The codon-optimization of cfaE gene and evaluating its high expression capacity and conserved immunogenicity in Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is the most common cause of children diarrhea in the world. Adhesion of ETEC to small intestine is an important virulence trait. One of the most prevalent colonization factors (CFs) in human is CFA/I fimbriae and CfaE which is the required binding factor for adhesion of ETEC to intestinal mucosa.We optimized cfaE gene codons according to codon bias of E. coli to achieve a high level of recombinant protein expression. The optimized gene was expressed in E. coli and rCFaE protein was used for mice immunization. Blocking activity of the obtained antibody was examined by microplate agglutination inhibition test. SDS-PAGE analysis indicated that the optimized sequence of cfaE produces a suitable amount of rCFaE in comparison with native gene sequence. This optimized rCFaE protein could induces strong humoral response in mice and the antibody obtained against rCFaE inhibited the adhesion of ETEC to human group A erythrocytes. It is concluded that codon optimization is a useful approach for obtaining large quantities of recombinant rCFaE protein. With regard to the results of hemagglutination inhibition test, codon optimization and increased production of recombinant protein expressed in E. coli did not affect the immunogenicity potential of CFaE.     

Enhancement of Th1 immune response by CD8α dendritic cells loaded with heat shock proteins enriched tumor extract in tumor-bearing mice

The discovery of dendritic cells (DCs) as professional antigen presenting cells has opened up new possibilities for their use in the development of tumor vaccines. We investigated the effect of the CD8α⁺ DCs loaded with heat-treated tumor lysate (HTL) as a vaccine in tumor immunotherapy. The HTL loaded CD8α⁺ DCs, TL loaded CD8α⁺ DCs and unloaded CD8α⁺ DCs were subcutaneously injected in the fibrosarcoma-bearing mice. The splenocyte proliferation and the shifting of Th1/Th2 response were measured. The results indicated a significant increase in the lymphocytes proliferation and the IFN-γ production in the test group of mouse splenocytes. According to the results, HTL loaded CD8α⁺ DCs vaccine significantly decreased tumor growth and longer survival than the other immunized animals. These findings show that anti-tumor immune response against the fibrosarcoma can be induced by HTL loaded CD8α⁺ DCs and may provide a useful therapeutic model for development of approaches to tumor treatments.     

Comparison of different solvents for extraction of polyhydroxybutyrate from Cupriavidus necator

Polyhydroxybutyrate (PHBs) have attracted much attention due to their biodegradability and biocompatibility properties. The main drawback to the commercial production of them is their high cost. The recovery of PHB from bacterial cytoplasm significantly increases total processing costs. Efficient, economical, and environment‐friendly extraction of PHB from cells is required for its industrial production. In the present study, several nonhalogenated organic solvents (ethylene carbonate, dimethyl sulfoxide, dimethyl formamide, hexane, propanol, methanol, and acetic acid) were examined for their efficacy regarding recovery at different temperatures from culture broth containing Cupriavidus necator cells. The highest recovery percentage (98.6%) and product purity (up to 98%) were seen to be those of ethylene carbonate‐assisted extraction at 150°C within 60 min of incubation time. Average molecular weight of the recovered PHB (1.3 × 10⁶) was not significantly affected by the extraction solvent and conditions. The melting point of PHB extracted using ethylene carbonate was measured to be 176.2°C with an enthalpy of fusion of 16.8% and the corresponding degree of crystallinity of 59.2%. NMR and GC analyses confirmed that the extracted biopolymer was PHB. The presented strategy can help researchers to reduce the cost to obtain the final product.     

In silico prediction of exposure amino acid sequences of outer inflammatory protein A of Helicobacter pylori for surface display on Eschierchia coli

BACKGROUND:

Outer inflammatory protein A (OipA) is an outer membrane protein of Helicobacter pylori that is involved in inducing IL-8 and intracellular signaling. In this study, we have predicted exposure amino acid sequences of OipA for insertion in permissive sites of CstH subunit of Eschierchia coli CS3 pilli for bacterial surface display.

MATERIALS AND METHODS:

Databases: National Center for Biotechnology Institute and Protein Data Bank. Servers: PHD, SABLE, GOR 4, SignalP3.0, TBBpred, PRODIV-TMHMM, TMRPres2D, CPH Models, PHYRE, GETAREA, VADAR, Pep state and pep window. Software: Swiss PDB viewer and Discovery studio.

RESULTS:

In silico prediction of exposure amino acid sequences of OipA led to detection of six sequences of amino acid, 76-87, 106-112, 170-182, 222-230, 242-258, and 278-290. These sequences inserted between amino acid sequences 66-67, 100-101, and 109-110 of CstH that were predicted by Eskandari et al. as permissive sites of CstH.

CONCLUSION:

OipA has the ability to induce IL-8 from gastric epithelial cells and some papers are mentioned that this outer membrane protein involve to attachment and intracellular signaling. Receptor of OipA and adhesion motifs on this protein is unknown. Detection of exposure motifs aids to recognition of adhesion motifs and receptor of OipA on gastric epithelial cells. In this study, we have predicted exposure amino acid sequences for insert to subunit CstH of CS3 pilli E. coli for surface display.     

Cloning, Expression, Purification and Toxicity Evaluation of Helicobacter pylori Outer Inflammatory Protein A

The Helicobacter pylori outer membrane proteins play an important role in pathogenesis; the outer inflammatory protein A (OipA) is one of these proteins which play the main role in the development of inflammation. In this study, purification of recombinant H. pylori OipA was performed by Ni–NTA affinity chromatography. Gastric carcinoma epithelial cells (AGS cell) were treated by different concentrations of recombinant OipA for various lengths of time and cell viability was evaluated by the viability assay. Statistical analysis showed that OipA had toxic effects on AGS cells in a concentration of 500 ng/ml after 24 and 48 h, and this toxic dose was 256 ng/ml after 72 h. OipA had direct toxic effects on gastric epithelial cells and the toxicity was observed to depend on time and dose of H. pylori exposure. Attachment of H. pylori to gastric epithelial cells is a key part in the pathogenesis and enables H. pylori to damage the epithelial cells with OipA.     

Mesenchymal stem cells alter the frequency and cytokine profile of natural killer cells in abortion-prone mice

Natural killer cells, which play a pivotal role in the establishment and maintenance of normal pregnancy, are the most abundant leukocytes at the fetomaternal interface that their subsets frequencies and cytokine profile are influential factors in the preservation of the decidual tolerogenic microenvironment. Any imbalance in NK cells' frequency and functions could be associated with pregnancy failure. Mesenchymal stem cells (MSCs) are shown to have immunomodulatory effects on NK cells and their cytokine profile. The purpose of this study is to evaluate the impact of MSCs therapy on the cytokine profiles and subpopulations of NK cells in a murine model of recurrent pregnancy loss. Adipose-derived MSCs were injected intraperitoneally to the abortion-prone mice on Day 4.5 of gestation. The abortion rate was determined after MSCs administration and the frequency and cytokine profiles of the different subsets of NK cells were determined using the flow cytometry. Our results showed that, in abortion-prone mice, the frequency of CD49b⁺ NK cells was significantly higher than normal pregnant mice that decreased after therapy. We also demonstrated that MSCs downregulated the production of IFN-γ and upregulated IL-4 and IL-10 production by uNK cells. These findings indicate that MSCs can decrease the infiltration of CD49b⁺ NK cells to the fetomaternal interface and modulate the cytokine profile of NK cells from inflammatory to tolerogenic profile and thereby improve the tolerogenic microenvironment at the fetomaternal interface in benefit of pregnancy maintenance.     

The new locally endemic genus Yazdana (Caryophyllaceae) and patterns of endemism highlight the high conservation priority of the poorly studied Shirkuh Mountains (central Iran)

Although mountain ranges are often recognized as global biodiversity hotspots with a high level of endemism, diversity and biogeographic connections of isolated and weakly explored mountains remain poorly understood. This is also the case for Shirkuh Mts. in central Iran. Here, Yazdana shirkuhensis gen. & spec. nov. (Caryophylleae, Caryophyllaceae) is described and illustrated from the high alpine zone of this mountain. Molecular phylogenetic analyses of nuclear and plastid DNA sequence data show that Y. shirkuhensis is related to Cyathophylla and Heterochroa (tribe Caryophylleae). The newly described genus and species accentuate Shirkuh Mts. as a center of endemism, which harbors a high number of narrowly distributed species, mostly in high elevations reaching alpine habitats. As this area is currently not protected, a conservation priority is highlighted for high elevations of Shirkuh Mts.     

Seed-coat microsculpturing and its systematic application in Isatis (Brassicaceae) and allied genera in Iran

In order to examine the systematic application of seed-coat microsculpturing in Isatis, seed surfaces of 23 species (41 populations) in four genera of tribe Isatideae were examined using scanning electron microscopy (SEM). Eight types of basic ornamentation patterns were recognized among the studied specimens. Of these, the reticulate–areolate type was the most common and was found in the genera Isatis, Pachypterygium, Samerari and Tauscheria and 15 species (e.g., I. cappodocica, I. kotschyana and I. tinctoria). The reticulate type, the second most frequent, occurred in 7 species while other types each were represented by only one or 2 species. Although different populations of a given species show similar seed-surface sculpturing in most cases, in some polymorphic species like I. cappadocica and I. kotschyana these patterns were variable among populations. To some extent the variation corresponds to infraspecific taxa for some species, but the differences are not significant enough to be useful in the delimitation of the subspecies recognized by previous workers. Moreover, seed-coat characters do not support the separation of genera Isatis, Pachypterygium, Sameraria and Tauscheria.     

Plant community responses to environmentally friendly piste management in northeast Iran

It is well‐known that pistes have adverse effects on alpine ecosystems. Previous studies urged that pistes should be installed and managed in the ways to minimize negative impacts on natural habitats. However, the impacts of this type of management on the plant communities are not widely studied. The aim of this study was to examine species composition and biodiversity changes in an environmentally friendly managed piste in northeast Iran. This piste has been established in a previously degraded alpine landscape. For the vegetation survey, we sampled 44 within and 28 off‐piste plots. Except for the piste management, other environmental factors were similar between the piste and off‐piste plots. Dominant species were determined, and variation in community composition of the two areas was visualized. Also, native species, phylogenetic, and functional Hill diversity of the two areas were compared. The results showed that there was a moderate differentiation in the species composition of the piste and off‐piste. Two palatable species (i.e., Bupleurum falcatum and Melica persica) were dominant in the piste and were not recorded in the off‐piste. The diversity calculations results showed that the species diversity of the piste was higher than that of the off‐piste. Phylogenetic diversity at the level of frequent and dominant plants showed a similar result. The piste had a higher functional diversity in terms of functional richness, and functional diversity of frequent and dominant plants. Our findings imply, after 10 years, species, phylogenetic, and functional diversity of the piste is significantly improved. Environmentally friendly piste management (EFPM) induced species composition change that led to emerging species that were absent in the off‐piste. We can conclude that EFPM led to restoration of a degraded landscape. Long‐term impacts of EFPM are still unknown, therefore, caution should be undertaken regarding the installation of new environmentally friendly pistes in other areas.