Effects of Plant-Based Extract Mixture on Alcohol Metabolism and Hangover Improvement in Humans: A Randomized, Double-Blind, Paralleled, Placebo-Controlled Clinical Trial.

Hangovers are uncomfortable physiological symptoms after alcohol consumption caused by acetaldehyde, a toxic substance in which alcohol is metabolized by alcohol dehydrogenase (ADH). Rapid alcohol and acetaldehyde decomposition are essential to alleviate alcohol handling symptoms. This study investigated the effects of HY_IPA combined with Mesembryanthemum crystallinum, Pueraria lobata flower, and Artemisia indica on alleviating hangovers. A randomized, double-blind, parallel-group, placebo-controlled clinical study was conducted on 80 individuals with hangover symptoms. Alcohol intake was 0.9 g/bw with 40% whiskey, adjusted proportionately to body weight. The Acute Hangover Scale total score was 5.24 ± 5.78 and 18.54 ± 18.50 in the HY_ IPA and placebo groups, respectively (p < 0.0001). All nine indicators of the hangover symptom questionnaire were significantly improved in the HY_IPA group (p < 0.01). Blood alcohol and acetaldehyde concentrations rapidly decreased from 30 min in the HY_IPA group (p < 0.05). ADH and acetaldehyde dehydrogenase (ALDH) activities in the blood of the HY_IPA group were significantly higher than those in the placebo group at 0, 1, and 2 h after alcohol consumption (p < 0.01). The rapid hangover relief was due to increased ADH and ALDH. Therefore, HY_IPA effectively relieves hangover symptoms by decomposing alcohol and acetaldehyde when consumed before alcohol consumption.

Protective Effects of Cudrania tricuspidata Against Helicobacter pylori-Induced Inflammation in C57BL/6 Mice.

Helicobacter pylori modulates the host inflammatory response, resulting in chronic gastritis, which contributes to gastric cancer pathogenesis. We verified the effect of Cudrania tricuspidata on H. pylori infection by inhibiting H. pylori-induced inflammatory activity. Five-week-old C57BL/6 mice (n = 8) were administered C. tricuspidata leaf extract (10 or 20 mg/kg per day) for 6 weeks. An invasive test (campylobacter-like organism [CLO]) and noninvasive tests (stool antigen test [SAT] and H. pylori antibody enzyme-linked immunosorbent assay) were performed to confirm the eradication of H. pylori. To evaluate the anti-inflammatory effect of C. tricuspidata, pro-inflammatory cytokines levels and inflammation scores were measured in mouse gastric tissue. C. tricuspidata significantly decreased the CLO score and H. pylori immunoglobulin G antibody optical density levels at both 10 and 20 mg/kg per day doses (P < .05). C. tricuspidata decreased the H. pylori antibody levels in a concentration-dependent manner, increased negative responses to SAT by up to 37.5%, and inhibited the pro-inflammatory cytokines interleukin (IL; IL-1ß, IL-6, 1L-8, and tumor necrosis factor alpha). C. tricuspidata also relieved gastric erosions and ulcers and significantly reduced the inflammation score (P < .05). We measured rutin in C. tricuspidata extract as a standard for high-performance liquid chromatography. C. tricuspidata leaf extract showed anti-H. pylori activity through the inhibition of inflammation. Our findings suggest that C. tricuspidata leaf extract is potentially an effective functional food material against H. pylori.

Performance of a Machine Learning-Based Methicillin Resistance of Staphylococcus aureus Identification System Using MALDI-TOF MS and Comparison of the Accuracy according to SCCmec Types.

The prompt presumptive identification of methicillin-resistant Staphylococcus aureus (MRSA) using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can aid in early clinical management and infection control during routine bacterial identification procedures. This study applied a machine learning approach to MALDI-TOF peaks for the presumptive identification of MRSA and compared the accuracy according to staphylococcal cassette chromosome mec (SCCmec) types. We analyzed 194 S. aureus clinical isolates to evaluate the machine learning-based identification system (AMRQuest software, v.2.1, ASTA: Suwon, Korea), which was constructed with 359 S. aureus clinical isolates for the learning dataset. This system showed a sensitivity of 91.8%, specificity of 83.3%, and accuracy of 87.6% in distinguishing MRSA. For SCCmec II and IVA types, common MRSA types in a hospital context, the accuracy was 95.4% and 96.1%, respectively, while for the SCCmec IV type, it was 21.4%. The accuracy was 90.9% for methicillin-susceptible S. aureus. This presumptive MRSA identification system may be helpful for the management of patients before the performance of routine antimicrobial resistance testing. Further optimization of the machine learning model with more datasets could help achieve rapid identification of MRSA with less effort in routine clinical procedures using MALDI-TOF MS as an identification method.

Development and metabolic profiling of a postbiotic complex exhibiting antibacterial activity against skin microorganisms and anti-inflammatory effect on human keratinocytes.

Beyond probiotics, the interest in the application of postbiotics to various fields has been growing. We aimed to develop a novel postbiotic complex (PC) with antibacterial and anti-inflammatory properties. Through antibacterial activity testing against Staphylococcus aureus or Cutibacterium acnes, a PC [a mixture of cell-free supernatants (postbiotics) from probiotic Lactobacillus helveticus (HY7801) and Lactococcus lactis (HY449)] was developed. Anti-inflammatory activity of the PC was investigated using HaCaT keratinocytes treated with S. aureus or C. acnes. PC significantly decreased IL-8 levels and increased hyaluronic acid levels in HaCaT cells cultured with S. aureus or C. acnes. GC-MS based metabolic profiling suggested 2-hydroxyisocaproic acid, hypoxanthine, succinic acid, ornithine, and γ-aminobutyric acid as potential contributing metabolites for the antibacterial and anti-inflammatory effects of PC. The PC developed in this study could be utilized in food, cosmetics, and pharmaceutical products as an alternative or complementary resources of probiotics. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01123-x.

Lactobacillus casei HY2782 and Pueraria lobata Root Extract Complex Ameliorates Particulate Matter-Induced Airway Inflammation in Mice by Inhibiting Th2 and Th17 Immune Responses.

This study aimed to investigate the effects of Lactobacillus casei HY2782 and Pueraria lobata root extract complex (HY2782 complex) in mitigating airway inflammation resulting from exposure to particulate matter ≤2.5 µm in diameter (PM2.5) in an animal model. Chronic inflammatory airway disease is associated with Th2-related cytokines interleukin (IL)-4, IL-5, and IL-13 and Th17-related cytokine IL-17A, which are the major contributors to allergy and asthma. Results indicated that PM2.5 elevates allergen-related airway inflammation and respiratory hyperresponsiveness in C57BL/6 mice. The HY2782 complex significantly reduced Th2/Th17-derived cytokines IL-4, IL5, IL-13, and IL-17A; immunoglobulin E; and leukotriene C4 in bronchoalveolar lavage fluid (BALF) and serum. Furthermore, the HY2782 complex was associated with the modulation of oxidative stress-related genes. Administration of the HY2782 complex resulted in a markedly reduced number of neutrophils and eosinophil infiltration in BALF. Histopathological observation of lung tissue also showed reduced inflammatory cell infiltration into airways and surrounding tissue. The HY2782 complex may be a promising candidate for the preventive therapy of allergic diseases and airway inflammation caused by PM2.5 inhalation.

Effects of Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032 on Overweight and the Gut Microbiota in Humans: Randomized, Double-Blinded, Placebo-Controlled Clinical Trial.

Obesity and overweight are closely related to diet, and the gut microbiota play an important role in body weight and human health. The aim of this study was to explore how Lactobacillus curvatus HY7601 and Lactobacillus plantarum KY1032 supplementation alleviate obesity by modulating the human gut microbiome. A randomized, double-blind, placebo-controlled study was conducted on 72 individuals with overweight. Over a 12-week period, probiotic groups consumed 1 × 1010 colony-forming units of HY7601 and KY1032, whereas the placebo group consumed the same product without probiotics. After treatment, the probiotic group displayed a reduction in body weight (p < 0.001), visceral fat mass (p < 0.025), and waist circumference (p < 0.007), and an increase in adiponectin (p < 0.046), compared with the placebo group. Additionally, HY7601 and KY1032 supplementation modulated bacterial gut microbiota characteristics and beta diversity by increasing Bifidobacteriaceae and Akkermansiaceae and decreasing Prevotellaceae and Selenomonadaceae. In summary, HY7601 and KY1032 probiotics exert anti-obesity effects by regulating the gut microbiota; hence, they have therapeutic potential for preventing or alleviating obesity and living with overweight.

Sulfated syndecan 1 is critical to preventing cellular senescence by modulating fibroblast growth factor receptor endocytosis.

Cellular senescence can be triggered by various intrinsic and extrinsic stimuli. We previously reported that silencing of 3'-phosphoadenosine 5'-phosphosulfate synthetase 2 (PAPSS2) induces cellular senescence through augmented fibroblast growth factor receptor 1 (FGFR1) signaling. However, the exact molecular mechanism connecting heparan sulfation and cellular senescence remains unclear. Here, we investigated the potential involvement of heparan sulfate proteoglycans (HSPGs) in augmented FGFR1 signaling and cellular senescence. Depletion of several types of HSPGs revealed that cells depleted of syndecan 1 (SDC1) exhibited typical senescence phenotypes, and those depleted of PAPSS2-, SDC1-, or heparan sulfate 2-O sulfotransferase 1 (HS2ST1) showed decreased FGFR1 internalization along with hyperresponsiveness to and prolonged activation of fibroblast growth factor 2 (FGF2)-stimulated FGFR1- v-akt murine thymoma viral oncogene homolog (AKT) signaling. Clathrin- and caveolin-mediated FGFR1 endocytosis contributed to cellular senescence through the FGFR1-AKT-p53-p21 signaling pathway. Dynasore treatment triggered senescence phenotypes, augmented FGFR1-AKT-p53-p21 signaling, and decreased SDC1 expression. Finally, the replicatively and prematurely senescent cells were characterized by decreases of SDC1 expression and FGFR1 internalization, and an increase in FGFR1-AKT-p53-p21 signaling. Together, our results demonstrate that properly sulfated SDC1 plays a critical role in preventing cellular senescence through the regulation of FGFR1 endocytosis.

Protective effect of Lactobacillus casei HY2782 against particulate matter toxicity in human intestinal CCD-18Co cells and Caenorhabditis elegans.

OBJECTIVES: To investigate the preventive effect of Lactobacillus casei HY2782 on toxicity induced by particulate matter (PM, inhalable particles less than 10 µm in diameter) in human intestinal CCD-18Co cells and a model animal Caenorhabditis elegans. RESULTS: L. casei HY2782 treatment prevented PM-induced intestinal cell death via cellular reactive oxygen species production and membrane disruption attenuation. PM significantly decreased the total number of eggs laid and the body bending activity of C. elegans, demonstrating PM toxicity. L. casei HY2782 treatment restored the reproductive toxicity and decline in locomotion activity induced by PM in C. elegans. Overall, L. casei HY2782 attenuated PM toxicity in vitro in cultured intestinal cells and in vivo in the model nematode. CONCLUSION: Our study provides a potential clue for developing L. casei HY2782 probiotics that attenuate PM-induced cellular and physiological toxicity; however, further in-depth preclinical trials using mammalian animal models and clinical trials are required.

Integrin α6ß4-Src-AKT signaling induces cellular senescence by counteracting apoptosis in irradiated tumor cells and tissues.

Cellular senescence refers to an irreversible growth arrest that is triggered by various intrinsic and extrinsic stresses. Many recent studies have demonstrated that cellular senescence plays a crucial role in the regression of tumors exposed to ionizing radiation (IR), but the underlying mechanism remains unknown. Here we show that the activation of integrin ß4 is essential for IR-induced cellular senescence. IR treatment results in the phosphorylation of integrin ß4 at tyrosine residue 1510, leading to activation of the integrin α6ß4-Src-AKT signaling pathway. We further reveal that the IR-induced phosphorylation of integrin ß4 is regulated by the cholesterol content and membrane fluidity. We also find that IR-induced p53-caspase signaling is independent of integrin α6ß4-Src-AKT signaling. Finally, we show that siRNA- or inhibitor-mediated blockade of integrin α6ß4-Src-AKT signaling switches the post-irradiation fate from senescence to apoptosis, under p53 activated condition, in both cancer cells and tumor tissues of xenograft mice. On the basis of our finding that, integrin α6ß4 is specifically activated and acts primarily to induce premature senescence in irradiated cancer cells, we propose that this integrin may be a valuable target and biomarker for radiotherapy.

mTOR kinase leads to PTEN-loss-induced cellular senescence by phosphorylating p53.

Loss of PTEN, the major negative regulator of the PI3K/AKT pathway induces a cellular senescence as a failsafe mechanism to defend against tumorigenesis, which is called PTEN-loss-induced cellular senescence (PICS). Although many studies have indicated that the mTOR pathway plays a critical role in cellular senescence, the exact functions of mTORC1 and mTORC2 in PICS are not well understood. In this study, we show that mTOR acts as a critical relay molecule downstream of PI3K/AKT and upstream of p53 in PICS. We found that PTEN depletion induces cellular senescence via p53-p21 signaling without triggering DNA damage response. mTOR kinase, a major component of mTORC1 and mTORC2, directly binds p53 and phosphorylates it at serine 15. mTORC1 and mTORC2 compete with MDM2 and increase the stability of p53 to induce cellular senescence via accumulation of the cell cycle inhibitor, p21. In embryonic fibroblasts of PTEN-knockout mice, PTEN deficiency also induces mTORC1 and mTORC2 to bind to p53 instead of MDM2, leading to cellular senescence. These results collectively demonstrate for the first time that mTOR plays a critical role in switching cells from proliferation signaling to senescence signaling via a direct link between the growth-promoting activity of AKT and the growth-suppressing activity of p53.

WIG1 is crucial for AGO2-mediated ACOT7 mRNA silencing via miRNA-dependent and -independent mechanisms.

RNA-binding proteins (RBPs) are involved in mRNA splicing, maturation, transport, translation, storage and turnover. Here, we identified ACOT7 mRNA as a novel target of human WIG1. ACOT7 mRNA decay was triggered by the microRNA miR-9 in a WIG1-dependent manner via classic recruitment of Argonaute 2 (AGO2). Interestingly, AGO2 was also recruited to ACOT7 mRNA in a WIG1-dependent manner in the absence of miR-9, which indicates an alternative model whereby WIG1 controls AGO2-mediated gene silencing. The WIG1-AGO2 complex attenuated translation initiation via an interaction with translation initiation factor 5B (eIF5B). These results were confirmed using a WIG1 tethering system based on the MS2 bacteriophage coat protein and a reporter construct containing an MS2-binding site, and by immunoprecipitation of WIG1 and detection of WIG1-associated proteins using liquid chromatography-tandem mass spectrometry. We also identified WIG1-binding motifs using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation analyses. Altogether, our data indicate that WIG1 governs the miRNA-dependent and the miRNA-independent recruitment of AGO2 to lower the stability of and suppress the translation of ACOT7 mRNA.

Caveolin-1 deficiency induces premature senescence with mitochondrial dysfunction.

Paradoxical observations have been made regarding the role of caveolin-1 (Cav-1) during cellular senescence. For example, caveolin-1 deficiency prevents reactive oxygen species-induced cellular senescence despite mitochondrial dysfunction, which leads to senescence. To resolve this paradox, we re-addressed the role of caveolin-1 in cellular senescence in human diploid fibroblasts, A549, HCT116, and Cav-1-/- mouse embryonic fibroblasts. Cav-1 deficiency (knockout or knockdown) induced cellular senescence via a p53-p21-dependent pathway, downregulating the expression level of the cardiolipin biosynthesis enzymes and then reducing the content of cardiolipin, a critical lipid for mitochondrial respiration. Our results showed that Cav-1 deficiency decreased mitochondrial respiration, reduced the activity of oxidative phosphorylation complex I (CI), inactivated SIRT1, and decreased the NAD+ /NADH ratio. From these results, we concluded that Cav-1 deficiency induces premature senescence via mitochondrial dysfunction and silent information regulator 2 homologue 1 (SIRT1) inactivation.

Acyl-CoA thioesterase 7 is involved in cell cycle progression via regulation of PKCζ-p53-p21 signaling pathway.

Acyl-CoA thioesterase 7 (ACOT7) is a major isoform of the ACOT family that catalyzes hydrolysis of fatty acyl-CoAs to free fatty acids and CoA-SH. However, canonical and non-canonical functions of ACOT7 remain to be discovered. In this study, for the first time, ACOT7 was shown to be responsive to genotoxic stresses such as ionizing radiation (IR) and the anti-cancer drug doxorubicin in time- and dose-dependent manners. ACOT7 knockdown induced cytostasis via activation of the p53-p21 signaling pathway without a DNA damage response. PKCζ was specifically involved in ACOT7 depletion-mediated cell cycle arrest as an upstream molecule of the p53-p21 signaling pathway in MCF7 human breast carcinoma and A549 human lung carcinoma cells. Of the other members of the ACOT family, including ACOT1, 4, 8, 9, 11, 12, and 13 that were expressed in human, ACOT4, 8, and 12 were responsive to genotoxic stresses. However, none of those had a role in cytostasis via activation of the PKCζ-p53-p21 signaling pathway. Analysis of the ACOT7 prognostic value revealed that low ACOT7 levels prolonged overall survival periods in breast and lung cancer patients. Furthermore, ACOT7 mRNA levels were higher in lung cancer patient tissues compared to normal tissues. We also observed a synergistic effect of ACOT7 depletion in combination with either IR or doxorubicin on cell proliferation in breast and lung cancer cells. Together, our data suggest that a low level of ACOT7 may be involved, at least in part, in the prevention of human breast and lung cancer development via regulation of cell cycle progression.

Radiation-inducible miR-770-5p sensitizes tumors to radiation through direct targeting of PDZ-binding kinase.

Radiotherapy represents the most effective non-surgical modality in cancer treatment. MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression, and are involved in many biological processes and diseases. To identify miRNAs that influence the radiation response, we performed miRNA array analysis using MCF7 cells at 2, 8, and 24 h post irradiation. We demonstrated that miR-770-5p is a novel radiation-inducible miRNA. When miR-770-5p was overexpressed, relative cell number was reduced due to increased apoptosis in MCF7 and A549 cells. Transcriptomic and bioinformatic analyses revealed that PDZ-binding kinase (PBK) might be a possible target of miR-770-5p for regulation of radiosensitivity. PBK regulation mediated by direct targeting of miR-770-5p was demonstrated using luciferase reporter assays along with wild-type and mutant PBK-3'untranslated region constructs. Radiation sensitivity increased and decreased in miR-770-5p- and anti-miR-770-5p-transfected cells, respectively. Consistent with this result, transfection of short interfering RNA against PBK inhibited cell proliferation, while ectopic expression of PBK restored cell survival from miR-770-5p-induced cell death. In addition, miR-770-5p suppressed tumor growth, and miR-770-5p and PBK levels were inversely correlated in xenograft model mice. Altogether, these data demonstrated that miR-770-5p might be a useful therapeutic target miRNA that sensitizes tumors to radiation via negative regulation of PBK.

ARAP, a Novel Adaptor Protein, Is Required for TCR Signaling and Integrin-Mediated Adhesion.

A novel adaptor protein was identified by analyzing phosphotyrosine proteomes from membrane rafts of activated T cells. This protein showed sequence similarity to a well-known T cell adaptor protein, adhesion and degranulation-promoting adaptor protein (ADAP); therefore, the novel protein was designated activation-dependent, raft-recruited ADAP-like phosphoprotein (ARAP). Suppression of ARAP impaired the major signaling pathways downstream of the TCR. ARAP associated with the Src homology 2 domain of Src homology 2-containing leukocyte protein of 76 kDa via the phosphorylation of two YDDV motifs in response to TCR stimulation. ARAP also mediated integrin activation but was not involved in actin polymerization. The results of this study indicate that a novel T cell adaptor protein, ARAP, plays a unique role in T cells as a part of both the proximal activation signaling and inside-out signaling pathways that result in integrin activation and T cell adhesion.

Identification of novel therapeutic targets in the secretome of ionizing radiation­induced senescent tumor cells.

Cellular senescence is a state of irreversible growth arrest that can be triggered by multiple mechanisms, including telomere shortening, the epigenetic derepression of the INK4α/ARF locus and DNA damage. Senescence has been considered a tumor­suppressing mechanism that permanently arrests cells at risk for malignant transformation. However, accumulating evidence shows that senescent cells have deleterious effects on the tissue microenvironment. Some of these effects could be attributed to the senescence­associated secretory phenotype that has the ability to promote tumor progression. However, secreted proteins from senescent tumor cells and their effects on the tumor microenvironment due to ionizing radiation (IR) exposure have not yet been fully elucidated. In the present study, we analyzed cytokines secreted from IR­induced senescent MCF7 cells by using cytokine microarrays and confirmed by western blot analysis that increased secretion of osteoprotegerin (OPG), midkine (MDK) and apolipoprotein E3 (ApoE3) occurs in these cells. Invasive, migratory and wound­healing activities were observed in MDA­MB­231 and MCF­10A cells following treatment with recombinant human OPG, MDK and ApoE3 proteins. Additionally, tube­formation activity was assessed in OPG­, MDK­ and ApoE3­treated human umbilical vein endothelial cells (HUVECs). We found that OPG, MDK and ApoE3 affected cell motility and tube­formation activity. Since OPG markedly affected cell motility, we examined the effect of senescent conditioned media containing neutralizing OPG antibodies on migration and wound­healing activity. Our results demonstrated that IR­induced senescent tumor cells influence the tumor microenvironment by increasing the production of cytokines, such as OPG, MDK and ApoE3. Furthermore, these data suggest that OPG is likely a promising target capable of reducing the deleterious effects on the tumor microenvironment during radiation therapy.

Effect of extremely low frequency magnetic fields on cell proliferation and gene expression.

Owing to concerns regarding possible effects of extremely low frequency magnetic fields (ELF-MF) on human health, many studies have been conducted to elucidate whether ELF-MF can induce modifications in biological processes. Despite this, controversies regarding effects of ELF-MF are still rife. In this study, we investigated biological effects of ELF-MF on MCF10A, MCF7, Jurkat, and NIH3T3 cell lines. ELF-MF with a magnetic flux density of 1 mT at 60 Hz was employed to stimulate cells for 4 or 16 h, after which the effects of ELF-MF on cell proliferation, cell death, cell viability, and DNA synthesis rates were assessed. Whereas Jurkat and NIH3T3 cells showed no consistent variation in cell number, cell viability, and DNA synthesis rate, MCF10A and MCF7 cells showed consistent and significant decreases in cell number, cell viability, and DNA synthesis rates. However, there was no effect of ELF-MF on cell death in any of tested cell lines. Next, to investigate the effect of ELF-MF on gene expression, we exposed MCF7 cells to 2 mT at 60 Hz for 16 h and examined transcriptional responses by using gene expression array. We found a gene, PMAIP1, that exhibited statistically significant variation using two-fold cut-off criteria and certified its expression change by using semi-quantitative and quantitative reverse transcription polymerase chain reaction. From these results, we concluded that ELF-MF could induce the delay of cell cycle progression in MCF7 and MCF10A cells in a cell context-specific manner and could up-regulate PMAIP1 in MCF7 cells.

Evaluation of premature senescence and senescence biomarkers in carcinoma cells and xenograft mice exposed to single or fractionated irradiation.

The purpose of the present study was to elucidate whether premature senescence contributes to the outcome of radiotherapy (RT) and to validate senescence biomarkers in vitro and in vivo. Cultured human cancer cell lines and xenografted mice were exposed to single (SR; 2, 6 or 12 Gy) or fractionated radiation (FR; 3 x 2 Gy or 6 x 2 Gy), and premature senescence was assessed using senescence-associated ß-galactosidase (SA-ß-Gal) activity, hypophosphorylation of pRb and p21 accumulation. A variety of senescence-associated biomarkers including cathepsin D (CD), the eukaryotic translation elongation factors eEF1A1, eEF1B2, decoy receptor 2 and Dec1 were further validated in vivo or in vitro. We demonstrated the beneficial tumor suppressive role of ionizing radiation (IR)-induced premature senescence in vitro and in vivo. FR inhibited tumor growth via induction of premature senescence as effectively as an equivalent SR dose (≥6 Gy). In addition, CD and eEF1 were valuable biomarkers of cellular senescence in either SR- or RF-exposed carcinoma cells or xenograft mice. Our results suggest that 2 Gy of a conventional RT regime could achieve a better clinical outcome if premature senescence could be increased through an improved understanding of its molecular action mechanism.

Investigation of Parental Transmission of RUNX2 Single Nucleotide Polymorphism and Its Association With Nonsyndromic Cleft Lip With or Without Palate.

OBJECTIVE: To investigate the association and parental transmission of RUNX2 single nucleotide polymorphisms (SNPs) with risk of nonsyndromic cleft lip with or without cleft palate (NS-CL±P). DESIGN: Four RUNX2 SNPs in 142 Korean NS-CL±P families (nine cleft lip, 26 cleft lip and alveolus, and 107 cleft lip and palate; 76 trios and 66 dyads) were genotyped. The minor allele frequency, heterozygosity, and chi-square test for Hardy-Weinberg equilibrium at each SNP were computed between parents. Pairwise linkage disequilibrium was computed as D' and r(2) for all SNPs. Both allelic and genotypic transmission disequilibrium tests (TDTs) were performed for individual SNPs using a family-based association test program. Sliding windows of haplotypes consisting of two to four SNPs were tested using a haplotype-based association test program. Genotypic odds ratios (GORs) were calculated from conditional logistic regression models. Parent-of-origin effects were assessed using transmission asymmetry test and parent-of-origin likelihood ratio test. RESULTS: The family-based TDT showed significant evidence of linkage and association at rs1934328 (P = .001). In the haplotype analysis, two, three, and four haplotypes containing rs1934328 revealed significant associations (P = .0017, P = .0022, and P = .0020, respectively). The genotypes A/T and T/T at rs1934328 were significantly associated with NS-CL±P compared with the genotype A/A (GOR = 2.75, 95% confidence interval [CI] = 1.39-5.45, P =0.0019 in the dominant model; GOR = 5.38, 95% CI = 1.34-21.68, P = .0046 in the additive model). However, no parent-of origin effect was observed. CONCLUSION: These findings suggest possible involvement of RUNX2-rs194328 in the etiology of NS-CL±P in Korean cleft-parent trios without excess parental transmission.

Low stability and a conserved N-glycosylation site are associated with regulation of the discoidin domain receptor family by glucose via post-translational N-glycosylation.

Cell-surface expression of the discoidin domain receptor (DDR) tyrosine kinase family in high molecular mass form was controlled sensitively by the glucose concentration through a post-translational N-glycosylation process. Cycloheximide time-course experiments revealed that the high-molecular-mass forms of DDR1 and DDR2 were significantly less stable than control receptor tyrosine kinases. Site-directed mutational analysis of the consensus N-glycosylation sites of the DDRs revealed that mutations of asparagine 213 of DDR2 and asparagine 211 of DDR1, a conserved N-glycosylation site among vertebrate DDRs, inhibited the generation of the high-molecular-mass isoform. Taken together, these results suggest a mechanism to control the activity of the DDR family by regulating its cell-surface expression. Due to low stability, the steady-state population of functional DDR proteins in the cell surface depends sensitively on its maturation process via post-translational N-glycosylation, which is controlled by the glucose supply and the presence of a conserved N-glycosylation site.