Invitro and Invivo Analysis of Human Milk Lactic Acid Bacteria Isolates for Their Anti-hypercholesterolemia Actions.

The aim of this study was to evaluate the cholesterol lowering ability of Lactic Acid Bacteria (LAB) isolated from human breast milk under in vitro and in vivo conditions. Six LAB isolates namely Lacticaseibacillus casei 1A, Lactobacillus gasseri 5A, Enterococcus faecium 2C, Limosilactobacillus fermentum 3D, Pediococcus acidilactici 1C, and Lactiplantibacillus plantarum 7A, were examined for their bile resistance, bile salt hydrolase activity, cholesterol assimilation and viability in cholesterol rich; DeMan Rogosa and Sharpe broth, simulated gastric, small and upper intestinal conditions. During in vivo experiments, two putative LAB isolates were orally gavage to BALB/c mice, fed with normal basal and cholesterol rich (HCD) diets, daily for a period of 4 weeks. Blood serum analysis including total serum cholesterol, triglycerides, high-density and low-density lipoprotein (LDL) cholesterol levels and total fecal LAB counts of the animals were determined. The isolates in study showed bile resistance and bile salt hydrolysis activity, while significant differences (P < 0.05) were seen in their cholesterol assimilation ability. L. gasseri 5A (195.67%) and L. plantarum 7A (193.78%) displayed highest cholesterol removal percentages, respectively. Animals in HCD, fed with L. gasseri 5A and L. plantarum 7A showed decreased levels of total cholesterol and LDL, compared to the control groups. In HCD group liver weight was increased, while fecal LAB counts were decreased. No changes were observed in behavior or body weight in all experimental groups. In conclusion, L. gasseri 5A and L. plantarum 7A isolated from human breast milk demonstrates significant hypocholesterolaemic actions in vitro and in vivo and might be considered a promising candidates for preventing hypercholesterolemia in man and animals.

Cloning, expression and molecular analysis of recombinant Netrin-A protein of Lucilia sericata Meigen (Diptera: Calliphoridae) larvae.

Objectives: Lucilia sericata (Diptera: Calliphoridae) is used in larval therapy for wound healing. Netrin-A is an enzyme secreted from the salivary glands of these larvae, and has a central role in neural regeneration and angiogenesis. This study aimed to produce the recombinant Netrin-A protein from Lucilia sericata larvae by the baculovirus expression vector system in the Sf9 insect cell line. Methods: The coding sequence of Netrin-A was cloned, amplified in the pTG19 vector, and then cloned in the pFastBac HTA vector. It was then transformed into DH10Bac, and the recombinant Bacmid was subsequently transfected into Sf9 cells. The recombinant Netrin-A was purified by Ni-NTA agarose. The evaluation was done using SDS-PAGE and western blot, respectively. Finally, its concentration was calculated with the Bradford assay. Results: The molecular weight of this protein was 52 kDa with 404 amino acids. The signal peptide was located between amino acids 24 and 25. The concentration of Netrin-A was calculated to be 48.8 µg/ml. It reaffirmed the characterized gene codes of Lucilia sericata Netrin-A in a previous study. Conclusions: The generation of recombinant Netrin-A could be used in larval therapy, and as a biomarker in certain diseases. The netrin-A of Lucilia sericata was unprecedentedly cloned and expressed in a eukaryotic cell line. Given that this larva is FDA-approved, and non-pathogenic, it conduces to research on the development of maggot therapy in future.

Effect of formalin percentage, incubation time and temperature on Clostridium chauvoei culture inactivation and immunogenicity.

OBJECTIVES: In order to find the optimal inactivation conditions for Clostridium chauvoei culture, different factors were investigated and the immunogenicity of inactivated cultures was studied. METHODS: C. chauvoei was cultured with different formalin percentages (0.3, 0.5 or 0.7% V/V), inactivation temperatures (37 °C or room temperature) and incubation times (one or two weeks). Sterility tests were performed and residual formaldehyde and pH were measured. Rabbits were immunized twice with inactivated cultures and sera were used for detection of immune response. RESULTS: In the one-week experiment, 0.5 and 0.7% formalin inactivated the bacteria after one week, and the percentage of 0.3 inactivated after three weeks. The residual formaldehyde at weeks 1 and 8 was not significantly different. In the two-week experiment, cultures treated with 0.3 and 0.5% formalin were inactivated after four weeks, and those with 0.7% formalin were inactivated after three weeks. Residual formaldehyde at week 8 differed significantly from that of week 1. Residual formaldehyde was affected by incubation temperature since it was lower at 37 °C than in room temperature. Also, a significant effect was observed for formalin on pH, as higher formalin contents led to lower pH values of the cultures. ELISA showed the lowest antibody titer achieved by 0.7% formalin group. Antibody titer was not different between 0.3 and 0.5% formalin. CONCLUSIONS: The best condition for inactivation of C. chauvoei was considered as one-week incubation with 0.5% formalin at 37 °C, leading to a high antibody response.

Identification, molecular characterization, and in silico structural analysis of larval salivary glands Netrin-A as a potent biomarker from Lucilia sericata (Diptera: Calliphoridae).

The greenbottle blowfly Lucilia sericata (L. sericata) is increasingly used in larval therapy of chronic wounds. Netrins as bifunctional proteins are in the superfamily of Laminins secreted from larval salivary glands. The Netrin protein has a significant instructive role in axon guidance, causing neuronal outgrowth, angiogenesis, and cell migration. It seems to be crucial in wound healing and acts as a potential biomarker in diagnosing some clinical diseases. This survey aimed to identify molecular features and analyze in silico structural configuration of Netrin-A in L. sericata larvae. The larvae were reared under standard maggotarium conditions. The nucleic acid sequence of L. sericata Netrin-A (LSN-A) was then identified using rapid amplification of circular DNA ends (RACE) and rapid amplification of genomic ends (RAGE). Parts of the Netrin-A gene, including the middle, 3'-, and 5'-ends, were identified, TA cloned in pTG19 plasmid, and transferred into DH5ɑ Escherichia coli. Each part was sequenced and assembled using SeqMan software. This gene structure was further subjected to in silico analysis. The DNA of LSN-A was identified to be 2407 bp, while its mRNA sequence was recognized as 2115 bp by Oligo0.7 software. It translated the Netrin-A protein with 704 amino acid residues. Its estimated molecular weight was 78.6 kDa. Sequencing of this fragment and its BLAST analysis revealed laminin-based high (95%) similarity with the mRNA sequence of Lucilia cuprina Netrin-A. The 3-D structure of Netrin-A drawn by SWISS-MODEL exhibited its partial resemblance to the reference molecule Netrin-1 of Homo sapiens. This study supports the molecular and structural analyses of LSN-A protein, which could lead to wound treatment. Ultimately, it can be an effective candidate to ameliorate injury. Our next attempt is to produce LSN-A recombinant protein for use in biomedical sciences.

Genetic Polymorphisms in MicroRNA-196a2 and the Risk of Human Abortion Related to Mycoplasma hominis.

Mutations in some miRNAs are associated with human recurrent pregnancy loss (RPL). In parallel, Mycoplasma spp. are one of the most common infections in pregnant women. The objective of this study was to identify the relationship between miRNA196a-2 gene polymorphism and Mycoplasma hominis (M. hominis) infection as a possible cause of human abortion. A total of 160 cervical swab specimens were collected from women (80 samples with at least one abortion as case, and 80 samples without abortion as control). A PCR-based method using 16S rRNA gene and tetra primer amplification refractory mutation system-polymerase chain (Tetra-ARMS-PCR) were used to identify the presence of M. hominis infections and miRNA196a-2 genotypes of studied women, respectively. Results showed that 22.5% of women with abortion and 7.5% of women without abortion were infected with M. hominis, thereby suggesting a significant difference between the two groups (P < 0.05). Tetra-ARMSPCR indicated that no significant difference in frequency of genotypes existed between women experimenting abortion and control group. Independently to the presence of M. hominis infection, a significant difference (P < 0.05) was observed in genotypic frequencies of miRNA196a-2 between RPL women and those with one abortion. Estimation of the Odds Ratios indicated that the chance of recurrent abortions in TT genotypes of miRNA196a-2 was about three times more likely than CC in non-infected individuals and about five times more likely than CC in M. hominis-infected patients. Our results proposed the role of miRNA196a-2 genotypes in RPL either in M. hominis-infected or non-infected individuals.

Molecular Characterization of a New Nosema bombycis Strain Detected in Iranian Silkworm Farms.

PURPOSE: Pebrine as the most dangerous disease of silkworm mostly caused by Nosema species has caused huge economic losses. There is no information on the species and the genomic sequences of the pebrine-causing microsporidia in Iran. METHODS: In the present research, we tried to determine the sequences of two regions of rDNA using molecular methods. First, infected larvae and mother moths were collected from several farms in the north of Iran for identification and molecular characterization of microsporidian isolates. After extracting the spores and genomic DNA from the collected samples, two fragments of internal transcribed spacer (ITS) rDNA and small subunit (SSU) rDNA were amplified and sequenced, and registered in NCBI database and then, the phylogenetic tree was drawn. RESULTS: Results showed the obtained sequences (ITS rDNA: Accession No. MZ322002 and SSU rDNA: Accession No. MZ314703) represent a new strain of Nosema bombycis, which differs from the sequences deposited in the NCBI. CONCLUSION: The new N. bombycis strain identified in our study will help in control and management of the pebrine disease by specific detection of the infectious agent.

Evaluation of cellular and humoral immune response in chickens immunized with flagellin-adjuvanted inactivated newcastle disease virus.

The aim of the present study was to evaluate the potential effect of flagellin as adjuvant in Newcastle disease virus (NDV) vaccine on the cellular and humoral immunity in chickens. Fifty-six specific pathogen-free chickens were assigned to seven groups of eight chickens and immunized twice with a two-week interval, intramuscularly. Group 1, received phosphate buffered saline as control (C), groups 2, 3, 4, 5, 6 and 7 were immunized with inactivated NDV [Ag], Ag + full FliC protein [AgF], Ag + truncated Flic protein [AgT], Ag + native Flic protein [AgN], commercial NDV vaccine [Vac] and Vac + N [VacN], respectively. After 45 days, spleen and bursa of Fabricius samples were collected and analyzed by flow cytometry and responses in control/vaccinated chickens were studied by immunophenotyping. Humoral response was also, evaluated by ELISA during the experiment. Results showed that immunized chickens with Ag + flagellin proteins had significantly higher frequency of circulating CD3+, CD4+ and CD8+ T cells in bursa of Fabricius in AgF, AgT and AgN, respectively, compared with other groups. Similar results were observed for spleen; however, the highest frequency of circulating CD3+, CD4+ and CD8+ T cells belonged to AgT and AgF, respectively. ELISA results showed that all flagellin-adjuvanted groups had higher antibody titers than other groups with the highest antibody response in VacN. It can be concluded that flagellin may induce both humoral and cellular immune responses against ND and is suggested for use as an efficient adjuvant.

Monitoring of synthetic insecticides resistance and mechanisms among malaria vector mosquitoes in Iran: A systematic review.

BACKGROUND: In Iran, the prospect of malaria control relies mainly on insecticides used against the genus Anopheles (Diptera: Culicidae) as important vectors of malaria, arboviruses, and so on. Only eight out of 30 malaria mosquito vectors (Anopheles species) have been examined for insecticide resistance in Iran. This study aimed to review articles related to the incremental trend in insecticide resistance and their mechanisms among anopheline malaria vectors in Iran. METHODS: A literature review was conducted based on such search engines as Iran doc, Web of Science, SID, PubMed, Scopus, and Google Scholar websites using the following keywords: "Anopheles," "Malaria," "Resistance," "Vectors," "Insecticide Resistance," and "Iran" for data collection. Published papers in English or Persian covering 1980 to 2020 were reviewed. RESULTS: A total of 1125 articles were screened, only 16 of which were filtered to be pertinent in this review. While most of the mosquito vectors of malaria, such as Anopheles stephensi, were resistant to DDT, dieldrin, malathion, and becoming less susceptible to deltamethrin and other synthetic pyrethroid insecticides, few like Anopheles fluviatilis s. l. were susceptible to all insecticides. A disseminating trend in insecticide resistance among different anopheline mosquito vector species was evident. Metabolic and insecticide target-site resistance mechanisms were involved with organochlorines and pyrethroids, respectively. CONCLUSIONS: Insecticide resistance is becoming a severe scourge to the effectiveness of vector-borne disease management measures. This event is especially critical in developing and marginalized communities that applied chemical-based vector elimination programs for malaria; therefore, it is crucial to monitor insecticide resistance in malaria vectors in Iran using biochemical and molecular tools.

Frequency of pyrethroid resistance in human head louse treatment: systematic review and meta-analysis.

Head lice (Pediculus humanus capitis) are one of the most common insects causing infestations in humans worldwide, and infestation is associated with adverse socio-economic and public health effects. The development of genetic insensitivity (e.g., target site insensitivity = knockdown resistance or kdr) to topical insecticides has impaired effective treatment. Therefore, this study was undertaken to review and meta-analyze the frequency of pyrethroid resistance in treated head louse populations from the beginning of 2000 to the end of June 2021 worldwide. In order to accomplish this, all English language articles published over this period were extracted and reviewed. Statistical analyses of data were performed using fixed and random effect model tests in meta-analysis, Cochrane, meta-regression and I2 index. A total of 24 articles from an initial sample size of 5033 were accepted into this systematic review. The mean frequency of pyrethroid resistance was estimated to be 76.9%. In collected resistant lice, 64.4% were homozygote and 30.3% were heterozygote resistant. Globally, four countries (Australia, England, Israel, and Turkey) have 100% kdr gene frequencies, likely resulting in the ineffectiveness of pyrethrin- and pyrethroid-based pediculicides. The highest resistance recorded in these studies was against permethrin. This study shows that pyrethroid resistance is found at relatively high frequencies in many countries. As a result, treatment with current insecticides may not be effective and is likely the cause of increased levels of infestations. It is recommended that resistance status be evaluated prior to insecticide treatment, to increase efficacy.

TITLE: Fréquence de la résistance aux pyréthroïdes dans le traitement du pou de tête chez l'homme : revue systématique et méta-analyse. ABSTRACT: Les poux de tête (Pediculus humanus capitis) sont l'un des insectes les plus courants à l'origine d'infestations chez l'homme dans le monde, et l'infestation est associée à des effets socio-économiques et de santé publique néfastes. Le développement d'une insensibilité génétique (par exemple, l'insensibilité au site cible = résistance knockdown ou kdr) aux insecticides topiques a altéré l'efficacité de leur traitement. Par conséquent, cette étude a été entreprise pour examiner et méta-analyser la fréquence de la résistance aux pyréthroïdes dans les populations de poux de tête étudiées du début 2000 à la fin juin 2021 dans le monde. Pour ce faire, tous les articles en anglais publiés au cours de cette période ont été extraits et examinés. Les analyses statistiques des données ont été effectuées à l'aide de tests de modèles à effets fixes et aléatoires dans la méta-analyse, Cochrane, méta-régression et indice I2. Un total de 24 articles provenant d'un échantillon initial de 5033 ont été acceptés dans cette revue systématique. La fréquence moyenne de la résistance aux pyréthroïdes a été estimée à 76,9 %. Chez les poux résistants collectés, 64,4 % étaient homozygotes résistants et 30,3 % étaient hétérozygotes résistants. À l'échelle mondiale, quatre pays (Australie, Angleterre, Israël et Turquie) ont des fréquences de gène kdr de 100 %, ce qui entraîne probablement une inefficacité des pédiculicides à base de pyréthrine et de pyréthrinoïde. La résistance la plus élevée enregistrée dans ces études était celle contre la perméthrine. Cette étude montre que la résistance aux pyréthroïdes est trouvée à des fréquences relativement élevées dans de nombreux pays. En conséquence, le traitement avec les insecticides actuels peut ne pas être efficace et est probablement la cause d'une augmentation des niveaux d'infestation. Il est recommandé d'évaluer le statut de résistance avant le traitement insecticide, pour augmenter son efficacité.

Comparison of flagellin and an oil-emulsion adjuvant in inactivated Newcastle disease vaccine in stimulation of immunogenic parameters.

The present study was designed to investigate the potential application of native (N) and recombinant (truncated modified [tmFliC] and full-length [flFliC]) flagellin proteins along with inactivated Newcastle disease virus (NDV). Fifty six SPF chickens were immunized twice with PBS (control), inactivated NDV (Ag), inactivated NDV/flFliC (AgF), inactivated NDV/tmFliC (AgT), inactivated NDV/N (AgN), commercial vaccine containing Montanide (Vac) and Vac/N (VacN), with a two-week interval. Blood was collected weekly and spleens were harvested after chickens were sacrificed. Interleukin-6 (IL-6) and tumor necrotic factor-α (TNF-α) gene expression in peripheral blood mononuclear cells were analyzed by Real-Time PCR. Antibody response was assessed by haemagglutination inhibition (HI). Cellular activity was quantified by MTT assay. Results showed that the most IL-6 and TNF-α gene expression was observed in AgF group (P < 0.01). The lowest gene expression among vaccinated groups was observed in Ag group for IL-6 and Ag and Vac group for TNF-α. The highest HI titer was observed in Vac, VacN, AgF and AgT groups. The AgF group showed the highest cellular activity (P < 0.01). In conclusion, flagellin-adjuvanted groups showed a pro-inflammatory effect and acted similarly to or better than the Vac group. Hence, flagellin can be proposed as a potential adjuvant for ND vaccine.

Are Epitopic Sites of 3AB and 3D Nonstructural Proteins Sufficient for Detection of Foot and Mouth Disease?

An efficient method for detection of foot and mouth disease (FMD) and, particularly, differentiation of vaccinated from infected animals is the use of nonstructural (NS) proteins as antigens in Enzyme-Linked Immunosorbent Assay (ELISA) Kits. In this study, only epitopic regions of 3AB and 3D NS proteins were used for recombinant protein production, as a cost-effective method instead of peptide synthesis, for application in in-house ELISA diagnostic kits. Specific primers were designed according to the antigenic regions of 3AB (C-terminus of 3A and the whole 3B) and 3D (N-terminus) proteins, and the polymerase chain reaction (PCR) amplification was performed. Purified amplicons were cloned into pET21a (+) vectors and then transformed into Escherichia coli (BL21). Thereafter, bacteria were induced with 1 mM isopropyl ß-d-1-thiogalactopyranoside (IPTG) for expression of antigenic proteins. Antigenic 3AB protein was expressed in soluble form, but 3D protein was extracted from the bacterial lysate. Protein expression was confirmed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses. An indirect ELISA was developed for each protein, and the diagnostic sensitivity and specificity were determined. The 3AB-ELISA showed higher sensitivity and specificity than 3D-ELISA (95.24% and 100%, compared with 90.48% and 88.71%, respectively). The epitopic 3AB-ELISA developed here can be used for detection and differentiation of FMD infected from vaccinated animals, but the epitopic 3D-ELISA showed lower efficiency in screening for FMD status.

Effect of flagellin on inhibition of infectious mechanisms by activating opsonization and salmonella flagellum disruption.

Some serovars of salmonella cause huge global diseases such as enteric fever and invasive non typhoidal Salmonella disease. Flagellin as a key antigenic component of salmonella, can induce humoral and cellular immunity responses. In this research, we performed an opsonophagocytic killing assay (OPKA) as an important mechanism of the host-defense system, for salmonella to study the activity of anti-sera of native FliC, truncated modified recombinant FliC (tmFliC) and full length recombinant FliC proteins (flFliC). Also, the potency of antibodies for inhibiting bacterial movement was evaluated by traditional and newly-designed motility inhibition assay methods. Results showed both recombinant FliC anti-sera and native FliC (nFliC) anti-serum had the ability to opsonize Salmonella typhimurim, which led to bacterial clearance by mice macrophages. Also, inhibition of bacterial motility was observed for all anti-sera. Anti-nFliC and anti-flFliC sera showed higher effects on Salmonella typhimurim motility than that of tmFliC. In traditional method, about 88%, 86% and 80% inhibition were observed by using 5% nFliC, anti-flFliC and anti-tmFliC sera, respectively. In the newly-designed method using SIM (Sulfide indole motility) medium, results confirmed the traditional method for motility inhibition. Our findings suggest that salmonella fliC as a protective antigen may disrupt the flagellum apparatus activity.

Skimmed milk as an alternative for IPTG in induction of recombinant protein expression.

Cost-effectiveness is an important issue in biotechnological manufacturing industry and using alternative cheap materials with the same benefits has been noticed in most literatures. Isopropyl ß-d-1-thiogalactopyranoside (IPTG), a well-known chemical element for induction of protein expression, has several disadvantages such as high expense and toxicity. In this study, we aimed to introduce skimmed milk as an alternative material for protein expression by induction of lac operon. In this way, Escherichia coli BL21 (DE3) bacteria were induced using 1 mM IPTG or 1.0% (w/v) skimmed milk. Protein purification was performed using Ni-NTA (nickel-nitrilotriacetic acid) for His-tagged recombinant proteins and protein purity was evaluated by SDS-PAGE. Results showed high level of recombinant protein expression using skimmed milk, and interestingly, the growth rate of bacteria improved. Our findings suggested that skimmed milk can be a suitable alternative for induction of recombinant protein expression, which has advantages such as more availability and affordability, in comparison to IPTG supplementation.

Molecular characterization of the netrin-1 UNC-5 receptor in Lucilia sericata larvae.

Larval therapy with Lucilia sericata is a promising strategy in wound healing. Axon guidance molecules play vital roles during the development of the nervous system and also regulate the capacity of neuronal restoration in wound healing. Netrin-1, one of the proteins that larvae secrete, plays a useful role in cell migration and nerve tissue regeneration. The UNC-5 receptor combines with a netrin-1 signal and transmits the signal from one side of the membrane to the other side, initiating a change in cell activity. In the current study, we identified the full length of the UNC-5 receptor mRNA in L. sericata using different sets of primers, including exon junction and specific region primers. The coding sequence (CDS) of the UNC-5 receptor was sequenced and identified to include 633 base-pair nucleic acids, and BLAST analysis on its nucleotide sequence revealed 96% identity with the Lucilia cuprina netrin-1 UNC-5 receptor. The protein residue included 210 amino acids (aa) and coded for a protein with 24 kD weight. This gene lacked the signal peptide. Furthermore, the UPA domain is conserved in UNC-5. It lied at the interval of 26-131 aa. We identified the CDS of netrin-1 UNC-5 receptor in L. sericata. It could be applied to research activities implementing a new essential component design in wound healing.

Cytotoxic effects of Pseudocerastes persicus venom and its HPLC fractions on lung cancer cells.

BACKGROUND: Several studies have pointed out that certain snake venoms contain compounds presenting cytotoxic activities that selectively interfere with cancer cell metabolism. In this study, Pseudocerastes persicus venom and its fractions were investigated for their anticancer potential on lung cancer cells. METHODS: Lung cancer cells (A549) and normal fibroblast cells (Hu02) were treated with the P. persicus venom and its HPLC fractions and the cell cytotoxic effects were analyzed using MTT and lactate dehydrogenase release assays. Apoptosis was determined in venom-treated cell cultures using caspase-3 and caspase-9 assay kits. RESULTS: The treatment of cells with HPLC fraction 21 (25-35 kDa) of P. persicus venom resulted in high LDH release in normal fibroblast cells and high caspase-3 and caspase-9 activities in lung cancer cells. These results indicate that fraction 21 induces apoptosis in cancer cells, whereas necrosis is predominantly caused by cell death in the normal cells. Fraction 21 at the final concentration of 10 µg/mL killed approximately 60% of lung cancer cells, while in normal fibroblast cells very low cell cytotoxic effect was observed. CONCLUSION: HPLC fraction 21 at low concentrations displayed promising anticancer properties with apoptosis induction in the lung cancer cells. This fraction may, therefore, be considered a promising candidate for further studies.

Effect of C-Terminus Modification in Salmonella typhimurium FliC on Protein Purification Efficacy and Bioactivity.

Recombinant flagellin (FliC) has shown low efficacy in purification because of inclusion bodies formation and aggregation. We hypothesized preserving TLR5 binding site of FliC and removing some amino acids could be responsible for aggregation and solubility improvement. Hence, a bioinformatics study was performed to find hotspots in aggregate formation. Protein modeling was carried out by SWISS-MODEL and I-TASSER servers and models were compared by MATRAS server and Chimera 1.11.2. Gene modification was carried out based on bioinformatics studies. Genes, (truncated modified fliC (tmFliC) and full-length fliC (flFliC)), were cloned and expressed in pET-21a vector. Protein purification was carried out using HIS-Tag method. Proliferation assay and also induction of IL-8 in HEK293 cells were performed to confirm bioactivity function of tmFliC. Bioinformatics results showed that partial deletion of C-terminus may increase solubility without unfavorable effect on TLR5 recognition. Also, model comparison showed that this protein may preserve 3D structure. In addition, GlobPlot server demonstrated that tmFliC formed its globular domains which were important in TLR5 recognition. As we expected, high purification efficacy for tmFliC compared with flFliC was also obtained in experimental studies and a proper function for tmFliC was observed. The tmFliC enhanced cell proliferation in HEK293 cells compare with control after 24 h. Also, IL-8 level was increased with stimulation by tmFliC after 24 h. In conclusion, reducing hydrophobicity in C-terminus and deleting necessary amino acids for filament formation may increase protein solubility.

Introducing a cost-effective method for purification of bioactive flagellin from several flagellated gram-negative bacteria.

The objective of this study was to introduce a simple and cheap method for purification of flagellin. So, flagellin proteins of Salmonella typhimurium (S. typhimurium), Escherichia coli (E. coli), Citrobacter freundii (C. freundii) and Salmonella typhi (S. typhi) were purified by a modified simple method. Bacterial cultures were precipitated by centrifugation. Precipitates were washed twice and flagellin proteins were detached by shaking vigorously (in PBS pH = 2), and then flagellin proteins were precipitated by ammonium sulfate saturation. Evaluation of purification efficiency and concentration were examined by SDS-PAGE and Bradford assay. Polyclonal antibodies were produced against S. typhimurium FliC and cross-reactivity of anti-S. typhimurium was assessed against other flagellins. Bioactivity of flagellins was evaluated by cell proliferation and IL-8 protein expression assay in HEK293 cells, and also, IL-6 and TNF-α genes expression in chicken cells. Results showed a single band for flagellin proteins of all bacteria on %10 SDS-PAGE, which concentration ranged from 150 to 400 µg/mL. All flagellin proteins increased cell proliferation, and IL-8 levels were increased after treatment by flagellins and levels of IL-6 and TNF-α were increased after treatment with S. typhimurium FliC. All flagellin proteins showed cross-reactivity with antibodies. Findings showed that application of our method, not only reduced time and cost, but also, the purified flagellin proteins had acceptable bioactivity.

Immunostimulatory effects of truncated and full-length flagellin recombinant proteins.

Problems regarding purification efficacy in recombinant technologies is due to the protein structure. Experimental manipulation of genes and the subsequent proteins may overcome this issue. In order to improve production efficacy and maintain immunestimulatory effect of flagellin, the Toll-like Receptor 5 (TLR5) ligand and a potent adjuvant, we performed a bioinformatic study to find the best model for FliC manipulation. Truncated modified FliC (tmFliC) and full length FliC (flFliC) genes were cloned and expressed in pET-21a vector and protein purification was carried out using an improved His-Tag method. Polyclonal antibodies were generated against flFliC and tmFliC in New Zealand white rabbits. IgG response to the recombinant proteins was determined by ELISA. Cross-reactivity assay was performed by ELISA for all proteins and bacteria. Immunogenicity of tmFliC and flFliC was evaluated in chicken cells, and expression level of tumor necrotic factor-α (TNF-α) and interleukin-6 (IL-6) were relatively analyzed by Real-Time-PCR. Results showed high purification efficacy for tmFliC. Antibody titer of tmFliC was significantly higher than that of flFliC. In addition, the cross-reactivity assay for both proteins and Salmonella was positive which indicates similar epitopic regions. Stimulation of both FliCs significantly increased TNF-α and IL-6 expression in peripheral blood mononuclear cells (PBMCs) and splenocytes, with higher effect observed with flFliC. IL-8 protein level increased after 6 and 24 h stimulation with different concentrations of tmFliC and flFliC. These results suggest that the aimed gene modification in fliC gene produces a bioactive immunostimulant type of flagellin which upregulates TLR5 downstream genes as well as in flFliC.

Cytotoxic effects of Pseudocerastes persicus venom and its HPLC fractions on lung cancer cells

Several studies have pointed out that certain snake venoms contain compounds presenting cytotoxic activities that selectively interfere with cancer cell metabolism. In this study, Pseudocerastes persicus venom and its fractions were investigated for their anticancer potential on lung cancer cells. Methods: Lung cancer cells (A549) and normal fibroblast cells (Hu02) were treated with the P. persicus venom and its HPLC fractions and the cell cytotoxic effects were analyzed using MTT and lactate dehydrogenase release assays. Apoptosis was determined in venom-treated cell cultures using caspase-3 and caspase-9 assay kits. Results: The treatment of cells with HPLC fraction 21 (25-35 kDa) of P. persicus venom resulted in high LDH release in normal fibroblast cells and high caspase-3 and caspase-9 activities in lung cancer cells. These results indicate that fraction 21 induces apoptosis in cancer cells, whereas necrosis is predominantly caused by cell death in the normal cells. Fraction 21 at the final concentration of 10 μg/mL killed approximately 60% of lung cancer cells, while in normal fibroblast cells very low cell cytotoxic effect was observed. Conclusion: HPLC fraction 21 at low concentrations displayed promising anticancer properties with apoptosis induction in the lung cancer cells. This fraction may, therefore, be considered a promising candidate for further studies.(AU)

Evolution and species-specific conservation of toll-like receptors in terrestrial vertebrates.

Toll-like receptors (TLRs) are known for their essential roles in promotion of innate immunity and induction of adaptive immunity through recognition of pathogen-associated molecular patterns (PAMPs) or danger associated molecular patterns (DAMPs). TLR genes are excellent models for the study of the selective pressure enforced by microorganisms on the host genome. Phylogenetic analyses have shown that interactions between pathogens and immune systems have changed during evolution. Selective pressure for maintenance of specific pathogen recognition has led to evolution of TLRs under both positive and purifying selection. However, intracellular and cell-surface TLRs have been affected differently due to their variation in conservational constraints. In this review, we summarize some of the main studies on the influence of selection on shaping the evolution of several TLR gene families in different terrestrial vertebrate species. We also describe the effect of evolution on the function of different TLRs and their specific conservations in these species and show similarities and differences in evolutionary patterns of TLR orthologs among species as well as among TLR gene families.