ABSTRACT
Objective: umbilical cord blood is used for transplantation purposes in regenerative medicine of hematological disorders. MicroRNAs are important regulators of gene expression that control both physiological and pathological processes such as cancer development and incidence. There is a new relation between p53 [tumor suppressor gene] and miR-145 [suppressor of cell growth] upregulation. In this study, we have assessed how adipose-derived stem cells [ADSCs] affect the expansion of hematopoietic stem cells [HSCs], as well as miR-145 and p53 expressions
Materials and Methods: in this experimental study, we cultured passage-3 isolated human ADSCs as a feeder layer. Flow cytometry analysis confirmed the presence of ADSC surface markers CD73, CD90, CD105. Ex vivo cultures of cordblood CD34+ cells were cultured under the following 4 culture conditions for 7 days: i. Medium only supplemented with cytokines, ii. Culture on an ADSCs feeder layer, iii. Indirect culture on an ADSCs feeder layer [Thin Cert[] plate with a 0.4 ?m pore size], and iv. Control group analyzed immediately after extraction. Real-time polymerase chain reaction [PCR] was used to determine the expressions of the p53 and miR-145 genes. Flow cytometry analysis of cells stained by annexin V and propidium iodide [PI] was performed to detect the rate of apoptosis in the expanded cells
Results: ADSCs tested positive for mesenchymal stem cell [MSC] markers CD105, CD90, and CD73, and negative for HSC markers CD34 and CD45. Our data demonstrated the differentiation potential of ASCs to osteoblasts by alizarin red and alkaline phosphatase staining. MTT assay results showed a higher proliferation rate of CD34+cells directly cultured on the ADSCs feeder layer group compared to the other groups. Direct contact between HSCs and the feeder layer was prevented by a microporous membrane p53 expression increased in the HSCs group with indirect contact of the feeder layer compared to direct contact of the feeder layer. p53 significantly downregulated in HSCs cultured on ADSCs, whereas miR-145 significantly upregulated in HSCs cultured on ADSCs
Conclusion: ADSCs might increase HSCs proliferation and self-renewal through miR-145, p53, and their relationship
ABSTRACT
Objective: ANRIL is an important antisense noncoding RNA gene in the INK4 locus [9p21.3], a hot spot region associated with multiple disorders including coronary artery disease [CAD], type 2 diabetes mellitus [T2DM] and many different types of cancer. It has been shown that its expression is dysregulated in a variety of immune-mediated diseases. CAD is a major problem in T2DM patients and the cause of almost 60% of deaths in these patients worldwide. The aim of the present study was to compare the expression level of ANRIL between T2DM patients with and without CAD
Materials and Methods: In this case-control study, we examined ANRIL expression in peripheral blood mononuclear cell samples by quantitative reverse transcriptionpolymerase chain reaction [RT-qPCR] in 64 T2DM patients with and without CAD [33 CAD+ and 31 CADpatients respectively, established by coronary angiography]
Results: Expression analysis revealed that ANRIL was up regulated [2.34-Fold, P=0.012] in CAD+ versus CAD diabetic patients. Data from receiver operating characteristic [ROC] curve analysis has shown that ANRIL could act as a potential biomarker for detecting CAD in diabetic patients
Conclusion: The expression level of ANRIL is associated with presence of CAD in diabetic patients and could be considered as a potential peripheral biomarker
ABSTRACT
Background: The cyclin E2 [CYCE2] is an important regulator in the progression and development of NSCLC, and its ectopic expression promoted the proliferation, invasion, and migration in several tumors, including Non-Small Cell Lung Cancer [NSCLC]. However, the upregulation of CYCE2 in NSCLC cells suggested that it has a key role in tumorigenicity. In addition, the RAS family proteins as oncoproteins were activated in many major tumor types and its suitability as the therapeutic target in NSCLC was proposed. Considering the crucial role of microRNAs, it was hypothesized that altered expression of hsa-miR-30d-5p and hsa-let-7b might provide a reliable diagnostic tumor marker for diagnosis of NSCLC
Method: Real-time RT-PCR approach could evaluate the expression alteration of hsa-miR-30d-5p and hsa-let-7b and it was related to the surgically resected tissue of 24 lung cancer patients and 10 non-cancerous patients. The miRNAs expression was associated with clinicopathological features of the patients
Results: Hsa-miR-30d showed a significant downregulation [p=0.0382] in resected tissue of NSCLC patients compared with control group. Its expression level could differentiate different stages of malignancies from each other. The ROC curve analysis gave it an AUC=0.73 [p=0.037] which was a good score as a reliable biomarker. In contrast, hsa-let-7b was significantly overexpressed in tumor samples [p=0.03]. Interestingly, our findings revealed a significant association of hsa-let-7b in adenocarcinoma tumors, compared to Squamous Cell Carcinomas [SCC] [p<0.05]. Also, analysis of ROC curve of hsa-let-7b [AUC=0.74, p-value=0.042] suggests that it could be as a suitable biomarker for NSCLC
Conclusion: Together, these results suggest a possible tumor suppressor role for hsa-miR-30d in lung tumor progression and initiation. Moreover, upregulation of hsa-let-7b was associated with the tumor type
ABSTRACT
Background: Familial hypercholesterolemia [FH] is a frequent autosomal dominant disorder of lipoprotein metabolism. This disorder is generally caused by mutations in low-density lipoprotein receptor [LDLR], apolipoprotein B 100 [APOB], and proprotein convertase subtilisin/kexin type 9 [PCSK9] genes. In the present study, we aimed at identifying the common LDLR and APOB gene mutations in an Iranian population
Methods: Eighty unrelated Iranian patients with FH entered the study, based on Simon Broome diagnostic criteria. All samples were screened for two common APOB gene mutations, including R3500Q and R3500W, by the means of ARMS-PCR and PCR- RFLP assays, respectively. In addition, exons 3, 4, 9, and 10 of LDLR gene were sequenced in all patients
Results: A novel mutation in exon 3 [C95W] and a previously described mutation in exon 4 [D139H] of LDLR gene were found. Three previously reported polymorphisms in LDLR gene as well as three novel polymorphisms were detected in the patients. However, in the studied population, no common mutations were observed in APOB gene
Conclusion: The results of our study imply that the genetic basis of FH in Iranian patients is different from other populations
Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Apolipoprotein B-100 , Receptors, LDL , GeneticsABSTRACT
Background: cancer is one of the most important factors which affect the human health .RNA interference [RNAi] based therapies have been developed and showed significant results in this regard. One of the most important problems of siRNA i system is its low stability. In order to overcome to this weakness, venous types of hairpin RNA or shRNA was introduced Over expressed colorectal cancer gene [OCC-I], which has several transcripts is considered as a hallmark of colorectal cancer. This gene has vital role in the development and progression of cancer through the cell signaling pathways, which are involved in cell proliferation. In the present study, the RNAi system was utilized in order to reduce the expression of specific transcript of OCC-1 gene
Methods: the silencer structure was designed and cloned in the expression vector pRNA-H1.l / Neo which has a promoter for RNApolIII. Plasmid was transferred into the colorectal cancer cell line, and the expression of transcript was compared to the control samples at 24 and 72 h post transfection by Real-Time PCR
Results: the expression of transcript was reduced about 25 % at 72 h post transfection following shRNA transfection, compared to the control sample
Conclusion: the designed shRNA structure successfully reduced the expression of target gene; accordingly, it can be used in future studies in order to investigate the effect of these transcripts on development and progression of colorectal cancer
ABSTRACT
Background: The human OCT4 gene, responsible for pluripotency and self-renewal of Embryonic Stem [ES] and Embryonic Carcinoma [EC] cells, can generate several transcripts [OCT4A, OCT4B-variant 2, OCT4B-variant 3, OCT4B-variant 5, OCT4B1, OCT4 B2 and OCT4B3] by alternative splicing and alternative promoters. OCT4A that is responsible for ES and EC cell stemness properties is transcribed from a promoter upstream of Exon1a in those cells. The OCT4B group variants [OCT4B-varianT[2], OCT4Bvariant3, OCT4B-variant5, OCT4B1, OCT4B2 and OCT4B3] are transcribed from a different promoter located in intron 1 and some of them respond to the cell stresses, but cannot sustain the ES/EC cell self-renewal. However, the exact function of OCT4B group variants is still unclear
Methods: In the present study, we employed RT-PCR and sequencing approaches to explore different forms of OCT4 transcripts
Results: Our data revealed that the OCT4B group variants [OCT4B-varianT[2], OCT4 B-variant3, OCT4B1, OCT4B2 and OCT4B3] have longer 5' UTR in the human bladder carcinoma cell line of 5637
Conclusion: These OCT4 variants undergo alternative splicing in their 5' UTR which might exert regulatory roles in transcription and translation mechanisms
ABSTRACT
Background: Alternative splicing is an important mechanism that regulates gene expression and function in human cells. OCT4, a crucial pluripotency marker in embryonic stem/carcinoma cells generates several spliced variants in different cell types and cancers. The expression of OCT4 in cancers has been challenged in many studies. The existence of several OCT4 spliced variants and absence of specific discriminating primers is the main reason of this controversy. Therefore, using specific primers and discriminating OCT4 variants from each other might help to reduce these discrepancies in carcinogenesis and stem cell researches
Methods: 17 various human cancer, pluripotent and normal cells were cultured and their RNAs were extracted. Related cDNAs were synthesized and the expression pattern of OCT4variants was investigated by RT-PCR assay. PCR products were cloned into pTZ57R/T vector and their authenticity was confirmed by DNA sequencing
Results: Expression pattern of OCT4 variants [OCT4A, OCT4B and OCT4B1] was analyzed by RT-PCR assay and the authenticity of PCR products was confirmed by DNA sequencing. A novel spliced variant of OCT4 was discovered and named as OCT4B3. This variant was very similar to OCT4B2 transcript except that 207-nt of exon 1b is lost. Moreover, the expression pattern of OCT4B3 variant was investigated in 17 human cell types, where its expression was only found in astrocytoma and bladder cancer cell types 1321N1 and 5637, respectively
Conclusion: OCT4 variants are differentially expressed in various human cancer cell lines. Moreover, a novel variant of OCT4, OCT4B3, was detected in two human cancer cell lines of bladder carcinoma [5637] and brain astrocytoma [1321N1] for the first time
ABSTRACT
OBJECTIVES: Ovarian hormones have been shown to regulate body weight, intra-abdominal fat accumulation and plasma level of cytokines. The aim of this study was to investigate the effect of estrogen replacement therapy on visceral adipose tissue, plasma level of apelin, lipid profiles, and glucose in ovariectomized (OVX) rats. METHODS: Thirty female Wistar rats were divided into OVX (n = 20) and sham (n = 10) groups. OVX rats were subdivided into estrogen replacement therapy (OVX+est; n = 10) receiving 17 β-estradiol valerates (30 µg/kg, s.c., 5 day/week, for eight weeks), and vehicle control group receiving sesame oil same as experiment group (OVX+ses oil; n = 10). After the treatments, all groups were sacrificed and blood samples were collected, visceral fats were taken from the abdominal cavity and weighed immediately. Apelin were measured using enzyme-linked immunosorbent assay kits. Lipid profiles and glucose were measured using the enzymatic colorimetric method. Data were analyzed with one-way analysis of variance and (P < 0.05) determined as the statistical significance level. RESULTS: After eight weeks, body weight, body mass index (BMI), visceral fat, apelin and lipid profiles (P < 0.01) were increased significantly in OVX rats compared to sham group. Treatment with estrogen leads to significant reduction in body weight and BMI (P < 0.05), there was no significant change in serum apelin level in OVX+est rats compared to OVX+ses. CONCLUSIONS: These results suggest that estradiol replacement therapy successfully attenuated some of the metabolic syndrome components, and apelin does not probably stand as a mediator of these physiological functions.
Subject(s)
Animals , Female , Humans , Rats , Abdominal Cavity , Blood Glucose , Body Mass Index , Body Weight , Cytokines , Enzyme-Linked Immunosorbent Assay , Estradiol , Estrogen Replacement Therapy , Estrogens , Glucose , Intra-Abdominal Fat , Methods , Plasma , Rats, Wistar , Sesame Oil , ValeratesABSTRACT
Aims: This study is designed to determine the frequency of Legionella pneumophila in cold and warm water as well as water containers of newborn incubators in Guilan province hospitals, Iran, using amplification of the macrophage infectivity protein gene (mip gene) by PCR. Study Design: Cross sectional study. Place and Duration of Study: The present study was performed in the Cellular and Molecular Research Center, Guilan University of Medical Sciences between June 2011 and July 2012 Methodology: Samples were collected directly in sterile containers, concentrated in centrifuge, transferred to yeast extract broth containing L- cysteine, Fe2+, Glycin and vancomycin and incubated for 3-4 days. DNA was extracted by using the boiling method and PCR was performed to search Legionella and mip gene using two pairs of primers. Contamination with other bacteria was evaluated in all negative samples using universal primers of 16S rRNA gene. Results: About 8.5% of the samples had L. pneumophila including 11% of the incubators and 5.8% of both hot and cold tap water. The mip gene was found in 2.8% of the samples. One third of the incubator and one half of the hot water habited L. pneumophila had the mip gene but it was not found in cold tap water samples. About 87.2% of the negative samples showed bacterial contamination as revealed by PCR with primers of 16S rRNA gene. Conclusions: This study indicates that in spite of using distilled water for incubators, L. pneumophila contamination is considerable and other bacterial contamination is very high. It may be related to the length of time that water remains in an incubator container which is a predisposing factor for both biofilm formation and the growth of water microflora. It seems that the high temperature of hot water system and the high rate of free residual chlorine in tap water system are the main causes of low rate of Legionella contamination but are ineffective on contamination with other bacteria.
ABSTRACT
In the present study, the possible role of a potent cannabinoid agonist, WIN55, 212-2 in the dorsal hippocampus on pain and memory performance has been evaluated. Animals were cannulated in CA1 region of the hippocampus using sterotaxic apparatus. Ten days after recovery, animals were trained in passive avoidance learning (PAL), and immediately received different doses of WIN 55212-2 (0.1, 0.25 and 0.5µg/rat), and were tested 24 h after the training. In the second part of the experiment animals received either WIN 55212-2 (0.5µg/rat) or saline respectively. Tail flick latency was measured three times with 10 minutes interval 30 minutes and 24 hours after the infusion into the CA1. Results indicate that post-training intra-CA1 administration of WIN55, 212-2 (0.25 and 0.5µg/rat).
ABSTRACT
The purpose of the present study was to determine the role of medial prefrontal cortex [mPFC] dopaminergic system in fear conditioning response considering individual differences. Animals were initially counterbalanced and classified based on open field test, and then were given a single infusion of the dopamine agonist, amphetamine [AMPH] and antagonist, clozapine [CLZ] into the medial prefrontal cortex. Rats received tone-shock pairing in a classical fear conditioning test and then exposed to the tone alone. Freezing responses were measured as conditioned fear index. The results showed that both AMPH and CLZ infusion in mPFC reduced the expression of conditioned fear. This finding indicates that elevation or reduction in the dopaminergic activity is associated with the decrease of fear responses, despite preexisting individual-typological differences
Subject(s)
Male , Animals, Laboratory , Prefrontal Cortex , Fear , Dopamine Agonists , Amphetamine , Clozapine , Rats, WistarABSTRACT
Leptospirosis is a common zoonosis throughout the world and common in the flat area of Guilan, Iran, with seasonal incidence, especially in rice farmers. Clinical diagnosis of leptospirosis is difficult, because its symptoms are similar to several acute infective diseases. Serological assays are important in diagnosis of the disease and microscopic agglutination test [MAT] is a gold standard, however, it is not a routine test in diagnostic laboratories. Thus, a simple and reliable test is a necessity. In this study, we evaluated a latex agglutination test using native strains of leptospires. A number of 98 positive cases and 54 negative cases which were screened by MAT, along with 30 sera of other diseases as control samples, were examined by latex agglutination test, using an antigenic suspension [whole antigen], which was extracted from 4 common native strains. False positive and false negative rate were 15 and 12 consequently. Sensivity, specificity, positive predictive value, negative predictive value, and accuracy were 89.0%, 84.5%, 86.7%, 87.2%, and 87.0% respectively. Regarding the considerable rate of sensivity and specificity of the test which is compatible to other performed studies, in addition to the simple performance test, does not need a complex laboratory facility, which may also be carried out in rural regions, therefore, this test is valuable for primary screening
Subject(s)
Humans , Latex Fixation Tests , Sensitivity and Specificity , Predictive Value of TestsABSTRACT
Leptospirosisis a very common zoonosis in the world. Culture is low sensitive with high rate false negative. So, serological assays are best alternative way for its diagnosis. Microscopic agglutination test [MAT] is gold standard but performing it requires a panel of some standard strains and need periodic subculturing of them, and also requires double sera with at least two weeks interval to investigate seroconversion. Furthermore, other serological methods should be investigated. The aim of this study was to evaluate an in-house IgM-ELISA developed by using antigen extracted from endemic isolates. 14 endemic isolates belonged to the serogroups: Icterohaemorrahgia, Pomona, Hardjo, and Gripotyphosa, were inoculated in EMJH to take well grown cultures. Whole antigen was extracted from each culture by Freezing-Thawing method in distilled water. Same amount of extraction of each culture with same OD number in 550nm were mixed together and were used for coating Elisa plates. Antihuman IgM conjugated with alkaline phosphatase were used in this assay. We used a commercial quantitative IgM-ELISA [SERION ELISA classic] for cut off determination. MAT was used for confirmation positive and negative cases. Sera with titer >/= 1:100 in MAT and positive criteria in commercial quantitative IgM-ELISA were considered as positive cases. 98 positive cases and 54 negative cases were chosen by screening 200 sera of patients suspected to leptospirosis by using MAT and commercial quantitative IgM-ELISA. We also used 30 sera of patients affected by hepatitis B, salmonelosis, and brucellosis as control cases. 88 of 98 positive cases were positive [false negative=10], 1 of 54 negative and all control case were negative [false positive =1] in the test. Sensitivity, specificity, PPV, NPV, and accuracy of the test were evaluated :99.0%, 89.1%, 90.75, 98.8%, and 94.25, respectively. ELISA for measuring specific IgM to leptospires antigen[s] could be a good alternative to MAT, which is not a routine diagnostic assay to perform in clinical diagnostic laboratories and only is reliable when there is paired sera. Sensitivity and specificity of the assay is dependent to several factors, especially to the type of antigen coated on plates, quality of assay materials, and also to the time of sampling. Sera of days >/= 6 of the disease has enough antibodies to measure and a common antigen extracted from several common pathogenic leptospires, especially from endemic isolates, could be more helpful to increase accuracy of the assay