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.

UNASSIGNED: The aim of the study was to study the role of nanobacteria in the formation of renal calculi and the underlying mechanism.
UNASSIGNED: A total of 90 clean Wistar male rats were randomly divided into a negative control group, an experimental group, and an interference group. From the end of the first week of modelling, 10 consecutive times once a week, 3 rats in each group were randomly selected to measure the biochemical blood markers and urine metabolism. After sacrifice, the formation of kidney stones was assessed by observing the ultrastructure of the kidney by electron microscopy and pathohistology. Finally, the expression of calcium-sensitive receptor (CaSR) and claudin-14 protein in the kidney tissue was examined by western blotting.
UNASSIGNED: Compared with the control group, the gross structure of the kidney was changed in the model group. At the fourth week of modelling, the rats in the nanobacteria group had significantly enlarged kidneys and increased kidney-to-body ratio, and the difference had statistical significance (p < 0.05). The colour of the kidney profile was dark, the structure of the skin pulp was less clear, and the accumulation of yellowish particles was observed at the junction of the cortical pulp. The creatinine, uric acid, urea nitrogen, and urinary calcium of the rats in the nanobacteria group began to increase at the third week, and the difference between the third and eighth week had statistical significance (p < 0.05). However, the difference between the 3 groups had no statistical significance after the eighth week. At the fourth week, we observed the formation of calculi, which were mainly distributed in the renal tubules and surrounding tissues. The kidney stone formation rate was 52.4% in the nanobacteria group and 27.8% in the interference group, and the difference had statistical significance (p < 0.05). Ultrastructure observations revealed that from the fourth week, the renal tissues in the nanobacteria group showed expanded renal tubules, swollen renal tubular epithelium, granular degeneration, shedding and lymphocyte infiltration of renal tubular epithelial cells, and a small amount of calcium salt crystals in renal tubules. At the third week, the expression of CaSR and Claudin-14 protein in the nanobacteria group increased, and the difference had statistical significance (p < 0.05). The expression of CaSR and Claudin-14 was positively correlated with urinary calcium (p < 0.05).
UNASSIGNED: The formation of renal calculi began in the fourth week after the model was established, and the crystals were mostly located in the renal tubules. During the formation of renal calculi, the renal tubular epithelial cells were damaged, showing granular degeneration and small amounts of calcium salt crystals, accompanied by a few renal tubules beginning to expand and epithelial swelling, granular degeneration, necrosis and shedding of renal tubular epithelial cells, lymphocyte infiltration in the renal interstitium, and small amounts of calcium salt crystals in the renal tubules, which aggravated with time. The serum creatinine, serum uric acid, urea nitrogen, and urinary calcium levels increased with time from the third week and returned to normal after the eighth week. The expression of CaSR and Claudin-14 protein was upregulated and positively correlated with the 24-h urinary calcium excretion value.

During durst storms, also biological material is transported from arid areas such as the Sahara Desert. In the present work, rain samples containing significant amounts of mineral dust have been collected in Granada during different red rain episodes. Biological features (bacteria, biofilm, pollen grain and fungal spore) as well as size-particle distribution and mineralogical composition were studied by SEM. Nanobacteria were observed for the first time in red rain samples. A preliminary metabarcoding analysis was performed on three red rain samples. Here, Bacillota made up 18 % and Pseudomonadota 23 % of the whole prokaryotic community. The fungal community was characterized by a high abundance of Ascomycota and, dependent on the origin, the presence of Chytridiomycota. By means of 16S rRNA sequencing, 18 cultivable microorganisms were identified. In general, members of the phyla Pseudomonadota and Bacillota made up the majority of taxa. Some species, such as Peribacillus frigoritolerans and Bacillus halotolerans were isolated during three different red rain episodes. Generally, red rain carries a wide variety of microorganisms, being their ecosystem and health effects largely unknown.

Kidney stones have been associated with an increased risk of chronic kidney diseases, end-stage renal failure. This study is devoted to isolate nanobacteria from patients with active urolithiasis and investigate the in vitro and in vivo antinanobacterial activity of some antibiotics alone or in combination with extracts of irradiated herbs from certain medicinal plants. Nanobacteria were detected using scanning (SEM) and transmission (TEM) electron microscopy, protein electrophoresis (SDS-PAGE) and DNA profile. The antimicrobial susceptibility of some biofilm-producing nanobacterial isolates was evaluated. The effect of medicinal plant extracts on growth was tested. A combination treatment between the most potent extracts and antibiotics was tested on biofilm production, protein profile, release of 260 nm absorbing material, protein content, and ultrastructure of the strongest biofilm producers. In vivo study of nanobacteria and its treatment by the most potent agents was evaluated on male rats. Renal function was measured in serum; histological examination and oxidative stress parameters were determined in kidney tissues. Results showed that streptomycin, trimethoprim/sulfamethoxazole, doxycycline, and water extracts of irradiated khella at 6 kGy had antinanobacterial activity. Meanwhile, the synergistic effect of the aqueous extract of irradiated Khella and doxycycline showed higher inhibition activity on microbial growth and biofilm production. They affected dramatically the strength of its cell membrane and subsequently its ultrastructure. Moreover, these results are confirmed by ameliorations in renal function and histological alterations. It could be concluded that the combination of DO and an aqueous extract of irradiated khella has an antinephrotoxic effect against nanobacteria-induced renal toxicity.

BACKGROUND: The differences in protein expression of calcium sensitive receptor (CaSR) and claudin-14 in a kidney stone model established by nanobacteria (NB) and ethylene glycol (EG) were compared.
METHODS: Ninety Wistar male rats were randomly divided into the NB group, the EG group, and the blank control group (NC group), with 30 rats in each group. Three rats of each group were sacrificed every week after injection. Histopathology was used to evaluate the stone formation of each group. The expression of CaSR and claudin-14 protein was detected by immunohistochemistry every week.
RESULTS: There was formation of bright crystals in the kidneys of the EG group and the NB group, but not the NC group. At the 3rd week, the expression of CaSR and claudin-14 in the kidney tissue of the EG group began to increase while that in the NB group increased at the 4th week. The expression of CaSR and claudin-14 protein in the EG group was stronger than that in the NB group. Meanwhile, CaSR was expressed in the NC group but did not change significantly. Claudin-14 was not expressed in the NC group.
CONCLUSIONS: Our results indicate that the traditional EG kidney stone modeling method is more rapid than the NB kidney stone modeling method, with a high stone formation rate, and the CaSR and claudin-14 protein expression levels are higher. Meanwhile, the NB used to establish the kidney stone model was isolated from patients with kidney stones, which may imitate the process of natural formation of kidney stones of patients. Therefore, the results of our research are more conducive to related research on the etiology of stones.

Cyanobacterial blooms affect biotic interactions in aquatic ecosystems, including those involving heterotrophic bacteria. Ultra-small microbial communities are found in both surface water and groundwater and include diverse heterotrophic bacteria. Although the taxonomic composition of these communities has been described in some environments, the involvement of these small cells in the fate of environmentally relevant molecules has not been investigated. Here, we aimed to test if small-sized microbial fractions from a polluted urban lagoon were able to degrade the cyanotoxin microcystin (MC). We obtained cells after filtration through 0.45 as well as 0.22 μm membranes and characterized the morphology and taxonomic composition of bacteria before and after incubation with and without microcystin-LR (MC-LR). Communities from different size fractions (< 0.22 and < 0.45 μm) were able to remove the dissolved MC-LR. The originally small-sized cells grew during incubation, as shown by transmission electron microscopy, and changed in both cell size and morphology. The analysis of 16S rDNA sequences revealed that communities originated from < 0.22 and < 0.45 μm fractions diverged in taxonomic composition although they shared certain bacterial taxa. The presence of MC-LR shifted the structure of < 0.45 μm communities in comparison to those maintained without toxin. Actinobacteria was initially dominant and after incubation with MC-LR Proteobacteria predominated. There was a clear enhancement of taxa already known to degrade MC-LR such as Methylophilaceae. Small-sized bacteria constitute a diverse and underestimated fraction of microbial communities, which participate in the dynamics of MC-LR in natural environments.

Kidney stone disease is one of the oldest diseases known to medicine; however, the mechanisms of stone formation and development remain largely unclear. Over the past decades, a variety of theories and strategies have been developed and utilized in the surgical management of kidney stones, as a result of recent technological advances. Observations from the authors and other research groups suggest that there are five entirely different main mechanisms for kidney stone formation. Urinary supersaturation and crystallization are the driving force for intrarenal crystal precipitation. Randall\'s plaques are recognized as the origin of calcium oxalate stone formation. Sex hormones may be key players in the development of nephrolithiasis and may thus be potential targets for new drugs to suppress kidney stone formation. The microbiome, including urease‑producing bacteria, nanobacteria and intestinal microbiota, is likely to have a profound effect on urological health, both positive and negative, owing to its metabolic output and other contributions. Lastly, the immune response, and particularly macrophage differentiation, play crucial roles in renal calcium oxalate crystal formation. In the present study, the current knowledge for each of these five aspects of kidney stone formation is reviewed. This knowledge may be used to explore novel research opportunities and improve the understanding of the initiation and development of kidney stones for urologists, nephrologists and primary care.

A total of 108 blood samples obtained from 28 male and 80 female patients diagnosed with ME were diluted in sterile, Ringer\'s Solution and forced (by suction) through 0.2 µm filters. Of the 28 male samples, 4 yielded filterable bacteria and of the 80 female samples, 18 gave filterable bacteria; as a result, of the total of 124 samples, 22 yielded FB. Filterable (0.4 and 0.2, but not 0.1micron filterable) bacteria were also isolated from the nose throat and skin of paediatric patients and from the throat and skin of staff at an emergency paediatric hospital. The highest percentage of bacterial passage occurred through the largest (0.4 µm) pores. The results show that ultrasmall bacteria occur in ME patients and in paediatric patients and nurses. The potential pathogenic role of such filterable bacteria is briefly discussed.

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.

Sialolithiasis is the primary etiology for parotid and submandibular swelling, potentially resulting in discomfort, bacterial infections, and hospitalization. The etiology of sialolith formation is unknown. Currently, the proposed etiologies range from inflammation, coalescence of organic molecules, sialomicrolith formation, pH changes, and biofilm formation. In this study, we performed a descriptive analysis of images obtained through electron microscopy of sialoliths. Based on our findings and descriptive analysis, we hypothesize that sialolith formation is likely multifactorial and begins with biofilm formation. Biofilm formation then triggers a host immune response, and it is the interaction of biofilm with host immune cells and calcium nanoparticles that forms the nidus and creates a favorable environment for calcium precipitation.
Sialoliths were extracted from patients and imaged under light and scanning electron microscopy. Specimens for light microscopy were prepared using a diamond saw. Specimens for electron microscopy were freeze-fractured, thus providing an undisturbed view of the core of the sialolith.
We were able to identify clear evidence of biofilm caves at the core of each sialolith. These biofilm caves were complex with the presence of bacteria and dehydrated extrapolysaccharide matrix, host cells (immune cells, platelets and erythrocytes), and calcium nanoparticles.
The etiology of sialolith formation is likely multifactorial. We propose that biofilm formation within a single salivary gland or duct leads to local ductal injury, which results in the influx of host immune cells that interact with the biofilm and calcium nanoparticles, creating a scaffold upon which further calcium deposition can occur.
NA Laryngoscope, 130:69-74, 2020.