A RecA-mediated exon profiling method

We have developed a RecA-mediated simple, rapid and scalable method for identifying novel alternatively spliced full-length cDNA candidates. This method is based on the principle that RecA proteins allow to carry radioisotope-labeled probe DNAs to their homologous sequences, resulting in forming triplexes. The resulting complex is easily detected by mobility difference on electrophoresis. We applied this exon profiling method to four selected mouse genes as a feasibility study. To design probes for detection, the information on known exonic regions was extracted from public database, RefSeq. Concerning the potentially transcribed novel exonic regions, RNA mapping experiment using Affymetrix tiling array was performed. As a result, we were able to identify alternative splice variants of Thioredoxin domain containing 5, Interleukin1β, Interleukin 1 family 6 and glutamine-rich hypothetical protein. In addition, full-length sequencing demonstrated that our method could profile exon structures with >90% accuracy. This reliable method can allow us to screen novel splice variants from a huge number of cDNA clone set effectively

Effects of acidic calcium phosphate concentration on setting reaction and tissue response to β-tricalcium phosphate granular cement

Beta-tricalcium phosphate granular cement (β-TCP GC), consisting of β-TCP granules and an acidic calcium phosphate (Ca-P) solution, shows promise in the reconstruction of bone defects as it sets to form interconnected porous structures, i.e., β-TCP granules are bridged with dicalcium phosphate dihydrate (DCPD) crystals. In this study, the effects of acidic Ca-P solution concentration (0–600 mmol/L) on the setting reaction and tissue response to β-TCP GC were investigated. The β-TCP GC set upon mixing with its liquid phase, based on the formation of DCPD crystals, which bridged β-TCP granules to one another. Diametral tensile strength of the set β-TCP GC was relatively the same, at approximately 0.6 MPa, when the Ca-P concentration was 20–600 mmol/L. Due to the setting ability, reconstruction of the rat’s calvarial bone defect using β-TCP GC with 20, 200, and 600 mmol/L Ca-P solution was much easier compared to that with β-TCP granules without setting ability. Four weeks after the reconstruction, the amount of new bone was the same, approximately 17% in both β-TCP GC and β-TCP granules groups. Cellular response to β-TCP granules and β-TCP GC using the 20 mmol/L acidic Ca-P solution was almost the same. However, β-TCP GC using the 200 and 600 mmol/L acidic Ca-P solution showed a more severe inflammatory reaction. It is concluded, therefore, that β-TCP GC, using the 20 mmol/L acidic Ca-P solution, is recommended as this concentration allows surgical techniques to be performed easily and provides good mechanical strength, and the similar cellular response to β-TCP granules

CAGE Basic/Analysis Databases: the CAGE resource for comprehensive promoter analysis

Cap-analysis gene expression (CAGE) Basic and Analysis Databases store an original resource produced by CAGE, which measures expression levels of transcription starting sites by sequencing large amounts of transcript 5′ ends, termed CAGE tags. Millions of human and mouse high-quality CAGE tags derived from different conditions in >20 tissues consisting of >250 RNA samples are essential for identification of novel promoters and promoter characterization in the aspect of expression profile. CAGE Basic Database is a primary database of the CAGE resource, RNA samples, CAGE libraries, CAGE clone and tag sequences and so on. CAGE Analysis Database stores promoter related information, such as counts of related transcripts, CpG islands and conserved genome region. It also provides expression profiles at base pair and promoter levels. Both databases are based on the same framework, CAGE tag starting sites, tag clusters for defining promoters and transcriptional units (TUs). Their associations and TU attributes are available to find promoters of interest. These databases were provided for Functional Annotation Of Mouse 3 (FANTOM3), an international collaboration research project focusing on expanding the transcriptome and subsequent analyses. Now access is free for all users through the World Wide Web at

Synergistic effect of surface phosphorylation and micro-roughness on enhanced osseointegration ability of poly(ether ether ketone) in the rabbit tibia

This study was aimed to investigate the osseointegration ability of poly(ether ether ketone) (PEEK) implants with modified surface roughness and/or surface chemistry. The roughened surface was prepared by a sandblast method, and the phosphate groups on the substrates were modified by a two-step chemical reaction. The in vitro osteogenic activity of rat mesenchymal stem cells (MSCs) on the developed substrates was assessed by measuring cell proliferation, alkaline phosphatase activity, osteocalcin expression, and bone-like nodule formation. Surface roughening alone did not improve MSC responses. However, phosphorylation of smooth substrates increased cell responses, which were further elevated in combination with surface roughening. Moreover, in a rabbit tibia implantation model, this combined surface modification significantly enhanced the bone-to-implant contact ratio and corresponding bone-to-implant bonding strength at 4 and 8 weeks post-implantation, whereas modification of surface roughness or surface chemistry alone did not. This study demonstrates that combination of surface roughness and chemical modification on PEEK significantly promotes cell responses and osseointegration ability in a synergistic manner both in vitro and in vivo. Therefore, this is a simple and promising technique for improving the poor osseointegration ability of PEEK-based orthopedic/dental implants

Epithelial EP4 plays an essential role in maintaining homeostasis in colon

Colonic epithelial cells comprise the mucosal barrier, and their dysfunction promotes microbial invasion from the gut lumen and induces the development of intestinal inflammation. The EP4 receptor is known to mediate the protective effect of prostaglandin (PG) E2 in the gastrointestinal tract; however, the exact role of epithelial EP4 in intestinal pathophysiology remains unknown. In the present study, we aimed to investigate the role of epithelial EP4 in maintaining colonic homeostasis by characterizing the intestinal epithelial cell-specific EP4 knockout (EP4 cKO) mice. Mice harboring the epithelial EP4 deletion showed significantly lower colonic crypt depth and lower numbers of secretory cell lineages, as well as impaired epithelial cells in the colon. Interestingly, EP4-deficient colon epithelia showed a higher number of apoptotic cells. Consistent with the defect in mucosal barrier function of colonic epithelia and secretory cell lineages, EP4 cKO colon stroma showed enhanced immune cell infiltration, which was accompanied by increased production of inflammatory cytokines. Furthermore, EP4-deficient colons were susceptible to dextran sulfate sodium (DSS)-induced colitis. Our study is the first to demonstrate that epithelial EP4 loss resulted in potential "inflammatory" status under physiological conditions. These findings provided insights into the crucial role of epithelial PGE2/EP4 axis in maintaining intestinal homeostasis

Clusters of internally primed transcripts reveal novel long noncoding RNAs

Non- protein- coding RNAs ( ncRNAs) are increasingly being recognized as having important regulatory roles. Although much recent attention has focused on tiny 22- to 25- nucleotide microRNAs, several functional ncRNAs are orders of magnitude larger in size. Examples of such macro ncRNAs include Xist and Air, which in mouse are 18 and 108 kilobases ( Kb), respectively. We surveyed the 102,801 FANTOM3 mouse cDNA clones and found that Air and Xist were present not as single, full- length transcripts but as a cluster of multiple, shorter cDNAs, which were unspliced, had little coding potential, and were most likely primed from internal adenine- rich regions within longer parental transcripts. We therefore conducted a genome- wide search for regional clusters of such cDNAs to find novel macro ncRNA candidates. Sixty- six regions were identified, each of which mapped outside known protein- coding loci and which had a mean length of 92 Kb. We detected several known long ncRNAs within these regions, supporting the basic rationale of our approach. In silico analysis showed that many regions had evidence of imprinting and/ or antisense transcription. These regions were significantly associated with microRNAs and transcripts from the central nervous system. We selected eight novel regions for experimental validation by northern blot and RT- PCR and found that the majority represent previously unrecognized noncoding transcripts that are at least 10 Kb in size and predominantly localized in the nucleus. Taken together, the data not only identify multiple new ncRNAs but also suggest the existence of many more macro ncRNAs like Xist and Air

Protocol for a multicentre, prospective observational study of elective neck dissection for clinically node-negative oral tongue squamous cell carcinoma (END-TC study)

Introduction: In early-stage oral tongue squamous cell carcinoma (OTSCC), elective neck dissection (END) is recommended when occult lymph node metastasis is suspected; however, there is no unanimous consensus on the risks and benefits of END in such cases. The management of clinically node-negative (cN0) OTSCC remains controversial. This study, therefore, aimed to evaluate the efficacy of END and its impact on the quality of life (QoL) of patients with cN0 OTSCC. Methods and analysis: This is a prospective, multicentre, nonrandomised observational study. The choice of whether to perform END at the same time as resection of the primary tumour is based on institutional policy and patient preference. The primary endpoint of this study is 3-year overall survival. The secondary endpoint are 3-year disease-specific survival, 3-year relapse-free survival and the impact on patient QoL. Propensity score-matching analysis will be performed to reduce selection bias. Ethics and dissemination: This study was approved by the Clinical Research Review Board of the Nagasaki University. The protocol of this study was registered at the University Hospital Medical Information Network Clinical Trials Registry. The datasets generated during the current study will be available from the corresponding author on reasonable request. The results will be disseminated internationally, through scientific and professional conferences and in peer-reviewed medical journals

Gene Organization in Rice Revealed by Full-Length cDNA Mapping and Gene Expression Analysis through Microarray

Rice (Oryza sativa L.) is a model organism for the functional genomics of monocotyledonous plants since the genome size is considerably smaller than those of other monocotyledonous plants. Although highly accurate genome sequences of indica and japonica rice are available, additional resources such as full-length complementary DNA (FL-cDNA) sequences are also indispensable for comprehensive analyses of gene structure and function. We cross-referenced 28.5K individual loci in the rice genome defined by mapping of 578K FL-cDNA clones with the 56K loci predicted in the TIGR genome assembly. Based on the annotation status and the presence of corresponding cDNA clones, genes were classified into 23K annotated expressed (AE) genes, 33K annotated non-expressed (ANE) genes, and 5.5K non-annotated expressed (NAE) genes. We developed a 60mer oligo-array for analysis of gene expression from each locus. Analysis of gene structures and expression levels revealed that the general features of gene structure and expression of NAE and ANE genes were considerably different from those of AE genes. The results also suggested that the cloning efficiency of rice FL-cDNA is associated with the transcription activity of the corresponding genetic locus, although other factors may also have an effect. Comparison of the coverage of FL-cDNA among gene families suggested that FL-cDNA from genes encoding rice- or eukaryote-specific domains, and those involved in regulatory functions were difficult to produce in bacterial cells. Collectively, these results indicate that rice genes can be divided into distinct groups based on transcription activity and gene structure, and that the coverage bias of FL-cDNA clones exists due to the incompatibility of certain eukaryotic genes in bacteria

Protocol for a multicentre, prospective observational study of elective neck dissection for clinically node-negative oral tongue squamous cell carcinoma (END-TC study)

Introduction In early-stage oral tongue squamous cell carcinoma (OTSCC), elective neck dissection (END) is recommended when occult lymph node metastasis issuspected; however, there is no unanimous consensus on the risks and benefits of END in such cases. The management of clinically node-negative (cN0) OTSCCremains controversial. This study, therefore, aimed to evaluate the efficacy of END and its impact on the quality of life (QoL) of patients with cN0 OTSCC.Methods and analysis This is a prospective, multicentre, nonrandomised observational study. The choice of whether to perform END at the same time as resection of the primary tumour is based on institutional policy and patient preference. The primary endpoint of this study is 3-year overall survival. The secondary endpoints are3-year disease-specific survival, 3-year relapse-free survival and the impact on patient QoL. Propensity score-matching analysis will be performed to reduce selection bias.Ethics and dissemination This study was approved by the Clinical Research Review Board of the Nagasaki University. The protocol of this study was registered at the University Hospital Medical Information Network Clinical Trials Registry. The datasets generated during the current study will be available from the correspondingauthor on reasonable request. The results will be disseminated internationally, through scientific and professional conferences and in peer-reviewed medical journals