Effects of calcium phosphate and phosphorus-dissolving bacteria on microbial structure and function during Torreya Grandis branch waste composting.

BACKGROUND BURKHOLDERIA: is a phosphorus solubilizing microorganism discovered in recent years, which can dissolve insoluble phosphorus compounds into soluble phosphorus. To investigate the effects of Burkholderia and calcium phosphate on the composting of Torreya grandis branches and leaves, as well as to explain the nutritional and metabolic markers related to the composting process. METHODS: In this study, we employed amplicon sequencing and untargeted metabolomics analysis to examine the interplay among phosphorus (P) components, microbial communities, and metabolites during T. grandis branch and leaf waste composting that underwent treatment with calcium phosphate and phosphate-solubilizing bacteria (Burkholderia). There were four composting treatments, 10% calcium phosphate (CaP) or 5 ml/kg (1 × 108/ml Burkholderia) microbial inoculum (WJP) or both (CaP + WJP), and the control group (CK). RESULTS: The results indicated that Burkholderia inoculation and calcium phosphate treatment affected the phosphorus composition, pH, EC, and nitrogen content. Furthermore, these treatments significantly affected the diversity and structure of bacterial and fungal communities, altering microbial and metabolite interactions. The differential metabolites associated with lipids and organic acids and derivatives treated with calcium phosphate treatment are twice as high as those treated with Burkholderia in both 21d and 42d. The results suggest that calcium phosphate treatment alters the formation of some biological macromolecules. CONCLUSION: Both Burkholderia inoculation and calcium phosphate treatment affected the phosphorus composition, nitrogen content and metabolites of T. grandis branch and leaf waste compost.These results extend our comprehension of the coupling of matter transformation and community succession in composting with the addition of calcium phosphate and phosphate-solubilizing bacteria.

Effects on calcium phosphate homeostasis after sodium-glucose cotransporter 2 inhibitor in patients with advanced chronic kidney disease and type 2 diabetes mellitus.

BACKGROUND: The effects of sodium-glucose cotransporter 2 inhibitors (SGLT2i) on calcium phosphate homeostasis in patients with type 2 diabetes mellitus (T2DM) and chronic kidney disease (CKD) remain uncertain. METHODS: A retrospective observational cohort study of patients with T2DM at CKD stage G3b-5ND who received SGLT2i as compared to control from 1 January 2015 through 31 December 2021 was recruited. Propensity score assignment at 1:3 ratio by logistic regression was done. All patients were followed for 12 months. Outcomes were changes in phosphate level. RESULTS: We analyzed 1,450 SGLT2i users and 4,350 control subjects. At the 12th month, SGLT2i users had a slower increase in phosphate levels (absolute change: -0.01 ± 0.28 vs + 0.14 ± 0.34 mmol/L; percentage change: -0.74 % ± 25.56 vs + 10.88 ± 28.15 %, P for both < 0.001). The proportion of patients with high phosphate was lower with SGLT2i (8.2 % vs 24.6 % increase). In the generalized estimating equation, SGLT2i was linked to a longitudinal reduction in phosphate (B -0.039, P<0.001). CONCLUSIONS: SGLT2i can effectively slow down the progression of phosphate retention in advanced CKD with T2DM.

Species-level characterization of gut microbiota and their metabolic role in kidney stone formation using full-length 16S rRNA sequencing.

The critical role of the human gut microbiota in kidney stone formation remains largely unknown, due to the low taxonomic resolution of previous sequencing technologies. Therefore, this study aimed to explore the gut microbiota using high-throughput sequencing to provide valuable insights and identify potential bacterial species and metabolite roles involved in kidney stone formation. The overall gut bacterial community and its potential functions in healthy participants and patients were examined using PacBio sequencing targeting the full-length 16S rRNA gene, coupled with stone and statistical analyses. Most kidney stones comprised calcium oxalate and calcium phosphate (75%), pure calcium oxalate (20%), and calcium phosphate and magnesium phosphate (5%), with higher content of Ca (130,510.5 ± 108,362.7 ppm) followed by P (18,746.4 ± 23,341.2 ppm). The microbial community structure was found to be weaker in patients' kidney stone samples, followed by patients' stool samples, than in healthy participants' stool samples. The most abundant bacterial species in kidney stone samples was uncultured Morganella, whereas that in patient and healthy participant stool samples was Bacteroides vulgatus. Similarly, Akkermansia muciniphila was significantly enriched in patient stool samples at the species level, whereas Bacteroides plebeius was significantly enriched in kidney stone samples than that in healthy participant stool samples. Three microbial metabolic pathways, TCA cycle, fatty acid oxidation, and urea cycle, were significantly enriched in kidney stone patients compared to healthy participants. Inferring bacteria at the species level revealed key players in kidney stone formation, enhancing the clinical relevance of gut microbiota.

Genomic insights into organic acid production and plant growth promotion by different species of phosphate-solubilizing bacteria.

Bacteria can solubilize phosphorus (P) through the secretion of low-molecular-weight organic acids and acidification. However, the genes involved in the production of these organic acids are poorly understood. The objectives of this study were to verify the calcium phosphate solubilization and the production of low-molecular-weight organic acids by diverse genera of phosphate solubilizing bacterial strains (PSBS); to identify the genes related to the synthesis of the organic acids in the genomes of these strains and; to evaluate growth and nutrient accumulation of maize plants inoculated with PSBS and fertilized with Bayóvar rock phosphate. Genomic DNA was extracted for strain identification and annotation of genes related to the organic acids production. A greenhouse experiment was performed with five strains plus 150 mg dm- 3 P2O5 as Bayóvar rock phosphate (BRP) to assess phosphate solubilization contribution to maize growth and nutrition. Paraburkholderia fungorum UFLA 04-21 and Pseudomonas anuradhapurensis UFPI B5-8A solubilized over 60% of Ca phosphate and produced high amounts of citric/maleic and gluconic acids in vitro, respectively. Eleven organic acids were identified in total, although not all strains produced all acids. Besides, enzymes related to the organic acids production were found in all bacterial genomes. Plants inoculated with strains UFPI B5-6 (Enterobacter bugandensis), UFPI B5-8A, and UFLA 03-10 (Paenibacillus peoriae) accumulated more biomass than the plants fertilized with BRP only. Strains UFLA 03-10 and UFPI B5-8A increased the accumulation of most macronutrients, including P. Collectively, the results show that PSBS can increase maize growth and nutrient accumulation based on Bayóvar rock phosphate fertilization.

Post-mortem recrystallization of biogenic amorphous calcium carbonate guided by the inherited macromolecular framework.

In contrast to abiotically formed carbonates, biogenetic carbonates have been observed to be nanocomposite, organo-mineral structures, the basic build-blocks of which are particles of quasi-uniform size (10-100 nm) organized into complex higher-order hierarchical structures, typically with highly controlled crystal-axis alignments. Some of these characteristics serve as criteria for inferring a biological origin and the state of preservation of fossil carbonate materials, and to determine whether the biomineralization process was biologically induced or controlled. Here we show that a calcium storage structure formed by the American lobster, a gastrolith initially consisting of amorphous calcium carbonate (ACC) and amorphous calcium phosphate (ACP), post-mortem can crystallize into (thus secondary) calcite with structural properties strongly influenced by the inherited organic matrix. This secondary calcite meets many structural criteria for biominerals (thus called the biomorphic calcite), but differs in trace element distributions (e.g., P and Mg). Such observations refine the capability to determine whether a fossil carbonates can be attributed to biogenic processes, with implications for the record of life on Earth and other terrestrial planets.

Recombinant human enamelin produced in Escherichia coli promotes mineralization in vitro.

BACKGROUND: Enamelin is an enamel matrix protein that plays an essential role in the formation of enamel, the most mineralized tissue in the human body. Previous studies using animal models and proteins from natural sources point to a key role of enamelin in promoting mineralization events during enamel formation. However, natural sources of enamelin are scarce and with the current study we therefore aimed to establish a simple microbial production method for recombinant human enamelin to support its use as a mineralization agent. RESULTS: In the study the 32 kDa fragment of human enamelin was successfully expressed in Escherichia coli and could be obtained using immobilized metal ion affinity chromatography purification (IMAC), dialysis, and lyophilization. This workflow resulted in a yield of approximately 10 mg enamelin per liter culture. Optimal conditions for IMAC purification were obtained using Ni2+ as the metal ion, and when including 30 mM imidazole during binding and washing steps. Furthermore, in vitro mineralization assays demonstrated that the recombinant enamelin could promote calcium phosphate mineralization at a concentration of 0.5 mg/ml. CONCLUSIONS: These findings address the scarcity of enamelin by facilitating its accessibility for further investigations into the mechanism of enamel formation and open new avenues for developing enamel-inspired mineralized biomaterials.

Calcium phosphate controls nucleation and growth of calcium oxalate crystal phases in kidney stones.

Kidney stone disease is a serious disease due to the severe pain it causes, high morbidity, and high recurrence rate. Notably, calcium oxalate stones are the most common type of kidney stone. Calcium oxalate appears in two forms in kidney stones: the stable phase, monohydrate (COM), and the metastable phase, dihydrate (COD). Particularly, COM stones with concentric structures are hard and difficult to treat. However, the factor determining the growth of either COM or COD crystals in the urine, which is supersaturated for both phases, remains unclear. This study shows that calcium phosphate ingredients preferentially induce COM crystal nucleation and growth, by observing and analyzing kidney stones containing both COM and COD crystals. The forms of calcium phosphate are not limited to Randall's plaques (1-2 mm size aggregates, which contain calcium phosphate nanoparticles and proteins, and form in the renal papilla). For example, aggregates of strip-shaped calcium phosphate crystals and fields of dispersed calcium phosphate microcrystals (nano to micrometer order) also promote the growth of concentric COM structures. This suggests that patients who excrete urine with a higher quantity of calcium phosphate crystals may be more prone to forming hard and troublesome COM stones.

Genomic insight of phosphate solubilization and plant growth promotion of two taxonomically distinct winter crops by Enterobacter sp. DRP3.

AIMS: Study of rhizospheric microbiome-mediated plant growth promotional attributes currently highlighted as a key tool for the development of suitable bio-inoculants for sustainable agriculture purposes. In this context, we have conducted a detailed study regarding the characterization of phosphate solubilizing potential by plant growth-promoting bacteria that have been isolated from the rhizosphere of a pteridophyte Dicranopteris sp., growing on the lateritic belt of West Bengal. METHODS AND RESULTS: We have isolated three potent bacterial strains, namely DRP1, DRP2, and DRP3 from the rhizoids-region of Dicranopteris sp. Among the isolated strains, DRP3 is found to have the highest phosphate solubilizing potentiality and is able to produce 655.89 and 627.58 µg ml-1 soluble phosphate by solubilizing tricalcium phosphate (TCP) and Jordan rock phosphate, respectively. This strain is also able to solubilize Purulia rock phosphate moderately (133.51 µg ml-1). Whole-genome sequencing and further analysis of the studied strain revealed the presence of pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase gdh gene along with several others that were well known for their role in phosphate solubilization. Further downstream, quantitative reverse transcriptase PCR-based expression study revealed 1.59-fold upregulation of PQQ-dependent gdh gene during the solubilization of TCP. Root colonization potential of the studied strain on two taxonomically distinct winter crops viz. Cicer arietinum and Triticum aestivum has been checked by using scanning electron microscopy. Other biochemical analyses for plant growth promotion traits including indole acetic acid production (132.02 µg ml-1), potassium solubilization (3 mg l-1), biofilm formation, and exopolymeric substances productions (1.88-2.03 µg ml-1) also has been performed. CONCLUSION: This study highlighted the active involvement of PQQ-dependent gdh gene during phosphate solubilization from any Enterobacter group. Moreover, our study explored different roadmaps for sustainable farming methods and the preservation of food security without endangering soil health in the future.

Unraveling the role of carboxylate groups and elastin particle size in medial calcification.

While it is known that calcium phosphate (CaP) minerals deposit in elastin-rich medial layers of arteries during medial calcification, their nucleation and growth sites are still debated. Neutral carbonyl groups and carboxylate groups are possible candidates. Also, while it is known that elastin degradation leads to calcification, it is unclear whether this is due to formation of new carboxylate groups or elastin fragmentation. In this work, we disentangle effects of carboxylate groups and particle size on elastin calcification; in doing so, we shed light on CaP mineralization sites on elastin. We find carboxylate groups accelerate calcification only in early stages; they mainly function as Ca2+ ion chelation sites but not calcification sites. Their presence promotes formation (likely on Ca2+ ions adsorbed on nearby carbonyl groups) of CaP minerals with high calcium-to-phosphate ratio as intermediate phases. Larger elastin particles calcify slower but reach similar amounts of CaP minerals in late stages; they promote direct formation of hydroxyapatite and CaP minerals with low calcium-to-phosphate ratio as intermediate phases. This work provides new perspectives on how carboxylate groups and elastin particle size influence calcification; these parameters can be tuned to study the mechanism of medial calcification and design drugs to inhibit the process.

Osteopontin stabilization and collagen containment slows amorphous calcium phosphate transformation during human aortic valve leaflet calcification.

Calcification of aortic valve leaflets is a growing mortality threat for the 18 million human lives claimed globally each year by heart disease. Extensive research has focused on the cellular and molecular pathophysiology associated with calcification, yet the detailed composition, structure, distribution and etiological history of mineral deposition remains unknown. Here transdisciplinary geology, biology and medicine (GeoBioMed) approaches prove that leaflet calcification is driven by amorphous calcium phosphate (ACP), ACP at the threshold of transformation toward hydroxyapatite (HAP) and cholesterol biomineralization. A paragenetic sequence of events is observed that includes: (1) original formation of unaltered leaflet tissues: (2) individual and coalescing 100's nm- to 1 µm-scale ACP spherules and cholesterol crystals biomineralizing collagen fibers and smooth muscle cell myofilaments; (3) osteopontin coatings that stabilize ACP and collagen containment of nodules preventing exposure to the solution chemistry and water content of pumping blood, which combine to slow transformation to HAP; (4) mm-scale nodule growth via ACP spherule coalescence, diagenetic incorporation of altered collagen and aggregation with other ACP nodules; and (5) leaflet diastole and systole flexure causing nodules to twist, fold their encasing collagen fibers and increase stiffness. These in vivo mechanisms combine to slow leaflet calcification and establish previously unexplored hypotheses for testing novel drug therapies and clinical interventions as viable alternatives to current reliance on surgical/percutaneous valve implants.

Ecofriendly solidification of sand using microbially induced calcium phosphate precipitation.

This study introduces microbiologically induced calcium phosphate precipitation (MICPP) as a novel and environmentally sustainable method of soil stabilization. Using Limosilactobacillus sp., especially NBRC 14511 and fish bone solution (FBS) extracted from Tuna fish bones, the study was aimed at testing the feasibility of calcium phosphate compounds (CPCs) deposition and sand stabilization. Dynamic changes in pH and calcium ion (Ca2+) concentration during the precipitation experiments affected the precipitation and sequential conversion of dicalcium phosphate dihydrate (DCPD) to hydroxyapatite (HAp), which was confirmed by XRD and SEM analysis. Sand solidification experiments demonstrated improvements in unconfined compressive strength (UCS), especially at higher Urea/Ca2+ ratios. The UCS values obtained were 10.35 MPa at a ratio of 2.0, 3.34 MPa at a ratio of 1.0, and 0.43 MPa at a ratio of 0.5, highlighting the advantages of MICPP over traditional methods. Microstructural analysis further clarified the mineral composition, demonstrating the potential of MICPP in environmentally friendly soil engineering. The study highlights the promise of MICPP for sustainable soil stabilization, offering improved mechanical properties and reducing environmental impact, paving the way for novel geotechnical practices.

The Rise in Tubular pH during Hypercalciuria Exacerbates Calcium Stone Formation.

In calcium nephrolithiasis (CaNL), most calcium kidney stones are identified as calcium oxalate (CaOx) with variable amounts of calcium phosphate (CaP), where CaP is found as the core component. The nucleation of CaP could be the first step of CaP+CaOx (mixed) stone formation. High urinary supersaturation of CaP due to hypercalciuria and an elevated urine pH have been described as the two main factors in the nucleation of CaP crystals. Our previous in vivo findings (in mice) show that transient receptor potential canonical type 3 (TRPC3)-mediated Ca2+ entry triggers a transepithelial Ca2+ flux to regulate proximal tubular (PT) luminal [Ca2+], and TRPC3-knockout (KO; -/-) mice exhibited moderate hypercalciuria and microcrystal formation at the loop of Henle (LOH). Therefore, we utilized TRPC3 KO mice and exposed them to both hypercalciuric [2% calcium gluconate (CaG) treatment] and alkalineuric conditions [0.08% acetazolamide (ACZ) treatment] to generate a CaNL phenotype. Our results revealed a significant CaP and mixed crystal formation in those treated KO mice (KOT) compared to their WT counterparts (WTT). Importantly, prolonged exposure to CaG and ACZ resulted in a further increase in crystal size for both treated groups (WTT and KOT), but the KOT mice crystal sizes were markedly larger. Moreover, kidney tissue sections of the KOT mice displayed a greater CaP and mixed microcrystal formation than the kidney sections of the WTT group, specifically in the outer and inner medullary and calyceal region; thus, a higher degree of calcifications and mixed calcium lithiasis in the kidneys of the KOT group was displayed. In our effort to find the Ca2+ signaling pathophysiology of PT cells, we found that PT cells from both treated groups (WTT and KOT) elicited a larger Ca2+ entry compared to the WT counterparts because of significant inhibition by the store-operated Ca2+ entry (SOCE) inhibitor, Pyr6. In the presence of both SOCE (Pyr6) and ROCE (receptor-operated Ca2+ entry) inhibitors (Pyr10), Ca2+ entry by WTT cells was moderately inhibited, suggesting that the Ca2+ and pH levels exerted sensitivity changes in response to ROCE and SOCE. An assessment of the gene expression profiles in the PT cells of WTT and KOT mice revealed a safeguarding effect of TRPC3 against detrimental processes (calcification, fibrosis, inflammation, and apoptosis) in the presence of higher pH and hypercalciuric conditions in mice. Together, these findings show that compromise in both the ROCE and SOCE mechanisms in the absence of TRPC3 under hypercalciuric plus higher tubular pH conditions results in higher CaP and mixed crystal formation and that TRPC3 is protective against those adverse effects.

Ca(H2PO4)2 and MgSO4 activated nitrogen-related bacteria and genes in thermophilic stage of compost.

This study was conducted to investigate the effects of Ca(H2PO4)2 and MgSO4 on the bacterial community and nitrogen metabolism genes in the aerobic composting of pig manure. The experimental treatments were set up as control (C), 1% Ca(H2PO4)2 + 2% MgSO4 (CaPM1), and 1.5% Ca(H2PO4)2 + 3% MgSO4 (CaPM2), which were used at the end of composting for potting trials. The results showed that Ca(H2PO4)2 and MgSO4 played an excellent role in retaining nitrogen and increasing the alkali-hydrolyzed nitrogen (AN), available phosphorus (AP), and available potassium (AK) contents of the composts. Adding Ca(H2PO4)2 and MgSO4 changed the microbial community structure of the compost. The microorganisms associated with nitrogen retention were activated. The complexity of the microbial network was enhanced. Genetic prediction analysis showed that the addition of Ca(H2PO4)2 and MgSO4 reduced the accumulation of nitroso-nitrogen and the process of denitrification. At the same time, despite the reduction of genes related to nitrogen fixation, the conversion of ammonia to nitrogenous organic compounds was promoted and the stability of nitrogen was increased. Mantel test analysis showed that Ca(H2PO4)2 and MgSO4 can affect nitrogen transformation-related bacteria and thus indirectly affect nitrogen metabolism genes by influencing the temperature, pH, and organic matter (OM) of the compost and also directly affected nitrogen metabolism genes through PO43- and Mg2+. The pot experiment showed that composting with 1.5% Ca(H2PO4)2 + 3% MgSO4 produced the compost product that improved the growth yield and nutrient content of cilantro and increased the fertility of the soil. In conclusion, Ca(H2PO4)2 and MgSO4 reduces the loss of nitrogen from compost, activates nitrogen-related bacteria and genes in the thermophilic phase of composting, and improves the fertilizer efficiency of compost products. KEY POINTS: • Ca(H2PO4)2 and MgSO4 reduced the nitrogen loss and improved the compost effect • Activated nitrogen-related bacteria and altered nitrogen metabolism genes • Improved the yield and quality of cilantro and fertility of soil.

Role of amelogenin phosphorylation in regulating dental enamel formation.

Amelogenin (AMELX), the predominant matrix protein in enamel formation, contains a singular phosphorylation site at Serine 16 (S16) that greatly enhances AMELX's capacity to stabilize amorphous calcium phosphate (ACP) and inhibit its transformation to apatitic enamel crystals. To explore the potential role of AMELX phosphorylation in vivo, we developed a knock-in (KI) mouse model in which AMELX phosphorylation is prevented by substituting S16 with Ala (A). As anticipated, AMELXS16A KI mice displayed a severe phenotype characterized by weak hypoplastic enamel, absence of enamel rods, extensive ectopic calcifications, a greater rate of ACP transformation to apatitic crystals, and progressive cell pathology in enamel-forming cells (ameloblasts). In the present investigation, our focus was on understanding the mechanisms of action of phosphorylated AMELX in amelogenesis. We have hypothesized that the absence of AMELX phosphorylation would result in a loss of controlled mineralization during the secretory stage of amelogenesis, leading to an enhanced rate of enamel mineralization that causes enamel acidification due to excessive proton release. To test these hypotheses, we employed microcomputed tomography (µCT), colorimetric pH assessment, and Fourier Transform Infrared (FTIR) microspectroscopy of apical portions of mandibular incisors from 8-week old wildtype (WT) and KI mice. As hypothesized, µCT analyses demonstrated significantly higher rates of enamel mineral densification in KI mice during the secretory stage compared to the WT. Despite a greater rate of enamel densification, maximal KI enamel thickness increased at a significantly lower rate than that of the WT during the secretory stage of amelogenesis, reaching a thickness in mid-maturation that is approximately half that of the WT. pH assessments revealed a lower pH in secretory enamel in KI compared to WT mice, as hypothesized. FTIR findings further demonstrated that KI enamel is comprised of significantly greater amounts of acid phosphate compared to the WT, consistent with our pH assessments. Furthermore, FTIR microspectroscopy indicated a significantly higher mineral-to-organic ratio in KI enamel, as supported by µCT findings. Collectively, our current findings demonstrate that phosphorylated AMELX plays crucial mechanistic roles in regulating the rate of enamel mineral formation, and in maintaining physico-chemical homeostasis and the enamel growth pattern during early stages of amelogenesis.

ß-tricalcium phosphate enhanced biomineralization of Cd2+ and Pb2+ by Sporosarcina ureilytica HJ1 and Sporosarcina pasteurii HJ2.

Microbiologically induced CaCO3 precipitation (MICP) has been proposed as a potential bioremediation method to immobilize contaminating metals. In this study, carbonate mineralizing bacteria HJ1 and HJ2, isolated from heavy metal contaminated soil, was employed for Cd2+ and Pb2+ immobilization with or without ß-tricalcium phosphate addition. Compared with the only treatments amended with strains, the combined application of ß-tricalcium phosphate and HJ1 improved the immobilization rates of Cd and Pb by 1.49 and 1.70 times at 24 h, and the combined application of ß-tricalcium phosphate and HJ2 increased the immobilization rates of Cd and Pb by 1.25 and 1.79 times. The characterization of biomineralization products revealed that Cd2+ and Pb2+ primarily immobilized from the liquid phase as CdCO3 and PbCO3, and the addition of ß-tricalcium phosphate facilitated the formation of Ca4.03Cd0.97(PO4)3(OH) and Pb3(PO4)2. Also, the calcium source was related to the speciation of carbonate precipitation and improved the Cd and Pb remediation efficiency. This research demonstrated the feasibility and effectiveness of MICP combined with ß-tricalcium phosphate in immobilization of Cd and Pb, which will provide a fundamental basis for future applications of MICP to mitigate soil heavy metal pollutions.

Sex-specific Stone-forming Phenotype in Mice During Hypercalciuria/Urine Alkalinization.

Sex differences in kidney stone formation are well known. Females generally have slightly acidic blood and higher urine pH when compared with males, which makes them more vulnerable to calcium stone formation, yet the mechanism is still unclear. We aimed to examine the role of sex in stone formation during hypercalciuria and urine alkalinization through acetazolamide and calcium gluconate supplementation, respectively, for 4 weeks in wild-type (WT) and moderately hypercalciuric [TRPC3 knockout [KO](-/-)] male and female mice. Our goal was to develop calcium phosphate (CaP) and CaP+ calcium oxalate mixed stones in our animal model to understand the underlying sex-based mechanism of calcium nephrolithiasis. Our results from the analyses of mice urine, serum, and kidney tissues show that female mice (WT and KO) produce more urinary CaP crystals, higher [Ca2+], and pH in urine compared to their male counterparts. We identified a sex-based relationship of stone-forming phenotypes (types of stones) in our mice model following urine alkalization/calcium supplementation, and our findings suggest that female mice are more susceptible to CaP stones under those conditions. Calcification and fibrotic and inflammatory markers were elevated in treated female mice compared with their male counterparts, and more so in TRPC3 KO mice compared with their WT counterparts. Together these findings contribute to a mechanistic understanding of sex-influenced CaP and mixed stone formation that can be used as a basis for determining the factors in sex-related clinical studies.

Extracellular histones promote calcium phosphate-dependent calcification in mouse vascular smooth muscle cells.

Vascular calcification, a major risk factor for cardiovascular events, is associated with a poor prognosis in chronic kidney disease (CKD) patients. This process is often associated with the transformation of vascular smooth muscle cells (VSMCs) into cells with osteoblast-like characteristics. Damage-associated molecular patterns (DAMPs), such as extracellular histones released from damaged or dying cells, are suspected to accumulate at calcification sites. To investigate the potential involvement of DAMPs in vascular calcification, we assessed the impact of externally added histones (extracellular histones) on calcium and inorganic phosphate-induced calcification in mouse VSMCs. Our study found that extracellular histones intensified calcification. We also observed that the histones decreased the expression of VSMC marker genes while simultaneously increasing the expression of osteoblast marker genes. Additionally, histones treated with DNase I, which degrades dsDNA, attenuated this calcification, compared with the non-treated histones, suggesting a potential involvement of dsDNA in this process. Elevated levels of dsDNA were also detected in the serum of CKD model mice, underlining its potential role in vascular calcification in CKD. Our findings suggest that extracellular histones could play a pivotal role in the vascular calcification observed in CKD.

Anatomical Brushite-Coated Mg-Nd-Zn-Zr Alloy Cage Promotes Cervical Fusion: One-Year Results in Goats.

In this study, an anatomical brushite-coated Mg-Nd-Zn-Zr alloy cage was fabricated for cervical fusion in goats. The purpose of this study was to investigate the cervical fusion effect and degradation characteristics of this cage in goats. The Mg-Nd-Zn-Zr alloy cage was fabricated based on anatomical studies, and brushite coating was prepared. Forty-five goats were divided into three groups, 15 in each group, and subjected to C2/3 anterior cervical decompression and fusion with tricortical bone graft, Mg-Nd-Zn-Zr alloy cage, or brushite-coated Mg-Nd-Zn-Zr alloy cage, respectively. Cervical radiographs and computed tomography (CT) were performed 3, 6, and 12 months postoperatively. Blood was collected for biocompatibility analysis and Mg2+ concentration tests. The cervical spine specimens were obtained at 3, 6, and 12 months postoperatively for biomechanical, micro-CT, scanning electron microscopy coupled with energy dispersive spectroscopy, laser ablation-inductively coupled plasma-time-of-flight mass spectrometry, and histological analysis. The liver and kidney tissues were obtained for hematoxylin and eosin staining 12 months after surgery for biosafety analysis. Imaging and histological analysis showed a gradual improvement in interbody fusion over time; the fusion effect of the brushite-coated Mg-Nd-Zn-Zr alloy cage was comparable to that of the tricortical bone graft, and both were superior to that of the Mg-Nd-Zn-Zr alloy cage. Biomechanical testing showed that the brushite-coated Mg-Nd-Zn-Zr alloy cage achieved better stability than the tricortical bone graft at 12 months postoperatively. Micro-CT showed that the brushite coating significantly decreases the corrosion rate of the Mg-Nd-Zn-Zr alloy cage. In vivo degradation analysis showed higher Ca and P deposition in the degradation products of the brushite-coated Mg-Nd-Zn-Zr alloy cage, and no hyperconcentration of Mg was detected. Biocompatibility analysis showed that both cages were safe for cervical fusion surgery in goats. To conclude, the anatomical brushite-coated Mg-Nd-Zn-Zr alloy cage can promote cervical fusion in goats, and the brushite-coated Mg-Nd-Zn-Zr alloy is a potential material for developing absorbable fusion cages.

Positive effect of inoculation with an Aspergillus strain on phosphorus and iron nutrition plus volatile organic compounds in rice.

We explored the potential of a fungal strain Aspergillus costaricensis KS1 for modulating growth and nutrient mobilization in rice. At laboratory conditions, there was a decline in pH of the medium on inoculation with the strain and the production of citric acid was observed under broth conditions. Similarly, there was higher solubilization of tricalcium phosphate and siderophore production in liquid medium on inoculation with the strain. The effect of inoculation of KS1 was studied in rice and higher growth and yield were observed on inoculation compared to control. The content of phosphorus and iron in stem and roots of KS1 inoculated plants was higher in comparison with uninoculated control. There was also increased availability of phosphorus and iron content in soil grown with KS1 inoculated plants. In addition, inoculation with strain resulted in a higher content of volatile organic compounds such as linoleic acid, linolenic acid, and ethyl isoallocholate in stem of rice. A. costaricensis KS1 can be used for improving phosphorus and iron nutrition and impart tolerance against stresses in rice.

Enhancing cranial defect repair in rats: investigating the effect of combining Total Flavonoids from Rhizoma Drynariae with calcium phosphate/collagen scaffolds.

OBJECTIVE: To investigate the therapeutic efficacy of total flavonoids of Rhizoma Drynariae (TFRD) in conjunction with a calcium phosphate/collagen scaffold for the repair of cranial defects in rats. METHODS: The subjects, rats, were segregated into four groups: Control, TFRD, Scaffold, and TFRD + Scaffold. Cranial critical bone defects, 5 mm in diameter, were artificially induced through precise drilling. Post-surgery, at intervals of 2, 4, and 8 weeks, micro-CT scans were conducted to evaluate the progress of skull repair. Hematoxylin-eosin and Masson staining techniques were applied to discern morphological disparities, and immunohistochemical staining was utilized to ascertain the expression levels of local osteogenic active factors, such as bone morphogenetic protein 2 (BMP-2) and osteocalcin (OCN). RESULTS: Upon examination at the 8-week mark, cranial defects in the Scaffold and TFRD + Scaffold cohorts manifested significant repair, with the latter group displaying only negligible foramina. Micro-CT examination unveiled relative to its counterparts, and the TFRD + Scaffold groups exhibited marked bone regeneration at the 4- and 8-week intervals. Notably, the TFRD + Scaffold group exhibited substantial bone defect repair compared to the TFRD and Scaffold groups throughout the entire observation period, while histomorphological assessment demonstrated a significantly higher collagen fiber content than the other groups after 2 weeks. Immunohistochemical analysis further substantiated that the TFRD + Scaffold had augmented expression of BMP-2 at 2, 4 weeks and OCN at 2 weeks relative to other groups. CONCLUSIONS: The synergistic application of TFRD and calcium phosphate/collagen scaffold has been shown to enhance bone mineralization, bone plasticity, and bone histomorphology especially during initial osteogenesis phases.