Factors related to children's caries: a structural equation modeling approach

published_or_final_versio

Methylation profiling of Epstein-Barr virus immediate-early gene promoters, BZLF1 and BRLF1 in tumors of epithelial, NK- and B-cell origins

<p>Abstract</p> <p>Background</p> <p>Epstein-Barr virus (EBV) establishes its latency in EBV-associated malignancies, accompanied by occasionally reactivated lytic cycle. Promoter CpG methylation of EBV genome plays an essential role in maintaining viral latency. Two immediate-early (IE) genes, BZLF1 and BRLF1, induce the switch from latent to lytic infection. Studies of methylation-dependent binding of BZLF1 and BRLF1 to EBV promoters have been well reported, but little is known about the methylation status of <it>BZLF1 </it>and <it>BRLF1 </it>promoters (Zp and Rp) in tumor samples.</p> <p>Methods</p> <p>We evaluated the methylation profiles of Zp and Rp by methylation-specific PCR (MSP) and bisulfite genomic sequencing (BGS), as well as <it>BZLF1 </it>and <it>BRLF1 </it>expression by semiquantitative reverse transcription (RT)-PCR in tumors of epithelial, NK- and B-cell origins.</p> <p>Results</p> <p>We found that both Zp and Rp were hypermethylated in all studied EBV-positive cell lines and tumors of lymphoid (B- or NK cell) or epithelial origin, while unmethylated Zp and Rp alleles were detected in cell lines expressing <it>BZLF1 </it>and <it>BRLF1</it>. Following azacytidine treatment or combined with trichostatin A (TSA), the expression of <it>BZLF1 </it>and <it>BRLF1 </it>was restored along with concomitant promoter demethylation, which subsequently induced the reactivation of early lytic gene <it>BHRF1 </it>and late lytic gene <it>BLLF1</it>.</p> <p>Conclusions</p> <p>Hypermethylation of Zp and Rp mediates the frequent silencing of <it>BZLF1 </it>and <it>BRLF1 </it>in EBV-associated tumors, which could be reactivated by demethylation agent and ultimately initiated the EBV lytic cascade.</p

Localisation of RNAs into the germ plasm of vitellogenic xenopus oocytes

We have studied the localisation of mRNAs in full-grown Xenopus laevis oocytes by injecting fluorescent RNAs, followed by confocal microscopy of the oocyte cortex. Concentrating on RNA encoding the Xenopus Nanos homologue, nanos1 (formerly Xcat2), we find that it consistently localised into aggregated germ plasm ribonucleoprotein (RNP) particles, independently of cytoskeletal integrity. This implies that a diffusion/entrapment-mediated mechanism is active, as previously reported for previtellogenic oocytes. Sometimes this was accompanied by localisation into scattered particles of the “late”, Vg1/VegT pathway; occasionally only late pathway localisation was seen. The Xpat RNA behaved in an identical fashion and for neither RNA was the localisation changed by any culture conditions tested. The identity of the labelled RNP aggregates as definitive germ plasm was confirmed by their inclusion of abundant mitochondria and co-localisation with the germ plasm protein Hermes. Further, the nanos1/Hermes RNP particles are interspersed with those containing the germ plasm protein Xpat. These aggregates may be followed into the germ plasm of unfertilized eggs, but with a notable reduction in its quantity, both in terms of injected molecules and endogenous structures. Our results conflict with previous reports that there is no RNA localisation in large oocytes, and that during mid-oogenesis even germ plasm RNAs localise exclusively by the late pathway. We find that in mid oogenesis nanos1 RNA also localises to germ plasm but also by the late pathway. Late pathway RNAs, Vg1 and VegT, also may localise into germ plasm. Our results support the view that mechanistically the two modes of localisation are extremely similar, and that in an injection experiment RNAs might utilise either pathway, the distinction in fates being very subtle and subject to variation. We discuss these results in relation to their biological significance and the results of others

Structure-Function Analysis of Barley NLR Immune Receptor MLA10 Reveals Its Cell Compartment Specific Activity in Cell Death and Disease Resistance

Plant intracellular immune receptors comprise a large number of multi-domain proteins resembling animal NOD-like receptors (NLRs). Plant NLRs typically recognize isolate-specific pathogen-derived effectors, encoded by avirulence (AVR) genes, and trigger defense responses often associated with localized host cell death. The barley MLA gene is polymorphic in nature and encodes NLRs of the coiled-coil (CC)-NB-LRR type that each detects a cognate isolate-specific effector of the barley powdery mildew fungus. We report the systematic analyses of MLA10 activity in disease resistance and cell death signaling in barley and Nicotiana benthamiana. MLA10 CC domain-triggered cell death is regulated by highly conserved motifs in the CC and the NB-ARC domains and by the C-terminal LRR of the receptor. Enforced MLA10 subcellular localization, by tagging with a nuclear localization sequence (NLS) or a nuclear export sequence (NES), shows that MLA10 activity in cell death signaling is suppressed in the nucleus but enhanced in the cytoplasm. By contrast, nuclear localized MLA10 is sufficient to mediate disease resistance against powdery mildew fungus. MLA10 retention in the cytoplasm was achieved through attachment of a glucocorticoid receptor hormone-binding domain (GR), by which we reinforced the role of cytoplasmic MLA10 in cell death signaling. Together with our data showing an essential and sufficient nuclear MLA10 activity in disease resistance, this suggests a bifurcation of MLA10-triggered cell death and disease resistance signaling in a compartment-dependent manner

DNA barcoding and a precise morphological comparison revealed a cryptic species in the Nippolachnus piri complex (Hemiptera: Aphididae: Lachninae)

Nippolachnus is a small Palaearctic-Oriental genus of very characteristic aphids that live on the leaves of woody Rosaceae. One species, N. piri, has hitherto been regarded to be widely distributed and relatively polyphagous. Members of this genus are considered to be easy to recognize due to the absence of the ocular tubercle and triommatidia on the head. We conducted research on the morphology and generic characters of Nippolachnus piri complex using scanning electron microscopy (for the first time) and DNA barcoding. We analyzed N. piri populations on Pyrus and other plants (Eriobotrya, Rhaphiolepis and Sorbus) in Japan and the Republic of Korea. Specifically, a high genetic divergence value was found between the N. piri populations associated with different host plants. SEM investigation of the head capsule revealed that a triommatidium is present under the compound eye, despite their lack of an ocular tubercle. We propose Nippolachnus micromeli Shinji, 1924 stat. nov. as a cryptic species in the N. piri complex based on a morphological comparison, DNA barcoding and different host-plant associations. Illustrations and descriptions of studied species are given. Morphological keys to the apterae and alatae of all known species of the genus Nippolachnus are also provided

RMDAP: A Versatile, Ready-To-Use Toolbox for Multigene Genetic Transformation

Background: The use of transgenes to improve complex traits in crops has challenged current genetic transformation technology for multigene transfer. Therefore, a multigene transformation strategy for use in plant molecular biology and plant genetic breeding is thus needed. Methodology/Principal Findings: Here we describe a versatile, ready-to-use multigene genetic transformation method, named the Recombination-assisted Multifunctional DNA Assembly Platform (RMDAP), which combines many of the useful features of existing plant transformation systems. This platform incorporates three widely-used recombination systems, namely, Gateway technology, in vivo Cre/loxP and recombineering into a highly efficient and reliable approach for gene assembly. RMDAP proposes a strategy for gene stacking and contains a wide range of flexible, modular vectors offering a series of functionally validated genetic elements to manipulate transgene overexpression or gene silencing involved in a metabolic pathway. In particular, the ability to construct a multigene marker-free vector is another attractive feature. The built-in flexibility of original vectors has greatly increased the expansibility and applicability of the system. A proof-ofprinciple experiment was confirmed by successfully transferring several heterologous genes into the plant genome. Conclusions/Significance: This platform is a ready-to-use toolbox for full exploitation of the potential for coordinate regulation of metabolic pathways and molecular breeding, and will eventually achieve the aim of what we call ‘‘one-sto

The Function of Heterodimeric AP-1 Comprised of c-Jun and c-Fos in Activin Mediated Spemann Organizer Gene Expression

BACKGROUND:Activator protein-1 (AP-1) is a mediator of BMP or FGF signaling during Xenopus embryogenesis. However, specific role of AP-1 in activin signaling has not been elucidated during vertebrate development. METHODOLOGY/PRINCIPAL FINDINGS:We provide new evidence showing that overexpression of heterodimeric AP-1 comprised of c-jun and c-fos (AP-1(c-Jun/c-Fos)) induces the expression of BMP-antagonizing organizer genes (noggin, chordin and goosecoid) that were normally expressed by high dose of activin. AP-1(c-Jun/c-Fos) enhanced the promoter activities of organizer genes but reduced that of PV.1, a BMP4-response gene. A loss of function study clearly demonstrated that AP-1(c-Jun/c-Fos) is required for the activin-induced organizer and neural gene expression. Moreover, physical interaction of AP-1(c-Jun/c-Fos) and Smad3 cooperatively enhanced the transcriptional activity of goosecoid via direct binding on this promoter. Interestingly, Smad3 mutants at c-Jun binding site failed in regulation of organizer genes, indicating that these physical interactions are specifically necessary for the expression of organizer genes. CONCLUSIONS/SIGNIFICANCE:AP-1(c-Jun/c-Fos) plays a specific role in organizer gene expression in downstream of activin signal during early Xenopus embryogenesis