Latent toxoplasmosis impairs learning and memory yet strengthens short-term and long-term hippocampal synaptic plasticity at perforant pathway-dentate gyrus, and Schaffer collatterals-CA1 synapses.

Investigating long-term potentiation (LTP) in disease models provides essential mechanistic insight into synaptic dysfunction and relevant behavioral changes in many neuropsychiatric and neurological diseases. Toxoplasma (T) gondii is an intracellular parasite causing bizarre changes in host's mind including losing inherent fear of life-threatening situations. We examined hippocampal-dependent behavior as well as in vivo short- and long-term synaptic plasticity (STP and LTP) in rats with latent toxoplasmosis. Rats were infected by T. gondii cysts. Existence of REP-529 genomic sequence of the parasite in the brain was detected by RT-qPCR. Four and eight weeks after infection, spatial, and inhibitory memories of rats were assessed by Morris water maze and shuttle box tests, respectively. Eight weeks after infection, STP was assessed in dentate gyrus (DG) and CA1 by double pulse stimulation of perforant pathway and Shaffer collaterals, respectively. High frequency stimulation (HFS) was applied to induce LTP in entorhinal cortex-DG (400 Hz), and CA3-CA1 (200 Hz) synapses. T. gondii infection retarded spatial learning and memory performance at eight weeks post-infection period, whereas inhibitory memory was not changed. Unlike uninfected rats that normally showed paired-pulse depression, the infected rats developed paired-pulse facilitation, indicating an inhibitory synaptic network disruption. T. gondii-infected rats displayed strengthened LTP of both CA1-pyramidal and DG-granule cell population spikes. These data indicate that T. gondii disrupts inhibition/excitation balance and causes bizarre changes to the post-synaptic neuronal excitability, which may ultimately contribute to the abnormal behavior of the infected host.

Rational Design and Production of Bioactive Analogs of Recombinant Human Keratinocyte Growth Factor (rhKGF) with Reduced Aggregation Propensity.

Recombinant human keratinocyte growth factor (rhKGF) is a highly aggregation-prone therapeutic protein. The present study aimed to reduce aggregation propensity of rhKGF by engineering the aggregation hotspots. Initially, 21 mutants were designed based on the previously-identified aggregation-prone regions (APRs) and then four of them including mutants No. 4 (L91K, I119K), 7 (V13S, L91K), 14 (L91D, I119D), and 21 (A51E) were selected based on molecular dynamics (MD) simulations for further experimental studies. The recombinantly produced rhKGF and mutants were analyzed regarding secondary structure, thermal stability, aggregation propensity, and biological activity. Far-UV CD spectroscopy showed that the mutants have similar secondary structure with rhKGF. A51E mutant showed enhanced stability and decreased monomer loss under heat stress suggesting its reduced aggregation propensity compared to rhKGF. Mutant No. 14 showed higher stability and less aggregation tendency than mutant No. 4 indicating that only mutations decreasing pI of rhKGF are effective in reducing its aggregation tendency. All of the mutants were at least as potent as rhKGF in stimulating proliferation of MCF-7 epithelial cells. Our results identified A51E as an equally potent, more stable, and less aggregation-prone analog of rhKGF which could be a promising alternative drug candidate for the commercially available rhKGF (Palifermin).

Enhancement of keratinocyte growth factor potential in inducing adipose-derived stem cells differentiation into keratinocytes by collagen-targeting.

Different growth factors can regulate stem cell differentiation. We used keratinocyte growth factor (KGF) to direct adipose-derived stem cells (ASCs) differentiation into keratinocytes. To enhance KGF bioavailability, we targeted KGF for collagen by fusing it to collagen-binding domain from Vibrio mimicus metalloprotease (vibrioCBD-KGF). KGF and vibrioCBD-KGF were expressed in Escherichia coli and purified to homogeneity. Both proteins displayed comparable activities in stimulating proliferation of HEK-293 and MCF-7 cells. vibrioCBD-KGF demonstrated enhanced collagen-binding affinity in immunofluorescence and ELISA. KGF and vibrioCBD-KGF at different concentrations (2, 10, and 20 ng/ml) were applied for 21 days on ASCs cultured on collagen-coated plates. Keratinocyte differentiation was assessed based on morphological changes, the expression of keratinocyte markers (Keratin-10 and Involucrin), and stem cell markers (Collagen-I and Vimentin) by real-time PCR or immunofluorescence. Our results indicated that the expression of keratinocyte markers was substantially increased at all concentrations of vibrioCBD-KGF, while it was observed for KGF only at 20 ng/ml. Immunofluorescence staining approved this finding. Moreover, down-regulation of Collagen-I, an indicator of differentiation commitment, was more significant in samples treated with vibrioCBD-KGF. The present study showed that vibrioCBD-KGF is more potent in inducing the ASCs differentiation into keratinocytes compared to KGF. Our results have important implications for effective skin regeneration using collagen-based biomaterials.

Quality by Design in Downstream Process Development of Romiplostim

Background: Background: Downstream processing of therapeutic recombinant proteins expressed as the inclusion bodies (IBs) in E. coli is quite challenging. This study aimed to use the quality by design approach for developing the multi-step downstream process of a structurally complex therapeutic Fc-Peptide fusion protein, romiplostim. Methods: Methods: For development of a successful downstream process, risk analysis and experimental designs were used to characterize the most critical quality attributes (CQAs) and effects of process parameters on these quality attributes. Results: Results: The solubilization of IBs was optimized by design of experiment on three parameters with a focus on solubility yield, which resulted in >75% increase of the target protein solubilization. The pH of sample was identified as CQA in anion exchange chromatography that might have an impact on achieving >85% host cell proteins removal and >90% host cell DNA reduction. In the refolding step, process parameters were screened. Cystine/cysteine ratio, pH, and incubation time identified as CPPs were further optimized using Box-Behnken analysis, which >85% of the target protein was refolded. The design space for further purification step by HIC was mapped with a focus on high molecular weight impurities. After polishing by gel filtration, the final product's biological activity showed no statistically significant differences among the groups received romiplostim and Nplate®, as the reference product. Conclusions: Conclusion: This research presents a precise and exhaustive model for mapping the design space in order to describe and anticipate the link between the yield and quality of romiplostim and its downstream process parameters.

Co-delivery of PLGA nanoparticles loaded with rSAG1 antigen and TLR ligands: An efficient vaccine against chronic toxoplasmosis.

Although vaccination is a promising approach for the control of toxoplasmosis, there is currently no commercially available human vaccine. Adjuvants such as delivery vehicles and immunomodulators are critical components of vaccine formulations. In this study, Poly (D, l-lactide-co-glycolide) (PLGA) nanoparticles were applied to serve as delivery system for both surface antigen-1 (SAG1), a candidate vaccine against toxoplasmosis and two TLR ligands, monophosphoryl lipid A (MPL) and imiquimod (IMQ), respectively. Compared to rSAG1 alone, CBA/J mice immunized with rSAG1-PLGA produced higher anti-SAG1 IgG antibodies titers. This response was increased by the co-administration of IMQ-PLGA (p < 0.01). Compared to IMQ-PLGA co-administration, MPL-PLGA co-administration further increased the humoral response (p < 0.01) and potentiated the Th1 humoral response. Compared to rSAG1 alone, rSAG1-PLGA, or rSAG1-PLGA mixed with IMQ-PLGA or MPL-PLGA similarly enhanced the cellular response characterized by the production of IFN-γ, IL-2, TNF-α and low levels of IL-5, indicating a Th1-biased immunity. The induced immune responses, led to significant brain cyst reductions (p < 0.01) after oral challenge with T. gondii cysts in mice immunized with either rSAG1-PLGA, rSAG1-PLGA + IMQ-PLGA, rSAG1-PLGA + MPL-PLGA formulations. Taken together the results indicated that PLGA nanoparticles could serve as a platform for dual-delivery of antigens and immunomodulators to provide efficacious vaccines against toxoplasmosis.

Valproic Acid Inhibits Chronic Toxoplasma Infection and Associated Brain Inflammation in Mice.

Individuals infected with Toxoplasma gondii are prone to psychobehavioral disorders, most notably schizophrenia and bipolar disorder. Valproic acid reportedly inhibits the proliferation of T. gondii tachyzoites in vitro. However, animals treated with the drug neither lived longer during acute infection nor had fewer brain cysts upon chronic infection. In this study, a quantitative real-time PCR (qPCR) method was applied to quantify copy numbers of BAG1 (a bradyzoite-specific protein), REP529 DNA (a repetitive DNA fragment of the parasite), and SAG1 (a highly expressed tachyzoite-specific surface protein) in the brains of chronically infected mice treated with valproic acid. The treatment inhibited the infection and decreased BAG1, SAG1, and REP529 copy numbers in mice brains (P < 0.0001), comparable to the effects of trimethoprim-sulfamethoxazole (TMP-SMZ), the common medication for toxoplasmosis treatment. Moreover, valproic acid decreased brain tumor necrosis factor alpha (TNF-α) expression (P < 0.0001) comparably to TMP-SMZ. Histological examination of mouse brains showed marked reductions in cyst establishment, perivascular infiltration of lymphocytes, and glial nodules to the same levels as those in the TMP-SMZ group. Our results provide direct evidence for the efficacy of valproic acid, a mood-stabilizing and antipsychotic drug, against chronic Toxoplasma infection. These results might help modulate therapeutic regimens for neuropsychiatric patients and aid in the design of more effective anti-Toxoplasma drugs.

The Role of Recombinant Fibroblast Growth Factor 1 in Enhancing the Angiogenesis in Random Cutaneous Flaps in Animal Model of Rat.

BACKGROUND: Randomized skin flaps have been used as a basic treatment modality for covering skin defects for a long time but they have always been in the risk of an inherent ischemia. Fibroblast growth factor 1 is a known angiogenic factor in in vitro studies which has shown conflicting results in in vivo investigation. We aimed to determine the effect of recombinant fibroblast growth factor on the angiogenesis rate of random cutaneous flap in animal model of rats. METHODS: This experimental study was conducted on 24 adult male rats randomized to 2 groups. In the first group FGF1 was injected subdermally in equally divided doses and distances of random flap surface in days 1, 3 and 5. In second group, normal saline was injected as control. Flap surgery was done on day 21 after first injection. The extent of necrosis and angiogenesis (mean vessel density) were assessed in day 14 after surgery. RESULTS: The mean percentage of clinically apparent necrosis was 35.2% (±10.5) in intervention (FGF1) group and 38.1% (±8.7) in control (normal saline), respectively. Mean vessel density was 86.20±5.6/mm2 in control group and 90.17±5.5/mm2 in intervention group, which showed no statistically significant difference. CONCLUSION: Mean vessel density and mean percentage of clinically apparent necrosis area were similar in 2 groups of rats with random cutaneous flaps receiving FGF1 or normal saline.

Recombinant pebulin protein, a type 2 ribosome-inactivating protein isolated from dwarf elder (Sambucus ebulus L.) shows anticancer and antifungal activities in vitro.

In this study, encoding sequence of a new type 2 RIP (pebulin) was isolated and cloned from dwarf elder (Sambucus ebulus L.) native to the northern regions of Iran. The nucleotide sequence of pebulin was ligated to the pET-28a(+) expression plasmid and cloned into the E. coli strain BL21 (DE3) in order to express heterologously of recombinant protein. The recombinant pebulin protein was mainly produced in the form of insoluble inclusion bodies probably because to absence of N-glycosylation process in E. coli. Therefore, in order to increase the expression of recombinant protein in soluble form, co-expression of the target protein with the pG-Tf2 chaperone plasmid and incubation of bacterial culture under low temperature were used to enhance solubility and accumulation of recombinant protein. After purification of the recombinant protein using affinity chromatography method, the bioactivity of pebulin was analyzed by hemagglutination, anticancer, and antifungal assays. The results of the hemagglutination assay showed that purified pebulin agglutinated erythrocytes in all human blood groups. In addition, pebulin considerably inhibited the proliferation of cancer cell lines MCF-7 and HT-29 in a time- and dose-dependent manner and indicated remarkably growth-inhibiting effect against the plant pathogenic fungi such as Alternaria solani and Fusarium oxysporum.

Development of a Quantitative Real-Time PCR Assay for Detection of Toxoplasma gondii in Brain Samples.

BACKGROUND: Toxoplasmosis is a worldwide-distributed infection that can cause serious diseases, mainly in congenitally infected and immunodeficient individuals. PCR assays play an indispensable role in the detection of Toxoplasma gondii in different biological samples. METHODS: This study was conducted in the Parasitology Department at Pasteur Institute of Iran (Tehran) during 2016-2018. We designed a highly sensitive quantitative real-time PCR (RT-qPCR) targeted REP-529, a noncoding repetitive DNA. We cloned the amplicon in a plasmid (pTZREP-529) and used it to generate the standard curve. The Toxoplasma RT-qPCR characteristics, i.e., detection limit, specificity, linear dynamic range, linearity, intra-, and inter-assay precisions, were determined. The detection limit of the assay was one plasmid copy number (PCN) per reaction (about 0.004 T. gondii genome), and the linear dynamic range was equal to 6 logs (1× 101 to 1× 107 PCN per reaction). RESULTS: The assay showed no signal when genomic DNA of Plasmodium falciparum, Leishmania major, and Trichomonas vaginallis were used. The standard curve was drawn using dilutions of pTZREP-529 plasmid spiked with genomic DNA from a mouse brain, and test characteristics were shown unaffected. Applying the Toxoplasma RT-qPCR, we showed brain cysts were significantly decreased in mice vaccinated with GRA2 antigen of Toxoplasma formulated in Monophosphoryl Lipid A (MPL) adjuvant. CONCLUSION: We have developed a quantitative, specific, and highly sensitive PCR for detecting T. gondii in biological samples.

Isolation and characterization of nanobodies against epithelial cell adhesion molecule as novel theranostic agents for cancer therapy.

Epithelial cell adhesion molecule (EpCAM) plays an important role in tumorigenesis. Camelids produce functional antibodies composed of heavy chains only that bind to their antigens via a single domain variable fragment known as nanobody. Nanobodies show multiple advantages over traditional monoclonal antibodies. Isolation of functional anti-EpCAM nanobodies (Nbs) was the main aim of this study. An immune nanobody library containing 108 members was constructed previously. Anti -EpCAM nanobodies were isolated from camel immune library using phage display. Four consecutive rounds of biopanning were performed on immobilized EpCAM. Four nanobodies (Nb4, Nb5, Nb22, and Nb23) with highest signal intensity in monoclonal phage ELISA were selected. Affinity of these selected nanobodies for EpCAM was in the nanomolar range. Selected nanobodies significantly inhibited proliferation of MCF-7 cells. The in vivo study revealed that a significant reduction in tumor size occurred when treated with nanobodies Nb4 and Nb5, after 14 days monitoring. Our data revealed that nanobodies Nb4 and Nb5 could be considered as attractive theranostic agents for EpCAM overexpressing cancers.

In silico analysis of different signal peptides for the secretory production of recombinant human keratinocyte growth factor in Escherichia coli.

BACKGROUND: The recombinant human truncated Keratinocyte growth factor (Palifermin) is the only FDA approved medicine for the treatment of oral mucositis. The Keratinocyte growth factor is a fairly unstable protein due to its high aggregation propensity and therefore its expression as a secretory protein may results in the production of a protein with more stability, higher solubility, better folding, enhanced biological activity, N-terminal authenticity and simplified downstream processing. OBJECTIVE: The aim of this study was in silico evaluation of 31 different secretory signal peptides to determine the best theoretical candidates for the secretory production of recombinant truncated human KGF in E. coli. METHODS: Thirty different prokaryotic signal peptides experimentally shown to be capable of recombinant protein secretion in E.coli, along with the native KGF signal peptide were selected for further investigations. The signal peptide sequences were retrieved from the UniProt database. The ability of SPs to act as a secretory leader peptide for rhKGF and the location of cleavage sites were predicted by SignalP 4.1. Physicochemical properties of the signal peptides, which may influence protein secretion, were analyzed by ProtParam and PROSOII. Furthermore, the mRNA secondary structure and Gibbs free energy profile of the selected SPs were analyzed in the fusion state with the rhKGF using Visual Gene Developer package. RESULTS AND CONCLUSION: Computational analysis of the physicochemical properties affecting protein secretion identified Sec-B dependent OmpC, Bla, and StaI and SRP dependent TolB signal peptides as the best theoretical candidates for the secretory production of recombinant truncated human KGF in E.coli.

Soluble expression of IGF1 fused to DsbA in SHuffle™ T7 strain: optimization of expression and purification by Box-Behnken design.

Production of insulin-like growth factor 1 (IGF1) in Escherichia coli mostly results in the formation of inclusion bodies. In the present study, IGF1 was fused to disulfide bond oxidoreductase A (DsbA) and expressed in SHuffle™ T7 strain, in order to obtain correctly folded protein. Soluble expression and IMAC purification of DsbA-IGF1 were optimized by applying the Box-Behnken design of response surface methodology. The optimization greatly increased concentration of soluble protein from 317 to 2600 mg/L, and IMAC yield from 400 to 1900 mg/L. Results of ANOVA showed induction OD600 and temperature had significant effects on the soluble protein expression while isopropyl-ß-d thiogalactoside, in the concentrations tested, displayed no significant effect. Moreover, the three parameters of the binding buffer including, pH, concentration of NaCl, and imidazole displayed significant effects on the IMAC yield. Then, purified DsbA-IGF1 was cleaved by human rhinovirus 3C protease, and authentic IGF1 was obtained in flow through of a subtractive IMAC. Final polishing of the protein by reversed-phase HPLC yielded IGF1 with purity of 96%. The quality attributes of purified IGF1 such as purity, identity, molecular size, molecular weight, secondary structure, and biological activity were assessed and showed to be comparable to the standard IGF1. The final yield of purified IGF1 was estimated to be 120 ± 18 mg from 1 L of the culture. Our results demonstrated a simple and easily scalable strategy for production of large amounts of bioactive IGF1 by rational designing soluble protein expression, and further optimization of expression and purification methods.

A-NGR fusion protein induces apoptosis in human cancer cells.

The NGR peptide is one of the well-known peptides for targeting tumor cells. It has the ability to target aminopeptidase N (CD13) on tumor cells or the tumor vascular endothelium. In this study, the NGR peptide was used for targeting A subunit of the Shiga toxin to cancer cells. The cytotoxic effect of the A-NGR fusion protein was assessed on HT1080, U937, HT29 cancer cells and MRC-5 normal cells. For this purpose, cells were treated with different concentrations of A-NGR (0.5-40 µg/ml). The evaluation of cell viability was achieved by MTT assay. Apoptosis was determined by annexin-V/PI double staining flow cytometry. Alterations in the mRNA expression of apoptosis - related genes were assessed by real time RT- PCR. The results showed that A-NGR fusion protein effectively inhibited the growth of HT1080 and U937 cancer cells in comparison to negative control (PBS) but for CD13-negative HT-29 cancer cells, only at high concentrations of fusion protein was inhibited growth recorded. On the other hand, A-NGR had little cytotoxic effect on MRC-5 normal cells. The flow cytometry results showed that A-NGR induces apoptosis. Furthermore, the results of real time RT-PCR revealed that A-NGR significantly increases the mRNA expression of caspase 3 and caspase 9. Conclusively, A-NGR fusion protein has the ability of targeting CD13-positive cancer cells, the cytotoxic effect on CD13-positive cancer cells as well as has low cytotoxic effect on normal cells.

A Dual-Type L2 11-88 Peptide from HPV Types 16/18 Formulated in Montanide ISA 720 Induced Strong and Balanced Th1/Th2 Immune Responses, Associated with High Titers of Broad Spectrum Cross-Reactive Antibodies in Vaccinated Mice.

E. coli-derived concatenated, multitype L2-conserved epitopes of human papillomavirus (HPV) L2 protein might represent a less expensive and pan-type vaccine alternative (compared to type-specific HPV L1 virus-like particles), if stable protein expression and strong immunogenicity features could be met. Herein, three dual-type- (DT-) HPV L2 fusion peptides comprising the three head-to-tail tandem repeats (multimers) of either HPV 16 epitope "17-36" or "69-81" or one copy (monomer) of 11-88 fused to the same residues of HPV 18 were constructed and expressed in E. coli. SDS-PAGE and Western blot analyses indicated the proper expression and stability of the E. coli-derived DT peptides. Mice immunized by formulation of the purified DT peptides and Freund's adjuvant (CFA/IFA) raised neutralizing antibodies (NAbs; the highest for DT: 11-88 peptide) which showed proper cross-reactivity to HPV types: 18, 16, 31, and 45 and efficiently neutralized HPV 18/16 pseudoviruses in vitro. Immunization studies in mice by formulation of the DT: 11-88 × 1 peptide with various adjuvants (alum, MF59, and Montanides ISA 720 and 50) indicated that Montanide adjuvants elicited the highest cross-reactive titers of NAbs and similar levels of IgG1 and IgG2a (switching towards balanced Th1/Th2 responses). The results implied development of low-cost E. coli-derived DT: 11-88 peptide formulated in human compatible ISA 720 adjuvant as a HPV vaccine.

Immunization of C57BL/6 Mice with GRA2 Combined with MPL Conferred Partial Immune Protection against Toxoplasma gondii

Background: We have previously reported that immunization with GRA2 antigen of Toxoplasma gondii induces protective immunity in CBA/J (H2k) and BALB/c mice (H2d). We aimed to examine whether immunization of a distinct strain of rodent with recombinant dense granule antigens (GRA2) combined with monophosphorryl lipid A (MPL) adjuvant elicits protective immune response against T. gondii. Methods: C57BL/6 (H2b haplotype) mice were immunized with GRA2, formulated in MPL adjuvant. Results: Strong humoral response, predominantly of IgG1 subclass and cellular response, IFN-γ, was detected at three weeks post immunization. Mice immunized with GRA2 had significantly (p < 0.01) fewer brain cysts than those in the adjuvant group, upon challenge infection. Despite the production of a strong antibody response, IFN-γ production and brain cyst reduction were not significant when the immunized mice were infected four months after the immunization. Conclusion: We can conclude that GRA2 immunization partially protects against T. gondii infection in C57BL/6 mice, though the potency and longevity of this antigen as a standalone vaccine may vary in distinct genetic backgrounds. This observation further emphasizes the utility of GRA2 for incorporation into a multi-antigenic vaccine against T. gondii.

In Vitro Evaluation of Vegf-Pseudomonas Exotoxin: A Conjugated on Tumor Cells.

BACKGROUND: Angiogenesis which occurs mandatory in solid tumors, is a critical step in malignancy progression. Vascular endothelial growth factor (VEGF) is mainly responsible for angiogenesis process and facilitates the formation of new vessels. Distribution of monoclonal antibodies against VEGF or VEGF receptor (VEGFR) into the solid tumors is limited because of their huge dimensions. Moreover, many investigations have demonstrated the usefulness of immunotoxins to halt angiogenesis in solid tumors. MATERIALS AND METHODS: We designed, expressed and evaluated the cytotoxicity of a novel nano-immunotoxin composed of VEGF splice variant containing 121 amino acids (VEGF121) and truncated the exotoxin A of Pseudomonas aeruginosa (PE38-KDEL). The fusion protein VEGF121-PE38 was successfully cloned and expressed in Escherichia coli, purified by Ni+ 2 affinity chromatography. The fusion protein was subsequently subjected to refolding using the reduced and oxidized glutathione. RESULTS: The expression level of the fusion protein reached to 1 mg/ml. The VEGF121-PE38 immunotoxin showed a 59 KDa MW which had cytotoxic effect on HUVEC and 293/KDR cells as low and high expressing VEGFR2 cells, respectively. But the cytotoxicity on 293/KDR was 100 folds more than that of VEGFR2 low expressing cell HUVEC. CONCLUSION: The designed immunotoxin showed more selectivity for higher VEGFR2 expressing cells in vitro.

Immunization of mice by a multimeric L2-based linear epitope (17-36) from HPV type 16/18 induced cross reactive neutralizing antibodies.

Current licensed and commercially available prophylactic human papillomavirus (HPV) vaccines (Cervarix and quadrivalent/nine valents Gardasil) are based on major capsid protein L1 virus-like particles (VLPs) production which are expensive and type specific. Minor capsid L2-RG1 linear epitope (17-36) is a known candidate for induction of cross-neutralizing antibodies to develop low-cost pan-HPV vaccines. Herein, we report construction and expression of a three tandem repeats of L2-RG1 derived from HPV16 and 18 fused with the same head to tail pattern (HPV16:17-36×3+ HPV18:17-36×3; hereafter termed dual-type fusion L2 peptide) in E. coli and provide the results of its immunogenicity in mice. SDS-PAGE and western blot analyses indicated proper expression of the peptide that could be further purified by Ni-NTA affinity chromatography via the located C-terminal 6xHis-tag. Mice immunized by formulation of the purified peptide and Freund adjuvant raised neutralizing antibodies which showed proper cross reactivity to HPV L2 (11-200) of types: 18, 16, 31 and 45 (which totally are responsible for 90% of cervical cancers) and efficiently neutralized HPV18/16 pseudoviruses in vitro. Our results imply the possibility of development of a simple, low-cost preventive HPV vaccine based on this dual-type fusion L2 peptide in bacterial expression system with broad spectrum.

SHuffle™ T7 strain is capable of producing high amount of recombinant human fibroblast growth factor-1 (rhFGF-1) with proper physicochemical and biological properties.

BACKGROUND: Human fibroblast growth factor-1 (FGF-1) has powerful mitogenic activities in a variety of cell types and plays significant roles in many physiological processes e.g. angiogenesis and wound healing. There is increasing demand for large scale production of recombinant human FGF-1 (rhFGF-1), in order to investigate the potential medical use. In the present study, we explored SHuffle™ T7 strain for production of rhFGF-1. METHODS: A synthetic gene encoding Met-140 amino acid form of human FGF-1 was utilized for expression of the protein in three different E. coli hosts (BL21 (DE3), Rosetta-gami™ 2(DE3), SHuffle™ T7). Total expressions and soluble/insoluble expression ratios of rhFGF-1 in different hosts were analyzed and compared. Soluble rhFGF-1 produced in SHuffle™ T7 cells was purified using one-step heparin-Sepharose affinity chromatography and characterized by a variety of methods for physicochemical and biological properties. RESULTS: The highest level of rhFGF-1 expression and maximum soluble/insoluble ratio were achieved in SHuffle™ T7 strain. Using a single-step heparin-Sepharose chromatography, about 1500mg of purified rhFGF-1 was obtained from one liter of the culture, representing purification yield of ∼70%. The purified protein was reactive toward anti-FGF-1 ployclonal antibody in immunoblotting. Mass spectrometry confirmed the protein had expected amino acid sequence and molecular weight. In reverse-phase high-performance liquid chromatography (RP-HPLC), the protein displayed the same retention time with the human FGF-1 standard, and purity of 94%. Less than 0.3% of the purified protein was comprised of oligomers and/or aggregates as judged by high-performance size-exclusion chromatography (HP-SEC). Secondary and tertiary structures of the protein, investigated by circular dichroism and intrinsic fluorescence spectroscopy methods, respectively, represented native folding of the protein. The purified rhFGF-1 was bioactive and stimulated proliferation of NIH 3T3 cells with EC50 of 0.84ng/mL. CONCLUSION: Although SHuffle™ T7 has been introduced for production of disulfide-bonded proteins in cytoplasm, we herein successfully recruited it for high yield production of soluble and bioactive rhFGF-1, a protein with 3 free cysteine and no disulfide bond. To our knowledge, this is the highest-level of rhFGF-1 expression in E. coli reported so far. Extensive physicochemical and biological analysis showed the protein had similar characteristic to authentic FGF-1.

Toxoplasmosis accelerates acquisition of epilepsy in rats undergoing chemical kindling.

Epilepsy is one of the most common neurologic disorders worldwide with no distinguishable cause in 60% of patients. One-third of the world population has been infected with Toxoplasma gondii. This intracellular parasite has high tropism for excitable cells including neurons. We assessed impact of acute and chronic T. gondii infection on epileptogenesis in pentylenetetrazole (PTZ) kindling model in male rats. T. gondii cysts were administered to rats by intraperitoneal (i.p.) injection. The presence of T. gondii cysts in the brain of rats was verified by hematoxylin-eosin staining. One and eight weeks after cysts injection, as acute and chronic phases of infection, PTZ (30mg/kg, i.p.) was injected to the rats every other day until manifestation of generalized seizures. Histologic findings confirmed cerebral toxoplasmosis in rats. The rats with acute or chronic Toxoplasma infection became kindled by lower number of PTZ injections (14.8±1 and 13.6±1 injections, respectively) compared to corresponding uninfected rats (18.7±1 and 16.9±1 injections, p<0.05). Toxoplasma infection increased the rate of kindling in rats. The chronically-infected rats achieved focal and also generalized seizures earlier than the rats with acute infection. Toxoplasmosis might be considered as a risk factor for acquisition of epilepsy.

Seroepidemiological study of Toxoplasma gondii infection in a population of Iranian epileptic patients.

Epilepsy is one of the most common neurologic disorders. Underlying cause of epilepsy is unknown in 60 % of the patients. Toxoplasma gondii is an intracellular parasite which is capable of forming tissue cysts in brain of chronically infected hosts including humans. Some epidemiological studies suggested an association between toxoplasmosis and acquisition of epilepsy. In this study we determined seroprevalence of latent Toxoplasma infection in a population of Iranian epileptic patients. Participants were classified in three groups as Iranian epileptic patients (IEP, n = 414), non-epileptic patients who had other neurologic disorders (NEP, n = 150), and healthy people without any neurologic disorders (HP, n = 63). The presence of anti-Toxoplasma IgG antibodies and IgG titer in the sera were determined by ELISA method. Anti-T. gondii IgG seroprevalence obtained 35.3 %, 34.7 % and 38.1 % in IEP, NEP and HP, respectively. The seroprevalence rate was not significantly different among the three groups (P = 0.88). Anti-T. gondii IgG titer was 55.7 ± 78, 52.4 ± 74 and 69.7 ± 92 IU/ml in IEP, NEP and HP, respectively. There was not any statistically significant difference in the antibody titer between the study groups (P = 0.32). The rate of T. gondii infection in epileptic patients was not higher than non-epileptic patients and healthy people in the Iranian population.