Optimized Refolding Buffers Oriented Humoral Immune Responses Versus PfGCS1 Self-Assembled Peptide Nanoparticle.

Developing a novel class of vaccine is pivotal for eliminating and eradicating malaria. Preceding investigations demonstrated partial blocking activity in malaria transmission against recombinant vaccine PfHAP2-GCS1 and conserved region of the cd loop. The effectiveness of immune response varies with the size and shape of the self-assembly of peptide nanoparticles (SAPNs) displaying antigen, affected by different components in refolding buffers. Plasmodium falciparum Generative Cell Specific 1 (PfGCS1), a promising malaria transmission-blocking vaccine (TBV) candidate, was expressed, purified, and followed by a four-step refolding process to form nanoparticles (PfGCS1-SAPNs). The influence of buffer components on the size and shape of SAPNs was investigated by DLS and FESEM. Furthermore, the immunogenicity of nanostructures was assessed in different mouse groups. The results showed that PfGCS1-SAPN was immunogenic and its administration with Poly (I:C), stimulated humoral and cellular responses in the mouse model. In the immunized mice groups, the level of IgG antibodies against PfGCS1-SAPN was significantly increased in different time points (second and third boost) and heterogeneous boosters. The various IgG-subclasses profile shifted to Th1, Th2, or Th1/Th2 mix responses in mice immunized with PfGCS1-SAPN refolded in different buffers, indicating a prerequisite for further investigations to optimize vaccine formulation to enhance and modulate Th1/cellular responses. Such studies pave the way to improve biophysical features related to the nanoparticles' size, shape, and conformational epitopes of candidate antigens and T- and B-cells presented on the superficial structure to elicit robust immune responses.

Comparative Analysis of Leishmania major Nucleoside Hydrolases Toward Selecting Multi-target Strategy.

PURPOSE: Leishmania causes multiple types of leishmaniasis in different parts of the world. It has a lack of metabolic machine to produce purine bases. Therefore, the parasite produces purine bases through the breakdown of nutritional nucleotides and it makes the nucleoside hydrolases (NHs) good drug targets. They have different substrate-preferring (SP) types. Our objectives were modeling and comparative analysis of these protein structures for Leishmania major. METHOD: In this work, available sequences for all SP types of L. major NH enzymes including inosine-uridine preferring NH (IUNH), inosine-guanosine preferring NH (IGNH), and inosine-adenosine-guanosine preferring NH (IAGNH) were used to make 24 structural models via SWISS-MODEL and LOMETS. After evaluating the structural models, three enzyme structures were finalized and used to analyze substrate-binding pockets. RESULTS: The three SP types of L. major NH enzymes that can breakdown purine nucleosides were highly different in terms of sequence, structure, and profile of interacting residues within the substrate-binding pockets. In this study, new enzyme structures have been presented for three SP types and they have been compared in different aspects and it indicated that they were very different from each other. CONCLUSION: Although, previously indicated that from these three SP types in genera other than Leishmania, the role of IGNH and IAGNH was greater than IUNH in supplying purine bases, till this work, just IUNH has been structurally studied and used in drug-design investigations for Leishmania. Therefore, we are offering to use all three SP types of NHs as multi-target strategy in anti-leishmaniosis drug-design studies.

In vitro and in silico characterization of a novel glutamate carboxypeptidase from Cohnella sp. A01.

Glutamate carboxypeptidase is a bacterial enzyme of metallopeptidase superfamily. This enzyme is an exo-peptidase that catalyzes the hydrolysis of glutamate residues at the C-terminus of folic acid. The rCP302 is a novel zinc ion-dependent recombinant glutamate carboxypeptidase derived from a thermophilic bacterium, Cohnella sp. A01 (PTCC No: 1921). By simulating the structure of rCP302, analyzing its activity in various environmental settings, and contrasting it with that of related enzymes, we wanted to evaluate the heterologous production, purification, and characterization of this enzyme. The bioinformatics study showed that rCP302 had maximum similarity to M20 family of metallopeptidases. The purified rCP302 molecular weight was about 41.6 kDa. The optimum temperature and pH for the catalytic activity of rCP302 were 50 °C and 7.2, respectively. Fluorescence spectroscopy data elucidated the secondary structure of rCP302 and determined conformational changes caused by alterations in ambient conditions. Using folate as a substrate, Km and specific activity values were calculated as 0.108 µM and 687 µmol/min/mg, respectively. The enzyme activity was strongly inhibited when EDTA sequestered zinc ions. The half-life of this enzyme at 30 °C was 2012 min. Regarding the ability of rCP302 to degrade folic acid, and its long half-life at 37 °C, the normal temperature of many mammals, this enzyme can be introduced for further study for use in the pharmaceutical industry.

Increased macrophage phagocytic activity with TLR9 agonist conjugation of an anti- Borrelia burgdorferi monoclonal antibody.

Borrelia burgdorferi (Bb) infection causes Lyme disease, for which there is need for more effective therapies. Here, we sequenced the antibody repertoire of plasmablasts in Bb-infected humans. We expressed recombinant monoclonal antibodies (mAbs) representing the identified plasmablast clonal families, and identified their binding specificities. Our recombinant anti-Bb mAbs exhibit a range of activity in mediating macrophage phagocytosis of Bb. To determine if we could increase the macrophage phagocytosis-promoting activity of our anti-Bb mAbs, we generated a TLR9-agonist CpG-oligo-conjugated anti-BmpA mAb. We demonstrated that our CpG-conjugated anti-BmpA mAb exhibited increased peak Bb phagocytosis at 12-24 h, and sustained macrophage phagocytosis over 60+ hrs. Further, our CpG-conjugated anti-BmpA mAb induced macrophages to exhibit a sustained activation morphology. Our findings demonstrate the potential for TLR9-agonist CpG-oligo conjugates to enhance mAb-mediated clearance of Bb, and this approach might also enhance the activity of other anti-microbial mAbs.

PPD in HBsAg vaccine formulation suppressed IFN-γ and IL-4 cytokine responses and induced long-lived humoral immune responses: Results from 220-day monitoring of specific IgG responses.

Objectives: Here, immune responses and long-lived IgG responses of HBsAg-Alum, HBsAg-MF59, as well as HBsAg-MF59 were compared when formulated with PPD. Materials and Methods: BALB/c mice were vaccinated subcutaneously three times with a two-week -interval. Then, specific IgG, long-lived IgG responses up to 220 days, and IgG1/IgG2a isotypes, and IFN-γ and IL-4 on spleen cell culture supernatant were assessed using ELISA. Results: IFN-γ cytokine response between MF59- and Alum-adjuvanted vaccines did not show a significant difference. HBsAg-Alum revealed an increase in IL-4 cytokine versus HBsAg-MF59 at borderline (P=0.0553). In addition, HBsAg-MF59+PPD 10 µg showed a significant decrease in IL-4 and IFN-γ cytokines versus HBsAg-MF59. Furthermore, HBsAg-MF59+PPD10 µg showed a significant increase in the IL-2/IL-4 ratio versus HBsAg-MF59 (P=0.0339). Specific IgG antibody showed a significant increase in HBsAg-MF59, as compared with HBsAg-Alum. Furthermore, HBsAg-MF59 plus PPD showed a significant increase in IgG responses versus HBsAg-MF59 and HBsAg-Alum groups. Long-lived IgG responses showed a significant increase in HBsAgMF59 versus HBsAg-Alum group and PPD in the HBsAg-MF59 vaccine formulation, resulting in a significant increase in IgG responses versus HBsAg-MF59 group. In addition, HBsAg-MF59 plus PPD suppressed IgG1 response versus HBsAg-Alum. However, HBsAg-MF59 showed a significant increase in IgG2α versus the HBsAg-Alum group (P=0.0190). Immunization with HBsAg-MF59+PPD (10 µg) showed a significant increase versus the HBsAg-MF59 group (P=0.0040). IgG2a/IgG1 ratio in HBsAg-MF59+PPD1µg and HBsAg-MF59+PPD10 µg groups showed a significant increase versus HBsAg-MF59 groups (P<0.0345). Conclusion: PPD leads to a more potent long-lived IgG responses in the HBsAg vaccine, highlighting its potential as a component of a complex adjuvant.

The effects of different thermal and chemical stresses on release of outer membrane vesicles (OMVs) by ClearColi™.

The highly immunogenic properties of outer membrane vesicles (OMVs), small spherical nanoparticles commonly released by Gram-negative bacteria, led to their application as vaccine candidate. ClearColi™ is an engineered Escherichia coli strain, which does not produce endotoxic response in humans and is useful for production of OMV-based vaccines. Therefore, producing ClearColi™ OMVs with high yield attracts particular interest. As stresses can be removed by OMVs, they may affect OMVs release. We aimed to investigate the effects of culture temperature, chemical (NaCl, ethanol, EDTA, D-cycloserine, polymyxin B, 1-octanol, and H2O2) and thermal stresses on release of ClearColi™ OMVs. Herein, the growth rate of ClearColi™ was decreased in the presence of all chemical stresses with the exception of H2O2. The optimum temperature for OMVs production was 37 â„ƒ and their release was not increased under thermal shock. The highest and lowest OMVs release was obtained in the presence of NaCl and H2O2, respectively. Electron microscopy images confirmed that the bilayer spherical-shaped OMVs were isolated under different stresses. Furthermore, SEM and DLS analysis demonstrated that OMVs released under EDTA stress are smaller than those released from untreated cultures. It can be concluded that chemical stresses have influence on the level of ClearColi™ OMVs production. However, changes in their content should be further investigated.

Decrescent role of recombinant HSP60 antibody against atherosclerosis in high-cholesterol diet immunized rabbits.

Objectives: Atherosclerosis is the main cause of cardiovascular disease (CVD) which has a key role in the development of coronary artery disease (CAD). Based on clinical studies, HSP60 is the only HSP that can cause atherosclerosis. In this paper, the expression level of HSP60 and the pathogenic degree of its cloned part was investigated in atherosclerosis condition. Materials and Methods: After the designation of the specific primers for HSP60, PCR was done by the Pfu enzyme. Subsequently, the PCR products were cloned into a prokaryotic expression vector pET-28a. The resultant recombinant vector was transferred in BL21 and purified. Purification of protein was done by the Nickel affinity column. After confirmation of Western blotting and HSP60 protein purification, purified protein concentration was measured by the Bradford method, and purity was analyzed by SDS PAGE 12%. New Zealand rabbits were tested as an animal model. At the next step, the recombinant protein was injected into the animal model that was on a fatty diet. Results: The prokaryotic expression plasmid pET28a-hps60 was successfully constructed, the HSP60 protein was expressed and purified in Escherichia coli BL21 (DE3). We found that the rabbit that was receiving the recombinant vaccine with the fatty diet showed a lower amount of fat deposition at the media endothelial level than the rabbit which received only the fatty diet. Conclusion: Taking recombinant protein concomitant with a fatty diet, causes betterment of atherosclerosis via decreasing aggregation of cholesterol and thickness of the endothelial media.

A review of the berberine natural polysaccharide nanostructures as potential anticancer and antibacterial agents.

Despite the promising medicinal properties, berberine (BBR), due to its relatively poor solubility in plasma, low bio-stability and limited bioavailability is not used broadly in clinical stages. Due to these drawbacks, drug delivery systems (DDSs) based on nanoscale natural polysaccharides, are applied to address these concerns. Natural polymers are biodegradable, non-immunogenic, biocompatible, and non-toxic agents that are capable of trapping large amounts of hydrophobic compounds in relatively small volumes. The use of nanoscale natural polysaccharide improves the stability and pharmacokinetics of the small molecules and, consequently, increases the therapeutic effects and reduces the side effects of the small molecules. Therefore, this paper presents an overview of the different methods used for increasing the BBR solubility and bioavailability. Afterwards, the pharmacodynamic and pharmacokinetic of BBR nanostructures were discussed followed by the introduction of natural polysaccharides of plant (cyclodextrines, glucomannan), the shells of crustaceans (chitosan), and the cell wall of brown marine algae (alginate)-based origins used to improve the dissolution rate of poorly soluble BBR and their anticancer and antibacterial properties. Finally, the anticancer and antibacterial mechanisms of free BBR and BBR nanostructures were surveyed. In conclusion, this review may pave the way for providing some useful data in the development of BBR-based platforms for clinical applications.

Design of a chitosan-based nano vaccine against epsilon toxin of Clostridium perfringens type D and evaluation of its immunogenicity in BALB/c mice.

BACKGROUND AND PURPOSE: Clostridium perfringens is an anaerobic, spore-forming, and pathogenic bacterium that causes intestinal diseases in humans and animals. In these cases, therapeutic intervention is challenging; because the disease progresses much rapidly. This bacterium can produce 5 main toxins (alpha, beta, epsilon, iota, and a type of enterotoxin) among which the epsilon toxin (ETX) is used for bioterrorism. This toxin can be prevented by immunization with specific immunogenic vaccines. In the present research, we aimed at developing a recombinant chitosan-based nano-vaccine against ETX of C. perfringens and evaluate its effects on the antibody titration against epsilon toxin in BALB/c mice as the vaccine model. EXPERIMENTAL APPROACH: The etx gene from C. perfringens type D was cloned and expressed in E. coli. After analysis by SDS-PAGE and western blotting, the expressed products were purified, and the obtained proteins were used for immunization in mice as a chitosan nanoparticle containing recombinant, purified ETX, and protein. FINDINGS/RESULTS: The results of ELISA showed that IgA antibody serum level increased sufficiently using recombinant protein with nanoparticle as an oral and injectable formulation. IgG antibody titers increased significantly after administrating the recombinant proteins with nanoparticles through both oral delivery and intravenous injection. CONCLUSION AND IMPLICATION: In conclusion, the recombinant ETX is suggested as a good candidate for vaccine production against diseases caused by ETX of C. perfringens type D.

Docosahexaenoic acid reverses the promoting effects of breast tumor cell-derived exosomes on endothelial cell migration and angiogenesis.

AIM: As a natural compound, docosahexaenoic acid (DHA) exerts anti-cancer and anti-angiogenesis functions through exosomes; however, little is known about the molecular mechanisms. MAIN METHODS: Breast cancer (BC) cells were treated with DHA (50 µM) and then tumor cell-derived exosomes (TDEs) were collected and characterized by electron microscopy, dynamic light scattering, and western blot analyses. By the time the cells were treated with DHA, RT-qPCR was used to investigate the expression of vascular endothelial growth factor (VEGF) and the selected pro- and anti-angiogenic microRNAs (miRNAs). The quantification of secreted VEGF protein was measured by enzyme-linked immunosorbent assay (ELISA). The effects of TDEs on endothelial cell angiogenesis were explored by transwell cell migration and in vitro vascular tube formation assays. KEY FINDINGS: DHA treatment caused a significant and time-dependent decrease in the expression and secretion of VEGF in/from BC cells. This also increased expression of anti-angiogenic miRNAs (i.e. miR-34a, miR-125b, miR-221, and miR-222) while decreased levels of pro-angiogenic miRNAs (i.e. miR-9, miR-17-5p, miR-19a, miR-126, miR-130a, miR-132, miR-296, and miR-378) in exosomes derived from DHA-treated BC cells, TDE (DHA+). While treatment with exosomes (100 µg/ml) obtained from untreated BC cells, TDE (DHA-), enhanced the expression of VEGF-A in human umbilical vein endothelial cells (HUVECs), incubation with DHA or TDE (DHA+) led to the significant decrease of VEGF-A transcript level in these cells. We indicated that the incubation with TDE (DHA+) could significantly decrease endothelial cell proliferation and migration and also the length and number of tubes made by HUVECs in comparison with endothelial cells incubated with exosomes obtained from untreated BC cells. SIGNIFICANCE: DHA alters angiogenesis by shifting the up-regulation of exosomal miRNA contents from pro-angiogenic to anti-angiogenic, resulting in the inhibition of endothelial cell angiogenesis. These data can help to figure out DHA's anti-cancer function, maybe its use in cancer therapy.

Albumin binding, anticancer and antibacterial properties of synthesized zero valent iron nanoparticles.

BACKGROUND: Nanoparticles (NPs) have been emerging as potential players in modern medicine with clinical applications ranging from therapeutic purposes to antimicrobial agents. However, before applications in medical agents, some in vitro studies should be done to explore their biological responses. AIM: In this study, protein binding, anticancer and antibacterial activates of zero valent iron (ZVFe) were explored. MATERIALS AND METHODS: ZVFe nanoparticles were synthesized and fully characterized by X-ray diffraction, field-emission scanning electron microscope, and dynamic light scattering analyses. Afterward, the interaction of ZVFe NPs with human serum albumin (HSA) was examined using a range of techniques including intrinsic fluorescence, circular dichroism, and UV-visible spectroscopic methods. Molecular docking study was run to determine the kind of interaction between ZVFe NPs and HSA. The anticancer influence of ZVFe NPs on SH-SY5Y was examined by MTT and flow cytometry analysis, whereas human white blood cells were used as the control cell. Also, the antibacterial effect of ZVFe NPs was examined on Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25922), and Staphylococcus aureus (ATCC 25923). RESULTS: X-ray diffraction, transmission electron microscope, and dynamic light scattering analyses verified the synthesis of ZVFe NPs in a nanosized diameter. Fluorescence spectroscopy analysis showed that ZVFe NPs spontaneously formed a complex with HSA through hydrogen bonds and van der Waals interactions. Also, circular dichroism spectroscopy study revealed that ZVFe NPs did not change the secondary structure of HSA. Moreover, UV-visible data presented that melting temperature (Tm) of HSA in the absence and presence of ZVFe NPs was almost identical. Molecular dynamic study also showed that ZVFe NP came into contact with polar residues on the surface of HSA molecule. Cellular assays showed that ZVFe NPs can induce cell mortality in a dose-dependent manner against SH-SY5Y cells, whereas these NPs did not trigger significant cell mortality against normal white bloods in the concentration range studied (1-100 µg/mL). Antibacterial assays showed a noteworthy inhibition on both bacterial strains. CONCLUSION: In conclusion, it was revealed that ZVFe NPs did not induce a substantial influence on the structure of protein and cytotoxicity against normal cell, whereas they derived significant anticancer and antibacterial effects.

Investigation of indolamine 2, 3 dioxygenase (IDO-1) gene expression by real-time PCR among patients with lung cancer.

INTRODUCTION: The aim of this study was to evaluate the expression of IDO-1 gene and cancerous grades of non-small cell lung cancer (NSCLC) and its subclasses among patients with lung cancer using real-time polymerase chain reaction (PCR). MATERIALS AND METHODS: A total of 35 clinical samples were collected from patients with NSCLC. To evaluate the IDO-1 gene after the extraction of RNA and complementary DNA (cDNA) synthesis using real-time PCR, the expression of the gene was investigated. The western blot analysis method was used for protein expression. RESULTS: The highest grade, IIIa grade included six patients (17.1%). Approximately 74% of adenocarcinoma cases were in T-categories of lung cancer and 25% of patients in IIIa grade. Patients in the IIA and IIB categories belong to the SCC subclass. Results showed that the expression of INDO 5.22 fold gene was more common in patients with lung cancer than NSCLC. Protein expression in western blot analysis in patients compared with normal 3.22 fold change increased. CONCLUSION: The evidence shows that IDO-1 is a key parameter that inhibits antitumor immune responses in humans. This study has added interesting data to the IDO community for analyzing the expression of cancerous human cancer cells and cancer tissue in humans. The results showed that IDO-1 not only participates in the process of escape from tumor immunity but can also contribute to the safety of the pretumor area. A wide variety of observed IDO-1 expression values ​​among patients may present serious barriers to the clinical performance of anti- IDO strategies at present.

Titanium oxide nanoparticles fabrication, hemoglobin interaction, white blood cells cytotoxicity, and antibacterial studies.

This study is focused on the fabrication and characterization of titanium oxide (TiO2) NPs. Afterwards; the interaction of TiO2 NPs with human hemoglobin (Hb) was investigated by FTIR spectroscopy, fluorescence spectroscopy, and molecular docking studies. Also, the cytotoxic effect of fabricated TiO2 NPs against human white blood cells (WBCs) was considered by MTT assay. The antibacterial effect of synthesized NPs was examined on Pseudomonas aeruginosa (ATCC 27853); Escherichia coli (ATCC 25922) and Staphylococcus aureus (ATCC 25923). TEM and DLS investigations showed that the synthesized TiO2 NPs have a narrow nano-sized distribution. XRD pattern of the fabricated NPs exhibited that the TiO2 NPs contain anatase phase. Similarity in amide I and II signal intensities showed that secondary structure of the adsorbed Hb is preserved. The intrinsic fluorescence study revealed that the fluorescence quenching of Hb was done by complex formation between Hb and TiO2 NPs trough the hydrogen bond and van der Waals interactions. Synchronous fluorescence spectroscopy determined that interaction of TiO2 NPs with Hb did not unfold the Hb structure in the vicinity of the Tyr and Trp residues. Molecular docking study depicted that Glu-95, Thr-134 and Tyr-140 are involved in the formation of hydrophilic bonds. MTT data and antibacterial assays indicated that TiO2 NPs endow distinguished antibacterial activities against Gram-negative and Gram positive strains at safe concentrations. This study may reveal that fabricated TiO2 NP can be used as a safe and potent antibacterial agent. Communicated by Ramaswamy H. Sarma.

A spectroscopic study on the absorption of carbonic anhydrase onto the nanoporous silica nanoparticle.

Herein, KIT-6 nanoporous silica nanoparticles were used as a solid support for immobilization of bovine carbonic anhydrase, isoform II (BCA II). The zeta potential study revealed that KIT-6 and BCA II provided negative (-13.58±1.95mV) and positive (4.23±0.72mV) charge distribution, respectively. Dynamic light scattering (DLS) analysis also showed that the hydrodynamic radius of KIT-6 is less than 100nm. In addition, the structural studies of free and immobilized BCA II against urea-induced denaturation were investigated by circular dichroism (CD) and fluorescence spectroscopy. CD studies showed that the absorbed BCA II, in comparison with the free enzyme, demonstrated higher stability against rising urea concentration. Fluorescence spectroscopy showed lower values of Stern- Volmer constant (KSV) for immobilized BCA II relative to free enzyme, reflecting the relative enzyme stability of BCA II after immobilization. Melting temperature (Tm) measurement of free and immobilized BCA II showed that immobilized enzyme had a more stable structure (Tm=71.9°C) relative to the free counterpart (Tm=64.7°C). In addition, the immobilized BCA II showed pronounced stability against pH and thermal deactivation. This study may provide new and complementary details regarding the design and development of enzymes in industrial applications.