Morphological, Thermal, and Mechanical Assessment of Polypropylene and Ammonium Phosphate Composites Enhanced with Lignosulfonate and Zirconium.

Polypropylene and ammonium phosphate (AP) composites were synthesized at a 25 wt% concentration. The changes in the morphological, thermal, and physical behavior of the composites were analyzed with the addition of lignosulfonate (LG) and zirconium phosphate (ZrP). Additionally, metallic zirconium was deposited onto lignosulfonate using the magnetron sputtering technique to develop polypropylene and zirconium-modified lignosulfonate (LGMod) composites. Thus, composites of PP/25AP, PP/25AP/8LG/5ZrP, and PP/25AP/8LGMod were synthesized. The synthesis involved mixing the materials in a Hake mixer, followed by compression molding. The composites were characterized by field emission scanning electron microscopy (SEM-EDS), a thermogravimetric analysis (TGA) with combustion parameters, a vertical burn test (UL-94), a thermal camera, and mechanical properties. All composites achieved a V2 rating according to UL-94 standards. The PP/25AP extinguishes flames more quickly compared to other materials, approximately 99.2% faster than PP and showed the lowest temperature variation and mass loss after burning. The PP/25AP/8LG/5ZrP composite exhibited a 7% higher rigidity and 84.5% better flame retardancy compared to pure PP. Additionally, substituting ZrP with LGMod led to a lower environmental impact and improved thermal properties, despite some mechanical disadvantages.

Synthetic Bone Blocks Produced by Additive Manufacturing in the Repair of Critical Bone Defects.

This study evaluated the efficacy of synthetic bone blocks, composed of hydroxyapatite (HA) or ß-tricalcium phosphate (B-TCP), which were produced by additive manufacturing and used for the repair of critical size bone defects (CSDs) in rat calvaria. Sixty rats were divided into five groups (n = 12): blood clot (CONTROL), 3D-printed HA (HA), 3D-printed ß-TCP (B-TCP), 3D-printed HA + autologous micrograft (HA+RIG), and 3D-printed ß-TCP + autologous micrograft (B-TCP+RIG). CSDs were surgically created in the parietal bone and treated with the respective biomaterials. The animals were euthanized at 30 and 60 days postsurgery for microcomputed tomography (micro-CT) histomorphometric, and immunohistochemical analysis to assess new bone formation. Micro-CT analysis showed that both biomaterials were incorporated into the animals' calvaria. The HA+RIG group, especially at 60 days, exhibited a significant increase in bone formation compared with the control. The use of 3D-printed bioceramics resulted in thinner trabeculae but a higher number of trabeculae compared with the control. Histomorphometric analysis showed bone islands in close contact with the B-TCP and HA blocks at 30 days. The HA blocks (HA and HA+RIG groups) showed statistically higher new bone formation values with further improvement when autologous micrografts were included. Immunohistochemical analysis showed the expression of bone repair proteins. At 30 days, the HA+RIG group had moderate Osteopontin (OPN) staining, indicating that the repair process had started, whereas other groups showed no staining. At 60 days, the HA+RIG group showed slight staining, similar to that of the control. Osteocalcin (OCN) staining, indicating osteoblastic activity, showed moderate expression in the HA and HA+RIG groups at 30 days, with slight expression in the B-TCP and B-TCP+RIG groups. The combination of HA blocks with autologous micrografts significantly enhanced bone repair, suggesting that the presence of progenitor cells and growth factors in the micrografts contributed to the improved outcomes. It was concluded that 3D-printed bone substitute blocks, associated with autologous micrografts, are highly effective in promoting bone repair in CSDs in rat calvaria.

Effect of a Bacillus velezensis and Lysinibacillus fusiformis-based biofertilizer on phosphorus acquisition and grain yield of soybean.

Introduction: Phosphate-solubilizing bacteria that function through acidification (organic acid synthesis) or mineralization (production of enzymes such as phytase and phosphatases) have been explored as a biotechnological alternative to enhance plant access to phosphorus (P) retained in organic and inorganic forms in agricultural soils. This study tested the hypothesis that applying a biofertilizer composed of a recognized phosphate-solubilizing bacterium (Bacillus velezensis - endophytic strain BVPS01) and an underexplored plant growth-promoting bacterium (Lysinibacillus fusiformis - endophytic strain BVPS02) would improve the growth and grain yield of Glycine max L. plants. Methods: Initial in vitro tests assessed the functional traits of these bacteria, and a mix of strains BVPS01 and BVPS02 was produced and tested under field conditions to evaluate its agronomic efficiency. Results: The results confirmed the hypothesis that the tested biofertilizer enhances the agronomic performance of G. max plants in the field. The B. velezensis strain (BVPS01) was found to be more effective than the L. fusiformis strain (BVPS02) in solubilizing phosphates via the phosphatase enzyme production pathway, indicated by the expression of the phoC and phoD genes. In contrast, L. fusiformis was more effective in solubilizing phosphates through organic acid and phytase-related pathways, in addition to synthesizing indole-3-acetic acid and increasing the mitotic index in the root meristem of G. max plants. These strains exhibited biological compatibility, and the formulated product based on these rhizobacteria enhanced root development and increased the number of nodules and flowers, positively affecting 1000-grain weight, grain yield, and grain P content. Discussion: Thus, the tested biofertilizer demonstrated potential to improve root growth and increase both the yield and quality of soybean crops, making it a sustainable and low-cost strategy.

Strategies to Increase the Phosphorus Content in the Soil Profile of Vineyards Grown in Subtropical Climates.

Phosphate fertilizers are applied to the soil surface, especially in vineyards in production in subtropical regions. Nowadays, phosphorus (P) is not incorporated into the soil to avoid mechanical damage to the root system in orchards. However, over the years, successive surface P applications can increase the P content only in the topsoil, maintaining low P levels in the subsurface, which can reduce its use by grapevines. For this reason, there is a need to propose strategies to increase the P content in the soil profile of established orchards. The study aimed to evaluate the effect of management strategies to (i) increase the P content in the soil profile; (ii) enhance the grape production; and (iii) maintain the grape must composition. An experiment on the 'Pinot Noir' grape in full production was carried out over three crop seasons. The treatments were without P application (C), P on the soil surface without incorporation (SP), P incorporated at 20 cm (IP20), P incorporated at 40 cm (IP40), and twice the P dose incorporated at 40 cm (2IP40). The P concentration in leaves at flowering and veraison, P content in the soil, grape production and its components, and chemical parameters of the grape must (total soluble solids, total polyphenols, total titratable acidity, total anthocyanins, and pH) were evaluated. The P concentration in leaves did not differ among the P application modes. The application of P associated with soil mobilization, especially at 20 cm depth, increased grape production. The P application modes did not affect the values of the chemical parameters of the grape must except for the total anthocyanins, which had the highest values when the vines were subjected to 2IP40. Finally, the P application and incorporation into the soil profile was an efficient strategy for increasing the grape production in full production vineyards.

Comparison of the physical properties of glass ionomer modified with silver phosphate/hydroxyapatite or titanium dioxide nanoparticles: in vitro study.

Glass ionomer cements (GICs) are the common materials employed in pediatric dentistry because of their specific applications in class I restorations and atraumatic restoration treatments (ART) of deciduous teeth in populations at high risk of caries. Studies show a limited clinical durability of these materials. Attempts have thus been made to incorporate nanoparticles (NPs) into the glass ionomer for improving resistance and make it like the tooth structure. An in vitro experimental study was conducted using the required samples dimensions and prepared based on the test being carried out on the three groups with or without the modification of light-cured glass ionomer. Samples were grouped as follows: control group (G1_C), 2% silver phosphate/hydroxyapatite NPs group (G2_SPH), and 2% titanium dioxide NPs group (G3_TiO2). The physical tests regarding flexural strength (n = 10 per group), solubility (n = 10 per group), and radiopacity (n = 3 per group) were performed. The data were analyzed by Shapiro Wilks test, and one-way analysis of variance (one-way ANOVA), and multiple comparisons by post hoc Tukey's test. The p-value of < 0.05 was considered significant. No statistically significant difference was observed between the control group (G1_C) and (G2_SPH) (p = 0.704) in the flexural strength test, however differences were found between G2_SPH and G3_TiO2 groups, ANOVA (p = 0.006); post hoc Tukey's test (p = 0.014). Pertaining to the solubility, G2_SPH obtained the lowest among the three groups, ANOVA (p = 0.010); post hoc Tukey's test (p = 0.009). The three study groups obtained an adequate radiopacity of >1 mm Al, respectively. The resin-modified glass ionomer cement (RMGIC) was further modified with 2% silver phosphate/hydroxyapatite NPs to improve the physical properties such as enhancing the solubility and sorption without compromising the flexural strength and radiopacity behavior of modified RMGIC. The incorporation of 2% titanium dioxide NPs did not improve the properties studied.

Transcriptional and Post-Translational Roles of Calcineurin in Cationic Stress and Glycerol Biosynthesis in Cryptococcus neoformans.

Stress management is an adaptive advantage for survival in adverse environments. Pathogens face this challenge during host colonization, requiring an appropriate stress response to establish infection. The fungal pathogen Cryptococcus neoformans undergoes thermal, oxidative, and osmotic stresses in the environment and animal host. Signaling systems controlled by Ras1, Hog1, and calcineurin respond to high temperatures and osmotic stress. Cationic stress caused by Na+, K+, and Li+ can be overcome with glycerol, the preferred osmolyte. Deleting the glycerol phosphate phosphatase gene (GPP2) prevents cells from accumulating glycerol due to a block in the last step of its biosynthetic pathway. Gpp2 accumulates in a phosphorylated form in a cna1Δ strain, and a physical interaction between Gpp2 and Cna1 was found; moreover, the gpp2Δ strain undergoes slow growth and has attenuated virulence in animal models of infection. We provide biochemical evidence that growth in 1 M NaCl increases glycerol content in the wild type, whereas gpp2Δ, cna1Δ, and cnb1Δ mutants fail to accumulate it. The deletion of cnb1Δ or cna1Δ renders yeast cells sensitive to cationic stress, and the Gfp-Gpp2 protein assumes an abnormal localization. We suggest a mechanism in which calcineurin controls Gpp2 at the post-translational level, affecting its localization and activity, leading to glycerol biosynthesis. Also, we showed the transcriptional profile of glycerol-deficient mutants and established the cationic stress response mediated by calcineurin; among the biological processes differentially expressed are carbon utilization, translation, transmembrane transport, glutathione metabolism, oxidative stress response, and transcription regulation. To our knowledge, this is the first time that this transcriptional profile has been described. These results have implications for pathogen stress adaptability.

Phenotypic and genomic characterization of phosphate-solubilizing rhizobia isolated from native Mimosa and Desmodium in Brazil.

The phosphate (P)-solubilizing potential of rhizobia isolated from active root nodules of Brazilian native Mimosa and Desmodium was assessed. Out of the 15 strains selected, five Paraburkholderia isolated from Mimosa spp. grown in rocky outcrops stood out. The Ca3(PO4)2-solubilizing efficiency of these strains ranged from 110.67 to 356.3 mgL-1, with less expressive results for FePO4 and Al(H2PO4)3, that might be attributed to the low solubility of these two P compounds. Paraburkholderia strains CNPSo 3281 and CNPSo 3076 were the most efficient siderophore producers (44.17 and 41.87 µMol EDTA) and two of the top FePO4 solubilizers. Acidification of the culture media was observed for all the strains and P sources. Regarding Ca3(PO4)2 solubilization, the main organic acids detected were glucuronic (an important component of rhizobia exopolysaccharides) and gluconic acids. Genomic analysis of P. nodosa CNPSo 3281 and CNPSo 3076 along with other phosphate-solubilizing Paraburkholderia species of the genus pointed out a conserved gene organization of phoUBR, pstSCAB, ppk and ppx. Greenhouse experiment revealed that P. nodosa CNPSo 3281 and CNPSo 3076 promoted maize growth under low P. Our results indicate the relevance of native rhizobia as multifunctional plant-associated bacteria and the rocky outcrops ecosystems as hotspots for bioprospection.

Tricalcium phosphate-loaded injectable hydrogel as a promising osteogenic and bactericidal teicoplanin-delivery system for osteomyelitis treatment: An in vitro and in vivo investigation.

Osteomyelitis is an inflammation of bone tissue usually caused by pyogenic bacteria. The most recurrent clinical approach consists of bone debridement followed by parenteral administration of antibiotics. However, systemic antibiotic treatment has limitations regarding absorption rate and bioavailability over time. The main challenge of osteomyelitis treatment consists of coupling the persistent infection treatment with the regeneration of the bone debrided. In this work, we developed an injectable drug delivery system based on poloxamer 407 hydrogel containing undoped Mg, Zn-doped tricalcium phosphate (ß-TCP), and teicoplanin, a broad-spectrum antibiotic. We evaluated how the addition of teicoplanin and ß-TCP affected the micellization, gelation, particle size, and surface charge of the hydrogel. Later, we studied the hydrogel degradation and drug delivery kinetics. Finally, the bactericidal, biocompatibility, and osteogenic properties were evaluated through in vitro studies and confirmed by in vivo Wistar rat models. Teicoplanin was found to be encapsulated in the corona portions of the hydrogel micelles, yielding a bigger hydrodynamics radius. The encapsulated teicoplanin showed a sustained release over the evaluated period, enough to trigger antibacterial properties against Gram-positive bacteria. Besides, the formulations were biocompatible and showed bone healing ability and osteogenic properties. Finally, in vivo studies confirmed that the proposed locally injected formulations yielded osteomyelitis treatment with superior outcomes than parenteral administration while promoting bone regeneration. In conclusion, the presented formulations are promising drug delivery systems for osteomyelitis treatment and deserve further technological improvements.

Sphingosine 1-phosphate protective effect on human proximal tubule cells submitted to an in vitro ischemia model: the role of JAK2/STAT3.

Acute kidney injury is a serious public health problem worldwide, being ischemia and reperfusion (I/R) the main lesion-aggravating factor that contributes to the evolution towards chronic kidney disease. Nonetheless, intervention approaches currently available are just considered palliative options. In order to offer an alternative treatment, it is important to understand key factors involved in the development of the disease including the rescue of the affected cells and/or the release of paracrine factors that are crucial for tissue repair. Bioactive lipids such as sphingosine 1-phosphate (S1P) have significant effects on the modulation of signaling pathways involved in tissue regeneration, such as cell survival, proliferation, differentiation, and migration. The main objective of this work was to explore the protective effect of S1P using human kidney proximal tubule cells submitted to a mimetic I/R lesion, via ATP depletion. We observed that the S1P pre-treatment increases cell survival by 50% and preserves the cell proliferation capacity of injured cells. We showed the presence of different bioactive lipids notably related to tissue repair but, more importantly, we noted that the pre-treatment with S1P attenuated the ischemia-induced effects in response to the injury, resulting in higher endogenous S1P production. All receptors but S1PR3 are present in these cells and the protective and proliferative effect of S1P/S1P receptors axis occur, at least in part, through the activation of the SAFE pathway. To our knowledge, this is the first time that S1PR4 and S1PR5 are referred in these cells and also the first indication of JAK2/STAT3 pathway involvement in S1P-mediated protection in an I/R renal model.

Employment of pqqE gene as molecular marker for the traceability of Gram negative phosphate solubilizing bacteria associated to plants.

Insoluble phosphorous compounds solubilization by soil bacteria is of great relevance since it puts available the phosphorus to be used by plants. The production of organic acids is the main microbiological mechanism by which insoluble inorganic phosphorus compounds are solubilized. In Gram negative bacteria, gluconic acid is synthesized by the activity of the holoenzyme glucose dehydrogenase-pyrroloquinoline quinine named GDH-PQQ. The use of marker genes is a very useful tool to evaluate the persistence of the introduced bacteria and allow to follow-up the effect of biotic and abiotic factors on these beneficial microorganisms in the soil. In previous studies we detected the presence of the pqqE gene in a great percentage of both non-culturable and culturable native soil bacteria. The objective of this study was to analyze the phylogeny of the sequence of pqqE gene and its potential for the study of phosphate solubilizing bacteria from pure and mixed bacterial cultures and rhizospheric soil samples. For this, the presence of the pqqE gene in the genome of phosphate solubilizing bacteria that belong to several bacteria was determined by PCR. Also, this gene was analyzed from mixed bacterial cultures and rhizospheric soil associated to peanut plants inoculated or not with phosphate solubilizing bacteria. For this, degenerate primers designed from several bacterial genera and specific primers for the genus Pseudomonas spp., designed in this study, were used. DNA template used from simple or mixed bacterial cultures and from rhizospheric soil samples was obtained using two different DNA extraction techniques. Results indicated that pqqE gene amplification product was found in the genome of all Gram negative phosphate solubilizing bacteria analyzed. It was possible to detect this gene in the DNA obtained from mixed cultures where these bacteria grew in interaction with other microorganisms and in that obtained from rhizospheric soil samples inoculated or not with these bacteria. The phylogenetic analysis indicated that pqqE gene is a conserved gene within related genera. In conclusion, pqqE gene could be a potential marker for the study of phosphate solubilizing bacterial populations.

Enhancing Phosphate Uptake and Antifungal Activity in Tomato Plants via Bacillus licheniformis Mutagenesis: Evaluating Growth Parameters.

The objective of the investigation was to improve phosphate solubilization in tomato plants by Bacillus licheniformis, a rhizobacterium that promotes plant growth. Ultraviolet (UV) radiation, Ethyl methanesulfonate (EMS) and Ethidium bromide (EtBr) mutagenesis produced twenty-one mutants. Phosphate solubilization was higher in the PM7 (physical mutant) (121.00 g mL-1) than in the wild type (82.00 g mL-1). PM7 showed high antifungal activity against Phytophthora capsici, Fusarium oxysporum and Dematophora necatrix besides increased siderophore production and HCN production. In a net-house experiment, PM7 improved root and shoot parameters, P assimilation and soil P availability in tomato plants. This study demonstrates the potential of PM7 as an effective rhizobacterium for enhancing nutrient availability and plant growth.

Inoculation with Bacillus megaterium CNPMS B119 and Bacillus subtilis CNPMS B2084 improve P-acquisition and maize yield in Brazil.

Phosphorus (P) is a critical nutrient for plant growth, yet its uptake is often hindered by soil factors like clay minerals and metal oxides such as aluminum (Al), iron (Fe), and calcium (Ca), which bind P and limit its availability. Phosphate-solubilizing bacteria (PSB) have the unique ability to convert insoluble P into a soluble form, thereby fostering plant growth. This study aimed to assess the efficacy of inoculation of Bacillus megaterium B119 (rhizospheric) and B. subtilis B2084 (endophytic) via seed treatment in enhancing maize yield, grain P content, and enzyme activities across two distinct soil types in field conditions. Additionally, we investigated various mechanisms contributing to plant growth promotion, compatibility with commercial inoculants, and the maize root adhesion profile of these strains. During five crop seasons in two experimental areas in Brazil, Sete Lagoas-MG and Santo Antônio de Goiás-GO, single inoculations with either B119 or B2084 were implemented in three seasons, while a co-inoculation with both strains was applied in two seasons. All treatments received P fertilizer according to plot recommendations, except for control. Both the Bacillus strains exhibited plant growth-promoting properties relevant to P dynamics, including phosphate solubilization and mineralization, production of indole-3-acetic acid (IAA)-like molecules, siderophores, exopolysaccharides (EPS), biofilms, and phosphatases, with no antagonism observed with Azospirillum and Bradyrizhobium. Strain B2084 displayed superior maize root adhesion compared to B119. In field trials, single inoculations with either B119 or B2084 resulted in increased maize grain yield, with relative average productivities of 22 and 16% in Sete Lagoas and 6 and 3% in Santo Antônio de Goiás, respectively. Co-inoculation proved more effective, with an average yield increase of 24% in Sete Lagoas and 11% in Santo Antônio de Goiás compared to the non-inoculated control. Across all seasons, accumulated grain P content correlated with yield, and soil P availability in the rhizosphere increased after co-inoculation in Santo Antônio de Goiás. These findings complement previous research efforts and have led to the validation and registration of the first Brazilian inoculant formulated with Bacillus strains for maize, effectively enhancing and P grain content.

Effect of the calcium silicate and sodium phosphate remineralizing products on bleached enamel.

Context and Aims: This study evaluated the effect of calcium silicate and sodium phosphate (CSSP) dentifrice and serum on the surface of enamel bleached with hydrogen peroxide (H2O2). Materials and Methods: A total of 160 bovine enamel slabs were bleached with 35% H2O2 and treated with sodium fluoride (NaF) dentifrice-GI, CSSP dentifrice-GII; CSSP dentifrice + CSSP serum-GIII, or NaF dentifrice + NaF gel-GIV. The dentifrices were applied using a brushing machine three times daily for 7 days. After brushing, sodium phosphate gel and CSSP serum were applied. The microhardness (KNH, n = 14), surface roughness (Ra, n = 14), energy dispersive spectroscopy (n = 6), and scanning electron microscopy (n = 6) were assessed at t0 (before bleaching), t1 (after bleaching), and t2 (after postbleaching treatments). Statistical Analysis Used: The data were subjected to a two-way analysis of variance and Bonferroni's test. Results: The KNH decreased at t1 (P < 0.001) but recovered at t2 for all treatments, although only GII showed restored baseline values (P = 0.0109). The surface roughness increased at t1 (P < 0.001) and reduced at t2 (P < 0.001) for all groups, with no significant differences among groups. Enamel composition and morphology did not differ after the treatments, except for silicon accumulation in GIII. Conclusions: Postbleaching treatment with CSSP dentifrice and serum yielded superior remineralizing effects on bleached enamel.

Galactose-1-phosphate inhibits cytochrome c oxidase and causes mitochondrial dysfunction in classic galactosemia.

Classic galactosemia is an inborn error of metabolism caused by mutations in the GALT gene resulting in the diminished activity of the galactose-1-phosphate uridyltransferase enzyme. This reduced GALT activity leads to the buildup of the toxic intermediate galactose-1-phosphate and a decrease in ATP levels upon exposure to galactose. In this work, we focused our attention on mitochondrial oxidative phosphorylation in the context of this metabolic disorder. We observed that galactose-1-phosphate accumulation reduced respiratory rates in vivo and changed mitochondrial function and morphology in yeast models of galactosemia. These alterations are harmful to yeast cells since the mitochondrial retrograde response is activated as part of the cellular adaptation to galactose toxicity. In addition, we found that galactose-1-phosphate directly impairs cytochrome c oxidase activity of mitochondrial preparations derived from yeast, rat liver, and human cell lines. These results highlight the evolutionary conservation of this biochemical effect. Finally, we discovered that two compounds - oleic acid and dihydrolipoic acid - that can improve the growth of cell models of mitochondrial diseases, were also able to improve galactose tolerance in this model of galactosemia. These results reveal a new molecular mechanism relevant to the pathophysiology of classic galactosemia - galactose-1-phosphate-dependent mitochondrial dysfunction - and suggest that therapies designed to treat mitochondrial diseases may be repurposed to treat galactosemia.

Rhizospheric Bacteria of Cover Legumes from Acidic Soils Are Capable of Solubilizing Different Inorganic Phosphates.

Due to its adsorption with aluminum and iron hydroxides, phosphorus viability is low in acidic soils; thus, the aim of this study was to isolate and identify bacteria from the rhizosphere of four legumes growing in acidic soils of the Cumbaza Sub-basin, San Martín, Peru, as well as to characterize their ability to solubilize aluminum phosphate and iron phosphate. The isolation process was conducted on TSA medium and the isolates were classified based on their origin and morphocolonial characteristics, with the bacillary shape being the most frequent, followed by cocci. To assess the solubilization of aluminum and iron phosphates, the liquid medium GELP was employed. Sixteen strains were selected, among which three stood out for their effectiveness in solubilizing AlPO4 (Sfcv-098-02, 22.65 mg L-1; Sfc-093-04, 26.50 mg L-1; and Sfcv-041-01-2, 55.98 mg L-1) and one for its ability to solubilize FePO4 (Sfcr-043-02, 32.61 mg L-1). These four strains were molecularly characterized, being identified as Enterobacter sp., Pseudomonas sp., and Staphylococcus sp. Additionally, a decrease in pH was observed in the reactions, with values ranging from 5.23 to 3.29, which enhanced the phosphate of solubilization. This suggests that the selected bacteria could be used to improve phosphorus availability in agricultural soils.

Competitive oxidation of key pentose phosphate pathway enzymes modulates the fate of intermediates and NAPDH production.

The oxidative phase of the pentose phosphate pathway (PPP) involving the enzymes glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconolactonase (6PGL), and 6-phosphogluconate dehydrogenase (6PGDH), is critical to NADPH generation within cells, with these enzymes catalyzing the conversion of glucose-6-phosphate (G6P) into ribulose-5-phosphate (Ribu5-P). We have previously studied peroxyl radical (ROO•) mediated oxidative inactivation of E. coli G6PDH, 6PGL, and 6PGDH. However, these data were obtained from experiments where each enzyme was independently exposed to ROO•, a condition not reflecting biological reality. In this work we investigated how NADPH production is modulated when these enzymes are jointly exposed to ROO•. Enzyme mixtures (1:1:1 ratio) were exposed to ROO• produced from thermolysis of 100 mM 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). NADPH was quantified at 340 nm, and protein oxidation analyzed by liquid chromatography with mass spectrometric detection (LC-MS). The data obtained were rationalized using a mathematical model. The mixture of non-oxidized enzymes, G6P and NADP+ generated ∼175 µM NADPH. Computational simulations showed a constant decrease of G6P associated with NADPH formation, consistent with experimental data. When the enzyme mixture was exposed to AAPH (3 h, 37 °C), lower levels of NADPH were detected (∼100 µM) which also fitted with computational simulations. LC-MS analyses indicated modifications at Tyr, Trp, and Met residues but at lower concentrations than detected for the isolated enzymes. Quantification of NADPH generation showed that the pathway activity was not altered during the initial stages of the oxidations, consistent with a buffering role of G6PDH towards inactivation of the oxidative phase of the pathway.

The effect of nutritional and oxidative stress on the metabolome of Trypanosoma cruzi.

Trypanosoma cruzi, a flagellated protozoan, is the causative agent of Chagas disease. The parasite has developed various mechanisms to get through its intricate life cycle and adapt to different evolutionary phases. T. cruzi proliferates in the insect vector's digestive tract as an epimastigote form, encountering fluctuating nutrient availability and oxidative stress caused by the digestion of red blood cells from the mammalian host blood meal. To unravel how the parasite's metabolism adapts to these changing conditions, we conducted an analysis of the chemical species present in epimastigote forms. This involved comparing cultured parasites with those subjected to nutritional deficiency or oxidative stress using untargeted metabolomics. We looked at 21 samples: seven biological copies of parasites that were actively growing, seven samples that were put in a medium without nutrients for 3 h, and seven samples that were treated with glucose oxidase for 30 min to make H2O2 continuously. Importantly, in all conditions, parasite viability was maintained when the samples were collected. Upon nutrient removal, we observed a substantial decrease in amino acids and carbohydrate metabolites, accompanied by the accumulation of fatty acids and steroids, with the predominance of inositol and sphingolipid metabolism, along with a simultaneous decrease in the levels of H2O2. In the presence of H2O2, a significant rise in components of the pentose pathway and specific amino acids such as methionine and serine occurred, along with pathways related to an increase in antioxidant species metabolism such as ribulose 5-phosphate and glyceric acid. Conversely, fatty acid and steroid levels decrease. We found no common increase in metabolites or lipids. In contrast, eight species (succinic acid, glutamic acid, valine, 2-hydroxyisocaproic acid, alanine, indolelactic acid, proline, and lanosterol) were consumed under both stresses. These findings underscore the rapid and distinct enrichment responses in amino acids, lipids, and carbohydrates required to cope with each different environmental condition. We concluded that T. cruzi presents a flexible metabolism that rapidly adapts to variable changes in the environment.

Phosphate uptake in PhoX: Molecular mechanisms.

PhoX is a high-affinity phosphate binding protein, present in Xanthomonas citri, a phytopathogen responsible for the citrus canker disease. Performing molecular dynamics simulations and different types of computational analyses, we study the molecular mechanisms at play in relation to phosphate binding, revealing the global functioning of the protein: PhoX naturally oscillates along its global normal modes, which allow it to explore both bound and unbound conformations, eventually attracting a nearby negative phosphate ion to the highly positive electrostatic potential on its surface, particularly close to the binding pocket. There, several hydrogen bonds are formed with the two main domains of the structure. Phosphate creates, in this way, a strong bridge that connects the domains, keeping itself between them, in a tight closed conformation, explaining its high binding affinity.

Ion release mechanisms in composites containing CaP particles and hydrophilic monomers.

OBJECTIVE: To investigate the effect of hydrophilic/permeable polymer matrices on water sorption/solubility (WS/SL), Ca2+ release, mechanical properties and hydrolytic degradation of composites containing dicalcium phosphate dihydrate (DCPD) particles. METHODS: Six composites were tested, all with 10 vol% of glass particles and either 30 vol% or 40 vol% DCPD. Composites containing 1BisGMA:1TEGDMA in mols (at both inorganic levels) were considered controls. Four materials were formulated where 0.25 or 0.5 of the BisGMA/TEGDMA was replaced by pyromellitic dianhydride glycerol dimethacrylate (PMGDM)/ polyethylene glycol dimethacrylate (PEGDMA). Composites were tested for degree of conversion (FTIR spectroscopy), WS/SL (ISO 4049) and Ca2+ release (inductively coupled plasma optical emission spectroscopy). Fracture toughness (FT) and biaxial flexural strength/modulus (BFS/FM) were determined after 24 h and 60 days in water. The contributions of diffusional and relaxational mechanisms to Ca2+ release kinetics were analyzed using the semi-empirical Salim-Peppas model. Data were analysed by ANOVA/Tukey test (alpha: 0.05). RESULTS: WS/SL was higher for composites containing PMGDM/PEGDMA compared to the controls (p < 0.001). Only at 40% DCPD the 0.5 PMGDM/PEGDMA composite showed statistically higher Ca2+ release than the control. Relaxation diffusion was the main release mechanism. Initial FT was not negatively affected by matrix composition. BFS (both DCPD fractions) and FM (30% DCPD) were lower for composites with hydrophilic/permeable networks (p < 0.01). After 60 days in water, composites with PMGDM/PEGDMA presented significant reductions in FT, while all composites had reductions in BFS/FM. SIGNIFICANCE: Increasing matrix hydrophilicity/permeability significantly increased Ca2+ release only at a high DCPD fraction.