Introduction of a novel 18S rDNA gene arrangement along with distinct ITS region in the saline water microalga Dunaliella

Comparison of 18S rDNA gene sequences is a very promising method for identification and classification of living organisms. Molecular identification and discrimination of different Dunaliella species were carried out based on the size of 18S rDNA gene and, number and position of introns in the gene. Three types of 18S rDNA structure have already been reported: the gene with a size of ~1770 bp lacking any intron, with a size of ~2170 bp consisting one intron near 5' terminus, and with a size of ~2570 bp harbouring two introns near 5' and 3' termini. Hereby, we report a new 18S rDNA gene arrangement in terms of intron localization and nucleotide sequence in a Dunaliella isolated from Iranian salt lakes (ABRIINW-M1/2). PCR amplification with genus-specific primers resulted in production of a ~2170 bp DNA band, which is similar to that of D. salina 18S rDNA gene containing only one intron near 5' terminus. Whilst, sequence composition of the gene revealed the lack of any intron near 5' terminus in our isolate. Furthermore, another alteration was observed due to the presence of a 440 bp DNA fragment near 3' terminus. Accordingly, 18S rDNA gene of the isolate is clearly different from those of D. salina and any other Dunaliella species reported so far. Moreover, analysis of ITS region sequence showed the diversity of this region compared to the previously reported species. 18S rDNA and ITS sequences of our isolate were submitted with accesion numbers of EU678868 and EU927373 in NCBI database, respectively. The optimum growth rate of this isolate occured at the salinity level of 1 M NaCl. The maximum carotenoid content under stress condition of intense light (400 μmol photon m-2 s-1), high salinity (4 M NaCl) and deficiency of nitrate and phosphate nutritions reached to 240 ng/cell after 15 days

Designing a light-activated recombinant alpha hemolysin for colorectal cancer targeting

Introduction: Colorectal cancer (CRC) is one of the main health burden worldwide, which can cause major economic and physiological problems along with relatively high rate of mortality. It is important to develop new methods for the localized delivery of recombinant protein therapeutics, in large part due to the failure of conventional therapies in most cases. Since E. coli Nissle 1917 (EcN) does not produce any virulence factors, here we used these bacteria with the light-activated promoter system to deliver therapeutic agents in the desired location and time. Methods: In this study, Staphylococcus aureus alpha hemolysin (SAH), after codon usage optimization, was cloned into blue light activating vector (pDawn) and transferred to EcN strain. Then, the functionality and cytotoxicity of secreted alpha hemolysin was evaluated in the SW480 colon cancer cell line by using different experiments, including blood agar test, flow cytometry analysis, and DAPI staining. Results: Our findings revealed that EcN can produce functional SAH under the blue light irradiation against SW480 cancer cells. Moreover, cytotoxicity assays confirmed the dose- and time-dependent toxicity of this payload (SAH) against SW480 cancer cells. Conclusion: Based on our results, EcN is proposed as an appropriate light-activated vehicle for delivery of anticancer agents to the target cancer cells/tissues

Efficient and stable transformation of Dunaliella pseudosalina by 3 strains of Agrobacterium tumefaciens

Introduction: Several platforms including mammalian, plant and insect cells as well as bacteria, yeasts, and microalgae are available for the production of recombinant proteins. Low efficiency of delivery systems, extracellular and intracellular degradation of foreign genes during transformation, difficulties in targeting and importing into the nucleus, and finally problems in integration into nuclear genome are the most bottlenecks of classical plasmids for producing recombinant proteins. Owing to high growth rate, no common pathogen with humans, being utilized as humans’ food, and capability to perform N-glycosylation, microalgae are proposed as an ideal system for such biotechnological approaches. Here, Agrobacterium tumefaciens is introduced as an alternative tool for transformation of the microalga Dunaliella pseudosalina. Methods: The transformation of gfp gene into the D. pseudosalina was evaluated by three strains including EHA101, GV3301 and GV3850 of A. tumefaciens. The integrating and expression of gfp gene were determined by PCR, RT-PCR, Q-PCR and SDS-PAGE analyses. Results: The T-DNA of pCAMBIA1304 plasmid was successfully integrated into the genome of the microalgal cells. Although all of the strains were able to transform the algal cells, GV3301 possessed higher potential to transform the microalgal cells in comparison to EHA101 and GV3850 strains. Moreover, the stability of gfp gene was successfully established during a course of two months period in the microalgal genome. Conclusion: Agrobacterium is introduced as a competent system for stable transformation of Dunaliella strains in order to produce eukaryotic recombinant proteins

Purification of a Novel Anti-VEGFR2 Single Chain Antibody Fragmentand Evaluation of Binding Affinity by Surface Plasmon Resonance

Purpose: The single-chain variable fragment (scFv) domain of antibodies is now considered asone of the therapeutic tools that can be produced by phage display technology (PDT). Antibodypurification is one of the most important steps in antibodies production. The aim of study waspurification and characterization of anti-VEGFR2 scFv antibody fragments.Methods: After the coating of vascular endothelial growth factor receptor 2 (VEGFR2) peptidein ELISA microplates, the phage display library of Tomlinson was used for antibody isolation.The targeted scFv was purified by chromatography using a zeolite-based column. The purity andfunctional assessment of purified scFv were evaluated by sodium dodecyl sulfate polyacrylamidegel electrophoresis (SDS-PAGE) and western blotting techniques, respectively. Affinity bindingwas evaluated by surface plasmon resonance (SPR).Results: The desired scFv was selected after four stages of biopanning. SDS-PAGE analysisshowed a 28 kDa scFv with high purity (>90%). The western bloting analysis confirmed thebinding of produced scFv antibody to the desired peptide. The affinity binding of scFv antibodyanalyzed by SPR was about 60 μM.Conclusion: In this study, the novel scFv antibody against VEGFR2 peptide was purified bychromatography column containing zeolite. Based on our findings the produced antibody maybe applied for diagnosis or targeting of VEGFR2 in antibody-based therapy strategies

Effect of A-769662, a direct AMPK activator, on Tlr-4 expression and activity in mice heart tissue

Objective(s): TLR-4 activates a number of inflammatory signaling pathways. Also, AMPK could be involved in anti-inflammatory signaling. The aim of this study was to identify whether stimulation of AMPK could inhibit LPS-induced Tlr-4 gene expression in mice hearts. Materials and methods: Heart AMPK activity and/or Tlr-4 expression was stimulated in different mice groups, using respectively IP injection of A-769662 (10 mg/kg) and LPS (2 mg/kg) or a combination of both agents. Moreover, compound-C (20 mg/kg), as an AMPK antagonist, was intraperitoneally co-administrated with both A-769662 and LPS in another group to investigate the role of AMPK activity on Tlr-4 regulation. After 8 hr, in addition to peripheral neutrophil cell count, myocardial p-AMPK, p-ACC as well as MyD88 protein contents and Tlr-4 expression was assessed by Western blotting and real-time qRT-PCR, respectively. TNF-α and IL-6 expression levels were also determined by ELISA. Results: LPS induced heart Tlr-4 expression (

Lactobacillus plantarum induces apoptosis in gastric cancer cells via modulation of signaling pathways in Helicobacter pylori

Introduction: Gastric cancer is considered the second prevalent cause of death around the world. This type of cancer is generally induced by Helicobacter pylori which could colonize within the gastric mucosa of the infected cases. To date, triple antibiotic therapy has routinely been utilized for controlling the H. pylori-induced infection. However, this strategy has been unsuccessful, in large part because of issues such as occurring point mutations in the H. pylori genome that can induce resistance to the antibiotics administered. Recently, it has been shown that different probiotics may have strong anti-cancer effects, in which they are capable of inhibiting H. pylori by both immunological and non-immunological mechanisms. Here, we aimed at finding possible anti-cancer impacts of the probiotic bacterium Lactobacillus plantarum on gastric cancer, AGS cells. Methods: The anti-cancer effects of the conditioned media of the locally isolated L. plantarum on the AGS cells were evaluated by different analyses such as flow cytometry, DNA ladder assay, DAPI staining, and RT-PCR. Results: Our findings showed that the conditioned media of L. plantarum can inhibit both H. pylori and AGS cells through up-/down-regulation of PTEN, Bax, TLR4, and AKT genes. The exudates of the probiotic L. plantarum bacteria can increase the expression of PTEN, Bax, and TLR4, and also decrease the expression of AKT gene. Conclusion: In agreement with different reports, our results proved the anti-cancer effects of the locally isolated L. plantarum through some immunological cell signaling pathways. Accordingly, it seems the probiotics could be considered as at least a complementary treatment for different types of malignancies

Probiotic or antibiotic: future challenges in medicine

Genetic and environmental factors can affect the intestinal microbiome and microbial metabolome. Among these environmental factors, the consumption of antibiotics can significantly change the intestinal microbiome of individuals and consequently affect the corresponding metagenome. The term 'probiotics' is related to preventive medicine rather than therapeutic procedures and is, thus, considered the opposite of antibiotics. This review discusses the challenges between these opposing treatments in terms of the following points: (i) antibiotic resistance, the relationship between antibiotic consumption and microbiome diversity reduction, antibiotic effect on the metagenome, and disease associated with antibiotics; and (ii) probiotics as living drugs, probiotic effect on epigenetic alterations, and gut microbiome relevance to hygiene indulgence. The intestinal microbiome is more specific for individuals and may be affected by environmental alterations and the occurrence of diseases

Probiotic assessment of Lactobacillus plantarum 15HN and Enterococcus mundtii 50H isolated from traditional dairies microbiota

Purpose: Probiotics are microorganisms, which show beneficial health effects on hosts once consumed in sufficient amounts. Among probiotic bacteria, the bioactive compounds from lactic acid bacteria (LAB) group can be utilized as preservative agents. LAB group can be isolated and characterized from traditional dairy sources. This study aimed to isolate, identify, and biologically characterize probiotic LAB strains from Iranian traditional dairy products. Methods: A total of 19 LAB strains were identified by sequencing of their 16S rRNA genes. They were examined for adherence to human intestinal Caco-2 cells and tolerance to low pH/high bile salts and simulated in vitro digestion conditions. Moreover, they were evaluated further to assess their ability to prevent the adhesion of Escherichia coli 026 to the intestinal mucosa, inhibitory functions against pathogens, and sensitivity to conventional antibiotics. Results: L. plantarum 15HN and E. mundtii 50H strains displayed ≥ 71% survival rates at low pH/high bile salts and ≥ 40% survival rates in digestive conditions. Their adherences to Caco-2 cells were 3.2×105 and 2.6×105 CFU mL-1 respectively and high values of antiadhesion capability were observed (≥36%). They inhibited the growth of 13 and 11 indicator pathogens respectively. Moreover, they were sensitive or semi-sensitive to seven and three out of eight antibiotics respectively. Conclusion: L. plantarum 15HN and E. mundtii 50H, which were isolated from shiraz product, displayed above-average results for all of the criteria. Therefore, they can be introduced as novel candidate probiotics that could be used in the food industry

Prétraitements antioxydants des cellules souches mésenchymateuses contre le stress oxydatif et le déficit respiratoire : Étude des mécanismes cellulaires et moléculaires

Chapter 1 is devoted to a literature review of the redox biology of stem cells and recent strategies for controlling oxidative stress and the fate of stem cells. In Chapter 2 we have chosen to present stem cells, the method of extraction and characterization of mesenchymal stem cells that we used in all of the thesis studies. In Chapters 3A and 3B are presented two research articles on the effect of antioxidants, NAC (N-acetylcysteine) and mito-TEMPO ((2- (2,2,6,6-Tetramethylpiperidin-1-oxyl -4-ylamino) -2-oxoethyl) triphenylphosphonium chloride), on stem cells subjected to oxidative stress induced by antimycin A as a blocker of the mitochondrial electron transport chain.In Chapter 4 we present a review of the literature with a critical discussion on new perspectives concerning the nesting of stem cells in hypoxic ischemic lesions and the reprogramming of stem cells in a new niche of dysfunction with complex macromolecular actors. The Sirtuins / HIF1 α interactions in the fate of implanted stem cells are discussed and a bioinformatic analysis of the interaction of the genes involved is provided.In Chapter 5A, we present a bibliographic analysis article on the role of piezoelectric channels or Piezo proteins, recently recognized as special ion channels mediating mechanical detection and mechanotransduction.In Chapter 5B we present an article reviewing and analyzing recent data on in vitro culture models used to study differentiation such as three-dimensional aggregates of pluripotent stem cells called embryoid bodies (EB).Finally, a conclusion on all the results obtained and a discussion on possible future directions are presented.Le Chapitre 1 est consacré à une revue de la littérature sur la biologie redox des cellules souches et des stratégies récentes de contrôle du stress oxydatif et du devenir des cellules souches. Dans le Chapitre 2 nous avons choisi de présenter les cellules souches, la méthode d’extraction et de caractérisation de cellules souches mésenchymateuses que nous avons utilisée dans toutes les études de la thèse. Dans les Chapitres 3A et 3B sont présentés deux articles travaux de recherche sur l'effet des antioxydants, le NAC (N-acétylcystéine) et le mito-TEMPO ((2-(2,2,6,6-Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride), sur des cellules souches soumises au stress oxydatif induit par l'antimycine A en tant que bloqueur de la chaîne de transport d'électrons mitochondriale. Au Chapitre 4 nous présentons une revue de littérature avec une discussion critique sur de nouvelles perspectives concernant la nidification des cellules souches dans les lésions ischémiques hypoxiques et la reprogrammation des cellules souches dans une nouvelle niche de dysfonctionnement avec des acteurs macromoléculaires complexes. Les interactions Sirtuins / HIF1 α dans le devenir des cellules souches implantées est discutée et une analyse bioinformatique de l’interaction des gènes impliqués est fournie. Dans le Chapitre 5A, nous présentons un article d’analyse bibliographique sur le rôle des canaux piézoélectriques ou protéines Piézo, récemment reconnus comme des canaux ioniques spéciaux assurant la médiation de la détection mécanique et de la mécanotransduction. Dans le Chapitre 5B nous présentons un article de revue et analyse des données récentes sur les des modèles de culture in vitro utilisés pour étudier la différenciation comme les agrégats tridimensionnels de cellules souches pluripotentes appelés corps embryoïdes (EB).Enfin, une conclusion sur l’ensemble des résultats obtenus et une discussion sur les possibles directions futures sont présentés