Essential amino acid starvation induces cell cycle arrest, autophagy, and inhibits osteogenic differentiation in murine osteoblast.

This study investigates the effects of essential amino acid (EAA) starvation on murine osteoblasts cells and the underlying mechanisms. We performed and observed the cell proliferation, autophagy, and osteogenic differentiation under deprivation of EAA in vitro. The results showed that EAA starvation resulted in cell cycle arrest via phosphorylation of the MAPK signaling pathway, leading to inhibition of cell proliferation and osteogenic differentiation. Additionally, the LKB1-AMPK signaling pathway was also found to be phosphorylated, inducing autophagy. These findings highlight the significant role of EAA in regulating cellular processes. Furthermore, this study contributes to our understanding of the effects of nutrient deprivation on cellular physiology and may aid in the development of novel therapeutic strategies for diseases associated with amino acid metabolism.

Intracellular glucose starvation inhibits osteogenic differentiation in human periodontal ligament cells.

BACKGROUND: Periodontal ligament cells (PDLCs), as mesenchymal cells in the oral cavity, are closely linked to periodontal tissue regeneration. However, the effect of local glucose deficiency on periodontal tissue regeneration, such as immediately post-surgery, remains unknown. OBJECTIVE: In the present study, we investigated the effect of a low-glucose environment on the proliferation and osteogenic differentiation of PDLCs. MATERIALS AND METHODS: We used media with five glucose concentrations (100, 75, 50, 25, and 0 mg/dL) and focused on the effects of a low-glucose environment on the proliferation, osteogenic differentiation, and autophagy of PDLCs. Additionally, we focused on changes in lactate production in a low-glucose environment and investigated the involvement of lactate with AZD3965, a monocarboxylate transporter-1 (MCT-1) inhibitor. RESULTS: The low-glucose environment inhibited PDLCs proliferation, migration, and osteogenic differentiation, and induced the expression of the autophagy-related factors LC3 and p62. Lactate and ATP production were decreased under low-glucose conditions. The addition of AZD3965 (MCT-1 inhibitor) in normal glucose conditions caused a similar trend as in low-glucose conditions on PDLCs. CONCLUSION: Our results suggest lactate production through glucose metabolism in the osteogenic differentiation of PDLCs. A low-glucose environment decreased lactate production, inhibiting cell proliferation, migration, and osteogenic differentiation and inducing autophagy in PDLCs.

Essential Amino Acid Starvation-Induced Oxidative Stress Causes DNA Damage and Apoptosis in Murine Osteoblast-like Cells.

Intracellular nutrient metabolism, particularly the metabolism of essential amino acids (EAAs), is crucial for cellular functions, including energy production and redox homeostasis. An EAA deficiency can lead to cellular dysfunction and oxidative stress. This study explores the mechanisms underlying cellular responses to EAA starvation, focusing on ROS-induced DNA damage and apoptosis. MC3T3-E1 cells were subjected to EAA starvation, and various assays were conducted to assess cell proliferation, survival, DNA damage, and apoptosis. The antioxidant N-acetylcysteine (NAC) was employed to block ROS formation and mitigate cellular damage. Gene expression and Western blot analyses were performed to elucidate molecular pathways. EAA starvation-induced ROS generation, DNA damage, and apoptosis in MC3T3-E1 cells. NAC administration effectively reduced DNA damage and apoptosis, highlighting the pivotal role of ROS in mediating these cellular responses during EAA deficiency. This study demonstrates that EAA starvation triggers ROS-mediated DNA damage and apoptosis, offering insights into the intricate interplay between nutrient deficiency, oxidative stress, and programmed cell death. NAC emerges as a potential therapeutic intervention to counteract these adverse effects.

Stem Cell Transplantation and Cell-Free Treatment for Periodontal Regeneration.

Increasing attention has been paid to cell-based medicines. Many in vivo and in vitro studies have demonstrated the efficacy of stem cell transplantation for the regeneration of periodontal tissues over the past 20 years. Although positive evidence has accumulated regarding periodontal regeneration using stem cells, the exact mechanism of tissue regeneration is still largely unknown. This review outlines the practicality and emerging problems of stem cell transplantation therapy for periodontal regeneration. In addition, possible solutions to these problems and cell-free treatment are discussed.

Detection of a major QTL related to smut disease resistance inherited from a Japanese wild sugarcane using GRAS-Di technology.

Smut disease of sugarcane causes considerable yield losses and the use of resistant varieties is the best control practice. Our group identified a Japanese wild sugarcane with highly smut disease resistance named 'Iriomote8'. In this study, we conducted QTL analysis for smut disease resistance using a mapping population derived from a resistant variety 'Yaenoushie', in which resistance is inherited from 'Iriomote8'. We identified 4813 non-redundant markers using GRAS-Di technology and developed a linkage map of mapping parents. We evaluated smut disease resistance of the mapping population by the inoculation test. Consequently, a large number of clones did not show the disease symptoms and the distribution of smut disease incidence tended to be "L shaped". Composite interval mapping detected an identical QTL for indices of smut disease incidence with a markedly high LOD score (26.6~45.6) at the end of linkage group 8 of 'Yaenoushie'. This QTL explained approximately 50% of the cases of smut disease incidence. In the mapping population, there were no correlations between the indices of smut disease incidence and other agronomic traits. In conclusion, this QTL could be used for marker-assisted selection to significantly improve smut disease resistance without negative effects on other agronomic traits.

Irradiation by high-intensity red light-emitting diode enhances human bone marrow mesenchymal stem cells osteogenic differentiation and mineralization through Wnt/ß-catenin signaling pathway.

Photobiomodulation therapy (PBMT) using a light-emitting diode (LED) has been employed for various photomedicine studies. The aim of this study was to determine the effects of a high-intensity red LED on the proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (BMSCs) and the related mechanism. BMSCs were subjected to high-intensity red LED (LZ1-00R205 Deep Red LED) irradiations for 0 to 40 s with energy densities ranging from 0 to 8 J/cm2. The distance from the LED to the cell layer was 40 mm. The spot size on the target was 4 cm2. Cell proliferation was measured at 3, 24, 48, and 72 h. The effects of LED irradiation on osteogenic differentiation and mineralization were examined with a particular focus on the Wnt/ß-catenin signaling pathway. The high-intensity red LED irradiations did not alter BMSC proliferation after 72 h. LED exposure of 6 J/cm2 (30 s) led to significant enhancements of osteogenic differentiation and mineralization. Additionally, the high-intensity LED irradiation induced activation of Wnt/ß-catenin. The effects of the high-intensity LED irradiation on BMSC osteogenic differentiation and mineralization were suppressed by treatment with the Wnt/ß-catenin inhibitor XAV939. P < 0.05 was considered significant. The results indicate that high-intensity red LED irradiation increases BMSC osteogenic differentiation and mineralization via Wnt/ß-catenin activation. Therefore, short duration irradiation with a portable high-intensity LED may be used as a potential approach in hard tissue regeneration therapy.

Augmentation of Bone Regeneration by Depletion of Stress-Induced Senescent Cells Using Catechin and Senolytics.

Despite advances in bone regenerative medicine, the relationship between stress-induced premature senescence (SIPS) in cells and bone regeneration remains largely unknown. Herein, we demonstrated that the implantation of a lipopolysaccharide (LPS) sustained-release gelatin sponge (LS-G) increases the number of SIPS cells and that the elimination of these cells promotes bone formation in critical-sized bone defects in the rat calvaria. Histological (hematoxylin-eosin and SA-ß-gal) and immunohistological (p16 and p21 for analyzing cellular senescence and 4-HNE for oxidation) staining was used to identify SIPS cells and elucidate the underlying mechanism. Bone formation in defects were analyzed using microcomputed tomography, one and four weeks after surgery. Parallel to LS-G implantation, local epigallocatechin gallate (EGCG) administration, and systemic senolytic (dasatinib and quercetin: D+Q) administration were used to eliminate SIPS cells. After LS-G implantation, SA-ß-gal-, p16-, and p21-positive cells (SIPS cells) accumulated in the defects. However, treatment with LS-G+EGCG and LS-G+D+Q resulted in lower numbers of SIPS cells than that with LS-G in the defects, resulting in an augmentation of newly formed bone. We demonstrated that SIPS cells induced by sustained stimulation by LPS may play a deleterious role in bone formation. Controlling these cell numbers is a promising strategy to increase bone regeneration.

Biological Effects of Shikonin in Human Gingival Fibroblasts via ERK 1/2 Signaling Pathway.

Shikonin, an active ingredient of Lithospermum erythrorhizon, exerts anti-inflammatory and antibacterial effects, and promotes wound healing. We investigated whether shikonin stimulated gingival tissue wound healing in human gingival fibroblasts (hGF). In addition, we evaluated the effects of shikonin on the mitogen-activated protein kinase (MAPK) signaling pathway, which has an important role in wound healing. hGF were subjected to primary culture using gingiva collected from patients. The cells were exposed to/treated with Shikonin at concentrations ranging from 0.01 to 100 µM. The optimal concentration was determined by cell proliferation and migration assays. Type I collagen and fibronectin synthesis, the gene expression of vascular endothelial growth factor (VEGF) and FN, and the phosphorylation of Extracellular signal-regulated kinase (ERK) 1/2 were investigated. Identical experiments were performed in the presence of PD98059 our data suggest, a specific ERK 1/2 inhibitor. Shikonin significantly promoted hGF proliferation and migration. Shikonin (1 µM) was chosen as the optimal concentration. Shikonin promoted type I collagen and FN synthesis, increased VEGF and FN expression, and induced ERK 1/2 phosphorylation. These changes were partially suppressed by PD98059. In conclusion, Shikonin promoted the proliferation, migration, type I collagen and FN synthesis, and expression of VEGF and FN via ERK 1/2 signaling pathway in hGFs. Therefore, shikonin may promote periodontal tissue wound healing.

Nasal double DNA adjuvant induces salivary FimA-specific secretory IgA antibodies in young and aging mice and blocks Porphyromonas gingivalis binding to a salivary protein.

BACKGROUND: We previously showed that nasal administration of a combination of dendritic cell (DC) targeted DNA plasmid expressing Flt3 ligand and CpG oligodeoxynucleotides 1826 as a mucosal adjuvant (double adjuvant, DA) provoked protective immunity in the upper respiratory tract of young adult and aging mice. Here, we investigated whether the nasal DA system induces secretory (S)IgA antibodies (Abs) toward recombinant fimbrillin (rFimA) of Porphyromonas gingivalis (P. gingivalis) in the saliva of young adult and aging mice. Further, we examined the functional applicability of rFimA-specific salivary SIgA Abs. METHODS: BALB/c mice (8- or 48-week-old) were nasally immunized with rFimA plus DA three times at weekly intervals. Control mice were nasally administered rFimA alone. Saliva samples were collected 1 week after the final immunization, and were subjected to rFimA-specific ELISA. To examine the functional applicability of rFimA-specific SIgA Abs, IgA-enriched saliva samples were subjected to an inhibition assay in order to assess the numbers of P. gingivalis cells bound to the salivary protein statherin. RESULTS: The 8- and 48-week-old mice administered nasal rFimA plus DA showed significantly increased levels of rFimA-specific SIgA Abs in saliva and elevated numbers of CD11c+ DCs in sublingual glands (SLGs), periglandular lymph nodes (PGLNs) and submandibular glands (SMGs) as well as nasopharyngeal-associated lymphoid tissues (NALT) compared to mice administered rFimA alone. Further, rFimA-specific SIgA Abs-containing saliva, in which IgG Abs of 8- and 48-week-old mice administered nasal rFimA plus DA were removed, significantly inhibited binding of P. gingivalis to the salivary protein. CONCLUSIONS: These findings show that this DA system could be an effective nasal vaccine strategy for the enhancement of P. gingivalis-specific protective immunity in the oral cavity of adolescents and older individuals.

Estrogen induces estrogen receptor alpha expression and hepatocyte proliferation in the livers of male mice.

Estrogens play pivotal roles in sexual development, growth, reproduction, and sex differentiation and have been implicated in a number of physiological processes in various tissues. Most of the effects of estrogens are mediated by the estrogen receptors alpha (ERα), beta (ERß), and G protein-coupled receptor 30 (GPR30). The liver is known to be a target tissue for estrogen signaling, but the physiological role of this signaling is not well characterized. Through analyses of an estradiol (E2)-treated hepatocyte cell line and mice, we showed that E2 signaling controls hepatocyte proliferation. Importantly, our data showed that the E2 signaling that is mediated through ERα is crucial for efficient liver regeneration after partial hepatectomy (PH). PH rapidly induced marked increases in circulating E2 and ERα transcripts in periportal hepatocytes, well before the onset of hepatocyte proliferation. Taken together, our results indicate that increased E2 is one of the initiating signals that trigger liver regeneration. We suggest that E2 treatment could be beneficial for stimulating liver regeneration in humans.

The expression of 11ß-hydroxysteroid dehydrogenase type 1 is increased in experimental periodontitis in rats.

BACKGROUND: The involvement of 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), which converts inactive glucocorticoids into active glucocorticoids intracellularly, in metabolic diseases and chronic inflammatory diseases has been elucidated. We recently reported that an increase in 11ß-HSD1 expression was associated with chronic periodontitis in humans irrespective of obesity. To further clarify the role of 11ß-HSD1 in chronic periodontitis, the expression of 11ß-HSD1 was investigated in experimental periodontitis model in rats. METHODS: Experimental periodontitis was induced by silk ligature of left maxillary second molars of 7-week-old male Wistar rats, and periodontal tissues were collected at day 3. The expression of 11ß-HSD1, 11ß-HSD2, and TNFα mRNA was examined using real time reverse transcription-polymerase chain reaction. The expression of TNFα was used as an indicator of inflammation. Thus, the rats in which the levels of TNFα mRNA were increased in the ligature-induced periodontitis compared with the control were analysed. RESULTS: The findings demonstrated that the expression of 11ß-HSD1 mRNA was significantly increased in experimental periodontitis compared with the control. The increase in the levels of 11ß-HSD1 mRNA in the ligature-induced periodontitis compared with the control was positively correlated with that of TNFα mRNA. On the other hand, the expression of 11ß-HSD2 mRNA, which inactivates glucocorticoids, was slightly decreased in experimental periodontitis. Therefore, the ratio of 11ß-HSD1 versus 11ß-HSD2 mRNA was significantly higher in experimental periodontitis than in the control. CONCLUSIONS: These results suggest that the increased expression of 11ß-HSD1, which would result in the increased levels of intracellular glucocorticoids, may play a role in the pathophysiology of experimental periodontitis.

Arginine-induced insulin secretion in endoplasmic reticulum.

Arginine, a semi-essential amino acid, is known as one of the most strongest insulin secretagogues in a glucose-dependent manner, but major mechanism is unknown. Arginine induced insulin secretion in mice as well as ß cell line, NIT-1, in which more than 90% of intracellular insulin is prionsulin without arginine cultivation. Arginine administration reduced prionsulin amount in 30 s, then insulin is secreted from NIT1 cells. These data indicated that the target factor(s) for arginine-induced insulin secretion located in endoplasmic reticulum (ER). We established the screening system for identifying the arginine mimetics. Brazilian propolis, not Chinese propolis, induced insulin secretion. To identify target factor(s) of arginine induced insulin secretion, our previous study was that nanobeads technology facilitated us to purify chemical-target factors. This time we chose the other way, proinsulin associating factor purification and arginine-immobilized agarose. Three proinsulin associating factors and 5 arginine interacting factors were identified. Among theses factors, Calnexin (CNX) was the only one factor, which belonged to both groups, suggesting that CNX might play a key role in arginine-induced insulin secretion in ER.

Transformation assay in Bhas 42 cells: a model using initiated cells to study mechanisms of carcinogenesis and predict carcinogenic potential of chemicals.

Transformation assays using cultured cells have been applied to the study of carcinogenesis. Although various cell systems exist, few cell types such as BALB/c 3T3 subclones and Syrian hamster embryo cells have been used to study chemically induced two-stage carcinogenesis. Bhas 42 cells were established as a clone by the transfection with the v-Ha-ras gene into mouse BALB/c 3T3 A31-1-1 cells and their subsequent selection based on their sensitivity to 12-O-tetradecanoylphorbol-13-acetate. Using Bhas 42 cells, transformed foci were induced by the treatment with nongenotoxic carcinogens, most of which act as tumor promoters. Therefore, Bhas 42 cells were considered to be a model of initiated cells. Subsequently, not only nongenotoxic carcinogens but also genotoxic carcinogens, most of which act as tumor initiators, were found to induce transformed foci by the modification of the protocol. Furthermore, transformation of Bhas 42 cells was induced by the transfection with genes of oncogenic potential. We interpret this high sensitivity of Bhas 42 cells to various types of carcinogenic stimuli to be related to the multistage model of carcinogenesis, as the transfection of v-Ha-ras gene further advances the parental BALB/c 3T3 A31-1-1 cells toward higher transforming potential. Thus, we propose that Bhas 42 cells are a novel and sensitive cell line for the analysis of carcinogenesis and can be used for the detection of not only carcinogenic substances but also gene alterations related to oncogenesis. This review will address characteristics of Bhas 42 cells, the transformation assay protocol, validation studies, and the various chemicals tested in this assay.

Reactivation from occult HBV carrier status is characterized by low genetic heterogeneity with the wild-type or G1896A variant prevalence.

BACKGROUND & AIMS: Individuals negative for hepatitis B surface antigen (HBsAg) but positive for antibodies to hepatitis B core antigen (anti-HBc) are at risk of hepatitis B virus (HBV) reactivation under immunosuppressive conditions. We investigated clinical features and viral genetics in patients with reactivation from occult HBV infection triggered by chemotherapy or immunosuppressive therapy. METHODS: Clinical courses of 14 individuals originally HBsAg-negative but anti-HBc-positive that experienced HBV reactivation were examined. Ultra-deep sequencing analysis of the entire HBV genome in serum was conducted. Prevalence of the G1896A variant in latently infected livers was determined among 44 healthy individuals that were HBsAg-negative but anti-HBc-positive. RESULTS: In 14 cases, HBV reactivation occurred during (n=7) and after (n=7) termination of immunosuppressive therapy. Ultra-deep sequencing revealed that the genetic heterogeneity of reactivated HBV was significantly lower in patients with reactivation from occult HBV carrier status compared with that in patients from HBsAg carrier status. The reactivated viruses in each case were almost exclusively the wild-type G1896 or G1896A variant. The G1896A variant was detected in 42.9% (6/14) of cases, including two cases with fatal liver failure. The G1896A variant was observed in the liver tissue of 11.4% (5/44) of individuals with occult HBV infection. CONCLUSIONS: Reactivation from occult HBV infection is characterized by low genetic heterogeneity, with the wild-type G1896 or G1896A variant prevalent.

The effects of synthetic oligopeptide derived from enamel matrix derivative on cell proliferation and osteoblastic differentiation of human mesenchymal stem cells.

Enamel matrix derivative (EMD) is widely used in periodontal tissue regeneration therapy. However, because the bioactivity of EMD varies from batch to batch, and the use of a synthetic peptide could avoid use from an animal source, a completely synthetic peptide (SP) containing the active component of EMD would be useful. In this study an oligopeptide synthesized derived from EMD was evaluated for whether it contributes to periodontal tissue regeneration. We investigated the effects of the SP on cell proliferation and osteoblast differentiation of human mesenchymal stem cells (MSCs), which are involved in tissue regeneration. MSCs were treated with SP (0 to 1000 ng/mL), to determine the optimal concentration. We examined the effects of SP on cell proliferation and osteoblastic differentiation indicators such as alkaline phosphatase activity, the production of procollagen type 1 C-peptide and osteocalcin, and on mineralization. Additionally, we investigated the role of extracellular signal-related kinases (ERK) in cell proliferation and osteoblastic differentiation induced by SP. Our results suggest that SP promotes these processes in human MSCs, and that ERK inhibitors suppress these effects. In conclusion, SP promotes cell proliferation and osteoblastic differentiation of human MSCs, probably through the ERK pathway.

Surface modification affects human gingival epithelial cell behavior on polyetheretherketone surfaces.

Gingival epithelial attachment to the abutment is important for the prevention of peri-implantitis. Polyetheretherketone (PEEK) has recently gained attention as an alternative material to titanium; however, it is biologically inert, which is disadvantageous for obtaining soft tissue sealing of the transmucosal part of the implant abutment. Therefore, ultraviolet (UV) irradiation, argon plasma irradiation, and buffing were selected as treatments to modify the PEEK surface. None of the treatments had any effect on the material's mechanical strength. The UV and plasma treatments did not significantly affect the surface morphology. Surface elemental analysis showed a decrease in carbon content and an increase in oxygen content and wettability for all treatments. Human gingival epithelial cell adhesion, proliferation, and the expression of adhesion proteins integrin ß4 and laminin 332, were increased. Surface modification to PEEK was suggested to enhance cell activity on PEEK.

Machine learning for genomic and pedigree prediction in sugarcane.

Sugarcane (Saccharum spp.) plays a crucial role in global sugar production; however, the efficiency of breeding programs has been hindered by its heterozygous polyploid genomes. Considering non-additive genetic effects is essential in genome prediction (GP) models of crops with highly heterozygous polyploid genomes. This study incorporates non-additive genetic effects and pedigree information using machine learning methods to track sugarcane breeding lines and enhance the prediction by assessing the degree of association between genotypes. This study measured the stalk biomass and sugar content of 297 clones from 87 families within a breeding population used in the Japanese sugarcane breeding program. Subsequently, we conducted analyses based on the marker genotypes of 33,149 single-nucleotide polymorphisms. To validate the accuracy of GP in the population, we first predicted the prediction accuracy of the best linear unbiased prediction (BLUP) based on a genomic relationship matrix. Prediction accuracy was assessed using two different cross-validation methods: repeated 10-fold cross-validation and leave-one-family-out cross-validation. The accuracy of GP of the first and second methods ranged from 0.36 to 0.74 and 0.15 to 0.63, respectively. Next, we compared the prediction accuracy of BLUP and two machine learning methods: random forests and simulation annealing ensemble (SAE), a newly developed machine learning method that explicitly models the interaction between variables. Both pedigree and genomic information were utilized as input in these methods. Through repeated 10-fold cross-validation, we found that the accuracy of the machine learning methods consistently surpassed that of BLUP in most cases. In leave-one-family-out cross-validation, SAE demonstrated the highest accuracy among the methods. These results underscore the effectiveness of GP in Japanese sugarcane breeding and highlight the significant potential of machine learning methods.

A Data-Driven Approach to Sugarcane Breeding Programs with Agronomic Characteristics and Amino Acid Constituent Profiling.

Sugarcane (Saccharum spp. hybrids) and its processed products have supported local industries such as those in the Nansei Islands, Japan. To improve the sugarcane quality and productivity, breeders select better clones by evaluating agronomic characteristics, such as commercially recoverable sugar and cane yield. However, other constituents in sugarcane remain largely unutilized in sugarcane breeding programs. This study aims to establish a data-driven approach to analyze agronomic characteristics from breeding programs. This approach also determines a correlation between agronomic characteristics and free amino acid composition to make breeding programs more efficient. Sugarcane was sampled in clones in the later stage of breeding selection and cultivars from experimental fields on Tanegashima Island. Principal component analysis and hierarchical cluster analysis using agronomic characteristics revealed the diversity and variability of each sample, and the data-driven approach classified cultivars and clones into three groups based on yield type. A comparison of free amino acid constituents between these groups revealed significant differences in amino acids such as asparagine and glutamine. This approach dealing with a large volume of data on agronomic characteristics will be useful for assessing the characteristics of potential clones under selection and accelerating breeding programs.