ORIGINAL_ARTICLE Truncated Core/NS3 Fusion Protein of HCV Adjuvanted with Outer Membrane Vesicles of Neisseria meningitidis Serogroup B: Potent Inducer of the Murine Immune System Background: A licensed vaccine against hepatitis C virus (HCV) has not become available to date. The stability and antigenicity of a targeted synthesized recombinant fusion protein consisting of a truncated core and NS3 (rC/N) of HCV had been predicted. Although safe antigens, recombinant proteins are not efficacious vaccines without adjuvants. The present study evaluated the immunogenicity of rC/N as a bipartite antigen accompanied by Neisseria meningitidis serogroup B outer membrane vesicles (NMB OMVs) in BALB/c mice. Methods: The NMB OMVs were produced and evaluated accurately. The administrations were as follows: rC/N-OMV, rC/N-Freund’s complete/incomplete adjuvant (CIA), rC/N-MF59, rC/N, OMV, MF59, and PBS. The production of Th1 (IFN-γ, IL-2)/Th2 (IL-4)/Th17 (IL-17) cytokines and granzyme B (cytotoxic indicator) by splenic mononuclear cells and the humoral concentration of total IgG/IgG1 (Th2)/IgG2a (Th1) in sera of mice were measured using mouse ELISA kits. Results: Concentrations of Th1/Th2/Th17 cytokines, granzyme B, and  immunoglobulins in the spleens and sera of immunized mice, which had received antigen plus each adjuvant (rC/N-OMV, rC/N-Freund’s CIA and rC/N-MF59), significantly raised compared to the controls (rC/N, OMV, MF59 and PBS). Th1-type responses were dominant over Th2-type responses in vaccinated mice with rC/N-OMV, and Th2 type responses increased dominantly in vaccinated mice with rC/N-MF59 (p < 0.05). Conclusion: NMB OMVs were able to increase Th1 immune responses dramatically more than MF59 and Freund’s CIA. The formulation of rC/N with NMB OMVs showed its ability to induce Th1, Th2, and Th17 immune responses. rC/N-NMB OMVs is a promising approach for the development of an HCV therapeutic vaccine. http://ibj.pasteur.ac.ir/article-1-2576-en.pdf 2018-10-03 235 245 10.29252/ibj.23.4.235 Adjuvants Hepatitis C virus Immunization Vaccines Soheila Hekmat 1 Department of Hepatitis and AIDs, Pasteur Institute of Iran, Tehran, Iran AUTHOR https://orcid.org/0000-0001-9092-2567 Seyed Mehdi Sadat 2 Department of Hepatitis and AIDs, Pasteur Institute of Iran, Tehran, Iran AUTHOR https://orcid.org/0000-0001-7739-179X Mohammad Mehdi Aslani 3 Department of Microbiology, Pasteur Institute of Iran, Tehran, Iran AUTHOR https://orcid.org/0000-0002-3013-6940 Mehdi Mahdavi 4 Recombinant Vaccine Research Center, Tehran University of Medical Sciences, Tehran, Iran AUTHOR Azam Bolhassani 5 Department of Hepatitis and AIDs, Pasteur Institute of Iran, Tehran, Iran AUTHOR https://orcid.org/0000-0001-7363-7406 Fateme Asgar Halvaee 6 Department of Hepatitis and AIDs, Pasteur Institute of Iran, Tehran, Iran AUTHOR Seyed Mohammad Mahdi Ghahari 7 Department of Hepatitis and AIDs, Pasteur Institute of Iran, Tehran, Iran AUTHOR Mohammad Reza Aghasadeghi 8 Department of Hepatitis and AIDs, Pasteur Institute of Iran, Tehran, Iran AUTHOR https://orcid.org/0000-0003-2186-0774 Seyed Davar Siadat 9 Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran AUTHOR https://orcid.org/0000-0002-6892-5603
ORIGINAL_ARTICLE Comparison of the Utility of Recombinant B8/2 Subunit of the Antigen B, Native Antigen, and a Commercial ELISA Kit in the Diagnosis of Human Cystic Echinococcosis Background: Cystic echinococcosis (CE) is a helminthic disease caused by the larval form of Echinococcus granulosus.  In the present study, the B8/2 subunit of antigen B (AgB) of E. granulosus was expressed in E. coli host and then applied in a diagnostic ELISA set up. Methods: The DNA sequence of AgB8/2 subunit from E. granulosus was extracted from the GenBank and codon-optimized. The target sequence was cloned in an expression vector (pGEX-4T-1). The produced antigen was used in an ELISA system, and its performance for the diagnosis of human hydatid cyst was evaluated, using sera from CE and non-CE patients, along with the sera from healthy subjects. Moreover, the diagnostic value of the recombinant protein was compared with native AgB, as well as with a commercial kit. Results: Antibodies to hydatid cyst were detected in 27 out of 30 patients corresponding to a sensitivity of 90% (95% CI: 73-98%). Cross-reaction with sera of non-CE subjects was seen in two cases resulted in a specificity of 93.5% (95% CI: 82-98%) for the test. A sensitivity of 87% and specificity of 90% were found for the native form of the antigen, while the ELISA commercial kit had a sensitivity of 97% and specificity of 95%. Conclusion: Our data show that rEgAgB8/2 is an appropriate source of antigen for the serological diagnosis of human hydatid cyst. Co-expression of the rEgAgB/2 along with other subunits of AgB may enhance the performances of these antigens for the serodiagnosis of human CE. http://ibj.pasteur.ac.ir/article-1-2594-en.pdf 2018-10-28 246 252 10.29252/ibj.23.4.246 Diagnosis Echinococcus granulosus Hydatid cyst Amir Savardashtaki 1 Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran AUTHOR https://orcid.org/0000-0003-1700-9426 Zohreh Mostafavi-Pour 2 Recombinant Proteins Laboratory, Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran AUTHOR https://orcid.org/0000-0002-3779-177X Farzaneh Arianfar 3 Recombinant Proteins Laboratory, Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran AUTHOR Bahador Sarkari 4 Department of Parasitology and Mycology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran AUTHOR https://orcid.org/0000-0003-2045-9057
ORIGINAL_ARTICLE Physicochemical Position-Dependent Properties in the Protein Secondary Structures Background: Establishing theories for designing arbitrary protein structures is complicated and depends on understanding the principles for protein folding, which is affected by applied features. Computer algorithms can reach high precision and stability in computationally designing enzymes and binders by applying informative features obtained from natural structures. Methods: In this study, a position-specific analysis of secondary structures (α-helix, β-strand, and tight turn) was performed to reveal novel features for protein structure prediction and protein design. Results: Our results showed that the secondary structures in the N-termini region tend to be more compact than C-termini. Decoying periodicity in length and distribution of amino acids in α-helices is deciphered using the curve-fitting methods. Compared with α-helix, β-strands do not show distinct periodicities in length. Also, significant differences in position-dependent distribution of physicochemical properties are shown in secondary structures. Conclusion: Position-specific propensities in our study underline valuable parameters that could be used by researchers in the field of structural biology, particularly protein design through site-directed mutagenesis. http://ibj.pasteur.ac.ir/article-1-2650-en.pdf 2018-12-23 253 261 10.29252/ibj.23.4.253 Algorithms Amino acids Physicochemical Protein structure Ehsan Saghapour 1 Department of Bioelectronic and Biomedical Engineering, School of Advanced Technologies in Medicine, Isfahan University of Medical Sciences, Isfahan, Iran AUTHOR https://orcid.org/0000-0002-5520-2115 Mohammadreza Sehhati 2 Department of Bioelectronic and Biomedical Engineering, School of Advanced Technologies in Medicine, Isfahan University of Medical Sciences, Isfahan, Iran AUTHOR https://orcid.org/0000-0002-7316-5645
ORIGINAL_ARTICLE Simvastatin Attenuates Hippocampal MMP-9 Expression in the Streptozotocin-Induced Cognitive Impairment Background: Matrix metalloproteinase-9 (MMP-9) expression has been implicated in molecular mechanisms of neurodegenerative disorders, and its abnormal level has been reported in Alzheimer’s disease (AD). Some protective mechanisms of statins against neurodegeneration might be mediated by the inhibition of MMP-9 expression. Here, we investigated the effect of simvastatin, on the hippocampal MMP-9 expression in the context of AD. Methods: We examined the influence of three-week simvastatin (5 mg/kg) administration on hippocampal MMP-9 expression in a rat model of cognitive decline induced by streptozotocin (STZ). Spatial long-term memory and MMP-9 expression were assessed by Morris water maze (MWM) test and quantitative polymerase chain reaction, respectively. Results: The results showed a decline in the learning and memory in STZ group when compared with the control group. The MMP-9 was up-regulated (1.41 ± 0.2 vs. 0.980 ± 0.02, p < 0.05), and cresyl violet staining showed hippocampal cell damage in STZ group compared with the control group. Simvastatin prevented the up-regulation of MMP-9 (1.05 ± 0.05 vs. 1.41 ± 0.2, p < 0.05), improved spatial memory impairment and attenuated hippocampal cell damage. Furthermore, we found a negative correlation (r = 0.77) between MMP-9 expression and cognitive function. Conclusion: Our findings suggest that the neuroprotective influence of simvastatin in battle to cognitive impairment is mediated in part by the modulation of MMP-9 expression. The reduction of MMP-9 expression in simvastatin-treated animals is in correlation with the improvement of cognitive functions. Understanding the protective mechanism of simvastatin will shed light on more efficient therapeutic modalities in AD. http://ibj.pasteur.ac.ir/article-1-2557-en.pdf 2018-09-16 262 271 10.29252/ibj.23.4.262 Alzheimer’s disease Matrix metalloproteinase-9 Simvastatin Soheila Adeli 1 Department of Neuroscience and Addiction Studies, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran AUTHOR https://orcid.org/0000-0002-3341-7270 Maryam Zahmatkesh 2 Department of Neuroscience and Addiction Studies, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran AUTHOR https://orcid.org/0000-0002-8950-893X Mitra Ansari Dezfouli 3 Department of Neuroscience and Addiction Studies, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran AUTHOR
ORIGINAL_ARTICLE Enhancement of Expression Level of Modified t-PA (TNKase) in Leishmania tarentolae by Induction System Background: The expression of bio-therapeutic proteins in mammalian cells, such as CHO, attains high homogeneity related to post-translational modifications. Although CHO remains the most popular cell line for bestselling biotherapeutic proteins on the market, there are still drawbacks such as expensive culture media, long time line, and high drug cost. Recently, researches on a novel Leishmania protozoan system have confirmed that this low level eukaryote could represent a competitive alternative to the mammalian cell lines. Methods: The full length of coding sequence of modified tPA TNKase tenecteplase) was synthesized and cloned into an inducible expression vector of L. tarentolae T7-TR cells. Results: The expression of the construct was driven by a Tet-inducible promoter. A Leishmania secretory signal sequence was also added to the expression cassette to facilitate the release of the recombinant protein into the medium. The secretory recombinant protein was analyzed and confirmed by SDS-PAGE and Western blot analyses. The expression level of TNKase in this novel system of L. tarentolae was 810 IU/mL after induction, which means that the percentage of expression increases two times compared to previous models in L. tarentolae. The TNKase activity was comparable with Activase. Conclusion: Our results suggested that expressed TNK (modified tPA) is functionally compatible with Activase regarding their effect on fibrinolysis. Given the post-translational modification similarities between mammalian and L. tarentolae, it is speculated that this system is capable of producing complex proteins such as tPA similar to mammalian system, with easier manipulation and non-expensive method. http://ibj.pasteur.ac.ir/article-1-2560-en.pdf 2018-09-17 272 279 10.29252/ibj.23.4.272 Leishmania tarentolae Tenecteplase Tissue plasminogen activator Mohammad Mehdi Attarpour Yazdi 1 Department of Microbiology, Faculty of Medicine, Shahed University, Tehran, Iran AUTHOR https://orcid.org/0000-0002-0338-4092 Nikky Tofighi 2 Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran AUTHOR Taraneh Rajaee 3 Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran AUTHOR Marzieh Ghahremanlou 4 Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran AUTHOR https://orcid.org/0000-0001-6495-5779 Ahmad Adeli 5 Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran AUTHOR Azam Bolhassani 6 Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran, Iran AUTHOR https://orcid.org/0000-0001-7363-7406 Mohammad Azizi 7 Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran AUTHOR https://orcid.org/0000-0003-1326-1755 Noushin Davoudi 8 Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran AUTHOR https://orcid.org/0000-0001-6114-4188
ORIGINAL_ARTICLE Hydroalcoholic Extract of Levisticum officinale Increases cGMP Signaling Pathway by Down-Regulating PDE5 Expression and Induction of Apoptosis in MCF-7 and MDA-MB-468 Breast Cancer Cell Lines Background: This study aimed to investigate Levisticum officinale hydroalcoholic extract (LOHE) effect on both cGMP signaling pathway and phosphodiesterase 5 (PDE5) gene expression pattern and to examine the role of LOHE in apoptosis induction of MCF-7 and MDA-MB-468 cell lines. Methods: The half maximal inhibitory concentration (IC50) of LOHE was examined in both cell lines using the MTT assay. Using IC50 values of LOHE on both cells, the type of cell death was detected by flowcytometric analysis.  The values of PDE5 and cGMP were evaluated by real-time PCR and ELISA methods, respectively. Results: The IC50 values were measured as 150 μg/ml for MDA-MB-468 and 200 μg/ml for MCF-7. At 12 hour of treatment, a significant decrease in the PDE5 expression and maximum increase in the amount of intracellular cGMP were observed (p < 0.05). However, these effects were more noticeable in MDA-MB-468 triple-negative cells. Conclusion: Our data suggest that LOHE extract could be a potential source for new strategies towards targeting both PDE5 and cGMP signaling pathways. http://ibj.pasteur.ac.ir/article-1-2604-en.pdf 2018-11-03 280 286 10.29252/ibj.23.4.280 Apoptosis Breast cancer Levisticum PDE5 Marzieh Lotfian Sargazi 1 Cellular and Molecular Research Center Zahedan University of Medical Sciences, Zahedan, Iran AUTHOR https://orcid.org/0000-0001-9825-249X Ramin Saravani 2 Cellular and Molecular Research Center Zahedan University of Medical Sciences, Zahedan, Iran AUTHOR https://orcid.org/0000-0003-1941-3617 Ali Shahraki 3 Department of Biology, Faculty of Science, University of Sistan and Baluchestan, Zahedan, Iran AUTHOR https://orcid.org/0000-0002-9455-0243
ORIGINAL_ARTICLE Pharmacokinetics and Biodistribution of Rhopalurus junceus Scorpion Venom in Tumor-Bearing Mice after Intravenous and Oral Administration Introduction: Rhopalurus junceus scorpion venom has shown potential for anticancer treatment. However, there are no scientific evidence about venom pharmacokinetic (PK) and biodistribution (BD) in tumor-bearing mice. Methods: 131I-labeled venom was administrated by intravenous (IV) and oral (PO) routes at the single dose of 12.5 mg/kg. Mice were sacrificed and blood samples, major organs, and tumor were taken at 10, 20, 40, 90, 180, 300, 480, and 1440 min. Results: For IV route, maximum peak concentration (Cmax), elimination half-lives, total body clearance (CL), distribution volume (Vd), mean residence time (MRT), and area under curve (AUC) were 21.77 ± 2.45 %Dosis·h/mL, 12.65 ± 2.1 h, 4.59 ± 0.23 mL/h, 83.80 ± 12 mL, 12.49 ± 2.71 h, and 21.77 ± 2.45 %Dosis·h/mL, respectively. For  PO route, they were 0.60 ± 0.07 %Dosis·h/mL, 9.33 ± 1.35 h, 36.94 ± 4.01 mL/h, 497.33 ± 30 mL, 12.40 ± 1.87 h, and 6.89 ± 1.18 %Dosis·h/mL, respectively. PK parameters (Cmax, CL, Vd, and AUC) showed significant differences between IV and PO routes. Bioavailability was 31.6 ± 4% for PO dose. Kidney, stomach, liver, and lung for IV and stomach, kidney, spleen, and lung for PO routes showed the major uptakes for 131I-labeled venom. In tumor tissue, after the maximum uptake for both routes, there was a consistent behavior of radioactivity respect to the major organs during the first 480 min. Conclusion: The PK and BD of R. junceus venom in mice depend on the administration route. These data represent a starting point for future experiments with this scorpion venom in experimental models of cancer. http://ibj.pasteur.ac.ir/article-1-2677-en.pdf 2019-07-15 287 296 10.29252/ibj.23.4.287 Intravenous Mice Oral administration Radioactivity Scorpion venom Alexis Díaz-García 1 Research Department, Laboratories of Biopharmaceuticals and Chemistries Productions (LABIOFAM), Havana, Cuba AUTHOR https://orcid.org/0000-0002-7784-7846 Gilmara Pimentel González 2 Radiopharmacy Laboratory, Oncology and Radiobiology National Institute, Havana, Cuba AUTHOR https://orcid.org/0000-0001-6345-5844 Tais Basaco Bernabeu 3 Radiopharmacy Laboratory, Oncology and Radiobiology National Institute, Havana, Cuba AUTHOR https://orcid.org/0000-0002-1315-5744 Juan C. Rodríguez Aurrecochea 4 Research Department, Laboratory of Experimental pathology, Oncology and Radiobiology National Institute, Havana, Cuba AUTHOR https://orcid.org/0000-0002-0708-5399 Hermis Rodríguez Sánchez 5 Microbiology Department, Cell culture Laboratory, Tropical Medicine Institute “Pedro Kourí”, Havana, Cuba AUTHOR https://orcid.org/0000-0002-7753-7055 Iraida Sánchez Monzón 6 Radiopharmacy Laboratory, Oncology and Radiobiology National Institute, Havana, Cuba AUTHOR https://orcid.org/0000-0000-0000-0000 Maikel Hernández Gómez 7 Radiopharmacy Laboratory, Oncology and Radiobiology National Institute, Havana, Cuba AUTHOR https://orcid.org/0000-0002-1891-4033 Caridad Rodríguez Torres 8 Research Department, Laboratories of Biopharmaceuticals and Chemistries Productions (LABIOFAM), Havana, Cuba AUTHOR https://orcid.org/0000-0002-1454-5904 Maria Regla Rodríguez Capote 9 Research Department, Laboratories of Biopharmaceuticals and Chemistries Productions (LABIOFAM), Havana, Cuba AUTHOR https://orcid.org/0000-0001-7322-4425 Irania Guevara Orellanes 10 Research Department, Laboratories of Biopharmaceuticals and Chemistries Productions (LABIOFAM), Havana, Cuba AUTHOR https://orcid.org/0000-0002-7994-3137
SHORT_COMMUNICATION The Inhibitory Effect of Mouse Gastric DNA on Amplification of Helicobacter pylori Genomic DNA in Quantitative PCR Background: Quantitation of Helicobacter pylori (Hp) in the gastric tissue is essential for assessment of vaccination/therapeutic regimens. Methods & Results: Here, the inhibitory effect of mouse gastric DNA (MgDNA) on amplification of Hp genomic DNA (HpDNA) was evaluated by spiking HpDNA with serial dilutions of MgDNA, which yielded concentrations of >10 ng/µl and >0.63-10 ng/µl of MgDNA, as inhibition and interference zones, respectively. Mice were then inoculated with varying doses of Hp and assessed at the inhibition-free concentration of 0.63 ng/µl. The average Hp copy numbers per microgram of gastric tissue discriminated mice having received high vs. low dose inoculums (p < 0.001). Secondly, Hp copy numbers were quantitated in immunized mice, which demonstrated significantly lower numbers, in reference to controls (p = 0.006). Conclusion: Our method, bypassing the inhibition and/or interference imposed by MgDNA, was able to quantitate gastric tissue-colonizing Hp, segregating mice inoculated with low vs. high doses of Hp, as well as those immunized from controls. http://ibj.pasteur.ac.ir/article-1-2494-en.pdf 2018-07-25 297 302 10.29252/ibj.23.4.297 Amplification Inhibition Interference Standard curve Quantitative PCR Mehdi Alikhani 1 HPGC Research Group, Medical Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran AUTHOR Ebrahim Shafaie 2 HPGC Research Group, Medical Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran AUTHOR Esmat Mirabzadeh Ardakani 3 Animal Sciences Lab, Department of Molecular Medicine, Pasteur Institute of Iran, Tehran, Iran AUTHOR https://orcid.org/0000-0003-3636-0780 Maryam Esmaeili 4 HPGC Research Group, Medical Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran AUTHOR Samaneh Saberi 5 HPGC Research Group, Medical Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran AUTHOR Mojgan Hatefi 6 HPGC Research Group, Medical Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran AUTHOR Marjan Mohammadi 7 HPGC Research Group, Medical Biotechnology Department, Pasteur Institute of Iran, Tehran, Iran AUTHOR https://orcid.org/0000-0002-5585-4519