Elimination of Specific miRNAs by Naked 14-nt sgRNAs

tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is a newly developed technology to suppress mammalian gene expression. TRUE gene silencing works on the basis of a unique enzymatic property of mammalian tRNase ZL, which is that it can recognize a pre-tRNA-like or micro-pre-tRNA-like complex formed between target RNA and artificial small guide RNA (sgRNA) and can cleave any target RNA at any desired site. There are four types of sgRNA, 5′-half-tRNA, RNA heptamer, hook RNA, and ∼14-nt linear RNA. Here we show that a 14-nt linear-type sgRNA against human miR-16 can guide tRNase ZL cleavage of miR-16 in vitro and can downregulate the miR-16 level in HEK293 cells. We also demonstrate that the 14-nt sgRNA can be efficiently taken up without any transfection reagents by living cells and can exist stably in there for at least 24 hours. The naked 14-nt sgRNA significantly reduced the miR-16 level in HEK293 and HL60 cells. Three other naked 14-nt sgRNAs against miR-142-3p, miR-206, and miR-19a/b are also shown to downregulate the respective miRNA levels in various mammalian cell lines. Our observations suggest that in general we can eliminate a specific cellular miRNA at least by ∼50% by using a naked 14-nt sgRNA on the basis of TRUE gene silencing

Phase II Trial of Preoperative Chemotherapy with Docetaxel, Cisplatin and S-1 for T4 Locally Advanced Gastric Cancer

The standard treatment for T4 locally advanced gastric cancer is gastrectomy with D2 lymph node dissection followed by adjuvant chemotherapy with S-1 for 12 months; however, prognostic outcome in Stage IIIb has been insufficient. It is expected that survival is improved by preoperative treatment with a triplet regimen of docetaxel, cisplatin and S-1 (divided DCS therapy). A multicenter Phase II study has been conducted to evaluate the safety and efficacy of two courses of preoperative chemotherapy followed by gastrectomy. Fifty-five patients are required for this study. The primary endpoint of the study is pathological response rate of primary lesions. Secondary endpoints are overall survival, disease-free survival, R0 resection rate and adverse events

Modulation of Gene Expression by Human Cytosolic tRNase ZL through 5′-Half-tRNA

A long form (tRNase ZL) of tRNA 3′ processing endoribonuclease (tRNase Z, or 3′ tRNase) can cleave any target RNA at any desired site under the direction of artificial small guide RNA (sgRNA) that mimics a 5′-half portion of tRNA. Based on this enzymatic property, a gene silencing technology has been developed, in which a specific mRNA level can be downregulated by introducing into cells a synthetic 5′-half-tRNA that is designed to form a pre-tRNA-like complex with a part of the mRNA. Recently 5′-half-tRNA fragments have been reported to exist stably in various types of cells, although little is know about their physiological roles. We were curious to know if endogenous 5′-half-tRNA works as sgRNA for tRNase ZL in the cells. Here we show that human cytosolic tRNase ZL modulates gene expression through 5′-half-tRNA. We found that 5′-half-tRNAGlu, which co-immunoprecipitates with tRNase ZL, exists predominantly in the cytoplasm, functions as sgRNA in vitro, and downregulates the level of a luciferase mRNA containing its target sequence in human kidney 293 cells. We also demonstrated that the PPM1F mRNA is one of the genuine targets of tRNase ZL guided by 5′-half-tRNAGlu. Furthermore, the DNA microarray data suggested that tRNase ZL is likely to be involved in the p53 signaling pathway and apoptosis

Gastric cancer treatment in Japan: 2008 annual report of the JGCA nationwide registry

The Japanese Gastric Cancer Association (JGCA) started a new nationwide gastric cancer registry in 2008. Approximately 50 data items, including surgical procedures, pathological diagnoses, and survival outcomes, for 12004 patients with primary gastric cancer treated in 2001 were collected retrospectively from 187 participating hospitals. Data were entered into the JGCA database according to the JGCA Classification of gastric carcinoma, 13th edition and the International Union Against Cancer (UICC) TNM Classification of malignant tumors, 5th edition by using an electronic data collecting system. Finally, data of 11261 patients with gastric resection were analyzed. The 5-year follow-up rate was 83.5%. The direct death rate was 0.6%. TNM 5-year survival rates (5YSRs)/JGCA 5YSRs were 91.8/91.9% for stage IA, 84.6/85.1% for stage IB, 70.5/73.1% for stage II, 46.6/51.0% for stage IIIA, 29.9/33.4% for stage IIIB, and 16.6/15.8% for stage IV. The proportion of patients more than 80 years old was 7.0%, and their 5YSR was 48.7%. Compared to the JGCA archived data, though the follow-up rate needs to be improved, these data suggest that the postoperative results of patients with primary gastric carcinoma have improved in those with advanced disease and in the aged population in Japan

Serum 25-Hydroxyvitamin D Concentrations and Season-Specific Correlates in Japanese Adults

Background: Several lines of evidence indicate an important role for vitamin D in the prevention of a range of diseases. Blood vitamin D levels show clear seasonal variation; however, data on the determinants of vitamin D status for each season are limited. We investigated the association between lifestyle and serum vitamin D concentration by season in Japanese workers. Methods: Subjects were 312 men and 217 women aged 21 to 67 years who worked in municipal offices in Northern Kyushu, Japan and participated in a periodic checkup in July or November. Multiple linear regression analysis was used to examine the association between serum 25-hydroxivitamin D concentrations and lifestyle factors for each season. Results: Mean serum 25-hydroxyvitamin D concentration was 27.4 ng/ml (68.4 nmol/L) and 21.4 ng/ml (53.4 nmol/L) for workers surveyed in July and November, respectively (P \u3c 0.001); the prevalence of vitamin D deficiency ( both sexes) and nonsmoking and physical activity (in men) were significantly associated with higher concentrations of serum 25-hydroxyvitamin D. In summer, fish/shellfish intake was associated with higher serum 25- hydroxyvitamin D concentrations in women. Conclusions: Vitamin D deficiency is common in Japanese workers during seasons with limited sunlight. The lifestyle correlates of favorable vitamin D status in November were physical activity, dietary vitamin D intake, and nonsmoking

A survey of green plant tRNA 3'-end processing enzyme tRNase Zs, homologs of the candidate prostate cancer susceptibility protein ELAC2

<p>Abstract</p> <p>Background</p> <p>tRNase Z removes the 3'-trailer sequences from precursor tRNAs, which is an essential step preceding the addition of the CCA sequence. tRNase Z exists in the short (tRNase Z^S) and long (tRNase Z^L) forms. Based on the sequence characteristics, they can be divided into two major types: bacterial-type tRNase Z^Sand eukaryotic-type tRNase Z^L, and one minor type, <it>Thermotoga maritima </it>(TM)-type tRNase Z^S. The number of tRNase Zs is highly variable, with the largest number being identified experimentally in the flowering plant <it>Arabidopsis thaliana</it>. It is unknown whether multiple tRNase Zs found in <it>A. thaliana </it>is common to the plant kingdom. Also unknown is the extent of sequence and structural conservation among tRNase Zs from the plant kingdom.</p> <p>Results</p> <p>We report the identification and analysis of candidate tRNase Zs in 27 fully sequenced genomes of green plants, the great majority of which are flowering plants. It appears that green plants contain multiple distinct tRNase Zs predicted to reside in different subcellular compartments. Furthermore, while the bacterial-type tRNase Z^Ss are present only in basal land plants and green algae, the TM-type tRNase Z^Ss are widespread in green plants. The protein sequences of the TM-type tRNase Z^Ss identified in green plants are similar to those of the bacterial-type tRNase Z^Ss but have distinct features, including the TM-type flexible arm, the variant catalytic HEAT and HST motifs, and a lack of the PxKxRN motif involved in CCA anti-determination (inhibition of tRNase Z activity by CCA), which prevents tRNase Z cleavage of mature tRNAs. Examination of flowering plant chloroplast tRNA genes reveals that many of these genes encode partial CCA sequences. Based on our results and previous studies, we predict that the plant TM-type tRNase Z^Ss may not recognize the CCA sequence as an anti-determinant.</p> <p>Conclusions</p> <p>Our findings substantially expand the current repertoire of the TM-type tRNase Z^Ss and hint at the possibility that these proteins may have been selected for their ability to process chloroplast pre-tRNAs with whole or partial CCA sequences. Our results also support the coevolution of tRNase Zs and tRNA 3'-trailer sequences in plants.</p

Engineered Models of Metastasis with Application to Study Cancer Biomechanics

Three-dimensional complex biomechanical interactions occur from the initial steps of tumor formation to the later phases of cancer metastasis. Conventional monolayer cultures cannot recapitulate the complex microenvironment and chemical and mechanical cues that tumor cells experience during their metastatic journey, nor the complexity of their interactions with other, noncancerous cells. As alternative approaches, various engineered models have been developed to recapitulate specific features of each step of metastasis with tunable microenvironments to test a variety of mechanistic hypotheses. Here the main recent advances in the technologies that provide deeper insight into the process of cancer dissemination are discussed, with an emphasis on three-dimensional and mechanical factors as well as interactions between multiple cell types