Inhibitory Effects of Loganin on Adipogenesis In Vitro and In Vivo.

Obesity is characterized by the excessive accumulation of mature adipocytes that store surplus energy in the form of lipids. In this study, we investigated the inhibitory effects of loganin on adipogenesis in mouse preadipocyte 3T3-L1 cells and primary cultured adipose-derived stem cells (ADSCs) in vitro and in mice with ovariectomy (OVX)- and high-fat diet (HFD)-induced obesity in vivo. For an in vitro study, loganin was co-incubated during adipogenesis in both 3T3-L1 cells and ADSCs, lipid droplets were evaluated by oil red O staining, and adipogenesis-related factors were assessed by qRT-PCR. For in vivo studies, mouse models of OVX- and HFD-induced obesity were orally administered with loganin, body weight was measured, and hepatic steatosis and development of excessive fat were evaluated by histological analysis. Loganin treatment reduced adipocyte differentiation by accumulating lipid droplets through the downregulation of adipogenesis-related factors, including peroxisome proliferator-activated receptor γ (Pparg), CCAAT/enhancer-binding protein α (Cebpa), perilipin 2 (Plin2), fatty acid synthase (Fasn), and sterol regulatory element binding transcription protein 1 (Srebp1). Loganin administration prevented weight gain in mouse models of obesity induced by OVX and HFD. Further, loganin inhibited metabolic abnormalities, such as hepatic steatosis and adipocyte enlargement, and increased the serum levels of leptin and insulin in both OVX- and HFD-induced obesity models. These results suggest that loganin is a potential candidate for preventing and treating obesity.

Effects of Loganin on Bone Formation and Resorption In Vitro and In Vivo.

Osteoporosis is a disease caused by impaired bone remodeling that is especially prevalent in elderly and postmenopausal women. Although numerous chemical agents have been developed to prevent osteoporosis, arguments remain regarding their side effects. Here, we demonstrated the effects of loganin, a single bioactive compound isolated from Cornus officinalis, on osteoblast and osteoclast differentiation in vitro and on ovariectomy (OVX)-induced osteoporosis in mice in vivo. Loganin treatment increased the differentiation of mouse preosteoblast cells into osteoblasts and suppressed osteoclast differentiation in primary monocytes by regulating the mRNA expression levels of differentiation markers. Similar results were obtained in an osteoblast-osteoclast co-culture system, which showed that loganin enhanced alkaline phosphatase (ALP) activity and reduced TRAP activity. In in vivo experiments, the oral administration of loganin prevented the OVX-induced loss of bone mineral density (BMD) and microstructure in mice and improved bone parameters. In addition, loganin significantly increased the serum OPG/RANKL ratio and promoted osteogenic activity during bone remodeling. Our findings suggest that loganin could be used as an alternative treatment to protect against osteoporosis.

Inhibitory Effect of Ulmus davidiana and Cornus officinalis Extracts on Osteoporotic Bone Loss In Vitro and In Vivo.

Background and Objectives: Traditional herbal medicines are becoming more popular as a complementary medication as they have the advantages of being mostly harmless and safe, causing fewer side-effects than conventional medications. Here, we demonstrate the inhibitory effects of the combination of Ulmus davidiana (UD) and Cornus officinalis (CO) extracts on osteoporotic bone loss. Materials and Methods: This study presented osteogenic effects in primary cultured osteoblasts, pre-osteoblastic MC3T3-E1 cell lines, and osteoclastogenic effects in osteoclasts derived from bone marrow monocytes, and finally, protective effects on bone loss in an ovariectomy (OVX)-induced osteoporotic animal model. Results: A significant increase in alkaline phosphatase (ALP) activity was observed following treatment with UD and CO mixtures (8:2, 7:3, and 5:5 ratios) and individual UD and CO extracts, with the highest ALP activity being detected for the treatment with UD and CO extracts at a 5:5 ratio. An optimal ratio of UD and CO (UC) extract promoted osteoblast differentiation in both pre-osteoblastic cells and primary osteoblasts by increasing osteoblastic markers such as Alpl, Runx2, and Bglap. However, treatment with the UC extract inhibited osteoclast differentiation with a decreased expression of osteoclastogenesis-related genes, including Ctsk, Acp5, Mmp9, and Nfatc1. In addition, UC treatment prevented osteoporotic bone loss in OVX mice and improved impaired skeletal structure parameters. Conclusions: This study suggests that combined UD and CO extracts may be a beneficial traditional medicine for the prevention of postmenopausal osteoporosis.

Neurofibromin Deficiency Causes Epidermal Growth Factor Receptor Upregulation through the Activation of Ras/ERK/SP1 Signaling Pathway in Neurofibromatosis Type 1-Associated Malignant Peripheral Nerve Sheet Tumor.

Neurofibromatosis type 1 (NF1) is an autosomal dominant human genetic disorder. The progression of benign plexiform neurofibromas to malignant peripheral nerve sheet tumors (MPNSTs) is a major cause of mortality in patients with NF1. Although elevated epidermal growth factor receptor (EGFR) expression plays a crucial role in the pathogenesis of MPNST, the cause of EGFR overexpression remains unclear. Here, we assessed EGFR expression levels in MPNST tissues of NF1 patients and NF1 patient-derived MPNST cells. We found that the expression of EGFR was upregulated in MPNST tissues and MPNST cells, while the expression of neurofibromin was significantly decreased. Manipulation of NF1 expression by NF1 siRNA treatment or NF1-GAP-related domain overexpression demonstrated that EGFR expression levels were closely and inversely correlated with neurofibromin levels. Notably, knockdown of the NF1 gene by siRNA treatment augmented the nuclear localization of phosphorylated SP1 (pSP1) and enhanced pSP1 binding to the EGFR gene promoter region. Our results suggest that neurofibromin deficiency in NF1-associated MPNSTs enhances the Ras/ERK/SP1 signaling pathway, which in turn may lead to the upregulation of EGFR expression. This study provides insight into the progression of benign tumors and novel therapeutic approaches for treatment of NF1-associated MPNSTs.

Anti-Osteoporotic Effect of Morroniside on Osteoblast and Osteoclast Differentiation In Vitro and Ovariectomized Mice In Vivo.

Bone remodeling is a continuous process of bone synthesis and destruction that is regulated by osteoblasts and osteoclasts. Here, we investigated the anti-osteoporotic effects of morroniside in mouse preosteoblast MC3T3-E1 cells and mouse primary cultured osteoblasts and osteoclasts in vitro and ovariectomy (OVX)-induced mouse osteoporosis in vivo. Morroniside treatment enhanced alkaline phosphatase activity and positively stained cells via upregulation of osteoblastogenesis-associated genes in MC3T3-E1 cell lines and primary cultured osteoblasts. However, morroniside inhibited tartrate-resistant acid phosphatase activity and TRAP-stained multinucleated positive cells via downregulation of osteoclast-mediated genes in primary cultured monocytes. In the osteoporotic animal model, ovariectomized (OVX) mice were administered morroniside (2 or 10 mg/kg/day) for 12 weeks. Morroniside prevented OVX-induced bone mineral density (BMD) loss and reduced bone structural compartment loss in the micro-CT images. Taken together, morroniside promoted increased osteoblast differentiation and decreased osteoclast differentiation in cells, and consequently inhibited OVX-induced osteoporotic pathogenesis in mice. This study suggests that morroniside may be a potent therapeutic single compound for the prevention of osteoporosis.

Antiosteoarthritic Effect of Morroniside in Chondrocyte Inflammation and Destabilization of Medial Meniscus-Induced Mouse Model.

Osteoarthritis (OA) is a common degenerative disease that results in joint inflammation as well as pain and stiffness. A previous study has reported that Cornus officinalis (CO) extract inhibits oxidant activities and oxidative stress in RAW 264.7 cells. In the present study, we isolated bioactive compound(s) by fractionating the CO extract to elucidate its antiosteoarthritic effects. A single bioactive component, morroniside, was identified as a potential candidate. The CO extract and morroniside exhibited antiosteoarthritic effects by downregulating factors associated with cartilage degradation, including cyclooxygenase-2 (Cox-2), matrix metalloproteinase 3 (Mmp-3), and matrix metalloproteinase 13 (Mmp-13), in interleukin-1 beta (IL-1ß)-induced chondrocytes. Furthermore, morroniside prevented prostaglandin E2 (PGE2) and collagenase secretion in IL-1ß-induced chondrocytes. In the destabilization of the medial meniscus (DMM)-induced mouse osteoarthritic model, morroniside administration attenuated cartilage destruction by decreasing expression of inflammatory mediators, such as Cox-2, Mmp3, and Mmp13, in the articular cartilage. Transverse microcomputed tomography analysis revealed that morroniside reduced DMM-induced sclerosis in the subchondral bone plate. These findings suggest that morroniside may be a potential protective bioactive compound against OA pathogenesis.

Identification of rare missense mutations in NOTCH2 and HERC2 associated with familial central precocious puberty via whole-exome sequencing.

Objective: Genetic factors play a critical role in pubertal progression; however, mutations associated with central precocious puberty (CPP) have been reported only in four genes: KISS1, KISS1R, DLK1, and MKRN3. This study aimed to identify novel, potentially pathogenic variants in patients with familial CPP via whole-exome sequencing (WES).Methods: WES analysis was applied in 28 patients (25 girls and three boys) belonging to 14 families, wherein all siblings were diagnosed with CPP. Data analysis aimed to select only very rare variants (minor allele frequency <1%). Nonsense, splice-site, and frameshift variants were considered the most ideal candidate variants. Additionally, non-synonymous missense variants predicted as being deleterious using in silico analysis tools were further considered.Results: The analysis of exome sequencing data resulted in the identification of rare mutations in two promising candidate genes (NOTCH2 and HERC2) in a family. Siblings with CPP exhibited two heterozygous missense mutations (p. Leu15Phe in NOTCH2 and p. Arg4081His in HERC2). Moreover, their parents without history of CPP had a missense variant in either NOTCH2 or HERC2.Conclusions: We identified new candidate genes with potential roles in pubertal development. Digenic inheritance of the two genetic mutations associated with the Notch signaling pathway may have a synergistic effect resulting in CPP.

Scopolin Prevents Adipocyte Differentiation in 3T3-L1 Preadipocytes and Weight Gain in an Ovariectomy-Induced Obese Mouse Model.

Obesity is prevalent in modern human societies. We examined the anti-obesity effects of scopolin on adipocyte differentiation in preadipocyte 3T3-L1 cells and weight loss in an ovariectomy (OVX)-induced obese mouse model. Scopolin inhibited adipocyte differentiation and lipid accumulation in the preadipocyte cells by suppressing the transcription of adipogenic-related factors, including adiponectin (Adipoq), peroxisome proliferator-activated receptor gamma (Pparg), lipoprotein lipase (Lpl), perilipin1 (Plin1), fatty acid-binding protein 4 (Fabp4), glucose transporter type 4 (Slc2a4), and CCAAT/enhancer-binding protein alpha (Cebpa). In OVX-induced obese mice, administration of scopolin promoted the reduction of body weight, total fat percentage, liver steatosis, and adipose cell size. In addition, the scopolin-treated OVX mice showed decreased serum levels of leptin and insulin. Taken together, these findings suggest that the use of scopolin prevented adipocyte differentiation and weight gain in vitro and in vivo, indicating that scopolin may be a potential bioactive compound for the treatment and prevention of obesity in humans.

Osteoprotective Effects of Loganic Acid on Osteoblastic and Osteoclastic Cells and Osteoporosis-Induced Mice.

Osteoporosis is a common disease caused by an imbalance of processes between bone resorption by osteoclasts and bone formation by osteoblasts in postmenopausal women. The roots of Gentiana lutea L. (GL) are reported to have beneficial effects on various human diseases related to liver functions and gastrointestinal motility, as well as on arthritis. Here, we fractionated and isolated bioactive constituent(s) responsible for anti-osteoporotic effects of GL root extract. A single phytochemical compound, loganic acid, was identified as a candidate osteoprotective agent. Its anti-osteoporotic effects were examined in vitro and in vivo. Treatment with loganic acid significantly increased osteoblastic differentiation in preosteoblast MC3T3-E1 cells by promoting alkaline phosphatase activity and increasing mRNA expression levels of bone metabolic markers such as Alpl, Bglap, and Sp7. However, loganic acid inhibited osteoclast differentiation of primary-cultured monocytes derived from mouse bone marrow. For in vivo experiments, the effect of loganic acid on ovariectomized (OVX) mice was examined for 12 weeks. Loganic acid prevented OVX-induced bone mineral density loss and improved bone structural properties in osteoporotic model mice. These results suggest that loganic acid may be a potential therapeutic candidate for treatment of osteoporosis.

Antiadipogenic Effects of Mixtures of Cornus officinalis and Ribes fasciculatum Extracts on 3T3-L1 Preadipocytes and High-Fat Diet-Induced Mice.

Medicinal plants have been used worldwide as primary alternative healthcare supplements. Cornus officinalis (CO) and Ribes fasciculatum (RF) are traditional medicinal plants applied in East Asia to treat human diseases such as hepatitis, osteoporosis, oxidative stress and allergy. The aim of this study was to examine the anti-obesity effect of CO and RF on preadipocyte 3T3-L1 cells in vitro and high-fat diet (HFD)-induced obesity mice in vivo. Combination treatment of CO and RF in differentiated 3T3-L1 cells inhibited adipocyte differentiation through downregulation of adipogenesis-associated genes such as CCAAT/enhancer-binding protein alpha (Cebpa), fatty acid binding protein 4 (Fabp4), peroxisome proliferator-activated receptor gamma (Pparg) and sterol regulatory element binding protein (Srebp1). In vivo animal models showed that a mixture of CO and RF inhibited HFD-induced weight gain, resulting in decreased abdominal visceral fat tissues and fatty hepatocyte deposition. In addition, CO+RF treatment decreased HFD-induced adipogenesis-associated genes in abdominal white fat tissue. These results suggest that administration of a CO and RF mixture prevented adipocyte differentiation and lipid accumulation in preadipocyte cells and HFD-induced body weight in obesity mice. Therefore, combined therapy of CO and RF may be a protective therapeutic agent against obesity.

Antiobesity Effects of Gentiana lutea Extract on 3T3-L1 Preadipocytes and a High-Fat Diet-Induced Mouse Model.

Obesity is one of the most common metabolic diseases resulting in metabolic syndrome. In this study, we investigated the antiobesity effect of Gentiana lutea L. (GL) extract on 3T3-L1 preadipocytes and a high-fat-diet (HFD)-induced mouse model. For the induction of preadipocytes into adipocytes, 3T3-L1 cells were induced by treatment with 0.5 mM 3-isobutyl-1-methylxanthine, 1 mM dexamethasone, and 1 µg/mL insulin. Adipogenesis was assessed based on the messenger ribonucleic acid expression of adipogenic-inducing genes (adiponectin (Adipoq), CCAAT/enhancer-binding protein alpha (Cebpa), and glucose transporter type 4 (Slc2a4)) and lipid accumulation in the differentiated adipocytes was visualized by Oil Red O staining. In vivo, obese mice were induced with HFD and coadministered with 100 or 200 mg/kg/day of GL extract for 12 weeks. GL extract treatment inhibited adipocyte differentiation by downregulating the expression of adipogenic-related genes in 3T3-L1 cells. In the obese mouse model, GL extract prevented HFD-induced weight gain, fatty hepatocyte deposition, and adipocyte size by decreasing the secretion of leptin and insulin. In conclusion, GL extract shows antiobesity effects in vitro and in vivo, suggesting that this extract can be beneficial in the prevention of obesity.

Cirsium japonicum var. maackii and apigenin block Hif-2α-induced osteoarthritic cartilage destruction.

Although Hif-2α is a master regulator of catabolic factor expression in osteoarthritis development, Hif-2α inhibitors remain undeveloped. The aim of this study was to determine whether Cirsium japonicum var. maackii (CJM) extract and one of its constituents, apigenin, could attenuate the Hif-2α-induced cartilage destruction implicated in osteoarthritis progression. In vitro and in vivo studies demonstrated that CJM reduced the IL-1ß-, IL-6, IL-17- and TNF-α-induced up-regulation of MMP3, MMP13, ADAMTS4, ADAMTS5 and COX-2 and blocked osteoarthritis development in a destabilization of the medial meniscus mouse model. Activation of Hif-2α, which directly up-regulates MMP3, MMP13, ADAMTS4, IL-6 and COX-2 expression, is inhibited by CJM extract. Although cirsimarin, cirsimaritin and apigenin are components of CJM and can reduce inflammation, only apigenin effectively reduced Hif-2α expression and inhibited Hif-2α-induced MMP3, MMP13, ADAMTS4, IL-6 and COX-2 expression in articular chondrocytes. IL-1ß induction of JNK phosphorylation and IκB degradation, representing a critical pathway for Hif-2α expression, was completely blocked by apigenin in a concentration-dependent manner. Collectively, these effects indicate that CJM and one of its most potent constituents, apigenin, can lead to the development of therapeutic agents for blocking osteoarthritis development as novel Hif-2α inhibitors.

Synergistic Neuroprotective Effect of Schisandra chinensis and Ribes fasciculatum on Neuronal Cell Death and Scopolamine-Induced Cognitive Impairment in Rats.

Mild cognitive impairment (MCI) is considered as a transitional stage between aging and Alzheimer's disease. In the present study, we examined the protective effect of Schisandra chinensis (SC) and Ribes fasciculatum (RF) on neuronal cell death in vitro and scopolamine-induced cognitive impairment in Sprague Dawley® rats in vivo. A mixture of SC and RF extracts (SC+RF) significantly protected against hydrogen peroxide-induced PC12 neuronal cell death. The neuroprotective effect of SC+RF on scopolamine-induced memory impairment in rats was evaluated using the passive avoidance test and the Morris water maze test. In the passive avoidance test, SC+RF-treated rats showed an increased latency to escape, compared to the scopolamine-treated rats. Moreover, SC+RF treatment significantly reduced escape latency in water maze test, compared to treatment with scopolamine alone. To verify the long-term memory, we performed probe test of water maze test. As a result, rat treated with SC+RF spent more time in the target quadrant. Consistent with enhancement of memory function, the brain derived neurotrophic factor (BDNF) and its downstream molecules (pERK, pATK, and pCREB) are increased in SC+RF treatment in hippocampal area compared with scopolamine treated group. These results suggest that a mixture of SC and RF extracts may be a good therapeutic candidate for preventing mild cognitive impairment.

Anti-Osteoporotic Effects of Kukoamine B Isolated from Lycii Radicis Cortex Extract on Osteoblast and Osteoclast Cells and Ovariectomized Osteoporosis Model Mice.

Osteoporosis is an abnormal bone remodeling condition characterized by decreased bone density, which leads to high risks of fracture. Previous study has demonstrated that Lycii Radicis Cortex (LRC) extract inhibits bone loss in ovariectomized (OVX) mice by enhancing osteoblast differentiation. A bioactive compound, kukoamine B (KB), was identified from fractionation of an LRC extract as a candidate component responsible for an anti-osteoporotic effect. This study investigated the anti-osteoporotic effects of KB using in vitro and in vivo osteoporosis models. KB treatment significantly increased the osteoblastic differentiation and mineralized nodule formation of osteoblastic MC3T3-E1 cells, while it significantly decreased the osteoclast differentiation of primary-cultured monocytes derived from mouse bone marrow. The effects of KB on osteoblastic and osteoclastic differentiations under more physiological conditions were also examined. In the co-culture of MC3T3-E1 cells and monocytes, KB promoted osteoblast differentiation but did not affect osteoclast differentiation. In vivo experiments revealed that KB significantly inhibited OVX-induced bone mineral density loss and restored the impaired bone structural properties in osteoporosis model mice. These results suggest that KB may be a potential therapeutic candidate for the treatment of osteoporosis.

Identification of a rare homozygous c.790C>T variation in the TFB2M gene in Korean patients with autism spectrum disorder.

Mitochondrial dysfunction and subsequent enhanced oxidative stress is implicated in the pathogenesis of autism spectrum disorder (ASD). Mitochondrial transcription factor B2 (TFB2M) is an essential protein in mitochondrial gene expression. No reports have described TFB2M mutations and variations involved in any human diseases. We identified a rare homozygous c.790C>T (His264Tyr) variation in TFB2M gene in two Korean siblings with ASD by whole-exome sequencing. The roles of the TFB2M variation in the pathogenesis of ASD were investigated. Patient fibroblasts revealed increased transcription of mitochondrial genes and mitochondrial function in terms of ATP, membrane potential, oxygen consumption, and reactive oxygen species (ROS). Overexpression of the TFB2M variant in primary-cultured fibroblasts demonstrated significantly increased transcription of mitochondrial genes and mitochondrial function compared with overexpression of wild-type TFB2M. Molecular dynamics simulation of the TFB2M variant protein suggested an increase in the rigidity of the hinge region, which may cause alterations in loading and/or unloading of TFB2M on target DNA. Our results suggest that augmentation of mitochondrial gene expression and subsequent enhancement of mitochondrial function may be associated with the pathogenesis of ASD in Korean patients.

Antiadipogenic Effects of Loganic Acid in 3T3-L1 Preadipocytes and Ovariectomized Mice.

Obesity is caused by an excess storage of body fat, resulting from a chronic imbalance between energy intake and expenditure. Gentiana lutea L. (GL) root has been reported to reduce lipid accumulation in the aortic wall of diabetic rats. Here, we performed fractionation and isolation of the bioactive constituent(s) that may be responsible for the antiadipogenic effects of the GL root extract. A single compound, loganic acid, was identified as a candidate component in the 30% ethanol extract of GL. Loganic acid treatment significantly decreased the adipocyte differentiation of 3T3-L1 preadipocytes in a dose-dependent manner. The expression of key adipogenesis-related genes such as adiponectin (Adipoq), peroxisome proliferator-activated receptor gamma (Pparg), lipoprotein lipase (Lpl), perilipin1 (Plin1), fatty acid binding protein 4 (Fabp4), glucose transporter type 4 (Slc2a4), CCAAT/enhancer-binding protein alpha (Cebpa), and tumor necrosis factor-alpha (Tnf) were significantly reduced following treatment with loganic acid. In vivo experiments in an ovariectomy-induced obesity mouse model showed that loganic acid (oral administration with 10 and 50 mg/kg/day) significantly inhibited body weight gain, total fat increase, fatty hepatocyte deposition in the liver, and adipocyte enlargement in the abdominal visceral fat tissues. These results suggest that loganic acid in the GL root extract has antiadipogenic effects in vitro and in vivo. Loganic acid may be beneficial for the prevention and treatment of obesity, particularly in menopausal obese women.

Effects of Dihydrophaseic Acid 3'-O-ß-d-Glucopyranoside Isolated from Lycii radicis Cortex on Osteoblast Differentiation.

Our previous study showed that ethanol extract of Lyciiradicis cortex (LRC) prevented the loss of bone mineral density in ovariectomized mice by promoting the differentiation of osteoblast linage cells. Here, we performed fractionation and isolation of the bioactive compound(s) responsible for the bone formation-enhancing effect of LRC extract. A known sesquiterpene glucoside, (1'R,3'S,5'R,8'S,2Z,4E)-dihydrophaseic acid 3'-O-ß-d-glucopyranoside (abbreviated as DPA3G), was isolated from LRC extract and identified as a candidate constituent. We investigated the effects of DPA3G on osteoblast and osteoclast differentiation, which play fundamental roles in bone formation and bone resorption, respectively, during bone remodeling. The DPA3G fraction treatment in mesenchymal stem cell line C3H10T1/2 and preosteoblast cell line MC3T3-E1 significantly enhanced cell proliferation and alkaline phosphatase activity in both cell lines compared to the untreated control cells. Furthermore, DPA3G significantly increased mineralized nodule formation and the mRNA expression of osteoblastogenesis markers, Alpl, Runx2, and Bglap, in MC3T3-E1 cells. The DPA3G treatment, however, did not influence osteoclast differentiation in primary-cultured monocytes of mouse bone marrow. Because osteoblastic and osteoclastic precursor cells coexist in vivo, we tested the DPA3G effects under the co-culture condition of MC3T3-E1 cells and monocytes. Remarkably, DPA3G enhanced not only osteoblast differentiation of MC3T3-El cells but also osteoclast differentiation of monocytes, indicating that DPA3G plays a role in the maintenance of the normal bone remodeling balance. Our results suggest that DPA3G may be a good candidate for the treatment of osteoporosis.

TIMP-1 modulates chemotaxis of human neural stem cells through CD63 and integrin signalling.

We recently reported that hNSCs (human neural stem cells) have the interesting characteristic of migration towards an intracranial glioma. However, the molecules and mechanisms responsible for tumour tropism are unclear. In the present study, we used microarray and proteomics analyses to identify a novel chemoattractant molecule, TIMP-1 (tissue inhibitor of metalloproteinase-1), secreted from human brain tumour tissues. We demonstrate that TIMP-1 significantly enhances hNSC adhesion and migration in a cell culture system. These effects were critically dependent on CD63, as shRNA-mediated ablation of CD63 expression attenuated the response. TIMP-1 significantly increased the number of FAs (focal adhesions) and cytoskeletal reorganization for cell migration in hNSCs, whereas knockdown of CD63 resulted in decreased hNSC spreading, FAs and migration, even after TIMP-1 treatment. In addition, TIMP-1 binding to CD63 activated ß1 integrin-mediated signalling through Akt and FAK phosphorylation, leading to pattern changes in distribution of vinculin and F-actin (filamentous actin). Furthermore, inactivation of ß1 integrin by use of a blocking antibody or inhibition of PI3K (phosphoinositide 3-kinase) signalling impaired the migration of hNSCs towards TIMP-1. Collectively, our results underline TIMP-1 as a novel and effective key regulator of CD63 and ß1 integrin-mediated signalling, which regulates hNSC adhesion and migration.

Identification of the rare compound heterozygous variants in the NEB gene in a Korean family with intellectual disability, epilepsy and early-childhood-onset generalized muscle weakness.

We examined a Korean family with complex phenotypes characterized by intellectual disability, epilepsy and early-childhood-onset generalized muscle weakness. Since we did not find any abnormality using several conventional genetic tests for detection of chromosomal aberrations, gene copy number variations and mitochondrial gene mutations, we aimed to identify disease-causing genetic alteration(s) in this family. We conducted whole-exome sequencing (WES) in this family. After filtering the WES data, we compared five exome sequences of two affected siblings, one unaffected sibling and the unaffected parents, and we determined the allele frequency of the identified variants in an Asian population. Finally, we selected one candidate variant pair which is unique in the patients and corresponds to an autosomal recessive genetic model. The two affected siblings had the same compound heterozygous variation in the NEB gene encoding nebulin, which was composed of two different missense variants: c.2603T>C (p.L868P) in exon 27 and c.21340C>T (p.R7114W) in exon 143. We confirmed these variations by Sanger sequencing. On the basis of the fundamental role of nebulin in the brain and skeletal muscles, we concluded that this compound heterozygous NEB variation may be a sound candidate for the disease-causing mutation in this family. Since the patients are characterized by generalized muscle weakness together with neurodevelopmental phenotypes, it is suggested that NEB mutations could manifest more diverse phenotypes than those previously described.

Identification of KMT2D and KDM6A mutations by exome sequencing in Korean patients with Kabuki syndrome.

Kabuki syndrome (KS) (OMIM#147920) is a multiple congenital anomaly/mental retardation syndrome. Recently, pathogenic variants in KMT2D and KDM6A were identified as the causes of KS in 55.8-80.0% of patients. To elucidate further the molecular characteristics of Korean patients with KS, we screened a cohort of patients with clinically defined KS for mutations in KMT2D and KDM6A. Whole-exome sequencing and direct sequencing for validation were performed in 12 patients with a clinical suspicion of KS. KMT2D and KDM6A mutations were identified in 11 (91.7%) patients. No recurrent mutation was observed, and 10 out of the 11 mutations found were novel. KMT2D mutations were detected in 10 patients, including four small deletions or insertions and four nonsense and two missense mutations. One girl had a novel splice-site mutation in KDM6A. Each patient had a unique individual mutation. This is the first report of mutational analysis via exome sequencing in Korean patients with KS. Because the mutation-detection rate was high in this study, rigorous mutation analysis of KMT2D and KDM6A may be an important tool for the early diagnosis and genetic counseling of Korean patients with KS.