BACKGROUND AND PURPOSE
Lumbar facet synovial cysts are a cause of back pain and radiculopathy with facet joint degeneration, the most common cause for cyst formation. Typically, LFSCs are T2 hyperintense on MR imaging, but the signal intensity is variable. Treatment options include percutaneous rupture and surgical resection. This study evaluates the relationship between LFSC signal intensity on MR imaging and outcomes as it relates to percutaneous rupture success and need for subsequent surgery.
MATERIALS AND METHODS
A retrospective review of 110 patients who underwent CT fluoroscopic-guided rupture of symptomatic LFSCs was performed. The LFSCs were characterized by their T2 signal intensity on MR imaging and divided into 3 groups: high, intermediate, and low T2 signal intensity. The rates of successful cyst rupture and need for subsequent surgery were recorded.
Percutaneous LFSC rupture was technically successful in 87% of all cases. Cyst rupture was successful in 89% and 90% of high and intermediate signal intensity cysts, respectively, and in 65% of low signal intensity cysts (P = .017, .030). High signal intensity cysts had lower postprocedural surgical rates (29%) when compared with intermediate and low signal cyst as a group (P = .045).
T2 hyperintense and intermediate signal intensity LFSCs are easier to rupture, perhaps because the cysts contain a higher proportion of fluid and are less gelatinous or calcified than T2 hypointense cysts. Patients with T2 hyperintense LFSCs are less likely to need surgery.
Pancreatic cancer is a multiple genetic disorder with many mutations identified during the progression. Two mouse pancreatic cancer cell lines were established which showed different phenotype in vivo: a non-metastatic cell line, Panc02, and a highly metastatic cell line, Panc02-H7, a derivative of Panc02. In order to investigate whether the genetic mutations of key genes in pancreatic cancer such as KRAS, TP53 (p53), CDKN2A (p16), SMAD4, ZIP4, and PDX-1 contribute to the phenotypic difference of these two mouse pancreatic cancer cells, we sequenced the exonic regions of these key genes in both cell lines and in the normal syngeneic mouse pancreas and compared them with the reference mouse genome sequence. The exons of KRAS, SMAD4, CDKN2A (p16), TP53 (p53), ZIP4, and PDX-1 genes were amplified and the genotype of these genes was determined by Sanger sequencing. The sequences were analyzed with Sequencher software. A mutation in SMAD4 was identified in both cell lines. This homozygote G to T mutation in the first position of codon 174 (GAA) generated a stop codon resulting in the translation of a truncated protein. Further functional analysis indicates that different TGF-β/SMAD signaling pathways were involved in those two mouse cell lines, which may explain the phonotypic difference between the two cells. A single nucleotide polymorphism (SNP) in KRAS gene (TAT to TAC at codon 32) was also identified in the normal pancreas DNA of the syngenic mouse and in both derived tumoral Panc02 and Panc02-H7 cells. No mutation or SNP was found in CDKN2A (p16), TP53 (p53), ZIP4, and PDX-1 genes in these two cell lines. The absence of mutations in genes such as KRAS, TP53, and CDKN2A, which are considered as key genes in the development of human pancreatic cancer suggests that SMAD4 might play a central and decisive role in mouse pancreatic cancer. These results also suggest that other mechanisms are involved in the substantial phenotypic difference between these two mouse pancreatic cancer cell lines. Further studies are warranted to elucidate the molecular pathways that lead to the aggressive metastatic potential of Panc02-H7.
CDKN2A (p16); genomic sequence; KRAS; mouse pancreatic cancer; PDX-1; SMAD4; TP53 (p53); ZIP4
Our previous studies have shown that the inhibition of phosphatidylinositol 3-kinase (PI3K) or mTOR complex 1 can obviously promote the Coxsackievirus B3 (CVB3)-induced apoptosis of HeLa cells by regulating the expression of proapoptotic factors. To further illustrate it, Homo sapiens eIF4E-binding protein 1 (4EBP1), p70S6 kinase (p70S6K), Akt1 and Akt2 were transfected to HeLa cells, respectively. And then, we established the stable transfected cell lines. Next, after CVB3 infection, apoptosis in different groups was determined by flow cytometry; the expressions of Bim, Bax, caspase-9 and caspase-3 were examined by real-time fluorescence quantitative PCR and western blot analysis; the expression of CVB3 mRNA and viral capsid protein VP1 were also analyzed by real-time fluorescence quantitative PCR, western blot analysis and immunofluorescence, respectively. At the meantime, CVB3 replication was observed by transmission electron microscope. We found that CVB3-induced cytopathic effect and apoptosis in transfected groups were more obvious than that in controls. Unexpectedly, apoptosis rate in Akt1 group was higher than others at the early stage after viral infection and decreased with the viral-infected time increasing, which was opposite to other groups. Compared with controls, the expression of CVB3 mRNA was increased at 3, 6, 12 and 24 h postinfection (p. i.) in all groups. At the meantime, VP1 expression in 4EBP1 group was higher than control during the process of infection, while the expressions in the other groups were change dynamically. Moreover, overexpression of 4EBP1 did not affect the mRNA expressions of Bim, Bax, caspase-9 and caspase-3; while protein expressions of Bim and Bax were decreased, the self-cleavages of caspase-9 and caspase-3 were stimulated. Meanwhile, overexpression of p70S6K blocked the CVB3-induced Bim, Bax and caspase-9 expressions but promoted the self-cleavage of caspase-9. In the Akt1 group, it is noteworthy that the expressions of Bim protein were higher than controls at 3 and 6 h p. i. but lower at 24 h p. i., and the expression of Bax protein were higher at 6 and 24 h p. i., while their mRNA expressions were all decreased. Furthermore, overexpression of Akt1 stimulated the procaspase-9 and procaspase-3 expression but blocked their self-cleavages. Overexpression of Akt2, however, had little effect on Bim, Bax and caspase-3, while prevented caspase-9 from self-cleavage at the late stage of CVB3 infection. As stated above, our results demonstrated that overexpression of 4EBP1, p70S6K, Akt1 or Akt2 could promote the CVB3-induced apoptosis in diverse degree via different mediating ways in viral replication and proapoptotic factors in BcL-2 and caspase families. As 4EBP1, p70S6K and Akt are the important substrates of PI3K and mammalian target of rapamycin (mTOR), we further illustrated the role of PI3K/Akt/mTOR signaling pathway in the process of CVB3-induced apoptosis.
overexpression; 4EBP1; p70S6K; Akt; coxsackievirus b3; apoptosis
Expression of eukaryotic genes during development requires complex spatial-temporal regulation. This complex regulation is often achieved through the coordinated interaction of transcription regulatory elements in the promoters of the target genes. The identification and mapping of regulatory elements in genome scale is crucial to understand how gene expression is regulated. Chromatin immunoprecipitation is a standard method for assessing the occupancy of DNA binding proteins in vivo in their native chromatin context using antibodies. However, standard chromatin immunoprecipitation procedure is time consuming, labor intensive and not suited for analyzing many samples simultaneously.
Recently, we have developed a simple ChIP protocol that requires fewer steps and less hands-on time. This protocol is compatible with both 96-well plate and single tube formats, and enables higher sensitivity and more reliable performance, as compared to conventional approaches.
We have successfully used this protocol to map various clinically relevant chromatin marks and controls across several cell types to quantitatively measure chromatin states. This analysis included a variety of marks corresponding to repressed, poised and active promoters, strong and weak enhancers, putative insulators, transcribed regions, as well as large-scale repressed and inactive domains. This study demonstrates the utility of this approach for the characterization of model cellular systems in perturbation studies with chemical probes.
In several mammalian species, the configuration of germinal vesicle (GV) chromatin correlates with the developmental competence of oocytes. Yet, no study has been published on the configuration of GV chromatin in ferret, nor is it known whether a specific configuration predicts meiotic competence in this species, in spite of the potential importance of ferret cloning to the study of human disease and to species conservation efforts. Here, we report on an analysis of the chromatin configuration in ferret GV oocytes and on how they correlate with meiotic development. Three distinct configurations were identified based on the degree of chromatin condensation: (1) fibrillar chromatin (FC), featuring strands of intertwined chromatin occupying most of the visible GV region; (2) intermediate condensed chromatin (ICC), characterized by dense, irregular chromatin masses throughout the GV; and (3) condensed chromatin (CC), which is highly compact and centered around the nucleolus. We also found that chromatin configuration was related to the extent of association with cumulus cells in cumulus–oocyte complexes; CC-configured oocytes were most often surrounded by a compact cumulus layer and also a compact corona but FC-configured oocytes were associated with neither. In addition, increasing chromatin condensation corresponded to an increase in oocyte diameter. Finally, following in vitro culture, significantly more CC-configured oocytes underwent maturation to meiotic metaphase II than did FC- or ICC-configured oocytes. We conclude that, in ferret, chromatin condensation is related to the sequential achievement of meiotic competencies during oocyte growth and differentiation, and thus can be used as a predictor of competence.
To investigate the effect of acylated ghrelin on the activation of TLR4/NF-κB signaling pathway induced by palmitic acid in human monocyte-derived (THP-1) macrophages, THP-1 macrophages were cultured for 12 h by palmitic acid with various concentrations. The THP-1 macrophages was pretreated by acylated ghrelin at different doses for 4 h before cultivated by palmitic acid (200 μmol/L) for 12 h. We observed the level of TLR4, NF-κB p65 phosphorylation in THP-1 macrophages and TNF-α, IL-1β in culture supernatant. TLR4 mRNA was measured by real-time PCR. TLR4 protein and NF-κB p65 phosphorylation was measured by western blotting. The expression of TNF-α and IL-1β was detected by ELISA. Compared to the THP-1 macrophages without palmitic acid, the level of TLR4 mRNA protein and NF-κB p65 phosphorylation and the expression of TNF-α and IL-1β increased after treatment by palmitic acid in a dose-dependent fashion (P < 0.05). Compared to the THP-1 macrophages with palmitic acid (200 μmol/L), the level of the pervious substances decreased after preadministration by acylated ghrelin in a dose-dependent fashion. So, we make a conclusion that acylated ghrelin can regulate the activation of TLR4/NF-κB signaling pathway and inhibit the release of inflammatory cytokines in THP-1 macrophages which are stimulated by palmitic acid in a dose-dependent fashion.
Rapamycin impaired glucose tolerance and insulin sensitivity. Our previous study demonstrated that rapamycin significantly increases the expression of gastric ghrelin, which is critical in the regulation of glucose metabolism. Here, we investigated whether ghrelin contributes to derangements of glucose metabolism induced by rapamycin.
The effects of rapamycin on glucose metabolism were examined in mice receiving ghrelin receptor antagonist or with ghrelin receptor gene deletion. Changes in Glut4, JNK, and pS6 were investigated by immnuofluorescent staining or Western. Related hormones were detected by radioimmuno-assay kits.
Rapamycin impaired glucose metabolism and insulin sensitivity not only in normal C57BL/6J mice but also in both obese mice induced by high fat diet and db/db mice. This was accompanied by elevation of plasma acylated ghrelin. Rapamycin significantly increased the levels of plasma acylated ghrelin in normal C57BL/6J mice, high fat diet induced obese mice, and db/db mice. Elevation in plasma acylated ghrelin and derangements of glucose metabolism upon administration of rapamycin was significantly correlated. The deterioration in glucose homeostasis induced by rapamycin was blocked by D-Lys3-GHRP-6, a ghrelin receptor antagonist, or by deletion of ghrelin receptor gene. Ghrelin receptor antagonism and ghrelin receptor gene deletion blocked the up-regulation of JNK activity, and GLUT4 expression and translocation in the gastrocnemius muscle induced by rapamycin.
The current study demonstrates that ghrelin contributes to derangements of glucose metabolism induced by rapamycin via altering the expression and translocation of GLUT4 in muscles.
Ghrelin; glucose metabolism; rapamycin
Formalin-fixed, paraffin-embedded (FFPE) tumour tissue represents an immense but mainly untapped resource with respect to molecular profiling. The DASL (cDNA-mediated Annealing, Selection, extension, and Ligation) assay is a recently described, RT–PCR-based, highly multiplexed high-throughput gene expression platform developed by Illumina specifically for fragmented RNA typically obtained from FFPE specimens, which enables expression profiling. In order to extend the utility of the DASL assay for breast cancer, we have custom designed and validated a 512-gene human breast cancer panel.
The RNA from FFPE breast tumour specimens were analysed using the DASL assay. Breast cancer subtype was defined from pathology immunohistochemical (IHC) staining. Differentially expressed genes between the IHC-defined subtypes were assessed by prediction analysis of microarrays (PAM) and then used in the analysis of two published data sets with clinical outcome data.
Gene expression signatures on our custom breast cancer panel were very reproducible between replicates (average Pearson's R2=0.962) and the 152 genes common to both the standard cancer DASL panel (Illumina) and our breast cancer DASL panel were similarly expressed for samples run on both panels (average R2=0.877). Moreover, expression of ESR1, PGR and ERBB2 corresponded well with their respective pathology-defined IHC status. A 30-gene set indicative of IHC-defined breast cancer subtypes was found to segregate samples based on their subtype in our data sets and published data sets. Furthermore, several of these genes were significantly associated with overall survival (OS) and relapse-free survival (RFS) in these previously published data sets, indicating that they are biomarkers of the different breast cancer subtypes and the prognostic outcomes associated with these subtypes.
We have demonstrated the ability to expression profile degraded RNA transcripts derived from FFPE tissues on the DASL platform. Importantly, we have identified a 30-biomarker gene set that can classify breast cancer into subtypes and have shown that a subset of these markers is prognostic of OS and RFS.
breast cancer; DASL assay; bead array; formalin-fixed, paraffin-embedded (FFPE); relapse-free survival (RFS); overall survival (OS)
Polycomb protein histone methyltransferase enhancer of Zeste homologe 2 (EZH2) is frequently overexpressed in human malignancy and is implicated in cancer cell proliferation and invasion. However, it is largely unknown whether EZH2 has a role in modulating DNA damage response. Here, we show that EZH2 is an important determinant of cell fate decision in response to genotoxic stress. EZH2 depletion results in abrogation of both cell cycle G1 and G2/M checkpoints, directing DNA damage response toward predominant apoptosis in both p53-proficient and p53-deficient cancer cells, but not in normal cells. Mechanistically, EZH2 regulates DNA damage response in p53 wild-type cells mainly through transcriptional repression of FBXO32, which binds to and directs p21 for proteasome-mediated degradation, whereas it affects p53-deficient cells through regulating Chk1 activation by a distinct mechanism. Furthermore, pharmacological depletion of EZH2 phenocopies the effects of EZH2 knockdown on cell cycle checkpoints and apoptosis. These data unravel a crucial role of EZH2 in determining the cancer cell outcome following DNA damage and suggest that therapeutic targeting oncogenic EZH2 might serve as a strategy for improving conventional chemotherapy in a given malignancy.
EZH2; apoptosis; DNA damage; checkpoint
Solid rationales are still present for the identification of synthetic ligands to simultaneously target multiple PPAR subtypes for the treatment of T2DM. The purpose of this study was to characterize the in vitro and in vivo differential effects of chiglitazar, a non-TZD type of PPAR pan-agonist currently in phase III clinic development in China, from PPARγ-selective agonist like rosiglitazone. Chiglitazar showed transactivating activity in each PPARα, γ, and δ subtype and upregulated the expression of PPARα and/or PPARδ downstream genes involved in the key processes of lipid metabolism and thermogenesis. Comparable blood glucose lowering effect was observed between chiglitazar and rosiglitazone, but chiglitazar did not significantly increase the body weight in KKAy and fat pad weight in db/db mice. Chiglitazar had high distribution in liver, pancreas, and skeleton muscles but was less present in kidney, heart, and adipose in rats. Heart weight increase was not observed in rats treated with chiglitazar for 6 months at a dose as high as 45 mg kg−1. The in vitro and in vivo differential features of chiglitazar are informative and encouraging for the further development of this synthetic ligand for the potential use in T2DM.
In this study, the roles of p53 in impaired spermatogenic male germ cells of p53-deficient medaka were investigated by analyzing histological changes, and gene expressions of 42Sp50, Oct 4 and vitellogenin (VTG2) by RT-PCR or in situ hybridization in the testes. We found that a small number of oocyte-like cells (testis–ova) differentiated spontaneously in the cysts of type A and early type B spermatogonia in the p53-deficient testes, in contrast to the wild-type (wt) testes in which testis–ova were never found. Furthermore, ionizing radiation (IR) irradiation increased the number of testis–ova in p53-deficient testes, increased testis–ova size and proceeded up to the zygotene or pachytene stages of premature meiosis within 14 days after irradiation. However, 28 days after irradiation, almost all the testis–ova were eliminated presumably by p53-independent apoptosis, and spermatogenesis was restored completely. In the wt testis, IR never induced testis–ova differentiation. This is the first study to demonstrate the pivotal role of the p53 gene in the elimination of spontaneous testis–ova in testes, and that p53 is not indispensable for the restoration of spermatogenesis in the impaired testes in which cell cycle regulation is disturbed by IR irradiation.
sex differentiation; testis–ova; p53; irradiation; apoptosis; medaka
When a tensile strain is applied to a film supported on a compliant substrate, a pattern of parallel cracks can channel through both the film and substrate. A linear-elastic fracture-mechanics model for the phenomenon is presented to extend earlier analyses in which cracking was limited to the film. It is shown how failure of the substrate reduces the critical strain required to initiate fracture of the film. This effect is more pronounced for relatively tough films. However, there is a critical ratio of the film to substrate toughness above which stable cracks do not form in response to an applied load. Instead, catastrophic failure of the substrate occurs simultaneously with the propagation of a single channel crack. This critical toughness ratio increases with the modulus mismatch between the film and substrate, so that periodic crack patterns are more likely to be observed with relatively stiff films. With relatively low values of modulus mismatch, even a film that is more brittle than the substrate can cause catastrophic failure of the substrate. Below the critical toughness ratio, there is a regime in which stable crack arrays can be formed in the film and substrate. The depth of these arrays increases, while the spacing decreases, as the strain is increased. Eventually, the crack array can become deep enough to cause substrate failure.
A sensitive and reliable method of liquid chromatography–electrospray ionization/tandem mass spectrometry (LC-ESI/MS/ MS) was developed and validated for determining 1,3-dimethylamylamine (1,3-DMAA) and 1,4-dimethylamylamine (1,4-DMAA) in geranium plants (Pelargonium graveolens). The sample was extracted with 0.5 M HCl and purified by liquid-liquid partition with hexane. The parameters for reverse-phase (C18) LC and positive ESI/MS/MS were optimized. The matrix effect, specificity, linearity, precision, accuracy and reproducibility of the method were determined and evaluated. The method was linear over a range of 0.10–10.00 ng/mL examined, with R2 of 0.99 for both 1,3-DMAA and 1,4-DMAA. The recoveries from spiked concentrations between 5.00–40.00 ng/g were 85.1%–104.9% for 1,3-DMAA, with relative standard deviation (RSD) of 2.9%–11.0%, and 82.9%–101.8% for 1,4-DMAA, with RSD of 3.2%–11.7%. The instrument detection limit was 1–2 pg for both DMAAs. The quantification limit was estimated to be 1–2 ng/g for the plant sample. This method was successfully applied to the quantitative determination of 1,3- and 1,4-DMAA in both geranium plant and geranium oil.
1,3-dimethylamylamine; 1,4-dimethylamylamine; geranium (Pelargonium graveolens); liquid chromatography-tandem mass spectrometry (LC/MS/MS)
Neuroblastoma (NB) is a common childhood malignant tumor of the neural crest-derived sympathetic nervous system. In NB the frequent loss of heterozygosity (LOH) on chromosome 1p raises the possibility that this region contains tumor-suppressor genes whose inactivation contributes to tumorigenesis. The human homolog of the Drosophila neural fate determination gene CASZ1, a zinc-finger transcription factor, maps to chromosome 1p36.22, a region implicated in NB tumorigenesis. Quantitative real-time PCR analysis showed that low-CASZ1 expression is significantly correlated with increased age (≥18 months), Children's Oncology Group high-risk classification, 1p LOH and MYCN amplification (all P<0.0002) and decreased survival probability (P=0.0009). CASZ1 was more highly expressed in NB with a differentiated histopathology (P<0.0001). Retinoids and epigenetic modification agents associated with regulation of differentiation induced CASZ1 expression. Expression profiling analysis revealed that CASZ1 regulates the expression of genes involved in regulation of cell growth and developmental processes. Specific restoration of CASZ1 in NB cells induced cell differentiation, enhanced cell adhesion, inhibited migration and suppressed tumorigenicity. These data are consistent with CASZ1 being a critical modulator of neural cell development, and that somatically acquired disruption of normal CASZ1 expression contributes to the malignant phenotype of human NB.
CASZ1; neuroblastoma; tumor suppressor; transcription factor; developmental gene; chromosome 1p
Stereocilia of the inner ear play an integral role in the mechanotransduction of sound. Their structural support is derived from actin filaments and actin-binding proteins. We have identified a novel actin-binding protein, 2E4-kaptin (KPTN), which appears to be involved in this structural network. Using double label immunofluorescence, we now show that KPTN extends beyond the barbed ends of actin filaments at the tips of stereocilia, and using cloned human cDNA, we mapped KPTN to chromosome 19q13.4. A combination of FISH, radiation hybrid mapping and YAC screening localized KPTN between markers D19S412 and NIB1805, making this gene an excellent functional and positional candidate for DFNA4, a form of autosomal dominant non-syndromic hearing loss. We identified a second family with inherited deafness that also maps to the DFNA4 region. To screen KPTN for deafness-causing mutations, we first determined its genomic structure and then completed a mutational analysis by direct sequencing and SSCP in affected family members. Although no deafness-causing mutations were identified in the coding region, KPTN remains an excellent candidate gene for hearing loss; by synteny, its murine orthologue also remains a candidate gene for the Nijmegan waltzer (nv) mouse mutant, which has vestibular defects and a variable sensorineural hearing loss.
The human blood platelet circulates in the blood as a non-adherent disk. Upon receiving signals of blood vessel damage, the platelet reorganizes its actin cytoskeleton which transforms it into a spiky dynamic adherent glue. This transformation involves a temporal sequence of four morphologically distinct steps that can be reproducible in vitro. The actin dynamics that underlie these shape changes depend on a large number of actin-binding proteins. Maintenance of the discoid shape requires actin-binding proteins that inhibit these reorganizations, whereas transformation involves other proteins, some to disassemble old filaments and others to polymerize new ones. F-actin-affinity chromatography identified a large set of actin-binding proteins including VASP, Arp2 and 2E4/kaptin. Recent discoveries show that VASP inhibits filament disassembly and Arp2/3 is required to polymerize new filaments. Morphological analysis of the distribution of these actin-binding proteins in spread platelets together with biochemical measurements of their interactions with actin lead to a model of interactions with actin that mediate shape change.
Platelet activation; Shape change; Actin polymerization; Cytoskeleton; Actin-binding proteins; Arp2/3; VASP; 2E4/kaptin; Gelsolin; ADF/cofilin; Lamellipodia; Filopodia
The family of polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) is responsible for the altered glycosylation in cancer. The purpose of our study was to investigate the clinical significance of two isoforms, GalNAc-T6 and -T3, and their correlation with the prognosis of pancreatic cancer.
Immunohistochemistry was used to analyse GalNAc-T6 and -T3 expressions in 70 clinicopathologically characterised pancreatic cancer cases.
Positive expressions of GalNAc-T6 and -T3 were immunohistochemically identified in 51% (36 of 70) and in 77% (54 of 70) of patients, respectively. A close relationship was noted between GalNAc-T6 positive expression and pathological well/moderate differentiated type (P=0.001), small tumour size (P=0.044), absence of vascular invasion (P=0.009), and low stage of the American Joint Committee on Cancer systems (P=0.043). The expression of GalNAc-T3 significantly correlated with good differentiation (P=0.001), but not with other clinicopathologic features. Furthermore, univariate and multivariate analyses revealed that GalNAc-T6 expression was an independent prognosis indicator for the disease, whereas GalNAc-T3 expression had no impact on clinical outcome, even though 33 of 36 GalNAc-T6-positive cases also had a positive expression of GalNAc-T3 (P=0.001, r=0.356).
Both GalNAc-T6 and -T3 expressions correlated significantly with tumour differentiation, whereas only GalNAc-T6 expression predicted prognosis in pancreatic cancer.
pancreatic cancer; GalNAc-T6; GalNAc-T3; prognosis; metastasis
The potential for human disease treatment using human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells (iPSCs), also carries the risk of added genomic instability. Genomic instability is most often linked to DNA repair deficiencies, which indicates that screening/characterization of possible repair deficiencies in pluripotent human stem cells should be a necessary step prior to their clinical and research use. In this study, a comparison of DNA repair pathways in pluripotent cells, as compared to those in non-pluripotent cells, demonstrated that DNA repair capacities of pluripotent cell lines were more heterogeneous than those of differentiated lines examined and were generally greater. Although pluripotent cells had high DNA repair capacities for nucleotide excision repair, we show that ultraviolet radiation at low fluxes induced an apoptotic response in these cells, while differentiated cells lacked response to this stimulus, and note that pluripotent cells had a similar apoptotic response to alkylating agent damage. This sensitivity of pluripotent cells to damage is notable since viable pluripotent cells exhibit less ultraviolet light-induced DNA damage than do differentiated cells that receive the same flux. In addition, the importance of screening pluripotent cells for DNA repair defects was highlighted by an iPSC line that demonstrated a normal spectral karyotype, but showed both microsatellite instability and reduced DNA repair capacities in three out of four DNA repair pathways examined. Together, these results demonstrate a need to evaluate DNA repair capacities in pluripotent cell lines, in order to characterize their genomic stability, prior to their pre-clinical and clinical use.
Dendritic trafficking and translation of BDNF transcripts play a key role in mediating synaptic plasticity. Recently, we demonstrated that siRNA-mediated knockdown of translin, an RNA binding protein, impairs KCl-induced dendritic trafficking of BDNF mRNA in cultured hippocampal neurons. We have now assessed whether translin deletion impairs dendritic trafficking of BDNF mRNA in hippocampal neurons in vivo. We have found that translin and its partner protein, trax, undergo dendritic translocation in response to treatment with pilocarpine, a pro-convulsant muscarinic agonist that increases dendritic trafficking of BDNF mRNA in hippocampal neurons. In translin knockout mice, the basal level of dendritic BDNF mRNA is decreased in CA1 pyramidal neurons. However, translin deletion does not block pilocarpine’s ability to increase dendritic trafficking of BDNF mRNA indicating that the requirement for translin in this process varies with the stimulus employed to drive it. Consistent with this inference, we found that dendritic trafficking of BDNF mRNA induced by bath application of recombinant BDNF in cultured hippocampal neurons, is not blocked by siRNA-mediated knockdown of translin. Taken together, these in vivo and in vitro findings indicate that dendritic trafficking of BDNF mRNA can be mediated by both translin-dependent and -independent mechanisms.
trax; hippocampus; pilocarpine; CA1 pyramidal neurons; dendritic translation
The regulation of both mitochondrial dynamics and apoptosis is key for maintaining the health of a cell. Bcl-2 family proteins, central in apoptosis regulation, also have roles in the maintenance of the mitochondrial network. Here we report that Bax and Bak participate in the regulation of mitochondrial fusion in mouse embryonic fibroblasts, primary mouse neurons and human colon carcinoma cells. To assess how Bcl-2 family members may regulate mitochondrial morphogenesis, we determined the binding of a series of chimeras between Bcl-xL and Bax to the mitofusins, mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2). One chimera (containing helix 5 (H5) of Bax replacing H5 of Bcl-xL (Bcl-xL/Bax H5)) co-immunoprecipitated with Mfn1 and Mfn2 significantly better than either wild-type Bax or Bcl-xL. Expression of Bcl-xL/Bax H5 in cells reduced the mobility of Mfn1 and Mfn2 and colocalized with ectopic Mfn1 and Mfn2, as well as endogenous Mfn2 to a greater extent than wild-type Bax. Ultimately, Bcl-xL/Bax H5 induced substantial mitochondrial fragmentation in healthy cells. Therefore, we propose that Bcl-xL/Bax H5 disturbs mitochondrial morphology by binding and inhibiting Mfn1 and Mfn2 activity, supporting the hypothesis that Bcl-2 family members have the capacity to regulate mitochondrial morphology through binding to the mitofusins in healthy cells.
Bax; mitochondria fusion; Bak; apoptosis; Mfn2
The correlation and competition between antiferromagnetism and superconductivity are one of the most fundamental issues in high temperature superconductors. Superconductivity in high temperature cuprate superconductors arises from suppressing an antiferromagnetic (AFM) Mott insulator1 while in iron-pnictide superconductors arises from AFM semimetals and can coexist with AFM orders23456789. This difference raises many intriguing debates on the relation between the two classes of high temperature superconductors. Recently, superconductivity at 32 K has been reported in iron-chalcogenide superconductors AxFe2−ySe2 (A = K, Rb, and Cs)101112. They have the same structure as that of iron-pnictide 122-system131415. Here, we report electronic and magnetic phase diagram of KxFe2−ySe2 system as a function of Fe valence. We find a superconducting phase sandwiched between two AFM insulating phases. The two insulating phases are characterized by two distinct superstructures caused by Fe vacancy orders with modulation wave vectors of q1 = (1/5, 3/5, 0) and q2 = (1/4, 3/4, 0), respectively.
The period homolog genes Per1, Per2 and Per3 are important components of the circadian clock system. In addition to their role in maintaining circadian rhythm, these genes have been linked to mood disorders, stress response and vulnerability to addiction and alcoholism. In this study, we combined high-resolution sequence analysis and quantitative trait locus (QTL) mapping of gene expression and behavioral traits to identify Per3 as a compelling candidate for the interaction between circadian rhythm, alcohol and stress response. In the BXD family of mouse strains, sequence variants in Per3 have marked effects on steady-state mRNA and protein levels. As a result, the transcript maps as a cis-acting expression QTL (eQTL). We found that an insertion/deletion (indel) variant in a putative stress response element in the promoter region of Per3 causes local control of transcript abundance. This indel results in differences in protein binding affinities between the two alleles through the Nrf2 transcriptional activator. Variation in Per3 is also associated with downstream differences in the expression of genes involved in circadian rhythm, alcohol, stress response and schizophrenia. We found that the Per3 locus is linked to stress/anxiety traits, and that the basal expression of Per3 is also correlated with several anxiety and addiction-related phenotypes. Treatment with alcohol results in increased expression of Per3 in the hippocampus, and this effect interacts with acute restraint stress. Our data provide strong evidence that variation in the Per3 transcript is causally associated with and also responsive to stress and alcohol.
alcohol response; expression QTL; Per3; stress