Cell Journal

Objective: Glioblastoma multiforme (GBM), one of the most common and aggressive malignant brain tumors, is highly resistant to radiotherapy. Numerous approaches have been pursued to find new radiosensitizers. We used a picogreen and colonogenic assay to appraise the DNA damage and cell death in a spheroid culture of GBM cells caused by iodine-131 (I-131) beta radiation in the presence of topotecan (TPT). Materials and Methods: U87MG cells were cultured as spheroids with approximate diameters of 300 μm. Cells were treated with beta radiation of I-131 (at a dose of 2 Gy) and/ or TPT (1 μg/ml for 2 hours). The numbers of cells that survived were compared with untreated cells using a colonogenic assay. In addition, we evaluated possible DNA damages by the picogreen method. The relation between DNA damage and cell death was assessed in the experimental study of groups. Results: The findings showed that survival fraction (SF) in the I-131+TPT group (39%) was considerably less than the I-131 group (58.92%; p
The spectrum obtained from samples of the irradiated and control groups of cells treated with PicoGreen solution, at an excitation wave length of 485 nm and emission wave length of 600 nm (Taken by a Shimadzu spectroflourometer).
Calibration curve obtained by the amount of fluorescence intensities of control group and DNase-treated group ≈1.79 change in amount of fluorescence intensity, a 1% break will occur.
Spectrum of electrons released in the medium obtained by MCNP4c code.
The passage of ionizing radiation in living cells creates clusters of damaged nucleotides in DNA. In this study, DNA strand breaks induced by the beta particle of iodine-131 (I-131), have been determined experimentally and compared to Monte Carlo simulation results as a theoretical method of determining(131)I damage. In this experimental study, in order to create single strand breaks (SSB) and double strand breaks (DSB) in the DNA, glioblastoma (GBM) cells were exposed to 10 mCi I-131, at a dose of 2 Gy. Damage of irradiated cells were evaluated quantitatively by the Fast Micromethod assay. The energy spectrum of electrons released in cells were obtained by the macroscopic Monte Carlo code (MCNP4c) and used as an input of the micro Monte Carlo code (MCDS). The percent of damage induced in cells was analyzed by Mann-Whitney test. A significant reduction (p<0.05) in fluorescence intensity in irradiated cells compared to control cells as determined by the Fast Micromethod assay represented induced SSB and DSB damages in the DNA of irradiated cells. Comparison of experimental and theoretical results showed that the difference between the percentages of SSB per Gy was about 7.4% and DSB was about 1% per Gy. The differences in experimental and theoretical results may be due to the algorithm of applied codes. Since the Fast Micromethod and other experimental techniques do not provide information about the amount of detailed and complex damages of DNA-like base damages, the applied Monte Carlo codes, due to their capability to predict the amount of detailed damages that occur in the DNA of irradiated cells, can be used in in vitro experiments and radiation protection areas.
To assess relative biological effectiveness (RBE) of (131)I radiation relative to (60)Co gamma rays in glioblastoma spheroid cells. : In this experimental study, glioblastoma spheroid cells were exposed to (131)I radiation and (60)Co gamma rays. Radiation induced DNA damage was evaluated by alkaline comet assay. Samples of spheroid cells were treated by radiation from (131)I for four different periods of time to find the dose-response equation. Spheroid cells were also exposed by 200 cGy of (60)Co gamma rays as reference radiation to induce DNA damage as endpoint. Resulted RBE of (131)I radiation relative to (60)Co gamma rays in 100 µm giloblastoma spheroid cells was equal to 1.16. The finding of this study suggests that (131)I photons and electrons can be more effective than (60)Co gamma rays to produce DNA damage in glioblastoma spheroid cells.
Androgen stimulation of prostate cancer antigen 3 (PCA3) (A), Prostate specific antigen (PSA) mRNA (B) and, microRNA-141 (miR-141) (C) in prostate cancer cells and their release into medium. Following hormone depletion for 48 hours LNCaP cells were treated with 0, 1, 10, and 100 nM dihydrotestosterone (DHT) for 24 hours and RNA molecules quantified in quantitative polymerase chain reaction (qPCR). GAPDH was used as the reference gene for PCA3 and PSA-mRNA while miR-16 was used as the reference gene for miR-141. Results of five independent experiments were evaluated. Basal level (0 nM DHT) of each molecule was taken 1, and changes were expressed as 'fold changes'. Median and maximum values are shown.  
Plasma levels of prostate cancer antigen 3 (PCA3) (A) and prostate specific antigen (PSA) mRNA (B), and micro- RNA-141 (miR-141) (C) in prostate cancer (Pca) patients. Total RNA was isolated from plasma, and following cDNA synthesis, each RNA molecule was relatively quantified. Expression levels in healthy individuals were taken as 1 and changes in patients were expressed as 'fold changes'.  
Prostate cancer antigen 3 (PCA3) and microRNA-141 (miR-141) are emerging molecules in prostate cancer (PCa) pathogenesis and have been shown to be involved in androgen signaling. In this original research, we designed an experimental cell model with androgen-sensitive LNCaP cells to comparatively assess the extent of androgen responsiveness of PCA3-mRNA and miR-141 along with prostate specific antigen (PSA)mRNA and their release into culture medium. These molecules were also measured in the plasma of the patients with early PCa which is considered to be analogous to androgenresponsive cells. In this experimental study, LNCaP cells were exposed to androgen ablation for 48 hours and treated then with dihydrotestosterone (DHT) for 24 hours. Expression of all three RNA molecules in cells, culture medium or plasma was measured by quantitative polymerase chain reaction (qPCR). Our results show that DHT differentially affects the expression of these molecules. PCA3 was the most evidently induced molecule (up to 400-fold, p<0.001), while the effect was moderate for PSA-mRNA (up to 30-fold, p<0.001). In contrast, the stimulation of miR-141 was much weaker (up to 1.5-fold, p>0.05). With regard to the release into culture medium, a similar picture was observed except for PCA3. PCA3 was below the detection level despite its high stimulation. DHT treatment led to a significant release of PSA-mRNA (up to 12-fold). Similar to its induction pattern in LNCaP cells, miR-141 was released at a limited quantity into the medium (up to 1.7- fold, p=0.07). In plasma, only PCA3 differed significantly between the patients and healthy subjects (p=0.001). Our findings indicate that PCa-related RNA molecules respond differentially to androgen stimulation suggesting differential regulation by androgens.
shown on 1% Agarose gel. The marker is a 100bp ladder.
Amplification of VDR gene fragment. PCR products are
shown on 1% Agarose gel. The marker is a 100bp ladder
Restrictions digest of PCR product. The first well
shows the pattern of heterozygote (A/G), the second well
shows the pattern of homozygote for A/A, and the third well
shows the pattern of homozygote for G/G. The fourth well is
a 100bp DNA ladder
Osteoporosis is a bone disorder that reduces bone mineral density (BMD) and leads to bone fracture. In addition to different factors, gene polymorphisms have been revealed to be associated with osteoporosis. In this study, we investigated the association between the BsmI polymorphism of vitamin D receptor (VDR) gene (rs1544410) and BMD in a population of Iranian women. In this case control study, clinical risk factors for osteoporosis were obtained from the participants through a questionnaire for a case-control study. The World Health Organisation (WHO) criteria were applied for the diagnosis of the disease. Peripheral blood samples were obtained from 146 pre- and or postmenopausal Iranian women aged between 35 and 71 years (53.53 ± 9.8). The study population was classified for BMD into normal and osteoporotic groups, who matched for age, pregnancy status, menstrual condition, and body mass index (BMI). The BMD of the lumbar spine (L1-4) and femoral neck was measured. Polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) was performed to detect and analyze the genotype. The frequencies of AA and GG were significantly different between the two groups (p value<0.05), with the first genotype being higher in the patients and the second being higher in the normal group. The GG genotype was significantly associated with increased BMD in the lumbar spine (p value<0.05) but non-significant in the femoral neck (p value>0.05). BsmI polymorphism of VDR gene has a significant association with BMD in the lumbar spine and may have a minor effect on the proximal femur BMD in Iranian women.
The clavulanic acid regulatory gene (claR) is in the clavulanic acid biosynthetic gene cluster that encodes ClaR. This protein is a putative regulator of the late steps of clavulanic acid biosynthesis. The aim of this research is the molecular cloning of claR, isolated from the Iranian strain of Streptomyces clavuligerus (S. clavuligerus). In this experimental study, two different strains of S. clavuligerus were used (PTCC 1705 and DSM 738), of which there is no claR sequence record for strain PTCC 1705 in all three main gene banks. The specific designed primers were subjected to a few base modifications for introduction of the recognition sites of BamHI and ClaI. The claR gene was amplified by polymerase chain reaction (PCR) using DNA isolated from S. clavuligerus PTCC 1705. Nested-PCR, restriction fragment length polymorphism (PCR-RFLP), and sequencing were used for molecular analysis of the claR gene. The confirmed claR was subjected to double digestion with BamHI and ClaI. The cut claR was ligated into a pBluescript (pBs) vector and transformed into E. coli. The entire sequence of the isolated claR (Iranian strain) was identified. The presence of the recombinant vector in the transformed colonies was confirmed by the colony-PCR procedure. The correct structure of the recombinant vector, isolated from the transformed E. coli, was confirmed using gel electrophoresis, PCR, and double digestion with restriction enzymes. The constructed recombinant cassette, named pZSclaR, can be regarded as an appropriate tool for site directed mutagenesis and sub-cloning. At this time, claR has been cloned accompanied with its precisely selected promoter so it could be used in expression vectors. Hence the ClaR is known as a putative regulatory protein. The overproduced protein could also be used for other related investigations, such as a mobility shift assay.
Effects of IL-1α on chondrocyte morphology in explant culture. Explants were assessed by invert microscope. A. Cartilage
explant from the control group after 28 days. B. Cartilage explant cultured for 3 days in the presence of IL-1α. C. Cartilage
explant cultured for 7 days in the presence of IL-1α. The edges of the cartilage explants started to become translucent. D. Cartilage
explant cultured for 14 days in the presence of IL-1α. E and F Cartilage explant cultured for 21 days in the presence of
IL-1α. At day 21, the cell membranes began to show a vesicular appearance and the cytoplasm showed a granular appearance
. At day 28 of culture, chondrocytes showed a granular black point appearance (G and H). Magnification×400 for images A, B,
E-H; ×200 for images C and D.
Effects of IL-1α on tissue characteristics in bovine nasal explants. The samples were sectioned and stained with H&E. A.
Section from the control group after 28 days. B. Section from a cartilage explants cultured for 3 days in the presence of IL-1α.
C. Section from a cartilage explant cultured for 7 days in the presence of IL-1α. D. Section from a cartilage explant cultured for
14 days in the presence of IL-1α. E. Section from a cartilage explant cultured for 21 days in the presence of IL-1α. F. Section
from a cartilage explant cultured for 28 days in the presence of IL-1α. Magnification × 400.
Effects of IL-1α on proteoglycan degradation. The samples were stained with Alcian blue. A. Section from control group
after 28 days. B. Section from a cartilage explant cultured for 3 days in the presence of IL-1α. C. Section from a cartilage
explant cultured for 7 days in the presence of IL-1α. D. Section from a cartilage explant cultured for 14 days in the presence
of IL-1α. E. Section from a cartilage explant cultured for 21 days in the presence of IL-1α. F. Section from a cartilage explant
cultured for 28 days in the presence of IL-1α. Magnification ×400.
Comparison of lactate levels in media in bovine nasal
cartilage (BNC) explants after exposure to IL-1α and in
the control group for 28 days. Data represents absorbance.
*P<0.001 compared with control group.
A study of the histological events under interleukin-1α (IL-lα) induction of bovine nasal cartilage (BNC) could result in useful data to better understand the mechanisms involved in tissue breakdown in joint diseases. The aim of this study was to investigate the effects of IL-lα on chondrocyte phenotype and extracellular matrix (ECM) changes in BNC explants. In this experimental study, samples were divided into two groups. Group I (control group) BNC explants were cultured only in Dulbecco's modified Eagle's medium (DMEM). In group II, BNC explants were treated with IL-lα (10 ng/ ml) for 28 days. Then, samples were harvested on culture days 3, 7, 14, 21 and 28 and chondrocyte morphology and ECM alterations were assessed by invert microscopy and histology by hematoxylin and eosin (H&E) and Alcian blue. Cell viability was evaluated by the lactate dehydrogenase (LDH) assay test. Data were analyzed by the t test and p<0.05 was considered significant. IL-lα induced significant morphological changes in cartilage. In the presence of IL-lα, most chondrocytes transformed into a fibroblast-like morphology with a granular black point appearance. An increase in the cell: matrix ratio was observed and there were decreased numbers of chondrocytes.IL-lα induced breakdown of ECM. We observed partial degradation of ECM between days 7-14 and complete degradation occurred between days 21-28 of culture. The LDH levels increased. IL-1α induced morphological changes in chondrocytes and increased destruction of cartilage ECM. There was a parallel correlation between proteoglycan degradation and changes in chondrocyte morpholgy.
World Cancer Day (WCD) would be celebrated each year through the world on 4(th) of February, at Tuesday, to remember all the efforts done by the WHO, united nations, governmental and non-governmental health organizations regarding making the strategy to fight against cancer and delivering the real message about this epidemic sickness and its treatments containing its precautionary measures by uniting all the people a day on global basis.In brief, cancer is a large group of different disorders, all involving unregulated cell growth. In malignancy, cells divide and grow uncontrollably, forming malignant tumors, and invade neighboring parts of the body. The tumor may also spread to more distant parts of the body through the lymphatic system or blood stream.It has been observed that most of the malignancy cases and cancer deaths happen in less developed areas of the world. If it is not controlled, this situation may get worse by 2030. Therefore, it is very crucial to get control over such condition throughout of the world. The main aim of WCD are to aid, save millions of preventable deaths every year by raising alertness and education about malignancy, and pressing to governments throughout the world to take action against this disease.The day is also a key chance for everybody affected by cancer to work jointly to ensure that world leaders stick to the promises, they made at the United Nations summit in relation to reducing the impact of cancer. During this occasion celebration persons are promoted well about their healthy lifestyles, balanced diet, regular physical activity and weight management, using antioxidants in order to diminish the risk of happening of malignancies. They are encouraged to get rid of their unhealthy diet and physical immobility too.This day, also is celebrated to plan certain new strategies and implement various new programs which help to alert more people about this disorder. This event is organized on yearly basis under the supervision of Union for International Cancer Control (UICC) and other leading health organizations participating for cancer fighting. Also, WCD, is celebrated to make attentive the normal people about the risk factors and preventive methods of the cancer to get prevented or its prompt recognition. The theme of WCD of 2014 is "Debunk the Myths". Generally, people misery from the cancer are detested by the normal people in the society and behaved like an untouched person. There are some other social myths related to the malignancy that normal people believe that they, would got cancer if they would contact or live with the person having malignancy. The day is also distinguished as well to eradicate such type of the social myths related to the malignancy.Hence, it is celebrated to make the concern about all the reality of the malignancy, such as its symptoms, causing factors and treatment. On the other hand, some of the activities organized at this day to indicate people that, the person with malignancy, should not be treated distinctly. They must have equal rights to live similar a normal person in the society and any relation should not be altered for them.They must be fulfilled their wishes by their relatives even if they have less chances of existence. It is very essential to make them feel better like a normal person and should not make them sense that they are given some treatment for existence as they are dying. They should, feel self-respect and find a normal environment in their home and society. It is indispensable to remember that, normal individual must avoid being over-sympathetic to them.
Expression of IL10 as an IL-27 biological activity assay. Panel A shows the results of real time-PCR. IL-10 is overex- pressed in T cell-COS7/IL-27 compared with T cell-COS7. GAPDH served as the endogenous reference gene. Panel B shows the level of IL10 expression in T cells cultured with COS-7/IL-27 increased 5-fold compared with COS-7 by real time PCR. 
Autoimmune diseases precede a complex dysregulation of immune system. Thelper17 and IL-17 have central role in initiation of inflammation and subsequent autoimmune disease. IL-27 significantly controls autoimmune diseases by Th17 and IL-17suppressing. In the present study we created genetic engineered mesenchymal stem cell mediating with lentiviral vectors for releasing IL-27 as a good vehicle ex-vivo gene therapy for reduction of inflammation and autoimmune diseases. Mesenchymal stem cells (MSCs) were isolated from lipoasprate and characterized by differentiation. Two subunits of IL-27 (p28 and EBI3) were cloned in pCDH-513B-1 lentiviral vector. Expression of p28 and EBI3 was determined by real time PCR technique. MSCs were transduced by pCDH-CMV-p28-IRES-EBI3-EF-copGFP-Pur lentiviral vector and bioassay of IL-27 was evaluated by IL-10 expression. The cell differentiation confirmed truly isolation of MSCs from lipoaspirate. Restriction enzyme digestion and sequencing verified that p28 and EBI3 successfully were cloned in pCDH-513B-1 lentiviral vector. Real time PCR showed the high expression level of IL-27 and IL-10 expression as well as accurate activity of IL-27. The results showed transduction of functional IL-27 to AD-MSCs by mean of lentiviral vector. The lentiviral vector showed no effect on the MSC characteristics.
Percentages of plasmacytoid dendritic cells (pDCs) in peripheral blood mononuclear cells (PBMCs) measured by flow cytometry. The A region points to lymphocyte and monocyte regions. In B region, all CD123pos cells were selected. In the C region, ILT3pos cells and CD14+CD16neg cells were gated. As ILT3pos cells (or CD85kpos cells) were gated, all basophils (CD85kneg) were excluded; monocytes and DCs (CD85kpos) were restricted. CD14+CD16neg cells were selected to exclude all monocytes. Then, ILT3pos and CD123pos cells were selected, pointing to the D region. 
Supernatant IFN-α concentration/plasmacytoid dendritic cell (pDC) in different culture conditions: I. control, II. CpG alone, III. IL-29 alone and IV. CpG and IL-29. Based on increasing IFN-α secretion, three groups were identifiable. A pronounced tendency of increased se - cretion of IFN-α in response to CpG or CpG and IL-29 (violet triangles) was observed in group C (volunteers 8, 9, 10, 11). Only a slight increase in IFN-α secretion was ob - served in group B (volunteers 4, 5, 6). A negligible change in IFN-α secretion was noted in group A (volunteers 1, 2, 3). However, regardless of the difference in the amount of IFN-α secretion all volunteers showed approximately dou - ble the amount of IFN-α secretion in condition IV com - pared with condition II. 
The effect of interleukin (IL)-29, a new therapeutic agent similar to type I interferons (IFNs), on IFN-α secretion of human plasmacytoid dendritic cells (pDCs) has not been studied. Therefore, in this study, we aimed to clarify the effect of IL-29 on IFN-α secretion of pDCs using human peripheral blood mononuclear cells (PBMCs) in the presence of cytosine-phosphate-guanosinemotif-containing oligodeoxy nucleotides (CpG). In this experimental and prospective study, PBMCs were ob- tained from 11 healthy volunteers and divided into four culture conditions: I. control, II. CpG treatment, III. IL-29 treatment and IV. CpG plus IL-29 treatment. The amount of IFN-α secretion was measured from each culture supernatant by flow cytometry using the flowcytomix apparatus (eBioscience, Vienna, Austria). Fractional IFN-α production of the cultured PBMCs was measured by intracellular staining using the cytomics FC 500 system (Beckman Coulter, Brea, CA, USA) with CXP Software. The mean ± standard deviation (SD) of supernatant IFN-α secretion per pDC/μL was 5.7 ± 9.3 pg/mL/count/µL for condition I, 1071.5 ± 1026.6 pg/mL/count/µL for condition II, 14.1 ± 21.1 pg/mL/count/µL for condition III, and 1913.9 ± 1525.9 pg/mL/count/µL for condition IV. There were statistically significant differences between conditions I and II as well as betweenconditions II and IV. Intracellular IFN-α production was only detectable in the pDC fraction from one culture; the production amount was similar between the cells treated with CpG and those treated with CpG plus IL-29. Natural killer (NK) cell production of IFN-α was observed in two out of three cultures and one culture showed IFN- α production in the monocyte fraction. IL-29 alone did not show any effect on IFN-α secretion of PBMCs. However, the addition of CpG along with IL-29 enhanced IFN-α secretion of PBMCs. Given that pDCs are the major secretors of IFN-α in peripheral blood, this result has suggested the possibility that IL-29 has an enhancing effect in human pDC IFN-α secretion. Although the supernatant IFN-α secretion was not directly correlated with pDCs's intracellular IFN-α production in this study, prolonged incubation of pDC and other PB subsets with CpG or IL-29 for over 4 hours could be applied in future studies. These studies would help to elucidate the mechanism of action of IL-29 in human pDCs associated with viral infections.
Capture of an image from the microscope camera using Comet Score software. 
Phase contrast micrograph of U87MG cells in the monolayer culture with ×10 magni fi cation. 
Growth curve of U87MG cell line in the monolayer cul- tures. An average of nine counts was used to de fi ne each point. Mean ± SEM of three experiments. 
Distribution of DNA migrations (stages 0 to 4) among U87MG cells of 350 μ m spheroids after pretreatment for 67 hours (one volume doubling time) with 250 μ M 2ME2 and treatment for the next volume doubling time with 2ME2, IUdR and 60 Co gamma radiation. Data based on the analysis of 100 cells per slide, triplicate slides per samples. 
Images of single cell gel electrophoresis (comet assay) of U87MG cells of 350 μm spheroids after pretreatment for 67 hours (one volume doubling time) with 250 μM 2ME2 and treatment for the next volume doubling time with 2ME2, IUdR and 60 Co gamma radiation. Samples as follows: A. control, samples B to F were pretreated with 250μM 2ME2 and then treated as follows: B. vehicle, C. 2ME2, D. 2ME2 + IUdR, E. 2ME2 + 60 Co, F. 2ME2 + IUdR + 60 Co.
In this study, we investigated the combined effect of 2-Methoxyestradiol (2ME2) and (60)Co on the cytogenetic damage of iododeoxyuridine (IUdR) in the spheroid model of U87MG glioblastoma cancer cell lines by alkaline comet assay. U87MG cells were cultured as spheroids with diameters of 350 µm. As control, the spheroids of one plate were not treated. Other cultures were pretreated with 2ME2 (250 µM) for one volume doubling time (1 VDT). After this time, the subsequent treatments were performed according to the following groups: Vehicle (this sample was not treated in the 2(nd) VDT) Treated with 2ME2 (250 µM) for 1 VDT Treated simultaneously with 2ME2 (250 µM) and IUdR (1 µM) for 1 VDT Treated with 2ME2 (250 µM) for 1 VDT then irradiated with (60)Co (2 Gy) Treated simultaneously with 2ME2 (250 µM) and IUdR (1 µM) for 1 VDT then irradiated with (60)Co (2 Gy) Then the DNA damage was evaluated using the alkaline comet assay method. The results showed that 2ME2 in combination with gamma irradiation of (60)Co significantly (p<0.001) increased the DNA damage by IUdR as compared to the control group. Thus the combination of these two agents increased the cytogenetic effects of IUdR in the spheroid culture model of U87MG glioblastoma cell lines. By inhibiting the HIF-1α protein and preventing the G0 phase arrest, 2ME2 causes an increased progression into S phase and increases the IUdR absorption. Then the DNA damage in the spheroid cells increases as the uptake of IUdR is increased. These results suggest that the combined use of 2ME2 and (60)Co can increase the radiosensitization effect of IUdR.
The spice Zingiber officinale or ginger possesses antioxidant activity and neuroprotective effects. The effects of this traditional herbal medicine on 3,4-methylenedioxymethamphetamine (MDMA) induced neurotoxicity have not yet been studied. The present study considers the effects of Zingiber officinale on MDMA-induced spatial memory impairment and apoptosis in the hippocampus of male rats. In this experimental study, 21 adult male Sprague Dawley rats (200-250 g) were classified into three groups (control, MDMA, and MDMA plus ginger). The groups were intraperitoneally administered 10 mg/kg MDMA, 10 mg/kg MDMA plus 100 mg/kg ginger extract, or 1 cc/kg normal saline as the control solution for one week (n=7 per group). Learning memory was assessed by Morris water maze (MWM) after the last administration. Finally, the brains were removed to study the cell number in the cornu ammonis (CA1) hippocampus by light microscope, Bcl-2 by immunoblotting, and Bax expression by reverse transcription polymerase chain reaction (RT-PCR). Data was analyzed using SPSS 16 software and a one-way ANOVA test. Escape latency and traveled distances decreased significantly in the MDMA plus ginger group relative to the MDMA group (p<0.001). Cell number increased in the MDMA plus ginger group in comparison to the MDMA group. Down-regulation of Bcl-2 and up-regulation of Bax were observed in the MDMA plus ginger group in comparison to the MDMA group (p<0.05). Our findings suggest that ginger consumption may lead to an improvement of MDMA-induced neurotoxicity.
Clinico-pathological characteristics of patients with gastric cancer. Samples characteristics
miR-302b and U6 snRNA expression in AGS gastric cancer and NT2 human embryonic cancer
cell lines. A and C; Representative amplification plots of mir-302b (A) and U6 snRNA (internal control,
C) for NT2 and AGS cell lines. Note that the expression of miR-302b is significantly higher (~500x) in
NT2 cells compared to that of AGS cells. B and D; The corresponding melt curves of miR-302b and U6
miR-302b (B) and U6 snRNA (D) the PCR products confirmed the specificity of the primers to amplify
exact targets in both cell lines.
MiR-302b expression in tumor vs. non-tumor gastric samples. In each sample, the expression
level of miR-302b is normalized to that of U6 snRNA, as an internal control. The level of
expression of each sample is also calibrated to that of the least expressed sample. A. Histograms
show the mean values of miR-302b's relative expression in tumor and non-tumor samples, with
confidence interval as an error bar. Note the expression of miR-302b is significantly downregulated
in tumor samples compared to their non-tumor counterparts (p=0.001). B. Comparative
expression of miR-302b in different grades of gastric samples. Note that only the relative expression
of miR-302b in tumors with high grad of malignacy is significantly down-regulated
(p=0.009). C. The relative expression of miR-302b in individual samples, distributed in high and
low grade groups.
ROC curve analysis for testing the sensitivity and specificity
of miR-302b expression to discriminate between tumor
and non-tumor states of the samples. Area under curve
(AUC=0.63) shows that data from miR-302b expression does
not have high ability to correctly identify and distinguish tumor
versus non-tumor groups of gastric samples.
microRNAs (miRNAs) are a new class of non-coding RNAs involved in regulating various biological processes including proliferation, differentiation, and apoptosis, among others. Alterations in miRNA expression are reported in several human cancers, which suggests their potential roles in tumor initiation and progression. Members of the miR-302 cluster are highly expressed in embryonic stem cells (ESC), where they regulate cell self-renewal and pluripotency. Based on the cancer stem cell (CSC) hypothesis, mis-expression of such genes might contribute to tumorigenicity. This study aims to find a potential link between the expression level of human/homo sapiens miR-302b (has-miR- 302b) and tumor/grade state of gastric tissues. A matched based case-control study was conducted that included tumor and matched marginal non-tumor surgical specimens from 34 patients diagnosed with gastric adenocarcinoma. Randomly selected samples were obtained from the Iran National Tumor Bank. cDNA synthesis was carried out on total RNA, by using the miRCURY LNA(TM) Universal RT microRNA PCR Kit. Real-time reverse transcriptionpolymerase chain reaction (RT-PCR) assays were performed with specific LNA(TM) primers and SYBR Green master mix. The human embryonic carcinoma cell line, NTERA2 (NT2) and a human gastric adenocarcinoma cell line, AGS, were used to optimize the PCR reactions. A comparative evaluation of miR-302b expression in tumor and non-tumor gastric samples was performed by either paired t test or Wilcoxon non-parametric test. The ability of miR-302b to discriminate tumor from non-tumor gastric samples was evaluated using the area under the receiver operating characteristic (ROC) curve. According to our data, miR-302b expression (normalized to that of the U6 snRNA housekeeping gene) in the pluripotent cell line NT2 was more than 500 times greater than that of the AGS cell line. The level of expression was even lower in tumor and non-tumor gastric tissue samples. The data further revealed a down-regulation of miR-302b in gastric tumor samples (p=0.001), particularly in high-grade adenocarcinoma (p=0.009). However, ROC analysis data demonstrated a low sensitivity and specificity of miR-302b expression to discriminate between the tumor and non-tumor state of the samples (AUC=0.63). Despite the upregulation of some hESC-specific genes in tumors, our data revealed a down-regulation of miR-302b in high-grade tumors. This data suggested a potential tumor-suppressor role for miR-302b in tumorigenesis of gastric tissue.
Finding cell sources for cartilage tissue engineering is a critical procedure. The purpose of the present experimental study was to test the in vitro efficacy of the beta-tricalcium phosphate-alginate-gelatin (BTAG) scaffold to induce chondrogenic differentiation of human umbilical cord blood-derived unrestricted somatic stem cells (USSCs). In this experimental study, USSCs were encapsulated in BTAG scaffold and cultured for 3 weeks in chondrogenic medium as chondrogenic group and in Dulbecco's Modified Eagle's Medium (DMEM) as control group. Chondrogenic differentiation was evaluated by histology, immunofluorescence and RNA analyses for the expression of cartilage extracellular matrix components. The obtain data were analyzed using SPSS version 15. Histological and immunohistochemical staining revealed that collagen II was markedly expressed in the extracellular matrix of the seeded cells on scaffold in presence of chondrogenic media after 21 days. Reverse transcription-polymerase chain reaction (RT-PCR) showed a significant increase in expression levels of genes encoded the cartilage-specific markers, aggrecan, type I and II collagen, and bone morphogenetic protein (BMP)-6 in chondrogenic group. This study demonstrates that BTAG can be considered as a suitable scaffold for encapsulation and chondrogenesis of USSCs.
Objective: Multiple sclerosis (MS) is a chronic autoimmune disease due to demyelination of the central nervous system. It is believed that cytokines are involved in the pathogenesis of MS. The interleukin-2 (IL2) gene is powerful functional candidate that is involved in immune regulation and operation. In this study, for the first time, we investigated the effect of -475 A/T and -631 G/A IL2 polymorphisms on MS disease in Iranian patients. Materials and methods: In this case-control study, 100 MS patients (mean age: 32.95 ± 6.51 years, age range: 20-42 years) selected according to McDonald criteria, and 100 ethnically, sex and age matched healthy controls (mean age: 29 ± 7.8 years, age range: 20-52 years) with no personal or family history of autoimmune diseases were studied. The restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) method was applied to define different alleles and genotypes of IL2 promoter single nucleotide polymorphism -475 A/T as well as -631 G/A among individuals. χ(2) was calculated and Fisher's exact test was applied to analyze the obtained data. The value of p < 0.05 was considered significantly . Results: Evaluation of the -475 IL2 revealed that T allele and A/T genotype are present in 2% and 4% of MS patients, respectively, whereas T allele was absent in control samples. The comparison between alleles and genotypes in MS patients and healthy controls was not significant (p=0.1). For the -631 position, 1% and 2% of MS patients carried A allele and A/G heterozygote genotypes, respectively. All control samples had G allele and G/G genotype. The differences between patients and controls were not significant (p=0.4). Moreover, our results showed a very low frequency of T at -475 and A at -631 IL2 position in each of the two groups. Conclusion: Both -475 and -631 IL2 polymorphisms were higher in MS patients as compared to controls, but the frequency differences were not significant. Based on these data, it is suggested that the -475 and -631 IL2 polymorphisms as functional promoter position may be involved in IL2 expression and regulation. To find out the exact effect of the mentioned SNPs on susceptibility to MS, study on a larger sample size is suggested.
Mean distribution score of breast cancer oncogene (c-Myc) signal in 4T1 breast cancer mouse model under the effect of neem.Data are expressed as mean ± standard deviation.
a = Significant difference with CC group at p<0.05.
b = Significant difference with C250 group at p<0.05.
c = Significant difference with C500 group at p<0.05.
Mean intensity score of breast cancer oncogene (c-Myc) signal in 4T1 breast cancer mouse model under effect of neem.
Data are expressed as mean ± standard deviation.
a = Significant difference with CC group at p<0.05.
A. The negative control in situ RT-PCR with omission of primer showed no positive signal. B. The positive control in situ RT-PCR with no DNase treatment. The brownish color of the nucleus is very strong and intense. C. In situ RT-PCR detection of β-actin mRNA in breast tumour tissue of the CC group. A strong brown staining was observed in positive cells. D. In situ RT-PCR detection of β-actin mRNA in breast tumour tissue of the C250 group. A strong brown staining was observed in positive cells. E. In situ RT-PCR detection of β-actin mRNA in breast tumour tissue of the C500 group. A strong brown staining was observed in positive cells. F. In situ RT-PCR detection of β-actin mRNA in breast tumour tissue of the CT group. A strong brown staining was observed in positive cells. G. In situ RT-PCR detection of c-Myc mRNA in breast tumour tissue of the CC group. A strong brown staining was observed in positive cells. H. In situ RT-PCR detection of c-Myc mRNA in breast tumour tissue of the C250 group. A mild brown staining was observed in positive cells. I. In situ RT-PCR detection of c-Myc mRNA in breast tumour tissue of C500 group. A mild brown staining was observed in positive cells. J. In situ RT-PCR detection of c-Myc mRNA in breast tumour tissue of CT group. A mild brown staining was observed in positive cells. Magnification: x 200 for all slides.
Breast cancer is the most common cause of cancer-related deaths in women both worldwide and in Malaysia. Azadirachta indica (A. Juss), commonly known as neem, is one of the most versatile medicinal plants that has gained worldwide prominence due to its medicinal properties. However, the anticancer effect of ethanolic neem leaf extract against breast cancer has not been documented. The purpose of the present study is to investigate the effect of neem leaf extract on c-Myc oncogene expression in 4T1 breast cancer BALB/c mice. In this experimental study, A total of 48 female BALB/c mice were divided randomly into four groups of 12 mice per group: i.cancer control (CC) treated with 0.5% Tween 20 in PBS, ii. 0.5 µg/mL tamoxifen citrate (CT), iii. 250 mg/kg neem leaf extract (C250), and iv. 500 mg/kg neem leaf extract (C500). in situ reverse transcription polymerase chain reaction (in situ RT-PCR) was applied to evaluate suppression of c-Myc oncogene expression in breast cancer tissue. The C500 group showed significant (p<0.05) suppression of c-Myc oncogene expression compared to the CC group. c-Myc was found to be down regulated under the effect of 500 mg/kg ethanolic neem leaf extract.
Azadirachta indica (Neem) has been used traditionally for many centuries. Some impressive therapeutic qualities have been discovered. However, the therapeutic effect of neem leaf extract in 4T1 breast cancer has not been documented. The purpose of the present study is to investigate the therapeutic effect of ethanolic Neem leaf extract in an in vivo 4T1 breast cancer model in mice. A total of 84 female BALB/c mice were divided randomly into 7 groups (3 non-cancerous groups and 4 cancerous groups) consisting of 12 mice per group. The 3 non-cancerous groups were normal mice treated with 0.5% of Tween 20 in phosphate buffer saline (PBS) (NC), 250 mg/kg Neem (N250) or 500 mg/kg Neem (N500). The 4 cancerous groups were; cancer controls treated with 0.5% of Tween 20 in PBS (CC), and cancerous mice treated with 0.5 µg/mL tamoxifen citrate (CT), 250 mg/kg Neem leaf extract (CN 250) or 500 mg/kg Neem leaf extract (CN 500). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were used to evaluate apoptosis (cell death) in the breast cancer tissues. SPSS software, version 14 was used for statistical analysis. Statistical significance was defined as p≤0.05. Non parametric analysis of variance (ANOVA) was performed with the Kruskal Wallis test for the TUNEL assays. Parametric data among the groups was compared using ANOVA. TUNEL assays showed that the CN 250 and CN 500 groups had a higher incidence of apoptosis compared with the cancer controls. The findings showed that neem leaf extract induces apoptosis in 4T1 breast cancer BALB/c mice.
Comparison of long-scale fluorescent profiles of the FITC- and Alexa Fluor 568- conjugated ANM. Images were captured every 30 seconds followed by analysis of the data by ImageJ software.
Comparison of the fluorescence of FITC- and Alexa Fluor 568- conjugated ANM using ICC staining of bovine sertoli cells.
Analysis of the photostability of FITC- and Alexa Fluor 568-conjugated ANM by continuous illumination of ICC staining of bovine sertoli cells. Images were captured every 5 seconds followed by analysis of the data by ImageJ software.
Synthetic fluorescent dyes that are conjugated to antibodies are useful tools to probe molecules. Based on dye chemical structures, their photobleaching and photostability indices are quite diverse. It is generally believed that among different fluorescent dyes, Alexa Fluor family has greater photostability than traditional dyes like fluorescein isothiocyanate (FITC) and Cy5. Alexa Fluor 568 is a member of Alexa Fluor family presumed to have superior photostability and photobleahing profiles than FITC. In this experimental study, we conjugated Alexa Fluor 568 and FITC dyes to a mouse anti-human nestin monoclonal antibody (ANM) to acquire their photobleaching profiles and photostability indices. Then, the fluorophore/antibody ratios were calculated using a spectrophotometer. The photobleaching profiles and photostability indices of conjugated antibodies were subsequently studied by immunocytochemistry (ICC). Samples were continuously illuminated and digital images acquired under a fluorescent microscope. Data were processed by ImageJ software. Alexa Fluor 568 has a brighter fluorescence and higher photostability than FITC. Alexa Fluor 568 is a capable dye to use in photostaining techniques and it has a longer photostability when compared to FITC.
VDT curve of U87MG cell line in the spheroid cultures. The days 6 to 27 showing the log phase of curve were used to measure the VDT (63.49 ± 0.75 hour). The points indicate Mean ± SEM of 3 separate experiment. 
In radiation treatment, the irradiation which is effective enough to control the tumors is far exceeds of tolerance of normal tissues. Thus, to avoid such unfavorable outcomes; methods which sensitize the tumor cells to radiation are used. Iododeoxyuridine (IUdR) is a halogenated thymidine analogue known to be effective as a radiosensitizer in human cancer therapy.The potential for improving the efficacy of radiation therapy by combining it with hyperthermia is dependent upon the magnitude of the differential sensitization of the hyperthermic effects or upon a differential cytotoxicity of the radiation effects on the tumor cells.In this study we evaluated the combined effects of IUdR, hyperthermia and gamma rays of Co-60 on human glioblastoma spheroids culture. The cultured spheroids with 100μm diameter were treated by 1μM IUdR, 43˚ C hyperthermia for an hour and 2Gy gamma rays respectively. The DNA induced damages in cells were compared using alkaline comet assay method and dosimetry was performed by TLD-100. The results of the comet score were calculated as mean values±SEM by One-way Anova method. Comparison of DNA damages induced by IUdR and hyperthermia treatment on cultured cells following by ionizing radiation showed 2.67 and 1.92fold enhancement respectively in damages than radiation alone or radiation combined IUdR. Dosimetry results showed the accurate delivered dose to cells. Analyzing the comet tail moments of spheroids showed that the radiation treatments combined with hyperthermia and IUdR caused significant radiosensitization when compared to irradiation alone or irradiation with IUdR. These results suggest a potential clinical advantage of combining radiation with hyperthermia and indicate hyperthermia's effectiveness in induce cytotoxicity in tumor cells.
VDT curve of U87MG cell line in the spheroid cultures. The days 6 to 27 showing the log phase of curve were used to measure the VDT (63.49 ± 0.75 hour). The points indicate Mean ± SEM of 3 separate experiment. 
In radiation treatment, the irradiation which is effective enough to control the tumors far exceeds normal-tissues tolerance. Thus to avoid such unfavourable outcomes, some methods sensitizing the tumor cells to radiation are used. Iododeoxyuridine (IUdR) is a halogenated thymidine analogue that known to be effective as a radiosensitizer in human cancer therapy. Improving the potential efficacy of radiation therapy after combining to hyperthermia depends on the magnitude of the differential sensitization of the hyperthermic effects or on the differential cytotoxicity of the radiation effects on the tumor cells. In this study, we evaluated the combined effects of IUdR, hyperthermia and gamma rays of (60)Co on human glioblastoma spheroids culture.
Mean of cleavage rates of 2-cell embryos (to morula)
in three groups, cont; control (non-vitrified) group, vit1 ;
vitrification with 7.5% DMSO and 7.5% EG, vit2 ; vitrification
with 15% DMSO and 15% EG. a, b and c indicate the
significant differences among control, vit1 and vit2 (p<0.01).
The percentages of blastocyst formation of 2-cell embryos
in three groups, cont; control (non-vitrified) group, vit1 ;
vitrification with 7.5% DMSO and 7.5% EG, vit2 ; vitrification
with 15% DMSO and 15% EG. a, b and c indicate the significant
differences among control, vit1 and vit2 (p<0.05).
The relative quantification of Hsp72 after normalization
by Hprt1 in 2-cell embryo groups, cont; control (nonvitrified)
group, vit1 ; vitrification with 7.5% DMSO and
7.5% EG, vit2 ; vitrification with 15% DMSO and 15% EG.
a, b and c indicate the significant differences among control,
vit1 and vit2 (p<0.05).
Mean inverse Ct values of Hprt1 as the relevant
abundance of transcript 2-cell embryo groups, Ct; threshold
cycle, cont; control (non-vitrified) group, vit1; vitrification
with 7.5% DMSO and 7.5% EG, vit2 ; vitrification with 15%
DMSO and 15% EG. Bars are indicative of having no significant
The aim of the study was to compare the effects of two different concentrations of cryoprotectants by cryotopvitrification on survival, developmental capacity and Heat shock protein 72 (Hsp72) expression of two-cell mouse embryos. In this experimental study, transcript analysis of Hsp72 gene was performed on non-vitrified and vitrified 2-cell mouse embryos via a nested quantitative polymerase chain reaction (nqPCR) subsequent to normalization with Hprt1 as the reference gene. The different cryoprotectant combinations were 15% (vit1:7.5% of each ethylene glycol (EG) and dimethyl sulfoxide (DMSO), 30% (vit2:15% EG + 15% DMSO) and control group with no cryoprotectants. Vitrified and fresh 2-cell embryos were cultured to obtain cleavage and blastocyst formation rates. The results were analyzed via one-way analysis of variance and the mean values were compared with least significant difference (LSD) (p< 0.05). The relative expression of Hsp72 in vit2 (30% v/v) was significantly higher than vit1 (15% v/v). Survival rates were the same for both vitrification treatments and significantly lower than the control group. Cleavage and blastocyst rates in vit1 were significantly higher than vit2 while those in two vitrified groups were significantly lower than the control group. Our developmental data demonstrated that vit1 treatment (7.5% EG and 7.5% DMSO) was more efficient than vit2 (15% EG and 15% DMSO) in mouse embryos. The cryotopvitrification with two concentrations of cryoprotectants caused the relative changes of Hsp72 transcript level, but the stability of the gene in vit1 was significantly higher than vit2 and closer to the fresh 2-cell embryos.
Chromatograms from Finch TV DNA sequencing of the CYP2D6, novel polymorphism in intron 4. A. Wild type, B. Heterozygote and C. SNP. The ID of SNP in nucleotide NCBI is KF225465. SNP; Single nucleotide polymorphism, ID; Identification and NCBI; National Center for Biotechnology Information.  
Comparison of genotype and allele frequency of different ethnicities in Iranian participants 
CYP2D6, an enzyme, metabolizes a large number of commonly prescribed drugs. Variations in CYP2D6 gene encoding this enzyme have been associated with individual differences in drug metabolism rates. The purpose of our study was to identify some allelic variants of CYP2D6 gene and to detect defective CYP2D6 alleles, as part of a pharmacogenetic screening program. A prospective study was done on 120 participants referred to Royan Institute in 2013. Allele and genotype frequencies for polymorphism of CYP2D6 gene in exons 1 and 4 were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and sequencing on PCR products, respectively. We identified a novel variant of the gene encoding cytochrome P450 2D6 (CYP2D6) at position +90 of intron 4 by sequencing method. This novel polymorphism of CYP2D6 has been deposited in GeneBank(®) under the accession number KF225465 in Jun 2013. In the current study, we identified novel polymorphism in intron 4. This single nucleotide polymorphism (SNP) is known as +90G>A in the fourth intron.
Objective: Coronary artery disease (CAD) is a multi-factorial and heterogenic disease with atherosclerosis plaques formation in internal wall of coronary artery. Plaque formation results to limitation of the blood reaching to myocardium leading to appearance of some problems, such as ischemia, sudden thrombosis veins and myocardial infarction (MI). Several environmental and genetic factors are involved in prevalence and incident of CAD as follows: hypertension, high low density lipoprotein-cholesterol (LDL-C), age, diabetes mellitus, family history of early-onset heart disease and smoking. According to genome wide association studies (GWAS), five polymorphisms in the 9p21 locus seem to be associated with the CAD. We aimed to evaluate the remarkable association of two polymorphisms at 9p21 locus, rs1333049 and rs10757274, with CAD. Materials and Methods: This experimental study was conducted in Golestan, Aria Hospitals and Genetics Lab of Shahid Chamran University in the city of Ahvaz, Iran, in 2010- 2011. The collected blood samples belonging to 170 CAD patients (case group) and 100 healthy individuals (control group) were analyzed by tetra-primer amplification refractory mutation system (ARMS)-polymerase chain reaction (PCR) technique. The results were analyzed using software package used for statistical analysis (SPSS; SPSS Inc., USA) version 16. A value of p
Increase of Bax protein in the MDMA + SCH group.
Lane number 1: GCS, 2:MDMA + GCS, 3: Control, 4: MDMA, 5: MDMA + SCH, 6:SCH
Decreased Bcl-2 protein expression in the MDMA+SCH group. Lane number 1: SCH, 2: MDMA + SCH, 3: Control, 4: MDMA, 5: MDMA + GCS, 6: GCS.
Increased Bax protein in the MDMA + SCH group.
Bcl-2 protein expression in the MDMA+SCH group
Decreased Bcl-2 mRNA expression in MDMA+SCH group compared to MDMA group.
Ecstasy, also known as 3, 4-methylenedioxymethamphetamine (MDMA), is a psychoactive recreational hallucinogenic substance and a major worldwide recreational drug. There are neurotoxic effects observed in laboratory animals and humans following MDMA use. MDMA causes apoptosis in neurons of the central nervous system (CNS). Withdrawal signs are attenuated by treatment with the adenosine receptor (A2A receptor). This study reports the effects of glutamyl cysteine synthetase (GCS), as an A2A receptor agonist, and succinylcholine (SCH), as an A2A receptor antagonist, on Sprague Dawley rats, both in the presence and absence of MDMA. In this experimental study, we used seven groups of Sprague Dawley rats (200-250 g each). Each group was treated with daily intraperitoneal (IP) injections for a period of one week, as follows: i. MDMA (10 mg/kg); ii. GCS (0.3 mg/kg); iii. SCH (0.3 mg/kg); iv. GCS + SCH (0.3 mg/kg each); v. MDMA (10 mg/kg) + GCS (0.3 mg/kg); vi. MDMA (10 mg/kg) + SCH (0.3 mg/kg); and vi. normal saline (1 cc/kg) as the sham group. Bax (apoptotic protein) and Bcl-2 (anti-apoptotic protein) expressions were evaluated by striatum using RT-PCR and Western blot analysis. There was a significant increase in Bax protein expression in the MDMA+SCH group and a significant decrease in Bcl-2 protein expression in the MDMA+SCH group (p<0.05). A2A receptors have a role in the apoptotic effects of MDMA via the Bax and Bcl-2 pathways. An agonist of this receptor (GCS) decreases the cytotoxcity of MDMA, while the antagonist of this receptor (SCH) increases its cytotoxcity.
Chromosomal aberrations are common causes of multiple anomaly syndromes. Recurrent chromosomal aberrations have been identified by conventional cytogenetic methods used widely as one of the most important clinical diagnostic techniques. In this retrospective study, the incidences of chromosomal aberrations were evaluated in a six year period from 2005 to 2011 in Pardis Clinical and Genetics Laboratory on patients referred to from Mashhad and other cities in Khorasan province. Karyotyping was performed on 3728 patients suspected of having chromosomal abnormalities. The frequencies of the different types of chromosomal abnormalities were determined, and the relative frequencies were calculated in each group. Among these patients, 83.3% had normal karyotypes with no aberrations. The overall incidences of chromosomal abnormalities were 16.7% including sex and autosomal chromosomal anomalies. Of those, 75.1 % showed autosomal chromosomal aberrations. Down syndrome (DS) was the most prevalent autosomal aberration in the patients (77.1%). Pericentric inversion of chromosome 9 was seen in 5% of patients. This inversion was prevalent in patients with recurrent spontaneous abortion (RSA). Sex chromosomal aberrations were observed in 24.9% of abnormal patients of which 61% had Turner's syndrome and 33.5% had Klinefelter's syndrome. According to the current study, the pattern of chromosomal aberrations in North East of Iran demonstrates the importance of cytogenetic evaluation in patients who show clinical abnormalities. These findings provide a reason for preparing a local cytogenetic data bank to enhance genetic counseling of families who require this service.
The Results of eight-cell mouse embryos viability vitrified in DMSO/PROH solution after various storage durations P value No. of Embryos Test groups
Percentage of viability at the presence of DMSO and PROH as cryoprotectant for various storage durations . Error bars
show SE of mean values calculated for data obtained from different samples.
Percentage of chromosome abnormality at the presence of DMSO and PROH as cryoprotectant for various storage durations.
Error bars show SE of mean values calculated for data obtained from different samples.
Experiments were conducted to find the differences between post-thaw viability and chromosome aberrations in eight-cell mouse embryos at presence of dimethyl sulfoxide (DMSO) and 1, 2-propanediol (PROH) as croprotectants in different storage durations. In this case-control study, a total number of 720 mouse embryos from about 250 NMRI mice were vitrified with 30% PROH or DMSO; each diluted with a solution containing 30% ficol plus 0.5 M sucrose. Embryos were exposed to the solutions for 0.5 minute at 25℃ followed by cooling in liquid nitrogen, then after appropriate storage duration, they were rapidly warmed. Besides, there were 100 mouse embryos for each cryoprotectant group (totally 200 embryos) as control. Embryo survival was assessed by in vitro development, and chromosome abnormalities were analyzed by Giemsa staining. The proportion of mitotic abnormalities in PROH/DMSO vitrified embryos was significantly higher than unfrozen control group. This was confirmed also by a reduced viability of the embryos as judged by a culture at the blastocyst stage (p<0.05 in all test groups). It can be deduced that long term cryopreservation may result in chromosomal abnormalities and/or low viability.
RT-PCR analysis of the expression of VEGF and its receptors in the endometrium of both groups in WOI. RT- PCR; Reverse transcription- polymerase chain reaction, WOI; Window of implantation, URSA; Unexplained recur - rent spontaneous abortion, VEGF; Vascular endothelial growth factor, VEGFR1; Vascular endothelial growth fac - tor receptor 1 and VEGFR2; Vascular endothelial growth factor receptor 2. 
Results of real time PCR for normalized expression of VEGF, VEGFR1 and VEGFR2 in endometrium of both groups. URSA; Unexplained recurrent spontaneous abortion, VEGF; Vascular endothelial growth factor, VEGFR1; Vascular en - dothelial growth factor receptor 1 and VEGFR2; Vascular endothelial growth factor receptor 2. 
Unexplained recurrent spontaneous abortion (URSA) is one of the main complications of pregnancy which is usually defined as three or more consecutive pregnancy losses before the 20(th) week of gestation without a known cause. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor and shown, along with its receptors (VEGFR1, 2), to play important roles in several physiologic processes including reproduction. The aim of the present study was to analyze gene expression of VEGF and VEGF receptors in endometrium of patients with a history of URSA compared with normal fertile women. In addition, serum VEGF concentration was assessed and compared between the two groups at the same time. In this case control study, endometrial and blood samples were obtained between day 19(th)and 24(th) of menstrual cycle (window of implantation) from 10 women with a history of URSA (case group) and 6 fertile women who had at least one successful pregnancy (control group). Expression of VEGF and VEGFRs was studied by reverse transcription- polymerase chain reaction (RT-PCR) and then quantified by real time PCR. Normalization of expression levels was done by comparison with beta-actin expression level as an internal control. Relative VEGF, VEGFR1 and VEGFR2 expression quantities were compared between the two groups. Enzyme linked immunosorbent assay (ELISA) was used for serum VEGF assay. VEGF, VEGFR1 and VEGFR2 gene expression was detected in endometrial samples of both groups. The mean relative expression of VEGF gene was lower in the case group compared with control women, however, both VEGF receptors were expressed higher in endometrium of the case group. In addition, the serum level of VEGF was significantly higher in the case group compared with the controls. Alteration in gene expression of VEGF and its receptors in endometrium and changes of serum VEGF might play important roles in pathogenesis of unexplained RSA.
Ionocyte localization (by immunolocalization of Na+/K+-ATPase) in the skin and gill of Salmo trutta caspius
in the control group on the first day. (1A): The majority of ionocytes were observed on the yolk sac. (1B): ionocytes
were observed on gill arc and the lamellae had not developed yet. (1C): Some ionocytes located on dorsal part.
(1D): On the trunk, ionocytes mostly located on fins. (1E): Almost no ionocytes on the head skin.
A, Gill Arc; F, Gill Filament; E, Epidermis; IC, Ionocyte; YS, yolk sac; DF, dorsal fin; B, brain.
Ionocyte localization (by immunolocalization of Na+/K+-ATPase) in the gill of Salmo trutta caspius
in the control group on the seventh day. (2A): 7 day old Salmon trutta caspius alevins. (2B) and
(2C): ionocytes were observed on gill filaments at the base of lamellae.
A, Gill Arc; F, Gill Filament; IC, Ionocyte; L, gill lamellae.
SEM micrograph of the dorsal part of skin in 9
day alevins from the control group (3A) and high dose
(UHD) exposure groups (3B). (3A): apical pit of ionocytes
(microvili without pit (I) and pit (II)) and mucous
cells (deep pit without any ridge) located among pavement
cells. Mucus secretions were also seen. (3B): skin
severely destroyed and apical pit of ionocytes and mucous
cells are deformed; pavement cell microridges were
hardly observable. Many holes induced by mucus secretion
are present.
MC, Mucous cells; IC, Ionocyte; PC, Pavement cells; MR,
Micro-ridge; H, Hole induced by mucous secretion; B,
Boundary; MV, Microvili; N, Necrosis.
On a global scale, stratospheric ozone depletion has caused an increase in UV-B radiation reaching the earth's surface. Ultraviolet radiation has long been suspected to be harmful to aquatic organisms. In order to study ionocyte localization (by Na(+)/K(+)-ATPase immunolocalization) and the effects of UV radiation on the ionocytes of skin and gills, the alevins of Salmo trutta caspius were exposed to different doses of UV radiation [unit low doses (ULD) of: 60 µw/cm(2) UVC; 100 µw/cm(2) UVB and 40 µw/cm(2) UVA and unit high doses (UHD) of: 90 µw/cm(2) UVC; 130 µw/cm(2) UVB and 50 µw/cm(2) UVA] using two adjustable F8T5 UV-B, 302 nm lamps (Japan) for 15 minutes once a day in laboratory conditions. Alevins not subjected to UV exposure served as a control group. In both UV exposure groups, all the alevins died on the ninth day. No mortality was observed in the control group. The Na(+)/K(+)-ATPase immunolocalization study indicated that ionocytes were located, in lessening order, on the yolk sac, trunk, gills, opercula and rarely on the head skin. Immunohistochemical results showed significant reduction in the number of ionocytes on the yolk sac, with lesser reduction on the trunk in both UV exposure groups. In contrast, the number of immunofluorescence cells on the gill was significantly elevated. Our results also showed that the size of ionocytes was reduced on the trunk and yolk sac in the UV exposure groups, but not significantly. Deformation and destruction of ionocytes on the yolk sac and trunk were observed with scanning electron microscope (SEM) in the UV exposure groups. Our results showed that ionocytes were located mainly on the yolk sac, in lesser amounts on the trunk, gills and opercula, and rarely also on the head skin of alevins. UV radiation caused deformation and reduction in the number and size of ionocytes on the trunk and yolk sac. As the skin cells of trout alevins possess essential functions for respiration, osmoregulation, excretion and defense during this stage of life, the observed damage may have contributed to their suddenly mortality in the UV exposure condition.
Electrophoresis of ace gene amplified from V. cholerae on agarose gel (1% w/v). Lane 1. 1 kb DNA size marker, lanes 2, 3, 4. single expected band of ace (approximately 299 bp).
Agarose gel electrophoresis analysis of recombinant pET28a-ace. Lane 1. 1 kb DNA size marker, lane 2. double digestion of recombinant pET28a-ace with EcoRI, NdeI.
A. SDS-PAGE (15% w/v) analysis of expression product of pET28a-ace in E. coli BL21. Lane 1. protein marker, lanes 2, 3, 4. induction of pET28a-ace by treatment with 1mM IPTG (18 kDa). B. recombinant proteins purified by Ni-NTA column chromatograph. Lane 1. protein marker, lane 2. purified rAce protein (18kDa).
Western blot of the SDS-polyacrylamide gel prepared with anti-V. cholerae antibody. Antiserum was diluted 1:500. The 18 kDa proteins of the recombinant Ace were detected.
Rabbit ileal loop assay. Ileal tissues treated with recombinant Ace protein culture supernatants of the toxigenic V. cholerae 62013 (positive control). Sterile PBS was used as a negative control. Tube 1. fluid accumulation (FA) in negative control (ratio 0.5 ± 0.005), tube 2. significant hemorrhagic FA (ratio 1.25 ± 0.2) with recombinant Ace protein, tubes 3 and 4. other test substances, and tube 5. significant hemorrhagic FA (ratio 2 ± 0.2) with positive control.
Vibrio cholerae (V. cholerae) causes a potentially lethal disease named cholera. The cholera enterotoxin (CT) is a major virulence factor of V. cholerae. In addition to CT, V. cholerae produces other putative toxins, such as the zonula occludens toxin (Zot) and accessory cholera enterotoxin (Ace). The ace gene is the third gene of the V. cholerae virulence cassette. The Ace toxin alters ion transport, causes fluid accumulation in ligated rabbit ileal loops, and is a cause of mild diarrhea. The aim of this study is the cloning and overexpression of the ace gene into Escherichia coli (E. coli) and determination of some characteristics of the recombinant Ace protein. In this experimental study, the ace gene was amplified from V. cholerae strain 62013, then cloned in a pET28a expression vector and transformed into an E. coli (DH5 α) host strain. Subsequently, the recombinant vector was retransformed into E. coli BL21 for expression, induced by isopropythio-β-D-galctoside (IPTG) at a different concentration, and examined by SDS-PAGE and Western blot. A rabbit ileal loop experiment was conducted. Antibacterial activity of the Ace protein was assessed for E. coli, Stapylococcus aureus (S. aureus), and Pseudomonas aeruginosa (P. aeruginosa). The recombinant Ace protein with a molecular weight of 18 kDa (dimeric form) was expressed in E. coli BL21. The Ace protein showed poor staining with Coomassie blue stain, but stained efficiently with silver stain. Western blot analysis showed that the recombinant Ace protein reacted with rabbit anti-V. cholerae polyclonal antibody. The Ace protein had antibacterial activity at a concentration of ≥200 µg/ml and caused significant fluid accumulation in the ligated rabbit ileal loop test. This study described an E. coli cloning and expression system (E. coli BL21- pET-28a-ace) for the Ace protein of V. cholerae. We confirmed the antibacterial properties and enterotoxin activity of the resultant recombinant Ace protein.
Molecular systems orbiting around histone acetylation have been the centre of comprehensive investigations leading to the identification and functional characterization of the three major classes of actors involved in generating [acetyltransferases (HATs)], reading [bromodomains (BRDs)] and erasing [deacetylases (HDACs)] signalling to chromatin based on acetylation. In contrast, our knowledge of the molecular machinery managing signalling through histone acylations other than acetylation is very poor and only a few enzymes involved in their establishment and removal have been identified so far. All the acyl group donors are generated through cell metabolism and hence a critical question to address is how cell metabolism drives all these modifications and how it imposes a specific choice on the use of acyl group. Ac; Acetylation, Pr; Propionylation, Bu; Butyrylation, Hib; 2-hy- droxyisobutyrylation, Cr; Crotonylation, Su; Succinylation, Glu; Glutarylation and CBP/p300; CREB-binding protein/EP300.  
Histone acetylation, one of the first and best studied histone post-translational modifications (PTMs), as well as the factors involved in its deposition (writers), binding (readers) and removal (erasers), have been shown to act at the heart of regulatory circuits controlling essential cellular functions. The identification of a variety of competing histone lysine-modifying acyl groups including propionyl, butyryl, 2-hydroxyisobutyryl, crotonyl, malonyl, succinyl and glutaryl, raises numerous questions on their functional significance, the molecular systems that manage their establishment, removal and interplay with the well-known acetylation-based mechanisms. Detailed and large-scale investigations of two of these new histone PTMs, crotonylation and 2-hydroxyisobutyrylation, along with histone acetylation, in the context of male genome programming, where stage-specific gene expression programs are switched on and off in turn, have shed light on their functional contribution to the epigenome for the first time. These initial investigations fired many additional questions, which remain to be explored. This review surveys the major results taken from these two new histone acylations and discusses the new biology that is emerging based on the diversity of histone lysine acylations.
Many studies have focused on the epigenetic characteristics of donor cells to improve somatic cell nuclear transfer (SCNT). We hypothesized that the epigenetic status and chromatin structure of undifferentiated bovine adipose tissue-derived stem cells (BADSCs) would not remain constant during different passages. The objective of this study was to determine the mRNA expression patterns of DNA methyltransferases (DNMT1, DNMT3a, DNMT3b) and histone deacetyltransferses (HDAC1, HDAC2, HDAC3) in BADSCs. In addition, we compared the measured levels of octamer binding protein-4 expression (OCT4) and acetylation of H3K9 (H3K9ac) in BADSCs cultures and different passages in vitro. In this experimental study, subcutaneous fat was obtained from adult cows immediately post-mortem. Relative level of DNMTs and HDACs was examined using quantitative real time polymerase chain reaction (q-PCR), and the level of OCT4 and H3K9ac was analyzed by flow cytometry at passages 3 (P3), 5 (P5) and 7 (P7). The OCT4 protein level was similar at P3 and P5 but a significant decrease in its level was seen at P7. The highest and lowest levels of H3K9ac were observed at P5 and P7, respectively. At P5, the expression of HDACs and DNMTs was significantly decreased. In contrast, a remarkable increase in the expression of DNMTs was observed at P7. Our data demonstrated that the epigenetic status of BADSCs was variable during culture. The P5 cells showed the highest level of stemness and multipotency and the lowest level of chromatin compaction. Therefore, we suggest that P5 cells may be more efficient for SCNT compared with other passages.
Objective: The development of vertebrae is a complex phenomenon that is correlated with distinct morphological and biochemical alterations in the paraxial mesenchyme and glycoconjugates. The purpose of this study is to investigate the glycosylation pattern in paraxial mesenchyme-forming vertebrae by using the lectin histochemical technique. Materials and methods: In this descriptive-analytic study, B4G fixed paraffin sections of 9 to 15 day Balb/c mouse embryos were processed for histochemical studies using seven different HRP-labelled lectins: Glycin max (SBA), Maclura pomifera (MPA), Wistaria floribunda (WFA), Vicia villosa (VVA) which all of them are specific for N-acetylgalactosamine (GalNAc), Ulex europius (UEA1, binds to α-L-fucose), wheat germ agglutinin (WGA, binds to sialic acid), and Griffonia simplicifolia (GSA1-B4, binds to galactose terminal sugars). The sections were observed separately by three examiners who were blinded to the lectins. Grading was done according to the intensity of the tested lectins' reactions with the specimen, from negative (-) to severe (+++). Data was analysed with SPSS software (version 11.5) and the non-parametric Kruskal Wallis test; p<0.05 was considered significant. Results: Our findings showed that among the tested lectins, only GalNAc residue sensitive lectins showed regulated changes in paraxial mesenchyme. Reactions of WFA and MPA lectins with paraxial mesenchyme were severe on GD9. Reactions of WFA continued to GD15 constantly, while MPA reactions continued strongly to GD12, significantly decreased thereafter (p<0.001), and then disappeared. VVA and SBA bindings initiated weakly on GD10 and continued to GD12 without changing. These reactions increased significantly (p<0.001) thereafter, became severe to GD14, and later disappeared. The other tested lectins did not reveal regulated changes. Conclusion: According to these findings it can be concluded that only the GalNAc terminal sugar showed temporally regulated changes during the early embryonic development of vertebrae in mice. Therefore it most likely plays a key role (s) in the development of vertebrae, especially in the conversion of mesenchymal cells into chondroblasts. The other tested terminal sugars may have no role in this phenomenon.
Objective: Intra-peritoneal administration of riluzole has been shown to preserve the membrane properties and firing characteristics of Purkinje neurons in a rat model of cerebellar ataxia induced by 3-acetylpyridine (3-AP). However, the exact mechanism(s) by which riluzole restores the normal electrophysiological properties of Purkinje neurons is not completely understood. Changes in the conductance of several ion channels, including the BK channels, have been proposed as a neuro protective target of riluzole. In this study, the possible cellular effects of riluzole on Purkinje cells from 3-AP-induced ataxic rats that could be responsible for its neuro protective action have been investigated by computer simulations. Materials and methods: This is a computational stimulation study. The simulation environment enabled a change in the properties of the specific ion channels as the possible mechanism of action of riluzole. This allowed us to study the resulted changes in the firing activity of Purkinje cells without concerns about its other effects and interfering parameters in the experiments. Simulations were performed in the NEURON environment (Version 7.1) in a time step of 25 μs; analyses were conducted using MATLAB r2010a (The Mathworks). Data were given as mean ± SEM. Statistical analyses were performed by the student's t test, and differences were considered significant if p< 0.05. Results: The computational findings demonstrated that modulation of an individual ion channel current, as suggested by previous experimental studies, should not be considered as the only possible target for the neuro protective effects of riluzole to restore the normal firing activity of Purkinje cells from ataxic rats. Conclusion: Changes in the conductance of several potassium channels, including voltage- gated potassium (Kv1, Kv4) and big Ca(2+)-activated K(+) (BK) channels may be responsible for the neuro protective effect of riluzole against 3-AP induced alterations in the firing properties of Purkinje cells in a rat model of ataxia.
Nucleocytoplasmic translocation of actin. 1. Nuclear import of actin. Cofilin enters the nucleus through the import receptor, importin β. 2, 3. Nuclear export of actin. Actin can exit the nucleus in coupled with the actin binding protein, profilin, through the export receptor exportin 6 and/or through the exportin 1 receptor.  
Models for the function of actin in chromatin regulation. A. Actin is a component of ATP-dependent chromatin remodeling complexes (CRC) involved in transcriptional activation and B. Co-transcriptional recruitment of histone modifier elements to RNA polymerase II (RNAP II), mediated by the presence of heterogeneous nuclear ribonucleoproteins (hnRNPs). During transcription, actin can be recruited to the elongating transcription machinery and facilitate recruitment of histone acetyltransferase (HAT) to the active gene, enhancing the processivity of RNAP II.  
Over the past few decades, actin’s presence in the nucleus has been demonstrated. Actin is a key protein necessary for different nuclear processes. Although actin is well known for its functional role in dynamic behavior of the cytoskeleton, emerging studies are now highlighting new roles for actin. At the present time there is no doubt about the presence of actin in the nucleus. A number of studies have uncovered the functional involvement of actin in nuclear processes. Actin as one of the nuclear components has its own structured and functional rules, such as nuclear matrix association, chromatin remodeling, transcription by RNA polymerases I, II, III and mRNA processing. In this historical review, we attempt to provide an overview of our current understanding of the functions of actin in the nucleus.
In previous studies it has been emphasized that the site of morphine action may be either in the embryo or the placenta. In the present study, we attempt to identify the site of morphine action on the fetal section of Wistar rat placenta by using C(14)-morphine. IN THIS STUDY (EXPERIMENTAL), FEMALE WISTAR RATS (WEIGHTS: 170-200 g) were mated with male rats and their coupling times recorded. Experimental groups received daily doses of 0.05 mg/ml of C(14)-morphine in their drinking water. On the 9(th) and14(th) embryonic days, the pregnant rats were anesthetized and the placenta and uterus surgically removed. Placentas were fixed in 10% formalin for two weeks, then processed, sectioned in 5 µm and 25 µm thicknesses, and fixed on glass slides for further evaluation. The 25 µm sections were delivered to black and white film for three days. Films were processed and evaluated with a digital inverse microscope for possible radiological impression. The 5 µm sections were processed for hematoxylin and eosin (H&E) staining, and evaluated by light microscope and MOTIC software. Our results indicated that the site of action of C(14)-morphine was possibly located on the blood plexus of the fetal portion of the placenta. In addition, oral morphine consumption was shown to inhibit fetal and maternal placental development in the experimental groups. We conclude that morphine's effectiveness on the reduction of embryo growth and development may be via its effects on the blood plexus of the fetal section of the placenta.
It is believed that monocyte isolation methods and maturation factors affect the phenotypic and functional characteristics of resultant dendritic cells (DC). In the present study, we compared two monocyte isolation methods, including plastic adherence-dendritic cells (Adh-DC) and magnetic activated cell sorting- dendritic cells (MACS-DC), and their effects on phagocytic activity of differentiated immature DCs (immDCs). : In this experimental study, immDCs were generated from plastic adherence and MACS isolated monocytes in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) in five days. The phagocytic activity of immDCs was analyzed by fluorescein isothiocyanate (FITC)-conjugated latex bead using flow cytometry. One way ANOVA test was used for statistical analysis of differences among experimental groups, including Adh-DC and MACS-DC groups. We found that phagocytic activity of Adh-DC was higher than MACS-DC, whereas the mean fluorescence intensity (MFI) of phagocytic cells was higher in MACS-DC (p<0.05). : We concluded that it would be important to consider phagocytosis parameters of generated DCs before making any decision about monocyte isolation methods to have fully functional DCs.
The peroxisome proliferator-activated receptors (PPARs) are a group of nu- clear receptor proteins whose functions as transcription factors regulate gene expres- sions. PPARs play essential roles in the regulation of cellular differentiation, development, and metabolism (carbohydrate, lipid, protein), and tumorigenesis of higher organisms. This study attempts to determine the effect of baicalin, a PPARγ activator, on erythroid differentiation of cluster of differentiation 133(+)(CD133(+)) cord blood hematopoietic stem cells (HSCs). In this experimental study, in order to investigate the effects of the PPARγ agonists baicalin and troglitazone on erythropoiesis, we isolated CD133(+) cells from human umbilical cord blood using the MACS method. Isolated cells were cultured in erythroid-inducing medium with or without various amounts of the two PPARγ activa- tors (baicalin and troglitazone). Erythroid differentiation of CD133(+)cord blood HSCs were assessed using microscopic morphology analysis, flow cytometric analysis of erythroid surface markers transferrin receptor (TfR) and glycophorin A (GPA) and bycolony forming assay. Microscopic and flow cytometric analysis revealed the erythroid differentiation of CD133(+)cord blood HSCs under applied erythroid inducing conditions. Our flow cytometric data showed that the TfR and GPA positive cell population diminished significantly in the presence of either troglitazone or baicalin. The suppression of erythroid differentiation in response to PPARγ agonists was dose-dependent. Erythroid colony-forming ability of HSC decreased significantly after treatment with both PPARγ agonists but troglitazone had a markedly greater effect. Our results have demonstrated that PPARγ agonists modulate erythroid dif- ferentiation of CD133(+)HSCs, and therefore play an important role in regulation of normal erythropoiesis under physiologic conditions. Thus, considering the availability and applica- tion of this herbal remedy for treatment of a wide range of diseases, the inhibitory effect of baicalin on erythropoiesis should be noted.
Pedigree of patient's family.
A. Presence of c.617G>A heterozygous mutation in the patient. B. Absence of c.617G>A mutation in the patient's father. C. Absence of c.617G>A mutation in the patient's mother.
Pedigree of patient’s family.
A. Presence of c.617G>A heterozygous mutation in
the patient. B. Absence of c.617G>A mutation in the patient’s
father. C. Absence of c.617G>A mutation in the patient’s
Fibrodysplasia Ossificans Progressiva (FOP, MIM 135100) is a rare genetic disease that is often inherited sporadically in an autosomal dominant pattern. The disease manifests in early life with malformed great toes and, its episodic and progressive bone formation in skeletal muscle after trauma is led to extra-articular ankylosis. In this study, a 17 year-old affected girl born to a father with chemical injury due to exposure to Mustard gas during the Iran-Iraq war, and her first degree relatives were examined to find the genetic cause of the disease. The mutation c.617G>A in the Activin A receptor, type I (ACVR1) gene was found in all previously reported patients with FOP. Therefore, peripheral blood samples were taken from the patient and her first-degree relatives. DNA was extracted and PCR amplification for ACVR1 was performed. The sequencing of ACVR1 showed the existence of the heterozygous c.617G>A mutation in the patient and the lack of it in her relatives. Normal result of genetic evaluation in relatives of the patient, ruled out the possibility of the mutation being inherited from parents. Therefore, the mutation causing disease in the child, whether is a new mutation with no relation to the father's exposure to chemical gas, or in case of somatic mutation due to exposure to chemical gas, the mutant cells were created in father's germ cells and were not detectable in his blood sample.
The structure of the APC gene is partly demonstrating the intron 14 and exon 15. Horizontal arrows show the positions
of the overlapped fragments covering the nucleotide -286 in intron 14 and codon 1613 within exon 15.
DNA sequences of novel APC mutations. A. Chromatogram of c.3236C>G (p.Thr1079Ser) mutation. B. Chromatogram
of c.4468_4469dupCA (p.Phe1491IlefsX17) mutation.
Colorectal cancer (CRC) is one of the most common and aggressive cancers worldwide. The majority of CRC cases are sporadic that caused by somatic mutations. The Adenomatous Polyposis Coli (APC; OMIM 611731) is a tumor suppressor gene of Wnt pathway and is frequently mutated in CRC cases. This study was designed to investigate the spectrum of APC gene mutations in Iranian patients with sporadic colorectal cancer. In this descriptive study, Tumor and normal tissue samples were obtained from thirty randomly selected and unrelated sporadic CRC patients. We examined the hotspot region of the APC gene in all patients. Our mutation detection method was direct DNA sequencing. We found a total of 8 different APC mutations, including two nonsense mutations (c.4099C>T and c.4348C>T), two missense mutations (c.3236C>G and c.3527C>T) and four frame shift mutations (c.2804dupA, c.4317delT, c.4464_4471delATTACATT and c.4468_4469dupCA). The c.3236C>G and c.4468_4469dupCA are novel mutations. The overall frequency of APC mutation was 26.7% (8 of 30 patients). This mutation rate is lower in comparison with previous studies from other countries. The findings of present study demonstrate a different APC mutation spectrum in CRC patients of Iranian origin compared with other populations.
Objective: Tendon never returns to its complete biological and mechanical properties after repair. Bone marrow and, recently, adipose tissue have been used as sources of mesenchymal stem cells which have been proven to enhance tendon healing. In the present study, we compared the effects of allotransplantation of bone marrow derived mesenchymal stromal cells (BMSCs) and adipose derived stromal vascular fraction (SVF) on tendon mechanical properties after experimentally induced flexor tendon transection. Materials and methods: In this experimental study, we used 48 adult male New Zealand white rabbits. Twelve of rabbits were used as donors of bone marrow and adipose tissue, the rest were divided into control and treatment groups. The injury model was a unilateral complete transection of the deep digital flexor tendon. Immediately after suture repair, 4×10(6)cells of either fresh SVF from enzymatic digestion of adipose tissue or cultured BMSCs were intratendinously injected into tendon stumps in the treatment groups. Controls received phosphate-buffered saline (PBS). Immobilization with a cast was continued for two weeks after surgery. Animals were sacrificed three and eight weeks after surgery and tendons underwent mechanical evaluations. The differences among the groups were analyzed using the analysis of variance (ANOVA) test followed by Tukey's multiple comparisons test. Results: Stromal cell transplantation resulted in a significant increase in ultimate and yield loads, energy absorption, and stress of repairs compared to the controls. However, there were no statistically significant changes detected in terms of stiffness. In comparison, we observed no significant differences at the third week between SVF and BMSCs treated tendons in terms of all load related properties. However, at the eighth week SVF transplantation resulted in significantly increased energy absorption, stress and stiffness compared to BMSCs. Conclusion: The enhanced biomechanical properties of repairs in this study advocates the application of adipose derived SVF as an excellent source of multipotent cells instead of traditional BMSCs and may seem more encouraging in cell-based therapy for tendon injuries.
Flow cytometry analysis of C57BL/6 mice ADSCs
showing that they do not express CD31, and CD45, but express
CD73 and CD90. The white histograms show isotypematched
control staining.
Fluorescence images (first row) of a pooled corpus callosum single-cell suspensionfrom recipient mice 2
days after transplantation show PKH26+adipose mesenchymal stem cells (ADSCs; red color), with DAPI bluestained
nuclei, visualized among various cell types. ADSCs stained with PKH26 (A); nuclear staining with DAPI
(B); and merge them (C). Light (second row) and transmission electron micrographs (third and fourth rows)
showtransplantation of ADSCs facilitates remyelination in the corpus callosum of mice after cuprizone-induced
demyelination.The photomicrographs were taken from coronal (light micrographs) and sagittal sections (transmission
electron micrographs) of the corpus callosum of miceeuthanized 10 days after transplantation.(D-F): Myelin
content evaluated by luxol fast blue (LFB) staining. Corpus callosumof a control health mouse (D, delineated
by black lines); control vehicle (E); and transplanted group (F). (G-L): Electron micrographs show myelinated
and unmyelinated axons at 10 days after treatment. Electron micrograph magnifications: ×3000, ×72500. (G, J)
control health group; (H, K) control vehicle group; and (I, L) ADSCs transplantation group.Scale bars: A-C=100
μm; G-I=1 μm; and J-L=50 nm.
Percentage of myelinated axons in the corpus callosum (A); Mean of axon diameters (B); Mean of myelin sheath thickness (C);
and G-ratio (D). Quantitative analysis of the electron micrographs was performedwith Image tools J software. Results are mean ± SEM
off our different measurements for each experimental condition (*p<0.05).
Analysisof changes in cellular composition in the corpus callosum of cuprizone-induced demyelinated mice treated with
adipose mesenchymal stem cells (ADSCs) or vehicle alone. Mononuclear cells were isolated from the corpus callosum and the
frequencies of GFAP+ (astrocytes), Iba-1 (microglia), Olig2+ (oligodendroglial progenitor) and O4+ (oligodendrocytes) cellswere
determined by flow cytometryten days after transplantation. The respective isotype control is shown as a violet color.
Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS). Stem cell transplantation is a new therapeutic approach for demyelinating diseases such as MS which may promote remyelination. In this study, we evaluate the remyelinating potential of adipose mesenchymal stem cells (ADSCs) and their effect on neural cell composition in the corpus callosum in an experimental model of MS. This experimental study used adult male C57BL/6 mice. Cultured ADSCs were confirmed to be CD73(+),CD90(+), CD31(-),CD45(-), and labeled by PKH26. Animals were fed with 0.2% w/w cuprizone added to ground breeder chow ad libitum for six weeks. At day 0 after cuprizone removal, mice were randomly divided into two groups: the ADSCs-transplanted group and the control vehicle group (received medium alone). Some mice of the same age were fed with their normal diet to serve as healthy control group. Homing of ADSCs in demyelinated lesions was examined by fluorescent microscope. At ten days after transplantation, the mice were euthanized and their cells analyzed by luxol fast blue staining (LFB), transmission electron microscopy and flow cytometry. Results were analyzed by one-way analysis of variance (ANOVA). According to fluorescent cell labeling, transplanted ADSCs appeared to survive and exhibited homing specificity. LFB staining and transmission electron microscope evaluation revealed enhanced remyelination in the transplanted group compared to the control vehicle group. Flow cytometry analysis showedan increase in Olig2 and O4 cells and a decrease in GFAP and Iba-1 cells in the transplanted group. Our results indicate that ADSCs may provide a feasible, practical way for remyelination in diseases such as MS.
Primers used in real-time RT-PCR Product length (base pairs) Primer sequence (F, R, 5´3´) 5´3´) Gene
Proliferation of induced pluripotent stem cells (iPSCs) and adipose tissue derived mesenchymal stem cells (AT-MSCs) on tissue culture polystyrene (TCPS) over a 5 day culture period (asterisk shows significant difference, p<0.05). 
ALP activity of stem cells during osteogenic differentiation [asterisk shows significant difference between adipose tissue derived mesenchymal stem cells (AT-MSCs) and induced pluripotent stem cells (iPSCs) on each day, p<0.05]. 
Calcium content of stem cells during osteogenic differentiation [asterisk shows significant difference between AT-MSsC and iPSCs on each day at p<0.05]. 
Relative expression of Osteocalcin, Runx2 and Osteonectin in stem cells on days 7, 14 and 21 during osteogenic differentiation (asterisk shows significant difference between two groups on each day at p<0.05). 
Human induced pluripotent stem cells (iPSCs) have been shown to have promising capacity for stem cells therapy and tissue engineering applications. Therefore, it is essential to compare the ability of these cells with the commonly used mesenchymal stem cells (MSC) for bone tissue engineering in vitro. In the present study, the biological behavior and osteogenic capacity of the iPSCs were compared with MSC isolated from human adipose tissue (AT-MSC) using 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, Alizarin red staining, alkaline phosphatase (ALP) activity measurements, calcium content assay and common osteogenic-related genes. Data were reported as the mean ± SD. One-way analysis of variance (ANOVA) was used to compare the results. A p-value of less than 0.05 was considered statistically significant. According to the results, there was significant difference between the rate of proliferation of two stem cells, as iPSCs showed increased proliferation compared to AT-MSCs. During osteogenic differentiation, ALP activity and mineralization were demonstrated to be significantly higher in iPSCs. Although AT-MSC expressed higher levels of Runx2, iPSCs expressed higher amount of osteonection and osteocalcin during differentiation. Taking together, iPSCs showed a higher capacity for osteogenic differentiation and it hold promising potential for bone tissue engineering and cell therapy applications.
Scanning electron microscopy image of A(dry powder
of nZno) and B(dry powder of cZnO). Images show difference
between the size of nano and conventional form of ZnO
Comparison between anxiolytic effects of nano and conventional
ZnO (5, 10, 20 mg/kg). Each bar shows mean ± SEM,
*; p<0.05 in the treatment group compared to the control group,
+; p<0.05, ++; p<0.01 for comparison between equal doses.
The effect of morphine sulphate 6 m/kg and/or naloxone
hydrochloride 1 mg/kg alone and co-injected with
nZnO 5 mg/kg and/or cZnO 10 mg/kg on anxiety related
behaviors. Each bar is mean ± SEM. *; p<0.05, **; p<0.01
for the treatment group compared to the saline/saline control
group, ++; p<0.05, +++; p<0.01 compared to the naloxone/
saline control group, and ##; p<0.01, ###; p<0.001
compared to morphine/saline control group.
Effect of nZnO (5 mg/kg) and cZnO (10 mg/kg) in presence
of naloxone (1 μg/Rat) on anxiety-related behaviors and
locomotor activity. *; P<0.05 treatment group in comparison
with saline control group. Each bar is mean ± SEM.
Nano components are today's new wonder material. However, the safety or toxicity of these components in humans is not yet clear. In a previous study we indicated that nano ZnO (nZnO) has a stronger anxiolytic effect compared to the conventional Zno(cZnO). The present study was designed to evaluate the intraperitoneal administration of an opioidergic receptor agonist and antagonist of as well as the intra CA1 administration of an opioidergic receptor antagonist on the anxiolytic properties of nano and conventional ZnO in adult male Wistar rats. In this experimental study, rats received drugs via two modes of injection; intraperitoneal (IP.) and intra CA1 (intra hippocampus, CA1 area). Firstly, nZnO (5, 10, 20 mg/kg), cZnO (5, 10, 20 mg/kg), morphine 6mg/kg, and naloxone 1mg/kg were injected IP and naloxone 1µg/rat was injected intra CA1. Subsequently, morphine and naloxone (IP and intra CA1) were co-injected with the effective dose of nZnO and cZnO. An elevated plus maze was used to evaluate anxiety related behavior and anxiety parameters 30 minutes after the second injection. . The results indicated that the anxiolytic effects of nZnO 5mg/kg and cZnO 10 mg/kg were equal. When injected intraperitoneally, naloxone increased anxiety but did not inhibit the anxiolytic effect of nZnO and cZnO. The anxiolytic effects of morphine potentiated the anxiolytic effects of ZnO, particularly nZno. When introduced via intra CA1 injection naloxone alone had no effect on anxiety behaviors and did not inhibit the anxiolytic effect of nZnO. It seems that the opioidergic system activity involved in the anxiolytic effect of nano and conventional ZnO may operate through shared and unshared pathways.
Values of morphological parameters obtained from seminiferous tubules of control and experimental mice
Photomicrograph of seminiferous tubules in the control
group. The tubules are lined by germinal epithelium
and surrounded by interstitial connective tissue (×200, H&E
Photomicrograph of seminiferous tubules with wide
lumen lined by low height germinal epithelium in the experimental
group. The thick interstitial connective tissue is
seen around the tubules (×200, H&E staining).
Photomicrograph of seminiferous tubules lined
by cell series of germinal epithelium in the control group
(×400, H&E staining).
Photomicrograph of low germinal epithelium in seminiferous
tubule of the experimental group (×400, H&E staining).
Melatonin, the pineal gland hormone as a direct or indirect antioxidant and free radical scavenger, is involved in the process of both aging and age-related diseases. This study investigates the effects of melatonin on the histology of testicular seminiferous tubules in aged mice. Twenty male, white mice, aged 16 months, that weighed 20-23 gr were equally divided into control and experimental groups. The experimental group was intraperitoneally injected with a daily single dose of 10 mg/kg melatonin for 14 days. The control group received only saline. Six days after the last injection, all mice were sacrificed and the testes were excised and processed for light microscope observation. In the morphometric study, we evaluated testicular seminiferous tubule parameters such as height of germinal epithelium, seminiferous tubule diameter, thickness of interstitial connective tissue and spermatogenesis index (SI). SPSS software and student's t-test analyzed all parameters to assess the significance of changes between control and experimental groups. Melatonin-treated mice had seminiferous tubules with a wide lumen lined by low height germinal epithelium. The interstitial connective tissue thickened significantly in the experimental group (p<0.05), tubular diameter and germinal epithelium height decreased significantly (p<0.01), and the SI reduced compared to the control group (p<0.001). The results of this study showed the disadvantages of melatonin on seminiferous tubules of aged mice testes.
Diabetic neuropathy is the most common complication of diabetes mellitus affecting the nervous system. In this study, we investigated the in vivo effects of combined administration of 4-methylcatechol (4-MC) and progesterone (P) as a potential therapeutic tool for sciatic nerve function improvement and its role in histomorphological alterations in diabetic neuropathy in rats. Male adult rats were divided into 3 groups: sham operated control (CO), untreated diabetic (DM) and diabetic treated with progesterone and 4-methylcatechol (DMP4MC) groups. Diabetes was induced by a single dose injection of 55 mg/ kg streptozotocin (STZ). Four weeks after the STZ administration, the DMP4MC group was treated with P and 4-MC for 6 weeks. Then, following anesthesia, the animals' sciatic nerves were removed and processed for light and transmission electron microscopy (TEM) as well as histological evaluation. Diabetic rats showed a statistically significant reduction in motor nerve conduction velocity (MNCV), nerve blood flow (NBF), mean myelinated fiber (MF) diameters and myelin sheath thickness of the sciatic nerve after 10 weeks. In the sciatic nerve of the untreated diabetic group, endoneurial edema and increased number of myelinated fibers with myelin abnormalities such as infolding into the axoplasm, irregularity of fibers and alteration in myelin compaction were also observed. Treatment of diabetic rats with a combination of P and 4-MC significantly increased MNCV and NBF and prevented endoneurial edema and all myelin abnormalities. Our findings indicated that co-administration of P and 4-MC may prevent sciatic nerve dysfunction and histomorphological alterations in experimental diabetic neuropathy.
The nonlinear association between serum and salivary β 2 M.
The nonlinear association between serum and salivary
Β2-microglobulin (β2M) associated amyloidosis is an inevitable complication of chronic kidney disease (CKD). Testing β2M in the blood is invasive and expensive. On the other hand, oral fluid is a perfect medium to be explored for public health and disease surveillance. However, it has never been studied if salivary concentration of β2M reflects its concentration in the serum. Therefore, the current study aimed to examine the relationship between salivary and serum β2M in a sample of adult diabetic men with CKD. Among diabetic patients referred to the Nephrology Department of the Golestan Hospital of Ahvaz due to chronic kidney disease (CKD), 40 men not requiring renal replacement therapy were consecutively recruited for this cross-sectional study. Patients were excluded if they had any disease or were using any drugs that might affect the oral mucosa or saliva. The concentration of β2M was measured in both serum and saliva. The correlation between serum and salivary β2M was measured by calculating spearman's ρ. The Spearman's ρ for correlation between serum and salivary β2M was -0.017 (p=0.917), indicating lack of correlation. Serum and salivary creatinine (Spearman's ρ=0.54; p value<0.001) as well as serum and salivary urea nitrogen levels (Spearman's ρ=0.39; p value=0.014) were correlated. Salivary β2M levels poorly agreed with serum β2M levels, and thus may not be used as a surrogate for serum β2M in CKD patients who did not require replacement therapy.
Labeled motoneurons in L4-L6 segment of the spinal cord in the different groups after eight weeks
of treatment. Motor neuron cells in the ventral horn contain red colored particles (arrow) which indicate
retrograde DiI vesicles. The nucleus of the nerve cells is seen as a cavity (star). Control and epineural;
×200, autograft and NGC; ×400. Bars: 50 µm .
The mean number and mean percent of labeled cells in
different groups. There was a significant difference (p<0.01)
in the mean number of label cells in the epineural suture
group compared to the autograft and nerve guidance channel
groups and in the control group compared to the other
groups. The number of labelled cells in control rats was considered
to be 100%. A. The difference with epineural group is
significant (p<0.01), B. The difference with autograft group
is significant (p<0.01), C. The difference with NGC group is
significant (p<0.01).
Electron micrograph of spinal motoneuron cells eight weeks after sciatic nerve
transection in different groups. In the control group the cells can be seen to have normal
mitochondria (arrow) and euchromatin within the cell nucleus (magnification × 12400).
A few vacuoles are seen in a nerve cell from the epineural group (magnification × 12000).
The apoptotic morphology of the motoneurons with large vacuoles (V) can be observed in
the autograft group (magnification ×12000). The number of these vacuoles increased in the
NGC group (magnification ×20400). Bars=1 µm.
As effectiveness of the autologous graft in the repair of long nerve defects is very limited an effective substitute is needed. This study was conducted to determine the poled polyvinylidene fluoride (PVDF) tube as an alternative to nerve autograft. The left sciatic nerve was transected in 45 male Wistar rats. The animals were then divided randomly into three groups: in an epineural group the nerve was sutured end to end; in an autograft group a 10 mm piece of sciatic nerve was cut, rotated 180° and sutured in the nerve gap; and in a nerve guidance channel group (NGC), PVDF, tube containing nerve growth factor (NGF) and collagen gel was placed in the gap. In a control (n=15) group the sciatic nerve was exposed but not transected. To determine axonal regeneration, retrograde DiI tracer was injected into the gastrocnemius muscle. One week later, retrograde-labeled neurons were counted in the L4-L6 spinal segments and one way ANOVA analysis was performed to compare groups. Neuronal morphology changes were studied by electron microscopy. Significant statistical decreases in the mean number of labeled motoneurons were observed in all surgical groups compared to the control group; and in the autograft and the NGC groups compared to epinural suture group (p<0.01). No significant difference in the mean number of motoneurons was observed between the autograft and NGC groups. Chromatin condensation, dilated endoplasmic reticulum and large vacuoles were observed in the autograft and NGC groups. Regarding the positive effects of PVDF tube containing NGF and Collagen gel on the sciatic nerve regeneration, authors suggest that it may be useful in peripheral nerve repair.
MTT staining for assessing spinal cord slice viability. A. Freshly prepared slice (0 hour). B. Slice cultured for 6 hours (control). The viability of slices cultured for 6 hours in the presence of N/L typevoltage sensitive calcium channel blocker (loperamide hydrochloride, 100 µM),C. orNa+/Ca2+ exchanger inhibitor (bepridil hydrochloride, 20 µM), D. was considerably increased. Magnification: ×40.
The effect of N/L type voltage sensitive calcium channel blocker (loperamide hydrochloride) and Na+/Ca2+ exchanger inhibitor (bepridil hydrochloride) on the apoptosis of motor neurons. A. Normal motor neurons from freshly prepared slices (0 hour). B. Motor neurons from slices cultured for 6 hours (control) displayed morphological features of apoptosis. The application of loperamide hydrochloride, 100 µM, C. or bepridil hydrochloride, 20 µM, D. could prevent apoptosis in the motor neurons from slices cultured for 6 hours. Arrows point out motor neurons. Scale bar: 20 µm.
The effect of N/L type voltage sensitive calcium channel blocker (loperamide hydrochloride) and Na+/Ca2+ exchanger
inhibitor (bepridil hydrochloride) on motor neuron viability. The percentage of viable motor neurons was significantly increased
in slices exposed to loperamide hydrochloride, 100 µM, or bepridil hydrochloride, 20 µM, after 6 hours. Mean ± SD, n=12.
The apoptosis of motor neurons is a critical phenomenon in spinal cord injuries. Adult spinal cord slices were used to investigate whether voltage sensitive calcium channels and Na(+)/Ca(2+) exchangers play a role in the apoptosis of motor neurons. In this experimental research, the thoracic region of the adult mouse spinal cord was sliced using a tissue chopper and the slices were incubated in a culture medium in the presence or absence of N/L type voltage sensitive calcium channels blocker (loperamide, 100 µM) or Na(+)/Ca(2+) exchangers inhibitor(bepridil, 20 µM) for 6 hours. 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium (MTT) staining was used to assess slice viability while morphological features of apoptosis in motor neurons were studied using fluorescent staining. After 6 hours in culture, loperamideand bepridil not only increased slice viability, but also prevented motor neuron apoptosis and significantly increased the percentage of viable motor neurons in the ventral horns of the spinal cord. The results of this study suggest that voltage sensitive calcium channels and Na(+)/Ca(2+) exchanger might be involved in the apoptosis of motor neurons in adult spinal cord slices.
Auto-transplantation of adult mouse spermatogonia resulted
in spermatogenesis in recipient testis, The spermatogonial cells
were labeled with BrdU in vitro and transplanted into the recipient
testes via rete testis. Donor-derived spermatogonial cells were traced
in the recipient testes (A) two weeks. (C) 8 weeks after transplantation
elongated spermatids were detected within the testis (Arrows).
(E) Control group without adding primary antibody. The cells
showing nuclear BrdU staining were considered as transplanted cells.
(B, D, F) Phase contrast photographs. (Bar=50µm).
(A, C) A non-transplanted testis in 12 weeks after irradiation, the majority of the seminiferous tubules still contained only Sertoli cells. (B, D) Eight weeks after the auto-transplantation, more differentiated germ cell types are seen in seminiferous tubules. (Bar=1000 µm in A, B; Bar=500 µm in C, D).
We evaluated structural and functional changes of fresh and frozen-thawed adult mouse spermatogonial stem cells following auto-transplantation into gamma-irradiated testes. In this experimental research, the right testes from adult mice (n=25) were collected, then Sertoli and spermatogonial cells were isolated using two-step enzymatic digestion, lectin immobilization and differential plating. Three weeks after cultivation, the Bromodeoxyuridine (BrdU)-labeled spermatogonial cells were transplanted, via rete testis, into the other testis of the same mouse, which had been irradiated with 14Gy. The mice were transplanted with: fresh cells (control 1), fresh cells co-cultured with Sertoli cells (control 2), the frozen-thawed cells (experimental 1) and frozen-thawed cells co-cultured with Sertoli cells (experimental 2). The morphological changes between different transplanted testes groups were compared in 8 weeks after transplantation. The statistical significance between mean values was determined by Kruskal Wallis and one-way analysis of variance in efficiency of transplantation. The statistical analysis revealed significant increases in the mean percentage of testis weight and normal seminiferous tubules following spermatogonial stem cells transplantation in the recipient'fs testes. The normal seminiferous tubules percentage in the co-culture system with fresh cells and frozen-thawed groups were more than those in non-transplanted and fresh cell transplanted groups (p≤0.001). Our results demonstrated that spermatogonial stem cells in the colonies could result sperm production in the recipient's testes after autologous transplantation.
Morphological features of apoptosis in sensory neurons. Propidium iodide (red) and Hoechst 33342 (blue) staining
revealed morphological changes of apoptosis in dorsal root ganglion (DRG) sensory neurons. A. normal sensory neurons with
large cell body and nucleus from freshly prepared DRG (0 hour). Sensory neurons after 24 (B), 48 (C), 72 (D) and 96 hours
(E) displayed cell shrinkage as well as nuclear and chromatin condensation. Scale bar: 25 µm. Arrows show sensory neurons.
NA fragmentation during apoptosis of dorsal root ganglia (DRG) sensory neurons using TUNEL method. A. sensory
neurons from freshly dissected DRG (0 hour) appeared TUNEL negative. TUNEL positive sensory neurons after 24(B), 48(C),
72(D) and 96(E) hours in culture. Scale bar: 25 µm. Arrows show sensory neurons.
Caspase-dependent apoptosis in dorsal root ganglia (DRG) sensory neurons. A: Sensory neurons from freshly prepared
DRG (0 hour). B, C, D, and E: Sensory neurons from DRG cultured for 24, 48, 72 and 96 hours respectively (controls). B', C',
D' and E': Sensory neurons from cultured DRG in the presence of general caspase inhibitor (Z-VAD.fmk, 100 µM) after 24, 48,
72 and 96 hours. Scale bar: 25 µm. Arrows show sensory neurons.
: Immunolocalization of activated caspase-3 antibody
in dorsal root ganglia (DRG) sensory neurons. Sensory neurons were stained with activated caspase-3 antibody (green)
and counterstained with Hoechst 33342 (blue). (A-B).Weak
activated caspase-3 immunoreactivity in sensory neurons
from DRG at 0 hour with no sign of apoptotis. Sensory neurons from DRG cultured for 24 hours (C-D) and 48 hours
(E-F) displayed intense activated caspase-3 immunoreactivity both in the nucleus and the cytoplasm where the nuclei
showed apoptotic features. Scale bar: 25 µm. Arrows show
sensory neurons.
Sensory neurons in dorsal root ganglia (DRG) undergo apoptosis after peripheral nerve injury. The aim of this study was to investigate sensory neuron death and the mechanism involved in the death of these neurons in cultured DRG. In this experimental study, L5 DRG from adult mouse were dissected and incubated in culture medium for 24, 48, 72 and 96 hours. Freshly dissected and cultured DRG were then fixed and sectioned using a cryostat. Morphological and biochemical features of apoptosis were investigated using fluorescent staining (Propidium iodide and Hoechst 33342) and the terminal Deoxynucleotide transferase dUTP nick end labeling (TUNEL) method respectively. To study the role of caspases, general caspase inhibitor (Z-VAD.fmk, 100 μM) and immunohistochemistry for activated caspase-3 were used. After 24, 48, 72 and 96 hours in culture, sensory neurons not only displayed morphological features of apoptosis but also they appeared TUNEL positive. The application of Z-VAD.fmk inhibited apoptosis in these neurons over the same time period. In addition, intense activated caspase-3 immunoreactivity was found both in the cytoplasm and the nuclei of these neurons after 24 and 48 hours. Results of the present study show caspase-dependent apoptosis in the sensory neurons of cultured DRG from adult mouse.
Top-cited authors
Masoud Soleimani
  • Tarbiat Modares University
Saeid Abroun
  • Tarbiat Modares University
Shahnaz Razavi
  • Isfahan University of Medical Sciences
Maryam Shahhoseini
  • Royan Institute
Ashraf Moini
  • Tehran University of Medical Sciences