Study of interactions between DNA and aflatoxin B1 using electrochemical and fluorescence methods

In this study, a carbon paste electrode modified with N-butylpyridinium hexafluorophosphate (BPPF₆) ionic liquid and DNA was introduced as an electrochemical biosensor to study the interaction between DNA and aflatoxin B1 molecules. For this purpose, variations in oxidation peak current of guanine in various concentrations of aflatoxin B1 were measured by using the differential pulse voltammetry (DPV) method. According to this study, the binding constant of DNA–aflatoxin B1 was found to be 3.5 × 10⁶ M⁻¹. This modified electrode was also used for determination of low concentrations of aflatoxin B1 by using differential pulse voltammetry. A linear dynamic range from 8.00 × 10⁻⁸ to 5.91 × 10⁻⁷ M and a limit of detection of 2.00 × 10⁻⁸ M resulted from DPV measurements. To confirm our results, a fluorescence study was also performed. It resulted in a binding constant of 2.8 × 10⁶ M⁻¹, which is in good agreement with that obtained from electrochemical study.     

A Dual Tracer 18F-FCH/18F-FDG PET Imaging of an Orthotopic Brain Tumor Xenograft Model

Early diagnosis of low grade glioma has been a challenge to clinicians. Positron Emission Tomography (PET) using ¹⁸F-FDG as a radio-tracer has limited utility in this area because of the high background in normal brain tissue. Other radiotracers such as ¹⁸F-Fluorocholine (¹⁸F-FCH) could provide better contrast between tumor and normal brain tissue but with high incidence of false positives. In this study, the potential application of a dual tracer ¹⁸F-FCH/¹⁸F-FDG-PET is investigated in order to improve the sensitivity of PET imaging for low grade glioma diagnosis based on a mouse orthotopic xenograft model. BALB/c nude mice with and without orthotopic glioma xenografts from U87 MG-luc2 glioma cell line are used for the study. The animals are subjected to ¹⁸F-FCH and ¹⁸F-FDG PET imaging, and images acquired from two separate scans are superimposed for analysis. The ¹⁸F-FCH counts are subtracted from the merged images to identify the tumor. Micro-CT, bioluminescence imaging (BLI), histology and measurement of the tumor diameter are also conducted for comparison. Results show that there is a significant contrast in ¹⁸F-FCH uptake between tumor and normal brain tissue (2.65 ± 0.98), but with a high false positive rate of 28.6%. The difficulty of identifying the tumor by ¹⁸F-FDG only is also proved in this study. All the tumors can be detected based on the dual tracer technique of ¹⁸F-FCH/ ¹⁸F-FDG-PET imaging in this study, while the false-positive caused by ¹⁸F-FCH can be eliminated. Dual tracer ¹⁸F-FCH/¹⁸F-FDG PET imaging has the potential to improve the visualization of low grade glioma. ¹⁸F-FCH delineates tumor areas and the tumor can be identified by subtracting the ¹⁸F-FCH counts. The sensitivity was over 95%. Further studies are required to evaluate the possibility of applying this technique in clinical trials.     

Efficient enzymatic synthesis of 13 C,15N-labeled DNA for NMR studies

The power of heteronuclear NMR spectroscopy to study macromoleculesand their complexes has been amply demonstrated over the last decade. Theobstacle to routinely applying these techniques to the study of DNA has beenthe synthesis of ¹³C,¹⁵N-labeled DNA. Here wepresent a simple and efficient method to generate isotope-labeled DNA forNMR studies that is as easy as that for isotope labeling of RNA. The methodwas used to synthesize a uniformly¹³ C,¹⁵N-labeled 32-nucleotide DNA that binds tohuman basic fibroblast growth factor with high affinity and specificity.Isotope-edited experiments were applied to the¹³ C,¹⁵N-labeled DNA bound to unlabeled protein,and the ¹³ C,¹⁵N-labeled DNA was also examined incomplex with ¹⁵N-labeled protein. The NMR experiments showthat the DNA adopts a well-defined stable structure when bound to theprotein, and illustrate the potential of¹³ C,¹⁵N-labeled DNA for structural studies ofDNA–protein complexes.     

Cyclotron vs generator-produced 68Ga PSMA: a single-institution,prospective clinical trial

The clinical utility of gallium 68 (⁶⁸Ga)-PSMA PET for the diagnosis and management of prostate cancer is driven in part by radioisotope availability and production costs. This study evaluates the equivalence between the two manufacturing processes for ⁶⁸Ga-PSMA: ⁶⁸Ga-PSMA-cyclotron (from a solid target) and ⁶⁸Ga-PSMA-generator. A prospective, single-arm, single-institution non-randomized study was conducted where 16 patients with prostate adenocarcinoma underwent PET/CTs consecutively within 12 to 48 hours with each type of manufactured ⁶⁸Ga-PSMA between December 2020 and June 2021. The intraclass correlation coefficients suggested acceptable reliability in all lesion parameters (ICC > 0.70). Bland-Altman analysis demonstrated acceptable bias levels for all lesion parameters. Thereby ⁶⁸Ga-cyclotron (solid target) and ⁶⁸Ga-generator production methods tagged to the same PSMA ligand resulted in scans which were deemed to be equivalent in detecting PSMA+ lesions in our study. As cyclotron-produced, solid- target ⁶⁸Ga can be made in large (Ci) quantities, it is a promising tool for future application in ⁶⁸Ga-PSMA PET scans with the potential to decrease radiotracer production costs and increase isotope availability.     

Pulsed NMR studies on Na + binding to simple carbohydrates

Pulsed NMR spectroscopy has been used to study Na⁺ binding to several simple carbohydrates in aqueous solution. Changes in the ²³Na spin-lattice relaxation time (T₁) were monitored to indicate complex formation between sodium ions and a ligand. It was found that Na⁺ interacts with these hydroxy-compounds in a manner similar to other metal cations, but very weakly. Among the sugars investigated, $\text{c i s}$-inositol forms the strongest complexes with the stability constant about 1.2 M^?1 (if 1:1 complexes are assumed). A qualitative study of competition between Na⁺ and Ca²⁺ was done, indicating that both cations have the same binding sites.     

90Y-Labeled Anti-ROBO1 Monoclonal Antibody Exhibits Antitumor Activity against Small Cell Lung Cancer Xenografts

IntroductionROBO1 is a membrane protein that contributes to tumor metastasis and angiogenesis. We previously reported that ⁹⁰Y-labeled anti-ROBO1 monoclonal antibody (⁹⁰Y-anti-ROBO1 IgG) showed an antitumor effect against ROBO1-positive tumors. In this study, we performed a biodistribution study and radioimmunotherapy (RIT) against ROBO1-positive small cell lung cancer (SCLC) models.MethodsFor the biodistribution study, ¹¹¹In-labeled anti-ROBO1 monoclonal antibody (¹¹¹In-anti-ROBO1 IgG) was injected into ROBO1-positive SCLC xenograft mice via the tail vein. To evaluate antitumor effects, an RIT study was performed, and SCLC xenograft mice were treated with ⁹⁰Y-anti-ROBO1 IgG. Tumor volume and body weight were periodically measured throughout the experiments. The tumors and organs of mice were then collected, and a pathological analysis was carried out.ResultsAs a result of the biodistribution study, we observed tumor uptake of ¹¹¹In-anti-ROBO1 IgG. The liver, kidney, spleen, and lung showed comparably high accumulation of ¹¹¹In-labeled anti-ROBO1. In the RIT study, ⁹⁰Y-anti-ROBO1 IgG significantly reduced tumor volume compared with baseline. Pathological analyses of tumors revealed coagulation necrosis and fatal degeneration of tumor cells, significant reduction in the number of Ki-67-positive cells, and an increase in the number of apoptotic cells. A transient reduction of hematopoietic cells was observed in the spleen, sternum, and femur.ConclusionsThese results suggest that RIT with ⁹⁰Y-anti-ROBO1 IgG is a promising treatment for ROBO1-positive SCLC.     

A novel method to quantify H-ATPase-dependent Na transport across plasma membrane vesicles

To prevent sodium toxicity in plants, Na⁺ is excluded from the cytosol to the apoplast or the vacuole by Na⁺/H⁺ antiporters. The secondary active transport of Na⁺ to apoplast against its electrochemical gradient is driven by plasma membrane H⁺-ATPases that hydrolyze ATP and pump H⁺ across the plasma membrane. Current methods to determine Na⁺ flux rely either on the use of Na-isotopes (²²Na) which require special working permission or sophisticated equipment or on indirect methods estimating changes in the H⁺ gradient due to H⁺-ATPase in the presence or absence of Na⁺ by pH-sensitive probes. To date, there are no methods that can directly quantify H⁺-ATPase-dependent Na⁺ transport in plasma membrane vesicles. We developed a method to measure bidirectional H⁺-ATPase-dependent Na⁺ transport in isolated membrane vesicle systems using atomic absorption spectrometry (AAS). The experiments were performed using plasma membrane-enriched vesicles isolated by aqueous two-phase partitioning from leaves of Populus tomentosa. Since most of the plasma membrane vesicles have a sealed right-side-out orientation after repeated aqueous two-phase partitioning, the ATP-binding sites of H⁺-ATPases are exposed towards inner side. Leaky vesicles were preloaded with Na⁺ sealed for the study of H⁺-ATPase-dependent Na⁺ transport. Our data implicate that Na⁺ movement across vesicle membranes is highly dependent on H⁺-ATPase activity requiring ATP and Mg²⁺ and displays optimum rates of 2.50 μM Na⁺ mg^− 1 membrane protein min^− 1 at pH 6.5 and 25 °C. In this study, for the first time, we establish new protocols for the preparation of sealed preloaded right-side-out vesicles for the study of H⁺-ATPase-dependent Na⁺ transport. The results demonstrate that the Na⁺ content of various types of plasma membrane vesicle can be directly quantified by AAS, and the results measured using AAS method were consistent with those determined by the previous established fluorescence probe method. The method is a convenient system for the study of bidirectional H⁺-ATPase-dependent Na⁺ transport with membrane vesicles.     

NAD<Superscript>+</Superscript> Treatment Induces Delayed Autophagy in Neuro2a Cells Partially by Increasing Oxidative Stress

NAD⁺ plays important roles in various biological processes. In this study, we reported that treatment of NAD⁺ induces delayed autophagy in Neuro2a cells. Moreover, the effects of NAD⁺ on the autophagy in the cells appear to be, at least partially, mediated by oxidative stress. However, nicotinamide, a degradation product of NAD⁺, does not affect the autophagy. Our experiments have further indicated that the NAD⁺-induced autophagy contributes to the NAD⁺-induced decrease in the survival of these cells. In summary, our study has provided the first evidence that NAD⁺ treatment induces autophagy in cancer cells such as Neuro2a cells, which contributes to the NAD⁺-induced decrease in cancer cell survival.     

Maximum values of Ni2+, Cu2+, Pb2+ and Zn2+ in the biomonitor Tillandsia capillaris (Bromeliaceae): Relationship with cell membrane damage

Although several plants belonging to the Bromeliaceae family have been used as heavy metal accumulators in biomonitoring studies, their accumulation ability has not been investigated. The present study obtained the accumulation rates of Ni²⁺, Cu²⁺, Pb²⁺ and Zn²⁺ in leaves of Tillandsia capillaris and revealed their effects on lipid peroxidation by measuring the Malondialdehyde content (MDA). Leaves of T. capillaris were exposed to different metallic solutions of Cu²⁺, Ni²⁺, Pb²⁺ and Zn²⁺ cations. After this exposure period, the accumulation of these ions was measured by Total Reflection X-Ray Fluorescence (TXRF) analysis with Synchrotron Radiation, and the MDA content was calculated. Data sets were evaluated by a one-way analysis of variance (ANOVA) and a fitted regression hyperbola model. The results showed significant differences in the accumulation efficiencies of the cations under study. In addition, the enrichment factor (EF) estimated for these cations was higher for Ni²⁺, suggesting a greater affinity of the plant with this element. Over time, all the metals under study caused significant increases in the MDA content, indicating their toxicity effects even in the most diluted solutions used in this study.     

Effect of Cystone on urinary composition and stone formation over a one year period

Kidney stones are a common problem for which inadequate prevention exists. We recruited ten recurrent kidney stone formers with documented calcium oxalate stones into a two phased study to assess safety and effectiveness of Cystone^®, an herbal treatment for prevention of kidney stones. The first phase was a randomized double-blinded 12 week cross over study assessing the effect of Cystone^® vs. placebo on urinary supersaturation. The second phase was an open label one year study of Cystone^® to determine if renal stone burden decreased, as assessed by quantitative and subjective assessment of CT. Results revealed no statistically significant effect of Cystone^® on urinary composition short (6 weeks) or long (52 weeks) term. Average renal stone burden increased rather than decreased on Cystone^®. Therefore, this study does not support the efficacy of Cystone^® to treat calcium oxalate stone formers. Future studies will be needed to assess effects on stone passage, or on other stone types.     

Evolutionary analysis of prokaryotic heat-shock transcription regulatory protein σ³²

Heat-stress to any living cell is known to trigger a universal defense response, called heat-shock response, with rapid induction of tens of different heat-shock proteins. Bacterial heat-shock genes are transcribed by the σ³²-bound RNA polymerase instead of the normal σ⁷⁰-bound RNA polymerase. In this study, the diversity in sequence, variation in secondary structure and function amongst the different functional regions of the proteobacterial σ³² family of proteins, and their phylogenetic relationships have been analyzed. Bacterial σ³² proteins can be subdivided into different functional regions which are referred to as regions 2, 3, and 4. There is a great deal of sequence conservation among the functional regions of proteobacterial σ³² family of proteins though some mutations are also present in these regions. Region 2 is the most conserved one, while region 4 has comparatively more variable sequences. In the present work, we tried to explore the effects of mutations in these regions. Our study suggests that the sequence diversities due to natural mutations in the different regions of proteobacterial σ³² family lead to different functions. So far, this study is the first bioinformatic approach towards the understanding of the mechanistic details of σ³² family of proteins using the protein sequence information only. This study therefore may help in elucidating the hitherto unknown molecular mechanism of the functionalities of σ³²family of proteins.     

Glucagon like-peptide-1 receptor is covalently modified by endogenous mono-ADP-ribosyltransferase

Our previous study revealed a mono-ADP-ribosyltransferase mediated in vitro mono-ADP-ribosylation of IC₃ peptide, a peptide with sequence corresponded to third intracellular loop of glucagon like-peptide-1 (GLP-1) receptor. Furthermore, Arg³⁴⁸ was shown to be modified amino acid residue although its mutation did not eliminate mono-ADP-ribosylation completely. In order to further study the signaling mechanisms of GLP-1 receptor, we took on lease a possibility that an alternative site of enzymatic modification exist so mono-ADP-ribosylation of Cys³⁴¹ was hypothesized. The results confirmed both Arg³⁴⁸ and Cys³⁴¹ as a site of mono-ADP-ribosylation where Arg³⁴⁸ is modified predominantly. Sum of mono-ADP-ribosylation rate of both single IC₃ mutants coincided with IC₃ rate. What is in vivo role of Cys³⁴¹ mono-ADP-ribosylation is entirely speculative but our study represents an important step toward a complete understanding of signaling via GLP-1 receptor.     

The use of hair as an indicator of occupational 14C contamination

This paper presents a study in which the specific activity of ¹⁴C in hair has been investigated as an easily determined bio-indicator of the integrated ¹⁴C exposure (over several months). The study includes 28 Swedish workers handling ¹⁴C-labelled compounds, or working in a ¹⁴C-enriched environment. Hair samples from personnel at a Swedish nuclear power plant showed very low levels of ¹⁴C contamination, if any. In contrast, personnel at the investigated research departments showed ¹⁴C levels in hair of up to 60% above the natural specific activity of ¹⁴C. Much higher levels, up to 80 times the natural specific activity of ¹⁴C, were found in hair from individuals working at a pharmaceutical research laboratory. This contamination was, however, not solely an internal contamination. There were indications that most of the ¹⁴C in the hair originated from airborne ¹⁴C-compounds, which were adsorbed onto the hair. The difficulties in removing this external ¹⁴C contamination prior to analysis are discussed, as are the possibilities of using accelerator mass spectrometry to analyse various types of samples for retrospective dose assessment.     

Biodistribution and Elimination Study of Fluorine-18 Labeled Nε-Carboxymethyl-Lysine following Intragastric and Intravenous Administration

Background

N^ε-carboxymethyl-lysine (CML) is a major advanced glycation end-product (AGEs) widely found in foods. The aim of our study was to evaluate how exogenous CML-peptide is dynamically absorbed from the gastrointestinal tract and eliminated by renal tubular secretion using microPET imaging.

Methods

The present study consisted of three investigations. In study I, we synthesized the imaging tracer ¹⁸F-CML by reacting N-succinimidyl 4-¹⁸F-fluorobenzoate (¹⁸F-SFB) with CML. In study II, the biological activity of ¹⁸F-CML was evaluated in RAW264.7 cells and HepG2 cells. In study III, the biodistribution and elimination of AGEs in ICR mice were studied in vivo following tail vein injection and intragastric administration of ¹⁸F-CML.

Result

The formation of ¹⁸F-CML was confirmed by comparing its retention time with the corresponding reference compound ¹⁹F-CML. The radiochemical purity (RCP) of ¹⁸F-CML was >95%, and it showed a stable character in vitro and in vivo. Uptake of ¹⁸F-CML by RAW264.7 cells and HepG2 cells could be inhibited by unmodified CML. ¹⁸F-CML was quickly distributed via the blood, and it was rapidly excreted through the kidneys 20 min after tail vein injection. However, ¹⁸F-CML was only slightly absorbed following intragastric administration. After administration of ¹⁸F-CML via a stomach tube, the radioactivity was completely localized in the stomach for the first 15 min. At 150 min post intragastric administration, intense accumulation of radioactivity in the intestines was still observed.

Conclusions

PET technology is a powerful tool for the in vivo analysis of the gastrointestinal absorption of orally administered drugs. ¹⁸F-CML is hardly absorbed by the gastrointestinal tract. It is rapidly distributed and eliminated from blood following intravenous administration. Thus, it may not be harmful to healthy bodies. Our study showed the feasibility of noninvasively imaging ¹⁸F-labeled AGEs and was the first to describe CML-peptide gastrointestinal absorption by means of PET.     

Investigating proline puckering states in diproline segments in proteins

In the current study, the puckering states of the Proline ring occurring in diproline segments (^LPro‐^LPro) in proteins has been investigated with a segregation made on the basis of cis and trans states for the Pro‐Pro peptide bond and the conformational states for the diproline segment to investigate the effects of conformation of the diproline segment on the corresponding puckering state of the Proline ring in the segment if any. The value of the endocyclic ring torsional angles of the pyrrolidine ring has been used for calculating and visualizing various puckering states using a proposed new sign convention (+/?) nomenclature. The results have been compared to that obtained in a previous study on peptides from this group. In this study, quite interestingly, the Planar (G) conformation that was present in 14.3% of the cases in peptides, appears to be nearly a rare conformation in the case of proteins (1.9%). The present study indicates that the (C^γ‐exo/C^γ‐exo), (C^γ‐exo/Twisted C^γ‐exo‐C^β‐endo) and (Twisted C^γ‐endo‐C^β‐exo/Twisted C^γ‐endo‐C^β‐exo) categories are the most preferred combinations. For Proline rings in proteins, the states C^γ‐exo, Twisted C^γ‐exo‐C^β‐endo and Twisted C^γ‐endo‐C^β‐exo are the most preferred states. Within diproline segments, the pyrrolidine ring conformations do not show a strong co‐relation to the backbone conformation in which they are observed. It is likely that five‐membered rings have a considerable plasticity of structure and are readily deformed to accommodate a variety of energetically preferred backbone conformations. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 605–610, 2013.     

Variation in chemical composition of the River Frome, England, from 1965 to 1972

This study covers a 7-year period from 1965 to 1972, and shows the variation in flow, water chemistry and throughput of nutrients. The annual discharge of the River Frome varied considerably from year to year, ranging from a mean value of 4.48 m³ sec⁻¹ in 1971 to one of 8.38 m³ sec⁻¹ in 1966. The mean annual nutrient losses calculated over all 7 years of study from the River Frome catchment were: Nitrate 11.4 kg ha⁻¹, phosphorus 0.49 kg ha⁻¹ and potassium 8.4 kg ha⁻¹. There has been an increase in annual throughput (in years of similar flow) of 21% in phosphate and 41% in nitrate from 1965 to 1972.     

Four compartment mammillary model applied to the pharmacokinetics of a spiroarsorane administered orally to rabbits

Rekik et al.¹ dealt with the pharmacokinetics of two spiroarsoranes molecules^1,2 administered intravenously or orally to rabbits. For the intravenous study they used an open two-compartment model. For the oral study they admitted that they were unable to fit the data to a model. In this paper the plasma concentration profile of molecule 2 after oral administration using a four mammillary open compartment model is described. This type of model (which requires a fourth degree equation) has been previously described². Here it is applied to a concrete situation.     

Metabolism of the alpha-Pyridone Ring of Ricinine in Ricinus communis L

Chemically synthesized ricinine-3,5-¹⁴C was used to study the metabolism of this alkaloid in the plant which produces it, Ricinus communis L. In a time course study, ricinine-3,5-¹⁴C was administered to a series of castor plants (Ricinus communis L.) and the radioactivity recovered in the ricinine samples showed a decrease with increase in time. It was also observed that the alkaloid was translocated to the seed. The in vivo conversion of ricinine-3,5-¹⁴C to respiratory ¹⁴CO₂ occurred in both light and dark and indicated that the α-pyridone ring of ricinine could be degraded.     

Comparative Immune Phenotypic Analysis of Cutaneous Squamous Cell Carcinoma and Intraepidermal Carcinoma in Immune-Competent Individuals: Proportional Representation of CD8+ T-Cells but Not FoxP3+ Regulatory T-Cells Is Associated with Disease Stage

Squamous Cell Carcinoma (SCC) is a type of non-melanoma skin cancer prevalent in immune-suppressed transplant recipients and older individuals with a history of chronic sun-exposure. SCC itself is believed to be a late-stage manifestation that can develop from premalignant lesions including Intraepidermal Carcinoma (IEC). Notably, while SCC regression is rare, IEC typically regresses in response to immune modifying topical treatments, however the underlying immunological reasons for these differential responses remain unclear. This study aimed to define whether IEC and SCC are associated with distinct immune profiles. We investigated the immune cell infiltrate of photo-damaged skin, IEC, and SCC tissue using 10-colour flow cytometry following fresh lesion digest. We found that IEC lesions contain higher percentages of CD3⁺ T-cells than photo-damaged skin, however, the abundance of CD3⁻CD56⁺ Natural Killer (NK) cells, CD11c⁺HLA-DR⁺ conventional Dendritic Cells (cDC), BDCA-2⁺HLA-DR⁺ plasmacytoid DC (pDC), FoxP3⁺ Regulatory T-cells (T-reg), Vα24⁺Vβ11⁺ invariant NKT-cells, and γδ Tcells did not alter with disease stage. Within the total T-cell population, high percentages of CD4⁺ T-cells were associated with SCC, yet CD8⁺ T-cells were less abundant in SCC compared with IEC. Our study demonstrates that while IEC lesions contain a higher proportion of T-cells than SCC lesions in general, SCC lesions specifically display a lower abundance of CD8⁺ T-cells than IEC. We propose that differences in CD8⁺ T-cell abundance contribute critically to the different capacity of SCC and IEC to regress in response to immune modifying topical treatments. Our study also suggests that a high ratio of CD4⁺ T-cells to CD8⁺ T-cells may be a immunological diagnostic indicator of late-stage SCC development in immune-competent patients.     

Cell cytoskeleton and proliferation study for the RANKL-induced RAW264.7 differentiation

Although document studies (including ours) have been reported the achieved in vitro osteoclastic cellular model establishment from the RAW264.7 cell lineage, there was no study directly reported that American Type Culture Collection (ATCC) cell bank has various RAW264.7 cell lineages. Besides that, for our knowledge there was only one study compared the two different RAW264.7^TIB-71 and RAW264.7^CRL-2278 cell lineages for their osteoclastic differentiation, and they concluded that the RAW264.7^CRL-2278 demonstrated to generate much osteoclast than RAW264.7^TIB-71. However, on the contrary to their results we noticed the fusion of RAW264.7^TIB-71 in our previous studies was much compromising. Therefore, we try to explore the two cell lineages for their properties in osteoclastic differentiation with an in-depth cellular cytoskeletal study. Our current study has showed that comparing to the RAW264.7^CRL-2278, RAW264.7^TIB-71 demonstrated a much higher efficacies for RANKL-stimulated osteoclastic differentiation. Besides that, in our depth cytoskeletal studies, we found that the RANKL-induced RAW264.7^TIB-71 cells could finally differentiate into mature osteoclasts. However, regardless the various pre-treatment conditions, there was no mature osteoclast formed in RANKL-induced RAW264.7^CRL-2278 cell lineage.