Prosthetic and orthodontic rehabilitation of a patient with severe maxillary central incisor root resorption associated with impacted maxillary canines.

OBJECTIVE: This case report describes the orthodontic and prosthetic rehabilitation of a patient with resorption of the roots of the maxillary central incisors due to the ectopic maxillary canines. CLINICAL CONSIDERATIONS: A 16-year-old woman presented with severe resorption of the roots of the maxillary central incisors due to the ectopic maxillary canines. The impacted canines were orthodontically tracted with a lingual arch-supporting temporary central incisors and vertical elastics, and, undesirable root proximity was later corrected by moving the canines distally 1.5 mm apart. Gingival replacement cords were placed into the gingival sulcus of the canines, and tooth preparation was performed along with rotary gingival curettage of the interdental papilla. Convex form was provided for the mesial and labio-distal subgingival contour of the restorations. CONCLUSIONS: The creeping attachment of the interdental papilla was successfully achieved by the orthodontic arrangement of interdental distance and the prosthetic stimulus via the retraction cord, rotary curettage, and convex mesial subgingival contours. In addition, selective retraction of the labio-distal gingiva by overcontoured restorations moved the gingival zenith position (GZP) distally. Finally, the canine crown morphology and gingival level mimicked the central incisors. CLINICAL SIGNIFICANCE: This clinical report introduces a treatment workflow of to recover the esthetic disturbance due to severe root resorption of the maxillary central incisors associated with impacted maxillary canines. The present orthodontic and prosthetic procedure can improve both hard and soft tissue esthetics and could be used in similar cases, such as malformed teeth and tooth autotransplantation or transposition with disturbances in the interdental papilla height or the GZP.

Direct projection from the lateral habenula to the trigeminal mesencephalic nucleus in rats.

Trigeminal mesencephalic nucleus (Vmes) neurons are primary afferents conveying deep sensation from the masticatory muscle spindles or the periodontal mechanoreceptors, and are crucial for controlling jaw movements. Their cell bodies exist in the brain and receive descending commands from a variety of cortical and subcortical structures involved in limbic (emotional) systems. However, it remains unclear how the lateral habenula (LHb), a center of negative emotions (e.g., pain, stress and anxiety), can influence the control of jaw movements. To address this issue, we examined whether and how the LHb directly projects to the Vmes by means of neuronal tract tracing techniques in rats. After injections of a retrograde tracer Fluorogold in the rostral and caudal Vmes, a number of neurons were labeled in the lateral division of LHb (LHbl) bilaterally, whereas a few neurons were labeled in the medial division of LHb (LHbm) bilaterally. After injections of an anterograde tracer, biotinylated dextranamine (BDA) in the LHbl, a small number of labeled axons were distributed bilaterally in the rostral and caudal levels of Vmes, where some labeled axonal boutons contacted the cell body of rostral and caudal levels of Vmes neurons bilaterally. After the BDA injection into the LHbm, however, no axons were labeled bilaterally in the rostral and caudal levels of Vmes. Therefore, the present study for the first time demonstrated the direct projection from the LHbl to the Vmes and the detailed projection patterns, suggesting that jaw movements are modulated by negative emotions that are signaled by LHbl neurons.

A simple and robust method for establishing homogeneous mouse epiblast stem cell lines by wnt inhibition.

Epiblast stem cells (EpiSCs) are pluripotent stem cells derived from epiblasts of postimplantation mouse embryos, and thus provide a useful model for studying "primed" pluripotent states. Here, we devised a simple and robust technique to derive high-quality EpiSCs using an inhibitor of WNT secretion. Using this method, we readily established EpiSC lines with high efficiency and were able to use whole embryonic portions without having to separate the epiblast from the visceral endoderm (VE). Expression analyses revealed that these EpiSCs maintained a homogeneous, undifferentiated status, yet showed high potential for differentiation both in vitro and in teratomas. Unlike EpiSCs derived by the original protocol, new EpiSC lines required continuous treatment with the Wnt inhibitor, suggesting some intrinsic differences from the existing EpiSCs. The homogeneous properties of this new version of EpiSCs should facilitate studies on the establishment and maintenance of a "primed" pluripotent state, and directed differentiation from the primed state.

Large, male germ cell-specific hypomethylated DNA domains with unique genomic and epigenomic features on the mouse X chromosome.

To understand the epigenetic regulation required for germ cell-specific gene expression in the mouse, we analysed DNA methylation profiles of developing germ cells using a microarray-based assay adapted for a small number of cells. The analysis revealed differentially methylated sites between cell types tested. Here, we focused on a group of genomic sequences hypomethylated specifically in germline cells as candidate regions involved in the epigenetic regulation of germline gene expression. These hypomethylated sequences tend to be clustered, forming large (10 kb to ~9 Mb) genomic domains, particularly on the X chromosome of male germ cells. Most of these regions, designated here as large hypomethylated domains (LoDs), correspond to segmentally duplicated regions that contain gene families showing germ cell- or testis-specific expression, including cancer testis antigen genes. We found an inverse correlation between DNA methylation level and expression of genes in these domains. Most LoDs appear to be enriched with H3 lysine 9 dimethylation, usually regarded as a repressive histone modification, although some LoD genes can be expressed in male germ cells. It thus appears that such a unique epigenomic state associated with the LoDs may constitute a basis for the specific expression of genes contained in these genomic domains.

Generation of a novel germline stem cell line expressing a germline-specific reporter in the mouse.

Germline stem (GS) cells are stem cell lines derived from postnatal male germline cells. Remarkably, GS cells can form functional spermatozoa when transplanted into infertile host mouse testes, indicating that GS cells have spermatogonial stem cell (SSC) activity. As GS cells are the only type with SSC activity, they are most suitable for in vitro studies on male germ cell differentiation. However, GS cells can deviate from the germ cell state to become other types of cells, depending on culture conditions. Therefore, it is desirable to have a monitor system to ensure that GS cells are kept at the germ cell state in culture. Here, we established GS cell lines from neonatal testes of transgenic mice that express the fluorescent protein, Venus, whose gene expression is driven by the promoter of Mvh (mouse Vasa homolog), a gene highly specific to mammalian germ cells. This novel cell line has genuine GS cell properties equivalent to existing GS lines, including the ability to generate viable offspring. This Mvh-Venus GS cell line, to our knowledge, is the first one expressing a germ cell-specific reporter. This valuable resource should provide new opportunities for studies on male germ cell differentiation.

Incomplete X-inactivation initiated by a hypomorphic Xist allele in the mouse.

X chromosome inactivation (X-inactivation) in female mammals is triggered by differential upregulation of the Xist gene on one of the two X chromosomes and subsequent coating of the X in cis with its non-coding transcripts. Although targeted mutation has clearly shown that Xist is essential for X-inactivation in cis, the molecular mechanism by which Xist RNA induces chromosome silencing is largely unknown. Here, we demonstrate that an Xist mutant generated previously in mouse by gene targeting, Xist(IVS), is unique in that it partially retains the capacity to silence the X chromosome. Although Xist(IVS) is differentially upregulated and its mutated transcript coats the X chromosome in cis in embryonic and extra-embryonic tissues, X-inactivation thus initiated does not seem to be fully established. The state of such incomplete inactivation is probably unstable and the mutated X is apparently reactivated in a subset of extra-embryonic tissues and, perhaps, early epiblastic cells. Xist(IVS), which can be referred to as a partial loss-of-function mutation, would provide an opportunity to dissect the molecular mechanism of Xist RNA-mediated chromosome silencing.

Impeding Xist expression from the active X chromosome improves mouse somatic cell nuclear transfer.

Cloning mammals by means of somatic cell nuclear transfer (SCNT) is highly inefficient because of erroneous reprogramming of the donor genome. Reprogramming errors appear to arise randomly, but the nature of nonrandom, SCNT-specific errors remains elusive. We found that Xist, a noncoding RNA that inactivates one of the two X chromosomes in females, was ectopically expressed from the active X (Xa) chromosome in cloned mouse embryos of both sexes. Deletion of Xist on Xa showed normal global gene expression and resulted in about an eight- to ninefold increase in cloning efficiency. We also identified an Xist-independent mechanism that specifically down-regulated a subset of X-linked genes through somatic-type repressive histone blocks. Thus, we have identified nonrandom reprogramming errors in mouse cloning that can be altered to improve the efficiency of SCNT methods.

Natural antisense transcripts are co-expressed with sense mRNAs in synaptoneurosomes of adult mouse forebrain.

Natural antisense transcripts and overlapping sense transcripts are expressed in a variety of tissues, including adult mouse brain. Here we show that a subset of mRNA-like sense-antisense transcript pairs are co-expressed within synaptoneurosomes of adult mouse forebrain, a subcellular fraction that is enriched in pinched-off dendritic spines of pyramidal neurons. Several of these pairs involve mRNAs that have been implicated in synaptic functions and in Alzheimer disease pathways. This study provides evidence that a new class of noncoding RNAs (natural antisense transcripts) are expressed near synapses, and encourages further studies of their roles in neuronal function.

Comparative expression analysis uncovers novel features of endogenous antisense transcription.

Increasing numbers of sense-antisense transcripts (SATs), which are transcribed from the same chromosomal location but in opposite directions, have been identified in various eukaryotic species, but the biological meanings of most SATs remain unclear. To improve understanding of natural sense-antisense transcription, we performed comparative expression profiling of SATs conserved among humans and mice. Using custom oligo-arrays loaded with probes that represented SATs with both protein-coding and non-protein-coding transcripts, we showed that 33% of the 291 conserved SATs displayed identical expression patterns in the two species. Among these SATs, expressional balance inversion of sense-antisense genes was mostly observed in testis at a tissue-specific manner. Northern analyses of the individual conserved SAT loci revealed that: (i) a smeary hybridization pattern was present in mice, but not in humans, and (2) small RNAs (about 60 to 80 nt) were detected from the exon-overlapping regions of SAT loci. In addition, further analyses showed marked alteration of sense-antisense expression balance throughout spermatogenesis in testis. These results suggest that conserved SAT loci are rich in potential regulatory roles that will help us understand this new class of transcripts underlying the mammalian genome.

Recapitulation of in vivo gene expression during hepatic differentiation from murine embryonic stem cells.

Hepatic differentiation at the molecular level is poorly understood, mainly because of the lack of a suitable model. Recently, using adherent monoculture conditions, we demonstrated the direct differentiation of hepatocytes from embryonic stem (ES) cells. In this study, we exploited the direct differentiation model to compare the gene expression profiles of ES cell-derived hepatocytes with adult mouse liver using DNA microarray technology. The results showed that the ES cell-derived hepatocyte gene expression pattern is very similar to adult mouse liver. Through further analysis of gene ontology categories for the 232 most radically altered genes, we found that the significant categories related to hepatic function. Furthermore, through the use of small interfering RNA technology in vitro, hepatocyte nuclear factor 3beta/FoxA2 was identified as having an essential role in hepatic differentiation. These results demonstrate that ES cell-derived hepatocytes recapitulate the gene expression profile of adult mouse liver to a significant degree and indicate that our direct induction system progresses via endoderm differentiation. In conclusion, our system closely mimics in vivo hepatic differentiation at the transcriptional level and could, therefore, be useful for studying the molecular basis of hepatocyte differentiation per se.

Cloud point extraction and preconcentration for the gas chromatography of phenothiazine tranquilizers in spiked human serum.

Cloud point extraction was successfully applied to the preconcentration of phenothiazine derivatives, such as pericyazine (PC), chlorpromazine (CP) and fluphenazine (FUL), for gas chromatography (GC). Phenothiazine derivatives were separated from surfactants by passing the surfactant-rich phase through a cation exchange column after cloud point extraction, permitting the determination of the phenothiazine derivatives extracted in the surfactant-rich phase by GC. The optimal condition for the cloud point extraction of phenothiazine derivatives was also investigated using Triton X-100, Triton X-114, and PONPE10. Triton X-114 provided the most efficient recovery of phenothiazine derivatives among the surfactants used. The addition of sodium chloride and excess ammonia to the sample solution resulted in a decrement of the recovery of the phenothiazine derivatives. The proposed method was applied to the determination of phenothiazine derivatives in spiked human serum by GC. The recoveries of PC, CP, and FUL in spiked human serum were 95.1%, 87.1%, and 84.7%, respectively.

Collection, mapping, and annotation of over 28,000 cDNA clones from japonica rice.

We collected and completely sequenced 28,469 full-length complementary DNA clones from Oryza sativa L. ssp. japonica cv. Nipponbare. Through homology searches of publicly available sequence data, we assigned tentative protein functions to 21,596 clones (75.86%). Mapping of the cDNA clones to genomic DNA revealed that there are 19,000 to 20,500 transcription units in the rice genome. Protein informatics analysis against the InterPro database revealed the existence of proteins presented in rice but not in Arabidopsis. Sixty-four percent of our cDNAs are homologous to Arabidopsis proteins.