He resulting biofilms were sonicated for 1 second at output level 1 (output power, 25 W; oscillating frequency, 28 kHz; tip diameter, 2.5 mm) with a Handy Ultrasonic Disruptor (UR-20P, Tomy Seiko, Tokyo, Japan). Genomic DNA was isolated from biofilms formed by P. gingivalis and the CFU value (see above) was determined using real-time PCR, as described previously [19,42]. The data represent the mean 6 standard error of the mean of three separate experiments performed in duplicate for each strain.Supporting InformationFigure S1 Quantification of mean thickness and Felypressin manufacturer average substratum coverage from CLSM observation. Fluorescent images of CLSM (Figures 2A and 2B) were quantified using Imaris software and the mean thickness of cells (A) and that of exopolysaccharide (B), and average substratum coverage of cells (C) and that of exopolysaccharide (D) per field were calculated. The experiment was repeated independently three times. Data are presented as average of 8 fields per sample along with the standard errors of the mean. Statistical analysis was performed using a Welch’s t test. *P,0.001 in comparison with the wild type strain. (TIF)Author Contributions Statistical analysisThe significance of intergroup differences of all data was analyzed using Welch’s t tests. A P value,0.001 was considered to indicate statistical significance.Designed the method used in analysis of CLSM: MK. Conceived and designed the experiments: RY YN. Performed the experiments: RY MY YA HM. Analyzed the data: RY MY YA HM. Contributed reagents/ materials/analysis tools: SE MH. Wrote the paper: RY.
Retroviruses insert a double-stranded DNA copy of their genome non-specifically into the genome of the host and thereby act as insertional mutagens that can disrupt gene regulation or cause the production of an altered gene product [1]. The integrated retrovirus, termed the provirus, contains strong transcription-regulatory signals that can induce or enhance the expression of nearby genes. When such affected genes are involved in cell survival and proliferation, their deregulated expression may contribute to tumorigenesis [2]. In tumors caused by retroviral insertions, the proviruses constitute a tag allowing the identification of candidate genes with a possible role in tumorigenesis. By this approach several recent studies have contributed to the discovery of new proto-oncogenes and also, when performed in genetically modified mice expanded our knowledge on oncogene cooperativity [3?]. Moreover, insertional mutagenesis is a concern in gene therapy by retroviral vectors [5]. By another CI-1011 development, evidence is also emerging that endogenous retroviruses of mice and humans may contribute to oncogenesis by the activation of nearby genes without any need for new retroviral insertions in somatic cells [6]. Based on the analysis of somatic integrations selected during malignant transformation various types of virus-induced gene activation have been proposed. By the process known as enhancerinsertion a provirus increases the production of a normal transcript of an adjacent target gene [2]. In these cases, proviruses are often found outside the transcription unit of the target gene and in many cases upstream of the target gene and in the opposite transcriptional orientation. Other types of insertional mutagenesis result in the formation of chimeric RNA species containing viral and host sequences. One example of this is promoter insertion in which proviruses are integrated in the same.He resulting biofilms were sonicated for 1 second at output level 1 (output power, 25 W; oscillating frequency, 28 kHz; tip diameter, 2.5 mm) with a Handy Ultrasonic Disruptor (UR-20P, Tomy Seiko, Tokyo, Japan). Genomic DNA was isolated from biofilms formed by P. gingivalis and the CFU value (see above) was determined using real-time PCR, as described previously [19,42]. The data represent the mean 6 standard error of the mean of three separate experiments performed in duplicate for each strain.Supporting InformationFigure S1 Quantification of mean thickness and average substratum coverage from CLSM observation. Fluorescent images of CLSM (Figures 2A and 2B) were quantified using Imaris software and the mean thickness of cells (A) and that of exopolysaccharide (B), and average substratum coverage of cells (C) and that of exopolysaccharide (D) per field were calculated. The experiment was repeated independently three times. Data are presented as average of 8 fields per sample along with the standard errors of the mean. Statistical analysis was performed using a Welch’s t test. *P,0.001 in comparison with the wild type strain. (TIF)Author Contributions Statistical analysisThe significance of intergroup differences of all data was analyzed using Welch’s t tests. A P value,0.001 was considered to indicate statistical significance.Designed the method used in analysis of CLSM: MK. Conceived and designed the experiments: RY YN. Performed the experiments: RY MY YA HM. Analyzed the data: RY MY YA HM. Contributed reagents/ materials/analysis tools: SE MH. Wrote the paper: RY.
Retroviruses insert a double-stranded DNA copy of their genome non-specifically into the genome of the host and thereby act as insertional mutagens that can disrupt gene regulation or cause the production of an altered gene product [1]. The integrated retrovirus, termed the provirus, contains strong transcription-regulatory signals that can induce or enhance the expression of nearby genes. When such affected genes are involved in cell survival and proliferation, their deregulated expression may contribute to tumorigenesis [2]. In tumors caused by retroviral insertions, the proviruses constitute a tag allowing the identification of candidate genes with a possible role in tumorigenesis. By this approach several recent studies have contributed to the discovery of new proto-oncogenes and also, when performed in genetically modified mice expanded our knowledge on oncogene cooperativity [3?]. Moreover, insertional mutagenesis is a concern in gene therapy by retroviral vectors [5]. By another development, evidence is also emerging that endogenous retroviruses of mice and humans may contribute to oncogenesis by the activation of nearby genes without any need for new retroviral insertions in somatic cells [6]. Based on the analysis of somatic integrations selected during malignant transformation various types of virus-induced gene activation have been proposed. By the process known as enhancerinsertion a provirus increases the production of a normal transcript of an adjacent target gene [2]. In these cases, proviruses are often found outside the transcription unit of the target gene and in many cases upstream of the target gene and in the opposite transcriptional orientation. Other types of insertional mutagenesis result in the formation of chimeric RNA species containing viral and host sequences. One example of this is promoter insertion in which proviruses are integrated in the same.
