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

Bilayer Amniotic Membrane/Nano-fibrous Fibroin Scaffold Promotes Differentiation Capability of Menstrual Blood Stem Cells into Keratinocyte-Like Cells

The skin provides a dynamic barrier separating and protecting human body from the exterior world, and then immediate repair and rebuilding of the epidermal barrier is crucial after wound and injury. Wound healing without scars and complete regeneration of skin tissue still remain as a clinical challenge. The demand to engineer scaffolds that actively promote regeneration of damaged areas of the skin has been increased. In this study, menstrual blood-derived stem cells (MenSCs) have been induced to differentiate into keratinocytes-like cells in the presence of human foreskin-derived keratinocytes on a bilayer scaffold based on amniotic membrane and silk fibroin. Based on the findings, newly differentiated keratinocytes from MenSCs successfully expressed the keratinocytes specific markers at both mRNA and protein levels judged by real-time PCR and immunostaining techniques, respectively. We could show that the differentiated cells over bilayer composite scaffolds express the keratinocytes specific markers at higher levels when compared with those cultured in conventional 2D culture system. Based on these findings, bilayer amniotic membrane/nano-fibrous fibroin scaffold represents an efficient natural construct with broad applicability to generate keratinocytes from MenSCs for stem cell-based skin wounds healing and regeneration. © 2017, Springer Science+Business Media, LLC, part of Springer Nature

Regulation of luteinizing hormone receptor in hippocampal neurons following different long-lasting treatments of castrated adult rats

218-227The aim of this study was to investigate the effects of different Luteinizing hormone (LH) and steroid hormones levels on LH receptor (LHR) expression in the hippocampal cells. Rats (24 males and 24 females) were assigned to four groups: one control and three experimental [gonadectomy (GDX), gonadectomy + gonadotropin releasing hormone analogue (GDX+GnRHa) and GDX+GnRHa+estradiol (E2) or testosterone (T)] independently for each gender. All experimental rats were gonadectomized; then GnRHa was administrated to GDX+GnRHa group, and GnRHa plus steroid hormone to GDX+GnRHa+E2 or T group in both genders for four-month. LHR mRNA expression and its protein level in hippocampal cells were measured using QRT-PCR and Western blotting. Quantification of mRNA revealed a decrease in LHR transcripts level in GDX+GnRHa group of females. A significant change was observed between GDX groups and GDX+GnRHa+E2 or T versus GDX+GnRHa group in females. High levels of LH decreased significantly the immature isoform of LHR in GDX group compared to control group in both genders, but low LH concentrations in GDX+GnRHa group induced immature LHR isoform production only in females. Therefore increased LH concentration induces production of incomplete LHR transcripts in hippocampal cells and decreases immature LHR at the protein level. This implies that LH decreases the efficiency of translation through either producing non-functional LHR molecules or preventing their translation

Rotavirus VP7, VP4 and VP6 genotypes co-circulating in Tehran, Iran, between 2003 and 2004

Rotaviruses were detected by enzyme-linked immunosorbent assay (ELISA) in 92 out of 374 faecal samples collected between November 2003 and October 2004 at the Markaz Tebbi Koudakan Hospital, Tehran, Iran, from children aged 6 months to 5 years. Analysis of clinical and disease severity data showed a significant association between rotavirus infection and diarrhoea, vomiting and severe dehydration. Ninety-two samples (64 rotavirus ELISA-positive and 28 ELISA-negative samples) were sent to the Enteric Virus Unit, Virus Reference Department, Centre for Infection, Health Protection Agency, UK for rotavirus characterization by G-typing, P-typing and subgrouping (SG) using reverse transcriptase (RT)–PCR, semi-nested PCR and sequencing methods. In this study, both common and uncommon rotavirus genotypes were detected. The most prevalent types were G1P[8], SGII (59·2%) followed by G9P[8] SGII (15·5%) which has not been previously reported from Iran. Unusual genotypes G1P[10] SGI (2·8%) and G12P[8] SGII (1·4%) and strains derived from reassortment between common co-circulating genotypes such as G1P[4] SGII represented 5·6% of strains. Mixed infections with combinations of G1+G4P[8] SGII and G1+G9P[8] SGII were also found. This contrasts with previous reports from Iran in which a small number of common rotavirus strains (G1 and G4) were found. This study highlights the need for continued surveillance and characterization of rotaviruses to take account of the rapid evolution and introduction of novel rotaviruses into the human population