Adipose tissue has a central role in the regulation of energy balance and homoeostasis. There are two main types of adipose tissue: WAT (white adipose tissue) and BAT (brown adipose tissue). WAT from certain depots, in response to appropriate stimuli, can undergo a process known as browning where it takes on characteristics of BAT, notably the induction of UCP1 (uncoupling protein 1) expression and the presence of multilocular lipid droplets and multiple mitochondria. How browning is regulated is an intense topic of investigation as it has the potential to tilt the energy balance from storage to expenditure, a strategy that holds promise to combat the growing epidemic of obesity and metabolic syndrome. This review focuses on the transcriptional regulators as well as various proteins and secreted mediators that have been shown to play a role in browning. Emphasis is on describing how many of these factors exert their effects by regulating the three main transcriptional regulators of classical BAT development, namely PRDM16 (PR domain containing 16), PPARγ (peroxisome proliferator-activated receptor γ) and PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1α), which have been shown to be the key nodes in the regulation of inducible brown fat.
beige fat; browning; peroxisome-proliferator-activated receptor γ (PPARγ); peroxisome-proliferator-activated receptor γ coactivator 1-α (PGC-1α); PR domain containing 16 (PRDM16); white adipocytes; 4E-BP1, eukaryotic translation initiation factor 4E binding protein 1; ANP, atrial natriuretic peptide; ATF2, activating transcription factor 2; BAT, brown adipose tissue; BMP7, bone morphogenetic protein 7; C/EBP, CCAAT/enhancer-binding protein; Cidea, cell death-inducing DFFA-like effector a; CNP, cardiac natriuretic peptide; COX2, cyclo-oxygenase 2; CtBP, C-terminal-binding protein; EBF2, early B cell factor-2; eIF4E, eukaryotic translation initiation factor 4E; Elovl3, elongation of very long chain fatty acids (FEN1/Elo2, SUR4/Elo3, yeast)-like 3; FGF21, fibroblast growth factor 21; FoxC2, forkhead box protein C2; MAPK, mitogen-activated protein kinase; miRNA, microRNA; Myf5, myogenic factor 5; NPRC, natriuretic peptide receptor C; PGC-1α, peroxisome-proliferator-activated receptor γ coactivator 1α; PGI2, prostacyclin; PKA, protein kinase A (=cAMP-dependent protein kinase); PKG, protein kinase G (=cGMP-dependent protein kinase); PPARγ, peroxisome-proliferator-activated receptor gamma; PRDM16, PR domain containing 16; pRb, retinoblastoma protein; SRC-1, steroid receptor coactivator-1; SVF, stromal vascular fraction; TBX15, T-box 15; TFAM, mitochondrial transcription factor A; TIF2, transcriptional intermediary factor-2; TRPV4, transient receptor potential vanilloid 4; UCP1, uncoupling protein 1; WAT, white adipose tissue
Visualization of the blood vessels can provide valuable morphological information for diagnosis and therapy strategies for cardiovascular disease. Intravascular ultrasound (IVUS) is able to delineate internal structures of vessel wall with fine spatial resolution. However, the developed IVUS is insufficient to identify the fibrous cap thickness and tissue composition of atherosclerotic lesions. Novel imaging strategies have been proposed, such as increasing the center frequency of ultrasound or using a modulated excitation technique to improve the accuracy of diagnosis. Dual-mode tomography combining IVUS with optical tomography has also been developed to determine tissue morphology and characteristics. The implementation of these new imaging methods requires an open system that allows users to customize the system for various studies. This paper presents the development of an IVUS system that has open structures to support various imaging strategies. The system design is based on electronic components and printed circuit board, and provides reconfigurable hardware implementation, programmable image processing algorithms, flexible imaging control, and raw RF data acquisition. In addition, the proposed IVUS system utilized a miniaturized ultrasound transducer constructed using PMN-PT single crystal for better piezoelectric constant and electromechanical coupling coefficient than traditional lead zirconate titanate (PZT) ceramics. Testing results showed that the IVUS system could offer a minimum detectable signal of 25 μV, allowing a 51 dB dynamic range at 47 dB gain, with a frequency range from 20 to 80 MHz. Finally, phantom imaging, in vitro IVUS vessel imaging, and multimodality imaging with photoacoustics were conducted to demonstrate the performance of the open system.
Deletion of ribosomal protein L32 genes resulted in a nonsexual flocculation of fission yeast. Nonsexual flocculation also occurred when two other ribosomal protein genes, rpl21-2 and rpl9-2, were deleted. However, deletion of two nonribosomal protein genes, mpg and fbp, did not cause flocculation. Overall transcript levels of rpl32 in rpl32-1Δ and rpl32-2Δ cells were reduced by 35.9% and 46.9%, respectively, and overall ribosome levels in rpl32-1Δ and rpl32-2Δ cells dropped 31.1% and 27.8%, respectively, compared to wild-type cells. Interestingly, ribosome protein expression levels and ribosome levels were also reduced greatly in sexually flocculating diploid YHL6381/WT (h+/h−) cells compared to a mixture of YHL6381 (h+) and WT (h−) nonflocculating haploid cells. Transcriptome analysis indicated that the reduction of ribosomal levels in sexual flocculating cells was caused by more-extensive suppression of ribosomal biosynthesis gene expression, while the reduction of ribosomal levels caused by deleting ribosomal protein genes in nonsexual flocculating cells was due to an imbalance between ribosomal proteins. We propose that once the reduction of ribosomal levels is below a certain threshold value, flocculation is triggered.
Zebrafish can fully regenerate their myocardium after ventricular resection without evidence of scars. This extraordinary regenerative ability provides an excellent model system to study the activation of the regenerative potential for human heart tissue. In addition to the morphology, it is vital to understand the cardiac function of zebrafish. To characterize adult zebrafish cardiac function, an ultrasound biomicroscope (UBM) was customized for real-time imaging of the zebrafish heart (about 1 mm in diameter) at a resolution of around 37 µm. Moreover, we developed an image segmentation algorithm to track the cardiac boundary and measure the dynamic size of the zebrafish heart for further quantification of zebrafish cardiac function. The effectiveness and accuracy of the proposed segmentation algorithm were verified on a tissue-mimicking phantom and in vivo zebrafish echocardiography. The quantitative evaluation demonstrated that the accuracy of the proposed algorithm is comparable to the manual delineation by experts.
Micro-ultrasound is an invaluable imaging tool for many clinical and preclinical applications requiring high resolution (approximately several tens of micrometers). Imaging systems for micro-ultrasound, including single-element imaging systems and linear-array imaging systems, have been developed extensively in recent years. Single-element systems are cheaper, but linear-array systems give much better image quality at a higher expense. Annular-array-based systems provide a third alternative, striking a balance between image quality and expense. This paper presents the development of a novel programmable and real-time annular-array imaging platform for micro-ultrasound. It supports multi-channel dynamic beamforming techniques for large-depth-of-field imaging. The major image processing algorithms were achieved by a novel field-programmable gate array technology for high speed and flexibility. Real-time imaging was achieved by fast processing algorithms and high-speed data transfer interface. The platform utilizes a printed circuit board scheme incorporating state-of-the-art electronics for compactness and cost effectiveness. Extensive tests including hardware, algorithms, wire phantom, and tissue mimicking phantom measurements were conducted to demonstrate good performance of the platform. The calculated contrast-to-noise ratio (CNR) of the tissue phantom measurements were higher than 1.2 in the range of 3.8 to 8.7 mm imaging depth. The platform supported more than 25 images per second for real-time image acquisition. The depth-of-field had about 2.5-fold improvement compared to single-element transducer imaging.
Next generation sequencing has dramatically increased our ability to localize disease-causing variants by providing base-pair level information at costs increasingly feasible for the large sample sizes required to detect complex-trait associations. Yet, identification of causal variants within an established region of association remains a challenge. Counter-intuitively, certain factors that increase power to detect an associated region can decrease power to localize the causal variant. First, combining GWAS with imputation or low coverage sequencing to achieve the large sample sizes required for high power can have the unintended effect of producing differential genotyping error among SNPs. This tends to bias the relative evidence for association toward better genotyped SNPs. Second, re-use of GWAS data for fine-mapping exploits previous findings to ensure genome-wide significance in GWAS-associated regions. However, using GWAS findings to inform fine-mapping analysis can bias evidence away from the causal SNP toward the tag SNP and SNPs in high LD with the tag. Together these factors can reduce power to localize the causal SNP by more than half. Other strategies commonly employed to increase power to detect association, namely increasing sample size and using higher density genotyping arrays, can, in certain common scenarios, actually exacerbate these effects and further decrease power to localize causal variants. We develop a re-ranking procedure that accounts for these adverse effects and substantially improves the accuracy of causal SNP identification, often doubling the probability that the causal SNP is top-ranked. Application to the NCI BPC3 aggressive prostate cancer GWAS with imputation meta-analysis identified a new top SNP at 2 of 3 associated loci and several additional possible causal SNPs at these loci that may have otherwise been overlooked. This method is simple to implement using R scripts provided on the author's website.
As next-generation sequencing (NGS) costs continue to fall and genome-wide association study (GWAS) platform coverage improves, the human genetics community is positioned to identify potentially causal variants. However, current NGS or imputation-based studies of either the whole genome or regions previously identified by GWAS have not yet been very successful in identifying causal variants. A major hurdle is the development of methods to distinguish disease-causing variants from their highly-correlated proxies within an associated region. We show that various common factors, such as differential sequencing or imputation accuracy rates and linkage disequilibrium patterns, with or without GWAS-informed region selection, can substantially decrease the probability of identifying the correct causal SNP, often by more than half. We then describe a novel and easy-to-implement re-ranking procedure that can double the probability that the causal SNP is top-ranked in many settings. Application to the NCI Breast and Prostate Cancer (BPC3) Cohort Consortium aggressive prostate cancer data identified new top SNPs within two associated loci previously established via GWAS, as well as several additional possible causal SNPs that had been previously overlooked.
The Tat protein of HIV-1 has several well-known properties, such as nucleocytoplasmic trafficking, transactivation of transcription, interaction with tubulin, regulation of mitotic progression, and induction of apoptosis. Previous studies have identified a couple of lysine residues in Tat that are essential for its functions. In order to analyze the functions of all the lysine residues in Tat, we mutated them individually to alanine, glutamine, and arginine. Through systematic analysis of the lysine mutants, we discovered several previously unidentified characteristics of Tat. We found that lysine acetylation could modulate the subcellular localization of Tat, in addition to the regulation of its transactivation activity. Our data also revealed that lysine mutations had distinct effects on microtubule assembly and Tat binding to bromodomain proteins. By correlation analysis, we further found that the effects of Tat on apoptosis and mitotic progression were not entirely attributed to its effect on microtubule assembly. Our findings suggest that Tat may regulate diverse cellular activities through binding to different proteins and that the acetylation of distinct lysine residues in Tat may modulate its interaction with various partners.
Alzheimer's disease (AD) is a progressive neurodegenerative disorder that accounts for the major cause of dementia, and the increasing worldwide prevalence of AD is a major public health concern. Increasing epidemiological studies suggest that diet and nutrition might be important modifiable risk factors for AD. Dietary supplementation of antioxidants, B vitamins, polyphenols, and polyunsaturated fatty acids are beneficial to AD, and consumptions of fish, fruits, vegetables, coffee, and light-to-moderate alcohol reduce the risk of AD. However, many of the results from randomized controlled trials are contradictory to that of epidemiological studies. Dietary patterns summarizing an overall diet are gaining momentum in recent years. Adherence to a healthy diet, the Japanese diet, and the Mediterranean diet is associated with a lower risk of AD. This paper will focus on the evidence linking many nutrients, foods, and dietary patterns to AD.
We used a whole blood assay to characterize the immune system’s response following cardiopulmonary bypass (CPB) in children to identify the risk for postoperative infections. We assessed the impact of CPB on histone methylation as a potential mechanism for altering gene expression necessary for the immune system’s capacity to defend against infections.
We prospectively enrolled patients <18 years old undergoing heart surgery requiring CPB at C.S. Mott Children’s Hospital. Blood was obtained from patients prior to CPB, on CPB and on postoperative days 1, 3 and 5. Ex vivo LPS-induced TNF-α production measured the capacity of the immune system. Serum cytokines were measured using a multiplex assay. Chromatin Immunoprecipitation (ChIP) to detect histone modifications at the interleukin (IL)-10 promoter was performed on circulating mononuclear cells from a subgroup of patients.
We enrolled 92 patients and postoperative day 1 samples identified a subpopulation of immunocompetent patients at low risk for infections with a specificity of 93% (C.I. 83–98%) and a negative predictive value of 88% (C.I. 77–95%; p=0.006). Patients classified as immunoparalyzed had serum IL-10 levels 2.4 fold higher than the immunocompetent group (mean 14.3 ± 18.3 pg/ml vs. 6.0 ± 5.0 pg/ml, p=0.01). In a subgroup of patients, we identified a greater percent of the “gene on” epigenetic signature, H3K4me3, associated with the IL-10 promoter following CPB.
Our data demonstrate that immunophenotyping patients after CPB can predict their risk of developing postoperative infections. Novel mechanistic data suggest that CPB impacts epigenetic alterations in IL-10 gene regulation.
Cardiopulmonary Bypass; Immunoparalysis; Post-translational histone modification; Interleukin-10; Epigenetics; Sepsis
Background: Congenital heart disease (CHD) is one of the most common human birth defects. The etiology and pathogenesis of CHD are complex and involve several genes as well as multiple changes in signaling pathways. The aim of this study was to identify potential pathological mutations in the Homeobox C9 (Hoxc9) gene in 350 Chinese children with CHD to further understand the etiology of CHD. Method: Sequence analysis of the Hoxc9 gene in 350 nonsyndromic patients with CHD Result: We did not identify any nonsynonymous variants in the coding regions of Hoxc9 in the patients with CHD. We found one synonymous variant c.C564T (p. his188his) in one ventricular septal defect patient. We also identified four previously reported polymorphisms (rs56368105, rs12817092, rs34079606, and rs2241820) in CHD. Conclusions: We did not find any diagnostic alterations in the coding regions of Hoxc9 among the patients with CHD. Nevertheless, to our knowledge, this is the first study of Hoxc9 in nonsyndromic CHD and has expanded our overall knowledge of the etiology of this disease.
Over 40 missense mutations in the human SCN1A sodium channel gene are linked to an epilepsy syndrome termed genetic epilepsy with febrile seizures plus (GEFS+). Inheritance of GEFS+ is dominant but the underlying cellular mechanisms remain poorly understood. Here we report knock-in of a GEFS+ SCN1A mutation (K1270T) into the Drosophila sodium channel gene, para, causes a semi-dominant temperature-induced seizure phenotype. Electrophysiological studies of GABAergic interneurons in the brains of adult GEFS+ flies reveal a novel cellular mechanism underlying heat-induced seizures: the deactivation threshold for persistent sodium currents reversibly shifts to a more negative voltage when the temperature is elevated. This leads to sustained depolarizations in GABAergic neurons and reduced inhibitory activity in the central nervous system. Further, our data indicate a natural temperature-dependent shift in sodium current deactivation (exacerbated by mutation) may contribute to febrile seizures in GEFS+ and perhaps normal individuals.
Fission yeast cells express Rpl32-2 highly while Rpl32-1 lowly in log phase; in contrast, expression of Rpl32-1 raises and reaches a peak level while Rpl32-2 is downregulated to a low basic level when cells enter into stationary phase. Overexpression of Rpl32-1 inhibits cell growth while overexpression of Rpl32-2 does not. Deleting rpl32-2 impairs cell growth more severely than deleting rpl32-1 does. Cell growth impaired by deleting either paralog can be rescued completely by reintroducing rpl32-2, but only partly by rpl32-1. Overexpression of Rpl32-1 inhibits cell division, yielding 4c DNA and multiple septa, while overexpressed Rpl32-2 promotes it. Transcriptomics analysis proved that Rpl32 paralogs regulate expression of a subset of genes related with cell division and stress response in a distinctive way. This functional difference of the two paralogs is due to their difference of 95th amino acid residue. The significance of a competitive inhibition between Rpl32 paralogs on their expression is discussed.
Chicken interferon α (ChIFN-α) and ChIFN-β are type I IFNs that are important antiviral cytokines in the innate immune system. In the present study, we identified the virus-induced expression of ChIFN-α and ChIFN-β in chicken fibroblast DF-1 cells and systematically evaluated the antiviral activities of recombinant ChIFN-α and ChIFN-β by cytopathic-effect (CPE) inhibition assays. We found that ChIFN-α exhibited stronger antiviral activity than ChIFN-β in terms of inhibiting the replication of vesicular stomatitis virus, Newcastle disease virus and avian influenza virus, respectively. To elucidate the mechanism of differential antiviral activities between the two ChIFNs, we measured the relative mRNA levels of IFN-stimulated genes (ISGs) in IFN-treated DF-1 cells by real-time PCR. ChIFN-α displayed greater induction potency than ChIFN-β on several ISGs encoding antiviral proteins and MHC-I, whereas ChIFN-α was less potent than ChIFN-β for inducing ISGs involved in signaling pathways. In conclusion, ChIFN-α and ChIFN-β presented differential induction potency on various sets of ISGs, and the stronger antiviral activity of ChIFN-α is likely attributed to the greater expression levels of downstream antiviral ISGs.
Previously, it has been shown that GPI proteins are required for cell wall synthesis and organization in Aspergillus fumigatus, a human opportunistic pathogen causing life-threatening invasive aspergillosis (IA) in immunocompromised patients. Blocking GPI anchor synthesis leads to severe phenotypes such as cell wall defects, increased cell death, and attenuated virulence. However, the mechanism by which these phenotypes are induced is unclear. To gain insight into global effects of GPI anchoring in A. fumigatus, in this study a conditional expression mutant was constructed and a genome wide transcriptome analysis was carried out. Our results suggested that suppression of GPI anchor synthesis mainly led to activation of phosphatidylinositol (PtdIns) signaling and ER stress. Biochemical and morphological evidence showed that autophagy was induced in response to suppression of the GPI anchor synthesis, and also an increased necroptosis was observed. Based on our results, we propose that activation of PtdIns3K and increased cytosolic Ca2+, which was induced by both ER stress and PtdIns signaling, acted as the main effectors to induce autophagy and possible necroptosis.
The efficacy of radiation therapy for lung cancer is limited by radiation-induced lung toxicity (RILT). Although tumor necrosis factor-alpha (TNF-α) signaling plays a critical role in RILT, the molecular regulators of radiation-induced TNF-α production remain unknown. We investigated the role of a major TNF-α regulator, Tristetraprolin (TTP), in radiation-induced TNF-α production by macrophages. For in vitro studies we irradiated (4 Gy) either a mouse lung macrophage cell line, MH-S or macrophages isolated from TTP knockout mice, and studied the effects of radiation on TTP and TNF-α levels. To study the in vivo relevance, mouse lungs were irradiated with a single dose (15 Gy) and assessed at varying times for TTP alterations. Irradiation of MH-S cells caused TTP to undergo an inhibitory phosphorylation at Ser-178 and proteasome-mediated degradation, which resulted in increased TNF-α mRNA stabilization and secretion. Similarly, MH-S cells treated with TTP siRNA or macrophages isolated from ttp (−/−) mice had higher basal levels of TNF-α, which was increased minimally after irradiation. Conversely, cells overexpressing TTP mutants defective in undergoing phosphorylation released significantly lower levels of TNF-α. Inhibition of p38, a known kinase for TTP, by either siRNA or a small molecule inhibitor abrogated radiation-induced TNF-α release by MH-S cells. Lung irradiation induced TTPSer178 phosphorylation and protein degradation and a simultaneous increase in TNF-α production in C57BL/6 mice starting 24 h post-radiation. In conclusion, irradiation of lung macrophages causes TTP inactivation via p38-mediated phosphorylation and proteasome-mediated degradation, leading to TNF-α production. These findings suggest that agents capable of blocking TTP phosphorylation or stabilizing TTP after irradiation could decrease RILT.
Rho family guanosine triphosphatases (GTPases), such as RhoA, Cdc42, and Rac1, play a fundamental role in various cellular processes. The activation of Rho proteins is catalyzed by guanine nucleotide-exchange factors (GEFs), which promote the exchange of GDP for GTP. The precise mechanisms regulating the activation of Rho proteins are not fully understood. Herein, we demonstrate that RhoA activity is regulated by cylindromatosis (CYLD), a deubiquitinase harboring multiple functions. In addition, we find that RhoA-mediated cytoskeletal rearrangement, chromosome separation, and cell polarization are altered in CYLD-depleted cells. Mechanistically, CYLD does not interact with RhoA; instead, it interacts with and deubiquitinates leukemia-associated RhoGEF (LARG). Our data further show that CYLD-mediated deubiquitination of LARG enhances its ability to stimulate the GDP/GTP exchange on RhoA. These data thus identify LARG as a new substrate of CYLD and provide novel insights into the regulation of RhoA activation. Our results also suggest that the LARG-RhoA signaling pathway may play a role in diverse CYLD-mediated cellular events.
As a novel epigenetic mechanism, histone H3 methylation at R17 and R26, which is mainly catalyzed by coactivator-associated protein arginine methyltransferase 1 (CARM1), has been reported to modulate the transcription of key pluripotency factors and to regulate pluripotency in mouse embryos and mouse embryonic stem cells (mESCs) in previous studies. However, the role of CARM1 in human embryonic stem cells (hESCs) and the regulatory mechanism that controls CARM1 expression during ESCs differentiation are presently unknown. Here, we demonstrate that CARM1 plays an active role in the resistance to differentiation in hESCs by regulating pluripotency genes in response to BMP4. In a functional screen, we identified the miR-181 family as a regulator of CARM1 that is induced during ESC differentiation and show that endogenous miR-181c represses the expression of CARM1. Depletion of CARM1 or enforced expression of miR-181c inhibits the expression of pluripotency genes and induces differentiation independent of BMP4, whereas overexpression of CARM1 or miR-181c inhibitor elevates Nanog and impedes differentiation. Furthermore, expression of CARM1 rescue constructs inhibits the effect of miR-181c overexpression in promoting differentiation. Taken together, our findings demonstrate the importance of a miR-181c-CARM1 pathway in regulating the differentiation of hESCs.
Study on long non-coding RNAs (lncRNAs) has been promoted by high-throughput RNA sequencing (RNA-Seq). However, it is still not trivial to identify lncRNAs from the RNA-Seq data and it remains a challenge to uncover their functions.
We present a computational pipeline for detecting novel lncRNAs from the RNA-Seq data. First, the genome-guided transcriptome reconstruction is used to generate initially assembled transcripts. The possible partial transcripts and artefacts are filtered according to the quantified expression level. After that, novel lncRNAs are detected by further filtering known transcripts and those with high protein coding potential, using a newly developed program called lncRScan. We applied our pipeline to a mouse Klf1 knockout dataset, and discussed the plausible functions of the novel lncRNAs we detected by differential expression analysis. We identified 308 novel lncRNA candidates, which have shorter transcript length, fewer exons, shorter putative open reading frame, compared with known protein-coding transcripts. Of the lncRNAs, 52 large intergenic ncRNAs (lincRNAs) show lower expression level than the protein-coding ones and 13 lncRNAs represent significant differential expression between the wild-type and Klf1 knockout conditions.
Our method can predict a set of novel lncRNAs from the RNA-Seq data. Some of the lncRNAs are showed differentially expressed between the wild-type and Klf1 knockout strains, suggested that those novel lncRNAs can be given high priority in further functional studies.
The influenza A virus matrix 1 protein (M1) shuttles between the cytoplasm and the nucleus during the viral life cycle and plays an important role in the replication, assembly, and budding of viruses. Here, a leucine-rich nuclear export signal (NES) was identified specifically for the nuclear export of the M1 protein. The predicted NES, designated the Flu-A-M1 NES, is highly conserved among all sequences from the influenza A virus subtype, but no similar NES motifs are found in the M1 sequences of influenza B or C viruses. The biological function of the Flu-A-M1 NES was demonstrated by its ability to translocate an enhanced green fluorescent protein (EGFP)-NES fusion protein from the nucleus to the cytoplasm in transfected cells, compared to the even nuclear and cytoplasmic distribution of EGFP. The translocation of EGFP-NES from the nucleus to the cytoplasm was not inhibited by leptomycin B. NES mutations in M1 caused a nuclear retention of the protein and an increased nuclear accumulation of NEP during transfection. Indeed, as shown by rescued recombinant viruses, the mutation of the NES impaired the nuclear export of M1 and significantly reduced the virus titer compared to titers of wild-type viruses. The NES-defective M1 protein was retained in the nucleus during infection, accompanied by a lowered efficiency of the nuclear export of viral RNPs (vRNPs). In conclusion, M1 nuclear export was specifically dependent on the Flu-A-M1 NES and critical for influenza A virus replication.
The nuclear export of the influenza A virus ribonucleoprotein (vRNP) is crucial for virus replication. As a major component of the vRNP, nucleoprotein (NP) alone can also be shuttled out of the nucleus by interacting with chromosome region maintenance 1 (CRM1) and is therefore hypothesized to promote the nuclear export of the vRNP. In the present study, three novel nuclear export signals (NESs) of the NP—NES1, NES2, and NES3—were identified as being responsible for mediating its nuclear export. The nuclear export of NES3 was CRM1 dependent, whereas that of NES1 or NES2 was CRM1 independent. Inactivation of these NESs led to an overall nuclear accumulation of NP. Mutation of all three NP-NESs significantly impaired viral replication. Based on structures of influenza virus NP oligomers, these three hydrophobic NESs are found present on the surface of oligomeric NPs. Functional studies indicated that oligomerization is also required for nuclear export of NP. Together, these results suggest that the nuclear export of NP is important for virus replication and relies on its NESs and oligomerization.
We present a compendium of N-ethyl-N-nitrosourea (ENU)-induced mouse mutations, identified in our laboratory over a period of 10 years either on the basis of phenotype or whole genome and/or whole exome sequencing, and archived in the Mutagenetix database. Our purpose is threefold: 1) to formally describe many point mutations, including those that were not previously disclosed in peer-reviewed publications; 2) to assess the characteristics of these mutations; and 3) to estimate the likelihood that a missense mutation induced by ENU will create a detectable phenotype.
In the context of an ENU mutagenesis program for C57BL/6J mice, a total of 185 phenotypes were tracked to mutations in 129 genes. In addition, 402 incidental mutations were identified and predicted to affect 390 genes. As previously reported, ENU shows strand asymmetry in its induction of mutations, particularly favoring T to A rather than A to T in the sense strand of coding regions and splice junctions. Some amino acid substitutions are far more likely to be damaging than others, and some are far more likely to be observed. Indeed, from among a total of 494 non-synonymous coding mutations, ENU was observed to create only 114 of the 182 possible amino acid substitutions that single base changes can achieve. Based on differences in overt null allele frequencies observed in phenotypic vs. non-phenotypic mutation sets, we infer that ENU-induced missense mutations create detectable phenotype only about 1 in 4.7 times. While the remaining mutations may not be functionally neutral, they are, on average, beneath the limits of detection of the phenotypic assays we applied.
Collectively, these mutations add to our understanding of the chemical specificity of ENU, the types of amino acid substitutions it creates, and its efficiency in causing phenovariance. Our data support the validity of computational algorithms for the prediction of damage caused by amino acid substitutions, and may lead to refined predictions as to whether specific amino acid changes are responsible for observed phenotypes. These data form the basis for closer in silico estimations of the number of genes mutated to a state of phenovariance by ENU within a population of G3 mice.
N-ethyl-N-nitrosourea; Mouse; C57BL/6J; Mutagenesis; Genetic screen; PolyPhen-2; Strand asymmetry; Phenotype
Estrogens play an important role in modulating the morphology and function of temporomandibular joints (TMJs), which is suggested to act via estrogen receptors (ERs). The present study was to investigate the expression of aggrecan, collagen type II (Col II), Col X, aromatase, ERα and ERβ in degenerative changes of mandibular condylar cartilage.
Forty male and 40 female 8-week-old rats were enrolled in this study. In experimental groups, the disordered occlusion was created by moving the first molars mesially and the third ones distally. Immunohistochemistry and real-time PCR were performed at the end of the second or fourth week.
Degenerative changes, characterized by interrupted continuity of hypertrophic layer, pyknotic and eosinophilic lesion with few nuclei, areas filled with eosinophilic nuclei, were observed in more joints from female experimental groups than male ones. However, thickening changes in hypertrophic layer were only found in male experimental groups. The gene expression of Col II, Col X and aggrecan increased in 4-wk male experimental subgroup (both P < 0.01), but decreased in 2-wk and 4-wk female subgroups (P < 0.05). The gene expression of ERα decreased in 2-wk male and female experimental subgroups (both P < 0.01), however, that of ERβ increased except the 2-wk female experimental subgroup (all P < 0.01). The expression of aromatase decreased in both male and female experimental subgroups (all P<0.01).
Mandibular condylar cartilage responses differently to the disordered occlusion in male and female rats. The levels of locally synthesized estrogen, ERα and ERβ may have limited attribution, if any, to the sex-specific cartilage response.
MicroRNAs (miRNAs) are a new class of small, endogenous RNAs that play a regulatory role in various biological and metabolic processes by negatively affecting gene expression at the post-transcriptional level. While the number of known Arabidopsis and rice miRNAs is continuously increasing, information regarding miRNAs from woody plants such as citrus remains limited. Solexa sequencing was performed at different developmental stages on both an early flowering mutant of trifoliate orange (precocious trifoliate orange, Poncirus trifoliata L. Raf.) and its wild-type in this study, resulting in the obtainment of 141 known miRNAs belonging to 99 families and 75 novel miRNAs in four libraries. A total of 317 potential target genes were predicted based on the 51 novel miRNAs families, GO and KEGG annotation revealed that high ranked miRNA-target genes are those implicated in diverse cellular processes in plants, including development, transcription, protein degradation and cross adaptation. To characterize those miRNAs expressed at the juvenile and adult development stages of the mutant and its wild-type, further analysis on the expression profiles of several miRNAs through real-time PCR was performed. The results revealed that most miRNAs were down-regulated at adult stage compared with juvenile stage for both the mutant and its wild-type. These results indicate that both conserved and novel miRNAs may play important roles in citrus growth and development, stress responses and other physiological processes.
Melastatin-like transient receptor potential channel 2 (TRPM2) is an oxidant-sensitive and cationic non-selective channel that is expressed in mammalian vascular endothelium. Here we investigated the functional role of TRPM2 channels in hydrogen peroxide (H2O2)-induced cytosolic Ca2+ ([Ca2+]i) elavation, whole-cell current increase, and apoptotic cell death in murine heart microvessel endothelial cell line H5V. A TRPM2 blocking antibody (TM2E3), which targets the E3 region near the ion permeation pore of TRPM2, was developed. Treatment of H5V cells with TM2E3 reduced the [Ca2+]i rise and whole-cell current change in response to H2O2. Suppressing TRPM2 expression using TRPM2-specific short hairpin RNA (shRNA) had similar inhibitory effect. H2O2-induced apoptotic cell death in H5V cells was examined using MTT assay, DNA ladder formation analysis, and DAPI-based nuclear DNA condensation assay. Based on these assays, TM2E3 and TRPM2-specific shRNA both showed protective effect against H2O2-induced apoptotic cell death. TM2E3 and TRPM2-specific shRNA also protect the cells from tumor necrosis factor (TNF)-α-induced cell death in MTT assay. In contrast, overexpression of TRPM2 in H5V cells resulted in an increased response in [Ca2+]i and whole-cell currents to H2O2. TRPM2 overexpression also aggravated the H2O2-induced apoptotic cell death. Downstream pathways following TRPM2 activation was examined. Results showed that TRPM2 activity stimulated caspase-8, caspase-9 and caspase-3. These findings strongly suggest that TRPM2 channel mediates cellular Ca2+ overload in response to H2O2 and contribute to oxidant-induced apoptotic cell death in vascular endothelial cells. Down-regulating endogenous TRPM2 could be a means to protect the vascular endothelial cells from apoptotic cell death.