Astaxanthin-loaded alginate-chitosan gel beads activate Nrf2 and pro-apoptotic signalling pathways against oxidative stress.

Oxidative stress (OS) plays a crucial role in disease development. Astaxanthin (ATX), a valuable natural compound, may reduce OS and serve as a treatment for diseases like neurodegenerative disorders and cancer. Nuclear factor-erythroid 2-related factor 2 (Nrf2) regulates antioxidant enzymes and OS management. We evaluated ATX's antioxidant activity via Alg-CS/ATX gel beads in vitro. ATX-encapsulated alginate-chitosan (Alg-CS/ATX) gel beads were synthesized and structurally/morphologically characterized by SEM, FT-IR, and XRD. Their biological effects were examined in human umbilical vein endothelial cells (HUVECs) treated with H2O2 through MTT assay, Annexin V/PI, cell cycle studies, and western blotting. Alg-CS effectively carried ATX, with high capacity and reduced pore size. Alg-CS/ATX displayed an 84% encapsulation efficiency, maintaining stability for 30 days. In vitro studies showed a 1.4-fold faster release at pH 5.4 than at neutral pH, improving ATX's therapeutic potential. HUVECs treated with Alg-CS/ATX showed enhanced viability via increased Nrf2 expression. Alg-CS gel beads exhibit significant potential as a biocompatible vehicle for delivering ATX to combat OS with considerable opportunity for clinical applications.

Microalgae as an Efficient Vehicle for the Production and Targeted Delivery of Therapeutic Glycoproteins against SARS-CoV-2 Variants.

Severe acute respiratory syndrome-Coronavirus 2 (SARS-CoV-2) can infect various human organs, including the respiratory, circulatory, nervous, and gastrointestinal ones. The virus is internalized into human cells by binding to the human angiotensin-converting enzyme 2 (ACE2) receptor through its spike protein (S-glycoprotein). As S-glycoprotein is required for the attachment and entry into the human target cells, it is the primary mediator of SARS-CoV-2 infectivity. Currently, this glycoprotein has received considerable attention as a key component for the development of antiviral vaccines or biologics against SARS-CoV-2. Moreover, since the ACE2 receptor constitutes the main entry route for the SARS-CoV-2 virus, its soluble form could be considered as a promising approach for the treatment of coronavirus disease 2019 infection (COVID-19). Both S-glycoprotein and ACE2 are highly glycosylated molecules containing 22 and 7 consensus N-glycosylation sites, respectively. The N-glycan structures attached to these specific sites are required for the folding, conformation, recycling, and biological activity of both glycoproteins. Thus far, recombinant S-glycoprotein and ACE2 have been produced primarily in mammalian cells, which is an expensive process. Therefore, benefiting from a cheaper cell-based biofactory would be a good value added to the development of cost-effective recombinant vaccines and biopharmaceuticals directed against COVID-19. To this end, efficient protein synthesis machinery and the ability to properly impose post-translational modifications make microalgae an eco-friendly platform for the production of pharmaceutical glycoproteins. Notably, several microalgae (e.g., Chlamydomonas reinhardtii, Dunaliella bardawil, and Chlorella species) are already approved by the U.S. Food and Drug Administration (FDA) as safe human food. Because microalgal cells contain a rigid cell wall that could act as a natural encapsulation to protect the recombinant proteins from the aggressive environment of the stomach, this feature could be used for the rapid production and edible targeted delivery of S-glycoprotein and soluble ACE2 for the treatment/inhibition of SARS-CoV-2. Herein, we have reviewed the pathogenesis mechanism of SARS-CoV-2 and then highlighted the potential of microalgae for the treatment/inhibition of COVID-19 infection.

Toxicity of microwave-synthesized silver-reduced graphene oxide nanocomposites to the microalga Chlorella vulgaris: Comparison with the hydrothermal method synthesized counterparts.

The increased applications of nanomaterials in industry and biomedicine have resulted in a rising concern about their possible toxic impacts on living organisms. It has been claimed that the phytosynthesized nanomaterials have lower toxicity in comparison to their chemically synthesized counterparts. Therefore, it is important to evaluate their toxic effects on the environment. In the present study, we investigated the toxic effects of microwave-synthesized silver-reduced graphene oxide nanocomposites (MS-Ag-rGO) on Chlorella vulgaris. Algal cells were treated by 1, 2, 4 and 6 mg L-1 MS-Ag-rGO for 24 h. The obtained data with three replicates were examined using analysis of variance. Analysis of different growth parameters revealed that MS-Ag-rGO possessed significant dose-dependent toxic effect on C. vulgaris. Scanning electron microscope and fluorescence microscope images of the treated cells established morphological shrinkages and alteration in position of nucleoli. Moreover, reduction in the phenol and flavonoid contents, enhancement of H2O2 content, changes in the antioxidant enzymes activity and decreases in the growth parameters as well as photosynthetic pigments quantities confirmed the toxicity of MS-Ag-rGO to the C. vulgaris cells. Our findings revealed that MS-Ag-rGO possessed higher toxicity on C. vulgaris than Ag-rGO synthesized by hydrothermal technique.

Evaluating the Toxic Impacts of Cadmium Selenide Nanoparticles on the Aquatic Plant Lemna minor.

Cadmium selenide nanoparticles (CdSe NPs) were synthesized by an easy and simple method and their properties were assessed by XRD, TEM and SEM techniques. The effects of CdSe NPs as well as Cd2+ ions on Lemna minor plants were investigated. The absorption of CdSe NPs by the plants had some adverse consequences that were assessed by a range of biological analyses. The results revealed that both CdSe NPs and the ionic form of cadmium noticeably caused toxicity in L. minor. Morphological parameters as well as peroxidase (POD) activity were deteriorated. In contrast, the activities of some other antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)) as well as the contents of total phenol and flavonoids went up. Taken all together, it could be implied that CdSe NPs as well as Cd2+ were highly toxic to plants and stimulated the plant defense system in order to scavenge produced reactive oxygen species (ROS).

Are the green synthesized nanoparticles safe for environment? A case study of aquatic plant Azolla filiculoides as an indicator exposed to magnetite nanoparticles fabricated using microwave hydrothermal treatment and plant extract.

It has been claimed that the green synthesized NPs possess no toxicity in comparison to the NPs fabricated via conventional protocols like reduction by sodium borohydride. Therefore, it is necessary to test the toxic effects of NPs on environment. In the current study, we report the binding of Fe3O4 NPs to galate ions containing biomaterial namely "galate bio-capping agent". The bio-capping agent is simply mixed with the Fe3+ cations at pH 8 to produce negatively charged bio-capped Fe3O4 NPs. Finally, the toxic effects of the Fe3O4 NPs were investigated on some growth and developmental indices of the aquatic plant species Azolla filiculoides. The relative frond number and relative growth rate were calculated after treatment of plants with different concentrations of bio-capped Fe3O4 NPs. In addition, the content of phenolics as well as antioxidant enzymes' activity including superoxide dismutase and peroxidase were assessed. The Fe3O4 NPs led to growth reduction and significant changes in total phenol and flavonoid content as well as in antioxidant enzymes' activity. All these findings confirm reactive oxygen species formation due to the nanoparticle toxicity. In consequence, the enzymatic and non-enzymatic antioxidant defense systems of plant were stimulated against oxidative stress.

Stable transformation of Spirulina (Arthrospira) platensis: a promising microalga for production of edible vaccines.

The planktonic blue-green microalga Spirulina (Arthrospira) platensis possesses important features (e.g., high protein and vital lipids contents as well as essential vitamins) and can be consumed by humans and animals. Accordingly, this microalga gained growing attention as a new platform for producing edible-based pharmaceutical proteins. However, there are limited successful strategies for the transformation of S. platensis, in part because of an efficient expression of strong endonucleases in its cytoplasm. In the current work, as a pilot step for the expression of therapeutic proteins, an Agrobacterium-based system was established to transfer gfp:gus and hygromycin resistance (hygr) genes into the genome of S. platensis. The presence of acetosyringone in the transfection medium significantly reduced the transformation efficiency. The PCR and real-time RT-PCR data confirmed the successful integration and transcription of the genes. Flow cytometry and ß-glucuronidase (GUS) activity experiments confirmed the successful production of GFP and the enzyme. Moreover, the western blot analysis showed a ~ 90 kDa band in the transformed cells, indicating the successful production of the GFP:GUS protein. Three months after the transformation, the gene expression stability was validated by histochemical, flow cytometry, and hygromycin B resistance analyses.

Toxicity of ZnSe nanoparticles to Lemna minor: Evaluation of biological responses.

A clear consequence of the increasing application of nanotechnology is its adverse effect on the environment. Semiconductor nanoparticles are among engineered nanomaterials that have been considered recently for their specific characteristics. In the present work, zinc selenide nanoparticles (ZnSe NPs) were synthesized and characterized by XRD, TEM, DLS and SEM. Biological aspects related to the impact of nanoparticles and Zn2+ ions were analyzed on the aquatic higher plant Lemna minor. The localization of ZnSe NPs in the root cells of L. minor was determined by TEM and fluorescence microscopy. Then, the entrance of ZnSe NPs into the plant cells was evaluated by a range of biological tests. The outcomes revealed that both the NPs and the ionic forms noticeably poisoned L. minor. In one hand, growth parameters and physiological indices such as photosynthetic pigments content were decreased. On the other hand, the activities of some antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)), as well as the contents of nonenzymatic antioxidants (phenols and flavonoids) were elevated. Taken together, high concentration of ZnSe NPs and Zn2+ triggered phytotoxicity which in turn provoked the plants' defense system. The changes in antioxidant activities confirmed a higher toxicity by Zn2+ ions in comparison with ZnSe NPs. It means that the considered ions are more hazardous to the living organisms than the nanoparticles.

Effects of TiO2 nanoparticles on the aquatic plant Spirodela polyrrhiza: Evaluation of growth parameters, pigment contents and antioxidant enzyme activities.

Plants are essential components of all ecosystems and play a critical role in environmental fate of nanoparticles. However, the toxicological impacts of nanoparticles on plants are not well documented. Titanium dioxide nanoparticles (TiO2-NPs) are produced worldwide in large quantities for a wide range of purposes. In the present study, the uptake of TiO2-NPs by the aquatic plant Spirodela polyrrhiza and the consequent effects on the plant were evaluated. Initially, structural and morphological characteristics of the used TiO2-NPs were determined using XRD, SEM, TEM and BET techniques. As a result, an anatase structure with the average crystalline size of 8nm was confirmed for the synthesized TiO2-NPs. Subsequently, entrance of TiO2-NPS to plant roots was verified by fluorescence microscopic images. Activity of a number of antioxidant enzymes, as well as, changes in growth parameters and photosynthetic pigment contents as physiological indices were assessed to investigate the effects of TiO2-NPs on S. polyrrhiza. The increasing concentration of TiO2-NPs led to the significant decrease in all of the growth parameters and changes in antioxidant enzyme activities. The activity of superoxide dismutase enhanced significantly by the increasing concentration of TiO2-NPs. Enhancement of superoxide dismutase activity could be explained as promoting antioxidant system to scavenging the reactive oxygen species. In contrast, the activity of peroxidase was notably decreased in the treated plants. Reduced peroxidase activity could be attributed to either direct effect of these particles on the molecular structure of the enzyme or plant defense system damage due to reactive oxygen species.

Evaluation of antioxidant enzymes activities and identification of intermediate products during phytoremediation of an anionic dye (C.I. Acid Blue 92) by pennywort (Hydrocotyle vulgaris).

The potential of pennywort (Hydrocotyle vulgaris) for phytoremediation of C.I. Acid Blue 92 (AB92) was evaluated. The effects of various experimental parameters including pH, temperature, dye concentration and plant weight on dye removal efficiency were investigated. The results showed that the optimal condition for dye removal were pH 3.5 and temperature 25 degree C. Moreover, the absolute dye removal enhanced with increase in the initial dye concentration and plant weight. Pennywort showed the same removal efficiency in repeated experiments (four runs) as that obtained from the first run (a 6-day period). Therefore, the ability of the plant in consecutive removal of AB92 confirmed the biodegradation process. Accordingly, a number of produced intermediate compounds were identified. The effect of treatment on photosynthesis and antioxidant defense system including superoxide dismutase, peroxidase and catalase in plant roots and leaves were evaluated. The results revealed a reduction in photosynthetic pigments content under dye treatments. Antioxidant enzyme responses showed marked variations with respect to the plant organ and dye concentration in the liquid medium. Overall, the increase in antioxidant enzyme activity under AB92 stress in the roots was much higher than that in the leaves. Nevertheless, no significant increase in malondialdehyde content was detected in roots or leaves, implying that the high efficiency of antioxidant system in the elimination of reactive oxygen species. Based on these results, pennywort was founded to be a capable species for phytoremediation of AB92-contaminated water, may be effective for phytoremediation dye-contaminated polluted aquatic ecosystems.

Floral ontogeny in Astragalus compactus (Leguminosae: Papilionoideae: Galegeae): variable occurrence of bracteoles and variable patterns of sepal initiation.

Comparative studies of floral ontogeny represent a growing field that promise to provide new insights on floral evolution. Floral ontogenetic information has been used successfully in Leguminosae for re-examining phylogenetic relationships at different levels. Using epi-illumination light microscopy, we present original ontogenetic data in Astragalus compactus, which was chosen because of its unusual arrangement of inflorescence and variable occurrence of bracteoles on flowers. Based on our results, uncommon ontogeny of the inflorescence led to the arrangement of flowers in four different positions. Variation was observed not only in the presence of bracteoles, but also in the order of sepal initiation in flowers of the same inflorescence. Surprisingly, besides the widely stated unidirectional pattern, bidirectional, sequential and an atypical unreported order were observed. High degree of overlapping between whorls and formation of two types of common primordia also were found. The variable occurrence of bracteoles suggests that the species is in an intermediate state towards fully lacking of bracteoles. We propose that the variability of the sequence of sepal initiation is possibly a consequence of the function of mechanical forces generated by surrounding leaves. Relationships between mechanical force and auxin signalling are discussed.

Using physicochemical and biological parameters for the evaluation of water quality and environmental conditions in international wetlands on the southern part of Lake Urmia, Iran.

The Kani Barazan and Yadegarlou wetlands in the southern part of Lake Urmia (Iran) have been substantially modified due to human activities and anthropogenic use. In recent years, freshwater-based eco-biological studies to recognize the quality of water resources have been greatly expanded. Microalgae and Cyanophyta are considered important bioindicators for the evaluation of water quality and wetland health worldwide. Herein, 22 microalgae and 5 Cyanophyta genera were identified in both wetlands, in which Cyanophyta has mainly caused blooms. Principal components analysis (PCA) was carried out based on links between the distribution of microalgae and Cyanophyta with physical and chemical parameters. The data showed that depth, turbidity, and the temperature had a significant influence on the microalga and Cyanophyta communities in both wetlands. Based on the biological properties, it seems that the Kani Barazan and Yadegarlou international wetlands experience meso-eutrophic conditions. The integration of the physical, chemical and biological parameters with the water quality index (WQI) revealed that both wetlands were polluted as a consequence of human activities. Moreover, a close relationship between WQI and the biological parameters was documented. Thus, we concluded that microalgae and Cyanophyta communities, their abundance patterns, and water quality changes could provide valuable data for the conservation of the Kani Barazan and Yadegarlou international wetlands.

Comparison of the toxicity of pure and samarium-doped zinc oxide nanoparticles to the green microalga Chlorella vulgaris.

Although doping of various rare earth elements such as samarium on zinc oxide nanoparticles (ZnO NPs) can noticeably improve their photocatalytic performance, it may enhance their toxicity to living organisms. Thus, the toxic impacts of samarium-doped ZnO NPs (Sm/ZnO NPs) on different organisms should be carefully evaluated. In this study, an eco-toxicological experimentation system using the green microalga Chlorella vulgaris was established to determine the potential toxicity of ZnO and Sm/ZnO NPs synthesized by polymer pyrolysis method. Accordingly, growth parameters, oxidative stress biomarkers, and morphological features of the algal cells were analyzed. Both ZnO and Sm/ZnO NPs induced a concentration-dependent cytotoxicity by reducing the cell growth, decreasing photosynthetic pigment contents, and causing deformation in the cellular morphology. Moreover, generation of excessive H2O2, increased activity of superoxide dismutase and ascorbate peroxidase, and reduction in total phenolic and flavonoid contents were observed. Catalase activity was inversely influenced by the NPs in a way that its activity significantly increased at the concentrations of 20 and 25 mg L-1 of ZnO NPs, but was lessened by all supplemented dosages (5-25 mg L-1) of Sm/ZnO NPs. Altogether, the obtained results revealed that Sm-doping can play a significant role in ZnO NP-induced toxicity on C. vulgaris cells.

Impacts of oxidants and antioxidants on the emergence and progression of Alzheimer's disease.

The brain shows a high sensitivity to oxidative stress (OS). Thus, the maintenance and homeostasis of the brain, in particular neural cells, regarding the reduction-oxidation (redox) situation is crucial for the regular function of the central nervous systems (CNS). The imbalance between the reactive oxygen species (ROS) and the cellular mechanism(s) might lead to the emergence of OS, resulting in possible cell death and tissue damages, and initiating neurodegenerative disorders (NDDs). Characterized by the cytoplasmic growth of neurofibrillary tangles and extracellular ß-amyloid plaques, Alzheimer's disease (AD) is a complex NDD that causes dementia in adult life with severe manifestations. Nuclear factor erythroid 2-related factor 2 (NRF2) is a key transcription factor that regulates the functional expression of OS-related genes and the functionality of endogenous antioxidants in response to ROS. In the case of oxidative damage, NRF2 is transferred to the nucleus and attached to the antioxidant response element (ARE), which can subsequently enhance the functional expression of the cell-protecting genes. In this review, we impart on the key mechanisms engaged in the generation of active and reactive species of endogenous and exogenous oxidants and discuss the antioxidants as the defense system of neural cells regarding the NRF2-ARE signaling path in the CNS.

Qualitative and quantitative analysis of diosmin content of hyssop (Hyssopus officinalis) in response to salinity stress.

Hyssop (Hyssopus officinalis L.) is a perennial subshrub, which is distributed across the eastern Mediterranean region to central Asia. One of the most important bioactive compounds of hyssop is diosmin, a flavone glycoside of diosmetin, with application in the field of cardiovascular therapy. Salinity as one of the most essential environmental stress factors is able to alter secondary metabolite content in plants. Therefore, we aimed to investigate the effect of salinity on the levels of total flavonoid content and diosmin in hyssop. Accordingly, salinity stress was imposed by watering plants with four different concentrations of sodium chloride (NaCl) (50, 100, 150 and 200 mM) for 4 weeks. High-performance liquid chromatography (HPLC) method was used for purification of diosmin from dried leaves and measurement of it in dried shoots. Nuclear magnetic resonance (NMR) spectroscopy was applied for determination of the structure of diosmin. The obtained results showed that high salinity levels lead to a higher amount of total flavonoid and diosmin content in treated plants. Although alteration in diosmin content was not significant in treatments up to 100 mM NaCl, higher amounts of diosmin were observed in 150 and 200 mM NaCl salinity levels. We concluded that the contents of total flavonoid and diosmin were significantly elevated after exposure of hyssop plants to salt stress conditions.

Enhancement of In Vitro Production of Volatile Organic Compounds by Shoot Differentiation in Artemisia spicigera.

Callus initiation, shoot formation and plant regeneration were established for Artemisia spicigera, a traditional medicinal plant growing in Armenia, Middle-Anatolia and Iran, and producing valuable volatile organic compounds (VOCs) that are mostly represented by monoterpenoids. Optimal callus initiation and shoot production were obtained by culture of hypocotyl and cotyledon explants on MS medium comprising 0.5 mg L-1 naphthalene acetic acid (NAA) and 0.5 mg L-1 6-benzyladenine (BA). Consequently, the shoots were transferred onto the MS media supplemented with 1 mg L-1 of indole-3-butyric acid (IBA) or 1 mg L-1 of NAA. Both types of auxin induced root formation on the shoots and the resulting plantlets were successfully grown in pots. The production of VOCs in callus tissues and regenerated plantlets was studied by gas chromatography-mass spectrometry (GC-MS) analysis. Although the potential of undifferentiated callus to produce VOCs was very low, an increased content of bioactive volatile components was observed at the beginning of shoot primordia differentiation. Intriguingly, the volatiles obtained from in vitro plantlets showed quantitative and qualitative variation depending on the type of auxins used for the rooting process. The acquired quantities based on total ion current (TIC) showed that the regenerated plantlets using 1 mg L-1 NAA produced higher amounts of oxygenated monoterpenes such as camphor (30.29%), cis-thujone (7.07%), and 1,8-cineole (6.71%) and sesquiterpene derivatives, namely germacrene D (8.75%), bicyclogermacrene (4.0%) and spathulenol (1.49%) compared with the intact plant. According to these findings, in vitro generation of volatile organic compounds in A. spicigera depends on the developmental stages of tissues and may enhance with the formation of shoot primordia and regeneration of plantlets.

Potential of the genetically transformed root cultures of Plumbago europaea for biomass and plumbagin production.

Plumbago europaea L. is the main source of plumbagin which is a well-known pharmacological active compound. In this investigation, genetically transformed roots of P. europaea were obtained by improving some factors affecting the efficiency of Agrobacterium rhizoigenes-mediated transformation such as explant type, A. rhizoigenes strain, bacterial infection period, co-cultivation period and acetosyringone concentration. The leaf, hypocotyl and stem explants from in vitro grown plantlets were infected with bacterial strains (A4, ATCC15834, MSU440 and A13). The highest transformation rate of 69.3% was achieved after 7-9 days by inoculating A. rhizogenes MSU440 strain onto the 3-week-old stem explants followed by a co-cultivation period of 2 days on a medium containing 100 µM acetosyringone. To investigate the existence of the rolB gene, polymerase chain reaction was carried out using specific primers. Effects of growth media (MS, 1/2 MS, MS-B5 and ½ MS-B5), different sucrose concentrations and illumination on biomass production and plumbagin biosynthesis in P. europaea hairy root cultures were analyzed using stem explants after infection with MSU440 strain. ½ MS-B5 liquid medium containing 30 g L-1 sucrose incubated in the dark resulted in the efficient biomass production of transformed hairy roots (12.5 g fresh weight, 1.8 g dry weight) with 3.2 mg g-1 DW plumbagin accumulation. This procedure provides a framework for large-scale cultivation of hairy roots for plumbagin production. This is the first report describing the establishment of P. europaea hairy root culture with special emphasis on plumbagin production.

Towards a new avenue for producing therapeutic proteins: Microalgae as a tempting green biofactory.

Most of the recent approved therapeutic proteins are multi-subunit biologics, which need glycosylation and disulfide bridges for their correct conformation and biological functions. Currently, there exist many protein-based drugs that are mostly produced in the Chinese hamster ovary (CHO) cells. However, this expression system appears to associate with some limitations both in upstream and downstream processing steps, including low growth rate, sensitivity to different stresses and pathogens, and time-consuming purification processes. Some microalgae species offer a suitable expression system for the production of a wide range of recombinant proteins due to their key features such as fast-growing rate, having no common pathogens with the human, being used as the human food, and providing the possibility for the large-scale production in the closed/controlled bioreactors. More importantly, the protein biosynthesis machinery of some microalgae seems to be relatively similar to those of the human and animal cells. In fact, microalgae can assemble fully functional complex proteins that can be safely used in humans. In this review, we provide comprehensive insights into the currently used expression systems for the production of therapeutic proteins and discuss the essential features of the microalgae as a novel protein expression platform.

Effect of Ag-doping on cytotoxicity of SnO2 nanoparticles in tobacco cell cultures.

SnO2 nanoparticles (NPs) are promising materials for electrochemical, catalytic, and biomedical applications due to their high photosensitivity, suitable stability characteristics, wide band gap energy potential, and low cost. Doping SnO2 NPs with metallic elements such as Ag has been used to improve their efficiency. Despite their commercial importance, the current literature lacks investigations to determine their toxic effects on plant systems. In this study, SnO2 and Ag/SnO2 NPs were synthesized using polymer pyrolysis method and characterized by means of XRD, TEM, SEM, EDX, and DLS techniques. Subsequently, the toxicity of the synthesized NPs on cell viability, cell proliferation, and a number of oxidative stress markers were measured in tobacco cell cultures. SnO2 and Ag/SnO2 NPs were found to be polygonal in shape with the size range of 10-30 nm. Both NPs induced cytotoxicity by reducing the cell viability and cell proliferation in a dose-dependent manner. Furthermore, the generation of H2O2, phenolics, flavonoids, and increased activities of superoxide dismutase (SOD) and peroxidase (POD) were observed. According to the results, Ag-doping played a key role in the induction of toxicity in tobacco cell cultures. The obtained results confirmed that SnO2 and Ag/SnO2 NPs induced cytotoxicity in tobacco cells through oxidative stress.

Designing a new generation of expression toolkits for engineering of green microalgae; robust production of human interleukin-2.

Introduction: Attributable to some critical features especially the similarity of the protein synthesis machinery between humans and microalgae, these microorganisms can be utilized for the expression of many recombinant proteins. However, low and unstable gene expression levels prevent the further development of microalgae biotechnology towards protein production. Methods: Here, we designed a novel "Gained Agrobacterium -2A plasmid for microalgae expression" (named GAME plasmid) for the production of the human interleukin-2 using three model microalgae, including Chlamydomonas reinhardtii, Chlorella vulgaris , and Dunaliella salina . The GAME plasmid harbors a native chimeric hsp70/Int-1/rbcS2 promoter, the microalgae specific Kozak sequence, a novel hybrid 2A peptide, and Int-1 and Int-3 of the rbcS2 gene in its expression cassette. Results: The obtained data confirmed that the GAME plasmid can transform the microalgae with high transformation frequency. Molecular and proteomic analyses revealed the stable and robust production of the hIL-2 by the GAME plasmid in the microalgae. According to the densimetric analysis, the microalgae can accumulate the produced protein about 0.94% of the total soluble protein content. The ELISA data confirmed that the produced hIL-2 possesses the same conformation pattern with the acceptable biological activity found naturally in humans. Conclusion: Most therapeutic proteins need post-translational modifications for their correct conformation, biological function, and half-life. Accordingly, microalgae could be considered as a cost-effective and more powerful platform for the production of a wide range of recombinant proteins such as antibodies, enzymes, hormones, and vaccines.

Cell Suspension Culture of Plumbago europaea L. Towards Production of Plumbagin.

BACKGROUND: Plumbagin is as an important bioactive secondary metabolite found in the roots of Plumbago spp. The only one species, Plumbago europaea L., grows wild in Iran. The therapeutic use of plumbagin is limited due to its insufficient supply from the natural sources as the plants grow slowly and take several years to produce quality roots. OBJECTIVES: To develop an efficient protocol for the establishment of callus and cell suspension cultures of P. europaea and to evaluate production of plumbagin in callus and cell suspension cultures of P. europaea for the first time. MATERIAL AND METHODS: Stems and leaves explants were cultured on agar solidified (7% w/v) MS media, supplemented with different combination of 2, 4-D and Kin or 6-Benzylaminopurin (BA) for callus induction. The rapid growing calli were cultured in liquid Murashige and Skoog (MS) media in agitated condition for establishing cell suspension cultures of P. europaea. Moreover, the effects of light and dark conditions on the cell growth, cell viability and plumbagin production in cell suspension cultures of P. europaea were assessed. RESULTS: Friable calli were successfully induced using stem segments of P. europaea in semisolid MS medium supplemented with 1 mg.L-1 2, 4-Dichlorophenoxy acetic acid (2, 4-D) and 0.5 mg.L-1of kinetin (Kin). Optimal cell growth was obtained when the cells were grown in MS liquid media supplemented with 1 mg.L-1 2, 4-D and 0.5 mg.L-1 kinetin with an initial cell density of ~3×105 cells per ml incubated in the dark at 25 ± 1 °C. Growth curve revealed that the maximum cell growth rate (14.83×105 cells per ml) achieved on the day 18 and the highest plumbagin content (0.9 mg.g-1 Dry Cell Weight (DCW)) in the cells was obtained at the late exponential phase under dark condition which determined by High Performance Liquid Chromatography (HPLC) technique. Based on the obtained results, cell viability remained around 82.73% during the 18 days of cell culture in darkness. These suspension cultures showed continuous and stable production of plumbagin. CONCLUSIONS: Our study suggests that cell suspension cultures of P. europaea represent an effective system for biosynthesis and production of plumbagin as a valuable bioactive compound.