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Extracellular RNAs (exRNAs) are released by extracellular vesicles, small membranous nanoparticles secreted by all cell types. When transported into cells, exRNAs can modulate gene expression or cellular responses in the target cells since many small RNAs have regulatory functions. Indeed, it is widely acknowledged that endogenous exRNAs in the human body are related to various diseases. However, microbial exRNAs have been less studied, and their connection to host diseases has just begun to be explored. In this review, I will discuss analytical methods for exRNAs and the potential use of exRNAs as disease biomarkers. I also consider current progress in understanding the regulation of host mechanisms by microbial exRNAs as inter-kingdom communication, efforts to utilize extracellular vesicles as therapeutic vehicles loaded with engineered RNA cargos, and a putative connection between microbial exRNA-based regulation of host responses and human diseases such as Alzheimer’s. This overview aims to present novel insights into pathogenesis with regard to the function of microbial exRNAs as “disease-relevant travelers.” Impact statement The number of commensal bacteria in the body surpasses the number of actual human cells. Thus, various interactions between microbes and human cells constitute an inevitable phenomenon. Recent evidence has led to bacterial extracellular RNAs (exRNAs) being proposed as good candidates for microbe–host inter-kingdom communication tools as they can modulate the expression of host genes. However, research findings on the relevance of interactions between extracellular RNA and human diseases are still in their infancy. Nevertheless, substantial data suggest that microbial exRNAs are implicated in various human diseases both at local and distant sites. By exploring various scenarios for the involvement of microbial exRNAs in human diseases, we may better understand the role of exRNAs as “communication signals” for diseases and thereby develop novel therapeutic strategies by using them and their carrier extracellular vesicles.

The experimental animal model is still essential in the development of new anticancer drugs. We characterized mouse tumors derived from two-dimensional (2D) monolayer cells or three-dimensional (3D) spheroids to establish an in vivo model with highly standardized conditions. Primary cancer-associated fibroblasts (CAFs) were cultured from head and neck squamous cell carcinoma (HNSCC) tumor tissues and co-injected with monolayer cancer cells or spheroids into the oral mucosa of mice. Mice tumor blood vessels were stained, followed by tissue clearing and 3D Lightsheet fluorescent imaging. We compared the effect of exosomes secreted from 2D or 3D culture conditions on the angiogenesis-related genes in HNSCC cells. Our results showed that both the cells and spheroids co-injected with primary CAFs formed tumors. Interestingly, vasculature was abundantly distributed inside the spheroid-derived but not the monolayer-derived mice tumors. In addition, cisplatin injection more significantly decreased spheroid-derived but not monolayer-derived tumor size in mice. Additionally, exosomes isolated from co-culture media of FaDu spheroid and CAF upregulated angiogenesis-related genes in HNSCC cells as compared to exosomes from FaDu cell and CAF co-culture media under in vitro conditions. The mouse tumor xenograft model derived from 3D spheroids of HNSCC cells with primary CAFs is expected to produce reliable chemotherapy drug screening results given the robust angiogenesis and lack of necrosis inside tumor tissues.

Gram-negative bacterial extracellular vesicles (EVs), also known as outer membrane vesicles (OMVs), are secreted from bacterial cells and have attracted research attention due to their role in cell-to-cell communication. During OMV secretion, a variety of cargo such as extracellular RNA (exRNA) is loaded into the OMV. The involvement of exRNAs from a range of bacteria has been identified in several diseases, however, their mechanism of action has not been elucidated. We have recently demonstrated that OMVs secreted by the periodontopathogen Aggregatibacter actinomycetemcomitans can cross the blood–brain barrier (BBB) and that its exRNA cargo could promote the secretion of proinflammatory cytokines in the brain. However, it was unclear whether the brain immune cells could actually take up bacterial OMVs, which originate outside of the brain, in an appropriate immune response. In the present study, using monocyte-specific live CX3CR1-GFP mice, we visualized OMV-colocalized meningeal macrophages and microglial cells into which bacterial OMVs had been loaded and intravenously injected through tail veins. Our results suggested that meningeal macrophages uptake BBB-crossed OMVs earlier than do cortex microglia. BV2 cells (a murine microglia cell line) and exRNAs were also visualized after OMV treatment and their proinflammatory cytokine levels were observed. Interleukin (IL)-6 and NF-κB of BV2 cells were activated by A. actinomycetemcomitans exRNAs but not by OMV DNA cargo. Altogether, these findings indicate that OMVs can successfully deliver exRNAs into brain monocyte/microglial cells and cause neuroinflammation, implicating a novel pathogenic mechanism in neuroinflammatory diseases.

Background/aim Few studies have examined the effect of 2'-hydroxycinnamaldehyde (HCA) on head and neck squamous cell carcinoma (HNSCC) cell invasion. This study examined the role of BMP7 on the anti-migration and anti-invasion activity of HCA using HNSCC cells. Materials and methods Matrigel invasion and wound healing assays were conducted to investigate cell migration or invasion. BMP7 overexpression vector or siRNA mixture was used for transient regulation of gene expression. Results HCA attenuated HNSCC cell migration and spheroids Matrigel invasion without cytotoxicity. mRNA and protein expression of BMP7 increased with HCA treatment. Exogenous BMP7 overexpression without HCA treatment attenuated Matrigel invasion of cells. Furthermore, suppression of BMP7 by siRNA alleviated the inhibitory effect of HCA on the invasion of Matrigel by the cell, indicating that BMP7 is responsible for the anti-migration effect of HCA in HNSCC cells. Conclusion HCA treatment led to a remarkable up-regulation of BMP7, which resulted in the attenuation of HNSCC cell invasion.

The effect of oxytocin (OXT) on cancer invasion is controversial. Few studies have examined the effect of early growth response-1 (EGR1) on the invasion of head and neck squamous cell carcinoma (HNSCC). Here, we evaluated how EGR1 affects HNSCC cell migration through the molecular mechanism of OXT in exerting anti-invasion activity. Matrigel invasion and wound-healing assays were used to measure the in-vitro cell migration. The molecular mechanism of OXT was assessed by knockdown or overexpression of EGR1 in HNSCC cells. Three-dimensional (3-D) spheroids formation, followed by the image analysis for quantification was performed. OXT at 500 nmol/l increased mRNA and protein expression of E-cadherin without cytotoxicity. OXT upregulated mRNA and protein expression of EGR1 in 6 h. p53, phosphatase and tensin, and p21 expression was increased in an EGR1-dependent manner with OXT treatment. In addition, OXT significantly downregulated 3-D spheroids' formation according to spheroids' number and size. Our data showed that OXT downregulated HNSCC cell migration by EGR1 upregulation. OXT inhibited spheroids' formation of HNSCC cells under 3-D culture conditions.

Among the main bacteria implicated in the pathology of periodontal disease, Aggregatibacter actinomycetemcomitans (Aa) is well known for causing loss of periodontal attachment and systemic disease. Recent studies have suggested that secreted extracellular RNAs (exRNAs) from several bacteria may be important in periodontitis, although their role is unclear. Emerging evidence indicates that exRNAs circulate in nanosized bilayered and membranous extracellular vesicles (EVs) known as outer membrane vesicles (OMVs) in gram-negative bacteria. In this study, we analyzed the small RNA expression profiles in activated human macrophage-like cells (U937) infected with OMVs from Aa and investigated whether these cells can harbor exRNAs of bacterial origin that have been loaded into the host RNA-induced silencing complex, thus regulating host target transcripts. Our results provide evidence for the cytoplasmic delivery and activity of microbial EV-derived small exRNAs in host gene regulation. The production of TNF-α was promoted by exRNAs via the TLR-8 and NF-κB signaling pathways. Numerous studies have linked periodontal disease to neuroinflammatory diseases but without elucidating specific mechanisms for the connection. We show here that intracardiac injection of Aa OMVs in mice showed successful delivery to the brain after crossing the blood–brain barrier, the exRNA cargos increasing expression of TNF-α in the mouse brain. The current study indicates that host gene regulation by microRNAs originating from OMVs of the periodontal pathogen Aa is a novel mechanism for host gene regulation and that the transfer of OMV exRNAs to the brain may cause neuroinflammatory diseases like Alzheimer’s.—Han, E.-C., Choi, S.-Y., Lee, Y., Park, J.-W., Hong, S.-H., Lee, H.-J. Extracellular RNAs in periodontopathogenic outer membrane vesicles promote TNF-α production in human macrophages and cross the blood–brain barrier in mice.

BACKGROUND: The accumulation of oral bacterial biofilms is one of the primary etiological factors for oral diseases. Aronia melanocarpa extracts display general health benefits, including antimicrobial activities. This study evaluates the inhibitory effect of Aronia juice on oral streptococcal biofilm formation. RESULTS: Exposure to 1/10‐diluted Aronia juice for 1 min significantly decreased in vitro streptococcal biofilm formation (P

Background Periodontitis is an inflammatory disease of the tissues surrounding teeth that causes destruction of connective tissues. During the progress of periodontitis, osteoclasts are solely accountable for the resorption of alveolar bones that leads to the loss of teeth if not properly treated. Thus, the development of effective anti-resorptive therapies will greatly benefit the treatment of periodontitis patients. In the present study, we suggest an inhibitory effect of 6-shogaol, an ingredient of ginger, on osteoclast differentiation and bone resorption. Methods Mouse bone marrow cells were cultured in the presence of macrophage-colony stimulating factor and receptor activator of nuclear factor-κB ligand (RANKL) to investigate the effect of 6-shogaol on osteoclast differentiation and intracellular signaling pathways. 6-shogaol significantly reduced osteoclast differentiation, actin ring formation, and resorption. In the presence of 6-shogaol, osteoclast signaling including the RANKL-induced activation of mitogen-activated protein kinases, Ca2+ oscillation, generation of reactive oxygen species, and nuclear factor of activated T-cells, cytoplasmic 1 nuclear translocation was significantly inhibited in vitro. Furthermore, a ligature-induced periodontitis model in mice was used to determine the role of 6-shogaol in vivo. Results The administration of 6-shogaol prevented osteoclastogenesis and alveolar bone resorption induced by ligature. Furthermore, the ligature-induced number of macrophages and neutrophils as well as the expression of interleukin-1β and tumor necrosis factor-α were considerably lower in the periodontal tissues following shogaol injection. Conclusion These results confirm the anti-osteoclastogenic effect of 6-shogaol and suggest the possibility of application as an anti-resorptive strategy in periodontitis.

We investigated the role of exosomes in osteogenesis and the use of miRNA inhibitor-transfected exosomes to control osteogenic differentiation. RNA-sequencing (RNA-seq) of exosomal miRNAs revealed that growth condition of milieu of preosteoblast exosomes harbors high levels of let-7, which plays a critical role in osteogenesis regulation. We modified exosomes by transfecting let-7 inhibitor into exosomes under growth condition in MC3T3-E1 cells and revealed that exosomes whose let-7 was inactivated by engineering lost the ability to recover osteogenic differentiation. Genetically modified exosomes may serve as powerful biomaterials for developmental control, including of osteogenesis regulation.

Background Periodontitis is not only one of the most prevalent inflammatory diseases among adults, but also commonly linked to numerous systemic conditions including cardiovascular diseases, stroke, and diabetes. Although osteoclasts are responsible for the alveolar bone resorption during periodontitis pathogenesis, the development of pharmacologic strategies targeting these cells has not been vastly fruitful. Methods Bone marrow macrophages were cultured in the presence of macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL) to examine the direct effect of acalabrutinib on osteoclastogenesis. Ca2+ oscillation and nuclear localization of NFATc1 in osteoclast precursors were examined to determine the precise molecular mechanism. LPS-induced alveolar bone loss model was employed for studying effect in in vivo bone resorption. Results Acalabrutinib directly inhibited RANKL and LPS-induced in vitro osteoclast differentiation. In addition, acalabrutinib inhibited RANKL-induced phosphorylation of mitogen-activated protein kinases and reduced the expression of NF-κB. The inhibitory mechanism involved suppression of Ca2+ oscillation in osteoclast precursors resulting in the decreased NFATc1 expression and nuclear localization, which is a crucial prerequisite for osteoclastogenesis. The administration of acalabrutinib significantly reduced P. gingivalis lipopolysaccharide-induced alveolar bone erosion in mice. Conclusion These data indicate that acalabrutinib is an effective inhibitor of osteoclastogenesis both in vitro and in vivo, with a potential for a novel strategy against bone destruction by periodontitis.

Stromal cell-derived factor-1 (SDF-1) protein and its receptor, CXCR-4, play an important role in tissue repair and regeneration in various organs, including the bone. SDF-1 is indispensable for bone morphogenetic protein-2 (BMP-2)-induced osteogenic differentiation. However, SDF-1 is not needed after the osteogenic induction has been activated. Since the precise condition for the additive effects of combined DF-1 and BMP-2 in bone healing had not been fully investigated, we aimed to determine the optimal conditions for SDF-1- and BMP-2-mediated bone regeneration. We examined the in vitro osteoblastic differentiation and cell migration after sequential treatments with SDF-1 and BMP-2. Based on the in vitro additive effects of SDF-1 and BMP-2, the critical size defects of mice calvaria were treated with these cytokines in various sequences. Phosphate buffered saline (PBS)-, SDF-1-, or BMP-2-soaked collagen scaffolds were implanted into the calvarial defects (n=36). Periodic percutaneous injections of PBS or the cytokine SDF-1 and BMP-2 into the implanted scaffolds were performed on days 3 and 6, postoperatively. Six experimental groups were used according to the types and sequences of the cytokine treatments. After 28 days, the mice were euthanized and bone formation was evaluated with microcomputed tomography and histology. The molecular mechanism of the additive effect of SDF-1 and BMP-2 was evaluated by analyzing intracellular signal transduction through Smad and Erk phosphorylation. The in vitro experiments revealed that, among all the treatments, the treatment with BMP-2 after SDF-1 showed the strongest osteoblastic differentiation and enhanced cell migration. Similarly, in the animal model, the treatment with SDF-1 followed by BMP-2 treatment showed the highest degree of new bone regeneration than any other groups, including the one with continuous BMP-2 treatment. This new bone formation can be partially explained by the activation of Smad and Erk pathways and enhanced cell migration. These results suggest that sequential treatment with the cytokines, SDF-1 and BMP-2, may be a promising strategy for accelerating bone regeneration in critical size defects.

MicroRNAs are small non-coding RNAs that bind to the 3′-untranslated region of target mRNAs and have transcriptional or translational inhibitory function in eukaryotes. Before microRNAs were widely known, bacterial non-coding small RNAs around 50–200 nt in length were discovered whose mechanism of action resembled that of microRNAs. Recently, RNAs that are of similar size to or smaller than microRNAs have been discovered in bacteria and indeed, this class of small RNAs have been found throughout all domains of life. Moreover, recent findings suggest that these tiny RNAs can be released via extracellular vesicles (such as exosomes in eukaryotes and outer membrane vesicles in bacteria), which in turn heralds a new field of research, interkingdom communication. This review discusses two similar classes of small RNAs in evolutionarily distinct eukaryotes and bacteria. In addition to their biogenesis and regulation, we discuss small RNA vehicles and their secretion. Impact statement The possible endogenous functions of small RNAs such as regulatory small RNAs in bacteria and microRNAs in eukaryotes have been extensively studied since they were first discovered. However, their powerful functions should not be seen as limited to their cells of origin. Recently, several papers have demonstrated that small RNAs function as signaling molecules between cells. This is possible because small RNAs can be shuttled around after being incorporated into environmentally protective extracellular vesicles. It is now clearly plausible that secreted small RNAs can regulate other types of cells through biofluids. Given their “common molecule” status, the role of small RNAs in mediating bacteria-human crosstalk is an emerging and competitive area of genetic research. This review provides insight into the function of small RNAs in intercellular and even interkingdom communication.

Aim This study investigated the efficacy of the hypothalamic nonapeptide oxytocin (OT) by direct delivery to local defects using a microporous β-tricalcium phosphate (TCP) as the carrier for the future applications as a method to achieve predictable bone regeneration of large osseous defects requiring sinus bone graft and guided bone regeneration procedures for implant placement. Material and Methods Both the ectopic and new bone formation induced by the OT-loaded microporous β-TCP powder was histomorphometrically compared with unloaded β-TCP in a subcutaneous ectopic bone formation model and calvarial critical-sized defects (CSDs) in 45 rats. Results The OT-loaded β-TCP clearly enhanced ectopic bone formation compared with the unloaded control group. A High initial OT dose (250 μg) significantly increased ectopic bone formation at an early healing time-point compared with a lower OT dose (50 μg). The OT-loaded samples displayed greater new bone formation in the rat calvarial CSDs. Extensive new bone formation was achieved in the calvarial CSDs with the higher OT dose. Conclusion These results suggest that local OT delivery to bone substitute promotes new bone formation via an osteoinductive mode of action.

Osteoclasts are specialized cells for bone-resorption originated from precursors of macrophage/monocyte lineage. The receptor activator of NFκB ligand (RANKL) initiates osteoclast differentiation, in which nuclear factor of activated T cell cytoplasmic 1 (NFATc1) plays a key role as a master transcription factor. In the present report, we show that microRNA-124 (miR-124) regulates osteoclastogenesis of mouse bone marrow macrophages (BMMs) by suppressing NFATc1 expression. On the other hand, synthetic inhibitor that binds specifically to miR-124 enhanced osteoclast differentiation and NFATc1 expression. The overexpression of a constitutively active form of NFATc1 prevented the inhibitory effect of miR-124 on osteoclastogenesis. Finally, miR-124 also affected the proliferation and motility of osteoclast precursors, the latter coinciding with the reduced expression of RhoA and Rac1. These findings not only reveal unprecedented role of miR-124 in osteoclastogenesis but also suggest a novel mode of regulation of NFATc1 in osteoclasts.

Objectives The accumulation of oral bacterial biofilm is the main etiological factor of oral diseases. Recently, electrolyzed hydrogen-rich water (H-water) has been shown to act as an effective antioxidant by reducing oxidative stress. In addition to this general health benefit, H-water has antibacterial activity for disease-associated oral bacteria. However, little is known about the effect of H-water on oral bacterial biofilm. The objective of this study was to confirm the effect of H-water on streptococcal biofilm formation. Methods In vitro streptococcal biofilm was quantified using crystal violet staining after culture on a polystyrene plate. The effect of H-water on the expression of genes involved in insoluble glucan synthesis and glucan binding, which are critical steps for oral biofilm formation, was evaluated in MS. In addition, we compared the number of salivary streptococci after oral rinse with H-water and that with control tap water. Salivary streptococci were quantified by counting viable colonies on Mitis Salivarius agar-bacitracin. Results Our data showed that H-water caused a significant decrease in in vitro streptococcal biofilm formation. The expression level of the mRNA of glucosyltransferases (gtfB, gtfc, and gtfI) and glucan-binding proteins (gbpC, dblB) were decreased remarkably in MS after H-water exposure for 60 s. Furthermore, oral rinse with H-water for 1 week led to significantly fewer salivary streptococci than did that with control tap water. Conclusions Our data suggest that oral rinse with H-water would be helpful in treating dental biofilm-dependent diseases with ease and efficiency.

Objective Microbial aggregation around dental implants can lead to loss/loosening of the implants. This study was aimed at surface treating titanium microimplants with silver nanoparticles (AgNPs) to achieve antibacterial properties. Methods AgNP-modified titanium microimplants (Ti-nAg) were prepared using two methods. The first method involved coating the microimplants with regular AgNPs (Ti-AgNP) and the second involved coating them with a AgNP-coated biopolymer (Ti-BP-AgNP). The topologies, microstructures, and chemical compositions of the surfaces of the Ti-nAg were characterized by scanning electron microscopy (SEM) equipped with energy-dispersive spectrometer (EDS) and X-ray photoelectron spectroscopy (XPS). Disk diffusion tests using Streptococcus mutans, Streptococcus sanguinis, and Aggregatibacter actinomycetemcomitans were performed to test the antibacterial activity of the Ti-nAg microimplants. Results SEM revealed that only a meager amount of AgNPs was sparsely deposited on the Ti-AgNP surface with the first method, while a layer of AgNP-coated biopolymer extended along the Ti-BP-AgNP surface in the second method. The diameters of the coated nanoparticles were in the range of 10 to 30 nm. EDS revealed 1.05 atomic % of Ag on the surface of the Ti-AgNP and an astounding 21.2 atomic % on the surface of the Ti-BP-AgNP. XPS confirmed the metallic state of silver on the Ti-BP-AgNP surface. After 24 hours of incubation, clear zones of inhibition were seen around the Ti-BP-AgNP microimplants in all three test bacterial culture plates, whereas no antibacterial effect was observed with the Ti-AgNP microimplants. Conclusions Titanium microimplants modified with Ti-BP-AgNP exhibit excellent antibacterial properties, making them a promising implantable biomaterial.

The effect of icilin, a potent agonist of transient receptor potential ankyrin 1 (TRPA1) and TRPM8, on glycinergic transmission was examined in mechanically isolated rat medullary dorsal horn neurons by use of the conventional whole-cell patch-clamp technique. Icilin increased the frequency of glycinergic spontaneous miniature inhibitory post-synaptic currents (mIPSCs) in a dose-dependent manner. Either allyl isothiocyanate(AITC) or cinnamaldehyde, other TRPA1 agonists, also increased mIPSC frequency, but the extent of facilitation induced by AITC or cinnamaldehyde was less than that induced by icilin. However, menthol, a TRPM8 agonist, had no facilitatory effect on glycinergic mIPSCs. The icilin-induced increase in mIPSC frequency was significantly inhibited by either HC030031, a selective TRPA1 antagonist, or ruthenium red, a non-selective transient receptor potential channel blocker. Icilin failed to increase glycinergic mIPSC frequency in the absence of extracellular Ca(2+), suggesting that the icilin-induced increase in mIPSC frequency is mediated by the Ca(2+) influx from the extracellular space. In contrast, icilin still increased mIPSC frequency either in the Na(+) -free external solution or in the presence of Cd(2+), a general voltage-dependent Ca(2+) channel blocker. The present results suggest that icilin acts on pre-synaptic TRPA1-like ion channels, which are permeable to Ca(2+), to enhance glycinergic transmission onto medullary dorsal horn neurons. The TRPA1-like channel-mediated enhancement of glycinergic transmission in medullary dorsal horn neurons would contribute to the regulation of pain information from the peripheral tissues.

Ultra-small and well dispersed silver nanoparticles were prepared by mixing AgNO3 in PEI (polyethylenimine) solution. Particle size analysis and transmission electron microscopy confirmed that the average diameters of PEI/silver nanoparticles are 20-40 nm. The ultra-small silver nanoparticles had an antimicrobial activity in the aqueous solution at the concentration over 0.0105% by M.I.C test. Suggestively, the PEI/silver nanoparticles have potentials for development of wound dressings.

Ultrasmall superparamagnetic iron oxide nanoparticles (USPION) as magnetic resonance imaging were synthesized through a sonochemical co-precipitation method with poly (ethyleneimine) (PEI). The size of the USPION was controlled by poly (ethyleneimine) (PEI) contents, ultrasonic exposure time, and Na4OH concentration. The characteristics of PEI-USPION were studied by X-ray diffractometry, magnetic property measurement system, scanning electron microscopy, transmission electron microscopy, and particle size analysis. The PEI-USPION had an average size of 5~10nm with a narrow size distribution, a perfect magnetite crystal structure, and high saturation magnetization.

Molybdenum cofactor (Moco)-deficiency is a lethal autosomal recessive disease, for which until now no effective therapy is available. The biochemical hallmark of this disorder is the inactivity of the Moco-dependent sulfite oxidase, which results in elevated sulfite and diminished sulfate levels throughout the organism. In humans, Moco-deficiency results in neurological damage, which is apparent in untreatable seizures and various brain dysmorphisms. We have recently described a murine model for Moco-deficiency, which reflects all enzyme and metabolite changes observed in the patients, and an efficient therapy using a biosynthetic precursor of Moco has been established in this animal model. We now analyzed these mice in detail and excluded morphological brain damage, while expression analysis with microarrays indicates a massive cell death program. This neuronal damage appears to be triggered by elevated sulfite levels and is ameliorated in affected embryos by maternal clearance.

Substitution therapies for orphan genetic diseases, including enzyme replacement methods, are frequently hampered by the limited availability of the required therapeutic substance. We describe the isolation of a pterin intermediate from bacteria that was successfully used for the therapy of a hitherto incurable and lethal disease. Molybdenum cofactor (Moco) deficiency is a pleiotropic genetic disorder characterized by the loss of the molybdenum-dependent enzymes sulphite oxidase, xanthine oxidoreductase and aldehyde oxidase due to mutations in Moco biosynthesis genes. An intermediate of this pathway-'precursor Z'-is more stable than the cofactor itself and has an identical structure in all phyla. Thus, it was overproduced in the bacterium Escherichia coli, purified and used to inject precursor Z-deficient knockout mice that display a phenotype which resembles that of the human deficiency state. Precursor Z-substituted mice reach adulthood and fertility. Biochemical analyses further suggest that the described treatment can lead to the alleviation of most symptoms associated with human Moco deficiency.

Human molybdenum cofactor deficiency is a rare and devastating autosomal-recessive disease for which no therapy is known. The absence of active sulfite oxidase-a molybdenum cofactor-dependent enzyme-results in neonatal seizures and early childhood death. Most patients harbor mutations in the MOCS1 gene, whose murine homolog was disrupted by homologous recombination with a targeting vector. As in humans, heterozygous mice display no symptoms, but homozygous animals die between days 1 and 11 after birth. Biochemical analyis of these animals shows that molydopterin and active cofactor are undetectable. They do not possess any sulfite oxidase or xanthine dehydrogenase activity. No organ abnormalities were observed and the synaptic localization of inhibitory receptors, which was found to be disturbed in molybdenum cofactor deficient-mice with a Gephyrin mutation, appears normal. MOCS1(-/-) mice could be a suitable animal model for biochemical and/or genetic therapy approaches.

Streptococcus mutans and Streptococcus sobrinus are the major causative agents of human dental caries. Therefore, the removal or inhibition of these streptococcal biofilms is essential for dental caries prevention. In the present study, we evaluated the effects of ribose treatment alone or in combination with xylitol on streptococcal biofilm formation for both species. Furthermore, we examined the expression of genes responsible for dextran-dependent aggregation (DDAG). In addition, we investigated whether ribose affects the biofilm formation of xylitol-insensitive streptococci, which results from long-term exposure to xylitol. The viability of streptococci biofilms formed in a 24-well polystyrene plate was quantified by fluorescent staining with the LIVE/DEAD bacterial viability and counting kit, which was followed by fluorescence activated cell sorting analysis. The effects of ribose and/or xylitol on the mRNA expression of DDAG-responsible genes, gbpC and dblB, was evaluated by RT-qPCR. Our data showed that ribose and other pentose molecules significantly inhibited streptococcal biofilm formation and the expression of DDAG-responsible genes. In addition, co-treatment with ribose and xylitol decreased streptococcal biofilm formation to a further extent than ribose or xylitol treatment alone in both streptococcal species. Furthermore, ribose attenuated the increase of xylitol-insensitive streptococcal biofilm, which results in the reduced difference of biofilm formation between S. mutans that are sensitive and insensitive to xylitol. These data suggest that pentose may be used as an additive for teeth-protective materials or in sweets. Furthermore, ribose co-treatment with xylitol might help to increase the anti-cariogenic efficacy of xylitol.

There has been little evidence to support EGR1 and PTEN function on the EMT of cancer cells. We tried to evaluate how these genes affect cancer cell invasion and EMT through investigating the molecular mechanism(s) of 2'-benzoyloxycinnamaldehyde (BCA). Matrigel invasion and wound healing assay, and in vivo mice model were used to evaluate the effect of BCA on colon cancer cell migration. The molecular mechanism(s) of BCA were evaluated by knock-down or overexpression of EGR1 and PTEN. BCA at 50 nM increased E-cadherin and EGR1 expression without cytotoxicity. Cell migration was inhibited significantly by BCA both in vitro and in vivo. Moreover, BCA inhibits Snail and Vimentin expression, as well as β-catenin nuclear accumulation. Suppression of EGR1 by siRNA attenuated the inhibition of matrigel invasion by BCA, indicating that EGR1 is responsible for BCA effect. PTEN was upregulated by BCA treatment or EGR1 overexpression. In addition, shPTEN transfection stimulated EMT and cell invasion in vitro. Our data suggest that BCA leads to a remarkable upregulation of EGR1 expression, and that EMT and invasion is decreased via EGR1-dependent PTEN activation. These data showed a critical role of EGR1-PTEN signaling pathway in the EMT of colon cancer, as well as metastasis.

2′-Benzoyloxycinnamaldehyde (BCA) is a promising antitumor agent which induces cancer cells apoptosis via reactive oxygen species (ROS) generation. BCA shows more effective antiproliferation in MDA-MB-435 than in MCF-7 breast cancer cells. DJ-1 has been known to protect cells against oxidative stress as an antioxidant because of its cysteine residues sensitive to oxidative stress. In the present study, we evaluated the mechanism of DJ-1 for cell protection from oxidative stress after BCA treatment in MCF-7 cell. BCA upregulates the expression of DJ-1 in MCF-7 cells. However, DJ-1 expression decreased continuously for 24 h after BCA treatment in MDA-MB-435 cells. DJ-1 knockdown sensitized MCF-7 cells to BCA, on the contrary, DJ-1 overexpression induced MDA-MB-435 cells less sensitive to BCA. Confocal microscopic observation showed that only in MCF-7 cells BCA increased the overlapped signal between mitochondria and DJ-1 protein. Mitochondrial membrane potential (MMP) was decreased in MDA-MB-435 cells by BCA, and DJ-1 overexpression inhibited BCA-induced MMP decrease in these cells. On the contrary, DJ-1 knockdown in MCF-7 induced MMP perturbation by BCA. These findings suggest that DJ-1 upregulation protects MCF-7 cells from BCA via inhibiting mitochondrial damage.

Objective: To examine the effect of garlic extract on the biofilm formation by Streptococcus mutans on orthodontic wire and on glucosyltransferase gene expression. Materials and Methods: Growth inhibition of oral bacteria was tested after 50 µL of garlic extract was placed on an agar plate. The minimum inhibitory concentration (MIC) of garlic extract on S mutans growth was first determined. After cultivating streptococci in biofilm medium (BM)-sucrose with garlic extract and orthodontic wire, adenosine triphosphate (ATP) measurement and viable cell counting was performed from the bacteria attached on the wire. Scanning electron microscopy (SEM) analysis of morphology was observed on bacterial cells attached to orthodontic wire. The effect of garlic extract on gene expression was evaluated using quantitative real-time polymerase chain reaction (PCR) of glucosyltransferase. Results: Though garlic extract had a clear antibacterial effect on all microorganisms, it also enhanced S mutans attachment on orthodontic wire. Low concentration of garlic extract also increased glucosyltransferase gene expression of S mutans. Conclusions: Despite its antibacterial function, garlic extract increases biofilm formation by S mutans to orthodontic wire, likely through upregulation of glucosyltransferase expression. Garlic extract may thus play an important role in increased bacterial attachment to orthodontic wires.

2'-Benzoyloxycinnamaldehyde (BCA), one of the derivatives of 2'-hydroxycinnamaldehyde (HCA) isolated from the bark of Cinnamomum cassia, induces apoptosis in human cancer cells. We found that BCA induces stronger antiproliferative effects in K-ras-transformed cells (RK3E-ras) than in isogenic non-transformed cells (RK3E). Treatment of RK3E-ras with BCA resulted in increased ROS generation and depletion of intracellular glutathione, whereas BCA-treated RK3E showed no significant increase in the ROS level with concurrent increase in intracellular glutathione (GSH). Thiol antioxidants recovered cell proliferation inhibition caused by BCA in both cell lines, while non-thiol antioxidants failed to recover cell death. BCA decreased metallothionein (MT) expression in RK3E-ras, while inducing remarkable MT expression in RK3E. The increase of intracellular GSH in RK3E is partially caused by differential induction of γ-glutamylcysteine synthetase (γ-GCS) due to BCA treatment. To evaluate the upstream pathway for differential expression of γ-GCS and MT, we analyzed early DJ-1 (PARK7) and NF-E2 p45-related factor 2 (Nrf2) changes after BCA treatment. In RK3E, DJ-1 expression considerably increased for 3 h with concurrent induction of Nrf2, whereas in RK3E-ras cells BCA decreased these protein levels. Based on these findings, it seems that the therapeutic selectivity of BCA in RK3E-ras results from decreased thiol antioxidants via decreased DJ-1 and Nrf2 expression.

Current concepts of the pathogenesis of Parkinson's disease (PD) center on the formation of reactive oxygen species (ROS), and dopamine has been considered to be a major source of ROS. Recently, it has been shown in a postmortem study that nuclear translocation of nuclear factor-kappa B (NF-kappaB) was observed in dopaminergic neurons of patient with PD. However, its role is not known. The present study examined the possible role of NF-kappaB in ODA (auto-oxidized dopamine)-induced apoptosis to understand the process of PD. Using the electrophoretic mobility shift assay, it was found that ODA activated the DNA binding activity of NF-kappaB. Suppression of the transcriptional activity of NF-kappaB in PC12 cells by overexpression of a wild-type and a dominant negative mutant form (S32A/S36A) of inhibitor kappa B (IkappaB)-alpha led to increase of apoptotic cell death induced by treatment of ODA. In addition, overexpression of NF-kappaB in PC12 cells blocked ODA-induced cell death. However, JNK/SAPK activities, which mediate various stress signals, were similar among the parental, NF-kappaB- or dominant negative mutant IkappaB alpha-transfected cells. Therefore, these results suggest that activation of NF-kappaB during ODA-induced apoptosis may have a counteracting activity against the signals mediating apoptotic cell death and thereby delay the process of Parkinson's disease.

Current concepts of the pathogenesis of Parkinson's disease center on the formation of reactive oxygen species (ROS). Dopamine is one of the major sources of ROS. In this study, the molecular events during the dopamine-induced apoptosis in PC-12 cells were studied using auto-oxidized dopamine. Auto-oxidized-dopamine induced DNA fragmentation and activation of c-jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) faster and stronger than dopamine. Furthermore, N-acetylcysteine, an antioxidant, prevented the auto-oxidized dopamine-induced JNK/SAPK activation and DNA fragmentation. Meanwhile, Bcl-2 started to decrease after onset of apoptosis, and Bax was increased up to beginning of apoptosis, and thereafter decreased. Therefore, these results suggested that activation of JNK/SAPK and the decreased ratio of antiapoptotic Bcl-2 to proapoptotic Bax appear to be associated with the dopamine-induced apoptosis.