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Objective@#This research investigated the effects of human chorionic gonadotropin (HCG)-producing peripheral blood mononuclear cells (PBMCs) on the implantation rate and embryo attachment in mice. @*Methods@#In this experimental study, a DNA fragment of the HCG gene was cloned into an expression vector, which was transfected into PBMCs. The concentration of the produced HCG was measured using enzyme-linked immunosorbent assay. Embryo attachment was investigated on the co-cultured endometrial cells and PBMCs in vitro. As an in vivo experiment, intrauterine administration of PBMCs was done in plaque-positive female mice. Studied mice were distributed into five groups: control, embryo implantation dysfunction (EID), EID with produced HCG, EID with PBMCs, and EID with HCG-producing PBMCs. Uterine horns were excised to characterize the number of implantation sites and pregnancy rate on day 7.5 post-coitum. During an implantation window, the mRNA expression of genes was evaluated using real-time polymerase chain reaction. @*Results@#DNA fragments were cloned between the BamHI and EcoRI sites in the vector. About 465 pg/mL of HCG was produced in the transfected PBMCs. The attachment rate, pregnancy rate, and the number of implantation sites were substantially higher in the HCG-producing PBMCs group than in the other groups. Significantly elevated expression of the target genes was observed in the EID with HCG-producing PBMCs group. @*Conclusion@#Alterations in gene expression following the intrauterine injection of HCG-producing PBMCs, could be considered a possible cause of increased embryo attachment rate, pregnancy rate, and the number of implantation sites.
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Introduction: Hepatitis C virus [HCV] is a ?blood-borne pathogen, resulting in liver cirrhosis and liver cancer. Despite of many efforts in development of treatments for HCV, no vaccine has been licensed yet. The purpose of this study was ?to design and prepare a specific mRNA, without 5' cap and poly [A] tail transcribed in vitro capable of coding core protein and also to determine its functionality
Methods: Candidate mRNA was prepared by in vitro transcription of the designed construct consisting of ??5' and 3' untranslated regions of heat shock proteins 70 [hsp70] mRNA, T7 promoter, internal ribosome entry site [IRES] sequences of eIF4G related to human dendritic cells [DCs], and the ?Core gene of HCV. To design the modified mRNA, the ??5' cap and poly [A] tail structures were not considered. DCs were transfected by in vitro-transcribed messenger RNA [IVT-mRNA] and the expressions of green fluorescent protein [GFP], and Coregenes were determined by microscopic examination and Western blotting assay
Results: Cell transfection results showed that despite the absence of ??5' cap and poly [A] tail, the structure of the mRNA ?was stable. Moreover, the successful expressions of GFP and Coregenes were achieved
Conclusion: Our findings indicated the effectiveness of a designed IVT-mRNA harboring the Core gene of HCV in transfecting and expressing the antigens in DCs. Considering the simple and efficient protocol for the preparation of this IVT-mRNA and its effectiveness in expressing the gene that it carries, this IVT-mRNA could be suitable for development of an RNA vaccine against HCV
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Background: Genomic engineering of Escherichia coli is applied to design and produce recombinant proteins as the new drugs. The aim of this study was to CRISPR-Cas9 mediated capsule gene silencing in E. coli
Materials and Methods: We suppressed genes involved in capsule expression of E.coli by CRISPR cas9 process. The constructed E.coli was confirmed by microscopic smear, transmission electron microscopy and T7 phage influence assay
Results: The results were shown that the inhibition of capsule production was carried out successfully and there was not any capsule layer around the bacteria
Conclusion: E. coli without any capsule around may proper for replacement of it with other molecules in future
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Objective: Root resorption is a complication of orthodontic treatment and till date, there is a dearth of information regarding this issue. The aim of this study was to determine whether the expression of transforming growth factor-beta 1 [TGF-beta 1, an inflammatory cytokine] is related to orthodontic force. Moreover, if associated, the expression level may be helpful in differential diagnosis, control and ultimate treatment of the disease
Materials and Methods: In this experimental study, a total of 24 eight-week-old male Wistar rats were selected randomly. On day 0, an orthodontic appliance, which consisted of a closed coil spring, was ligated to the upper right first molar and incisor. The upper left first molar in these animals was not placed under orthodontic force, thus serving as the control group. On day 21, after anesthesia, the animals were sacrificed. The rats were then divided into two equal groups where the first group was subjected to histological evaluation and the second group to reverse transcriptase-polymerase chain reaction [RT-PCR]. Orthodontic tooth movement was measured in both groups to determine the influence of the applied force
Results: Statistical analysis of data showed a significant root resorption between the experimental group and control group [P<0.05], however, there was no significant difference in the expression level of the inflammatory cytokine, TGF-beta 1
Conclusion: Based on the findings of this study, we suggest that there is a direct relationship between orthodontic force and orthodontic induced inflammatory root resorption. In addition, no relationship is likely to exist between root resorption and TGF-beta 1 expression in the resorptive lacunae
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Background: Surface antigens [SAGs] of Toxoplasma gondii are known candidates for diagnostic tests and vaccines. The present study argues about the main necessary properties for determination and prediction of T-cell agretopes and B-cell epitopes of surface antigens of Toxoplasma gondii
Materials and Methods: Primary, secondary and tertiary structures of the proteins were analyzed by different methods. The three-dimensional structures were determined by use of ab initio method for prediction of discontinues epitopes. The agretopes and epitopes were predicted via several various web servers with different methods employed
Results: The results of in silico analyses showed that the regions 129-GAPAGRNNDGSSAPT-143 for protein p22, 234-SENPWQGNASSD-245 for protein p30 and 348-PGTEGESQAGT-358 for protein p43, have the highest immunogenic potential
Conclusion: We reached to three antigenic epitopes for cloning and protein expression. In following the purified polypeptide will be applied for diagnosis of Toxoplasma gondii
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Background: Visceral leishmaniasis [VL] or kala-azar is a parasitic disease caused by the species of Leishmania donovani complex. Mediterranean type of the disease is endemic in some parts of Iran and more than 95% of cases were reported in children up to 12 years of age. This study was performed to determine the seroprevalence of VL in the rural areas of the Dashti district from Bushehr province
Materials and Methods: In this cross-sectional study, a randomized cluster sampling method was used for the collection of blood samples from children up to 12 years old from rural areas of Dashti district. Before sampling; a questionnaire was filled out for each case. All the collected blood samples were examined after the serum separating by Direct Agglutination Test [DAT] for detection of anti-Leishmania infantum antibodies. The cutoff titers of >/=1: 3200 with specific clinical features were supposed to be considered as VL
Results: Altogether, 24 out of 1221 [1.96%] blood samples showed titers between 1:800 and 1:1600 which considered as suspicious cases. None of the suspicious cases had a history of kala-azar. None of 1221 collected blood samples showed anti Leishmania infantum [L. infantum] at titer >/=1:3200
Conclusion: This study confirms the circulation of L. infantum in Dashti district and highlights the sporadic pattern of VL in the studied areas which necessitates the surveillance system to be monitored by health authorities
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Background: Immunogenicity of Streptokinase, as a thrombolytic drug, has limited its clinical use. Elimination of the amino acid residues that are responsible for immunogenicity while don't affect the bioactivity of streptokinase is worthy. Recently, we modified the streptokinase through the elimination of 42 amino acids from its' C-terminal and assessed its bioactivity in vitro. In this study, bioactivity of the mutated-streptokinase determined and compared with those of commercially available streptokinase [Heberkinase] in rabbits with induced blood clot
Materials and Methods: Recombinant mutated streptokinase was purified and its lipopolysaccharide contained remove and evaluated by LAL test. Thrombolytic activity of drug was evaluated by rabbit jugular vein as in vivo thrombosis model. The thrombolytic property of the drug was evaluated with determining of D-dimer in plasma
Results: The results showed in vivo bioactivity of both truncated and commercial streptokinase [p<0.05]. This study showed an important influence of the 42 amino acids of C-terminal in bioactivity of the streptokinase
Conclusion: Clinical use of the r-streptokinase requires more modification to restore its' activity in vivo. This product may be a promising choice for clinical use after confirmation of its stability and non-immunogenicity
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Background: Visceral leishmaniasis [VL], caused by Leishmania infantum [L. infantum], is a life-threatening vector-borne parasitic disease is distributed in some parts of the world. The disease is endemic in some parts of Iran. This study was aimed to determine the seroprevalence of VL among children and domestic dogs [as a reservoir of the parasite] in Dehloran, west of Iran
Materials and Methods: This cross-sectional study was carried out in Dehloran County. The blood samples of 872 children up to 12 years old and 52 dogs were collected from 10 villages of Dehloran using randomly-clustered sampling method. Sera were separated from all peripheral blood samples and tested by direct agglutination test [DAT]. Anti-Leishmania infantum antibodies at titers of >/=1:800 and >/=1:80 were considered as Leishmania infantum infection in human and dog, respectively
Results: In general, among 872 human samples, 1.03% of samples had anti-Leishmania antibody with 1:1600 titers and 1.26% had 1:800 titers. In addition, from 52 dog samples, 21.15% of dogs had a titer of >/=1:320 and 25% had 1:80 and 1:160 titers
Conclusion: Our findings indicate that the seropositive dogs in the studied areas are considerable and L. infantum may be circulated between human and domestic dog in the studied area. Further study of isolation and molecular identification of Leishmania spp. is recommended
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Background: Cervical cancer is the second most common female cancer worldwide. Inhibitors of apoptosis proteins [IAPs] block apoptosis; therefore, therapeutic strategies targeting IAPs have attracted the interest of researchers in recent years. Apollon, a member of IAPs, inhibits apoptosis and cell death. RNA interference is a pathway in which small interfering RNA [siRNA] or shRNA [short hairpin RNA] inactivates the expression of target genes. The purpose of this study was to determine the effect of constructed shRNAs on apoptosis and growth inhibition through the suppression of apollon mRNA in HeLa cell line
Methods: Three shRNAs with binding ability to three different target sites of the first region of apollon gene were designed and cloned in pRNAin-H1.2/Neo vector. shRNA plasmids were then transfected in HeLa cells using electroporation. Down-regulation effects of apollon and the viability of HeLa cells were analyzed by RT-PCR, lactate dehydrogenase assay, and MTT assay, respectively. Also, the induction and morphological markers of apoptosis were evaluated by caspase assay and immunocytochemistry method
Results: The expression of shRNA in HeLa cells caused a significant decrease in the level of apollon mRNA1. In addition, shRNA1 effectively increased the mRNA level of Smac [as the antagonist of apollon], reduced the viability of HeLa cells and exhibited immunocytochemical apoptotic markers in this cell line
Conclusion: Apollon gene silencing can induce apoptosis and growth impairment in HeLa cells. In this regard, apollon can be considered a candidate therapeutic target in HeLa cells as a positive human papillomavirus cancer cell line
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Background: Luteinizing hormone [LH] was secreted by the stimulating cells of the testes and ovaries in the anterior pituitary gland. The application of this hormone is in the treatment of men and women with infertility and amenorrhea respectively
Materials and Methods: In the present study the alpha and beta subunits of human LH gene were cloned into the pEGFP-N1 expression vector and produced the recombinant LH hormone in Chinese hamster ovary [CHO] eukaryotic system
Results: Alpha and beta subunits of LH hormone were cloned between NheI and BamHI cut sites of pEGFP_N1 expression plasmid and confirmed by PCR. Hormone expression was evaluated in CHO cell line by Western blotting using the specific antibody
Conclusion: Alpha and beta subunits of LH hormone were expressed in CHO cell line perfectly
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Background and Aims: Some nanoparticles can be used in immunoassays to increase sensitivity. This study aimed to evaluate a novel nano-immunoassay based on bovine serum albumin nanoparticles [BSA NPs]
Materials and methods: At first, the nanostructure was synthesized, and then applied as a tag in the nano-immunoassay. Then the concentration of and beta;-subunit of human chorionic gonadotropin [and beta;HCG] in the clinical samples was quantified by traditional enzyme-linked immunosorbent assay [ELISA], and then checked by the nano-immunoassay
Results: The Pearson's correlation coefficient between ELISA and nano-immunoassay was high, i.e., 0.80. Relative sensitivity and specificity of this nano-immunoassay were reported 97.4% and 96.6%, respectively
Conclusions: BSA NPs can be applied in nano-immunoassays as a new structure, and as an example, and beta;HCG can be detected by this novel assay
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Background and Aims: although metal and metal oxide nanoparticles are used in different medical applications, they may have considerable toxicity on various cells, such as myocytes. Therefore, this study aimed to evaluate the toxicity of the naked and serum-treated silver nanoparticles [Ag NPs] and magnesium oxide nanoparticles [MgO NPs] on the cardiomyocytes
Materials and Methods: cardiomyocytes were separately exposed to different concentrations of the naked and serum-treated nanoparticles for 24 hours at 37degreeC. Then, MTT assay, cell metabolism assay and LDH assay were performed
Results: naked Ag NPs and MgO NPs had more toxicity than serum-treated nanoparticles. The highest cardiomyocyte toxicity was observed for naked Ag NPs, whereas the minimum toxicity was seen for the serum-treated MgO NPs
Conclusions: coating of nanoparticles with serum components leads to decrease in toxicity for cardiomyocytes, and MgO NPs have less toxicity on the myocytes than Ag NPs
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Extraction of blood genomic DNA is one of the main approaches for clinical and molecular biology studies. Although several methods have been developed for extraction of blood genomic DNA, most of these methods consume long time and use expensive chemicals such as proteinase K and toxic organic solvent such as phenol and chloroform. The objective of this study was to developed easy and safe method for DNA extraction from clotted and frozen whole blood. This method has many advantages: time reducing, using inexpensive materials, without phenol and chloroform, achieving of high molecular weight and good quality genomic DNA. DNA extraction was performed by two methods [new and phenol-chloroform method]. Then quantity and quality parameters were evaluated by 1% agarose gel electrophoresis, Nano drop analysis and efficiency of Polymerase Chain Reaction [PCR]. Extracted DNA from 500?L of blood samples were 457.7ng/microl and 212ng/microL and their purity [OD260/OD280] were 1.8 and 1.81 for new recommended and phenol-chloroform methods respectively. The PCR results indicated that D16S539 and CSF1PO loci were amplified. These results shown that this method is simple, fast, safe and most economical
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Interleukin [IL]-27 is a heterodimeric cytokine belonging to IL-12 and IL-23 families, secreted by antigen presenting cells [APCs]. The IL-27 is composed of 2 subunits: Epstein-Barr virus induced gene 3 [EBI3] and p28. IL-27 is an anti-inflammatory cytokine which has an inhibitory effect on Th17 population and suppress the IL-17 expression. It is suggested that IL-27 could be a potent drug candidate for treating auto immune diseases. The EBI3 and p28 subunits of human interleukin 27 were constructed into plasmid vectors; they sub-cloned into pETDuet-1 expression vector in restriction sites of BamHI, SacI and NotI. Subsequently the induction was carried out by 1mM IPTG and the recombinant proteins were confirmed by SDS-PAGE and Western Blot analysis using anti His-tag antibody. The EBI3 and p28 subunits of human interleukin 27were cloned into plasmid vectors. The 28 and 25kDa protein bands were observed on SDS-PAGE and finally confirmed by Western blot technique. In this research p28 and EBI3 proteins were produced successfully
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Infections are one of the correctable causes of infertility with low cost and cost effective treatment. The 50% of infertile cases is related to men in some way, and 30% of them are absolutely related to them. Mycoplasmas are the smallest microorganisms with capability of DNA replication. Present study is planned to compare the mycoplasma infection in infertile men and men with established fertility. 45 Semen samples were collected from case and control persons who referred to Royan Infertility and Fertility Institute between 2004 and 2005 and stored in -20 degree°C until time of test. DNA was extracted from semen using phenol chloroform. PCR reaction was done by mycoplasma specific primers. Mycoplasma genitalium gene was amplified in 6 [40%] cases from 15 infertile semen samples and 11 [36.6%] from 30 control semen samples. Probability of genital infection, at least, in these studies group, is very lower than other communities' reports
Subject(s)
Humans , Male , Infertility, Male , Fertility , SpermatozoaABSTRACT
Background: The homologous recombination [HR] is one of the conventional cloning methods for the production of recombinant DNA. It is a quick method for in vivo DNA cloning without using the high cost restriction enzymes. A few modifications in the cloning procedure can increase the efficiency of this method.
Materials and Methods: In this study, effect of heating on the rate of the IgG1 heavy chain gene cloning was investigated in the HR method and then it was compared with HR method without heating and traditional cloning method. For doing this comparison, three cloning methods including HR, HR with the heat treatment, and traditional cloning were used to clone the human IgG1 heavy chain into the pcDNA3.1[+] plasmid.
Results: The results showed that adding heat in the HR method converts insert and vector from the double strand DNA to the single strand DNA. This allows them to copulate with each other better and faster than the two other methods. The heat addition also helps the cell enzyme system for a faster and easier recombination and moreover it increases the cloning efficiency of the HR method in case of in vitro processing.
Conclusion: The results showed that giving heat in the HR method increases cloning rate 7.5% and this increase reaches 10% in comparison with the traditional method.
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Molecular farming has been considered as a secure and economical approach for production of biopharmaceuticals. Human TNF Related Apoptosis Inducing Ligand [TRAIL] as a promising biopharmaceutical candidate has been produced in different expression hosts. However, little attention has been paid to molecular farming of the TRAIL in spite of numerous advantages of plant expression systems. Therefore, in this study the cytoplasmic production of the TRAIL was tackled in Nicotiana tabacum using Agrobacterium tumefaciens LBA 4404. Initially, the desired coding sequence was obtained using PCR technique on the constructed human cDNA library. Afterward, the necessary requirements for expression of the TRAIL in plant cell system were provided through sub-cloning into 35S-CaMV [Cauliflower Mosaic Virus] helper and final 0179-pGreen expression vectors. Then, the final TRAIL-pGreen expression vector was cloned into A. tumefaciens LBA 4404. Subsequently, the N. tabacum cells were transformed through co-culture method and expression of the TRAIL was confirmed by western blot analysis. Finally, the recombinant TRAIL was extracted through chromatographic technique and biological activity was evaluated through MTT assay [Methylthiazol Tetrazolium Assay]. The result of western blot analysis indicated that only monomer and oxidized dimer forms of the TRAIL can be extracted from the N. tabacum cells. Moreover, the lack of trimeric assembly of the extracted TRAIL diminished its biological activity in sensitive A549 cell line. In conclusion, although N. tabacum cells can successfully produce the TRAIL, proper assembly and functionality of the TRAIL were unfavorable
Subject(s)
Humans , Nicotiana , Agrobacterium tumefaciensABSTRACT
Tumor necrosis factor-related apoptosis-inducing ligand [TRAIL], a member of TNF family, is an interesting ligand which selectively induces apoptosis in tumor cells and, therefore, it has been developed for cancer therapy. This ligand has been produced by various hosts such as E.coli. However, protein expression in E.coli cytoplasm leads to problems such as incorrect folding, reduction in biological activity, inclusion body formation, and sophisticated downstream. The aim of this study is to develop an expression system for the production of recombinant TRAIL secreted to the E.coli periplasm instead of cytoplasm. By using Overlapping Extension PCR, an OmpA signal sequence was fused to TRAIL cDNA and OmpA-TRAIL fragment was then cloned in pET-22b plasmid. This construct was confirmed by PCR and DNA sequencing. Promoter was induced in E.coli BL21 [DE3] and periplasmic expressed proteins were released using osmotic shock procedure. SDS-PAGE analysis showed that about 37% of recombinant TRAIL was transferred into the periplasm and its identity was confirmed by western blot analysis. Finally, the cytotoxic activity of TRAIL against HeLa cell line was confirmed by using MTT assay. The results demonstrate that our expression system may be useful for the production of TRAIL in the periplasmic space
Subject(s)
Periplasmic Proteins , Periplasm , Escherichia coliABSTRACT
The aim of this study was to detect the genotype of Fasciola spp. in Meshkin-Shahr, Ardabil Province, northwestern Iran in different hosts using PCR-RFLP. The parasite hosts included cattle, and sheep. Overall, 70 adult flukes from livers of slaughtered animals were collected from the abattoirs of aforementioned area. The included 35 samples from infected sheep and 35 samples from 35 infected cattle. PCR-RFLP and sequence analysis of the first nuclear ribosomal internal transcribed spacer [ITS 1] region from Fasciola species were used to conduct the study. The fragment of approximately 700bp in all of the Fasciola samples was amplified. PCR products of ITS 1 were subjected for digestion by restriction enzyme. RsaI restriction enzyme was selected for RFLP method that caused the separation specifically of Fasciola species. Amplicons with the sequences of F. hepatica had a pattern of about 360, 100, and 60 bp band size, whereas F. gigantica worms had a profile of 360, 170, and 60 bp in size, respectively. Results based on PCR-RFLP analysis were confirmed by sequence analysis of representative ITS 1 amplicons. No hybrid forms were detected in the present study. All sheep were infected with F. hepatica but cattle were infected with both species. Both species of Fasciola are present in Ardabil. The method described here can be valuable for identification of Fasciola species in endemic parts for fasciolosis, regions with intermediate species and in that overlapping distribution area
Subject(s)
Animals , Genotype , Sheep , Cattle , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , FascioliasisABSTRACT
Cryptosporidiosis is one of the most important parasitic infections in Iran which causes diarrhea in humans and animals. The identification of the Cryptosporidium species among humans is necessary. This study aims to identify species of Cryptosporidium isolated from patients that referred to three hospitals in Tehran based on the 18s rRNA gene by nested PCR-RFLP assay. In the first step of the present descriptive cross-sectional study, 1128 human fecal samples were collected from patients that referred to three hospitals [Ali Asgar, Mofid and Imam Khomeini] in Tehran. The samples were examined for Cryptosporidium by modified acid fast staining. In the second step, DNA of the positive samples were extracted, then gene of 18s rRNA was amplified by nested PCR in order to differentiate between species. The PCR products were subsequently digested by Vsp1 restriction enzyme and their sequences determined. The modified acid fast method detected 12 [1.06%] positive samples which was confirmed by a molecular technique. The 845bp fragment of 18s rRNA was digested by restriction enzymes. There were 10 samples identified as Cryptosporidium parvum that showed similar patterns on 2.5% agarose gel; 2 other samples were identified as Cryptosporidium homonis and Cryptosporidium andersoni based on the different patterns and sequence results. Although Cryptosporidium parvum is introduced as the major agent for cryptosporidiosis in humans, Cryptosporidium hominis and Cryptosporidium andersoni may also infect humans