increase in protein and plasmid yields of E. coli with optimized concentration of ampicillin as selection marker

Background: Escherichia coli is still the common host for ing and heterologous protein expression. Various strategies have been employed to increase protein expression in E. coli, but, it seems that external factors such as selection marker concentration can drastically affect the yield of protein and plasmid

Objectives: Alterations of protein expression and plasmid yields of E. coli in different concentrations of ampicillin, as selection marker, will be determined. In order to improve heterologous expression, the system will be redesigned and optimized

Materials and Methods: The expression cassette of codon optimized EGFP for E. coli was synthesized in pUC57. The pUC57-GFP was transformed into E. coli Top10F'. The expression of GFP was verified by SDS-PAGE and flow cytometry after induction by IPTG [0.5 mM] and incubation with 0, 100, 200 and 300 micro g.mL[-1] ampicillin. Plasmid copy numbers of samples were determined by Real-Time PCR on AMP gene using regression line of diluted standard curve

Results: GFP expressing clones formed fair green colonies on LB agar supplemented with 0.5 mM IPTG and showed fluorescence in FL1 filter of flow cytometry and an extra protein band on SDS-PAGE gel. The fluorescent intensity of GFP in 0, 100, 200 and 300 micro g.mL[-1] ampicillin in medium were 549.83, 549.78, 1443.52, 684.87, and plasmid copy numbers were 6.07x10[9], 3.21x10[9], 2.32x10[10] , 8.11x10[8], respectively. The plasmid yields were 55 ng.micro L[-1], 69 ng.micro L[-1], 164 ng.micro L[-1] and 41 ng.micro L[-1], respectively

Conclusion: Protein and plasmid yields of E. coli are variable in different concentrations of ampicillin and need to be optimized in newly designed expression systems. Protein and plasmid yield in the optimized concentration [200 micro g.mL[-1]] was significantly [p < 0.01] higher than other doses

Real-time PCR: an appropriate approach to confirm ssDNA generation from PCR product in SELEX process

Background: Aptamers are single stranded DNA [ssDNA] or RNA molecules. The potential of aptamers for binding to the different targets has made them be widely used as the preferred diagnostic and therapeutic tools. DNA aptamers present several advantages over the RNA oligonucleotides due to their higher stability, easier selection, and production. Selection of DNA aptamers which is facilitated through a systematic evolution of ligand by exponential enrichment [SELEX] method is much dependent on the successful conversion of double stranded DNA [dsDNA] to ssDNA

Objective: There are different methods available for ssDNA generation. While visualization of ssDNA is limited to the gelbased method, the method is not applicable in the initial rounds of SELEX due to more than 1015 different sequences. This study was designed to evaluate the efficiency of another technique for confirming the ssDNA generation in comparison to the polyacrylamide electrophoresis [PAGE] analysis

Materials and Methods: Real-time PCR was employed in the present study for PCR amplification of the initial library that was followed by enzymatic digestion of the dsDNA. Subsequently melting curve analysis was carried out to evaluate ssDNA generation from dsDNA. Moreover, PAGE analysis was performed and the results were compared with the melt curve analysis

Results: The melt curves, revealed dsDNA conversion to the ssDNA based on a significant reduction of Tm from 73.8 to 41.5 [degree]C. Applying PAGE analysis, it was not effectively feasible to show ssDNA generation from the corresponding initial dsDNA library, while, it was efficient enough to confirm ssDNA generation in accordance with the increasing the number of SELEX rounds

Conclusion: The present study has proven the applicability of the real-time PCR as a suitable confirmatory technique for validating ssDNA generation in the DNA aptamer selection process for the initial library preparation

Gene expression analysis of VEGF and its receptors and assessment of its serum level in unexplained recurrent spontaneous abortion

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

Altered Th17/Treg ratio in recurrent miscarriage after treatment with paternal lymphocytes and vitamin D3: a double-blind placebo-controlled study

Background: Recurrent miscarriage [RM] affects 2-5% of pregnant women. Paternal lymphocyte immunotherapy is a common treatment for RM patients but the outcome has not been consistent. Therefore, combined therapy with other immunosuppressive drugs such as 1a, 25-dihydroxy-vitamin-D3 [vitamin D3] may improve the outcome

Objectives: To investigate the effect of vitamin D3 on the balance of two essential T cells subsets, T helper [Th] 17 and T regulatory [Treg] cells, which regulate tolerance

Methods: The expression levels of CD4 and forkhead box protein 3 [FOXP3] in Treg cells, and the expression levels of CD4 and IL-17 in Th17 cells, were evaluated pre- and 3 months post-immunotherapy in RM patients treated with a combination of paternal lymphocytes and vitamin D3 compared with RM patients receiving lymphocyte immunotherapy alone

Results: Vitamin D3 therapy decreased the frequency of Th17 cells in addition to reducing the Th17/Treg ratio in peripheral blood of RM patients compared with the control group [p<0.05]

Conclusion: Considering that RM patients have a higher Th17/Treg ratio in peripheral blood, vitamin D3 may be a candidate therapeutic approach in this disease

Innate lymphoid cells and cytokines of the novel subtypes of helper T cells in asthma

BACKGROUND: In this study, the expression of interleukin-9 (IL-9), IL-17, IL-22, and IL-25 genes that might be the potential predisposing factors for asthma as well as count of innate lymphoid cells (ILCs) as another source of inflammatory cytokines have been evaluated. OBJECTIVE: The aim of this study was to evaluate the expression of newly identified helper T cells signature cytokines and amount of ILCs. METHODS: Blood and sputum samples from 23 patients with moderate to severe asthma and 23 healthy volunteers were collected. The types of allergens to which our patients were sensitive were defined using immunoblotting method. Gene expression of studied cytokines was evaluated using quantitative transcription-polymerase chain reaction and ILCs were counted by the flow cytometry method. RESULTS: In this research, the gene expressions of IL-9, IL-17, IL-22, and IL-25 were significantly higher in asthmatics, especially in the severe form of the disease. This increase was even higher in serum samples compared with sputum samples. Counting ILCs revealed their increase in comparison with normal people. CONCLUSION: We showed the importance of IL-25, IL-22, IL-17, and IL-9 cytokines in patients with asthma as their expression levels are increased and these increase are correlated with the severity of the disease. We also showed that the increased amount of ILCs in asthmatics could confirm their potential role in the immunopathogenesis of asthma as another source of inflammatory cytokines.

Variations in T-helper 17 and regulatory T cells during the menstrual cycle in peripheral blood of women with recurrent spontaneous abortion

Disorders in immune system regulation may result in pregnancy abnormalities such as recurrent spontaneous abortion [RSA]. This study aims to determine the ratio of regulatory T [Treg] and T helper [Th] 17 cells in unexplained RSA [URSA] women during proliferative and secretory phases of their menstrual cycles compared to healthy non-pregnant women. In this case control study, 25 women with URSA and 35 healthy, non-pregnant women were enrolled. The percentage of Th17 and Treg cells in participants peripheral blood were determined by flow cytometry. The percentage of Th17 cells and their related cytokines in serum [IL-17A] were higher in the proliferative and secretory phases of the menstrual cycles of URSA women compared to the control women. However, a lower percentage of Treg cells and their related cytokines in serum, transforming growth factor [TGF] beta1 and interleukin [IL]-10 were detected in the proliferative but not the secretory phase of the URSA group. The ratio of Th17/CD4+ Treg was higher in the URSA group than the control group. We observed an increased ratio of Th17/CD4+ Treg during the proliferative and secretory phases in URSA women. The imbalance between Th17 and Treg cells during the proliferative phase of menstrual cycles in the URSA group may be considered a cause for spontaneous abortion

Positive and negative regulation of Th17 cell differentiation: evaluating the impact of RORC2

Th17 cells are known to be involved in some types of inflammations and autoimmune disorders. RORC2 is the key transcription factor coordinating Th17 cell differentiation. Thus, blocking RORC2 may be useful in suppressing Th17-dependent inflammatory processes. The aim was to silence RORC2 by specific siRNAs in naive T cells differentiating to Th17. Time-dependent expression of RORC2 as well as IL-17 and IL-23R were considered before and after RORC2 silencing. In this experimental study, naive CD4+ T cells were isolated from human cord blood samples. Cytokines TGFbeta plus IL-6 and IL-23 were used to polarize the naive T cells to Th17 cells in X-VIVO 15 serum free medium. A mixture of three siRNAs specific for RORC2 was applied for blocking its expression. RORC2, IL-17 and IL-23R mRNA and protein levels were measured using qRT-PCR, ELISA and flow cytometry techniques. Pearson correlation and one-way ANOVA were used for statistical analyses. Significant correlations were obtained in time-dependent analysis of IL-17 and IL-23R expression in relation with RORC2 [R=0.87 and 0.89 respectively, p<0.05]. Silencing of RORC2 was accompanied with almost complete suppression of IL-17 [99.3%; p<0.05] and significant decrease in IL-23R gene expression [77.2%, p<0.05]. Our results showed that RORC2 is the main and the primary trigger for upregulation of IL-17 and IL-23R genes in human Th17 cell differentiation. Moreover, we show that day 3 could be considered as the key day in the Th17 differentiation process

Inhibitory activity of Avicennia marina, a medicinal plant in Persian folk medicine, against HIV and HSV

Avicennia marina [Avicenniaceae] is a species of mangrove tree used for treatment of small pox lesions in Persian folk medicine. The antiviral activity of methanol, ethanol, water, chloroform and n-hexane extracts was evaluated against HIV-1 and HSV. Methanol extract had the highest antiviral activity and the most polar fraction of this extract [fraction D] inhibited HSV with TI and SI values of 57.1 and 133; however, it showed mild activity against HIV with SI value of 6.25 [fraction 3]. The anti-HSV activity of active fraction was confirmed using FLASH-PCR. Phytochemical investigation revealed that fraction D encompasses flavonoids compounds. The time-of-addition study demonstrated that fraction D disturbs viral replication after penetrating to the cell. A. marina was endowed with fragments by which found to be able to inhibit replication of HSV after entry but did not show significant potency against HIV-1. This promotes further investigation in anti-HSV drug discovery

RORC2 gene silencing in human Th17 cells by siRNA: design and evaluation of highly efficient siRNA

RNA interference-based gene silencing has recently been applied as an efficient tool for functional gene analysis. RORC2 is the key transcription factor orchestrating Th17 cells differentiation, the cells that are known as the pathogenic elements in various autoimmune diseases. The aim of this study was to design efficient siRNAs specific for RORC2 and to evaluate different criteria affecting their functionality. Three siRNA duplexes specific for RORC2 mRNA were designed. Th17 cells were produced from IL-6 and IL-1 treated cord blood CD4[+] T cells. The T cells were transfected with three different designed siRNAs against RORC2 and the expression of RORC2 gene was measured using quantitative real time PCR. Different levels of RORC2 down regulation were observed in the presence of each of the designed siRNAs. Efficient siRNA with 91.1% silencing activity met the majority of the established bioinformatics criteria while the one with 46.6% silencing activity had more deviations from these criteria. The more bioinformatics criteria are considered, the more functionality were observed for silencing RORC2. However, the importance of the type of criteria per se should not be neglected. Although all recommended criteria are important for designing siRNA but their value is not the same

CD4[+] Foxp3[+] Treg and its ICOS[+] subsets in patients with myocardial infarction

Atherosclerosis is a multifactorial disorder with chronic inflammatory conditions in which immune cells play a significant role in its pathogenic process. Regulatory T cells [Treg], as a part of immune system, are involved in controlling autoimmune and inflammatory diseases. Quantitative and/or functional alteration of Tregs has been shown to play an atheroprotective role and may also promote plaque stabilization. To assess if inducible costimulatory molecule [ICOS] expression on one subtype of Treg cells with high suppressive potential correlates with the pathogenesis of atherosclerosis. Patients with myocardial infarction [MI] and/or stable angina [SA], diagnosed as atherosclerosis by angiography, and a group of individuals with normal coronary angiography [NCA] were recruited for the present study. Peripheral blood mononuclear cells [PBMCs] were prepared and the expression of ICOS, Foxp3 and CD4 molecules was tested by flowcytometry. The percentage of CD4[+] Foxp3[+] Treg cells was reduced in MI group compared to NCA and SA groups [p<0.005]. Evaluation of the two Treg subsets according to ICOS expression showed a decreased ICOS[+]/ICOS[-] Treg ratio in MI and SA groups compared to NCA individuals [p=0.002 and p=0.048, respectivly]. The present data indicate that Tregs and its ICOS[+] subsets are decreased in patients with MI or SA, suggesting a potential role for Treg in atherosclerosis progression or onset of acute coronary syndrome

effects of isoproterenol and propranolol on cytokine profile secretion by cultured tumor-infiltrating lymphocytes derived from colorectal cancer patients

Anti-tumor immunity and cytokine profiles have important roles in the development of cancer. Norepinephrine [NE] release due to sympathetic activation leads to a Th2 deviation via the beta-2 adrenergic receptor [beta -2AR] and could increase cancer progression. This study intends to determine the effects of isoproterenol [ISO; betaagonist] and propranolol [PRO; beta-antagonist] on the production of IFN-gamma, IL-4, and IL-17. Cytokine levels have been examined in tumor-infiltrating lymphocytes [TILs] and peripheral blood mononuclear cells [PBMCs] of patients with colorectal cancer [CRC]. The beta-2AR expression on lymphocyte subsets was also assessed. In this experimental study, TILs were isolated from fresh CRC tissue and patient PBMCs were obtained just prior to surgery. The cells were cultured in medium for 72 hours. Concomitantly, cells were stimulated with 10 micro g/ ml phytohemagglutinin [PHA] alone or in the presence of either 1 micro mol/L of PRO or 1 micro mol/L ISO. The concentration of cytokines in the supernatants was measured by ELISA. Three-color flow cytometry was used to determine the expression of beta-2AR on the lymphocyte subsets. Statistical analyses were performed via paired or independent t-test. Levels of IFN-gamma, IL-4 and IL-17 were elevated after PHA-stimulation of PBMCs and TILs. However, the elevation of IFN-gamma and IL-17 production by TILs in response to PHA was significantly lower than PBMCs. In the presence of ISO, the IFN-gamma/IL-4 ratio reduced in all groups, but this reduction was very low in TILs. Interestingly, the effects of PRO on cytokine production were, at least partially, comparable to those of ISO. Depressed levels of beta-2AR expression were demonstrated on CD4+IFN-gamma+ and CD4+IL-17+ lymphocytes in patients' PBMCs and TILs. This study has demonstrated the effects of ISO and PRO on cytokine production by TILs and determined beta-2AR expression on these cells. ISO failed to induce a shift toward the expected Th2 cytokine profile in CRC patients' TILs, which might be due to the downregulation of beta-2AR expression on TILs. Additionally, in this study, PRO induced a shift to a Th2 profile in PBMCs

Anti-streptokinase antibody detection before and immediately after streptokinase therapy in patients with myocardial infarction

Myocardial infarction [MI] is one of the most common and serious diseases resulting from coronary artery occlusion and major reduction in blood flow. Streptokinase as a thrombolytic is considered the first and most important therapeutic intervention for reperfusion following MI in most countries including Iran. Our previous study showed that, the prevalence of high antibody titers against streptokinase was 13.5% in the studied population from Iran, which was 2.5 times more common than data from other studies. To evaluate anti-streptokinase antibody titers before and immediately after streptokinase administration and its relation to reperfusion therapy success rates. A total of 200 patients with acute MI was selected. Antibodies against streptokinase were measured before and 2 days after administration of streptokinase. Before streptokinase administration and every hour after streptokinase administration, for 3 consecutive hours, an ECG was taken from each participant and changes were evaluated in relation to antibody levels. Out of 200 patients, 42 [21%] had high levels of antibody titer. Out of these 42 patients, 13 [6.5%] still had measurable levels of anti-streptokinase antibody after streptokinase administration. Our results show the ability of the antistreptokinase antibody to neutralize the effects of streptokinase