Heparin interacts with candidalysin and neutralizes its cytotoxicity to oral epithelial cells.

OBJECTIVES: Candidalysin is a peptide toxin produced by Candida albicans that causes damage to epithelial cells by destabilizing the plasma membrane. This study aimed to evaluate heparin's ability to neutralize candidalysin and protect epithelial cells from lysis. METHODS: The study was conducted using a human oral epithelial cell line and synthetic candidalysin. Cell damage was assessed by measuring lactate dehydrogenase release. Enzyme-linked immunosorbent assay and immunoblotting were used to determine cytokine concentrations and assess activation of intracellular signaling molecules and transcription factors, respectively. Flow cytometry was used to measure cell-bound candidalysin. RESULTS: Heparin diminished the cell-lytic activity of candidalysin and subsequent epithelial responses. Additionally, heparin inhibited the interaction between candidalysin and epithelial cells. Furthermore, polyacrylic acid, a synthetic polymer, mimicked the neutralizing effects of candidalysin. CONCLUSION: Our findings suggest that negatively charged polymers could be a potential therapeutic option for preventing the damage caused by candidalysin. Further research is needed to explore the effectiveness of other anionic polymers and their potential clinical applications.

Calcium phosphate-adsorbable and acid-degradable carboxylated polyrotaxane consisting of ß-cyclodextrins suppresses osteoclast resorptive activity.

Recently, the potential of ß-cyclodextrin-thread acid-degradable polyrotaxane (AdPRX) has been emphasized as a therapeutic agent for cholesterol-related metabolic disorders. In this study, we investigated whether carboxymethyl carbamate-modified AdPRX (CMC-AdPRX) can be used for adsorption to calcium phosphate to treat bone diseases. We first synthesized CMC-AdPRX and used it to coat the calcium phosphate plate. RAW264.7 cells were then differentiated into osteoclasts via a receptor activator of nuclear factor-κB ligand, and the number of osteoclasts and the area of absorption lacunae were determined. The number of tartrate-resistant acid phosphatase-positive multinucleated cells was reduced on the CMC-AdPRX-coated plate. The area of the absorption lacunae was smaller with CMC-AdPRX than with AdPRX, which was not carboxy-modified. Our results suggest that CMC-AdPRX can adsorb to calcium phosphate and act on differentiated osteoclasts to suppress their functional expression.

Differential roles of two DDX17 isoforms in the formation of membraneless organelles.

The RNA helicase, DDX17 is a member of the DEAD-box protein family. DDX17 has two isoforms: p72 and p82. The p82 isoform has additional amino acid sequences called intrinsically disordered regions (IDRs), which are related to the formation of membraneless organelles (MLOs). Here, we reveal that p72 is mostly localized to the nucleoplasm, while p82 is localized to the nucleoplasm and nucleoli. Additionally, p82 exhibited slower intranuclear mobility than p72. Furthermore, the enzymatic mutants of both p72 and p82 accumulate into the stress granules. The enzymatic mutant of p82 abolishes nucleolar localization of p82. Our findings suggest the importance of IDRs and enzymatic activity of DEAD-box proteins in the intracellular distribution and formation of MLOs.

Knockdown of sphingomyelin synthase 2 inhibits osteoclastogenesis by decreasing RANKL expression in mouse primary osteoblasts.

Sphingomyelin is a major lipid of the plasma membrane and is enriched in microdomains of the plasma membrane that are critical for signal transduction. However, the function of sphingomyelin in the cell membrane of osteoblasts has not been clarified. Therefore, we examined how sphingomyelin synthase 2 (SMS2) affects osteoclast differentiation by osteoblasts. We knocked down the expression of SMS2 with siRNA targeting the Sgms2 gene in mouse primary osteoblasts. The effects of SMS2 knockdown in osteoblasts were examined using polymerase chain reaction and western blotting. The knockdown of SMS2 suppressed the formation of TRAP-positive multinucleated cells by co-culture of osteoblasts and bone marrow cells compared to the control. We found that receptor activator of nuclear factor κB ligand (RANKL) mRNA expression was significantly reduced by 1,25(OH)2D3 stimulation in SMS2 siRNA osteoblasts. The knockdown of SMS2 repressed the expression of retinoid-X-receptor-α (RXRα) regardless of 1,25(OH)2D3 stimulation. TRAP-positive multinucleated cell formation was significantly reduced by RXRα siRNA in osteoblasts in a co-culture system. These results suggest that SMS2 regulates osteoclast differentiation by inducing RANKL expression via RXRα.

Intracellular dynamics of actin affects Borna disease virus replication in the nucleus.

Borna disease virus (BoDV) is a nonsegmented, negative-strand RNA virus that uniquely replicates and establishes persistent infection in cell nucleus. Recent studies have demonstrated the presence of actin in the nucleus and its role in intranuclear phenomena such as transcription and DNA repair. Although nuclear actin is involved in the life cycle of some intranuclear DNA viruses, the interaction between BoDV and nuclear actin has not been reported. In this study, we show that the inhibition of the nucleocytoplasmic transport of actin affects the replication of BoDV in the nucleus. The knockdown of a nuclear export factor of actin, exportin 6, results in the induction of structural aberration in intranuclear viral factories of BoDV. Furthermore, the inhibition of the nuclear export of actin promotes accumulation of viral matrix protein in the cytoplasm and periphery of the infected cells. These results suggest that the dynamics of actin affect the replication of BoDV by disturbing the structure of viral factories in the nucleus.

RNA interference-mediated knockdown of Smad1 inhibits receptor activator of nuclear factor κB ligand expression induced by BMP-2 in primary osteoblasts.

OBJECTIVE: BMP-2 induces osteoblast differentiation and activates osteoclast formation. Here, we investigated the role of Smad1, a molecule that signals downstream of BMP-2, in mediating the effects of BMP-2 on osteoclast differentiation induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in osteoblasts. DESIGN: The effects of 1,25(OH)2D3 and BMP-2 in osteoclasts were examined using polymerase chain reaction and Western blotting to measure changes in target gene and protein expression. Immunostaining was carried out to investigate the localization of the vitamin D receptor (VDR) in the nucleus in response to BMP-2. RESULTS: Stimulation with both 1,25(OH)2D3 and BMP-2 resulted in significantly greater osteoclast formation and receptor activator of nuclear factor κB ligand (RANKL) mRNA expression compared to stimulation with 1,25(OH)2D3 alone. In addition, expression of the VDR protein was increased, enhancing the activity of 1,25(OH)2D3. Interestingly, knockdown of Smad1 resulted in reduced osteoclast formation, RANKL mRNA expression, and VDR protein expression compared with control cells. Costimulation with 1,25(OH)2D3 and BMP-2 enhanced VDR localization in the nucleus. CONCLUSIONS: We found that BMP-2 induced Smad1 activation, thereby influencing the localization of VDR in the nucleus in the presence of 1,25(OH)2D3 and resulting in increased RANKL mRNA expression. These effects ultimately resulted in enhanced osteoclast differentiation.

Expression patterns of CD66a and CD117 in the mouse submandibular gland.

The epithelial tissue of the salivary gland consists of the acinar and ductal parts, the latter of which is further divided into the intercalated, striated and excretory duct segments and is the residential site for salivary stem/progenitor cells. In the present study, the expression patterns of two cell surface molecules, CD66a and CD117, were investigated in the adult mouse submandibular glands (SMG) by immunofluorescence microscopy. Combinations of the two molecules differentially marked several types of SMG epithelial cells, including acinar cells (CD66a-intense, CD117-negative), intercalated duct cells (CD66a-intense, CD117-positive), a subset of the striated and excretory duct cells (CD66a-weak, CD117-positive). Most of the CD117-positive ductal cells were negative for cytokeratin 5 and overlapped with the NKCC1-expressing cells. The CD117- and keratin 5-positive cells resided only in the excretory duct were suggested to correspond to the recently identified salivary stem cells. CD66a and CD117 may be useful markers to isolate several cell types consisting of SMG epithelium and to analyze their molecular and cellular nature. Our data also suggest that CD117-expressing epithelial cells of the gland include at least two distinct populations of the stem/progenitor cells.

A 3D sphere culture system containing functional polymers for large-scale human pluripotent stem cell production.

Utilizing human pluripotent stem cells (hPSCs) in cell-based therapy and drug discovery requires large-scale cell production. However, scaling up conventional adherent cultures presents challenges of maintaining a uniform high quality at low cost. In this regard, suspension cultures are a viable alternative, because they are scalable and do not require adhesion surfaces. 3D culture systems such as bioreactors can be exploited for large-scale production. However, the limitations of current suspension culture methods include spontaneous fusion between cell aggregates and suboptimal passaging methods by dissociation and reaggregation. 3D culture systems that dynamically stir carrier beads or cell aggregates should be refined to reduce shearing forces that damage hPSCs. Here, we report a simple 3D sphere culture system that incorporates mechanical passaging and functional polymers. This setup resolves major problems associated with suspension culture methods and dynamic stirring systems and may be optimal for applications involving large-scale hPSC production.

Genetic evidence points to an osteocalcin-independent influence of osteoblasts on energy metabolism.

The skeleton has been shown recently to regulate glucose metabolism through an osteoblast-specific hormone, osteocalcin, which favors ß-cell proliferation, insulin secretion, insulin sensitivity, and energy expenditure. An implication of this finding is that a decrease in osteoblast numbers would compromise glucose metabolism in an osteocalcin-dependent manner. To test this hypothesis, osteoblasts were inducibly ablated by cross-breeding transgenic mice expressing a tamoxifen-regulated Cre under the control of the osteocalcin promoter with mice in which an inactive form of the diphtheria toxin A chain was introduced into a ubiquitously expressed locus. Ablation of osteoblasts in adult mice profoundly affected glucose metabolism. In a manner similar to what is seen in the case of osteocalcin deficiency, a partial ablation of this cell population resulted in hypoinsulinemia, hyperglycemia, glucose intolerance, and decreased insulin sensitivity. However, and unlike what is seen in osteocalcin-deficient mice, osteoblast ablation also decreased gonadal fat and increased energy expenditure and the expression of resistin, an adipokine proposed to mediate insulin resistance. While administration of osteocalcin reversed (fully) the glucose intolerance and reinstated normal blood glucose and insulin levels, it only partially restored insulin sensitivity and did not affect the improved gonadal fat weight and energy expenditure in osteoblast-depleted mice. These observations not only strengthen the notion that osteoblasts are necessary for glucose homeostasis and energy expenditure but also suggest that in addition to osteocalcin, other osteoblast-derived hormones may contribute to the emerging function of the skeleton as a regulator of energy metabolism.

FoxO1 is a positive regulator of bone formation by favoring protein synthesis and resistance to oxidative stress in osteoblasts.

Osteoporosis, a disease of low bone mass, is associated with decreased osteoblast numbers and increased levels of oxidative stress within osteoblasts. Since transcription factors of the FoxO family confer stress resistance, we investigated their potential impact on skeletal integrity. Here we employ cell-specific deletion and molecular analyses to show that, among the three FoxO proteins, only FoxO1 is required for proliferation and redox balance in osteoblasts and thereby controls bone formation. FoxO1 regulation of osteoblast proliferation occurs through its interaction with ATF4, a transcription factor regulating amino acid import, as well as through its regulation of a stress-dependent pathway influencing p53 signaling. Accordingly, decreasing oxidative stress levels or increasing protein intake normalizes bone formation and bone mass in mice lacking FoxO1 specifically in osteoblasts. These results identify FoxO1 as a crucial regulator of osteoblast physiology and provide a direct mechanistic link between oxidative stress and the regulation of bone remodeling.

New attempt of ultrasonic tissue characterization: decreased chaos in myocardial echo in patients with dilated cardiomyopathy.

Ultrasonic radiofrequency (RF) signals returned from the myocardium may display complex behavior, including deterministic chaos, and such chaos may be a useful marker of the tissue properties that are not obtainable with myocardial integrated backscatter. Thus, RF signals were obtained from the myocardium by the transthoracic approach in seven healthy subjects and eight patients with dilated cardiomyopathy (DCM). They were analyzed by a time-delay embedding technique to construct a continuous 3-D trajectory. The attractor formed a thick ring-like structure consisting of a relatively empty, roughly circular, core region in all healthy subjects. In DCM patients, the ring-like structure is thinned with a distinct empty circular core region. When the relation between embedding dimension and averaged correlation dimension was compared, the correlation dimension reached a plateau at the value of 3.5 in healthy subjects, and at the value of 2.6 in DCM patients. In conclusion, chaotic behavior is prevalent in the RF signals for the healthy myocardium, and a decrease in such chaos may be indicative of the damaged myocardium in DCM patients.