ABSTRACT
Background: DNA isolation procedure can significantly influence the quantification of DNA by real time PCR specially when cell free DNA [cfDNA] is the subject. To assess the extraction efficiency, linearity of the extraction yield, presence of co-purified inhibitors and to avoid problems with fragment size relevant to cfDNA, development of appropriate External DNA Control [EDC] is challenging. Using non-human chimeric nucleotide sequences, an EDC was developed for standardization of qPCR for monitoring stability of cfDNA concentration in blood samples over time
Methods: A DNA fragment of 167 bp chimeric sequence of parvovirus B19 and pBHA designated as EDC fragment was designed. To determine the impact of different factors during DNA extraction processing on quantification of cfDNA, blood samples were collected from normal subjects and divided into aliquots with and without specific treatment. In time intervals, the plasma samples were isolated. The amplicon of 167 bp EDC fragment in final concentration of 1.1 pg/ 500 microl was added to each plasma sample and total DNA was extracted by an in house method. Relative and absolute quantification real time PCR was performed to quantify both EDC fragment and cfDNA in extracted samples
Results: Comparison of real time PCR threshold cycle [Ct] for cfDNA fragment in tubes with and without specific treatment indicated a decrease in untreated tubes. In contrast, the threshold cycle was constant for EDC fragment in treated and untreated tubes, indicating the difference in Ct values of the cfDNA is because of specific treatments that were made on them
Conclusions: Spiking of DNA fragment size relevant to cfDNA into the plasma sample can be useful to minimize the bias due to sample preparation and extraction processing. Therefore, it is highly recommended that standard external DNA control be employed for the extraction and quantification of cfDNA for accurate data analysis
ABSTRACT
Objective: the out-of-field/non-target effect is one of the most important phenomena of ionizing radiation that leads to molecular and cellular damage to distant non-irradiated tissues. The most important concern about this phenomenon is carcinogenesis many years after radiation treatment. In vivo mechanisms and consequences of this phenomenon are not known completely. Therefore, this study aimed to evaluate the oxidative damages to out-of-field lung tissues 24 and 72 hours after pelvic irradiation in rats
Materials and Methods: in this experimental- interventional study, Sprague-Dawleymale rats [n=49] were divided into seven groups [n=7/each group], including two groups of pelvis-exposed rats [out-of-field groups], two groups of whole body- exposed rats [scatter groups], two groups of lung-exposed rats [direct irradiation groups], and one control sham group. Out-of-field groups were irradiated at a 2×2 cm area in the pelvis region with 3 Gy using 1.25 MeV cobalt-60 gamma-ray source, and subsequently, malondialdehyde [MDA] and glutathione [GSH] levels as well as superoxide dismutase [SOD] activity in out-of-field lung tissues were measured. Results were compared to direct irradiation, control and scatter groups at 24 and 72 hours after exposure. Data were analyzed using Mann-Whitney U test
Results: SOD activity decreased in out-of-field lung tissue 24 and 72 hours after irradiation as compared with the controls and scatter groups. GSH level decreased 24 hours after exposure and increased 72 hours after exposure in the out-of-field groups as compared with the scatter groups. MDA level in out-of-field groups only increased 24 hours after irradiation
Conclusion: pelvis irradiation induced oxidative damage in distant lung tissue that led to a dramatic decrease in SOD activity. This oxidative stress was remarkable, but it was less durable as compared to direct irradiation
ABSTRACT
Background: Colorectal cancer [CRC] is one of the most common causes of cancer-related death in the world. The expression of N-myc downstream-regulated gene 2 [NDRG2] is down-regulated in CRC. The aim of this study was to investigate the effect of NDRG2 overexpression on cell proliferation and invasive potential of SW48 cells
Methods: SW48 cells were transfected with a plasmid overexpressing NDRG2. After stable transfection, the effect of NDRG2 overexpression on cell proliferation was evaluated by MTT assay. The effects of NDRG2 overexpression on cell migration, invasion and cell motility and matrix metalloproteinase 9 [MMP9] activities were also investigated using matrigel transwell assay, wound healing assay and gelatin zymography, respectively
Results: MTT assay showed that overexpression of NDRG2 caused attenuation of SW48 cell proliferation. Transwell and wound healing assay revealed that NDRG2 overexpression led to inhibition of migration, invasion, and motility of SW48 cells. The overexpression of NDRG2 also reduced the activity of secreted MMP-9
Conclusions: The results of this study suggest that NDRG2 overexpression inhibits proliferation and invasive potential of SW48 cells, which likely occurs via suppression of MMP-9 activity
ABSTRACT
Amylin and Salmon Calcitonin belong to the calcitonin family of peptides and have high affinity binding sites in the rat spinal cord. The aim of this study was to characterize receptors for Amylin and Salmon Calcitonin functionally in the spinal cord of rats. We assessed the expression of c-Fos in response to intraplantar formalin in the lumbar regions of the spinal cord in conscious rats. Amylin [0.05 nmoles] or Salmon Calcitonin [0.005 nmoles] was administered intrathecally [i.t.] 10 minutes before the start of the formalin test. Antagonists were injected intrathecally 10 minutes before the administration of either of the peptides. Two hours after formalin stimulation, rats pretreated intrathecally by either Amylin or Salmon Calcitonin, showed lower numbers of c-Fos immunoreactive nuclei in their lumbar spinal cord as compared to rats pretreated with saline. These effects were reversed upon co-administration of either of the Amylin antagonists AC187 or rat amylin[8-37], but not rat alpha-CGRP[8-37] A few cells with c-Fos immunoreactivity were found in the lumbar spinal cord of rats two hours after i.t. injection of saline, Amylin and/or Salmon Calcitonin. However, Fos-like immunoreactivity was increased in the lumbar spinal cord two hours after i.t. treatment of either of the antagonists AC187 and rat amylin[8-37], when compared to saline treated rats. Both Amylin and Salmon Calcitonin inhibit formalin induced c-Fos expression in the rat lumbar spinal cord when administered intrathecally. Effects of the two peptides were possibly produced by undefined receptors
ABSTRACT
The role of oxidative stress in endosulfan-induced reproductive toxicity has been implicated. This study was performed to evaluate the possible protective effect of vitamins E and C, against endosulfan-induced reproductive toxicity in rats. Fifty adult male Sprague-Dawley rats were randomly divided into five groups [n=10 each]. The groups included a control receiving vehicle, a group treated with endosulfan [10 mg/kg/day] alone, and three endosulfan-treated group receiving vitamin C [20 mg/kg/day], vitamin E [200 mg/kg/day], or vitamine C+vitamin E at the same doses. After 10 days of treatment, sperm parameters, plasma lactate dehydrogenase [LDH], plasma testosterone and malondialdehyde [MDA] levels in the testis were determined. Oral administration of endosulfan caused a reduction in the sperm motility, viability, daily sperm production [DSP] and increased the number of sperm with abnormal chromatin condensation. Endosulfan administration increased testis MDA and plasma LDH. Supplementation of vitamin C and vitamin E to endosulfan-treated rats reduced the toxic effect of endosulfan on sperm parameters and lipid peroxidation in the testis. Vitamin E was more protective than vitamin C in reducing the adverse effects of the endosulfan. The findings data suggest that administration of vitamins C and E ameliorated the endosulfan-induced oxidative stress and sperm toxicity in rat. The effect of vitamin E in preventing endosulfan-induced sperm toxicity was superior to that of vitamin C