Causes of bimodal melting curve:Asymmetric guaninecytosine (GC) distribution causing two peaks in melting curve and affecting their shapes

The aim of this study was to present a new situation in which a relatively single short PCR-product might show two separate peaks with sequence specific shapes at the dissociation curve. SYBR-Green I real-time RT-PCR was performed on Lhcgr-gene transcripts in rats. Different programs were used for melting curve simulation and estimating Tm. Statistical tests were performed to determine whether two peaks at the dissociation curve were belonging to a single template. A bimodal melting curve was observed in real-time RT-PCR on a short segment (169 bp) of Lhcgr gene with a single band in gel electrophoresis. Sequencing of the Cloned PCR-product was compatible with template sequence. Realtime PCR using the vector conveying interested sequence, showed again two peaks at dissociation curve. The GC-content of first 100 bases (75%) and last 69 bases (42%) were significantly different. DNA melting simulation programs also confirmed the bimodal pattern, although, their height and wideness were different to actual peaks. Due to the asymmetric GC distribution effect on dissociation curve in short sequences, it is highly recommended to use DNA melting simulation programs to predict the number of peaks in the melting curve when designating primers; however, predicted peak shapes are not always accurate.Key words: Asymmetric GC distribution, bimodal melting curve, DNA melting simulation, SYBR-green I realtime PCR

GSTM1 and GSTP1 polymorphisms and glutathione S-transferase activity: Iranian infertile men

"nBackground: Pi-GST and Mu-GST are subclasses of glutathione S-transferase that present on human sperm surface and play an important role against oxidative stress. Therefore, any defects in the enzyme activity may be associated with male infertility.In this study the polymorphisms of GSTM1 and GSTP1 in association with enzyme activity and sperm parameters were studied. "nMethods: This case-control study involved 95 men with oligoastenoteratozoospermia and 26 controls with normozoospermia. Semen analyses were carried out according to WHO guidelines. Blood DNA was extracted using salting out procedures. GSTM1 and GSTP1 polymorphisms gene were determined through PCR-RFLP and multiplex PCR, respectively. Finally, Glutathione S-transferase activity was measured. "nResults: Frequencies of GSTM1 null genotype in oligoastenoteratospermic and normospermic groups were 52.1% and 53.8% respectively. There were no statistically significant differences in sperm parameters and enzyme activity between GSTM1 null and positive genotypes in two groups. There were no statistically significant differences in glutathione S-transferase activity between oligoastenoteratospermia and normospermic groups (p>0.05). All the 121 men in this study had Ile/Ile genotypes at 105 codon of GSTP1. Frequency of normal homozygote (114Ala/Ala), heterozygote (114Ala/Val) and mutant homozygote (114Val/Val) genotypes in oligoastenoteratospermic group were 81.1%, 17.9% and 1.1% respectively but in the control group they were 88.5%, 11.5% and null. "nConclusions: Total glutathione S-transferase activity and sperm parameters were not affected by deficient Glutathione S-transferase activity in GSTM1 null genotype. Compensate activity of other sperm surface glutathione S-transferase isozymes, like GSTP1, may justify the cause

Patent foramen ovale

Patent foramen ovale (PFO) is the most common congenital heart abnormality of fetal origin and is present in approximately ∼25% of the worldwide adult population. PFO is the consequence of failed closure of the foramen ovale, a normal structure that exists in the fetus to direct blood flow directly from the right to the left atrium, bypassing the pulmonary circulation. PFO has historically been associated with an increased risk of stroke, the mechanism of which has been attributed to the paradoxical embolism of venous thrombi that shunt through the PFO directly to the left atrium. However, several studies have failed to show an increased risk of stroke in asymptomatic patients with a PFO, and the risk of stroke recurrence is low in patients who have had a stroke that may be attributed to a PFO. With the advent of transoesophageal and transthoracic echocardiography, as well as transcranial Doppler, a PFO can be routinely detected in clinical practice. Medical treatment with either antiplatelet or anticoagulation therapy is recommended. At the current time, closure of the PFO by percutaneous interventional techniques does not appear to reduce the risk of stroke compared to conventional medical treatment, as shown by three large clinical trials. Considerable controversy remains regarding the optimal treatment strategy for patients with both cryptogenic stroke and PFO. This Primer discusses the epidemiology, mechanisms, pathophysiology, diagnosis, screening, management and effects on quality of life of PFO