Recent success in the derivation of haploid embryonic stem cells (haESCs) from mouse via parthenogenesis and androgenesis has enabled genetic screening in mammalian cells and generation of gene-modified animals. However, whether haESCs can be derived from primates remains unknown. Here, we report the derivation of haESCs from parthenogenetic blastocysts of Macaca fascicularis monkeys. These cells, termed as PG-haESCs, are pluripotent and can differentiate to cells of three embryonic germ layers in vitro or in vivo. Interestingly, the haploidy of one monkey PG-haESC line (MPH1) is more stable compared with that of the other one (MPH2), as shown by the existence of haploid cells for more than 140 days without fluorescence-activated cell sorting (FACS) enrichment of haploid cells. Importantly, transgenic monkey PG-haESC lines can be generated by lentivirus- and piggyBac transposon-mediated gene transfer. Moreover, genetic screening is feasible in monkey PG-haESCs. Our results demonstrate that PG-haESCs can be generated from monkeys, providing an ideal tool for genetic analyses in primates.
embryonic stem cell; haploid cells; monkey
In HIV patients, antiretroviral medications trigger metabolic abnormalities, including insulin resistance. In addition, the inflammatory cytokine tumor necrosis factor-α (TNFα), which is elevated in human immunodeficiency virus encephalitis (HIVE), also induces insulin resistance and inflicts neuronal damage in vitro. In differentiated PC12 cells and rat cortical neurons, high glucose (HG; 25 mM) triggers reactive oxygen species (ROS) accumulation, contributing to the retraction of neuronal processes, with only a minimal involvement of neuronal apoptosis. In the presence of TNFα, HG-treated neurons undergo massive apoptosis. Because mammalian homolog of the Forkhead family of transcription factors, Forkhead box O transcription factor 3a (FOXO3a), controls ROS metabolism, we asked whether FOXO3a could affect the fate of differentiated neurons in the paradigm of HIVE. We observed FOXO3a nuclear translocation in HG-treated neuronal cultures, accompanied by partial loss of mitochondrial potential and gradual retraction of neuronal processes. Addition of TNFα to HG-treated neurons increased expression of the FOXO-dependent proapoptotic gene Bim, which resulted in extensive apoptotic death. Insulin-like growth factor-I (IGF-I) significantly lowered intracellular ROS, which was accompanied by IGF-I-mediated FOXO3a nuclear export and decrease in its transcriptional activity. The clinical relevance of these findings is supported by detection of nuclear FOXO3a in TUNEL-positive cortical neurons from HIVE, especially in brain areas characterized by elevated TNFα.
IGF-I; FOXO3a; ROS; neuronal apoptosis; HIV encephalitis
MiRNA primarily acts to repress gene expression at the post-transcriptional level through imperfect complementarity of its 5′ region to the “seed site” in the 3′ untranslated region of target mRNAs, with its “3′–supplementary site” and “center site” also playing important roles under certain circumstances. The aim of this study was to test if artificial miRNA mimics (miR-Mimics) that are designed to target the “centered sites” without “seed sites” complementarity are able to repress gene expression as natural miRNAs.
We designed miR-Mimics carrying centered-site matches (CS–miR-Mimics) or seed-site matches (SS–miR-Mimics) and siRNA to two antiapoptotic genes BCL2 and AKT1. We tested the gene targeting of these constructs using real-time RT-PCR and Western blot to quantify mRNA and protein levels of BCL2 and AKT1, respectively, luciferase reporter gene assay to investigate the interaction between miR-Mimics and their target sites, and cell survival assay to study the functional outcomes of the miR-Mimics.
We found that CS-miR-Mimic, SS-miR-Mimic and siRNA, all down regulated the mRNA and protein levels of their cognate target BCL2 or AKT1 in a concentration-dependent manner. Luciferase reporter gene assay further confirmed the functional interactions of CS–miR-Mimic, SS-miR-Mimic and siRNA with their target sites. We then observed that the miR-Mimics and siRNAs were all able to induce cell death, as indicated by the reduced survival rate of cells.
We have provided evidence for the feasibility of CS–miR-Mimics for post-transcriptional repression of genes, which can be designed to have reduced numbers of seed type off-target sites compared to the number of target sites from an average endogenous seed–site miRNA. CS–miR-Mimics may be a novel approach for miRNA research requiring miRNA gain-of-function.
Tau aggregation and amyloidogenesis are common hallmarks for neurodegenerative disorders called tauopathies. The molecular chaperone network constitutes the cellular defense against insults such as tau aggregation. However, chaperone effects on tau are dichotomous. Loss of tau’s microtubule-binding activity facilitates an inappropriate chaperone interaction that promotes an amyloidogenic tau conformation. Conversely, other chaperones are capable of promoting tau clearance. Here, we demonstrate that a critical contributor to tau triage is the DnaJ-binding domain of Hsp70 proteins. In particular, over-expression of the constitutive DnaJ, DnaJA1, mediated tau clearance, while knockdown facilitated tau accumulation. This clearance was not specific to distinct pathogenic tau species. The activity of DnaJA1 was attenuated by concomitant increases in Hsp70. Tau reductions facilitated by DnaJA1 were dependent on the integrity of lysines known to be poly-ubiquitinated in human Alzheimer’s brain. In vivo, DnaJA1 and tau levels were inversely correlated. The effects of DnaJA1 were partially specific: DnaJA1 reduced the levels of a polyQ protein but had no significant effect on α-synuclein levels.
These data suggest that DnaJA1 triages all tau species for ubiquitin-dependent clearance mechanisms. Moreover, the levels of DnaJA1 and Hsp70 seem to play against each other with regard to tau: as DnaJA1 levels increase, tau levels are reduced, but this can be prevented if Hsp70 levels are simultaneously induced. Thus, the DnaJ repertoire possibly represents a powerful set of genetic modifiers for tau pathogenesis. Further investigations, could provide new insights about triage decisions that facilitate or prevent amyloidogenesis of tau and other proteins associated with neurodegenerative disease.
High shear force critically regulates platelet adhesion and thrombus formation during ischemic vascular events. To identify genetic factors that influence platelet thrombus formation under high shear stress, we performed a genome-wide association study (GWAS) and confirmatory experiments in human and animal platelets.
Methods and Results
Closure times in the shear-dependent Platelet Function Analyzer (PFA)-100® were measured on healthy, non-diabetic European Americans (n=125) and African Americans (n=116). A GWAS significant association (p<5X10−8) was identified with 2 SNPs within the SVIL gene (chr 10p11.23) in African-Americans but not European Americans. Microarray analyses of human platelet RNA demonstrated the presence of SVIL isoform 1 (supervillin) but not muscle-specific isoforms 2 and 3 (archvillin, SmAV). SVIL mRNA levels were associated with SVIL genotypes (p≤0.02) and were inversely correlated with PFA-100 closure times (p<0.04) and platelet volume (p<0.02). Leukocyte-depleted platelets contained abundant levels of the ~205 kD supervillin polypeptide. To assess functionality, mice lacking platelet supervillin were generated and back-crossed onto a C57BL/6 background. Compared to controls, murine platelets lacking supervillin were larger by flow cytometry and confocal microscopy, and exhibited enhanced platelet thrombus formation under high shear, but not low shear, conditions.
We show for the first time that 1) platelets contain supervillin, 2) platelet thrombus formation in the PFA-100® is associated with human SVIL variants and low SVIL expression, and 3) murine platelets lacking supervillin exhibit enhanced platelet thrombus formation at high shear stress. These data are consistent with an inhibitory role for supervillin in platelet adhesion and arterial thrombosis.
genetics; genome-wide association study; murine model; platelets; thrombosis
Existence of inert biomass and its impact on biomass accumulation patterns and biofilter performance were investigated. Four biofilters were set up in parallel to treat gaseous toluene. Each biofilter operated under different inlet toluene loadings for 100 days. Two microbial growth models, one with an inert biomass assumption and the other without, were established and compared. Results from the model with the inert biomass assumption showed better agreement with the experimental data than those based on the model without the inert biomass assumption thus verifying that inert biomass accumulation cannot be ignored in the long-term operation of biofilters. According to the model with an inert biomass assumption, the ratio of active biomass to total biomass will decrease and the inert biomass will become dominant in total biomass after a period of time. Filter bed structure simulation results showed that the void fraction is more sensitive to biomass accumulation than the specific surface area. The final void fraction of the biofilters with the highest inlet toluene loading is only 67% of its initial level while the final specific surface area is 82%. Identification and quantification of inert biomass will give a better understanding of biomass accumulation in biofilters and will result in a more exact simulation of biomass change during long-term operations. Results also indicate that an ideal biomass control technique should be able to remove most inert biomass while simultaneously preserving as much active biomass as possible.
biofilter; microbial growth model; inert biomass; biomass control
Patients with stage I non-small cell lung cancer who have undergone complete surgical resection harbor a 30% risk for tumor recurrence. Thus, the identification of factors that are predictive for tumor recurrence is urgently needed. The aim of this study was to test the prognostic value of serum albumin levels on tumor recurrence in patients with stage I non-small cell lung cancer.
Stage I non-small cell lung cancer patients who underwent complete surgical resection of the primary tumor at Zhejiang Hospital were analyzed in this study. Serum albumin levels were measured before surgery and once again after surgery in 101 histologically diagnosed non-small cell lung cancer patients. Correlations between the pre- and post-operative serum albumin levels and various clinical demographics and recurrence-free survival rates were analyzed.
Patients with pre-operative hypoalbuminemia (<3.5 g/dl) had a significantly worse survival rate than patients with normal pre-operative serum albumin levels (≥3.5 g/dl) (p = 0.008). Patients with post-operative hypoalbuminemia had a worse survival rate when compared with patients with normal post-operative serum albumin levels (p = 0.001). Cox multivariate analysis identified pre-operative hypoalbuminemia, post-operative hypoalbuminemia and tumor size over 3 cm as independent negative prognostic factors for recurrence.
Serum albumin levels appear to be a significant independent prognostic factor for tumor recurrence in patients with stage I non-small cell lung cancer who have undergone complete resection. Patient pre-treatment and post-treatment serum albumin levels provide an easy and early means of discrimination between patients with a higher risk for recurrence and patients with a low risk of recurrence.
Prognostic Impact; Recurrence-Free Survival; Stage I Non-Small Cell Lung Cancer; Serum Albumin Level
Hyperlipidemia may lead to endothelial injury, due to its effects on homocysteine and vascular endothelial growth factor in the serum, and the mRNA expression levels of peroxisome proliferator-activated receptor-γ (PPARγ), and caspase-3 and -8 in the vascular wall. In order to prevent and mitigate the high-fat state that results from endothelial injury, this study examined the effect of folic acid (FA) and vitamin B12 (VB12) on the expression of PPARγ and caspase-3 and -8 mRNA in the abdominal aortas of rats with hyperlipidemia. Sixty 4-week-old healthy male Sprague Dawley rats were randomly divided into five groups (each n=12): the normal control (NC), high-fat diet (HL), FA, VB12 and FA+VB12 groups. Following one week of adaptive feeding, the FA, VB12 and FA+VB12 groups were subject to the intraperitoneal injection of FA (0.5 mg/day), VB12 (0.05 mg/day) and FA+VB12 (0.5 mg/day and 0.05 mg/day), respectively, while fed a high-fat diet. The rats in the NC group were injected intraperitoneally with 0.9% NaCl solution (0.5 ml/day) and fed a normal diet, whereas those in the HL group were fed a high-fat diet only. A reverse transcription-polymerase chain reaction (RT-PCR) assay demonstrated that at the end of week 12, the FA treatment had effectively increased the PPARγ mRNA level, while reducing the caspase-3 and -8 mRNA levels, compared with the high-fat diet treatment (P<0.05). The effect of FA on the expression of PPARγ and caspase-3 and -8 was enhanced when used in combination with VB12 (P<0.05). These results revealed that the application of FA, alone or in combination with VB12, improves and mitigates the high-fat state that results from endothelial injury.
hyperlipidemia; PPARγ; caspase-8; caspase-3; folic acid
Tumor formation constitutes a major obstacle to the clinical application of embryonic stem cell–derived (ESC-derived) cells. In an attempt to find major extracellular signaling and intrinsic factors controlling tumorigenicity and therapeutic functionality of transplanted ESC-derived retinal progenitor cells (ESC-RPCs), we evaluated multiple kinds of ESC-RPCs in a mouse retinal degeneration model and conducted genome-wide gene expression profiling. We identified canonical WNT signaling as a critical determinant for the tumorigenicity and therapeutic function of ESC-RPCs. The function of WNT signaling is primarily mediated by TCF7, which directly induces expression of Sox2 and Nestin. Inhibition of WNT signaling, overexpression of dominant-negative Tcf7, and silencing Tcf7, Sox2, or Nestin all resulted in drastically reduced tumor formation and substantially improved retinal integration and visual preservation in mice. These results demonstrate that the WNT signaling cascade plays a critical role in modulating the tumorigenicity and functionality of ESC-derived progenitors.
Substantia nigra pars reticulata (SNr) is a key basal ganglia output nucleus critical for movement control. Its γ-aminobutyric acid (GABA)-containing projection neurons intermingle with nigral dopamine (DA) neuron dendrites. Here we show that SNr GABA neurons co-express dopamine D1 and D5 receptor mRNAs and also mRNA for TRPC3 channels. Dopamine induced an inward current in these neurons and increased their firing frequency. These effects were mimicked by D1-like agonists, blocked by a D1-like antagonist. D1-like receptor blockade reduced SNr GABA neuron firing frequency and increased their firing irregularity. These D1-like effects were absent in D1 or D5 receptor knockout mice and inhibited by intracellularly applied D1 or D5 receptor antibody. These D1-like effects were also inhibited when the tonically active TRPC3 channels were inhibited by intracellularly applied TRPC3 channel antibody. Furthermore, stimulation of DA neurons induced a direct inward current in SNr GABA neurons that was sensitive to D1-like blockade. Manipulation of DA neuron activity and DA release and inhibition of dopamine reuptake affected SNr GABA neuron activity in a D1-like receptor-dependent manner. Taken together, our findings indicate that dendritically released dopamine tonically excites SNr GABA neurons via D1-D5 receptor co-activation that enhances constitutively active TRPC3 channels, forming an ultra-short SNc→SNr dopamine pathway that regulates the firing intensity and pattern of these basal ganglia output neurons.
basal ganglia; dopamine receptor; spontaneous firing; substantia nigra pars reticulata; TRP channel; Parkinson’s disease
Only B lymphocytes can express immunoglobulins according to the traditional immunological theories, and the expression of immunoglobulin G (IgG) messenger RNA (mRNA) and protein was found in certain human cancer cells recently. However, the expression pattern of IgG and its possible role in human urothelial carcinoma are still elusive. In this study, we investigated the expression of IgG in two human urothelial carcinoma cell lines, T24 and BIU-87, and in 56 cases of clinical urothelial carcinoma tissues. The mRNA of IgG was positively detected by in situ hybridization and reverse transcription PCR; furthermore, IgG protein was also positively detected by immunohistochemistry and Western blot. Moreover, blockade of tumor-derived IgG by either antihuman IgG antibody or antisense oligonucleotides increased cell apoptosis and inhibited cell growth in bladder cancer cell lines in vitro, and antihuman IgG antibody could suppress the growth of xenotransplant tumor in vivo. In addition, either antihuman IgG antibody or antisense oligonucleotides enhanced the sensitivity to mitomycin C in bladder cancer cell line T24. Furthermore, blockade of IgG in bladder cancer cell T24 resulted in upregulation of cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase. Our results indicated that bladder cancer cells were capable of expressing IgG, and blockade of IgG expression induced cell apoptosis through activation of caspase-dependent pathway. A novel potential targeted therapy for bladder cancer will be possibly developed based on these data.
Electronic supplementary material
The online version of this article (doi:10.1007/s13277-013-0717-z) contains supplementary material, which is available to authorized users.
Immunoglobin G; Antisense oligonucleotides; Urothelial carcinoma; Mitomycin C; Apoptosis
MicroRNAs (miRNAs), a class of highly conserved small non-coding RNA molecules, are known to play essential roles in central nervous system (CNS) by causing post-transcriptional gene silencing. There is much evidence that miRNAs have specific temporal and spatial expression patterns in the mammal brain, but little is known about the role of the region specificity for the gene regulatory networks of the brain. This study represents the first attempt to perform a profiling analysis of the differential expression of miRNAs between hippocampus and the Marginal division (MrD) of the neostriatum in the rat brain.
Microarray was used to detect the expression of 357 miRNAs in hippocampus and the MrD from three rats. A short-list of the most dysregulated 30 miRNAs per rat was generated for data analysis, and the miRNAs that were represented in two or three short-lists were then further analyzed. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) was employed to validate the aberrantly expressed miRNAs obtained from the miRNA microarray analysis. A family of 11 miRNAs demonstrated differential expression between the MrD and hippocampus in more than one rat. Amongst these, miR-383 was differentially expressed in all three rats and up-regulated to the largest degree in rat one, and the ten other miRNAs, let-7d*, miR-181b, miR-187, miR-195, miR-214, miR-382, miR-411, miR-466b, miR-592 and miR-1224 were differentially expressed in at least two rats. Of these ten, besides miR-382 and miR-411 which were up-regulated in one rat and down-regulated in another, the other eight miRNAs retained a uniform direction of regulation (up-regulation or down-regulation) between different specimens. When further examined by RT-PCR, the aberrantly expressed miRNAs, except miR-383 and let-7d*, demonstrated differential expression that significantly correlated with the microarray findings.
This study reported that the miRNA expression patterns in MrD was distinct from that of Hip, suggesting the role of miRNAs in the learning and memory function of the MrD probably different from hippocampus.
mRNA transcript; Oligo-microarray; Gene silence; Synapse formation; Learning and memory
A phase III clinical trial has already shown the survival benefits of postoperative chemotherapy in gastric cancer. However, there are limited published data concerning the elderly. This study aims to investigate the use of adjuvant chemotherapy for gastric cancer after D2 gastrectomy among the elderly and identify its impact on survival.
We retrospectively reviewed 360 patients who had undergone D2 gastrectomy, aged 65 years or older, with non-metastatic gastric cancer in a single institution. We analyzed the predictors and survival benefits of adjuvant chemotherapy use in the elderly. Further, we analyzed the survival benefits of adjuvant chemotherapy by dividing the patients into groups according to disease stages and chemotherapeutic regimens.
Among the 360 patients, only 34.7% of patients received adjuvant chemotherapy. Age, tumor location, lymph node involvement and tumor invasion were associated with the receipt of adjuvant chemotherapy. Adjuvant chemotherapy improved the overall survival for non-metastatic elderly patients (HR 0.60, 95%CI 0.42–0.83, P = 0.003). Significant survival benefits were found with adjuvant chemotherapy in stage III patients (HR 0.67, 95%CI 0.47–0.97, P = 0.033), but not in stage I patients or in stage II patients (HR 0.52, 95%CI 0.21–1.30 P = 0.161). Compared to adjuvant chemotherapy without platinum, no significant survival benefits were observed with platinum-containing chemotherapy (HR 0.84, 95%CI 0.49–1.45, P = 0.530). Besides adjuvant chemotherapy, other independent prognostic factors of survival included tumor location, tumor size, histologic grade, depth of tumor invasion, and lymph node status.
This study demonstrated the survival benefits of adjuvant fluoropyrimidine-based chemotherapy among the elderly patients with non-metastatic gastric cancer after D2 gastrectomy. However, due to the limitations of this study, further well-designed prospective studies with large populations are needed to confirm these findings and identify the patients that can tolerate and benefit from adjuvant chemotherapy.
Tumor necrosis factor-α (TNFα) released in the brain by HIV-activated macrophages/microglia is suspected to compromise neuronal survival. Previously, we have demonstrated that activated receptor for insulin-like growth factor I (IGF-IR) protects neurons from TNFαinduced neuronal damage (Wang et al.  J. Neurosci. Res. 83:7–18). Because TNFα triggers phosphorylation of insulin receptor substrate 1 (IRS-1) on serine residues (pS-IRS-1; Rui et al.  J. Clin. Invest. 107:181–189), and pS-IRS-1 binds integrins (Reiss et al.  Oncogene 20:490–500), we asked how these events affect neuronal processes. We show that β1-integrin and pS-IRS-1 colocalize in PC12 cells and in primary cortical neurons. TNFα treatment elevated membrane-associated pS-IRS-1, enhanced pSIRS-1 interaction with β1-integrin, and attenuated cell attachment to collagen IV. In contrast, IGF-I inhibited pS-IRS-1–β1-integrin complexes and improved cell attachment. The domain of IRS-1 involved in β1-integrin binding mapped between amino acids 426 and 740, and the expression of 426–740/IRS-1 mutant attenuated neuronal outgrowth. Our results indicate that TNFα facilitates the interaction of pS-IRS-1 and β1-integrin and destabilizes neuronal processes. IGF-I counteracts TNFα-mediated accumulation of pS-IRS-1–β1-integrin complexes supporting the stability of neuronal processes.
IRS-1; integrins; TNFα; IGF-I; neuronal damage
Generation of induced pluripotent stem cells (iPSCs) has opened new avenues for the investigation of heart diseases, drug screening and potential autologous cardiac regeneration. However, their application is hampered by inefficient cardiac differentiation, high interline variability, and poor maturation of iPSC-derived cardiomyocytes (iPS-CMs). To identify efficient inducers for cardiac differentiation and maturation of iPSCs and elucidate the mechanisms, we systematically screened sixteen cardiomyocyte inducers on various murine (m) iPSCs and found that only ascorbic acid (AA) consistently and robustly enhanced the cardiac differentiation of eleven lines including eight without spontaneous cardiogenic potential. We then optimized the treatment conditions and demonstrated that differentiation day 2-6, a period for the specification of cardiac progenitor cells (CPCs), was a critical time for AA to take effect. This was further confirmed by the fact that AA increased the expression of cardiovascular but not mesodermal markers. Noteworthily, AA treatment led to approximately 7.3-fold (miPSCs) and 30.2-fold (human iPSCs) augment in the yield of iPS-CMs. Such effect was attributed to a specific increase in the proliferation of CPCs via the MEK-ERK1/2 pathway by through promoting collagen synthesis. In addition, AA-induced cardiomyocytes showed better sarcomeric organization and enhanced responses of action potentials and calcium transients to β-adrenergic and muscarinic stimulations. These findings demonstrate that AA is a suitable cardiomyocyte inducer for iPSCs to improve cardiac differentiation and maturation simply, universally, and efficiently. These findings also highlight the importance of stimulating CPC proliferation by manipulating extracellular microenvironment in guiding cardiac differentiation of the pluripotent stem cells.
induced pluripotent stem cells; ascorbic acid; cardiomyocytes; cardiac progenitor cells
The novel compound JRS-15 was obtained through the chemical modification of xylocydine. JRS-15 exhibited much stronger cytotoxic and pro-apoptotic activity than its parent compound in various cancer cell lines, with IC50 values in HeLa, HepG2, SK-HEP-1, PC-3M and A549 cells ranging from 12.42 to 28.25 μM. In addition, it is more potent for killing cancer than non-cancerous cells. Mechanistic studies showed that JRS-15 treatment arrested cell cycle at the G1/S phase, which further triggered the translocation of Bax and Bak to the mitochondria, resulting in mitochondrial membrane potential (MMP) depolarization and the subsequent release of cytochrome c and the second mitochondria-derived activator of caspase (Smac). The sequential activation of caspase-9 and caspase-3/7 and the cleavage of poly (ADP-ribose) polymerase (PARP) were observed following these mitochondrial events. Caspase-8, an initiator caspase that is required to activate the membrane receptor-mediated extrinsic apoptosis pathway was not activated in JRS-15-treated cells. Further analysis showed that the levels of the anti-apoptotic proteins Bcl-xL and XIAP were significantly reduced upon JRS-15 treatment. Furthermore, the caspase-9 inhibitor z-LEHD-fmk, the pan-caspase inhibitor z-VAD-fmk, and Bcl-xL or XIAP overexpression all effectively prevented JRS-15-induced apoptosis. Taken together, these results indicate that JRS-15 induces cancer cell apoptosis by regulating multiple apoptosis-related proteins, and this compound may therefore be a good candidate reagent for anticancer therapy.
apoptosis; Bax; Bak; Bcl-xL; cell cycle; cytochrome c; JRS-15; Smac; XIAP
Stroke is a major cause of mortality and the leading cause of permanent disability. In this study, we adopted the classic middle cerebral artery occlusion(MCAO) stroke model to observe the therapeutic effects of coccomyxa gloeobotrydiformis(CGD) on ischemic stroke, and discuss the underlying mechanisms. Low dose (50 mg/kg.day) and high dose (100 mg/kg.day) concentrations of the drug CGD were intragastrically administrated separately for 8 weeks. Infarct volumes, neurologic deficits and degree of stroke-induced brain edema were measured 24 hours after reperfusion. Furthermore, oxidative stress related factors (SOD and MDA), mitochondrial membrane potential, and apoptosis regulatory factors (mitochondrial Cyt-C, Bcl-2, Bax, and caspase-3) were all investigated in this research. We found that CGD attenuated cerebral infarction, brain edema and neurologic deficits; CGD maintained the mitochondrial membrane potential and decreased mitochondrial swelling. It also prevented oxidative damage by reducing MDA and increasing SOD. In addition, CGD could effectively attenuate apoptosis by restoring the level of mitochondrial Cyt C and regulating the expression of Bcl-2, Bax and caspase 3. These results revealed that CGD has a therapeutic effect on ischemic stroke, possibly by inducing mitochondrial protection and anti-apoptotic mechanisms.
Coccomyxa gloeobotrydiformis (CGD); ischemic stroke; apoptosis; mitochondrion
Hypersensitivity diseases are associated with many severe human illnesses, including leprosy and tuberculosis. Emerging evidence suggests that the pathogenesis and pathological mechanisms of treating these diseases may be attributable to sphingolipid metabolism.
High performance liquid chromatography-tandem mass spectrometry was employed to target and measure 43 core sphingolipids in the plasma, kidneys, livers and spleens of BALB/c mice from four experimental groups: control, delayed-type hypersensitivity (DTH) model, DTH+triptolide, and control+triptolide. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to identify potential biomarkers associated with variance between groups. Relationships between the identified biomarkers and disease markers were evaluated by Spearman correlation.
As a treatment to hypersensitivity disease, triptolide significantly inhibit the ear swelling and recover the reduction of splenic index caused by DTH. The sphingolipidomic result revealed marked alterations in sphingolipid levels between groups that were associated with the effects of the disease and triptolide treatment. Based on this data, 23 potential biomarkers were identified by OPLS-DA, and seven of these biomarkers correlated markedly with the disease markers (p<0.05) by Spearman correlation.
These data indicate that differences in sphingolipid levels in plasma and tissues are related to DTH and treatment with triptolide. Restoration of proper sphingolipid levels may attribute to the therapeutic effect of triptolide treatment. Furthermore, these findings demonstrate that targeted sphingolipidomic analysis followed by multivariate analysis presents a novel strategy for the identification of biomarkers in biological samples.
Generalized vitiligo is the most common pigmentation disorder, the result of autoimmune loss of melanocytes from the skin and hair, with a high frequency of other autoimmune diseases in vitiligo patients and their relatives. We previously reported the linkage signals on chromosomes 1, 7, and 17 in Caucasian families with generalized vitiligo and associated autoimmune diseases and identified the risk loci of chromosomes 17 and 1 as NLRP1 (NALP1) and FOXD3, respectively. Here, we describe fine-scale genetic association analyses in two independent series of Caucasian multiplex families, refining localization of the chromosome 7 locus and a locus on chromosome 9. Three susceptibility signals, represented by single-nucleotide polymorphisms (SNPs) rs6960920 in 7p13, rs734930 in 7q11, and rs4744411 in 9q22, were significantly associated with vitiligo and other autoimmune diseases. We also detected significant three-way interaction effects of chromosome 7 SNP rs6960920, chromosome 9 SNP rs4744411, and NLRP1 SNP rs6502867 on both the vitiligo phenotype and an expanded autoimmune disease phenotype, and significant three-way interaction effects of both chromosome 7 SNPs and NLRP1 SNP rs6502867 on the vitiligo phenotype. These support the validity of the chromosomes 7 and 9 linkage/association signals and underscore the utility of gene-gene interaction analysis in characterizing the genetic effects of candidate association signals.
In previous linkage and genome-wide association studies we identified 17 susceptibility loci for generalized vitiligo. By a second genome-wide association study, meta-analysis, and independent replication study, we have now identified 13 additional vitiligo-associated loci, including OCA2-HERC2, a region of 16q24.3 containing MC1R, a region of chromosome 11q21 near TYR, several immunoregulatory loci including IFIH1, CD80, CLNK, BACH2, SLA, CASP7, CD44, IKZF4, SH2B3, and a region of 22q13.2 where the causal gene remains uncertain. Functional pathway analysis shows that most vitiligo susceptibility loci encode immunoregulatory proteins or melanocyte components that likely mediate immune targeting and genetic relationships among vitiligo, malignant melanoma, and normal variation of eye, skin, and hair color.
(De)acetylation of histone and non-histone proteins is an important post-translational modification affecting many cellular processes. Here we report that NuA4 acetylation of Sip2, one of three regulatory β subunits of Snf1 complex (yeast AMP-activated protein kinase), decreases as cells age. We used mutations at four acetylation sites, K12, 16, 17 and 256, to study acetyl-Sip2 function. Sip2 acetylation, controlled by antagonizing NuA4 acetyltransferase and Rpd3 deacetylase, enhances interaction with Snf1, the catalytic subunit of Snf1 complex. Sip2-Snf1 interaction inhibits Snf1 activity, thus decreasing phosphorylation of a downstream target, Sch9 (homolog of Akt/S6K), ultimately leads to slower growth but extends replicative lifespan. Sip2 acetylation mimetics are more resistant to oxidative stress. We further demonstrate that the anti-aging effect of Sip2 acetylation is independent of extrinsic nutrient availability and TORC1 activity. We propose a novel protein acetylation- phosphorylation cascade that regulates Sch9 activity, controls intrinsic aging and extends replicative lifespan in yeast.
Cyclin A-Cdk2, a cell cycle regulated Ser/Thr kinase, plays important roles in a variety of apoptoticprocesses. However, the mechanism of cyclin A-Cdk2 regulated apoptosis remains unclear. Here, we demonstrated that Rad9, a member of the BH3-only subfamily of Bcl-2 proteins, could be phosphorylated by cyclin A-Cdk2 in vitro and in vivo. Cyclin A-Cdk2 catalyzed the phosphorylation of Rad9 at serine 328 in HeLa cells during apoptosis induced by etoposide, an inhibitor of topoisomeraseII. The phosphorylation of Rad9 resulted in its translocation from the nucleus to the mitochondria and its interaction with Bcl-xL. The forced activation of cyclin A-Cdk2 in these cells by the overexpression of cyclin A,triggered Rad9 phosphorylation at serine 328 and thereby promoted the interaction of Rad9 with Bcl-xL and the subsequent initiation of the apoptotic program. The pro-apoptotic effects regulated by the cyclin A-Cdk2 complex were significantly lower in cells transfected with Rad9S328A, an expression vector that encodes a Rad9 mutant that is resistant to cyclin A-Cdk2 phosphorylation. These findings suggest that cyclin A-Cdk2 regulates apoptosis through a mechanism that involves Rad9phosphorylation.
Elevated expression of specific transposable elements (TEs) has been observed in several neurodegenerative disorders. TEs also can be active during normal neurogenesis. By mining a series of deep sequencing datasets of protein-RNA interactions and of gene expression profiles, we uncovered extensive binding of TE transcripts to TDP-43, an RNA-binding protein central to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Second, we find that association between TDP-43 and many of its TE targets is reduced in FTLD patients. Third, we discovered that a large fraction of the TEs to which TDP-43 binds become de-repressed in mouse TDP-43 disease models. We propose the hypothesis that TE mis-regulation contributes to TDP-43 related neurodegenerative diseases.
Melanoma antigen D1 (MAGED1) is a member of the type II melanoma antigen (MAGE) family. The down-regulation of MAGED1 expression has been shown in breast carcinoma cell lines and in glioma stem cells and may play an important role in apoptosis and anti-tumorigenesis. However, there is no report on its clinical role in colorectal cancer (CRC).
We examined the expression of MAGED1 by qPCR in colorectal cancer tissues and their adjacent non-tumorous tissues taken from 6 cases and performed Western blotting and IHC analyses. In addition, we analyzed MAGED1 expression in 285 clinicopathologically characterized colorectal cancer patients.
MAGED1 expression was significantly down-regulated in colorectal cancer tissues compared with adjacent non-tumorous tissues and was associated with clinical stage (p < 0.001), T classification (p = 0.001), N classification (p < 0.001), M classification (p < 0.001) and pathologic differentiation (p = 0.002). Patients with lower MAGED1 expression had a shorter survival time than those with higher MAGED1 expression. Univariate and multivariate analyses indicated that MAGED1 expression was an independent prognostic factors (p < 0.001).
MAGED1 may serve as a novel prognostic biomarker of human colorectal cancer.
MAGED1; Colorectal cancer; Melanoma antigen and prognosis