Polymorphic USP8 allele promotes Parkinson's disease by inducing the accumulation of α-synuclein through deubiquitination.

Parkinson's disease (PD) is one of the most common neuro-degenerative diseases characterized by α-synuclein accumulation and degeneration of dopaminergic neurons. Employing genome-wide sequencing, we identified a polymorphic USP8 allele (USP8D442G) significantly enriched in Chinese PD patients. To test the involvement of this polymorphism in PD pathogenesis, we derived dopaminergic neurons (DAn) from human-induced pluripotent stem cells (hiPSCs) reprogrammed from fibroblasts of PD patients harboring USP8D442G allele and their healthy siblings. In addition, we knock-in D442G polymorphic site into the endogenous USP8 gene of human embryonic stem cells (hESCs) and derived DAn from these knock-in hESCs to explore their cellular phenotypes and molecular mechanism. We found that expression of USP8D442G in DAn induces the accumulation and abnormal subcellular localization of α-Synuclein (α-Syn). Mechanistically, we demonstrate that D442G polymorphism enhances the interaction between α-Syn and USP8 and thus increases the K63-specific deubiquitination and stability of α-Syn . We discover a pathogenic polymorphism for PD that represent a promising therapeutic and diagnostic target for PD.

Genome re-sequencing and bioinformatics analysis of a nutraceutical rice.

The genomes of two rice cultivars, Nipponbare and 93-11, have been well studied. However, there is little available genetic information about nutraceutical rice cultivars. To remedy this situation, the present study aimed to provide a basic genetic landscape of nutraceutical rice. The genome of Black-1, a black pericarp rice containing higher levels of anthocyanins, flavonoids, and a more potent antioxidant capacity, was sequenced at ≥30 × coverage using Solexa sequencing technology. The complete sequences of Black-1 genome shared more consensus sequences with indica cultivar 93-11 than with Nipponbare. With reference to the 93-11 genome, Black-1 contained 675,207 single-nucleotide polymorphisms, 43,130 insertions and deletions (1-5 bp), 1,770 copy number variations, and 10,911 presence/absence variations. These variations were observed to reside preferentially in Myb domains, NB-ARC domains and kinase domains, providing clues to the diversity of biological functions or secondary metabolisms in this cultivar. Intriguingly, 496 unique genes were identified by comparing it with the genomes of these two rice varieties; among the genes, 119 genes participate in the biosynthesis of secondary metabolites. Furthermore, several unique genes were predicted to be involved in the anthocyanins synthesis pathway. The genome-wide landscape of Black-1 uncovered by this study represents a valuable resource for further studies and for breeding nutraceutical rice varieties.

Apigenin mediated protection of OGD-evoked neuron-like injury in differentiated PC12 cells.

Ischemic stroke has been confirmed to cause neuronal injury due to its insufficient supply of glucose and oxygen to brain tissue. Previous research has shown that oxidative stress, a result of excessive accumulation of reactive oxygen species (ROS), relates to pathophysiology of ischemic stroke, and causes oxidative damage to biomolecules, eventually leading to programmed cell death. Meanwhile, apigenin has been shown to exhibit antioxidant, anti-inflammatory, anti-cancer properties and neuroprotective action. Hence, this study was to investigate the potential mechanisms underlying the neural protection of apigenin on oxygen and glucose deprivation/reperfusion (OGD/R) induced neuronal injury in differentiated PC12 cells. Cells were pretreated with apigenin for 6 h, and then subjected to OGD for 12 h followed by reperfusion for 24 h. The results showed that OGD/R significantly decreased cell viability, mitochondrial membrane potential, mRNA levels of antioxidant and detoxifying enzymes and Nrf2 protein expression, while elevated the release of LDH, cell apoptosis, intracellular ROS level, P53 protein expression and upregulated its downstream genes in PC12 cells. However, apigenin effectively inhibited these undesirable changes induced by OGD/R. Our findings demonstrate that this compound attenuates OGD/R induced neuronal injury mainly by virtue of its anti-apoptosis and antioxidative properties via affecting the expression of Nrf2 and P53, and their downstream target gene transcription.

Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors.

Human embryonic stem cells (hESCs) hold enormous promise for regenerative medicine. Typically, hESC-based applications would require their in vitro differentiation into a desirable homogenous cell population. A major challenge of the current hESC differentiation paradigm is the inability to effectively capture and, in the long-term, stably expand primitive lineage-specific stem/precursor cells that retain broad differentiation potential and, more importantly, developmental stage-specific differentiation propensity. Here, we report synergistic inhibition of glycogen synthase kinase 3 (GSK3), transforming growth factor ß (TGF-ß), and Notch signaling pathways by small molecules can efficiently convert monolayer cultured hESCs into homogenous primitive neuroepithelium within 1 wk under chemically defined condition. These primitive neuroepithelia can stably self-renew in the presence of leukemia inhibitory factor, GSK3 inhibitor (CHIR99021), and TGF-ß receptor inhibitor (SB431542); retain high neurogenic potential and responsiveness to instructive neural patterning cues toward midbrain and hindbrain neuronal subtypes; and exhibit in vivo integration. Our work uniformly captures and maintains primitive neural stem cells from hESCs.

DNp73 improves generation efficiency of human induced pluripotent stem cells.

BACKGROUND: Recent studies have found that p53 and its' associated cell cycle pathways are major inhibitors of human induced pluripotent stem (iPS) cell generation. In the same family as p53 is p73, which shares sequence similarities with p53. However, p73 also has distinct properties of its own, such as two alternative promoters to express transactivation of p73 (TAp73) and N terminal deleted p73 (DNp73). Functionally, TAp73 acts similarly to p53 in tumor suppression. However, DNp73, on the other hand acts as an oncogene to suppress p53 and p73 induced apoptosis. Therefore, how can p73 have opposing roles in human iPS cell generation? RESULTS: Transcription factors, Oct4, Sox2, Klf4 and cMyc (4TF, Yamanaka factors) are used as basal conditions to generate iPS cells. In addition, the factor of DNp73(actually alpha splicing DNp73, DNp73α) is used to generate iPS cells. The experiment found that the addition of DNp73 gene increases human iPS cell generation efficiency by 12.6 folds in comparison to human fibroblast cells transduced with only the basal conditions. Also, iPS cells generated with DNp73 expression are more resistant to in vitro and in vivo differentiation. CONCLUSIONS: This study found DNp73, a family member of p53, is also involved in the human iPS cell generation. Specifically, that the involvement of DNp73 generates iPS cells that are more resistant to in vitro and in vivo differentiation. Therefore, this data may prove to be useful in future developmental studies and cancer researches.

Critical roles of the immunoglobulin intronic enhancers in maintaining the sequential rearrangement of IgH and Igk loci.

V(D)J recombination of immunoglobulin (Ig) heavy (IgH) and light chain genes occurs sequentially in the pro- and pre-B cells. To identify cis-elements that dictate this order of rearrangement, we replaced the endogenous matrix attachment region/Igk intronic enhancer (MiE(kappa)) with its heavy chain counterpart (Emu) in mice. This replacement, denoted EmuR, substantially increases the accessibility of both V(kappa) and J(kappa) loci to V(D)J recombinase in pro-B cells and induces Igk rearrangement in these cells. However, EmuR does not support Igk rearrangement in pre-B cells. Similar to that in MiE(kappa)(-/-) pre-B cells, the accessibility of V(kappa) segments to V(D)J recombinase is considerably reduced in EmuR pre-B cells when compared with wild-type pre-B cells. Therefore, Emu and MiE(kappa) play developmental stage-specific roles in maintaining the sequential rearrangement of IgH and Igk loci by promoting the accessibility of V, D, and J loci to the V(D)J recombinase.

A chemical platform for improved induction of human iPSCs.

The slow kinetics and low efficiency of reprogramming methods to generate human induced pluripotent stem cells (iPSCs) impose major limitations on their utility in biomedical applications. Here we describe a chemical approach that dramatically improves (200-fold) the efficiency of iPSC generation from human fibroblasts, within seven days of treatment. This will provide a basis for developing safer, more efficient, nonviral methods for reprogramming human somatic cells.

p53 induces differentiation of mouse embryonic stem cells by suppressing Nanog expression.

The tumour suppressor p53 becomes activated in response to upstream stress signals, such as DNA damage, and causes cell-cycle arrest or apoptosis. Here we report a novel role for p53 in the differentiation of mouse embryonic stem cells (ESCs). p53 binds to the promoter of Nanog, a gene required for ESC self-renewal, and suppresses Nanog expression after DNA damage. The rapid down-regulation of Nanog mRNA during ESC differentiation correlates with the induction of p53 transcriptional activity and Ser 315 phosphorylation. The importance of Ser 315 phosphorylation was revealed by the finding that induction of p53 activity is impaired in p53(S315A) knock-in ESCs during differentiation, leading to inefficient suppression of Nanog expression. The decreased inhibition of Nanog expression in p53(S315A) ESCs during differentiation is due to an impaired recruitment of the co-repressor mSin3a to the Nanog promoter. These findings indicate an alternative mechanism for p53 to maintain genetic stability in ESCs, by inducing the differentiation of ESCs into other cell types that undergo efficient p53-dependent cell-cycle arrest and apoptosis.

Important roles for E protein binding sites within the immunoglobulin kappa chain intronic enhancer in activating Vkappa Jkappa rearrangement.

The immunoglobulin kappa light chain intronic enhancer (iEkappa) activates kappa rearrangement and is required to maintain the earlier or more efficient rearrangement of kappa versus lambda (lambda). To understand the mechanism of how iEkappa regulates kappa rearrangement, we employed homologous recombination to mutate individual functional motifs within iE(kappa) in the endogenous kappa locus, including the NF-kappaB binding site (kappaB), as well as kappaE1, kappaE2, and kappaE3 E boxes. Analysis of the impacts of these mutations revealed that kappaE2 and to a lesser extent kappaE1, but not kappaE3, were important for activating kappa rearrangement. Surprisingly, mutation of the kappaB site had no apparent effect on kappa rearrangement. Comparable to the deletion of the entire iEkappa, simultaneous mutation of kappaE1 and kappaE2 reduces the efficiency of kappa rearrangement much more dramatically than either kappaE1 or kappaE2 mutation alone. Because E2A family proteins are the only known factors that bind to these E boxes, these findings provide unambiguous evidence that E2A is a key regulator of kappa rearrangement.

Generation of human-induced pluripotent stem cells in the absence of exogenous Sox2.

Induced pluripotent stem cell technology has attracted enormous interest for potential application in regenerative medicine. Here, we report that a specific glycogen synthase kinase 3 (GSK-3) inhibitor, CHIR99021, can induce the reprogramming of mouse embryonic fibroblasts transduced by only two factors, Oct4 and Klf4. When combined with Parnate (also named tranylcypromine), an inhibitor of lysine-specific demethylase 1, CHIR99021 can cause the reprogramming of human primary keratinocyte transduced with the two factors, Oct4 and Klf4. To our knowledge, this is the first time that human iPS cells have been generated from somatic cells without exogenous Sox2 expression. Our studies suggest that the GSK-3 inhibitor might have a general application to replace transcription factors in both mouse and human reprogramming.

Acetylation of mouse p53 at lysine 317 negatively regulates p53 apoptotic activities after DNA damage.

Posttranslational modifications of p53, including phosphorylation and acetylation, play important roles in regulating p53 stability and activity. Mouse p53 is acetylated at lysine 317 by PCAF and at multiple lysine residues at the extreme carboxyl terminus by CBP/p300 in response to genotoxic and some nongenotoxic stresses. To determine the physiological roles of p53 acetylation at lysine 317, we introduced a Lys317-to-Arg (K317R) missense mutation into the endogenous p53 gene of mice. p53 protein accumulates to normal levels in p53(K317R) mouse embryonic fibroblasts (MEFs) and thymocytes after DNA damage. While p53-dependent gene expression is largely normal in p53(K317R) MEFs after various types of DNA damage, increased p53-dependent apoptosis was observed in p53(K317R) thymocytes, epithelial cells from the small intestine, and cells from the retina after ionizing radiation (IR) as well as in E1A/Ras-expressing MEFs after doxorubicin treatment. Consistent with these findings, p53-dependent expression of several proapoptotic genes was significantly increased in p53(K317R) thymocytes after IR. These findings demonstrate that acetylation at lysine 317 negatively regulates p53 apoptotic activities after DNA damage.

Neuroprotective Effect of Optimized Yinxieling Formula in 6-OHDA-Induced Chronic Model of Parkinson's Disease through the Inflammation Pathway.

Parkinson's disease (PD) is characterized by progressive degeneration of dopaminergic neurons in the substantia nigra (SN)-striatum circuit, which is associated with glial activation and consequent chronic neuroinflammation. Optimized Yinxieling Formula (OYF) is a Chinese medicine that exerts therapeutical effect and antiinflammation property on psoriasis. Our previous study has proven that pretreatment with OYF could regulate glia-mediated inflammation in an acute mouse model of PD induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Given that PD is a chronic degeneration disorder, this study applied another PD animal model induced by striatal injection of 6-hydroxydopamine (6-OHDA) to mimic the progressive damage of the SN-striatum dopamine system in rats. The OYF was administrated in the manner of pretreatment plus treatment. The effects of the OYF on motor behaviors were assessed with the apomorphine-induced rotation test and adjusting steps test. To confirm the effect of OYF on dopaminergic neurons and glia activation in this model, we analyzed the expression of tyrosine hydroxylase (TH) and glia markers, ionized calcium-binding adapter molecule 1 (Iba-1), and glial fibrillary acidic protein (GFAP) in the SN region of the rat PD model. Inflammation-associated factors, including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), IL-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2), were further evaluated in this model and in interferon-γ- (INF-γ-) induced murine macrophages RAW264.7 cells. The results from the in vivo study showed that OYF reversed the motor behavioral dysfunction in 6-OHDA-induced PD rats, upregulated the TH expression, decreased the immunoreactivity of Iba-1 and GFAP, and downregulated the mRNA levels of TNF-α and COX-2. The OYF also trended to decrease the mRNA levels of IL-1ß and iNOS in vivo. The results from the in vitro study showed that OYF significantly decreased the mRNA levels of TNF-α, IL-1ß, IL-6, iNOS, and COX-2. Therefore, this study suggests that OYF exerts antiinflammatory effects, which might be related to the protection of dopaminergic neurons in 6-OHDA-induced chronic neurotoxicity.

Functional analysis of the roles of posttranslational modifications at the p53 C terminus in regulating p53 stability and activity.

Posttranslational modification of the tumor suppressor p53 plays important roles in regulating its stability and activity. Six lysine residues at the p53 C terminus can be posttranslationally modified by various mechanisms, including acetylation, ubiquitination, neddylation, methylation, and sumoylation. Previous cell line transfection studies show that ubiquitination of these lysine residues is required for ubiquitin-dependent degradation of p53. In addition, biochemical and cell line studies suggested that p53 acetylation at the C terminus might stabilize p53 and activate its transcriptional activities. To investigate the physiological functional outcome of these C-terminal modifications in regulating p53 stability and activity, we introduced missense mutations (lysine to arginine) at the six lysine residues (K6R) into the endogenous p53 gene in mouse embryonic stem (ES) cells. The K6R mutation prevents all posttranslational modifications at these sites but conserves the structure of p53. In contrast to conclusions of previous studies, analysis of p53 stability in K6R ES cells, mouse embryonic fibroblasts, and thymocytes showed normal p53 stabilization in K6R cells both before and after DNA damage, indicating that ubiquitination of these lysine residues is not required for efficient p53 degradation. However, p53-dependent gene expression was impaired in K6R ES cells and thymocytes in a promoter-specific manner after DNA damage, indicating that the net outcome of the posttranslational modifications at the C terminus is to activate p53 transcriptional activities after DNA damage.

Curative Anti-Inflammatory Properties of Chinese Optimized Yinxieling Formula in Models of Parkinson's Disease.

Parkinson's disease (PD) is marked by the progressive degeneration of dopaminergic neurons (DAN) accompanied by glial activation. Thus, inhibiting glial activation that occurs during this disease could be an effective method for treating PD. Optimized Yinxieling Formula (OYF), a Chinese medicinal formula, which is used to efficiently treat autoimmune disease psoriasis, has been proved to display potential immunomodulatory effects in inflammation-associated diseases. This study assessed the therapeutic benefits of OYF on glial-mediated neuroinflammation and neuroprotection in PD models in vitro and in vivo. First, the results showed that OYF significantly suppresses LPS-induced proinflammatory cytokine secretion and attenuates the overall inflammatory responses in BV-2 cells. Second, in vivo studies confirm that while the validity of our MPTP-induced PD mouse models possesses activated glia and significant neurobehavioral dysfunction, pretreatment with OYF prevents glial activation and ameliorates movement dysfunction in the MPTP-induced PD mouse models as evaluated by the pole and rotarod tests. Third, transcriptomic analyses were carried out to reveal the underlying molecular mechanism of the OYF treatment. Sixteen pathways were significantly upregulated in the OYF-treated PD model mice, including the cytokine-cytokine receptor interaction, cell adhesion molecules, coagulation, and complement cascades. Fifteen pathways were significantly downregulated in the OYF-treated PD model mice, such as the natural killer cell mediated cytotoxicity, hematopoietic cell lineage, phagosome, and others. These pathways share direct or indirect features of immunomodulation, suggesting that the physiological effects of OYF involve key roles of immune and inflammation regulations. Therefore, we prove that OYF is a useful immunomodulatory formula in developing prevention and treatment methods for neurodegenerative disease PD.

p53 switches off pluripotency on differentiation.

The role of p53 as "a guardian of the genome" has been well established in somatic cells. However, its role in pluripotent stem cells remains much more elusive. Here, we discuss research progress in understanding the role of p53 in pluripotent stem cells and in pluripotent stem cell-like cancer stem cells. The p53 protein, which plays a key role in embryonic stem cells, was first discovered in 2005. Landmark studies of p53-related reprogramming elucidated this protein's importance in induced pluripotent stem cells in 2009. The p53-related safety concerns in pluripotent stem cells have been raised in stem cell-based therapy although the use of iPSCs in therapeutic application is promising. Because cancer stem cells have profiles similar to those of pluripotent stem cells, we also describe potential strategies for studies in cancer stem cells and cancer treatments. The new discoveries of p53 family proteins in pluripotent stem cells have made possible stable progress in stem cell transplantation efficiency and safety, as well as treatment strategies targeting cancer stem cells based on pluripotent stem cell technology.

Stem Cells for Modeling and Therapy of Parkinson's Disease.

Parkinson's disease (PD) is the second most frequent neurodegenerative disease after Alzheimer's disease, which is characterized by a low level of dopamine being expressing in the striatum and a deterioration of dopaminergic neurons (DAn) in the substantia nigra pars compacta. Generation of PD-derived DAn, including differentiation of human embryonic stem cells, human neural stem cells, human-induced pluripotent stem cells, and direct reprogramming, provides an ideal tool to model PD, creating the possibility of mimicking key essential pathological processes and charactering single-cell changes in vitro. Furthermore, thanks to the understanding of molecular neuropathogenesis of PD and new advances in stem-cell technology, it is anticipated that optimal functionally transplanted DAn with targeted correction and transgene-free insertion will be generated for use in cell transplantation. This review elucidates stem-cell technology for modeling PD and offering desired safe cell resources for cell transplantation therapy.

[Experimental study on protective effects of curcumin on exaggerated extracellular matrix accumulation of pulmonary fibrosis rats].

OBJECTIVE: To study the protective effects of curcumin on exaggerated extracellular matrix accumulation of pulmonary fibrosis rats. METHOD: One hundred and forty-four male Sprague-Dawley rats were randomly divided into 6 groups (24 rats in each group). Rats in the model control group, positive medicine group, and high, moderate and low curcumin groups were injected with a single dose of bleomycin by trachea, and rats in sham-model control group with same volume normal saline. One day after the injection, curcumin solution of different dosages (200, 100, 50 mg x kg(-1) x d(-1)) was respectively given to rats in the high, moderate and low curcumin group daily by gastrogavage, while equal volume of normal saline was given to those in the sham-model control group and model control group, and an equal volume of prednisone (0.56 mg x kg(-1) x d(-1)) was given to those in positive medicine control group. On the 7, 14, 28 days, 8 rats per treatment group were randomly killed, the levels of III-collagen, IV-collagen, laminin and hyaluronic acid in the serum were determined, the determination of hydroxyproline in lung homogenates was analyzed, and the lung was incised to make pathological sections which were stained with HE and Mallory. RESULT: Curcumin could decreas the levels of III-collagen, IV-collagen, laminin and hyaluronic acid in the serum, and inhihit the proliferation of fibrous tissue. CONCLUSION: Curcumin may play its therapetuic role by leveling down the content of extracellular matrix in rats with pulmonary fibrosis induced by bleomycin.

Stem Cell Therapy and Immunological Rejection in Animal Models.

With their capability to undergo unlimited self-renew and to differentiate into various functional cells, human pluripotent stem cells, including embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), hold great promise in regenerative medicine to treat currently incurable diseases. Significant progress has been achieved in differentiating pluripotent stem cells into various functional cells, such as pancreatic ß cells, neural cells, hepatocytes, and cardiomyocytes. In addition, three hESC-based therapies to treat spinal cord injury, macular degeneration and type 1 diabetes have entered clinical trial. However, there remain several major bottlenecks that hinder the clinical trial of stem cell based therapy. One such key challenge is the immune rejection of cells derived from allogeneic hESCs. The challenge of immune rejection is mitigated by recent discovery of iPSCs, raising the hope that patient-specific hiPSCs can be differentiated into autologous cells for transplantation into the same patient without the concern of immune rejection. However, due to the oncogenic potential of the reprogramming factors and the reprogramming- induced DNA damage, there remain safety concerns about the cancer risk and immunogenicity of hiPSC-derived cells. This review discusses recent progress in our understanding of the immunogenicity of pluripotent stem cells and the development of new strategies to resolve this challenge.

Reprogramming with Small Molecules instead of Exogenous Transcription Factors.

Induced pluripotent stem cells (iPSCs) could be employed in the creation of patient-specific stem cells, which could subsequently be used in various basic and clinical applications. However, current iPSC methodologies present significant hidden risks with respect to genetic mutations and abnormal expression which are a barrier in realizing the full potential of iPSCs. A chemical approach is thought to be a promising strategy for safety and efficiency of iPSC generation. Many small molecules have been identified that can be used in place of exogenous transcription factors and significantly improve iPSC reprogramming efficiency and quality. Recent studies have shown that the use of small molecules results in the generation of chemically induced pluripotent stem cells from mouse embryonic fibroblast cells. These studies might lead to new areas of stem cell research and medical applications, not only human iPSC by chemicals alone, but also safe generation of somatic stem cells for cell based clinical trials and other researches. In this paper, we have reviewed the recent advances in small molecule approaches for the generation of iPSCs.

An Efficient Extraction Method for Fragrant Volatiles from Jasminum sambac (L.) Ait.

The sweet smell of aroma of Jasminum sambac (L.) Ait. is releasing while the flowers are blooming. Although components of volatile oil have been extensively studied, there are problematic issues, such as low efficiency of yield, flavour distortion. Here, the subcritical fluid extraction (SFE) was performed to extract fragrant volatiles from activated carbon that had absorbed the aroma of jasmine flowers. This novel method could effectively obtain main aromatic compounds with quality significantly better than solvent extraction (SE). Based on the analysis data with response surface methodology (RSM), we optimized the extraction conditions which consisted of a temperature of 44°C, a solvent-to-material ratio of 3.5:1, and an extraction time of 53 min. Under these conditions, the extraction yield was 4.91%. Furthermore, the key jasmine essence oil components, benzyl acetate and linalool, increase 7 fold and 2 fold respectively which lead to strong typical smell of the jasmine oil. The new method can reduce spicy components which lead to the essential oils smelling sweeter. Thus, the quality of the jasmine essence oil was dramatically improved and yields based on the key component increased dramatically. Our results provide a new effective technique for extracting fragrant volatiles from jasmine flowers.