Ribosome biogenesis is a fundamental and tightly regulated cellular process, including synthesis, processing, and assembly of rRNAs with ribosomal proteins. Protein arginine methyltransferases (PRMTs) have been implicated in many important biological processes, such as ribosome biogenesis. Two alternative precursor rRNA (pre-rRNA) processing pathways coexist in yeast and mammals; however, how PRMT affects ribosome biogenesis remains largely unknown. Here we show that Arabidopsis PRMT3 (AtPRMT3) is required for ribosome biogenesis by affecting pre-rRNA processing. Disruption of AtPRMT3 results in pleiotropic developmental defects, imbalanced polyribosome profiles, and aberrant pre-rRNA processing. We further identify an alternative pre-rRNA processing pathway in Arabidopsis and demonstrate that AtPRMT3 is required for the balance of these two pathways to promote normal growth and development. Our work uncovers a previously unidentified function of PRMT in posttranscriptional regulation of rRNA, revealing an extra layer of complexity in the regulation of ribosome biogenesis.
DNA methylation at the C-5 position of cytosine (5mC) is one of the best-studied epigenetic modifications and plays important roles in diverse biological processes. Iterative oxidation of 5mC by the ten-eleven translocation (Tet) family of proteins generates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC are selectively recognized and excised by thymine DNA glycosylase (TDG), leading to DNA demethylation. Functional characterization of Tet proteins has been complicated by the redundancy between the three family members. Using CRISPR/Cas9 technology, we generated mouse embryonic stem cells (ESCs) deficient for all three Tet proteins (Tet triple knockout [TKO]). Whole-genome bisulfite sequencing (WGBS) analysis revealed that Tet-mediated DNA demethylation mainly occurs at distally located enhancers and fine-tunes the transcription of genes associated with these regions. Functional characterization of Tet TKO ESCs revealed a role for Tet proteins in regulating the two-cell embryo (2C)-like state under ESC culture conditions. In addition, Tet TKO ESCs exhibited increased telomere-sister chromatid exchange and elongated telomeres. Collectively, our study reveals a role for Tet proteins in not only DNA demethylation at enhancers but also regulating the 2C-like state and telomere homeostasis.
A real-time PCR for detection and quantification of M. ovipneumoniae was developed using 9 recently sequenced M. ovipneumoniae genomes and primers targeting a putative adhesin gene p113. The assay proved to be specific and sensitive (with a detection limit of 22 genomic DNA) and could quantify M. ovipneumoniae DNA over a wide linear range, from 2.2 × 10(2) to 2.2 × 10(7) genomes.
With the exception of imprinted genes and certain repeats, DNA methylation is globally erased during preimplantation development. Recent studies have suggested that Tet3-mediated oxidation of 5-methylcytosine (5mC) and DNA replication-dependent dilution both contribute to global paternal DNA demethylation, but demethylation of the maternal genome occurs via replication. Here we present genome-scale DNA methylation maps for both the paternal and maternal genomes of Tet3-depleted and/or DNA replication-inhibited zygotes. In both genomes, we found that inhibition of DNA replication blocks DNA demethylation independently from Tet3 function and that Tet3 facilitates DNA demethylation largely by coupling with DNA replication. For both genomes, our data indicate that replication-dependent dilution is the major contributor to demethylation, but Tet3 plays an important role, particularly at certain loci. Our study thus defines the respective functions of Tet3 and DNA replication in paternal DNA demethylation and reveals an unexpected contribution of Tet3 to demethylation of the maternal genome.
In this paper, we report a novel polyol process to synthesize highly water-dispersible anatase titanium dioxide (TiO2) nanoparticles (?5 nm) by the introduction of inorganic oxidizing agent--KIO3. The obtained TiO2 nanoparticles are well dispersible in water at pH?5.0 and the resulting aqueous dispersion remains stable over months. The superior water-dispersibility of as-formed TiO2 is ascribed to the electrostatic repulsion from carboxylic acid group modified on TiO2 nanoparticles, which is the oxidation product of solvent diethylene glycol (DEG) by KIO3. Based on the characterization results, the formation processes of water-dispersibility TiO2 nanoparticles are proposed. Meanwhile, the synthesized TiO2 nanoparticles are found to be doped by iodine and exhibit excellent photocatalytic activity on degradation of rhodamine-B (RhB) under visible-light irradiation. The further tests demonstrate that the O(2-) is the main active species during photodegradation of RhB.
The selection of stably expressed reference genes is a prerequisite when evaluating gene expression, via real-time PCR, in cells in response to viral infections. The objective of our study was to identify suitable reference genes for mRNA expression analysis in chicken embryonic fibroblasts (CEF) after infection with avian leukosis virus subgroup J (ALV-J).
Protein ubiquitination is involved in most cellular processes. In Arabidopsis (Arabidopsis thaliana), ubiquitin-mediated protein degradation regulates the stability of key components of the circadian clock feedback loops and the photoperiodic flowering pathway. Here, we identified two ubiquitin-specific proteases, UBP12 and UBP13, involved in circadian clock and photoperiodic flowering regulation. Double mutants of ubp12 and ubp13 display pleiotropic phenotypes, including early flowering and short periodicity of circadian rhythms. In ubp12 ubp13 double mutants, CONSTANS (CO) transcript rises earlier than that of wild-type plants during the day, which leads to increased expression of FLOWERING LOCUS T. This, and analysis of ubp12 co mutants, indicates that UBP12 and UBP13 regulate photoperiodic flowering through a CO-dependent pathway. In addition, UBP12 and UBP13 regulate the circadian rhythm of clock genes, including LATE ELONGATED HYPOCOTYL, CIRCADIAN CLOCK ASSOCIATED1, and TIMING OF CAB EXPRESSION1. Furthermore, UBP12 and UBP13 are circadian controlled. Therefore, our work reveals a role for two deubiquitinases, UBP12 and UBP13, in the control of the circadian clock and photoperiodic flowering, which extends our understanding of ubiquitin in daylength measurement in higher plants.
Johnes disease (JD) is prevalent worldwide and has a significant impact on the global agricultural economy. In the present study, we evaluated the protective efficacy of a leuD (?leud) mutant and gained insight into differential immune responses after challenge with virulent M. avium subsp. paratuberculosis in a caprine colonization model. The immune response and protective efficacy were compared with those of the killed vaccine Mycopar. In vitro stimulation of peripheral blood mononuclear cells with johnin purified protein derivative showed that Mycopar and ?leuD generated similar levels of gamma interferon (IFN-?) but significantly higher levels than unvaccinated and challenged phosphate-buffered saline controls. However, only with ?leuD was the IFN-? response maintained. Flow cytometric analysis showed that the increase in IFN-? correlated with proliferation and activation (increased expression of CD25) of CD4, CD8, and ??T cells, but this response was significantly higher in ?leuD-vaccinated animals at some time points after challenge. Both Mycopar and ?leuD vaccines upregulated Th1/proinflammatory and Th17 cytokines and downregulated Th2/anti-inflammatory and regulatory cytokines at similar levels at almost all time points. However, significantly higher levels of IFN-? (at weeks 26 and 30), interleukin-2 (IL-2; week 18), IL-1b (weeks 14 and 22), IL-17 (weeks 18 and 22), and IL-23 (week 18) and a significantly lower level of IL-10 (weeks 14 and 18) and transforming growth factor ? (week 18) were detected in the ?leuD-vaccinated group. Most importantly, ?leuD elicited an immune response that significantly limited colonization of tissues compared to Mycopar upon challenge with wild-type M. avium subsp. paratuberculosis. In conclusion, the ?leuD mutant is a promising vaccine candidate for development of a live attenuated vaccine for JD in ruminants.
Johnes disease (JD), caused by Mycobacterium avium subsp paratuberculosis (MAP), occurs worldwide as chronic granulomatous enteritis of domestic and wild ruminants. To develop a cost effective vaccine, in a previous study we constructed an attenuated Salmonella strain that expressed a fusion product made up of partial fragments of MAP antigens (Ag85A, Ag85B and SOD) that imparted protection against challenge in a mouse model. In the current study we evaluated the differential immune response and protective efficacy of the Sal-Ag vaccine against challenge in a goat model as compared to the live attenuated vaccine MAP316F. PBMCs from goats vaccinated with Sal-Ag and challenged with MAP generated significantly lower levels of IFN-?, following in vitro stimulation with either Antigen-mix or PPD jhonin, than PBMC from MAP316F vaccinated animals. Flow cytometric analysis showed the increase in IFN-? correlated with a significantly higher level of proliferation of CD4, CD8 and ??T cells and an increased expression of CD25 and CD45R0 in MAP316F vaccinated animals as compared to control animals. Evaluation of a range of cytokines involved in Th1, Th2, Treg, and Th17 immune responses by quantitative PCR showed low levels of expression of Th1 (IFN-?, IL-2, IL-12) and proinflammatory cytokines (IL-6, IL-8, IL-18, TNF-?) in the Sal-Ag immunized group. Significant levels of Th2 and anti-inflammatory cytokines transcripts (IL-4, IL-10, IL-13, TGF-?) were expressed but their level was low and with a pattern similar to the control group. Over all, Sal-Ag vaccine imparted partial protection that limited colonization in tissues of some animals upon challenge with wild type MAP but not to the level achieved with MAP316F. In conclusion, the data indicates that Sal-Ag vaccine induced only a low level of protective immunity that failed to limit the colonization of MAP in infected animals. Hence the Sal-Ag vaccine needs further refinement to increase its efficacy.
Protein arginine methyltransferase 10 (PRMT10) is a type I arginine methyltransferase that is essential for regulating flowering time in Arabidopsis thaliana. We present a 2.6 Å resolution crystal structure of A. thaliana PRMT 10 (AtPRMT10) in complex with a reaction product, S-adenosylhomocysteine. The structure reveals a dimerization arm that is 12-20 residues longer than PRMT structures elucidated previously; as a result, the essential AtPRMT10 dimer exhibits a large central cavity and a distinctly accessible active site. We employ molecular dynamics to examine how dimerization facilitates AtPRMT10 motions necessary for activity, and we show that these motions are conserved in other PRMT enzymes. Finally, functional data reveal that the 10 N-terminal residues of AtPRMT10 influence substrate specificity, and that enzyme activity is dependent on substrate protein sequences distal from the methylation site. Taken together, these data provide insights into the molecular mechanism of AtPRMT10, as well as other members of the PRMT family of enzymes. They highlight differences between AtPRMT10 and other PRMTs but also indicate that motions are a conserved element of PRMT function.
Mycoplasma ovipneumoniae is associated with chronic nonprogressive pneumonia in both sheep and goats. Studies concerning its molecular pathogenesis, genetic analysis, and vaccine development have been hindered due to limited genomic information. Here, we announce the first complete genome sequence of this organism.
Uniform tellurium nanorod arrays (TNA) have been successfully deposited directly on Pt/FTO (F-doped SnO(2))/glass substrate through a facile surfactant-assisted approach, which involved chemical reduction of TeO(3)(2-) ions by hydrazine hydrate. The whole synthesis process is highly repeatable and performed simply by immersing the Pt/FTO/glass in the solution for a certain time. During the growth of TNA, Pt catalyzed the reduction of TeO(3)(2-) ions by hydrazine hydrate and Te nanoparticles were deposited firmly on the substrate at first. Then, under the regulation of the surfactant (cetyltrimethylammonium bromide, CTAB), the deposited Te grew into nanorod arrays and adhered firmly to the substrate. Similar Te nanorod arrays could also grow on a Pd substrate which has the same catalytic performance as that of Pt. The as-synthesized TNA could be used as a good template to synthesize platinum-and gold-coated nanorods through convenient galvanic replacement. As a demonstration of potential application, the gold/tellurium nanorods showed uniform surface-enhanced Raman scattering (SERS) using rhodamine 6G (Rh6G) as the analyte. This approach provides a simple route for the growth of standing Te nanorods on a substrate, which may be used for the synthesis of other standing one-dimensional materials through a similar mechanism.
Polycomb group (PcG)-mediated histone H3 lysine 27 trimethylation (H3K27me3) has a key role in gene repression and developmental regulation. There is evidence that H3K27me3 is actively removed in plants, but it is not known how this occurs. Here we show that RELATIVE OF EARLY FLOWERING 6 (REF6), also known as Jumonji domain-containing protein 12 (JMJ12), specifically demethylates H3K27me3 and H3K27me2, whereas its metazoan counterparts, the KDM4 proteins, are H3K9 and H3K36 demethylases. Plants overexpressing REF6 resembled mutants defective in H3K27me3-mediated gene silencing. Genetic interaction tests indicated that REF6 acts downstream of H3K27me3 methyltransferases. Mutations in REF6 caused ectopic and increased H3K27me3 level and decreased mRNA expression of hundreds of genes involved in regulating developmental patterning and responses to various stimuli. Our work shows that plants and metazoans use conserved mechanisms to regulate H3K27me3 dynamics but use distinct subfamilies of enzymes.
Platinum nanostructured networks (PNNs) can be synthesized through the chemical reduction of H(2)PtCl(6) by benzyl alcohol under microwave irradiation without the introduction of any surfactants, templates, or seeds. The synthesis route utilizes benzyl alcohol as both the reductant and the structure-directing agent, and thus, the process is particularly simple and highly repeatable. The formation of the PNN structure was ascribed to the collision-induced fusion of Pt nanocrystals owing to the cooperative functions of microwave irradiation and benzyl alcohol. Compared with a commercial Pt/C catalyst, the as-prepared PNNs possessed superior electrochemical activity and stability on the oxidation of methanol because of the unique 3D nanostructured networks and abundant defects formed during the assembly process. This study may provide a facile microwave-induced approach for the synthesis of other 3D nanostructured noble metals or their alloys.
Radiation-induced sarcoma in the head and neck (RISHN) is a rare condition whose clinical presentation and management remain difficult because of its low incidence. In this retrospective study, we analyzed the symptoms, diagnosis, and the treatment of 16,634 patients with head and neck disease, who received radiotherapy between 1960 and 2010 at the Affiliated Tumor Hospital and its predecessor, Guangxi Medical University, China. Among these patients, 16 with a first tumor of nasopharyngeal carcinoma (NPC) and 1 with squamous carcinoma of the tongue met the criteria of RISHN in the head and neck. Our epidemiological data showed that the incidence of RISHN rose from 0.06 to 0.17% from 1960 to 2010; the 3-year overall survival rate was 19.1%, and 3-year disease-free survival rate was 11.1%. The mean latency (SD) period was 93.2 (33) months. Based on the experiences at our institution, we suggest that RISHN is a rare complication after radiotherapy for head and neck tumors, especially NPC. Owing to its low incidence, it should not be a major factor affecting decisions about radiotherapy. Nevertheless, there may be a possibility of increasing incidence of RISHN after radiotherapy of NPC, as shown in our epidemiological results. Given the poor prognosis of RISHN, this possibility should be taken into serious consideration before determination of high-dose radiotherapy for patients with NPC and other head and neck tumors.
Protein arginine methylation, one of the most abundant and important posttranslational modifications, is involved in a multitude of biological processes in eukaryotes, such as transcriptional regulation and RNA processing. Symmetric arginine dimethylation is required for snRNP biogenesis and is assumed to be essential for pre-mRNA splicing; however, except for in vitro evidence, whether it affects splicing in vivo remains elusive. Mutation in an Arabidopsis symmetric arginine dimethyltransferase, AtPRMT5, causes pleiotropic developmental defects, including late flowering, but the underlying molecular mechanism is largely unknown. Here we show that AtPRMT5 methylates a wide spectrum of substrates, including some RNA binding or processing factors and U snRNP AtSmD1, D3, and AtLSm4 proteins, which are involved in RNA metabolism. RNA-seq analyses reveal that AtPRMT5 deficiency causes splicing defects in hundreds of genes involved in multiple biological processes. The splicing defects are identified in transcripts of several RNA processing factors involved in regulating flowering time. In particular, splicing defects at the flowering regulator flowering locus KH domain (FLK) in atprmt5 mutants reduce its functional transcript and protein levels, resulting in the up-regulation of a flowering repressor flowering locus C (FLC) and consequently late flowering. Taken together, our findings uncover an essential role for arginine methylation in proper pre-mRNA splicing that impacts diverse developmental processes.
The molecular diversity of the gene encoding the outer membrane protein A (OmpA) of Haemophilus parasuis has been unclear. In this study, the structural characteristics, sequence types, and genetic diversity of ompA were investigated in 15 H. parasuis reference strains of different serovars and 20 field isolates. Three nucleotide lengths of the complete open reading frame (ORF) of ompA were found: 1098 base pairs (bp), 1104 bp, and 1110 bp. The OmpA contained 4 hypervariable domains, mainly encoding the 4 putative surface-exposed loops, which makes it a potential molecular marker for genotyping. Western blot analysis showed that the recombinant OmpAs of serovars 4 and 5 could cross-react with antiserum to all 15 serovars. Hence, although ompA of H. parasuis exhibited high variation among serovars, this variation did not seem to affect the strong antigenic characteristics of OmpA.
In diverse eukaryotes, constitutively silent sequences, such as transposons and repeats, are marked by methylation at histone H3 lysine 9 (H3K9me). Although selective H3K9me is critical for maintaining genome integrity, mechanisms to exclude H3K9me from active genes remain largely unexplored. Here, we show in Arabidopsis that the exclusion depends on a histone demethylase gene, IBM1 (increase in BONSAI methylation). Loss-of-function ibm1 mutation results in ectopic H3K9me and non-CG methylation in thousands of genes. The ibm1-induced genic H3K9me depends on both histone methylase KYP/SUVH4 and DNA methylase CMT3, suggesting interdependence of two epigenetic marks--H3K9me and non-CG methylation. Notably, IBM1 enhances loss of H3K9me in transcriptionally de-repressed sequences. Furthermore, disruption of transcription in genes induces ectopic non-CG methylation, which mimics the loss of IBM1 function. We propose that active chromatin is stabilized by an autocatalytic loop of transcription and H3K9 demethylation. This process counteracts a similarly autocatalytic accumulation of silent epigenetic marks, H3K9me and non-CG methylation.
Histone methylation plays a fundamental role in regulating diverse developmental processes and is also involved in silencing repetitive sequences in order to maintain genome stability. The methylation marks are written on lysine or arginine by distinct enzymes, namely, histone lysine methyltransferases (HKMTs) or protein arginine methyltransferases (PRMTs). Once established, the methylation marks are specifically recognized by the proteins that act as readers and are interpreted into specific biological outcomes. Histone methylation status is dynamic; methylation marks can be removed by eraser enzymes, the histone demethylases (HDMs). The proteins responsible for writing, reading, and erasing the methylation marks are known mostly in animals. During the past several years, a growing body of literature has demonstrated the impact of histone methylation on genome management, transcriptional regulation, and development in plants. The aim of this review is to summarize the biochemical, genetic, and molecular action of histone methylation in two plants, the dicot Arabidopsis and the monocot rice.
Duck hepatitis A virus genotype C (DHAV-C), recognized recently, is one of the pathogens causing fatal duck viral hepatitis in ducklings, especially in Asia. To demonstrate the pathogenesis of the DHAV-C isolate, 3-day-old specific pathogen free ducklings were inoculated subcutaneously with a DHAV-C isolate and the clinical signs were observed. Virus distribution, histological and apoptotic morphological changes of various tissues were examined at different times post inoculation. The serial, characteristic changes included haemorrhage and swelling of the liver. Apoptotic cells and virus antigen staining were found in all of the tissues examined. Where more virus antigen staining was detected, there were more severe histopathological and apoptotic changes. The amount of virus antigen and the histological and apoptotic morphological changes agreed with each other and became increasingly severe with length of time after infection. Apoptotic cells were ubiquitously distributed, especially among lymphocytes, macrophages and monocytes in immune organs such as the bursa of Fabricius, thymus and spleen, and in liver, kidney and cerebral cells. Necrosis was also observed within 72 h post inoculation in all organs examined, except the cerebrum, and was characterized by cell swelling and collapsed plasma membrane. These results suggest that the recent outbreak of disease caused by DHAV-C virus is pantropic, causing apoptosis and necrosis of different organs. The apoptosis and necrosis caused by the DHAV-C field strain in this study is associated with pathogenesis and DHAV-C-induced lesions.
Electrochemical detection for point-of-care diagnostics is of great interest due to its high sensitivity, fast analysis time and ability to operate on a small scale. Herein, we report the fabrication of a nanoporous thin-film electrode and its application in the configuration of a simple and robust enzymatic biosensor. The nanoporous thin-film was formed in a planar gold electrode through an alloying/dealloying process. The nanoporous electrode has an electroactive surface area up to 40 times higher than that of a flat gold electrode of the same size. The nanoporous electrode was used as a substrate to build an enzymatic electrochemical biosensor for the detection of glucose in standard samples and control serum samples. The example glucose biosensor has a linear response up to 30 mM, with a high sensitivity of 0.50 ?A mM?¹ mm?², and excellent anti-interference ability against lactate, uric acid and ascorbic acid. Abundant catalyst and enzyme were stably entrapped in the nanoporous structure, leading to high stability and reproducibility of the biosensor. Development of such nanoporous structure enables the miniaturization of high-performance electrochemical biosensors for point-of-care diagnostics or environmental field testing.
A series of novel benzamides derivatives was designed and synthesized as HDAC inhibitors. Exploration of the structure-activity relationships resulted in compounds that are potent in vitro. In addition, the best compound 1a exhibited an acceptable pharmacokinetic profile with bioavailability in rat of 81% and could be considered as a candidate compound for further development.
Copper indium disulphide (CIS) nanocrystals (NCs) were prepared using a one-pot synthesis. The stoichiometry was optimized based on its current density as measured by photoelectrochemical (PEC) experiments at interfaces between NC films deposited on ITO and 0.1 M methyl viologen dichloride (MV(2+)) solution. This method also offers insight into the kinetics of the photoreaction. A copper poor sulphur rich starting ratio was found to produce a copper-rich, indium-poor and slightly sulphur rich material. Further NC characterization was performed with SEM and TEM to investigate the morphology and crystallinity of the 30-70 nm NCs. The oxidation states of the individual elements were determined to be I, III, and 2- for Cu, In and S, respectively. Characteristics of optimal as-prepared NCs were found to be compatible among high functioning absorbing layers.
Arabidopsis AtPRMT10 is a plant-specific type I protein arginine methyltransferase that can asymmetrically dimethylate arginine 3 of histone H4 with auto-methylation activity. Mutations of AtPRMT10 derepress FLOWERING LOCUS C (FLC) expression resulting in a late-flowering phenotype. Here, to further investigate the biochemical characteristics of AtPRMT10, we analyzed a series of mutated forms of the AtPRMT10 protein. We demonstrate that the conserved "VLD" residues and "double-E loop" are essential for enzymatic activity of AtPRMT10. In addition, we show that Arg54 and Cys259 of AtPRMT10, two residues unreported in animals, are also important for its enzymatic activity. We find that Arg13 of AtPRMT10 is the auto-methylation site. However, substitution of Arg13 to Lys13 does not affect its enzymatic activity. In vivo complementation assays reveal that plants expressing AtPRMT10 with VLD-AAA, E143Q or E152Q mutations retain high levels of FLC expression and fail to rescue the late-flowering phenotype of atprmt10 plants. Taken together, we conclude that the methyltransferase activity of AtPRMT10 is essential for repressing FLC expression and promoting flowering in Arabidopsis.
A series of novel bis-aryl ureas containing trifluoromethyl imidazolyl group targeting Raf kinase were designed and synthesized based on the lead compound of Sorafenib. All the prepared compounds were evaluated for their in vitro antiproliferative activities against three human cancer cell lines including MDA-MB-231 (breast), BGC-823 (gastric), and SMMC-7721 (liver). Several compounds from the series exhibited excellent antitumor activities against all three tested cancer lines. Further their inhibitory activities against Raf kinase were investigated, and three compounds (11c, 11d, and 11p) demonstrated better activities than contrast drug Sorafenib. Especially compound 11c was found to be a potent and selective Raf kinase inhibitor and could be considered as a candidate compound for further development.
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