Enhancement of DNA-transfection frequency by X-rays

&#8195;This study was conducted to evaluate the frequency of DNA transfection into human cells following X-ray irradiation. We transfected plasmid DNA (pSV2neo) into human cells, HeLa and PA-1, by either calcium phosphate precipitation or the lipofection method immediately after irradiating the cells with various doses of X-rays. The transfection frequency was evaluated by counting the number of G418-resistant colonies. When circular plasmid DNA was used, irradiation up to a dose of 2 Gy dose-dependently increased the transfection frequency, which reached a maximum of 5 to 10-fold that of the control unirradiated cells. When linear plasmid DNA was used, the transfection frequency was 2 times higher than that of circular DNA. All five of the clones that were randomly chosen expressed the transfected neo gene. In addition, the pSV2neo gene was randomly integrated into the genomic DNA of each clone. These findings indicate that X-ray treatment can facilitate foreign DNA transfer into human cells and that radiation-induced DNA breaks may promote the insertion of foreign DNA into host DNA. The enhancement of DNA transfection with X-rays may be instrumental in practicing gene therapy.</p

An Intronic Signal for Alternative Splicing in the Human Genome

An important level at which the expression of programmed cell death (PCD) genes is regulated is alternative splicing. Our previous work identified an intronic splicing regulatory element in caspase-2 (casp-2) gene. This 100-nucleotide intronic element, In100, consists of an upstream region containing a decoy 3′ splice site and a downstream region containing binding sites for splicing repressor PTB. Based on the signal of In100 element in casp-2, we have detected the In100-like sequences as a family of sequence elements associated with alternative splicing in the human genome by using computational and experimental approaches. A survey of human genome reveals the presence of more than four thousand In100-like elements in 2757 genes. These In100-like elements tend to locate more frequent in intronic regions than exonic regions. EST analyses indicate that the presence of In100-like elements correlates with the skipping of their immediate upstream exons, with 526 genes showing exon skipping in such a manner. In addition, In100-like elements are found in several human caspase genes near exons encoding the caspase active domain. RT-PCR experiments show that these caspase genes indeed undergo alternative splicing in a pattern predicted to affect their functional activity. Together, these results suggest that the In100-like elements represent a family of intronic signals for alternative splicing in the human genome.</p

An Intronic Signal for Alternative Splicing in the Human Genome

An important level at which the expression of programmed cell death (PCD) genes is regulated is alternative splicing. Our previous work identified an intronic splicing regulatory element in caspase-2 (casp-2) gene. This 100-nucleotide intronic element, In100, consists of an upstream region containing a decoy 3′ splice site and a downstream region containing binding sites for splicing repressor PTB. Based on the signal of In100 element in casp-2, we have detected the In100-like sequences as a family of sequence elements associated with alternative splicing in the human genome by using computational and experimental approaches. A survey of human genome reveals the presence of more than four thousand In100-like elements in 2757 genes. These In100-like elements tend to locate more frequent in intronic regions than exonic regions. EST analyses indicate that the presence of In100-like elements correlates with the skipping of their immediate upstream exons, with 526 genes showing exon skipping in such a manner. In addition, In100-like elements are found in several human caspase genes near exons encoding the caspase active domain. RT-PCR experiments show that these caspase genes indeed undergo alternative splicing in a pattern predicted to affect their functional activity. Together, these results suggest that the In100-like elements represent a family of intronic signals for alternative splicing in the human genome

Yeast functional assay of the p53 gene status in human cell lines maintained in our laboratory

We used a yeast functional assay (functional analysis of separated alleles in yeast: FASAY) to determine the p53 gene status of human cell lines maintained in our laboratory. This assay enables the researcher to score wild-type p53 expression on the basis of the ability of expressed p53 to transactivate the reporter gene HIS 3 via the p53-responsive GAL 1 promoter in Saccharomyces cerevisiae. The cell lines examined were ten hepatoma, two hepatoblastoma, three in vitro immortalized fibroblast, two osteosarcoma, a chondrosarcoma, an ovarian teratocarcinoma and a colon cancer cell line. Out of 20 cell lines, 11 cell lines had mutations in both alleles of the p53 gene, and another 8 cell lines had no mutation in the p53 gene. Thus, 55% of the cell lines examined had mutations in the p53. Interestingly, PA-1 cells had both the normal and the mutant p53 alleles, showing that FASAY is a useful method for detecting the wild-type and mutated p53 genes simultaneously. As for the three liver cell lines harboring HBsAg, there was no relationship between their p53 gene status and the presence of HBsAg. Two cell lines were normal for p53 status, while the other had a mutation of the p53 gene.</p

Comparison of cellular characteristics between human hepatoma cell lines with wild-type p53 and those with mutant-type p53 gene

Characteristics of human hepatoma cell lines with the wild-type p53 were compared with those of human hepatoma cell lines with the mutant-type p53. The p21 protein located downstream of p53 was expressed in cell lines with the wild-type p53 but was not expressed in cell lines with the mutant-type p53. As to other tumor suppressor genes such as p16 and p27, there was no difference in their expression between both types of cell lines. In addition, no marked difference was observed in the activities of CDK2 and CDK4 between cell lines with the wild-type and the mutant-type p53. Phosphorylated Rb protein was detected in all cell lines except the HLE line, indicating that this cell line may have a deletion of and/or a mutation of the Rb gene. These results indicate that abnormalities of tumor suppressor genes other than p53, p16, p27, and Rb may be involved in hepatocarcinogenesis. The population doubling time of the wild-type p53 cells was significantly longer than that of the mutant p53 cells. Neither type of cell line showed a specific chromosome distribution which would indicate karyotype instability. The cell lines expressing the wild-type p53 produced tumors at lower frequency than those with the mutant p53 gene. Although there was no significant difference in effects of TGF-&#946;1, EGF, cholera toxin, and db-cAMP on cell growth between the two types of cells, all three cell lines with the wild-type p53 were resistant to cytotoxicity of TNF-&#945;, while two of the three with the mutant p53 were very sensitive to its cytotoxic effects.</p

Cross-sectional survey of depressive symptoms and suicide-related ideation at a Japanese national university during the COVID-19 stay-home order

Background We aimed to estimate the prevalence of depressive symptoms as well as suicide-related ideation among Japanese university students during the stay-home order necessitated by the coronavirus disease 2019 pandemic in Japan, and offer evidence in support of future intervention to depression and suicide prevention strategies among college and university students. Methods The data for this cross-sectional study were derived from the Student Mental Health Survey conducted from May 20 to June 16, 2020 at a national university in Akita prefecture. Among the 5111 students recruited, 2712 participated in this study (response rate, 53%; mean age ± standard deviation, 20.5 ±3.5 years; men, 53.8%). Depressive symptoms were identified by using the Patient Health Questionnaire-9 (PHQ-9). Results The prevalence of moderate depressive symptoms based on a PHQ-9 score ≥10 and suicide-related ideation based on question 9 of PHQ-9 ≥1, which encompasses thoughts of both suicide and self-harm, was 11.7% and 6.7%, respectively. Multivariable logistic regression analyses showed that risk factors for depression included being a woman, smoking, alcohol consumption, and social network communication using either video or voice. For suicide-related ideation, alcohol consumption was the only risk factor. Exercise and having someone to consult about worries were associated with decreased risk of both depressive symptoms and suicide-related ideation. Conclusions Negative lifestyles of smoking and drinking, and being a woman, may be important risk factors for depressive symptoms, whereas exercise and having someone to consult about worries may be protective factors

NSSRs/TASRs/SRp38s function as splicing modulators via binding to pre-mRNAs

The genes for neural-salient serine/arginine-rich (NSSR) proteins 1 and 2 have been cloned from a neuronal differentiated embryocarcinoma cell line, P19. NSSRs contain an RNA recognition motif (RRM) at the N-terminal and several SR rich regions at the C-terminal resembling RS domains. We found that NSSRs associated with U1-70k, and determined the exon inclusion activity of NSSRs' C-terminals. First, the RRM was changed to the MS2 coat protein (MS2CP) and then, MS2 RNA stem-loops were inserted in the middle of the exon N of the clathrin light chain B minigene as an artificial exonic splicing enhancer to be recognized by the MS2CP. The modified exon N of the pre-mRNA was included by the MS2CP switched NSSR 1, but it was excluded by the MS2CP switched NSSR 2. Deletion analysis of the MS2CP switched NSSR1 suggested that the middle SR rich region was responsible for the activity of the modified exon N inclusion. Furthermore, the RRM domain of NSSRs recognized mRNAs. NSSRs were expressed in the nervous system, especially in cerebellar and hippocampal primordia, neopallial cortex, ventricular zone, retina, and olfactory epithelium and bulb at E15.5. Taken together, our results showed that NSSRs modulate alternative splicing via binding to pre-mRNAs during neural differentiation

Performance and Internal Flow of a Dental Air Turbine Handpiece

An air turbine handpiece is a dental abrasive device that rotates at high speed and uses compressed air as the driving force. It is characterized by its small size, light weight, and painless abrading due to its high-speed rotation, but its torque is small and noise level is high. Thus, to improve the performance of the air turbine handpiece, we conducted a performance test of an actual handpiece and a numerical analysis that modeled the whole handpiece; we also analyzed the internal flow of the handpiece. Results show that experimental and calculated values were consistent for a constant speed load method with the descending speed of 1 mm/min for torque and turbine output. When the tip of the blade was at the center of the nozzle, the torque was at its highest. This is likely because the jet from the nozzle entered the tip of the blade from a close distance that would not reduce the speed and exited along the blade

Prediction of tertiary structure of NSSRs' RNA recognition motif and the RNA binding activity

RNAs possess potentials to become excellent bio-material because of their biochemical and biological activities. For instance, most RNA splicings are catalyzed by machinery including their own RNAs or other RNAs. The eukaryote machineries for splicing of pre-mRNA, which are called spliceosomes, are flexible and accurate for separating substrate and catalytic RNAs. Although RNAs themselves catalyze the splicing, spliceosomes are supported by many proteins. Furthermore, a great accuracy is required for the alternative splicing because there are choices available, which must be regulated in tissue-specifically and developmentally manners. Neural-salient serine/arginine-rich (NSSR) proteins 1 and 2 are candidates for supporting the accuracy of the splicing. The features of their amino acid sequences suggest that NSSRs are SR proteins, which bind to pre-mRNA and determine the splicing site. Since SR proteins have a RNA recognition motif or motives (RRM or RRMs), which binds to RNA, we predicted the secondary and tertiary structures of NSSRs' RRM by comparing them to RRMs of other proteins. The predicted structure suggested that the RNA binding activity of NSSRs' RRM is similar to the poly A binding protein (PABP). Moreover, to detect the targets for NSSR, mRNAs were obtained by screening them from murine brains with bacterial recombinant NSSRs' RRM and microarray experiments were conducted using these mRNAs. The results suggested that NSSRs bind specifically to particular pre-mRNAs and regulate the alternative splicing of the binding pre-mRNA