OBJECTIVE: Calcifying nanoparticles (CNPs), referred to as nanobacteria (NB), are recognized to be associated with ectopic calcification. This study aims to isolate and culture CNPs from the dental plaque of patients with periodontal disease and investigate their possible role in unravelling the aetiology of periodontal disease.
METHODS: Supragingival and subgingival plaques were sampled from 30 periodontitis patients for CNPs isolation and culture. Alkaline phosphatase (ALP) content changes were tracked over time. Positive samples underwent thorough morphological identification via hematoxylin and eosin (HE) staining, Alizarin red S (ARS), and transmission electron microscopy (TEM). The chemical composition of CNPs analysis involved calcium (Ca) and phosphorus (P) content determination, Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD).
RESULTS: The subgingival plaque dental group exhibited a higher CNPs isolation rate at 36.67% (11/30) compared to the supragingival dental plaque group at 66.67% (20/30). ALP activity varied among the positive, negative and control groups. Morphological observation characterized the CNPs as round, oval, and ellipsoid particles with Ca deposits. Chemical analysis revealed the Ca/P ratio was 0.6753. Hydroxyl, methyl, carbonate, phosphate, hydrogen phosphate, and dihydrogen phosphate were detected by FTIR; the main chemical components detected by XRD were hydroxyapatite and tricalcium phosphate.
CONCLUSIONS: CNPs were found in periodontitis-related dental plaque and exhibited the potential to develop calcified structures resembling dental calculus. However, the potential involvement of ALP in CNPs formation requires deeper exploration, as does the precise nature of its role and the interrelation with periodontitis demand a further comprehensive investigation.

Candidate bacterial phylum Omnitrophota has not been isolated and is poorly understood. We analysed 72 newly sequenced and 349 existing Omnitrophota genomes representing 6 classes and 276 species, along with Earth Microbiome Project data to evaluate habitat, metabolic traits and lifestyles. We applied fluorescence-activated cell sorting and differential size filtration, and showed that most Omnitrophota are ultra-small (~0.2 μm) cells that are found in water, sediments and soils. Omnitrophota genomes in 6 classes are reduced, but maintain major biosynthetic and energy conservation pathways, including acetogenesis (with or without the Wood-Ljungdahl pathway) and diverse respirations. At least 64% of Omnitrophota genomes encode gene clusters typical of bacterial symbionts, suggesting host-associated lifestyles. We repurposed quantitative stable-isotope probing data from soils dominated by andesite, basalt or granite weathering and identified 3 families with high isotope uptake consistent with obligate bacterial predators. We propose that most Omnitrophota inhabit various ecosystems as predators or parasites.

Calciprotein particles (CPPs), which increasingly arise in the circulation during the disorders of mineral homeostasis, represent a double-edged sword protecting the human organism from extraskeletal calcification but potentially causing endothelial dysfunction. Existing models, however, failed to demonstrate the detrimental action of CPPs on endothelial cells (ECs) under flow. Here, we applied a flow culture system, where human arterial ECs were co-incubated with CPPs for 4 h, and a normolipidemic and normotensive rat model (10 daily intravenous injections of CPPs) to simulate the scenario occurring in vivo in the absence of confounding cardiovascular risk factors. Pathogenic effects of CPPs were investigated by RT-qPCR and Western blotting profiling of the endothelial lysate. CPPs were internalised within 1 h of circulation, inducing adhesion of peripheral blood mononuclear cells to ECs. Molecular profiling revealed that CPPs stimulated the expression of pro-inflammatory cell adhesion molecules VCAM1 and ICAM1 and upregulated transcription factors of endothelial-to-mesenchymal transition (Snail, Slug and Twist1). Furthermore, exposure to CPPs reduced the production of atheroprotective transcription factors KLF2 and KLF4 and led to YAP1 hypophosphorylation, potentially disturbing the mechanisms responsible for the proper endothelial mechanotransduction. Taken together, our results suggest the ability of CPPs to initiate endothelial dysfunction at physiological flow conditions.

Dietary phosphate overload induces chronic kidney disease (CKD), and calciprotein particles (CPPs), a form of nanoparticle comprising calcium phosphate and serum proteins, has been proposed to cause renal toxicity. However, the mechanism of CPP cytotoxicity in renal tubular cells is unknown. Here we show that in renal proximal tubular epithelial HK-2 cells, endocytosed CPPs accumulate in late endosomes/lysosomes (LELs) and increase their luminal pH by ~ 1.0 unit. This results in a decrease in lysosomal hydrolase activity and autophagic flux blockage without lysosomal rupture and reactive oxygen species generation. CPP treatment led to vulnerability to H2O2-induced oxidative stress and plasma membrane injury, probably because of autophagic flux blockage and decreased plasma membrane cholesterol, respectively. CPP-induced disruption of lysosomal homeostasis, autophagy flux and plasma membrane integrity might trigger a vicious cycle, leading to progressive nephron loss.

The process of kidney stone formation is complex and still not completely understood. Supersaturation and crystallization are the main drivers for the etiopathogenesis of uric acid, xanthine and cystine stones but this physicochemical concept fails to adequately explain the formation of calcium-based nephrolithiasis, which represents the majority of kidney stones. Contemporary concepts of the pathogenesis of calcium-based nephrolithiasis focus on a nidus-associated stone formation of calcium-based nephrolithiasis on Randall\'s plaques or on plugs of Bellini\'s duct. Randall\'s plaques originate from the interaction of interstitial calcium supersaturation in the renal papilla, vascular and interstitial inflammatory processes and mineral deposits of calcifying nanoparticles on the basal membrane of the thin ascending branch of the loop of Henle; however, plugs of Bellini\'s duct are assumed to be caused by mineral deposits on the wall of the collecting ducts. Aggregation and overgrowth are influenced by the interaction of matrix proteins with calcium supersaturated urine, by an imbalance between promoters and inhibitors of stone formation in the calyceal urine. Current research has elucidated many factors contributing to stone formation by revealing novel insights into the physiology of nephron and papilla, by analyzing vascular, inflammatory and calcifying processes in the renal medulla, by examining the proteome, the microbiome, promoters and inhibitors of stone formation in the urine and by conducting the first genome-wide association studies; however, more future research is mandatory to fill the gap of knowledge and hopefully, to obtain novel prophylactic, therapeutic and metaphylactic tools beyond the current state of knowledge.

Nanobacteria or calcifying nanoparticles are 80-500 nm sized nano-organisms that are physically associated with carbonate apatite mineral formations. They have been indicated in various diseases, including kidney stone formation, Alzheimer\'s disease, and atherosclerosis. Nanoparticles contain calcium and apatite-binding protein fetuin-A, a calcification inhibitor. However, recent evidence indicates that fetuin-A can form nucleation seeds or nidi that grow in size through ion sedimentation to become larger amorphous nanoparticles in the presence of excess calcium and apatite ions. Fetuin-A also functions as an inhibitor of meprin, a metalloproteinase implicated in inflammation and neurodegenerative diseases. During inflammation, meprin functions to regulate chemokine activity of monocyte chemotactic protein 1, which is associated with chronic inflammatory diseases, including atherosclerosis, renal inflammatory diseases, and multiple sclerosis (MS). In addition, calcium phosphate nanocrystals that contain fetuin-A are pro-inflammatory to macrophages and promote vascular smooth muscle cell mineralization, potentiating a vicious cycle of inflammation and calcification. Thus, mineral stress and inflammation appear to be associated with each other. Furthermore, fetuin-A deficient mice exhibited reduced experimental autoimmune encephalomyelitis severity. Thus, fetuin-A plays a direct role in the neuroinflammatory response. Indeed, the level of fetuin-A in cerebrospinal fluid has been defined as a biomarker of disease activity in MS. MS is a chronic, inflammatory, demyelinating, and neurodegenerative disease of the central nervous system (CNS) with an unknown etiology. The \"inside-out\" model of MS, supported by recent data, states that the initial axonal degeneration in the CNS occurs before demyelination, which then stimulates an auto-immune attack. It was shown very recently that influx of calcium from the extracellular space through nanoscale ruptures of the axonal plasma membrane predict axon degeneration in neuroinflammation. Calcium is an activator of calpains, proteases that function to break down the cytoskeleton, leading to neurodegeneration. Nanoruptures of the plasma membrane were suggested to occur at the early stages of axon damage, especially at nodes of Ranvier, which are devoid of myelin. Here, I propose that calcifying nanoparticles may have a role in the etiology and/or pathophysiology of MS. The initial event causing neurodegeneration may be due to the nanoparticles that have been suggested to easily cross the blood-brain barrier. Following this, the nanoparticles may create nanoruptures in the axonal membrane and also increase the calcium concentration around and within the neurons by forming nidi for calcification, eventually causing neurodegeneration. Nanoparticles can self-replicate; hence, they may represent an infectious causative agent for the development of MS.

Vascular calcification is a major contributor to increased cardiovascular mortality in chronic kidney disease (CKD). Recently, calciprotein particles (CPP) were identified to drive the calcification process. CPP may explain the effects of high phosphate on vascular calcification. Magnesium is a promising novel therapeutic approach to halt vascular calcification, because it inhibits CPP maturation and is associated with reduced cardiovascular mortality in CKD. We aim to examine the current evidence for the role of CPP in the calcification process and to explain how magnesium prevents calcification.
A recent meta-analysis concluded that reducing high phosphate levels in CKD patients does not associate with lowering cardiovascular mortality. Inhibition of CPP formation prevents phosphate-induced calcification in vitro. Consequently, delaying CPP formation and maturation may be a clinical approach to reduce calcification. Magnesium inhibits CPP maturation and vascular calcification. Clinical pilot studies suggest that magnesium is a promising intervention strategy against calcification in CKD patients.
CPP induce vascular calcification and are modulated by serum phosphate and magnesium concentrations. Magnesium is a strong inhibitor of CPP maturation and therefore, a promising therapeutic approach to reduce vascular calcification in CKD. Currently, several studies are being performed to determine the clinical outcomes of magnesium supplementation in CKD.

The N-TiO2/g-C3N4@diatomite (NTCD) composite has been prepared through a simple impregnation method, using titanium tetrachloride as precursor and urea as nitrogen-carbon source. Then the effects of calcination temperature on structure, surface property and photocatalytic activity of the catalysts were investigated. And XRD, TEM, XPS, FTIR and UV-vis diffuse adsorption spectroscopy were used to characterize the obtained powders. The photocatalytic activity of the NTCD was evaluated through the reduction of aqueous Cr (VI) under visible light irradiation (λ > 400 nm). The results demonstrated that the nano-TiO2 particles ranging from 15 to 30 nm in the crystal of anatase are well deposited on the surface of diatomite in the NTCD-500 which calcined at 500 °C for 2 h. Furthermore, the g-C3N4 with the lay thickness of 0.92 nm was attached to the surface of nano-TiO2. The N-doped TiO2 and g-C3N4 doped catalysts could co-enhance response in the visible light region and reduce band gap of NTCD-500 (Eg = 3.07 eV). And the NTCD-500 sample exhibited nearly 100% removal rate within 5 h for photocatalytic reduction of Cr (VI) which was higher activity than P25, crude TiO2@diatomite and g-C3N4@diatomite.

The use of nanotechnology for nanobacteria (or calcifying nanoparticles) treatment is a new creative approach. Use of selenium nanoparticles (SeNPs) as anti-nanobacterial agents might be considered as a bright promising approach due to their critical role in the inhibition of crystal growth and aggregation of calcium oxalate. Hence, in this study, we investigated the probable outcome of SeNPs inhibitory effects on growth of nanobacteria. Fragments of thirty urinary tract stones were chemically analyzed by X-ray diffraction (XRD) and urinary stones Kits for calcifying nanoparticles presence. Then powder of stone fragments were resuspended in Dulbecco\'s modified Eagle\'s medium (DMEM), sterilized by filtration and cultured in presence of 1, 5, 30, 60, and 90 μmol/L SeNPs concentrations. Besides, calcifying nanoparticles growth in the culture without SeNPs was measured spectrophotometrically. Also, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) analyses were used, where calcifying nanoparticles formation occurred. Results showed that in the culture without SeNPs, the positive calcifying nanoparticles detection was 60% while after adding SeNPs at 90 μmol/L, not any calcifying nanoparticles were observed. Further confirmation came out when Energy-dispersive X-ray (EDX) analysis showed calcium and phosphate peaks in the culture medium without any SeNPs while in the culture containing 90 μm/L SeNPs a decrease in calcium and other minerals was obvious. Therefore, SeNPs clearly restricted the growth of nanobacteria due to their inhibitory effects on calcium oxalate deposition.

BACKGROUND: Calcifying nanoparticles (NPs) have been proven to be associated with a variety of pathological calcification and previously detected in semen samples from patients with testicular microlithiasis (TM). The present study was designed to test the hypothesis if human-derived NPs could invade the seminiferous tubules and induce TM phenotype.
METHODS: The animals were divided into three groups. Normal saline (0.2 mL) was injected into the proximal right ductus deferens in group A as a control group. The experimental groups, B and C received Escherichia coli (106 cfu/mL, 0.2 mL) and human-derived NPs suspension (0.2 mL), respectively. Rats were euthanized in 2 batches at 2 and 4 weeks. Testicular pathology, ultrastructure and inflammatory mediators were assessed.
RESULTS: Chronic inflammatory changes were observed at 2 weeks in both groups B and C. Moreover, the innermost layer of sperm cells were structurally impaired and a zone of concentrically layered collagen fibers around the human NPs body was formed in the lumen of the seminiferous tubule in group C only, in which TM phenotype of remarkable calcification surrounded by cellular debris within the seminiferous tubules was built at 4 weeks.
CONCLUSIONS: The results obtained from our study suggested a potential pathogenic effect of NPs in the development of calcification within the seminiferous tubules, which should be addressed in the future studies.