Mining Tamarix ramosissima roots for endophytic growth promoting fungi to improve wheat root growth.

Endophytic fungi are commonly found in the root endosphere and can enhance plant growth through various mechanisms. The aim of this study was to isolate cultivable endophytic fungi associated with the roots of Tamarix ramosissima and to evaluate their plant growth promoting properties. About 35 isolated fungal endophytes belonging to the Ascomycota from four different genera were isolated from the endosphere of T. ramosissima: Alternaria, Aspergillus, Fusarium and Talaromyces. These fungal endophytes showed different abilities to solubilize phosphate and produce indole-3-acetic acid (IAA). The fungal isolates of T. allahabadensis (T3) and A. niger (T4) showed different efficiency in solubilizing phosphate. Almost all fungal isolates were able to produce IAA, and the highest value (0.699 µg/ml) was found in the isolate of F. solani (T11). Inoculation of wheat seeds with endophytic fungi significantly increased the initial growth of wheat roots. The results showed that inoculation with the endophytic fungus A. fumigatus T15 significantly increased root length by 75%. The extensive root system of T. ramosissima may be due to symbiosis with IAA-producing endophytic fungi, which enhance root development and water uptake in dry conditions. These fungi can also boost soil phosphorus levels, promoting plant growth.

Targeted delivery of 5-fluorouracil and shikonin by blended and coated chitosan/pectin nanoparticles for treatment of colon cancer.

Herein, 5-fluorouracil and shikonin (extracted from Fusarium tricinctum) were loaded in chitosan/pectin nanoparticle (CS/PEC-NPs), prepared by blending (B-CS/PEC-NPs) and coating (C-CS/PEC-NPs) methods. The nanoparticles characterized by Fourier Transform Infrared (FTIR), X-ray diffraction (XRD), Energy-dispersive X-ray (EDX), Scanning Electron Microscope (SEM) and Differential Light Scattering (DLS). Then, some properties of the nanoparticles such as drug release rate and the nanoparticles cytotoxicity were studied. The FTIR, XRD, EDX, SEM and DLS results showed that the nanoparticles synthesized properly with an almost spherical morphology, an average size of 82-93 nm for B-CS/PEC-NPs, an average diameter of below 100 nm (mostly 66-89 nm) for C-CS/PEC-NPs, and hydrodynamic diameter of 310-817 nm. The drug release results indicated the lower release rate of drugs for B-CS/PEC-NPs relative to C-CS/PEC-NPs at different pHs, high release rate of drugs for the nanoparticles in the simulated large intestinal fluids containing pectinase, and Korsmeyer-Peppas model for release of the drugs. The results showed more cytotoxicity of B-CS/PEC-NPs containing drugs, especially B-CS/PEC-NPs containing both drugs (B-CS/PEC/5-FU/SHK-NPs) after treating with pectinase (IC50 of 18.6 µg/mL). In conclusion, despite the limitation of C-CS/PEC-NPs for simultaneous loading of hydrophilic and hydrophobic drugs, B-CS/PEC-NPs showed suitable potency for loading and targeted delivery of the drugs.

Low UV radiation influenced DNA methylation, gene regulation, cell proliferation, viability, and biochemical differentiation in the cell suspension cultures of Cannabis indica.

The effect of low artificial Ultraviolet (UV) on the DNA methylation remains controversial. This study addresses how differential photoperiods of UV radiation affect the biochemical and molecular behaviors of Cannabis indica cell suspension cultures. The cell suspensions were illuminated with the compact fluorescent lamps (CFL), emitting a combination of 10% UVB, 30% UVA, and the rest visible wavelengths for 0, 4, 8, and 16 h. The applied photoperiods influenced cell morphological characteristics. The 4 h photoperiod was the most effective treatment for improving biomass, growth index and cell viability percentage while these indices remained non-significant in the 16 h treatment. The methylation-sensitive amplified polymorphism (MASP) assay revealed that the UV radiation was epigenetically accompanied by DNA hypermethylation. The light-treated cells significantly displayed higher relative expression of the cannabidiolic| acid synthase (CBDAS) and delta9-tetrahydrocannabinolic acid synthase (THCAS) genes about 4-fold. The expression of the olivetolic acid cyclase (OAC) and olivetol synthase (OLS) genes exhibited an upward trend in response to the UV radiation. The light treatments also enhanced the proline content and protein concentration. The 4 h illumination was significantly capable of improving the cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC) concentrations, in contrast with 16 h. By increasing the illumination exposure time, the activity of the phenylalanine ammonia-lyase (PAL) enzyme linearly upregulated. The highest amounts of the phenylpropanoid derivatives were observed in the cells cultured under the radiation for 4 h. Taken collective, artificial UV radiation can induce DNA methylation modifications and impact biochemical and molecular differentiation in the cell suspensions in a photoperiod-dependent manner.

Investigation the biological activities and the metabolite profiles of endophytic fungi isolated from Gundelia tournefortii L.

Endophytic fungi are microorganisms that are considered as a potential source of natural compounds, and can be applied in various industries. The aims of this research were molecular identification of endophytic fungi isolated from the Gundelia tournefortii stems, and investigation their biological activities as well as phenolic and fatty acid profile. Surface sterilized stems of G. tournefortii were placed on potato dextrose agar (PDA) to isolate the fungal endophytes. Genomic DNA was extracted by CTAB method, and PCR amplification was performed by ITS 1 and ITS 4 as primers. The enzyme production of endophytic fungi was determined based on the formation of a clear zone that appeared around the colonies of fungus. The anti-oxidant activity was evaluated by measuring the amount of free radicals DPPH. Also, the total phenol and flavonoid contents were measured obtained by Folin-Ciocalteu and aluminum chloride colorimetric methods, respectively. Moreover, the separation and identification of phenolic acids and fatty acids were done by HPLC and GC, respectively. Phylogenetic analysis was done based on the Internal Transcribed Spacer (ITS) region, and five isolates were identified as following: Aspergillus niger, Penicillium glabrum, Alternaria alternata, A. tenuissima, and Mucor circinelloides. Evaluation of the enzymatic properties showed that P. gabrum (31 ± 1.9 mm), and A. niger (23 ± 1.7) had more ability for producing pectinase and cellulase. The anti-oxidant activity of isolates showed that A. alternata extract (IC50 = 471 ± 29 µg/mL) had the highest anti-oxidant properties, followed by A. tenuissima extract (IC50 = 512 ± 19 µg/mL). Also, the extract of A. alternata had the greatest amount of total phenols and flavonoids contents (8.2 ± 0.4 mg GAL/g and 2.3 ± 0.3 mg QE/g, respectively). The quantification analysis of phenolic acid showed that rosmarinic acid, para-coumaric acid, and meta-coumaric acid (42.02 ± 1.31, 7.53 ± 0.19, 5.41 ± 0.21 mg/g, respectively) were the main phenolic acids in the studied fungi. The analysis of fatty acids confirmed that, in all fungi, the main fatty acids were stearic acid (27.9-35.2%), oleic acid (11.3-17.3%), palmitic acid (16.9-23.2%), linoleic acid (5.8-11.6%), and caprylic acid (6.3-10.9%). Our finding showed that endophytic fungi are a source of bioactive compounds, which could be used in various industries. This is the first report of endophytic fungi associated with G. tournefortii, which provides knowledge on their future use on biotechnological processes.

Silicon nanoparticles (SiNPs) stimulated secondary metabolism and mitigated toxicity of salinity stress in basil (Ocimum basilicum) by modulating gene expression: a sustainable approach for crop protection.

The underlying mechanisms through which silicon oxide nanoparticles (SiNPs) can confer salinity resistance to plants are poorly understood. This study explored the efficacy of supplementing nutrient solution with SiNPs (20-30 nm; 10 mg kg-1 soil) to stimulate metabolism and alleviate the risks associated with salinity (0.73 g kg-1 soil) in basil seedlings. For this purpose, variations in photosynthetic indices, proline osmoprotectant, antioxidant markers, phenylpropanoid metabolism, and transcriptional behaviors of genes were investigated. SiNPs increased shoot fresh weight (38%) and mitigated the risk associated with the salinity stress by 14%. SiNPs alleviated the inhibitory effects of salinity on the total chlorophyll concentration by 15%. The highest increase (twofold) in proline content was recorded in the SiNP-treated seedlings grown under salinity. The nano-supplement enhanced the activity of enzymatic antioxidants, including peroxidase (2.5-fold) and catalase (4.7-fold). SiNPs induced the expression of gamma-cadinene synthase (CDS) and caffeic acid O-methyltransferase (COMT) genes by 6.5- and 18.3-fold, respectively. SiNPs upregulated the eugenol synthase (EGS1) and fenchol synthase (FES) genes by six- and nine-fold, respectively. Salinity transcriptionally downregulated the geraniol synthase (GES) gene, while this gene displayed an upward trend in response to SiNPs by eight-fold. The nano-supplement transcriptionally stimulated the R-linalool synthase (LIS) gene by 3.3-fold. The terpinolene synthase (TES) gene displayed a similar trend to that of the GES gene. The highest expression (25-fold) of the phenylalanine ammonia-lyase (PAL) gene was recorded in seedlings supplemented with SiNPs. The physiological and molecular assessments demonstrated that employing SiNPs is a sustainable strategy for improving plant primary/secondary metabolism and crop protection.

Selenium nanoparticles conferred drought tolerance in tomato plants by altering the transcription pattern of microRNA-172 (miR-172), bZIP, and CRTISO genes, upregulating the antioxidant system, and stimulating secondary metabolism.

Drought stress is one of the major limiting factors for the production of tomato in Iran. In this study, the efficiency of selenate and Se nanoparticle (SeNP) foliar application on tomato plants was assessed to vestigate mitigating the risk associated with water-deficit conditions. Tomato plants were treated with SeNPs at the concentrations of 0 and 4 mg L-1; after the third sprays, the plants were exposed to water-deficit conditions. The foliar spraying with SeNPs not only improved growth, yield, and developmental switch to the flowering phase but also noticeably mitigated the detrimental risk associated with the water-deficit conditions. Gene expression experiments showed a slight increase in expression of microRNA-172 (miR-172) in the SeNP-treated plants in normal irrigation, whereas miR-172 displayed a downregulation trend in response to drought stress. The bZIP transcription factor and CRTISO genes were upregulated following the SeNP and drought treatments. Drought stress significantly increased the H2O2 accumulation that is mitigated with SeNPs. The foliar spraying with Se or SeNPs shared a similar trend to alleviate the negative effect of drought stress on the membrane integrity. The applied supplements also conferred drought tolerance through noticeable improvements in the non-enzymatic (ascorbate and glutathione) and enzymatic (catalase and peroxidase) antioxidants. The SeNP-mediated improvement in drought stress tolerance correlated significantly with increases in the activity of phenylalanine ammonia-lyase, proline, non-protein thiols, and flavonoid concentrations. SeNPs also improved the fruit quality regarding K, Mg, Fe, and Se concentrations. It was concluded that foliar spraying with SeNPs could mitigate the detrimental risk associated with the water-deficit conditions.

Methyl jasmonate conferred Arsenic tolerance in Thymus kotschyanus by DNA hypomethylation, stimulating terpenoid metabolism, and upregulating two cytochrome P450 monooxygenases.

Arsenic (As) is a highly cytotoxic element impairing normal cellular functions, and its bioremediation has become one of the environmental concerns. This study explored the molecular and physiological responses of thyme (Thymus kotschyanus) seedlings to incorporating As (0 and 10 mgl-1) and methyl jasmonate (MJ; 0 and 10 µM) into the culture medium. The MJ treatment reinforced root system and mitigated the As cytotoxicity risk. MJ contributed to hypomethylation, a potential adaptation mechanism for conferring the As tolerance. Two cytochrome P450 monooxygenases, including CYP71D178 and CYP71D180 genes, were upregulated in response to As and MJ. The MJ treatment contributed to up-regulation in the γ-terpinene synthase (TPS) gene, a marker gene in the terpenoid metabolism. The As presence reduced photosynthetic pigments (chlorophylls and carotenoids), while the MJ utilization alleviated the As toxicity. The MJ supplementation increased proline accumulation and soluble phenols. The application of MJ declined the toxicity sign of As on the concentration of proteins. The activities of peroxidase, catalase, and phenylalanine ammonia-lyase (PAL) enzymes displayed an upward trend in response to As and MJ treatments. Taken collective, MJ can confer the As tolerance by triggering DNA hypomethylation, regulating CYPs, and stimulating primary and secondary metabolism, especially terpenoid.

Alteration in the callogenesis, tropane alkaloid formation, and gene expression in Hyoscyamus niger under clinorotation.

This study aimed to investigate the effects of clinorotation induced by 2-D clinostat on the growth, tropane alkaloid production, gene expression, antioxidant capacity, and cellular defense responses in the callus tissue of Hyoscyamus niger. Callus induction was conducted by putting hypocotyl explants in the MS culture medium supplemented with 1 mgL-1 2,4-D and 1 mgL-1 BAP growth regulators. The sub-cultured calli were placed on a clinostat for 0, 3, 7, and 10 days (2.24 × 10-5 g on the edge of the callus ring). Clinorotation significantly increased callus fresh weight, dry weight, protein, carbohydrate, and proline contents compared to the control, and their maximum contents were obtained after 7 and 10 days. H2O2 level enhanced under clinorotation with a 76.3% rise after 10 days compared to control and positively affected the atropine (77.1%) and scopolamine (69.2%) productions. Hyoscyamine 6-beta hydroxylase and putrescine N-methyltransferase gene expression involved in the tropane alkaloid biosynthesis were upregulated markedly with 14.2 and 17.1-folds increase after 10 days of clinorotation, respectively. The expressions of jasmonic acid, mitogen-activated protein kinase, and ethylene-responsive element-binding transcription factor were upregulated, and the activity of peroxidase and catalase showed a 72.7 and 80% rise after 10 days. These findings suggest that microgravity can enhance callogenesis by stimulating the ROS level, which can impact the antioxidant enzymes, tropane alkaloid formation, and gene expression.

The effects of titanium dioxide (TiO2) nanoparticles on physiological, biochemical, and antioxidant properties of Vitex plant (Vitex agnus - Castus L).

Titanium dioxide nanoparticles (TiO2NPs) are widely used in agriculture in order to increase the yield and growth characteristics of plants. This study investigated the effects of TiO2NPs on photosynthetic pigments and several biochemical activities and antioxidant enzymes of the Vitex plant. Different concentrations of nanoparticles (0, 200, 400, 600 and 800 ppm) at five levels were sprayed on Vitex plants on the 30th day of the experiment. TiO2NPs at different concentrations had positive effects on root and shoot dry weight and a negative effect on leaf dry weight. The amount of chlorophyll increased with the concentration of TiO2NPs; however, the amount of chlorophyll b showed a decreasing trend while the total chlorophyll had a constant trend. The highest amount of soluble sugar was obtained in the treatment of 200 ppm nanoparticles. The application of TiO2NPs did not have any effect on the content of proline and soluble proteins of Vitex plant. The effects of foliar TiO2NPs, compared to the control, showed a significant increase in the activity of antioxidant enzymes. In general, TiO2NPs had a favorable effect on dry matter production and some antioxidant and biochemical properties of the Vitex plant.

Preparation, characterization, and Co-delivery of cisplatin and doxorubicin-loaded liposomes to enhance anticancer Activities.

Ovarian cancer stands as a leading cause of cancer-related deaths among women globally. This malignancy has hindered successful treatment attempts due to its inherent resistance to chemotherapy agents. The utilization of cisplatin and doxorubicin-loaded liposomes emerges as a strategically advantageous approach in the realm of biomedical applications. This strategy holds promise for augmenting drug efficacy, mitigating toxicity, refining pharmacokinetics, and facilitating versatile drug delivery while accommodating combination therapies. In pursuit of scholarly investigations, the eminent databases, including PubMed/MEDLINE, ScienceDirect, Scopus, and Google Scholar, were meticulously scrutinized. Within this study, a nano-liposomal formulation was meticulously designed to serve as a co-delivery system. This system was optimized by varying lipid concentrations, hydration time, and DSPC: cholesterol molar ratios to efficiently encapsulate and load doxorubicin (DOX) and cisplatin (CIS) to overcome drug resistance problems. The Lipo (CIS + DOX) formulation underwent rigorous characterization including dimensions, entrapment efficiencies and drug release kinetics. Notably, the entrapment efficiency of cisplatin and doxorubicin loaded liposomal nanoparticles was an impressive 85.29 ± 1.45 % and 73.62 ± 1.70 %, respectively. Furthermore, Lipo (CIS + DOX) drug release kinetics exhibited pH-dependent properties, with lower drug release rates at physiological pH (7.4) than acidic (pH 5.4). Subsequent cytotoxicity assays revealed the enhanced biocompatibility of dual-drug liposomes with HFF cells compared to free drug combinations. Impressively, CIS and DOX-loaded liposomes induced significant cytotoxicity against A2780 in comparison to free drugs and combinatorial free drugs. Furthermore, the CIS and DOX-loaded liposome showed induced apoptotic potential and cell cycle arrest in A2780 compared to CIS, DOX, and their combination (CIS + DOX). Combining CIS and DOX via liposomal nanoparticles introduces a promising therapeutic avenue for addressing ovarian cancer. These nano-scale carriers hold the potential for attenuating the untoward effects of singular drugs and their attendant toxicities.

Carboxylic acid-functionalized multiwalled carbon nanotubes (COOH-MWCNTs) improved production of atropine in callus of Datura inoxia by influencing metabolism, gene regulation, and DNA cytosine methylation; an in vitro biological assessment.

Atropine is a well-known tropane alkaloid commonly employed in medicine class called anticholinergics. This study intends to address biochemical and molecular responses of Datura inoxia calluses to fortifying culture medium with carboxylic acid-functionalized multi-walled carbon nanotubes (COOH-MWCNTs). The application of MWCNTs influenced callogenesis performance and biomass in a dose-dependent manner. The MWCNT at 5 mgL-1 resulted in the highest biomass of calluses by 57%. While, MWCNTs at high concentrations were accompanied by cytotoxicity. On the other hand, MWCNTs at concentrations above 100 mgL-1 exhibited cytotoxicity, decreased callogenesis performance, and reduced Atropine biosynthesis. The MWCNTs increased the activity of phenylalanine ammonia-lyase (PAL) and catalase enzymes. The concentrations of proline and soluble phenols displayed upward trends in response to using MWCNTs. According to the HPLC assessment, enriching culture medium with MWCNTs at 5 mgL-1 elicited Atropine production in calluses by 64%. The quantitative PCR assessment referred to the upregulation in the transcription of the PAL gene. The expression of ornithine decarboxylase (ODC) and putrescine N-methyltransferase 1 (PMT) genes were also upregulated in calluses cultured in a medium supplemented with MWCNTs. Methylation Sensitive Amplification Polymorphism (MSAP) technique indicated that employing MWCNTs altered the DNA methylation profile, reflecting epigenetic modification. Overall, engineering plant cells with MWCNTs as a nano-elicitor can be suggested for large-scale synthesis of industrially-valuable secondary metabolites.

Corona discharge plasma stimulated production of atropine in callus of Datura inoxia by DNA hypomethylation and gene regulation: a novel technology for plant cell and tissue culture.

Few investigations have tested the practical use of cold plasma as a novel technology to meet the requirements in the plant cell and tissue culture field. To fill the knowledge gap, we intend to respond to the question of whether plasma priming influenced DNA ultrastructure and the production of atropine (a tropane alkaloid) in Datura inoxia. Calluses were treated with the corona discharge plasma at time durations ranging from 0 to 300 s. Significant increases (about 60%) in biomass were observed in the plasma-primed calluses. The plasma priming of calluses enhanced the accumulation of atropine about 2-fold. The plasma treatments increased proline concentrations and soluble phenols. The drastic increases in the activity of the phenylalanine ammonia-lyase (PAL) enzyme resulted from the applied treatments. Likewise, the plasma treatment of 180 s upregulated the expression of the PAL gene by 8-fold. Also, the expression of the ornithine decarboxylase (ODC) and tropinone reductase I (TR I) genes were stimulated by 4.3-fold and 3.2-fold, respectively, in response to the plasma treatment. The putrescine N-methyltransferase gene displayed a similar trend to that of TR I and ODC genes following the plasma priming. Methylation sensitive amplification polymorphism method was employed to explore the plasma-associated epigenetic changes in DNA ultrastructure. The molecular assessment referred to DNA hypomethylation, validating an epigenetic response. This biological assessment study validates the hypothesis that plasma priming of callus is an efficient, cost-effective, and eco-friendly tool to enhance callogenesis efficiency, elicit metabolism, affect gene regulation, and modify chromatin ultrastructure in D. inoxia.

Synergetic effect of water deficit and arbuscular mycorrhizal symbiosis on the expression of aquaporins in wheat (Triticum aestivum L.) roots: insights from NGS RNA-sequencing.

Wheat (Triticum aestivum) is one of the most important crops in the world. This investigation was attempted to evaluate the transcriptional responses of aquaporins (AQPs) to the mycorrhizal inoculation and/or water deficit conditions in wheat to clarify how the arbuscular mycorrhizal symbiosis can contribute to the modulation of water homeostasis. The wheat seedlings were subjected to the water deficiency, and mycorrhizal inoculation using arbuscular fungus Funneliformis mosseae and Illumina RNA-Seq analyses confirmed that aquaporins expressed differentially in response to both the irrigation levels and mycorrhizal colonization. Results of this study showed that only 13% of the studied AQPs were responsive to water deficit with a tiny fraction (3%) being up-regulated. Mycorrhizal inoculation had a greater impact on the expression of AQPs with ca. 26% being responsive, ca. 4% of which were up-regulated. The samples with arbuscular mycorrhizal inoculation yielded more root and stem biomass. Water deficit and mycorrhizal inoculation caused different AQPs to be up-regulated. The effect of mycorrhizal inoculation on the expression of AQPs was intensified by applying water deficiency with 32% of studied AQPs being responsive, 6% of which up-regulated. We also found that the overexpression of three genes TaNIP1-10, TaNIP3-3, and TaNIP3-4 was chiefly triggered by mycorrhizal inoculation. Our results show that water deficit has a lower impact on the expression of aquaporins compared to what the arbuscular mycorrhizal inoculation has; water deficit and arbuscular mycorrhizal inoculation mainly cause the down-regulation of the aquaporins, and water deficit and the arbuscular inoculation have synergetic effects. These findings could improve our knowledge of how arbuscular mycorrhizal symbiosis can contribute to the modulation of water homeostasis. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01285-w.

Non-thermal plasma improved callogenesis performance and elicited the production of cannabinoids by modifying DNA methylome, expression of WRKY1 and ERF1B transcription factors, and expression of genes that contributed to the biosynthesis of cannabinoids.

The current decade has witnessed notable advancement towards the utilization of non-thermal (cold) plasma in multidisciplinary fields such as plant sciences. This study intends to validate whether cold plasma contributes to improving callogenesis performance and eliciting the production of cannabinoids in cannabis. The cannabis-derived calli were treated with plasma at different exposure times, including 0, 60, 120, and 180 s. The plasma priming improved the callogenesis performance and callus biomass by an average of 46.6%. The molecular assessment (MSAP method) validated how the plasma priming is epigenetically associated with variation in DNA methylome in the cannabis calli. The cold plasma treatments transcriptionally upregulated the expression of WRKY1 and ERF1B transcription factors by averages of 3.5- and 3.8-fold. The plasma treatment also stimulated the transcription of OLS, OAC, CBGAS, CBDAS, and THCAS genes involved in the biosynthesis of cannabinoids. The HPLC assessment proved the high potency of cold plasma to enhance the synthesis of cannabinoids, including Cannabigerol (CBG), Cannabidiol (CBD), and cannabinol (CBN). The plasma-primed calli contained higher concentrations of proteins (56%), proline (38%), and soluble phenols (40%). The activities of peroxidase and catalase enzymes showed a similar upward trend in response to the plasma. The profound increase in the concentrations of soluble sugars resulted from the plasma treatments. The plasma priming of calli contributed to the significant upregulation in the activity of the phenylalanine ammonia-lyase enzyme. This biological assessment study validates the high potency of plasma priming to elicit the biosynthesis of cannabinoids in cannabis calli.

Zinc oxide nanoparticles (ZnONPs) influenced seed development, grain quality, and remobilization by affecting the transcription of microRNA 171 (miR171), miR156, NAM, and SUT genes in wheat (Triticum aestivum): a biological advantage and risk assessment study.

Limited studies have been conducted on the role of microRNAs (miRs) and transcription factors in regulating plant cell responses to nanoparticles. This study attempted to address whether the foliar application of zinc oxide nanoparticles (ZnONPs; 0, 10, 25, and 50 mgL-1) can affect miRs, gene expression, and wheat grain quality. The seedlings were sprayed with ZnONPs (0, 10, 25, and 50 mgL-1) or bulk counterpart (BZnO) five times at 72 h intervals. The application of ZnONPs at 10 mgL-1 increased the number of spikelets and seed weight, while the nano-supplement at 50 mgL-1 was accompanied by severe restriction on developing spikes and grains. ZnONPs, in a dose-dependent manner, transcriptionally influenced miR156 and miR171. The expression of miR171 showed a similar trend to that of miR156. The ZnONPs at optimum concentration upregulated the NAM transcription factor and sucrose transporter (SUT) at transcriptional levels. However, the transcription of both NAM and SUT genes displayed a downward trend in response to the toxic dose of ZnONPs (50 mgL-1). Utilization of ZnONPs increased proline and total soluble phenolic content. Monitoring the accumulation of carbohydrates, including fructan, glucose, fructose, and sucrose, revealed that ZnONPs at 10 mgL-1 modified the source/sink communication and nutrient remobilization. The molecular and physiological data revealed that the expression of miR156 and miR171 is tightly linked to seed grain development, remobilization of carbohydrates, and genes involved in nutrient transportation. This study establishes a novel strategy for obtaining higher yields in crops. This biological risk assessment investigation also displays the potential hazard of applying ZnONPs at the flowering developmental phase.

Simulated microgravity contributed to modification of callogenesis performance and secondary metabolite production in CannabisIndica.

In vitro plant culture paves the way for meeting the industrial demand of pharmaceutically valuable secondary metabolites. This study intends to monitor how callus cells of Cannabis indica respond to the simulated microgravity (clinorotation; a Man-made technology). Callus initiation resulted from the culture of the leaf explant in a medium supplemented with kinetin (0.5 mgL-1) and 2, 4-D (2 mgL-1). Calli were treated with microgravity at three exposure times (0, 3, and 5 days). The microgravity treatments increased callus biomass about 2.5-fold. The clinorotation treatments transcriptionally induced the olivetolic acid cyclase (OAC) and olivetol synthase (OLS) genes about 6.2-fold. The tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) genes displayed a similar upward trend in response to microgravity. The applied treatments also stimulated the expression of the ethylene-responsive element-binding proteins (ERF1B) and WRKY1 transcription factors by an average of 7.6-fold. Moreover, the simulated microgravity triggered epigenetic modification in the DNA methylation profile. The HPLC-based assessment validated the high efficacy of the clinorotation treatments to increase the concentration of cannabinoids, including Cannabigerol (CBG) and Cannabidiol (CBD). However, the clinorotated calli contained a lower concentration of Tetrahydrocannabinol (THC) than the control group. The microgravity treatments increased concentrations of proline (79%), soluble sugars (61.3%), and proteins (21.4%) in calli. The biochemical assessment revealed that the clinorotation treatments slightly increased H2O2 concentration. The upregulation in the activities of peroxidase, catalase, and phenylalanine ammonia-lyase enzymes resulted from the microgravity treatments. Both HPLC and molecular assessments validated the significant efficacy of microgravity to enhance the production of cannabinoids.

Expression and characterization of hemagglutinin-neuraminidase protein from Newcastle disease virus in Bacillus subtilis WB800.

BACKGROUND: Newcastle disease virus (NDV) belongs to the genus Avaluvirus and Paramyxoviridae family, and it can cause acute, highly contagious Newcastle disease in poultry. The two proteins, haemagglutinin neuraminidase (HN) and Fusion (F), are the main virulence factor of the virus and play an essential role in immunogenicity against the virus. In most paramyxoviruses, the F protein requires HN protein to fuse the membrane, and HN proteins substantially enhance the viruses' fusion activity. RESULTS: The present study describes the successful cloning and expression of HN protein from NDV in Bacillus subtilis WB800 using the modified shuttle vector pHT43. HN coding sequence was cloned into the pGet II vector. It was then subcloned into the PHT43 shuttle vector and transferred to Escherichia coli for replication. The recombinant plasmid was extracted from E. coli and used to transform B. subtilis by electroporation. After induction of recombinant B. subtilis by IPTG, total cell protein and the protein secreted into the media were analysed through a time course using SDS-PAGE. The expressed HN protein was purified using cation exchange chromatography followed by metal affinity chromatography, using the 6× His epitope introduced at the carboxyl terminus of the recombinant protein. The accuracy of the PHT43-HN construct was confirmed by sequencing and enzymatic digestion. SDS-PAGE results showed that the recombinant HN protein was successfully expressed and secreted into the medium. Moreover, the purified HN protein showed neuraminidase activity with characteristics similar to the indigenous HN NDV protein. B. subtilis is a free endotoxin host that could be a favourite prokaryotic platform for producing the recombinant HN protein. CONCLUSION: The establishment of this expression and purification system has allowed us to explore further the biochemical characteristics of HN protein and obtain material that could be suitable for a new production of NDV candidate vaccine with high immunogenicity.

Molecular evaluation and genetic characterisation of Newcastle disease virus's haemagglutinin-neuraminidase protein isolated from broiler chickens in Iran.

BACKGROUND: Newcastle disease (ND) virus (NDV) is one of the major pathogens in poultry farms that causes severe economic damages to the poultry industry, especially broiler chicken and turkey farms. Despite the endemicity of ND and its many epidemics in the country, the nature of the Iranian strain of the Newcastle virus is still largely unknown. This study aimed to characterise and evaluate NDV isolates obtained from commercial poultry farms in Iran in 2019 through haemagglutinin-neuraminidase (HN) gene sequencing. METHOD: HN gene of each NDV isolate was amplified and sequenced using specific primers followed by phylogenetic analysis of full length of HN gene open reading frame and amino acid (aa) sequence of HN. RESULTS: Phylogenetic analysis of the HN gene showed that the virus is very closely related to genotypes VII and III. Analysis of HN gene nucleotide sequences showed that all isolates encode proteins with a length of 571 aa. CONCLUSION: Results of the present study are useful for a better understanding of molecular epidemiology of indigenous NDV strains and determining important molecular differences between fields and commonly used vaccine strains related to main immunogenic proteins.

Evaluation of the Potential Effects of Retinol and Alginate/Gelatin-Based Scaffolds on Differentiation Capacity of Mouse Mesenchymal Stem Cells (MSCs) into Retinal Cells.

Background and Objectives: Retinal stem cells (RSCs) resided in ciliary epithelium have shown to possess a high capacity to self-renew and differentiate into retinal cells. RSCs could be induced to differentiate when they are exposed to stimuli like natural compounds and suitable contexts such as biomaterials. The aim of this study was to examine the effects of Retinol and alginate/gelatin-based scaffolds on differentiation potential of mesenchymal stem cells (MSCs) originated from mouse ciliary epithelium. Methods and Results: MSCs were extracted from mouse ciliary epithelium, and their identity was verified by detecting specific surface antigens. To provide a three-dimensional in vitro culture system, 2% alginate, 0.5% gelatin and the mixed alginate-gelatin hydrogels were fabricated and checked by SEM. Retinol treatment was performed on MSCs expanded on alginate/gelatin hydrogels and the survival rate and the ability of MSCs to differentiate were examined through measuring expression alterations of retina-specific genes by ICC and qPCR. The cell population isolated from ciliary epithelium contained more than 93.4% cells positive for MSC-specific marker CD105. Alginate/gelatin scaffolds showed to provide an acceptable viability (over 70%) for MSC cultures. Retinol treatment could induce a high expression of rhodopsin protein in MSCs expanded in alginate and alginate-gelatin mixtures. An elevated presentation of Nestin, RPE65 and Rhodopsin genes was detected in retinol-treated cultures expanded on alginate and alginate-gelatin scaffolds. Conclusions: The results presented here elucidate that retinol treatment of MSCs grown on alginate scaffolds would promote the mouse ciliary epithelium-derived MSCs to differentiate towards retinal neurons.

Nutlet micromorphology and character evolution of Lappula species (Boraginaceae) and its systematic implications.

BACKGROUND: The macro/micro-morphology of nutlets in 11 species (and 22 accessions) of the Boraginaceae family was investigated using stereomicroscope and scanning electron microscopy to evaluate the taxonomic relevance of the traits. To evaluate the phylogenetic significance of the character evolution, phylogenetic analysis was carried out by comparing available DNA sequence data from GenBank with selected original nutlet data. RESULTS: The Rochelieae nutlets' shape varied from ovoid (ovoid, ovoid-triangular, and ovoid-rectangular) to pyramid. Six major patterns were recognized based on the nutlet ultrastructure characters. Rocheliae is characterized by a transition from "without appendage" to "with tubercles and prickles" on the nutlet disk, and also via a shift from "lack of prickles" to "glossy prickles". CONCLUSIONS: The results show that the nutlet ultrastructure pattern of Rochelieae is systematically informative at the genus level, but not at the species level. Findings demonstrated that glochid is not an ancestral trait but is a synapomorphy and the transition to this trait occurred in the genus Lappula. The close boundary of nutlet microstructures between L. barbata and L. microcarpa has been discussed.