A fluorescent chemical probe CDy9 selectively stains and enables the isolation of live naïve mouse embryonic stem cells.

Human and mouse embryonic stem cells (ESCs) differ in terms of their pluripotency status, i.e., naïve vs. primed. This affects various biological properties and leads to several technical hurdles for future clinical applications, such as difficulties in chimera formation, single-cell passaging, and gene editing. In terms of generating functional human tissues and organs via mammalian interspecies chimerism, a fluorescent chemical probe that specifically labels naïve ESCs would help to isolate these cells and monitor their conversion. This study demonstrates that the fluorescent chemical probe compound of designation yellow 9 (CDy9) selectively stains naïve, but not primed, mouse ESCs (mESCs). CDy9 entered cells via Slc13a5, a highly expressed membrane transporter in naïve mESCs. Fluorescence-based cell sorting based on CDy9 staining successfully separated naïve mESCs from primed mESCs. Mice generated using CDy9+ cells isolated during the conversion of mouse epiblast stem cells into naïve mESCs exhibited coat color chimerism. Furthermore, CDy9 specifically stained cells in the inner cell mass of mouse embryos. These findings suggest that CDy9 is a useful tool to isolate functional naïve mESCs.

Expression profiling of calcium induced genes in cultured human keratinocytes.

Terminal differentiation of skin keratinocytes is a vertically directed multi-step process that is tightly controlled by the sequential expression of a variety of genes. To examine the gene expression profile in calcium-induced keratinocyte differentiation, we constructed a normalized cDNA library using mRNA isolated from these calcium-treated keratinocytes. After sequencing about 10,000 clones, we were able to obtain 4,104 independent genes. They consisted of 3,699 annotated genes and 405 expressed sequence tags (ESTs). Some were the genes involved in constituting epidermal structures and others were unknown genes that are probably associated with keratinocytes. In particular, we were able to identify genes located at the chromosome 1q21, the locus for the epidermal differentiation complex, and 19q13.1, another probable locus for epidermal differentiation-related gene clusters. One EST located at the chromosome 19q13.1 showed increased expression by calcium treatment, suggesting a novel candidate gene relevant to keratinocyte differentiation. These results demonstrate the complexity of the transcriptional profile of keratinocytes, providing important clues on which to base further investigations of the molecular events underlying keratinocyte differentiation.

The epigenetic silencing of LIMS2 in gastric cancer and its inhibitory effect on cell migration.

Recent finding has shown that LIMS2 (also known as PINCH2) functions as a natural regulator of the LIMS1-ILK-parvin complex formation and is associated with cell spreading and migration via integrins at focal adhesions. Here, we report for the first time the epigenetic silencing of LIMS2 in gastric tumors. Downregulation of LIMS2 was detected in 91% (10 of 11) of gastric cancer cell lines by real-time quantitative RT-PCR and 80% (8 of 10) of the LIMS2-downregulated cell lines were associated with CpG island hypermethylation at a 5'-upstream region of LIMS2. Furthermore, LIMS2 was restored in its non-expressing cell lines after treatment with 5-Aza-dC and/or trichostatin A. Loss of expression of LIMS2 was also detected in 53% (51 of 96) of primary gastric tumors. This decrease in expression level significantly correlated with an increase of the CpG island hypermethylation. In addition, the methylation status in any normal-appearing gastric tissues was gradually increased in an age-dependent manner, suggesting that the positive methylation in normal-appearing gastric mucosa can be due to 'field cancerization effect' as an early event in gastric carcinogenesis. Moreover, the transient transfection of LIMS2-siRNA significantly stimulated cell migration in gastric cancer cells but had no effects on cell growth. These results suggest that the frequent inactivation of LIMS2 by epigenetic alteration in gastric cancer may be important in tumor progression events, such as invasion and metastasis. Thus, LIMS2 may be useful as a molecular biomarker and a therapeutic target by increasing its expression and activity in gastric cancer.

Clinical feature analysis of congenital optic nerve abnormalities.

PURPOSE: We investigated the clinical characteristics and associated ocular and systemic anomalies in young children with congenital optic nerve abnormalities, and evaluated the therapeutic results of treatment for strabismus and amblyopia. METHODS: A retrospective study was conducted using the medical records of patients who were diagnosed with congenital optic nerve abnormalities in our hospital between 1995 and 2004. RESULTS: This study involved 72 eyes of 51 young children (M : F = 25 : 26); 21 of these patients (41.2%) had bilateral abnormalities. The types and percentages of each abnormality were as follows: optic nerve hypoplasia, 47%; optic disc coloboma, 31.4%; morning glory syndrome, 11.8%; and myelinated nerve fiber, 9.8%. The first clinical manifestations noticed by parents were strabismus and decreased visual acuity. Occlusion therapy was carried out in 16 patients, and the visual acuities of two patients were improved by more than two lines of the Early Treatment Diabetic Retinopathy Study visual acuity chart. Accompanying strabismus was found in 70.6% of the patients; 14 patients underwent strabismic surgery, and 10 of these patients (41.2%) maintained a stable angle. CONCLUSIONS: The most common congenital optic nerve abnormality was optic nerve hypoplasia, and the associated systemic abnormalities found were optic nerve hypoplasia and disc coloboma. Patients with congenital optic nerve abnormalities usually have a generally poor visual prognosis, but we emphasize the importance of treatment for associated strabismus and amblyopia.

Transcriptome analysis of human gastric cancer.

To elucidate the genetic events associated with gastric cancer, 124,704 cDNA clones were collected from 37 human gastric cDNA libraries, including 20 full-length enriched cDNA libraries of gastric cancer cell lines and tissues from Korean patients. An analysis of the collected ESTs revealed that 97,930 high-quality ESTs coalesced into 13,001 clusters, of which 11,135 clusters (85.6%) were annotated to known ESTs. The analysis of the full-length cDNAs also revealed that 4862 clusters (51.7%) contained at least one putative full-length cDNA clone with an initiation codon, with the average length of the 5' UTR of 140 bp. A large number appear to have a diverse transcription start site (TSS). An examination of the TSS of some genes, such as TEGT and GAPD, using 5' RACE revealed that the predicted TSSs are actually found in human gastric cancer cells and that several TSSs differ depending on the specific gastric cell line. Furthermore, of the human gastric ESTs, 766 genes (9.5%) were present as putative alternatively spliced variants. Confirmation of the predicted spliced isoforms using RT-PCR showed that the predicted isoforms exist in gastric cancer cells and some isoforms coexist in gastric cell lines. These results provide potentially useful information for elucidating the molecular mechanisms associated with gastric oncogenesis.

The effects of laser photocoagulation on reopened macular holes, as assessed by optical coherence tomography.

PURPOSE: To evaluate the effects of laser photocoagulation on reopened macular holes. METHODS: This study involved 9 eyes from 9 patients who underwent laser photocoagulation coupled with fluid-gas exchange for reopened macular holes. The photocoagulation was performed at the center of the macular hole. Closure of the reopened hole was categorized by optical coherence tomography (OCT) according to the presence (type 1 closure) or absence (type 2 closure) of continuity in the foveal tissue. Best corrected visual acuity (BCVA), closure types, and complications were assessed. RESULTS: Upon final examination, all macular holes were found to have closed. Six eyes were classified as type 1 closure, and three were classified as type 2 closure. The mean BCVAs, before and after laser photocoagulation, were 0.11 and 0.31, respectively (P<.05). The eyes with type 1 closure were associated with shorter symptom durations and greater visual improvement than those with type 2 closure (P<.05). CONCLUSIONS: The combination of laser photocoagulation and fluid-gas exchange appears to be a safe and effective treatment for reopened macular holes. Early intervention should be encouraged to ensure complete hole closure and improved visual outcomes.