-graft-poly(ethylene glycol) as nucleating agent and dispersant was fabricated followed with DQS immobilization via a dynamic phenylborate ester bond, obtaining the DQS-loaded cerium oxide nanozyme (defined as Ce@PBD). Because of the ability to mimic the cascade processes of superoxide dismutase and catalase, Ce@PBD could scavenge excessive accumulated ROS, showing strong antioxidant and anti-inflammatory properties. Meanwhile, the P2Y2 receptors in the conjunctival cells could be stimulated by DQS in Ce@PBD, which can relieve the incompleteness and instability of the tear film. The animal experiments demonstrated that Ce@PBD significantly restored the defect of the corneal epithelium and increased the number of goblet cells, with the promotion of tear secretion, which was the best among commercial DQS ophthalmic solutions.

Dentin erosive wear is reduced by fluoride varnishes containing nanosized sodium trimetaphosphate in vitro.

This study evaluated the effect of fluoride varnishes containing micrometric or nanosized sodium trimetaphosphate (TMP) on dentin erosive wear in vitro. Bovine root dentin blocks were selected by surface hardness and randomly divided into five experimental groups/varnishes (n = 20/group): placebo, 5% sodium fluoride (NaF); 5% NaF+5% micrometric TMP; 5% NaF+2.5% nanosized TMP; and 5% NaF+5% nanosized TMP. Half of the surface of all blocks received a single application of the assigned varnish, with subsequent immersion in artificial saliva for 6 h. Varnishes were then removed and the blocks were immersed in citric acid (90 s, 4×/day, 5 days). After each erosive cycle, ten blocks of each group were immersed in a placebo dentifrice for 15 s (ERO), while the other ten blocks were subjected to abrasion by brushing (ERO+ABR). Dentin erosive wear was assessed by profilometry. Data were submitted to 2-way ANOVA and to the Holm-Sidak test (p<0.05). Dentin erosive wear was significantly higher for ERO+ABR than for ERO for all varnishes. TMP-containing varnishes promoted superior effects against dentin erosive wear compared with 5% NaF alone; and 5% nanosized TMP led to the lowest wear among all varnishes. In conclusion, the addition of TMP to conventional fluoride varnish (i.e., varnish containing only NaF) enhanced its protective effects against bovine root dentin erosion and erosion+abrasion. Additionally, the use of 5% nanosized TMP led to superior effects in comparison to 5% micrometric TMP, both for erosion and erosion+abrasion in vitro.

Dissolved organic phosphorus bond-class utilization by Synechococcus.

Dissolved organic phosphorus (DOP) contains compounds with phosphoester, phosphoanhydride, and phosphorus-carbon bonds. While DOP holds significant nutritional value for marine microorganisms, the bioavailability of each bond-class to the widespread cyanobacterium Synechococcus remains largely unknown. This study evaluates bond-class specific DOP utilization by Synechococcus strains from open and coastal oceans. Both strains exhibited comparable growth rates when provided phosphate, a phosphoanhydride [3-polyphosphate and 45-polyphosphate], or a DOP compound with both phosphoanhydride and phosphoester bonds (adenosine 5'-triphosphate). Growth rates on phosphoesters [glucose-6-phosphate, adenosine 5'-monophosphate, bis(4-methylumbelliferyl) phosphate] were variable, and neither strain grew on selected phosphorus-carbon compounds. Both strains hydrolyzed 3-polyphosphate, then adenosine 5'-triphosphate, and lastly adenosine 5'-monophosphate, exhibiting preferential enzymatic hydrolysis of phosphoanhydride bonds. The strains' exoproteomes contained phosphorus hydrolases, which combined with enhanced cell-free hydrolysis of 3-polyphosphate and adenosine 5'-triphosphate under phosphate deficiency, suggests active mineralization of phosphoanhydride bonds by these exoproteins. Synechococcus alkaline phosphatases presented broad substrate specificities, including activity toward the phosphoanhydride 3-polyphosphate, with varying affinities between strains. Collectively, these findings underscore the potentially significant role of compounds with phosphoanhydride bonds in Synechococcus phosphorus nutrition and highlight varied growth and enzymatic responses to molecular diversity within DOP bond-classes, thereby expanding our understanding of microbially mediated DOP cycling in marine ecosystems.

Growth and no-growth boundary of Clostridium perfringens in cooked cured meats - A logistic analysis and development of critical control surfaces using a solid growth medium.

This study was conducted to evaluate the effect of sodium nitrite (NaNO2, 100-200 ppm), sodium erythorbate (SE, 0-547 ppm), sodium tripolyphosphate (STPP, 0-0.5 %), and sodium chloride (NaCl, 2-3 %) on growth of C. perfringens using a solid growth medium and to develop a growth/no-growth boundary (critical control surface, or CCS) to prevent its growth in cooked cured meat under the optimal temperature condition. Melted Shahidi Ferguson Perfringens (SFP) agar, inoculated with a 3-strain spore cocktail and mixed with NaNO2, SE, STPP, and NaCl according to a Box-Behnken response surface experimental design, was dispersed in 96-well microplates and incubated anaerobically in an incubator programmed to remain at 4 °C for 24 h, heat to 80 °C in 1.75 h, quickly (0.5 h) cool to 46 °C (optimum temperature), and then maintain at 46 °C overnight. The plates were examined optically and visually for colony formation. Any well free of growth was designated as no-growth. Logistic regression was used to analyze the growth probability (P) as affected by NaNO2, SE, STPP, and NaCl and define a CSS as meeting the criterion of P < 1/96. The results showed that STPP and the interactions of SE with NaNO2 and NaCl could reduce the growth probability of C. perfringens in SFP agar. The validation of CCS with ground beef showed an accuracy of 96.3 % for no growth of C. perfringens in the inoculated samples. The results of this study proved that cured meat can be formulated with proper combinations of NaNO2, SE, STPP, and NaCl to prevent the growth of C. perfringens even under the optimum temperature condition, thus preventing food poisoning caused by the growth of this microorganism.

"One for two" strategy to construct an organic-inorganic polymer colloid for flame-retardant modification of flax fabric and rigid polyurethane foam.

Polymeric materials such as fabric and foam have high flammability which limits their application in the field of fire protection. To this end, an organic-inorganic polymer colloid constructed from carboxymethyl chitosan and ammonium polyphosphate was used to improve the flame retardancy of flax fabric (FF) and rigid polyurethane foam (RPUF) based on a "one for two" strategy. The modification processes of FF and RPUF relied on pad-dry-cure method and UV-curing technology, respectively, and the modified FF and RPUF were severally designated as CMC/APP-FF and RFR-RPUF. Flame retardancy studies showed that CMC/APP-FF and RFR-RPUF exhibited limiting oxygen index values as high as 39.4 % and 42.6 %, respectively, and both achieved self-extinguishing behavior when external ignition source was removed. Thermogravimetric analysis and cone calorimetry test confirmed that CMC/APP-FF and RFR-RPUF had good charring ability and demonstrated reduced peak heat release rate values of 90.1 % and 10.8 %, respectively, distinct from before they were modified. In addition, condensed phase analysis showed that after burning, CMC/APP-FF became an integration char structure, whereas RFR-RPUF turned into a sandwiched char structure. In summary, the "one for two" strategy reported in this work provides a new insight into the economical fabrication of flame-retardant polymeric materials.

Quantification of Polyphosphate in Environmental Planktonic Samples Using a Novel Fluorescence Dye JC-D7.

Polyphosphate (polyP) is found in plankton of diverse aquatic ecosystems and is important for plankton ecology and biogeochemical cycling. However, our knowledge of polyP in aquatic environments is hindered by a lack of data due to the limitations of quantification methods. The estimate of polyP in model organisms using phenol-chloroform extraction followed by enzymatic hydrolysis is complicated and fails for environmental samples. The commonly used 4',6-diamidino-2-phenylindole (DAPI) fluorescence method for environmental studies, on the contrary, severely overestimates polyP due to interference. In this paper, we develop a plankton lysis buffer to extract polyP and a quantification method using a novel polyP-specific fluorescence dye JC-D7. We test the methods using cultured algae and bacteria, as well as natural samples from marine and freshwater environments. We show that our plankton lysis extracts polyP with high recovery while requiring substantially less time and effort. Subsequent polyP quantification using JC-D7 fluorescence overcomes the interference encountered by the DAPI method and provides an accurate measurement of polyP down to <0.5 µmol L-1. This novel method enables more accurate quantification of polyP in aquatic environments and will profoundly enhance our knowledge of polyP, plankton ecology, and biogeochemistry.

A mammalian model reveals inorganic polyphosphate channeling into the nucleolus and induction of a hyper-condensate state.

Inorganic polyphosphate (polyP) is a ubiquitous polymer that controls fundamental processes. To overcome the absence of a genetically tractable mammalian model, we developed an inducible mammalian cell line expressing Escherichia coli polyphosphate kinase 1 (EcPPK1). Inducing EcPPK1 expression prompted polyP synthesis, enabling validation of polyP analytical methods. Virtually all newly synthesized polyP accumulates within the nucleus, mainly in the nucleolus. The channeled polyP within the nucleolus results in the redistribution of its markers, leading to altered rRNA processing. Ultrastructural analysis reveals electron-dense polyP structures associated with a hyper-condensed nucleolus resulting from an exacerbation of the liquid-liquid phase separation (LLPS) phenomena controlling this membraneless organelle. The selective accumulation of polyP in the nucleoli could be interpreted as an amplification of polyP channeling to where its physiological function takes place. Indeed, quantitative analysis of several mammalian cell lines confirms that endogenous polyP accumulates within the nucleolus.

Evaluating the antibacterial effect of meropenem-loaded chitosan/sodium tripolyphosphate (TPP) nanoparticles on Acinetobacter baumannii isolated from hospitalized patients.

BACKGROUND: Acinetobacter baumannii is a health threat due to its antibiotic resistance. Herein, antibiotic susceptibility and its association with the Toxin-antitoxin (TA) system genes in A. baumannii clinical isolates from Iran were investigated. Next, we prepared meropenem-loaded chitosan nanoparticles (MP-CS) and investigated their antibacterial effects against meropenem-susceptible bacterial isolates. METHODS: Out of 240 clinical specimens, 60 A. baumannii isolates were assessed. Antibiotic resistance of the isolates against conventional antibiotics was determined alongside investigating the presence of three TA system genes (mazEF, relBE, and higBA). Chitosan nanoparticles were characterized in terms of size, zeta potential, encapsulation efficiency, and meropenem release activity. Their antibacterial effects were assessed using the well diffusion method, minimum inhibitory concentration (MIC), and colony-forming unit (CFU) counting. Their cytotoxic effects and biocompatibility index were determined via the MTT, LDH, and ROS formation assays. RESULTS: Ampicillin, ceftazidime, and colistin were the least effective, and amikacin and tobramycin were the most effective antibiotics. Out of the 60 isolates, 10 (16.7%), 5 (8.3%), and 45 (75%) were multidrug-resistant (MDR), extensively drug-resistant (XDR), and pandrug-resistant (PDR), respectively. TA system genes had no significant effect on antibiotic resistance. MP-CS nanoparticles demonstrated an average size of 191.5 and zeta potential of 27.3 mV alongside a maximum encapsulation efficiency of 88.32% and release rate of 69.57%. MP-CS nanoparticles mediated similar antibacterial effects, as compared with free meropenem, against the A. baumannii isolates with significantly lower levels of meropenem. MP-CS nanoparticles remarkably prevented A549 and NCI-H292 cell infection by the A. baumannii isolates alongside demonstrating a favorable biocompatibility index. CONCLUSION: Antibiotic-loaded nanoparticles should be further designed and investigated to increase their antibacterial effect against A. baumannii and assess their safety and applicability in vivo settings.

Integration of N- and P- elements in sodium alginate aerogels for efficient flame retardant and thermal insulating properties.

In the field of building energy conservation, the development of biodegradable biomass aerogels with excellent mechanical performance, flame retardancy and thermal insulation properties is of particular importance. Here, a directional freeze-drying method was used for fabricating composite sodium alginate (SA) aerogels containing functionalized ammonium polyphosphate (APP) flame retardant. In particular, APP was coated with melamine (MEL) and phytic acid (PA) by a supramolecular assembly process. Through optimizing the flame retardant addition, the SA-20 AMP sample exhibited excellent flame retardant and thermal insulation properties, with the limiting oxygen index of 38.2 % and the UL-94 rating of V-0. Such aerogels with anisotropic morphology demonstrated a low thermal conductivity of 0.0288 (W/m·K) in the radial direction (perpendicular to the lamellar structure). In addition, as-obtained aerogels displayed remarkable water stability and mechanical properties, indicating significant potential for practical applications.

Development of Polyphosphate/Nanokaolin-Modified Alginate Sponge by Gas-Foaming and Plasma Glow Discharge Methods for Ultrarapid Hemostasis in Noncompressible Bleeding.

Effective bleeding management strategies in uncontrollable and noncompressible massive hemorrhage are becoming important in both clinical and combat situations. Here, a novel approach was developed to create a superporous and highly absorbable hemostatic sponge through a facile chemical gas-foaming method by cross-linking long-chain polyphosphate along with nanokaolin and Ca2+ in an alginate structure to synergistically activate the coagulation pathway. Natural kaolin obtained from the Marand mine in East Azarbaijan was converted into pseudohexagonal-shaped kaolin nanoparticles (30 to 150 nm) using ball milling followed by a newly developed glow discharge plasma treatment method. The obtained ultralight sponges (>90% porosity) exhibit ultrarapid water/blood absorption capacity (∼4000%) and excellent shape memory, which effectively concentrates coagulation factors. The results of in vitro tests demonstrated that the proposed sponges exhibited enhanced blood clotting ability (BCI < 10%) and superior cohesion with red blood cells (∼100) and platelets (∼80%) compared to commercially available hemostatic products. The in vivo host response results exhibited biosafety with no systemic and significant local inflammatory response by hematological, pathological, and biochemical parameter assessments. In a rat femoral artery complete excision model, the application of alginate/k/polyp nanocomposite sponges resulted in a complete hemostasis time of 60 s by significant reduction of hemostasis time (∼6.7-8.3 fold) and blood loss (∼2-2.8-fold) compared to commercially available hemostatic agents (P < 0.001). In conclusion, distinct physical characteristics accompanied by unique chemical composition multifunctional sponges activate hemostasis synergistically by triggering the XII, XI, X, IX, V, and II factors and the contact pathway and have the ability of rapid hemostasis in noncompressible severe bleeding.

Monitoring Ammonium Polyphosphate (APP) Biodegradation by Acinetobacter nosocomialis D-3 Using DAPI.

Ammonium polyphosphate (APP), a pivotal constituent within environmentally friendly flame retardants, exhibits notable decomposition susceptibility and potentially engenders ecological peril. Consequently, monitoring the APP concentration to ensure product integrity and facilitate the efficacious management of wastewater from production processes is of great significance. A fluorescent assay was devised to swiftly discern APP utilizing 4',6'-diamino-2-phenylindole (DAPI). With increasing APP concentrations, DAPI undergoes intercalation within its structure, emitting pronounced fluorescence. Notably, the flame retardant JLS-PNA220-A, predominantly comprising APP, was employed as the test substrate. Establishing a linear relationship between fluorescence intensity (F-F0) and JLS-PNA220-A concentration yielded the equation y = 76.08x + 463.2 (R2 = 0.9992), with a LOD determined to be 0.853 mg/L. The method was used to assess the degradation capacity of APP-degrading bacteria. Strain D-3 was isolated, and subsequent analysis of its 16S DNA sequence classified it as belonging to the Acinetobacter genus. Acinetobacter nosocomialis D-3 demonstrated superior APP degradation capabilities under pH 7 at 37 °C, with degradation rates exceeding 85% over a four-day cultivation period. It underscores the sensitivity and efficacy of the proposed method for APP detection. Furthermore, Acinetobacter nosocomialis D-3 exhibits promising potential for remediation of residual APP through environmental biodegradation processes.

A comprehensive comparison of microbial communities between aerobic granular sludge and flocculent sludge for nutrient removal in full-scale wastewater treatment plants.

Understanding the microbial community structure of sludge is crucial for improving the design, operation and optimisation of full-scale wastewater treatment plants (WWTPs). This study aimed to have a comprehensive comparison of microbial communities between aerobic granular sludge and flocculent sludge from two full-scale sequential batch reactors-based WWTPs with nutrient removal for the first time. To better understand key functional bacteria such as polyphosphate accumulating bacteria (PAOs), competitive bacteria such as glycogen accumulating bacteria (GAOs) and nitrifying bacteria for both nitrogen and phosphorus removal, another two full-scale WWTPs with only carbon (C) removal and C and nitrogen (N) removal were compared too. It was found that the richness and diversity of the microbial population in sludge increased with pollutant removal from only C, C and N, to C,N, P removal. For C, N P removal, granule structure led to a more diverse and rich microbial community structure than flocculent structure. Although more abundant nitrifying bacteria were enriched in granular sludge than flocculent sludge, the abundance of total putative PAOs was equivalent. However, the most typical putative PAOs such as Tetrasphaera and Candidatus Accumulibacter seemed to be more correlated with biological phosphorus removal performance, which might be more proper to be used as an indication for P removal potential. The higher abundance of GAOs in flocculent sludge with better phosphorus removal performance might suggest that further investigation is needed to understand the functions of GAOs. In addition, the equivalent abundances of PAOs in the WWTPs with only C removal and with C, N, and P removal, respectively, indicate that many newly reported putative PAOs might not contribute to P removal. This study provides insight into the microbial communities and functional bacteria in aerobic granular sludge and flocculent sludge in full-scale SBRs, which can provide microbes-informed optimisation of reactor operation for better nutrient removal.

Comparing two mucin secretagogues for the treatment of dry eye disease: a prospective randomized crossover trial.

This study aimed to compare the clinical efficacy and investigate patients' preferences for two mucin secretagogues in the treatment of dry eye disease (DED). Thirty patients with DED were randomly treated with either 3% diquafosol or 2% rebamipide ophthalmic solution for 4 weeks, followed by an additional 4-week treatment using the other eye drop after a 2-week washout period. Objective and subjective assessments, including the corneal and conjunctival staining score, tear breakup time (TBUT), Schirmer 1 test, tear osmolarity, tear matrix metalloproteinase-9 (MMP-9), lipid layer thickness (LLT) and ocular surface disease index (OSDI), were performed at baseline, 4 weeks, 6 weeks, and 10 weeks. Patient preferences were assessed based on four categories (comfort, efficacy, convenience, willingness to continue) using a questionnaire and the overall subjective satisfaction score for each drug was obtained at the end of the trial. In total, 28 eyes from 28 patients were included in the analysis. Both diquafosol and rebamipide significantly improved the OSDI (p = 0.033 and 0.034, respectively), TBUT (p < 0.001 and 0.026, respectively), and corneal (p < 0.001 and 0.001, respectively) and conjunctival (p = 0.017 and 0.042, respectively) staining after 4 weeks of treatment. An increase in Schirmer test scores was observed only after rebamipide treatment (p = 0.007). No significant changes were detected in tear osmolarity, MMP-9, and LLT following both treatments. The patients' preference was slightly greater for diquafosol (46.4%) than rebamipide (36.7%), presumably due to rebamipide's bitter taste. The self-efficacy of both drugs and overall satisfaction scores were comparable. These findings indicate that two mucin secretagogues showed comparable effects in ameliorating symptoms and improving signs (TBUT, corneal and conjunctival staining) in patients with DED.

C-terminal Poly-histidine Tags Alter Escherichia coli Polyphosphate Kinase Activity and Susceptibility to Inhibition.

In Escherichia coli, many environmental stressors trigger polyphosphate (polyP) synthesis by polyphosphate kinase (PPK1), including heat, nutrient restriction, toxic compounds, and osmotic imbalances. PPK1 is essential for virulence in many pathogens and has been the target of multiple screens for small molecule inhibitors that might serve as new anti-virulence drugs. However, the mechanisms by which PPK1 activity and polyP synthesis are regulated are poorly understood. Our previous attempts to uncover PPK1 regulatory elements resulted in the discovery of PPK1* mutants, which accumulate more polyP in vivo, but do not produce more in vitro. In attempting to further characterize these mutant enzymes, we discovered that the most commonly-used PPK1 purification method - Ni-affinity chromatography using a C-terminal poly-histidine tag - altered intrinsic aspects of the PPK1 enzyme, including specific activity, oligomeric state, and kinetic values. We developed an alternative purification strategy using a C-terminal C-tag which did not have these effects. Using this strategy, we were able to demonstrate major differences in the in vitro response of PPK1 to 5-aminosalicylic acid, a known PPK1 inhibitor, and observed several key differences between the wild-type and PPK1* enzymes, including changes in oligomeric distribution, increased enzymatic activity, and increased resistance to both product (ADP) and substrate (ATP) inhibition, that help to explain their in vivo effects. Importantly, our results indicate that the C-terminal poly-histidine tag is inappropriate for purification of PPK1, and that any in vitro studies or inhibitor screens performed with such tags need to be reconsidered in that light.

Polyphosphate attachment to lysine repeats is a non-covalent protein modification.

Polyphosphate (polyP) is a chain of inorganic phosphate that is present in all domains of life and affects diverse cellular phenomena, ranging from blood clotting to cancer. A study by Azevedo et al. described a protein modification whereby polyP is attached to lysine residues within polyacidic serine and lysine (PASK) motifs via what the authors claimed to be covalent phosphoramidate bonding. This was based largely on the remarkable ability of the modification to survive extreme denaturing conditions. Our study demonstrates that lysine polyphosphorylation is non-covalent, based on its sensitivity to ionic strength and lysine protonation and absence of phosphoramidate bond formation, as analyzed via 31P NMR. Ionic interaction with lysine residues alone is sufficient for polyP modification, and we present a new list of non-PASK lysine repeat proteins that undergo polyP modification. This work clarifies the biochemistry of polyP-lysine modification, with important implications for both studying and modulating this phenomenon. This Matters Arising paper is in response to Azevedo et al. (2015), published in Molecular Cell. See also the Matters Arising Response by Azevedo et al. (2024), published in this issue.

On the covalent nature of lysine polyphosphorylation.

Post-translational modifications of proteins (PTMs) introduce an extra layer of complexity to cellular regulation. Although phosphorylation of serine, threonine, and tyrosine residues is well-known as PTMs, lysine is, in fact, the most heavily modified amino acid, with over 30 types of PTMs on lysine having been characterized. One of the most recently discovered PTMs on lysine residues is polyphosphorylation, which sees linear chains of inorganic polyphosphates (polyP) attached to lysine residues. The labile nature of phosphoramidate bonds raises the question of whether this modification is covalent in nature. Here, we used buffers with very high ionic strength, which would disrupt any non-covalent interactions, and confirmed that lysine polyphosphorylation occurs covalently on proteins containing PASK domains (polyacidic, serine-, and lysine-rich), such as the budding yeast protein nuclear signal recognition 1 (Nsr1) and the mammalian protein nucleolin. This Matters Arising Response paper addresses the Neville et al. (2024) Matters Arising paper, published concurrently in Molecular Cell.