Generation of mutation-corrected induced pluripotent stem cell lines derived from adrenoleukodystrophy patient by using homology directed repair.

X-linked adrenoleukodystrophy (ALD) caused by the ABCD1 mutation, is the most common inherited peroxisomal disease. Previously, we generated an ALD patient-derived SCHi001-A iPSC model. In this study, we have performed the first genome editing of ALD patient-derived SCHi001-A iPSCs using homology-directed repair (HDR). The mutation site, c.1534G > A [GenBank: NM_000033.4], was corrected by introducing ssODN and the CRISPR/Cas9 system. The cell line exhibited normal iPSC plulipotency marker expression following genome editing. Mutation-corrected iPSCs from SCHi001-A iPSC line can be used in research into the pathophysiology of and therapeutics for ALD.

Sequence-specific prediction of the efficiencies of adenine and cytosine base editors.

Base editors, including adenine base editors (ABEs)1 and cytosine base editors (CBEs)2,3, are widely used to induce point mutations. However, determining whether a specific nucleotide in its genomic context can be edited requires time-consuming experiments. Furthermore, when the editable window contains multiple target nucleotides, various genotypic products can be generated. To develop computational tools to predict base-editing efficiency and outcome product frequencies, we first evaluated the efficiencies of an ABE and a CBE and the outcome product frequencies at 13,504 and 14,157 target sequences, respectively, in human cells. We found that there were only modest asymmetric correlations between the activities of the base editors and Cas9 at the same targets. Using deep-learning-based computational modeling, we built tools to predict the efficiencies and outcome frequencies of ABE- and CBE-directed editing at any target sequence, with Pearson correlations ranging from 0.50 to 0.95. These tools and results will facilitate modeling and therapeutic correction of genetic diseases by base editing.

Differential effects on sodium current impairments by distinct SCN1A mutations in GABAergic neurons derived from Dravet syndrome patients.

BACKGROUND: We investigated how two distinct mutations in SCN1A differentially affect electrophysiological properties of the patient-derived GABAergic neurons and clinical severities in two Dravet syndrome (DS) patients. MATERIALS AND METHODS: We established induced pluripotent stem cells from two DS patients with different mutations in SCN1A and subsequently differentiated them into forebrain GABAergic neurons. Functionality of differentiated GABAergic neurons was examined by electrophysiological recordings. RESULTS: DS-1 patient had a missense mutation, c.4261G > T [GenBank: NM_006920.4] and DS-2 patient had a nonsense frameshift mutation, c.3576_3580 del TCAAA [GenBank: NM_006920.4]. Clinically, contrary to our expectations, DS-1 patient had more severe symptoms including frequency of seizure episodes and the extent of intellectual ability penetration than DS-2 patient. Electrophysiologic recordings showed significantly lower sodium current density and reduced action potential frequency at strong current injection (>60 pA) in GABAergic neurons derived from both. Intriguingly, unique genetic alterations of SCN1A differentially impacted electrophysiological impairment of the neurons, and the impairment's extent corresponded with the symptomatic severity of the donor from which the iPSCs were derived. CONCLUSION: Our results suggest the possibility that patient-derived iPSCs may provide a reliable in vitro system that reflects clinical severities in individuals with DS.

Three-dimensional brain-like microenvironments facilitate the direct reprogramming of fibroblasts into therapeutic neurons.

Biophysical cues can improve the direct reprogramming of fibroblasts into neurons that can be used for therapeutic purposes. However, the effects of a three-dimensional (3D) environment on direct neuronal reprogramming remain unexplored. Here, we show that brain extracellular matrix (BEM) decellularized from human brain tissue facilitates the plasmid-transfection-based direct conversion of primary mouse embryonic fibroblasts into induced neuronal (iN) cells. We first show that two-dimensional (2D) surfaces modified with BEM significantly increase the generation efficiency of iN cells and enhance neuronal transdifferentiation and maturation. Moreover, in an animal model of ischaemic stroke, iN cells generated on the BEM substrates and transplanted into the brain led to significant improvements in locomotive behaviours. We also show that compared with the 2D BEM substrates, 3D BEM hydrogels recapitulating brain-like microenvironments further promote neuronal conversion and potentiate the functional recovery of the animals. Our findings suggest that 3D microenvironments can boost nonviral direct reprogramming for the generation of therapeutic neuronal cells.

Generation of two induced pluripotent stem cell (iPSC) lines from X-linked adrenoleukodystrophy (X-ALD) patients with adrenomyeloneuropathy (AMN).

X-linked adrenoleukodystrophy (X-ALD) is an inherited disorder caused by a mutation in the ATP-binding cassette transporter subfamily D member 1 (ABCD1) gene. We generated two induced pluripotent stem cell (iPSC) lines from X-ALD patients with adrenomyeloneuropathy (AMN) by Sendai virus containing OCT4, SOX2, KLF4 and c-MYC. Established iPSC lines expressed various pluripotency markers, had differentiation potential of three germ layers in vitro, had normal karyotype and retained ABCD1 mutation.

Isolated cerebellar variant of adrenoleukodystrophy with a de novo adenosine triphosphate-binding cassette D1 (ABCD1) gene mutation.

X-linked adrenoleukodystrophy (X-ALD) shows a wide range of phenotypic expression, but clinical presentation as an isolated lesion of the cerebellar white matter and dentate nuclei has not been reported. We report an unusual presentation of X-ALD only with an isolated lesion of the cerebellar white matter and dentate nuclei. The proband, a 37-year-old man presented with bladder incontinence, slurred speech, dysmetria in all limbs, difficulties in balancing, and gait ataxia. Brain magnetic resonance imaging showed an isolated signal change of white matter around the dentate nucleus in cerebellum. With high level of very long chain fatty acid, gene study showed a de novo mutation in exon 1 at nucleotide position c.277_296dup20 (p.Ala100Cysfs*10) of the adenosine triphosphate-binding cassette D1 gene. It is advised to consider X-ALD as a differential diagnosis in patients with isolated cerebellar degeneration symptoms.

Induced pluripotent stem cells for modeling of pediatric neurological disorders.

The pathophysiological mechanisms underlying childhood neurological disorders have remained obscure due to a lack of suitable disease models reflecting human pathogenesis. Using induced pluripotent stem cell (iPSC) technology, various neurological disorders can now be extensively modeled. Specifically, iPSC technology has aided the study and treatment of early-onset pediatric neurodegenerative diseases such as Rett syndrome, Down syndrome, Angelman syndrome. Prader-Willi syndrome, Friedreich's ataxia, spinal muscular atrophy (SMA), fragile X syndrome, X-linked adrenoleukodystrophy (ALD), and SCN1A gene-related epilepsies. In this paper, we provide an overview of various gene delivery systems for generating iPSCs, the current state of modeling early-onset neurological disorders and the ultimate application of these in vitro models in cell therapy through the correction of disease-specific mutations.

Licochalcone E has an antidiabetic effect.

Licochalcone E (lico E) is a retrochalcone isolated from the root of Glycyrrhiza inflata. Retrochalcone compounds evidence a variety of pharmacological profiles, including anticancer, antiparasitic, antibacterial, antioxidative and superoxide-scavenging properties. In this study, we evaluated the biological effects of lico E on adipocyte differentiation in vitro and obesity-related diabetes in vivo. We employed 3T3-L1 preadipocyte and C3H10T1/2 stem cells for in vitro adipocyte differentiation study and diet-induced diabetic mice for in vivo study. The presence of lico E during adipogenesis induced adipocyte differentiation to a significant degree, particularly at the early induction stage. Licochalcone E evidenced weak, but significant, peroxisome proliferator-activated receptor gamma (PPARγ) ligand-binding activity. Two weeks of lico E treatment lowered blood glucose levels and serum triglyceride levels in the diabetic mice. Additionally, treatment with lico E resulted in marked reductions in adipocyte size and increases in the mRNA expression levels of PPARγ in white adipose tissue (WAT). Licochalcone E was also shown to significantly stimulate Akt signaling in epididymal WAT. In conclusion, lico E increases the levels of PPARγ expression, at least in part, via the stimulation of Akt signals and functions as a PPARγ partial agonist, and this increased PPARγ expression enhances adipocyte differentiation and increases the population of small adipocytes, resulting in improvements in hyperglycemia and hyperlipidemia under diabetic conditions.

Ethanol extracts of Saururus chinensis suppress ovalbumin-sensitization airway inflammation.

AIM OF THE STUDY: The aerial part of Saururus chinensis has been used in folk medicine to treat several inflammatory diseases in China and Korea. Previously, our group reported that anti-asthmatic activity of an ethanol extract of Saururus chinensis (ESC) might occur, in part, via the inhibition of prostaglandin D(2) (PGD(2)) and leukotriene C(4) (LTC(4)) production, and degranulation reaction in vitro, as well as through the down-regulation of interleukin (IL)-4 and eotaxin mRNA expression in an in vivo ovalbumin-sensitization animal model. However, the effects of Saururus chinensis on eicosanoid generation, as well as Th2 cytokines and eotaxin production in an in vivo asthma model, have not been fully investigated. Moreover, it has not been determined whether ESC can ameliorate airway inflammation in vivo. In the present study, we investigated the therapeutic activity of Saururus chinensis on ovalbumin (OVA)-sensitized airway inflammation and its major phytochemical compositions. MATERIALS AND METHODS: Asthma was induced in BALB/c mice by ovalbumin-sensitization and inhalation. ESC (10-100 mg/kg) or dexamethasone (5 mg/kg), a positive control, was administered 7 times orally every 12 h from one day before the first challenge to 1 h before the second challenge. The recruitment of inflammatory cells and hyperplasia of goblet cells were evaluated by H&E and PAS staining. Levels of Th2 cytokines, eotaxin, PGD(2) and LTC(4) were measured to evaluate the anti-inflammatory activity of ESC in OVA-sensitized mice. Contents of major components were analyzed by HPLC using a reversed-phase C18 column. RESULTS: ESC (10 mg/kg) suppressed allergic airway inflammation by inhibition of the production of IL-4 (P<0.001), IL-5 (P<0.05), IL-13 (P<0.001), eotaxin (P<0.001), PGE(2) (P<0.001), LTC(4) (P<0.001) in lung extract and IgE level (P<0.001) in the serum. In addition, ESC (50 mg/kg) reduced the infiltration of inflammatory cells and hyperplasia of goblet cells in the lung tissues. The anti-inflammatory effect of ESC was comparable to that of the positive control drug, dexamethasone. Its major phytochemical composition includes manassantin A, B and sauchinone. CONCLUSIONS: These results suggest that ESC decreased inflammation and mucus secretion in the OVA-induced bronchial asthma model, and its anti-asthmatic activity may occur in part via the inhibition of Th2 cytokines and eotaxin protein expression, as well as through prostaglandin E(2) (PGE(2)) and leukotriene C(4) (LTC(4)) generation. This effects may be attributed particularly to the presence of manassantin A, B and sauchinone major component evidenced by a HPLC analysis.

The effects of Wnt inhibitors on the chondrogenesis of human mesenchymal stem cells.

The purpose of this study was to test the hypothesis that the inhibition of the Wnt pathway promotes chondrogenesis of human mesenchymal stem cells (hMSCs). To investigate its effect on early chondrogenesis, in vitro pellet cultures were prepared using MSCs at passage 3 at varying concentrations of 100, 200, and 300 ng/mL of either Dickkopf (DKK)-1 or secreted frizzled-related protein (sFRP)-1, and analyzed for chondrogenic gene and protein expressions after 3 and 6 days of culture. After that to study the effects on chondrogenesis at a longer term, 200 ng of sFRP-1 was challenged either in the presence or absence of transforming growth factor (TGF)-beta(3) to pellets of MSCs at passage 3 for 7 days. Then the pellets were cultured without sFRP-1 for 14 further days. For early chondrogenesis, both DKK-1 and sFRP-1 increased glycosaminoglycan synthesis as well as the gene and protein expressions of SOX-9 and type II collagen, more prominently by sFRP-1 than by DKK-1. However, after 21 days of in vitro chondrogenic culture under TGF-beta(3), sFRP-1 treatment did not further increase the gene expression of SOX-9 and type II collagen. The overall results of this study suggest that although the inhibitors of Wnt pathway promote early chondrogenesis of MSCs, they do not provide an ultimately enhancing role in the cartilage tissue engineering of MSCs.

Effect of dexamethasone on STAT6-dependent Ym1/2 expression in vivo and in vitro.

Ym1 and Ym2 (Ym1/2) are chitinase-like proteins and we reported previously that IL-4 induced Ym1/2 in mouse bone marrow-derived mast cells. In the present study, ovalbumin-induced asthmatic mice were used to investigate the effect of glucocorticoids on Ym1/2 expression. Ym1/2 were highly induced in bronchoalveolar lavage fluid (BALF) and the lung. Ym1/2 expression was completely inhibited by dexamethasone (Dex) in BALF and weakly inhibited in the lung. Primary cultured macrophages were used to investigate the inhibition of Ym1/2 expression at the cellular level. Although Dex pretreatment inhibited the Ym1/2 expression level in an animal model, it did not reduce IL-4 induction of Ym1/2 expression in vitro. Next, we tested whether Dex blocks IL-4 induced STAT6 signaling and found that it had no inhibitory effect on the phosphorylation level of STAT6 in macrophages. The luciferase reporter assay also revealed that Dex did not inhibit IL-4 induction of Ym1/2 promoter activity. These results indicate that the inhibitory effect of Dex on Ym1/2 protein expression in the murine model of asthma does not involve the STAT6 signaling pathway.

The naturally occurring flavolignan, deoxypodophyllotoxin, inhibits lipopolysaccharide-induced iNOS expression through the NF-kappaB activation in RAW264.7 macrophage cells.

Deoxypodophyllotoxin (DPT), a naturally occurring flavolignan with anti-inflammatory activity, was isolated from Anthriscus sylvestris HOFFM., and we examined its effects on the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated, murine macrophage-like RAW264.7 cells. Western blot analysis performed with specific anti-iNOS antibodies showed that a decrease in nitric oxide (NO) was accompanied by a decrease in the iNOS protein level. To clarify the mechanistic basis for DPT's ability to inhibit iNOS induction, we examined the effect of DPT on nuclear factor (NF)-kappaB transcriptional activity and DNA binding activity. DPT potently suppressed both reporter gene activity and DNA binding activity. These findings suggest that DPT in RAW264.7 cells abolished LPS-induced iNOS expression by inhibiting the transcription factor, NF-kappaB.

Inhibitory effect of synthetic C-C biflavones on various phospholipase A(2)s activity.

Several prototypes of C-C biflavones (a-f) were synthesized and evaluated their inhibitory activity against phospholipase A(2)s (PLA(2)s) activity. The synthetic C-C biflavones (a-f) showed rather different inhibitory activity against various PLA(2)s. Most synthetic C-C biflavonoids exhibited potent and broad inhibitory activity against various sPLA(2)s and cPLA(2) tested regardless of their structural array. In particular, of natural and synthetic biflavonoids tested, the synthetic C-C biflavonoid (d) only showed inhibitory activity against sPLA(2) X. None of the natural and synthetic biflavonoids tested showed inhibitory activity against sPLA(2) IB. Further chemical modification of these basic structures will be carried out in order to investigate the synthetic C-C biflavones which possess more selective inhibitory activity against isozymes of PLA(2).

Ochnaflavone, naturally occurring biflavonoid, inhibits phospholipase A2 dependent phosphatidylethanolamine degradation in a CCl4-induced rat liver microsome.

This study investigated the protective effects of a group IIA secretory phospholipase A2 (sPLA2-IIA) inhibitor, ochnaflavone, on the progression of carbon tetrachloride (CCl4)-induced acute liver injury in rat liver microsomes in vitro. When rat liver was incubated at 37 degrees C in the presence of CCl4, the level of phosphatidylethanolamine (PE) degradation increased markedly compared with the control. The rat 14 kDa platelet PLA2 antibody, R377, suppressed the degradation of PE. Pretreating the microsome with ochnaflavone (2-16 microM) reduced the level of PE degradation in a dose dependent manner. In addition, p-bromophenacy bromide (p-BPB), which is a PLA2 inhibitor, also inhibited PE degradation. However, the inhibitory activity was weaker than that of ochnaflavone. Further investigation showed that ochnaflavone not only inhibited the purified rat platelet sPLA2 activity in a dose dependent manner with an IC50 value of 3.45 microM, when arachidonyl PE was used as a substrate, but also inhibited lipid peroxidation in a dose dependent manner with an IC50 value of 7.16 microM. This result suggests that ochnaflavone prevents the progression of CCl4-induced PE hydrolysis by inhibiting the endogenous sPLA2 activity.

Effects of methyl gallate on arachidonic acid metabolizing enzymes: Cyclooxygenase-2 and 5-lipoxygenase in mouse bone marrow-derived mast cells.

Methyl gallate (MG) is a medicinal herbal product that is isolated from Paeonia lactiflora that inhibits cyclooxygenase-2 (COX-2) dependent phases of prostaglandin D2 (PGD2) generation in bone marrow-derived mast cells (BMMC) in a concentration-dependent manner with an IC50 values of 17.0 microM. This compound also found inhibited the COX-2-dependent conversion of the exogenous arachidonic acid to PGD2 in a dose-dependent manner with an IC50 values of 19.0 microM, using a COX enzyme assay kit. However, at concentrations up to 80 microM, MG did not inhibit COX-2 protein expression in BMMC, indicating that MG inhibits COX-2 activity directly. Furthermore, MG consistently inhibited the production of leukotriene C4 (LTC4) in a dose dependent manner, with an IC50 value of 5.3 microM. These results demonstrate that MG has a dual cyclooxygenase-2/5-lipoxygenase inhibitory activity, which might provide the basis for novel anti-inflammatory drugs.

Anti-inflammatory activity of Ailanthus altissima in ovalbumin-induced lung inflammation.

As part of an ongoing investigation to find bioactive medicinal herbs exerting anti-inflammation activity, the effect of an ethanol extract from the parts of Ailanthus altissima (Simaroubaceae) was evaluated in both in vitro and in in vivo system. The ethanol extract of A. altissima (EAa) inhibited generation of the cyclooxygenase-2 (COX-2) dependent phases of prostaglandin D2 in bone marrow-derived mast cells (BMMC) in a concentration-dependent manner with an IC50 value of 214.6 microg/ml. However, this compound did not inhibit COX-2 protein expression up to a concentration of 400 microg/ml in the BMMC, indicating that EAa directly inhibits COX-2 activity. In addition, EAa inhibited leukotriene C4 production with an IC50 value of 25.7 microg/ml. Furthermore, this compound inhibited degranulation reaction in a dose dependent manner, with an IC50 value of 27.3 microg/ml. Ovalbumin (OVA)-sensitized mice were orally pretreated with EAa before aerosol challenges. EAa reduced the eosinophil infiltration into the airway and the eotaxin, IL-4, and IL-13 mRNA expression levels. These results suggest that the anti-inflammation activity of A. altissima in OVA-induced lung inflammation may occur in part via the down regulation of T(H)2 cytokines and eotaxin transcripts as well as the inhibition of inflammatory mediators.

Deoxypodophyllotoxin (DPT) inhibits eosinophil recruitment into the airway and Th2 cytokine expression in an OVA-induced lung inflammation.

The effect of deoxypodophyllotoxin (DPT) isolated from Anthriscus sylvestris Hoffm. was evaluated in an IN VIVO animal model for antiasthmatic activity. DPT (1.0 to 5 mg/kg) was given orally to ovalbumin (OVA)/alum-induced asthmatic mice. DPT reduced the number of infiltrated eosinophils in bronchoalveolar lavage (BAL) fluid in a dose-dependent manner. Dexamethasone (5 mg/kg), which was used as a positive control, also strongly inhibited the number of infiltrated eosinophils. The effect of DPT on a transcript profile in a murine asthma model was determined by RT-PCR, which showed that DPT decreased the mRNA levels of the Th2 cytokines. Northern blot analysis showed that DPT also reduced both the eotaxin and arginase I mRNA levels in a dose-dependent manner.

Extraction and chromatography of carotenoids from pumpkin.

Vitamin A deficiency is a health problem in Southeast Asia that can be corrected by feeding orange fruits and vegetables such as mango. Pumpkin is a traditional Korean food that is easy to store and is already believed to have health benefits. We extracted carotenoids from pumpkin by liquid-liquid extraction and by supercritical fluid extraction. We measured carotenoids by reversed-phase chromatography with diode array detection. The major carotenoid in pumpkin (> 80%) is beta-carotene, with lesser amounts of lutein, lycopene, alpha-carotene and cis-beta-carotene. Pumpkin is a rich source of beta-carotene and might be useful for preventing Vitamin A deficiency.