Differences In DNA Levels Research Articles

Evaluating the Impact of Ultrasonic Irrigation on Bacterial Levels and Activity Following Chemomechanical Procedures

IntroductionThis single-arm interventional trial aimed to investigate the efficacy of ultrasonic irrigation as a supplementary disinfection approach after chemomechanical procedures (CMP) using molecular techniques based on ribosomal RNA (rRNA) and rRNA genes (referred to as DNA). MethodsSamples were collected from 35 single-rooted teeth with radiographic evidence of apical periodontitis. Samples were taken after gaining root canal access (S1), (S2), and ultrasonic irrigation (S3). DNA-targeted quantitative polymerase chain reaction using universal primers was used to estimate total bacterial levels, while rRNA-targeted quantitative polymerase chain reaction was used to assess bacterial activity. Ratios between rRNA and DNA levels were calculated to search for active bacteria in the samples (rRNA/DNA ≥ 1). Wilcoxon matched-pairs signed-rank test was used to compare the differences in DNA levels between samples and DNA and rRNA levels within samples (P < .05). ResultsDNA-based methods revealed a significant decrease in bacterial levels from S1 to S2 and S2 to S3 (both P < .05). Notably, 11 out of 35 (31.4%) root canals did not harbor bacterial DNA after CMP, whereas ultrasonic activation increased DNA-negative samples to 17 (48.6%). However, all DNA-positive samples were also positive for rRNA, with significantly higher rRNA than DNA levels (P < .05), indicating bacterial activity at the sampling time. ConclusionsUltrasonic irrigation improved the disinfection of root canals after CMP by reducing bacterial levels. However, persisting bacteria remained active in the root canals after CMP and ultrasonic irrigation.

The effect of temperature and storage time of cuccal swabs on FGA and D13S317 loci with the STR PCR method

The samples used for forensic DNA analysis in living individuals are usually blood and buccal swabs, however, blood collection requires an invasive method that can cause discomfort, thus a buccal swab can be a good choice for individuals examined, especially children. This study aimed to determine the effect of temperature and storage time of buccal swabs on the quantity of DNA as material for DNA examination in the forensic field. This study was a laboratory experiment to determine the effect after treatment. Buccal swab samples were 48 and divided into 2 temperature groups, namely room temperature (RT) and 4℃. The division of the temperature groups was also observed with time differences, namely 1, 3, 5, 7 days. EDNA extraction used the DNAzol method and DNA quantification used a Spectrophotometer. The PCR process was carried out with STR primers FGA and D13S317 loci. The visualization stage used acrylamide gel and silver staining.&#x0D; The results of this study prove that there is an effect of temperature and storage time of buccal swab samples. The longer the treatment time, the lower the DNA level. With statistical analysis, it is obtained p-value of &lt;0.005, it can be concluded that there are significant differences in DNA levels at the temperature and storage time treatments of the buccal swab sample. The results of DNA visualization at the FGA and D13S317 loci using the STR PCR method in this study can still be detected and can be used as a reference for examination in forensic cases.&#x0D;

Helping doctors pick the best antibiotic

In just 30 minutes, a new method can determine if the bacteria in a person’s urine sample are vulnerable to a certain antibiotic (Sci. Transl. Med. 2017, DOI: 10.1126/scitranslmed.aal3693). Such a test could help doctors better manage antibiotic use when treating urinary tract infections (UTIs) by allowing them to reserve valuable, potent drugs for when other drugs will not work. In the new method, Rustem F. Ismagilov of California Institute of Technology and colleagues incubate bacteria in a urine sample either with or without an antibiotic of choice for 15 minutes. By comparing the bacterial DNA levels between the two conditions, the researchers can determine if the infectious strain is susceptible to the drug. To detect such small differences in DNA levels, the team uses an optimized version of loop-mediated isothermal amplification on a microfluidic chip to count the copies of a certain piece of DNA from Escherichia coli—the most

Hepatitis B viral load in diagnosing different clinical stages of chronic hepatitis B in a tertiary care hospital in North Kerala

Background: The diagnosis of chronic hepatitis B (CHB) infection has progressed from serological to molecular diagnostic methods. The newer sensitive technique of quantitation of hepatitis B virus (HBV) DNA by real-time polymerase chain reaction (RT-PCR) has helped in understanding the clinical stages of CHB, deciding on treatment and monitoring treatment response. Aim: This study aimed to determine the HBV DNA load by quantitative RT-PCR in the various clinical stages of CHB. Materials and Methods: Blood samples of CHB patients from the Gastroenterology Department received from June 2014 to December 2014 in the Microbiology Department were subjected to quantitative PCR analysis for HBV DNA. However, to facilitate analysis, only those patients' samples where hepatitis B e antigen (HBeAg) and alanine aminotransferase (ALT) status were known were selected for the study. Statistical analysis was done using SPSS (PASW statistics 18) for windows software. Chi-square test was used to analyse the differences in DNA level between the study groups. Results: A total of 71 CHB patients were included in the study. Of these, 29 (40.8%) were inactive carriers (HBeAg-negative ALT normal) and 42 (59.2%) were chronic active hepatitis B patients (ALT elevated with HBeAg-positive and HBeAg-negative cases). HBeAg was positive in 26 (36.6%) and negative in 45 (63.3%) patients. Among the 45 HBeAg-negative patients, 16 (22.5%) had CHB. Of the 71 CHB patients, 61 (85.9%) had detectable viral load. Serum HBV DNA load of 16 patients who were HBeAg-negative was significantly lower (median 5.5 × 105) than that of 26 patients who were HBeAg-positive (median 2.4 × 108) and higher than the 29 inactive carriers (median 1.6 × 103). Based on HBV load, 14 CHB patients who were HBeAg-positive and seven who were HBeAg-negative were started on antiviral therapy. Conclusion: Quantitation of HBV DNA based on HBeAg and ALT status helps to determine the stages of CHB. It could play an important role in assessing the status of those patients who are HBeAg-negative and inactive carriers with respect to viral load, as the former require treatment. The major role of HBV DNA determination is to evaluate patients with CHB (HBeAg-positive or HBeAg-negative) and to decide on antiviral therapy.

English

OBJECTIVE: To establish the relationship between biochemical, serological and viral replication indices in an individual with incidentally detected asymptomatic HBsAg positive. METHOD: This cross-sectional study was undertaken on 73 asymptomatic HBsAg positive individuals between January 2010 and December 2011. They were divided into 5 groups on the basis of HBeAg status, ALT levels, and HBV DNA levels. RESULTS: There was statistically significant difference in HBV DNA levels and ALT between HBeAg positive and negative; and in ALT levels between cases in Group C and E and that between Groups C & D. There were significant difference in DNA levels between group D & group E and that between group C & group E. HBV DNA levels Co- related significantly with grade (r = 0.48, p 40 IU/ml) and those with normal ALT(<40 IU/ml). CONCLUSION: ALT and AST had a strong correlation with HBV DNA level. INTRODUCTION: Hepatitis B Virus (HBV) infection and its sequel are serious public health problems worldwide. Around 2 billion people are infected by hepatitis B virus (HBV) worldwide of which 400 million persons suffer chronic infection with HBV. 1 HBV infection is usually clinically in apparent. Approximately, 5-10% of infected adults develop chronic liver disease of varying severity. More than 90% of infants infected during the first year of life and 30-50% children infected between 1-4 yrs of age develop chronic infection. The risk of death from HBV related liver cancer and cirrhosis is approximately 25% in persons who become chronically infected during child hood.2, 3 15-40% of carriers will develop serious complications during lifetime like cirrhosis, decompensated liver disease or HCC. 1 75% of carriers are present in Asia. India has the second largest pool of HBsAg carriers after China. AIMS AND OBJECTIVES: This study was undertaken to establish the relationship between biochemical, serological, viral replication indices in an individual with incidentally detected asymptomatic HBsAg positive.

Effect of an injectable 3D scaffold for osteoblast differentiation depends on bead size

The objective of this study was to evaluate the effect of beta-tricalcium phosphate (beta-TCP) bead size on the behavior of KUSA/A1 mouse osteoblasts when the beta-TCP beads are used as the solid phase of a scaffold in which alginate was used as the gel phase. KUSA/A1 cells were loaded onto a three-dimensional (3D) scaffold fabricated from beta-TCP beads with diameters ranging from 300 to 500 microm (small beads), 500-700 microm (medium beads) and 700-850 microm (large beads); cells were cultured for 3, 7 and 14 days. Scanning electron microscope observations showed that each bead was connected in a network consisting of the alginate gel and KUSA/A1 cellular matrix that was tightly bonded to form a 3D structure. After 3 days, cells in the 3D scaffold with medium beads had a significantly higher alkaline phosphatase activity (ALP) than cells in the other scaffolds. However, by 7 and 14 days in culture there was no significant difference in DNA levels, ALP activity or osteocalcin expression. At 8 weeks, only the composite containing small beads and KUSA/A1 cells had turned completely into bone in vivo. Thus, bead size may influence the success of bone formation in this context.

Significant differences in DNA viral load between HIV-1 and HIV-2 infected patients

Gottlieb et al.[1] suggest that the significance of the lower viral load in HIV-2-infected patients compared with HIV-1-infected patients could be biased by different distributions of CD4 cell counts and that we failed to adjust for CD4 cell count and other potential confounding factors like age, sex and the duration of infection. In fact, our analysis of DNA levels was stratified by CD4 cell counts (fig. 1 in reference [2]) and showed lower levels of DNA viral load in HIV-2 compared with HIV-1 in the higher CD4 strata (≥300 cells/μl), but not in the lower CD4 stratum (<300 cells/μl). To complete this analysis, we have performed a multivariate linear regression analysis including age, sex and HIV RNA within each CD4 cell count stratum (Table 1). After adjustment for age the difference of HIV DNA between the two types of HIV persisted in the higher strata (≥300 cells/μl), but it was only at the limit of significance in both strata after adjustment for age and sex. The relationship between viral type and DNA load was not significant any more after adjustment for HIV RNA, suggesting that the difference was mainly explained by the relation between viral replication and amount of HIV DNA. Therefore, at a given level of replication, no difference in the DNA level is found between HIV-1 and HIV-2. These results were also confirmed when we took into account the difference in sensitivity of our PCR method by imputing DNA values of 5 copies/μg for HIV-1 and 10 copies/μg for HIV-2 when the sample was below the respective detection limits.Table 1: Multivariate linear regression analysis of factors associated with HIV DNA: ANRS CO5 HIV-2 cohort.Given the low detection rate of HIV-2 RNA in plasma, Gottlieb et al.[1] also questioned whether our population of HIV-2-infected patients was representative. Studies in Africa generally overrepresent patients with advanced HIV disease and detectable plasma viral load, as in the study by Gottlieb et al.[1] in Senegal, taken as an example. We recently published viro-immunological follow-up data among a subset of incident HIV-2 infected patients from the French ANRS CO5 HIV-2 cohort compared with a matched HIV-1-infected patient incident group. The HIV RNA load was detectable in 86% and 15% of HIV-1-patients and HIV-2-patients, respectively, with 4.11 log10 copies/ml for HIV-1 and 2.09 log10 copies/ml for HIV-2, in full agreement with the values reported in this HIV DNA load comparative study involving prevalent cases [3]. Furthermore, the 29% overall rate of detectable HIV-2 RNA in prevalent cases of the French HIV-2 cohort, with the limit of detection of 100 copies/ml, ranges from 8% for a CD4 cell count greater than 500 cells/μl, to 35% when in the range 300–500 cells/μl, and 62% when below 300 cells/μl. However, the main conflict between our data and those of Gottlieb et al.[1] concerns the level of HIV-1 DNA. Gottlieb et al.[1] report median HIV DNA of 1.45 log10/μg which is lower than that reported in other studies quantifying HIV-1 DNA in antiretroviral-naïve patients, using the same quantification method (Amplicor HIV-1 Monitor, Roche Molecular Systems, Inc., Branchburg, New Jersey, USA) [4–6]. Indeed, these studies report median values between 2.0 and 2.6 log10 of total HIV-1 DNA/μg. Assuming that the amount of target DNA initially present in the PCR tube follows Poisson's law, the claimed sensitivity of the Monitor test (1 copy/μg) is controversial as it is highly unlikely to detect 1 copy/PCR in 100% of cases [7]. If a sample with 1 copy/μg is systematically detectable, then the calibration standard is underestimated. Moreover, HIV-1 strains circulating in Senegal are highly divergent [8] and sometimes difficult to quantify with primers and probes corresponding to less highly conserved genome regions than those selected for our PCR method [9]. Our work shows that significant differences in total DNA levels exist between HIV-1 and HIV-2 and that they are highly related to viral replication and CD4+ cell count as expected. These differences should be considered as one of the many factors contributing to the clinical and epidemiological differences between these two infections.

Hydrodynamic compression of young and adult rat osteoblast‐like cells on titanium fiber mesh

Living bone cells are responsive to mechanical loading. Consequently, numerous in vitro models have been developed to examine the application of loading to cells. However, not all systems are suitable for the fibrous and porous three-dimensional materials, which are preferable for tissue repair purposes, or for the production of tissue engineering scaffolds. For three-dimensional applications, mechanical loading of cells with either fluid flow systems or hydrodynamic pressure systems has to be considered. Here, we aimed to evaluate the response of osteoblast-like cells to hydrodynamic compression, while growing in a three-dimensional titanium fiber mesh scaffolding material. For this purpose, a custom hydrodynamic compression chamber was built. Bone marrow cells were obtained from the femora of young (12-day-old) or old (1-year-old) rats, and precultured in the presence of dexamethasone and beta-glycerophosphate to achieve an osteoblast-like phenotype. Subsequently, cells were seeded onto the titanium mesh scaffolds, and subjected to hydrodynamic pressure, alternating between 0.3 to 5.0 MPa at 1 Hz, at 15-min intervals for a total of 60 min per day for up to 3 days. After pressurization, cell viability was checked. Afterward, DNA levels, alkaline phosphatase (ALP) activity, and extracellular calcium content were measured. Finally, all specimens were observed with scanning electron microscopy. Cell viability studies showed that the applied pressure was not harmful to the cells. Furthermore, we found that cells were able to detect the compression forces, because we did see evident effects on the cell numbers of the cells derived from old animals. However, there were no other changes in the cells under pressure. Finally, it was also noticeable that cells from old animals did not express ALP activity, but did show similar calcified extracellular matrix formation to the cells from young animals. In conclusion, the difference in DNA levels as reaction toward pressure, and the difference in ALP levels, suggest that the osteogenic properties of bone marrow-derived osteoblast-like cells are different with respect to the age of the donor.

Differential Effect of 5α-Reductase Inhibition and Castration on Androgen-Regulated Gene Expression in Rat Prostate

Castration reduces prostate size and causes intraprostatic testosterone (T) and dihydrotestosterone (DHT) to fall to very low levels. 5 alpha-Reductase inhibition also reduces prostate size, but results in a marked increase in intraprostatic T levels. To compare the effects of 5 alpha-reductase inhibition and castration on prostate physiology, male Sprague-Dawley rats were left intact, castrated, or given the selective 5 alpha-reductase inhibitor finasteride for up to 9 days. To be sure that finasteride itself did not directly affect gene expression, an additional group of rats was castrated and given finasteride for 4 days. The prostates were weighed, intraprostatic RNA, DNA, and androgen levels were measured, and mRNAs for two androgen-regulated genes, prostate steroid-binding protein (PSBP; an androgen-induced gene) and testosterone-repressed prostate message (TRPM-2), were quantitated by Northern and slot blot analyses. Finasteride caused a 95% reduction in intraprostatic DHT levels and a 10-fold increase in intraprostatic T levels. Finasteride, as expected, caused a pronounced decrease in prostate weight (45% on day 4). DNA content fell correspondingly (48% on day 4). Intraprostatic DNA (micrograms of DNA per gland) on day 4 was 328 +/- 53 in control rats, 171 +/- 10 in finasteride-treated rats (P less than 0.001 compared to controls), 115 +/- 2 in castrated rats (P less than 0.05 compared to finasteride), and 107 +/- 43 in finasteride-treated plus castrated rats (P = NS compared to castration alone). There were no significant differences in DNA levels among the groups when expressed per mg prostate tissue, indicating that mean prostate cell size was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular and tumoural heterogeneity of EGFR gene amplification in human malignant gliomas

It has been demonstrated that the epidermal growth factor receptor (EGFR) gene, the normal human counterpart of the viral erb-B oncogene is amplified and overexpressed in over 50% of human malignant gliomas (HMGs). In the present study, analysis of the immunohistological staining characteristics of 57 HMGs using an anti-EGFR monoclonal antibody (mab) showed positive staining in 65% of the tumours with large cellular and regional differences in staining pattern and intensity. Screening a smaller number of HMGs with molecular hybridization techniques revealed 10/21 glioblastomas (48%) amplified for the gene; of 11 glioblastomas studied by Northern blot hybridization, 7 tumours with gene amplification showed RNA overexpression, the remaining 4 without amplification did not. Regional differences in DNA levels were observed by Southern blot in 2 tumours: in one particular case, amplification and overexpression were found to be localized to one half of a single HMG, the other half showing neither EFGR gene amplification nor overexpression.