Nanobacteria Research Articles

Muramyl Dipeptide-Presenting Polymersomes as Artificial Nanobacteria to Boost Systemic Antitumor Immunity.

The clinical efficacy of cancer vaccines is closely related to immunoadjuvants that play a crucial role in magnifying and prolonging the immune response. Muramyl dipeptide (MDP), a minimal and conserved peptidoglycan found in almost all bacteria, can trigger robust immune activation by uniquely antagonizing the nucleotide-binding oligomerization domain 2 (NOD2) pathway. However, its effectiveness has been hindered by limited solubility, poor membrane penetration, and rapid clearance from the body. Here, we introduce MDP-presenting polymersomes as artificial nanobacteria (NBA) to boost the antitumor immune response. The NBA, featuring abundant MDP molecules, induces superior stimulation of immune cells including macrophages and bone marrow-derived dendritic cells (BMDCs) compared to free MDP, likely via facilitating immune cell uptake and cooperatively stimulating systemic NOD2 signaling. Importantly, systemic administration of NBA significantly enhances the chemo-immunotherapy of B16-F10 melanoma-bearing mice pretreated with doxorubicin by reversing the immunosuppressive tumor microenvironment. Furthermore, NBA carrying ovalbumin and B16-F10 cell lysates induces robust OVA-IgG antibody production and effectively inhibit tumor growth, respectively. The artificial nanobacteria hold great promise as a potent systemic immunoadjuvant for cancer immunotherapy.

A preliminary study on calcifying nanoparticles in dental plaque: Isolation, characterization, and potential mineralization mechanism.

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. 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). 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. 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.

Calcifying nanoparticles initiate the calcification process of mesenchymal stem cells in vitro through the activation of the TGF-β1/Smad signaling pathway and promote the decay of echinococcosis

Abstract The role of the calcifying nanoparticles (CNPs) in the calcification process of the outer cyst wall in hepatic cystic echinococcosis (HCE) remains unknown. CNPs were isolated from the tissues of the patients with HCE. Western blotting, alkaline phosphatase staining, and alizarin staining were performed to detect the cellular calcium ion deposition induced by the CNPs. CCK-8 and flow cytometry assays were conducted to determine the effect of CNPs on the apoptosis of mesenchymal stem cells (MSCs). Western blot experiments were performed to examine the expression levels of apoptosis-related factors and TGF-β1/Smad signaling pathway constituents. Treatment with CNPs induced the differentiation of MSCs. Calcium-related proteins, including OPN, BMP-2, and RUNX2, were upregulated after the CNP treatment. Similarly, CNP exposure increased the cellular calcium ion deposition in MSCs. In addition, the expression of Bax and Caspase-8 was elevated by the CNPs in MSCs. Treatment with CNPs promoted MSC apoptosis and inhibited the MSC growth. The TGF-β1/Smad signaling pathway was also activated after the CNP treatment. This study indicated that CNPs may play a critical role in initiating calcification of the outer cyst wall of HCE and promote the decay of echinococcosis, providing a new strategy for the treatment of hepatic echinococcosis.

Nanoparticulate System Based on Calcium-Crosslinked Carbomer Retards Percutaneous Drug Permeation: New Insight Into Skin Barrier Functions.

The stratum corneum poses a formidable barrier for dermal and transdermal delivery of drugs. Besides the stratum corneum barrier, the viable epidermis poses another challenge to pharmaceutical formulators. A drug is probably transdermally permeable if it rapidly crosses the epidermal secondary barrier, while stimulation of lamellar body secretion from granular cells and intracellular release of Ca++ from endoplasmic reticulum (ER) result in retardation. To evaluate the skin permeability of lidocaine HCl loaded in nanoparticles made of carbomer calcified with calcium gluconate, while figuring out the physiological mechanism that regulates the Ca++ related skin barrier function. Lidocaine hydrochloride was loaded in a nanoparticulate system based on calcified carbomer, fabricated by using a water-in-oil microemulsion as a precursor. In vitro release and percutaneous permeation testing were carried out to compare between calcified and non-calcified nanoparticles. In addition, comparison was also made between calcified nanoparticles using carbomer gels prepared at two pH values and at two different ratios of Ca++/carbomer. A unique structure of the calcified nanoparticles has been proposed, in which the carbomer nanoparticles are partially coated by gluconate ions through hydrogen bonding and partially through ionic interactions with calcium ions. Although the in vitro release data showed no difference between non-calcified and calcified carbomer nanoparticles, a calcium-related phenomenon of skin retardation has been revealed. It has been proposed that stimulation of lamellar body secretion from granular cells and Ca++ release from ER, which is elicited by the calcium gluconate-coated nanoparticles, result in dermal retardation of lidocaine.

SPOTLIGHT ON THE ASSOCIATION BETWEEN NANOBACTERIA AND SOME HUMAN DISEASES

Nanobacteria are the smallest cell-walled bacteria, recently discovered in human and commercial cell culture serum, also known as calcifying nanoparticles (CNPs). Nanobacteria are the name of a proposed class of living organisms particularly cell-walled microorganisms with a size much smaller than the generally accepted lower limit size for life (about 200nm). Recently, histopathological immunohistochemical staining, transmission electron microscopy (TEM), and calcific staining studies revealed the association of nanobacteria in kidney and gall stones. Nanobacteria have been appeared to contribute to different benign and malignant calcifications in the form of calcium phosphate crystals and contribute to malignant calcifications in breast and ovarian cancer. Nanobacteria also cause prostatic stones in patients with chronic pelvic pain syndrome due to chronic prostatitis and are present in dental and pulp calculus. The evidence that nanobacteria exist within the human body and are closely related to numerous sorts of diseases is now overpowering. However, future research is required to reveal their nature and impact on health and disease. This paper aimed to highlight the harmful role of nano-bacteria on human health and to describe the association of nanobacteria and their hypothesized relationship with some human diseases.

Potential Risk of Calcified Nanoparticles for Recurrent Urinary Tract Infection after Minimally Invasive Percutaneous Nephrolithotomy for Renal Calculi.

Among urolithiasis, urolithiasis is a more extensive and difficult to treat disease. In recent years, with the development of endoscopic equipment and related surgical methods, a new treatment, namely minimally invasive percutaneous nephrolithotomy, appears and is widely used in the treatment of renal calculi. It has been developed for more than 50 years since its appearance. Its advantages are a high stone clearance rate, small surgical trauma, fewer intraoperative and postoperative complications, and a short recovery time for postoperative patients. It is widely used in clinical treatment. The purpose of this paper is to study the potential risk factors of recurrence and the possibility of urinary tract infection in patients with renal calculi after minimally invasive percutaneous nephrolithotomy (MPCNL) with calcified nanoparticles, so as to obtain correct nursing knowledge and reduce recurrence and infection. In order to explore the renal calculi caused by calcified nanoparticles (CNPs), we established a rat model of renal calculi, injected CNPs into rats, collected experimental samples after a specified time, and determined NGAL, OPN and MCP-1 by enzyme-linked immunosorbent assay (ELISA) and compared them with the formation of crystallization. In the study of the possible influencing factors of septic shock after PCNL, we established a multivariate regression analysis and model, used statistical methods and professional statistical software, and used the method of classified data analysis to find out the influencing factors. After the experiment, we found that positive urine culture (P < 0.05) and preoperative urinary tract infection (P < 0.05) were significant influencing factors, and the total accuracy was 99.2%. At the same time, it is concluded that calcium nanoparticles can promote the retention, adhesion and aggregation of calcium salt crystals in the kidney, and then lead to the formation of stones.

Characterization of Biofilm Producer Nanobacteria Isolated from Kidney Stones of Some Egyptian Patients.

&lt;b&gt;Background and Objective:&lt;/b&gt; Nanobacteria (NB) appear to contribute to many calcifying diseases including kidney stones which represent a common problem with inadequate prevention exist. NB framing itself with a mineral coat that assists as a primary defence shield against the immune system, antibiotics. This study aims to collect and detect nanobes from different kidney stones from patients with active urolithiasis then investigated the anti-nano-bacterial activity of some antibiotics alone or in combination with extracts of irradiated herbs of certain medicinal plants which will represent a new approach to therapy for patients with kidney stones. &lt;b&gt;Materials and Methods:&lt;/b&gt; Total of 32 nanobes were isolated from 54 kidney stones. Fourier Transforms Infrared Spectroscopy (FTIR) revealed that calcium and phosphate are the main components of stones. Scanning Electron Microscopy (SEM) with Energy-dispersive X-ray spectroscopy (EDX) and Transmission Electron Microscope (TEM), showed that nanobes were Gram-ve cocci with size ranged from (375:600 nm). The biofilm production ability of nanobes was estimated qualitatively and quantitatively. &lt;b&gt;Results:&lt;/b&gt; The results revealed that all were strong biofilm producers. Further, the antibiotic susceptibility test indicates their resistance towards most of the tested antibiotics. Molecular identification of the strong biofilm producer isolates by ribosomal ribonucleic acid (rRNA) revealed that it is indicated by 85.37% to &lt;i&gt;Bartonella apis&lt;/i&gt; strain PEB0122. &lt;b&gt;Conclusion:&lt;/b&gt; The findings of the current study evidenced that combination treatment between Doxycycline (DO) and water extract of khella exhibited a significant reduction in biofilm formation ability of the strongest producers nanobes. Therefore, this treatment can play a role in enhancing public health, especially with patients who suffer from recurrent kidney stone formation.

Establishment of a male Wistar rat model of nanobacteriainduced kidney stones

Purpose: To establish a male Wistar rat model of nanobacteria (NB)-induced kidney stones.&#x0D; Methods: Sixty male Wistar rats were randomly divided into control group (NC group) given caudal vein injection of saline + saline gavage, and NB-induced stone group (NBS group) given caudal vein injection of NB + saline gavage.&#x0D; Results: Compared with NC, serum creatinine, blood uric acid, urea nitrogen and urinary calcium levels in NBS group increased between weeks 3 and 8 (p &lt; 0.05). Kidney index (kidney weight/body weight ratio) in the NBS group was higher than that in NC group from weeks 8-10. At week 8, urine pH and serum phosphorus in NBS group were higher than those in NC group (p &lt; 0.05). Between weeks 6 and 7, serum calcium in NBS group was higher than that in NC group (p &lt; 0.05). Calcium crystals in NBS rats were distributed mostly in the distal and proximal convoluted tubules. However, no such crystals were observed in NC rats. Similarly, no such pathological changes were seen in the renal tissue of NC group. Calculus analysis showed that stone formation was higher in NBS group than in NC group (p &lt; 0.05). There was no significant difference in micro-CT between the two groups (p ˃ 0.05).&#x0D; Conclusion: The successful establishment of the Wistar rat kidney stone model using NB cultured from urine of upper urinary tract stone patient is potentially useful for further etiological studies on kidney stone formation.

Calcium sensitive receptor and claudin-14 expression in kidney tissues of two kidney stone models.

IntoductionThe 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.Material and methodsNinety 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.ResultsThere 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.ConclusionsOur 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.

Preliminary Evaluation of Nanobacteria on Crystal Retention, CaSR, and Claudin‐14 Expression in HK‐2 Cells

To evaluate cell morphology, crystal adhesion, cell damage, Calcium sensitive receptor (CaSR), and Claudin protein‐14 (Claudin‐14) expression at different time intervals and explore the role of nanobacteria in the formation of urinary calculi. In this experiment, HK‐2 cells were cocultured with nanobacteria (NB) in the absence or presence of tetracycline (Tet). Cells treated with calcium oxalate monohydrate (COM) crystals were used as a positive control of urinary stone‐induced cell damage. After which, cell morphology was evaluated by hematoxylin‐eosin staining in comparison to untreated HK‐2 cells (negative control). Use different methods to assess cell damage, crystal adhesion, and protein expression. (The degree of cell damage, crystal adhesion, and protein expression were evaluated by various methods). It was found that the degree of cell damage observed in Tet + NB‐treated cells was significantly lower than that in NB‐treated cells. Lactate dehydrogenase (LDH) leakage was higher in COM‐exposed than in control cells (P &lt; 0.05). However, LDH release from both NB‐ and Tet + NB‐treated cells was significantly lower than from COM‐treated cells (P &lt; 0.05). The relative expression of CaSR and Claudin‐14 proteins was higher in NB, COM, and TET + NB cells than in control cells (P &lt; 0.001) and was lower in Tet + NB than in NB cells (P &lt; 0.01). And P &lt; 0.05 means that the difference was statistically significant, and P &lt; 0.001 means that there was a significant difference between the both things. From the cell morphology, the cell damage in the COM group was greater than that in the NB group, and the cell damage markers in the COM group and the NB group were elevated. NB caused damage to HK‐2 cells by inducing lipid peroxidation, and the degree of damage was increased in processing time. The adhesion of HK‐2 cells to COM crystals increased after injury and was proportional to the duration of NB coculture. NB upregulated the expression of CaSR and Claudin‐14 in HK‐2 cells.

Polyamino acid calcified nanohybrids induce immunogenic cell death for augmented chemotherapy and chemo-photodynamic synergistic therapy.

Background: Monotherapy for cancer treatment is limited by unstable efficacy and uncontrollable toxic side effects, while the multifunctional nanoplatform with complex preparation process cannot avoid the potential toxicity of each functional component.Methods: We exploited tumor-specific activated polyamino acid calcified nanoparticles (CHC NPs) as new-type oxidative stress amplification of anticancer drugs via building a safe and biodegradable multifunctional nanoplatform. Giving priority to chemotherapy, and synergizing chemodynamic therapy (CDT) with photodynamic therapy (PDT), this strategy was to achieve enhanced chemotherapy, simultaneously inducing immunogenic cell death and inhibiting tumor cell invasion.Results: Based on amorphous calcium carbonate, pH-responsive nanocarrier was prepared with classical chemotherapeutic drug 10-hydroxycamplothecin (HCPT) and photosensitizer Chlorin e6 (Ce6) to realize multifunctional nanotheranostics.Conclusion: Inventive calcified nanohybrids, where topoisomerase inhibited by HCPT to prevent DNA synthesis, the generation of •OH induced via Fenton reaction, along with a large amount of 1O2 produced by Ce6, might be a promising strategy for anti-tumor combination therapy in clinical translation.

Tetracycline attenuates calcifying nanoparticles-induced renal epithelial injury through suppression of inflammation, oxidative stress, and apoptosis in rat models.

Calcifying nanoparticles (CNPs) has been associated with the occurrence and development of kidney stones, but the exact mechanism is not clear. This study aimed to establish a rat model of CNP-induced renal epithelial injury and assess the efficacy of tetracycline in preventing this injury. Kidney stones from patients after percutaneous nephrolithotomy (PCNL) were collected to isolate and culture CNPs. Thirty Sprague-Dawley rats were divided into three groups: the sham group (G1), the CNP group (G2), and the CNP + tetracycline group (G3). Rats in G2 and G3 were given an intravenous injection of CNPs via the tail vein, while rats in G1 were given saline. Meanwhile, rats in G3 were given tetracycline by gavage twice a day at a dose of 25 mg/kg. After 8 weeks, the 24-h urine of all rats was collected, and all rats were sacrificed to obtain blood and kidneys. The results revealed that in G2, activities of antioxidant enzymes such as superoxide dismutase and catalase were significantly lower than those in G1, while malondialdehyde activity in G2 was significantly higher than that in G1 and both of them were inhibited by tetracycline co-treatment in G3. CNPs significantly increased expression of inflammatory cytokines, including monocyte chemotactic protein 1 and interleukin 6, which were largely alleviated in G3. CNPs significantly increased TUNEL-positive cells and the apoptosis activity of Bcl2-associated X protein but decreased B-cell lymphoma-2 level compared with that in G1, and was limited by tetracycline co-treatment in G3. Furthermore, CNPs led to notable renal tubular epithelial cell damage, hyaline cast formation, desquamation, swelling, vacuolization in histology, all of which were alleviated by tetracycline. Tetracycline can attenuate CNP-induced renal epithelial injury through suppression of inflammation, oxidative stress, and apoptosis.

Mechanism of nanobacteria promoting apoptosis of human gallbladder epithelial cells

Objective To observe the interaction between nanobacteria (NB) and human gallbladder epithelial cells, and explore the possible mechanism of porcelain gallbladder formation. Methods Absorbing bile from porcelain gallbladder and normal gallbladder to culture nanobacteria, the morphology of NB was observed under an electron microscope. Primary cultured human gallbladder epithelial cells were obtained by cutting out some normal gallbladders, and the expression of Cytokeratin-19 (CK19) was detected by immunofluorescence. NB of 1.0 Macfarland (MCF) was induced into human gallbladder epithelial cells at 0, 12, 24, 48 h. Cell damage and apoptosis rate were detected by Cell Counting Kit-8 (CCK-8), lactate dehydrogenase (LDH) kit and flow cytometry. Protein expression of B-lymphoma-2 (bcl-2), bcl-2 related X protein (bax), cytochrome C (Cyt-C), and cleaved Caspsae-9 were detected by Western blotting. Results The positive rate of NB in porcelain gallbladder and normal gallbladder bile was 88.9% and 15.0%. The difference was statistically significant (χ2=14.39, P<0.01). Under transmission electron microscopy, NB is round and unequal in size and has a diameter of about 80 nm. Immunofl-uorescence showed that CK19 protein was positive in primary cultured human gallbladder epithelial cells. NB induced human gallbladder epithelial cells at 0, 12, 24, 48 h, cell survival rate decreased significantly at 24, 48 h, the difference was statistically significant (F=305.501, P<0.01), the release of LDH began to increase significantly at 12 h, and the difference was statistically significant (F=5351.109, P<0.05). The apoptosis rate increased significantly at 24 h and 48 h, and the difference was statistically significant (F=597.457, P<0.01). The expression of bcl-2/bax was significantly decreased by Western blotting at 24 h and 48 h, and the difference was statistically significant (F=1 336.833, P<0.01). The relative expression of Cty-c was significantly increased at 12, 24, 48 h, and the difference was statistically significant (F=1 422.101, P<0.01). Cleved Caspsae-9 increased significantly at 24 h and 48 h, and the difference was statistically significant (F=2 348.061, P<0.01). Conclusion NB promotes apoptosis of human gallbladder epithelial cells through the mitochondrial pathway, which may be closely related to the formation of porcelain gallbladder. Key words: Nanobacteria; Human gallbladder epithelial cells; Apoptosis; Porcelain gallbladder

Dynamic study on the expression of CaSR in the formation of Nanobacteria renal stone

Objective To study the expression of CaSR in renal calculi by injecting nanoparticles into the tail vein and to investigate the role of CaSR in the formation of gallstones. Methods A total of 60 Wistar male rats were randomly divided into normal control group and nanobacterial group, with 30 rats in each group. The rats in the normal control group were given disposable intravenous injection with 1.2 mL of 0.9% sodium chloride solution via the tail vein, the rats in the nanobacterium group were given disposable intravenous injection with 1.2 mL of nanobacterial suspension via the tail vein at one time. Three rats in each group were sacrificed every week from the 1th to 10th week after injection. The gross morphology and crystal granule formation of the kidney were observed by using an anatomical microscope. Histopathological examination was performed every week to evaluate the stone formation in 3 male Wistar rats, and the expression of CaSR was detected by immunohistochemistry for 10 weeks. Results ①Pathological examination results: From the 4th to 10th week, the crystal particles were found in the renal tubules of Wistar male rats in the nanobacterial group, and mainly distributed in the proximal and distal convoluted tubules. from the 1th to 10th week, the Wistar rats in the control group had no crystal granules.②The results of immunohistochemistry: Persistent expression of CaSR in nanobacterial group from the 1th to 10 th week. From the 1th to 3th week, there was no significant difference between the nanobacterial group and the control group, but the expression intensity increased gradually and the expression intensity was significantly higher than that in the control group from the 4th to 10th week. From the 1th to 10th week, CaSR in the control group while also sustained expression, but its strength was no obvious change. From the 1th to 10th week, although the expression of CaSR was persistent in the control group, there was no significant change in its intensity. Conclusions The increased activity of CaSR may play an important role in the formation of Nanobacteria renal stone. Key words: Kidney Calculi; Nanostructures; Disease Models, Animal; Receptors, Calcium-Sensing

Neogene of crystal cell response in kidney stones: calcified nanoparticles

At present, it is thought that the inflammatory injury of the kidney plays a very important role in the formation of kidney stones in the crystal-cell reaction. In recent years, calcifying nanoparticles (CNP) is gradually considered as one of the initiators of the formation of kidney stones, and has become a research hotspot. This article reviews the role of calcified nanoparticles in the inflammatory response of renal calculi cells. Key words: Calcifying nanoparticles; Kidney stones; Inflammation

Antibiofilm effect of drug-free and cationic poly(D,L-lactide-co-glycolide) nanoparticles via nano-bacteria interactions.

Recently, nano-bio interactions and their biomedical impacts have drawn much attention, but nano-bacteria interaction and its function are unknown. Herein, we aim to synthesize drug-free and cationic nanoparticles (CNPs) and investigate CNP-bacteria interaction and its antibiofilm effect. The bioactivity of CNPs against Streptococcus mutans was examined by colony-forming units counting and scanning electron microscopy. CNP-bacteria interaction force was measured by atomic force microscopy. CNPs (217.7nm, 14.7mv) showed a concentration-dependent activity against bacteria. Particularly, CNPs at 200μg/ml completely inhibited planktonic bacterial growth and biofilm formation, and disrupted ∼70% mature biofilm. CNP-bacteria interaction force was up to 184nN. CNPs have great potentials for convenient local use for prevention and treatment of bacteria-related oral diseases.

Calcifying nanoparticles induce cytotoxicity mediated by ROS-JNK signaling pathways.

Calcifying nanoparticles (CNPs) play an important role in kidney stone formation, but the mechanism(s) are unclear. CNPs were isolated and cultured from midstream urine of patients with kidney stones. CNP morphology and characteristics were examined by electron microscopy and electrophoresis analysis. Chemical composition was analyzed using energy-dispersive X-ray microanalysis and Western blotting. Human renal proximal convoluted tubule cell (HK-2) cultures were exposed to CNPs for 0, 12 and 72h, and production of reactive oxygen species (ROS), mitochondrial membrane potential and apoptosis levels were evaluated. CNPs isolated from patients showed classical morphology, the size range of CNPs were 15-500nm and negative charge; they were found to contain fetuin-A. Exposure of HK-2 cells to CNPs induced ROS production, decreased mitochondrial membrane potential and decreased cell viability. Transmission electron microscopy showed that CNPs can enter the cell by phagocytosis, and micrographs revealed signs of apoptosis and autophagy. CNPs increased the proportion of apoptotic cells, down-regulated Bcl-2 expression and up-regulated Bax expression. CNPs also up-regulated expression of LC3-B, Beclin-1and p-JNK.CNPs are phagocytosed by HK-2 cells, leading to autophagy, apoptosis and ROS production, in part through activation of JNK signaling pathways. ROS and JNK pathways may contribute to CNP-induced cell injury and kidney stone formation.

Nanoparticle Tracking Analysis for the Enumeration and Characterization of Mineralo-Organic Nanoparticles in Feline Urine.

Urinary stone disease, particularly calcium oxalate, is common in both humans and cats. Calcifying nanoparticles (CNP) are spherical nanocrystallite material, and are composed of proteins (fetuin, albumin) and inorganic minerals. CNP are suggested to play a role in a wide array of pathologic mineralization syndromes including urolithiasis. We documented the development of a clinically relevant protocol to assess urinary CNP in 9 healthy cats consuming the same diet in a controlled environment using Nanoparticle Tracking Analysis (NTA®). NTA® is a novel method that allows for characterization of the CNP in an efficient, accurate method that can differentiate these particles from other urinary submicron particulates. The predominant nanoscale particles in feline urine are characteristic of CNP in terms of their size, their ability to spontaneously form under suitable conditions, and the presence of an outer layer that is rich in calcium and capable of binding to hydroxyapatite binders such as alendronate and osteopontin. The expansion of this particle population can be suppressed by the addition of citrate to urine samples. Further, compounds targeting exosomal surfaces do not label these particulates. As CNP have been associated with a number of significant urologic maladies, the method described herein may prove to be a useful adjunct in evaluating lithogenesis risk in mammals.

Cytotoxicity induced by nanobacteria and nanohydroxyapatites in human choriocarcinoma cells.

We explored the cytotoxic effects of nanobacteria (NB) and nanohydroxyapatites (nHAPs) against human choriocarcinoma cells (JAR) and the mechanisms of action underlying their cytotoxicity. JAR cells were co-cultured with NB and nHAPs for 48 h, and ultrastructural changes were more readily induced by NB than nHAPs. Autophagy in the plasma of JAR cells were observed in the NB group. The rate of apoptosis induced by NB was higher than that for nHAPs. The expression of Bax and FasR proteins in the NB group was stronger than that for the nHAP group. NB probably resulted in autophagic formation. Apoptosis was possibly activated via FasL binding to the FasR signaling pathway.

Comparison of Polymerase Chain Reaction and Immunologic Methods for the Detection of Nanobacterial Infection in Type-III Prostatitis

To compare the results of polymerase chain reaction (PCR) and immunologic methods for the detection of nanobacteria (NB) in the expressed prostatic secretions (EPSs) of patients with type-III prostatitis. In total, 150 patients with type-III prostatitis for whom conventional clinical treatment had failed were selected from September 2009 to April 2010. The EPS of each patient was divided into 3 parts, which were used for PCR analysis, indirect immunofluorescence staining (IIFS), and culture and subsequent indirect immunofluorescence staining (CIIFS). PCR analysis has a higher sensitivity than IIFS for the detection of NB in EPSs. Of 83 CIIFS-positive EPS samples, 79 (95.2%) were positive by PCR. Of 67 EPS samples that were negative by CIIFS, 60 (89.6%) were negative by PCR. The sensitivity of PCR for the detection of NB compared with the CIIFS method was 95.2%, with a specificity of 89.6%. The positive predictive value was 91.9%, and the negative predictive value was 93.8%. A comparative evaluation showed no statistically significant difference between PCR and CIIFS in the detection of NB in EPSs. A strong agreement in the positive and the negative results obtained by PCR and CIIFS for NB detection was found for all EPS samples. PCR analysis has a higher sensitivity than IIFS for NB detection in type-III prostatitis. PCR can detect nanobacterial infection in type-III prostatitis equally well as CIIFS and offers significant advantages for the rapid, simple, and economical detection of nanobacterial infection in type-III prostatitis.