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The development of next-generation sequencing (NGS) has dramatically increased the speed and volume of genetic analysis. Furthermore, the range of applications of NGS is rapidly expanding to include genome, epigenome (such as DNA methylation), metagenome, and transcriptome analyses (such as RNA sequencing and single-cell RNA sequencing). NGS enables genetic research by offering various sequencing methods as well as combinations of methods. Bone tissue is the most important unit supporting the body and is a reservoir of calcium and phosphate ions, which are important for physical activity. Many genetic diseases affect bone tissues, possibly because metabolic mechanisms in bone tissue are complex. For instance, the presence of specialized immune cells called osteoclasts in the bone tissue, which absorb bone tissue and interact with osteoblasts in complex ways to support normal vital functions. Moreover, the many cell types in bones exhibit cell-specific proteins for their respective activities. Mutations in the genes encoding these proteins cause a variety of genetic disorders. The relationship between age-related bone tissue fragility (also called frailty) and genetic factors has recently attracted attention. Herein, we discuss the use of genomic, epigenomic, transcriptomic, and metagenomic analyses in bone genetic disorders. [ABSTRACT FROM AUTHOR]

The hedgehog (Hh) family consists of numerous signaling mediators that play important roles at various stages of development. Thus, the Hh pathway is essential for bone tissue development and tumorigenesis. Gorlin syndrome is a skeletal and tumorigenic disorder caused by gain-of-function mutations in Hh signaling. In this review, we first present the phenotype of Gorlin syndrome and the relationship between genotype and phenotype in bone and craniofacial tissues, including the causative gene as well as other Hh-related genes. Next, the importance of new diagnostic methods using next-generation sequencing and multiple gene panels will be discussed. We summarize Hh-related genetic disorders, including cilia disease, and the genetics of Hh-related bone diseases. [ABSTRACT FROM AUTHOR]

Hajdu-Cheney syndrome (HCS) is a rare autosomal dominant manifestation of a congenital genetic disorder caused by a mutation in the NOTCH2 gene. NOTCH signaling has variations from NOTCH 1 to 4 and maintains homeostasis by determining and regulating the proliferation and differentiation of various cells. In HCS, the over-accumulated NOTCH2 causes abnormal bone resorption due to its continuous excessive signaling. HCS is characterized by progressive bone destruction, has complex wide-range clinical manifestations, and significantly impacts the patient's quality of life. However, no effective treatment has been established for HCS to date. There are genetic variants of NOTCH2 that have been reported in the ClinVar database of the U.S. National Institutes of Health. In total, 26 mutant variants were detected based on the American College of Medical Genetics and Genomics (ACMC). To date, there has been no comprehensive compilation of HCS mutations. In this review, we provide the most comprehensive list possible of HCS variants, nucleotide changes, amino acid definitions, and molecular consequences reported to date, following the ACMC guidelines. [ABSTRACT FROM AUTHOR]

Leptin deficiency enhances sensitivity of rats to alcoholic steatohepatitis through suppression of metallothionein. Am J Physiol Gastrointest Liver Physiol 287: G1078-G1085, 2004; doi:10.1152/ ajpgi.00107.2004.--Oxidative stress is stated to be a central mechanism of hepatocellular injury in alcohol-induced liver injury. Recent reports have shown that Kupffer cell dysfunction in the leptin-deficient state contributes partly to the increased sensitivity to endotoxin liver injury. Here we report that leptin also plays a key role in the development of alcoholic liver injury and that leptin signaling in hepatocytes is involved in cellular mechanisms that mediate ethanol-induced oxidative stress. We found that chronic ethanol feeding in leptin receptor-deficient Zucker (fa/fa) rats for 6 wk resulted in a much more severe liver injury and augmented accumulation of hepatic lipid peroxidation compared with control littermates. The hepatic induction of stress-response and antioxidant proteins, such as metallothionein (MT)-1 and -2, was significantly suppressed in fa/fa rats after chronic ethanol feeding. Zinc concentration in liver was also decreased in fa/fa rats, compared with control littermates. In primary cultured hepatocytes from fa/fa rats, incubation with ethanol significantly suppressed MT-1 and -2 expressions. Addition of leptin to leptin-deficient ob/ob mouse primary hepatocytes led to an increase in MT-1 and -2 mRNA levels and a decrease in oxidative stress after incubation with ethanol. In conclusion, leptin deficiency enhances sensitivity of rats to alcohol-induced steatohepatitis through hepatocyte-specific interaction of MT-1 and -2 and resultant exaggeration of oxidative stress in hepatocytes. These findings suggest that leptin resistance in hepatocytes is an important mechanism of alcohol-induced liver injury. oxidative stress; zinc; tumor necrosis factor-[alpha];

Acute ethanol exposure induces oxidative stress and apoptosis in primary rat hepatocytes. Previous data indicate that the mitochondrial permeability transition (MPT) is essential for ethanol-induced apoptosis. However, the mechanism by which ethanol induces the MPT remains unclear. In this study, we investigated the role of Bax, a proapoptotic Bcl-2 family protein, in acute ethanol-induced hepatocyte apoptosis. We found that Bax translocates from the cytosol to mitochondria before mitochondrial cytochrome c release. Bax translocation was oxidative stress dependent. Mitochondrial Bax formed a protein complex with the mitochondrial voltage-dependent anion channel (VDAC). Prevention of Bax-VDAC interactions by a microinjection of anti-VDAC antibody effectively prevented hepatocyte apoptosis by ethanol. In conclusion, these data suggest that Bax translocation from the cytosol to mitochondria leads to the subsequent formation of a Bax-VDAC complex that plays a crucial role in acute ethanol-induced hepatocyte apoptosis. alcoholic liver disease; mitochondria; oxidative stress; cytochrome c

The gelsolin, an 82 kDa actin-binding protein, regulates the motility of cells which help in hemostasis, inflammation, and wound healing. The gelsolin is not present in embryonic cells and the process of actin filament dynamics is not required for motility in the early embryogenesis. The transgenic gelsolinnull (Gsn-) mice were produced for the vivo experiments on the functioning of gelsolin. The Gsn-mice show less changed platelet shape and this led to more continuous bleeding.

Odontoblasts play critical roles in dentin formation and sensory transduction following stimuli on the dentin surface. Exogenous stimuli to the dentin surface elicit dentinal sensitivity through the movement of fluids in dentinal tubules, resulting in cellular deformation. Recently, Piezo1 channels have been implicated in mechanosensitive processes, as well as Ca2+ signals in odontoblasts. However, in human odontoblasts, the cellular responses induced by mechanical stimulation, Piezo1 channel expression, and its pharmacological properties remain unclear. In the present study, we examined functional expression of the Piezo1 channel by recording direct mechanical stimulation-induced Ca2+ signaling in dentin matrix protein 1 (DMP-1)-, nestin-, and dentin sialophosphoprotein (DSPP)-immunopositive human odontoblasts. Mechanical stimulation of human odontoblasts transiently increased intracellular free calcium concentration ([Ca2+]i). Application of repeated mechanical stimulation to human odontoblasts resulted in repeated transient [Ca2+]i increases, but did not show any desensitizing effects on [Ca2+]i increases. We also observed a transient [Ca2+]i increase in the neighboring odontoblasts to the stimulated cells during mechanical stimulation, showing a decrease in [Ca2+]i with an increasing distance from the mechanically stimulated cells. Application of Yoda1 transiently increased [Ca2+]i. This increase was inhibited by application of Gd3+ and Dooku1, respectively. Mechanical stimulation-induced [Ca2+]i increase was also inhibited by application of Gd3+ or Dooku1. When Piezo1 channels in human odontoblasts were knocked down by gene silencing with short hairpin RNA (shRNA), mechanical stimulation-induced [Ca2+]i responses were almost completely abolished. Piezo1 channel knockdown attenuated the number of Piezo1-immunopositive cells in the immunofluorescence analysis, while no effects were observed in Piezo2-immunopositive cells. Alizarin red staining distinctly showed that pharmacological activation of Piezo1 channels by Yoda1 significantly suppressed mineralization, and shRNA-mediated knockdown of Piezo1 also significantly enhanced mineralization. These results suggest that mechanical stimulation predominantly activates intracellular Ca2+ signaling via Piezo1 channel opening, rather than Piezo2 channels, and the Ca2+ signal establishes intercellular odontoblast-odontoblast communication. In addition, Piezo1 channel activation participates in the reduction of dentinogenesis. Thus, the intracellular Ca2+ signaling pathway mediated by Piezo1 channels could contribute to cellular function in human odontoblasts in two ways: (1) generating dentinal sensitivity and (2) suppressing physiological/reactional dentinogenesis, following cellular deformation induced by hydrodynamic forces inside dentinal tubules. [ABSTRACT FROM AUTHOR]

Background/Objectives: PTH plays an important role in bone remodeling, and different actions have been reported depending on its administration method. iPSCs are promising as a cell source for regeneration of periodontal tissue due to their ability of proliferation and pluripotency. However, the effects of PTH on iPSCs remain mostly unknown. The purpose of this study was to investigate in vitro effects of parathyroid hormone (PTH) on osteoblastic differentiation of induced pluripotent stem cells (iPSCs) in a 3D culture model. Materials and Methods: Following embryoid body (EB) induction from mouse iPSCs (miPSCs), dissociated cells (miPS‐EB‐derived cells) were seeded onto atelocollagen sponge (ACS) in osteoblast differentiation medium (OBM). Cell‐ACS constructs were divided into three groups: continuous treatment with human recombinant PTH (1‐34) (PTH‐C), intermittent PTH treatment (PTH‐I) or OBM control. To confirm the expression of PTH receptor‐1(PTH1R), the expression of Pth1r and cAMP production over time were assessed. Real‐time PCR was used to assess the expression of genes encoding osterix (Sp7), runt‐related transcription factor 2 (Runx2), collagen type 1 (Col1a1), and osteocalcin (Bglap) at different time points. Mineralization was assessed by von Kossa staining. Histochemical staining was used to analyze alkaline phosphatase (ALP) activity, and immunolocalization of SP7 and BGLAP was analyzed by confocal laser scanning microscopy (CLSM). Results: On days 7 and 14, expression of the Pth1r in miPS‐EB‐derived cells was increased in all groups. Production of cAMP, the second messenger of the PTH1R, tended to increase in the PTH‐I group compared with PTH‐C group on day 14. Expression of Col1a1 in the PTH‐I group on day 14 was significantly higher than other groups. There was a time‐dependent increase in the expression of Sp7 in all groups. On day 14, the expression level of Sp7 in the PTH‐I group was significantly higher than other groups. In von Kossa staining, the PTH‐I group showed higher level of staining compared with other groups on day 14, whereas the level was slightly attenuated in the PTH‐C group. In histochemical staining, ALP‐positive cells were significantly increased in the PTH‐I group compared with other groups on day 14. In CLSM analysis, the numbers of SP7‐ and BGLAP‐positive cells showed a gradual increase over time, and on day 14, a significantly greater SP7 expression was observed in the PTH‐I group than other groups. Conclusion: These results suggested that the intermittent PTH treatment promotes osteoblastic differentiation and mineralization of miPSCs in the ACS scaffold. [ABSTRACT FROM AUTHOR]

A missense mutation of the guanine nucleotide binding protein alpha stimulating activity polypeptide 1 (GNAS) gene, typically Arg201Cys or Arg201His (R201H/R201C), leads to constitutive activation of the Gsα-cyclic AMP (cAMP) signaling pathway that causes several diseases. However, no germline mutations of GNAS have been identified to date, likely due to their lethality, and no robust human cell models have been generated. Therefore, the aim of this study was to generate GNAS -mutated disease-specific induced pluripotent stem cells as a model for these diseases. We then analyzed the functionality of this induced pluripotent stem cell model and differentiated epithelial cells. We generated disease-specific induced pluripotent stem cells by introducing a mutation in GNAS with the clustered regularly interspaced short palindromic repeats (CRISPR) nickase method, which has lower off-target effects than the conventional CRISPR/Cas9 method. We designed the target vector to contain the R201H mutation in GNAS, which was transfected into human control induced pluripotent stem cells (Nips-B2) by electroporation. We confirmed the establishment of GNAS R201H - mutated (GNAS R201H/+) induced pluripotent stem cells that exhibited a pluripotent stem cell phenotype. We analyzed the effect of the mutation on cAMP production, and further generated teratomas for immunohistochemical analysis of the luminal epithelial structure. GNAS -mutated induced pluripotent stem cells showed significantly higher levels of intracellular cAMP, which remained elevated state for a long time upon hormonal stimulation with parathyroid hormone or adrenocorticotropic hormone. Immunohistochemical analysis revealed that several mucins, including MUC1, 2, and MUC5AC, are expressed in cytokeratin 18 (CK18)-positive epithelial cells. However, we found few CK18-positive cells in mutated induced pluripotent stem cell–derived teratoma tissues, and reduced MUCINs expression in mutated epithelial cells. There was no difference in CDX2 expression; however, mutated epithelial cells were positive for CEA and CA19-9 expression. GNAS R201H-mutated induced pluripotent stem cells and GNAS R201H-mutated epithelial cells have distinct phenotypic and differentiation characteristics. We successfully established GNAS R201H-mutated human induced pluripotent stem cells with increased cAMP production. Considering the differentiation potential of induced pluripotent stem cells, these cells will be useful as a model for elucidating the pathological mechanisms of GNAS -mutated diseases. [ABSTRACT FROM AUTHOR]

Background: Runt-related transcription factor 2 (RUNX2) haploinsufficiency causes cleidocranial dysplasia (CCD) which is characterized by supernumerary teeth, short stature, clavicular dysplasia, and osteoporosis. At present, as a therapeutic strategy for osteoporosis, mesenchymal stem cell (MSC) transplantation therapy is performed in addition to drug therapy. However, MSC-based therapy for osteoporosis in CCD patients is difficult due to a reduction in the ability of MSCs to differentiate into osteoblasts resulting from impaired RUNX2 function. Here, we investigated whether induced pluripotent stem cells (iPSCs) properly differentiate into osteoblasts after repairing the RUNX2 mutation in iPSCs derived from CCD patients to establish normal iPSCs, and whether engraftment of osteoblasts derived from properly reverted iPSCs results in better regeneration in immunodeficient rat calvarial bone defect models. Methods: Two cases of CCD patient-derived induced pluripotent stem cells (CCD-iPSCs) were generated using retroviral vectors (OCT3/4, SOX2, KLF4, and c-MYC) or a Sendai virus SeVdp vector (KOSM302L). Reverted iPSCs were established using programmable nucleases, clustered regularly interspaced short palindromic repeats (CRISPR)/Casderived RNA-guided endonucleases, to correct mutations in CCD-iPSCs. The mRNA expressions of osteoblast-specific markers were analyzed using quantitative reverse-transcriptase polymerase chain reaction. iPSCs-derived osteoblasts were transplanted into rat calvarial bone defects, and bone regeneration was evaluated using microcomputed tomography analysis and histological analysis. Results: Mutation analysis showed that both contained nonsense mutations: one at the very beginning of exon 1 and the other at the initial position of the nuclear matrix-targeting signal. The osteoblasts derived from CCD-iPSCs (CCD-OBs) expressed low levels of several osteoblast differentiation markers, and transplantation of these osteoblasts into calvarial bone defects created in rats with severe combined immunodeficiency showed poor regeneration. However, reverted iPSCs improved the abnormal osteoblast differentiation which resulted in much better engraftment into the rat calvarial bone defect. Conclusions: Taken together, these results demonstrate that patient-specific iPSC technology can not only provide a useful disease model to elucidate the role of RUNX2 in osteoblastic differentiation but also raises the tantalizing prospect that reverted iPSCs might provide a practical medical treatment for CCD. [ABSTRACT FROM AUTHOR]

Gorlin syndrome is an autosomal dominant inherited syndrome that predisposes a patient to the formation of basal cell carcinomas, odontogenic keratocysts, and skeletal anomalies. Causative mutations in several genes associated with the sonic hedgehog (SHH) signaling pathway, including PTCH1, have been identified in Gorlin syndrome patients. However, no definitive genotype—phenotype correlations are evident in these patients, and their clinical presentation varies greatly, often leading to delayed diagnosis and treatment. We generated iPSCs from four unrelated Gorlin syndrome patients with loss-of-function mutations in PTCH1 using the Sendai virus vector (SeVdp(KOSM)302). The patient-derived iPSCs exhibited basic iPSC features, including stem cell marker expression, totipotency, and the ability to form teratomas. GLI1 expression levels were greater in fibroblasts and patient-derived iPSCs than in the corresponding control cells. Patient-derived iPSCs expressed lower basal levels than control iPSCs of the genes encoding the Hh ligands Indian Hedgehog (IHH) and SHH, the Hh acetyltransferase HHAT, Wnt proteins, BMP4, and BMP6. Most of these genes were upregulated in patient-derived iPSCs grown in osteoblast differentiation medium (OBM) and downregulated in control iPSCs cultured in OBM. The expression of GLI1 and GLI2 substantially decreased in both control and patient-derived iPSCs cultured in OBM, whereas GLI3, SHH, and IHH were upregulated in patient-derived iPSCs and downregulated in control iPSCs grown in OBM. Activation of Smoothened by SAG in cells grown in OBM significantly enhanced alkaline phosphatase activity in patient-derived iPSCs compared with control iPSC lines. In summary, patient-derived iPSCs expressed lower basal levels than the control iPSCs of the genes encoding Hh, Wnt, and bone morphogenetic proteins, but their expression of these genes strongly increased under osteogenic conditions. These findings indicate that patient-derived iPSCs are hypersensitive to osteogenic induction. We propose that Hh signaling is constituently active in iPSCs from Gorlin syndrome patients, enhancing their response to osteogenic induction and contributing to disease-associated abnormalities. [ABSTRACT FROM AUTHOR]

Gorlin syndrome is a genetic disorder of autosomal dominant inheritance that predisposes the affected individual to a variety of disorders that are attributed largely to heterozygous germline patched1 (PTCH1) mutations. PTCH1 is a hedgehog (Hh) receptor as well as a repressor, mutation of which leads to constitutive activation of Hh pathway. Hh pathway encompasses a wide variety of cellular signaling cascades, which involve several molecules; however, no associated genotype–phenotype correlations have been reported. Recently, mutations in Suppressor of fused homolog (SUFU) or PTCH2 were reported in patients with Gorlin syndrome. These facts suggest that multi-layered mutations in Hh pathway may contribute to the development of Gorlin syndrome. We demonstrated multiple mutations of Hh-related genes in addition to PTCH1, which possibly act in an additive or multiplicative manner and lead to Gorlin syndrome. High-throughput sequencing was performed to analyze exome sequences in four unrelated Gorlin syndrome patient genomes. Mutations in PTCH1 gene were detected in all four patients. Specific nucleotide variations or frameshift variations of PTCH1 were identified along with the inferred amino acid changes in all patients. We further filtered 84 different genes which are closely related to Hh signaling. Fifty three of these had enough coverage of over ×30. The sequencing results were filtered and compared to reduce the number of sequence variants identified in each of the affected individuals. We discovered three genes, PTCH2, BOC, and WNT9b, with mutations with a predicted functional impact assessed by MutationTaster2 or PolyPhen-2 (Polymorphism Phenotyping v2) analysis. It is noticeable that PTCH2 and BOC are Hh receptor molecules. No significant mutations were observed in SUFU. Multi-layered mutations in Hh pathway may change the activation level of the Hh signals, which may explain the wide phenotypic variability of Gorlin syndrome. [ABSTRACT FROM AUTHOR]

Background: We have previously reported that repeated treatment of human periodontal ligament cells and murine pre-osteoblast MC3T3-E1 cells with transforming growth factor-beta 1 (TGF-β1) inhibited their osteoblastic differentiation because of decreased insulin-like growth factor-1 (IGF-1) secretion. We also found that IGF-1/PI3K signaling plays an important role in osteoblast differentiation induced by TGF-β1 treatment; however, the downstream signaling controlling this remains unknown. The aim of this current study is to investigate whether Akt activation is required for osteoblast differentiation. Methodology/Principal Findings: MC3T3-E1 cells were cultured in osteoblast differentiation medium (OBM) with or without 0.1 ng/mL TGF-β1. OBM containing TGF-β1 was changed every 12 h to provide repeated TGF-β1 administration. MC3T3-E1 cells were infected with retroviral vectors expressing constitutively active (CA) or dominant-negative (DN)-Akt. Alkaline phosphatase (ALP) activity and osteoblastic marker mRNA levels were substantially decreased by repeated TGF-β1 treatment compared with a single TGF-β1 treatment. However, expression of CA-Akt restored ALP activity following TGF-β1 treatment. Surprisingly, ALP activity increased following multiple TGF-β1 treatments as the number of administrations of TGF-β1 increased. Activation of Akt significantly enhanced expression of osteocalcin, but TGF-β1 treatment inhibited this. Mineralization of MC3T3-E1 cells was markedly enhanced by CA-Akt expression under all medium conditions. Exogenous IGF-1 restored the down-regulation of osteoblast-related gene expression by repeated TGF-β1 administration. However, in cells expressing DN-Akt, these levels remained inhibited regardless of IGF-1 treatment. These findings indicate that Akt activation is required for the early phase of osteoblast differentiation of MC3T3-E1 cells induced by TGF-β1. However, Akt activation is insufficient to reverse the inhibitory effects of TGF-β1 in the late stages of osteoblast differentiation. Conclusions: TGF-β1 could be an inducer or an inhibitor of osteoblastic differentiation of MC3T3-E1 cells depending on the state of Akt phosphorylation. Our results indicate that Akt is the molecular switch for TGF-β1-induced osteoblastic differentiation of MC3T3-E1 cells. [ABSTRACT FROM AUTHOR]

In this study, we developed a new method to stimulate osteogenic differentiation in tissue-nonspecific alkaline phosphatase (TNAP)-positive cells liberated from human induced pluripotent stem cells (hiPSCs)-derived embryoid bodies (EBs) with 14 days long TGF-β/IGF-1/FGF-2 treatment. TNAP is a marker protein of osteolineage cells. We analyzed and isolated TNAP-positive and E-cadherin-negative nonepithelial cells by fluorescence-activated cell sorting. Treating the cells with a combination of transforming growth factor (TGF)-β, insulin-like growth factor (IGF)-1, and fibroblast growth factor (FGF)-2 for 14 days greatly enhanced TNAP expression and maximized expression frequency up to 77.3%. The isolated cells expressed high levels of osterix, which is an exclusive osteogenic marker. Culturing these TNAP-positive cells in osteoblast differentiation medium (OBM) led to the expression of runt-related transcription factor 2, type I collagen, bone sialoprotein, and osteocalcin (OCN). These cells responded to treatment with activated vitamin D3 by upregulating OCN. Furthermore, in OBM they were capable of generating many mineralized nodules with strong expression of receptor activator of NF-kappaB ligand and sclerostin (SOST). Real-time RT-PCR showed a significant increase in the expression of osteocyte marker genes, including SOST, neuropeptide Y, and reelin. Scanning electron microscopy showed dendritic morphology. Examination of semi-thin toluidine blue-stained sections showed many interconnected dendrites. Thus, TNAP-positive cells cultured in OBM may eventually become terminally differentiated osteocyte-like cells. In conclusion, treating hiPSCs-derived cells with a combination of TGF-β, IGF-1, and FGF-2 generated TNAP-positive cells at high frequency. These TNAP-positive cells had a high osteogenic potential and could terminally differentiate into osteocyte-like cells. The method described here may reveal new pathways of osteogenesis and provide a novel tool for regenerative medicine and drug development. [ABSTRACT FROM AUTHOR]

Cancer and stem cells share the ability to silence tumor suppressors. We focused on Lefly, which encodes one of the most abundant tumor suppressors in embryonic stem (ES) cells and is not expressed in somatic cancer cells. We found that transforming growth factor β (TGF-β) induced demethylation of the Lefly B cytosine-phosphate-guanine (CpG) island and increased Lefly expression (10-200 times) in human pancreatic cancer cells and human liver cancer cells (PLC/PRF/5 and HLF). Expression of Cripto, another important factor in Nodal-Lefty signaling, was not increased after adding TGF-β. We generated reprogrammed cancer cells that revealed high expression of immature marker proteins, high proliferation, and the potential to express morphological patterns of ectoderm, mesoderm, and endoderm, suggesting that these cells may have cancer stem cell-like phenotypes. We investigated Lefly and found that reprogrammed human liver cancer cells (induced pluripotent cancer cells) displayed a much lower ability to express Lefly, although less Lefly B CpG methylation was also observed. We also found that a MEK inhibitor dramatically enhanced Lefly expression in human pancreatic cancers with mutated ras, whereas Lefly B CpG methylation was not decreased. These observations indicate that despite the demethylation of DNA strands in promoter regions of pluripotency-associated genes, including Lefly gene, Lefty expression was not induced well in reprogrammed cells. Of note was the fact that Lefty is abundantly expressed in human ES cells but not in induced pluripotent stem (iPS) cells. We thus think that reprogrammed cancer cells share the mechanism for expression of Lefty with iPS cells. This shared mechanism may contribute to the cancerous transformation of iPS cells. [ABSTRACT FROM AUTHOR]

Background: The malQ and glgP genes, respectively, annotated as putative 4-α-glucanotransferase and putative glycogen phosphorylase are located with a 29 nucleotide overlap on the Streptococcus mutans genome. We found that the glgP gene of this organism was induced with maltose, and the gene likely constituted an operon with the upstream gene malQ. This putative operon was negatively regulated with the malR gene located upstream from the malQ gene and a MalR-binding consensus sequence was found upstream of the malQ gene. S. mutans is not able to catabolize starch. However, this organism utilizes maltose degraded from starch in the presence of saliva amylase. Therefore, we hypothesized that the MalQ/GlgP system may participate in the metabolism of starch-degradation products. Methods: A DNA fragment amplified from the malQ or glgP gene overexpressed His-tagged proteins with the plasmid pBAD/HisA. S. mutans malQ and/or glgP mutants were also constructed. Purified proteins were assayed for glucose-releasing and phosphorylase activities with appropriate buffers containing maltose, maltotriose, maltodextrin, or amylodextrin as a substrate, and were photometrically assayed with a glucose-6- phosphate dehydrogenase-NADP system. Results: Purified MalQ protein released glucose from maltose and maltotriose but did not from either maltodextrin or amylodextrin. The purified GlgP protein did not exhibit a phosphorylase reaction with maltose or maltotriose but generated glucose-1-phosphate from maltodextrin and amylodextrin. However, the GlgP protein released glucose-1-phosphate from maltose and maltotriose in the presence of the MalQ protein. In addition, the MalQ enzyme activity with maltose released not only glucose but also produced maltooligosaccharides as substrates for the GlgP protein. Conclusion: These results suggest that the malQ gene encodes 4-α-glucanotransferase but not α-1,4- glucosidase activity. The malQ mutant could not grow in the presence of maltose as a carbon source, which suggests that the malQ gene is essential for the utilization of starch-degradation products. [ABSTRACT FROM AUTHOR]

Background: Streptococcus mutans surface-protein antigen (SpaP, PAc, or antigen I/II) has been well known to play an important role in initial attachment to tooth surfaces. However, strains with weak SpaP-expression were recently reported to be found in natural populations of S. mutans. The S. mutans gbpC-negative strain Z1, which we previously isolated from saliva and plaque samples, apparently expresses relatively low levels of SpaP protein compared to S. mutans strains MT8148 or UA159. Objective: To elucidate the mechanism for weak SpaP-expression in this strain, the spaP gene region in strain Z1 was amplified by polymerase chain reaction (PCR) and analyzed. Methods: Allelic exchange mutants between strains Z1 and UA159 involving the spaP gene region were constructed. The SpaP protein expressed in the mutants was detected with Coomasie Brilliant Blue (CBB)- staining and Western blot analysis following SDS-PAGE. Results: The 4689 bp spaP gene coding sequence for Z1 appeared to be intact. In contrast, a 20 bp nucleotide sequence appeared to be deleted from the region immediately upstream from the Z1 spaP gene when compared to the same region in UA159. The 216 bp and 237 bp intergenic fragments upstream from the spaP gene, respectively, from Z1 and UA159 were isolated, modified, and transformed into the other strain by allelic replacement. The resultant UA159-promoter region-mutant exhibited extremely weak SpaP-expression similar to that of strain Z1 and the Z1 complemented mutant expressed Spa protein levels like that of strain UA159. Conclusion: These results suggest that weak SpaP-expression in strain Z1 resulted from a 20 bp-deletion in the spaP gene promoter region. [ABSTRACT FROM AUTHOR]

ABSTRACT Background Methods Results Conclusions Odontogenic keratocyst (OKC) is a partial manifestation of Gorlin syndrome (GS), resulting from the abnormal activation of the hedgehog signaling pathway. OKC predominantly occurs in young adults and is mostly asymptomatic at the time of initial diagnosis. As OKC is asymptomatic, GS can be challenging to diagnose in certain instances. In this study, we attempted to identify asymptomatic GS from sporadic OKC cases using a previously developed gene panel for GS.Genomic DNA was extracted from patient samples. These DNA samples were analyzed using the AmpliSeq Custom DNA Panel (Illumina), which was specifically designed to target four previously established genes (PTCH1, PTCH2, SMO, and SUFU). Mutations from patients were predicted using tools, such as MutationTaster, CADD, and Polyphen‐2.Thirty‐one patients with OKC were included: 22 sporadic, 9 syndromic, 14 cases with dentigerous cysts, and 3 patients with orthokeratinized odontogenic cysts. One patient with sporadic OKC carried 50% genetic mutation in the cyst and blood, indicative of GS. PTCH1 mutations were found in one of the 14 patients with dentigerous cysts, 3 of the 17 first‐time sporadic cases, and all four recurrent cases. Resected OKC tissues revealed a PTCH1 mutation.We found one patient with GS from those diagnosed with sporadic OKC. Our findings suggest that PTCH1 mutations are associated with postoperative recurrence of OKC, implying that hedgehog‐related gene variations may contribute to jaw cyst development and improve the prognosis of OKC. [ABSTRACT FROM AUTHOR]

Gelsolin is a calcium-activated actin-binding protein with six subdomains. The N-terminal (G1) domain is essential for actin-filament-severing activity while other domains within G2-3 position the protein on the filament side allowing G1 to sever. In order to generate reagents capable of competitively inhibiting endogenous gelsolin and, potentially, other actin filament regulatory protein, we expressed several truncates of gelsolin in Escherichia coli, and analyzed how they affected the in vitro activity of two different actin-binding proteins, gelsolin and cofilin. A ca2+-sensitive truncate containing G2--6 inhibited the F-actin-depolymerizing activities of both gelsolin and cofilin, while a G2--3 truncate was less effective. Using two independent assays, our results support the idea that gelsolin truncates inhibit actin filament severing and do not markedly affect actin subunit dissociation kinetics. Cosedimentation assays in the presence of calcium demonstrate that the G2-6 truncate binds to F-action more strongly than the G2-3 truncate consistent with a protection mechanism by conformational change of F-actin and/or competitive binding to actin filaments which depends upon the presence of action binding domains. [ABSTRACT FROM AUTHOR]

Gorlin syndrome is a skeletal disorder caused by a gain of function mutation in Hedgehog (Hh) signaling. The Hh family comprises of many signaling mediators, which, through complex mechanisms, play several important roles in various stages of development. The Hh information pathway is essential for bone tissue development. It is also the major driver gene in the development of basal cell carcinoma and medulloblastoma. In this review, we first present the recent advances in Gorlin syndrome research, in particular, the signaling mediators of the Hh pathway and their functions at the genetic level. Then, we discuss the phenotypes of mutant mice and Hh signaling-related molecules in humans revealed by studies using induced pluripotent stem cells. [ABSTRACT FROM AUTHOR]