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Abstract Background Preserving plant genetic resources is essential for tackling global food security challenges. Effectively meeting future agricultural demands requires comprehensive and efficient assessments of genetic diversity in breeding programs and germplasm from gene banks. This research investigated the diversity of pheno-morphological traits, along with the fatty acid and tocopherol content and composition, in 135 double haploid lines of camelina. Results The number of sub-branches, siliques number of main-branch and sub-branch, and seeds number in siliques of the main-branch displayed notable coefficients of variation with values of 33.19%, 30.32%, 29.23%, and 23.81% respectively. Within the current investigation, the measurements of height, sub-branch number, and thousand seed weight varied from 53.50 to 86.50 cm, 3.50 to 14, and 0.73 to 1.52 g, respectively. The analysis unveiled that the average content of omega-3, omega-6, and omega-9 fatty acids in the examined lines was approximately 33%, 20%, and 17%, respectively. The total tocopherol content varied between 675 and 877 ppm, predominantly consisting of gamma-tocopherol, which accounted for over 95% of the total content. The fatty acid C18:2 displayed a markedly strong positive correlation with alpha-tocopherol (0.99**), while C18:3 exhibited positive correlations with gamma-tocopherol (0.98**) and total tocopherol (0.98**). Furthermore, a positive correlation was evident between C20:1 and delta-tocopherol (0.98**). The scrutinized lines, specifically lines 35, 72, 94, and 126 demonstrated notable attributes regarding yield and yield components. Conversely, in the realm of biochemical traits, lines 35, 66, 47, 30, 65, 135, 83, 27, 4, 77, 62, 81, and 93 stood out for their elevated potential. The gene expression analysis related to the tocopherol biosynthesis pathway revealed distinct expression patterns. Specifically, the VTE1 gene exhibited the highest level of expression. In contrast, the VTE3 gene displayed the lowest level of expression compared to other genes. Conclusions The study’s findings hold great potential for improving food security by enabling the selection of superior camelina parent plants based on specific traits. This approach can drive the development of high-yielding varieties with enhanced nutritional value and better-quality camelina oil.

Circular ribonucleic acids (CircRNAs) represent a category of noncoding RNAs characterized by a circular structure bonded through covalent bonds. Unlike linear RNAs, CircRNAs are not affected by exonucleases and exhibit more stable expression. Research suggests that CircRNAs may have the ability to be translated through the internal ribosome entry sites (IRESs) mechanism and it could enable CircRNAs to produce proteins despite lacking a 5' cap structure. They have gained attention due to their diverse functions, including acting as miRNA sponges, participating in complex gene expression regulation, and disease markers. CircRNAs play crucial roles in various biological processes, including cancer development and progression. These cyclic molecules have garnered significant interest for their diverse effects in humans. Furthermore, CircRNAs have been implicated in human genetic diseases, offering insights into potential diagnostic and therapeutic avenues. They are formed through back-splicing of pre-mRNA transcripts, resulting in covalently closed loops. Recent studies have highlighted the significance of CircRNAs in cancer biology, such as their involvement in modulating gene expression, acting as micro RNA (miRNA) sponges, and regulating cellular processes. There are some detection methods including computational models, experimental techniques, and advanced RNA-based and CLIP-Seq-based methods. This review could help to understand the key points regarding biogenesis, biological function, literature trend and biological function of CircRNAs.

Abstract Saffron (Crocus sativus L.) is being embraced as the most important medicinal plant and the commercial source of saffron spice. Despite the beneficial economic and medicinal properties of saffron, the regulatory mechanism of the correlation of TFs and genes related to the biosynthesis of the apocarotenoids pathway is less obvious. Realizing these regulatory hierarchies of gene expression networks related to secondary metabolites production events is the main challenge owing to the complex and extensive interactions between the genetic behaviors. Recently, high throughput expression data have been highly feasible for constructing co-regulation networks to reveal the regulated processes and identifying novel candidate hub genes in response to complex processes of the biosynthesis of secondary metabolites. Herein, we performed Weighted Gene Co-expression Network Analysis (WGCNA), a systems biology method, to identify 11 regulated modules and hub TFs related to secondary metabolites. Three specialized modules were found in the apocarotenoids pathway. Several hub TFs were identified in notable modules, including MADS, C2H2, ERF, bZIP, HD-ZIP, and zinc finger protein MYB and HB, which were potentially associated with apocarotenoid biosynthesis. Furthermore, the expression levels of six hub TFs and six co-regulated genes of apocarotenoids were validated with RT-qPCR. The results confirmed that hub TFs specially MADS, C2H2, and ERF had a high correlation (P

Abstract Micro RNAs (miRNAs, miRs) and relevant networks might exert crucial functions during differential host cell infection by the different Leishmania species. Thus, a bioinformatic analysis of microarray datasets was developed to identify pivotal shared biomarkers and miRNA-based regulatory networks for Leishmaniasis. A transcriptomic analysis by employing a comprehensive set of gene expression profiling microarrays was conducted to identify the key genes and miRNAs relevant for Leishmania spp. infections. Accordingly, the gene expression profiles of healthy human controls were compared with those of individuals infected with Leishmania mexicana, L. major, L. donovani, and L. braziliensis. The enrichment analysis for datasets was conducted by utilizing EnrichR database, and Protein–Protein Interaction (PPI) network to identify the hub genes. The prognostic value of hub genes was assessed by using receiver operating characteristic (ROC) curves. Finally, the miRNAs that interact with the hub genes were identified using miRTarBase, miRWalk, TargetScan, and miRNet. Differentially expressed genes were identified between the groups compared in this study. These genes were significantly enriched in inflammatory responses, cytokine-mediated signaling pathways and granulocyte and neutrophil chemotaxis responses. The identification of hub genes of recruited datasets suggested that TNF, SOCS3, JUN, TNFAIP3, and CXCL9 may serve as potential infection biomarkers and could deserve value as prognostic biomarkers for leishmaniasis. Additionally, inferred data from miRWalk revealed a significant degree of interaction of a number of miRNAs (hsa-miR-8085, hsa-miR-4673, hsa-miR-4743-3p, hsa-miR-892c-3p, hsa-miR-4644, hsa-miR-671-5p, hsa-miR-7106-5p, hsa-miR-4267, hsa-miR-5196-5p, and hsa-miR-4252) with the majority of the hub genes, suggesting such miRNAs play a crucial role afterwards parasite infection. The hub genes and hub miRNAs identified in this study could be potentially suggested as therapeutic targets or biomarkers for the management of leishmaniasis.

Abstract Background Quantifying gene expression is a critical aspect of applied genomics research across all organisms, and real-time PCR has emerged as a powerful tool for this purpose. However, selecting appropriate internal control genes for data normalization presents specific challenges. This study aimed to identify suitable reference genes for gene expression analysis under various conditions, encompassing salinity, low and high-temperature stresses, and different elicitor treatments. These treatments included titanium dioxide, cold plasma, 24-epibrassinolide, and melatonin, resulting in a total of 13 unique treatments and 148 treatment combinations applied to fenugreek plants. Results As per the analysis performed with the BestKeeper tool, EEF-1α, and GAPDH were recognized as the most stable reference genes under the majority of conditions. Furthermore, the GeNorm and NormFinder tools identified β-tubulin and EEF-1α as the most stable reference genes. The findings of this research demonstrated that, although the stability of three reference genes expression was acceptable in almost all evaluated treatments, fluctuations in their expression were observed under the treatments of cold stress with TiO2 NPs application, cold plasma application with salinity stress, and cold plasma application with high-temperature stress compared to others. Simultaneously, the GeNorm analysis results demonstrated that in the mentioned treatments, relying on only one reference gene is inadequate. To corroborate the results, we examined the expression profile of the SSR gene, a pivotal gene in diosgenin biosynthesis, under all investigated treatments and treatment combinations. The outcomes suggested that employing stable reference genes yielded highly consistent results. Conclusions The varying expression patterns of the target genes emphasize the crucial need for precise optimization of experimental conditions and selecting stable reference genes to achieve accurate results in gene expression studies utilizing real-time PCR. These findings offer valuable insights into the selection of appropriate reference genes for gene expression analysis under diverse conditions using real-time PCR.

MicroRNAs (miRNAs) are small (~22 nucleotides) non-coding endogenous RNA molecules that negatively regulate gene expression at the post-transcriptional level by degrading the target protein-coding mRNA genes or suppressing translation in plants, which consequently participate in a variety of biological and metabolic processes in both animals and plants. Detection of miRNAs is chiefly carried out by microarray, real-time-PCR, northern blot, and bioinformatics approaches. Bioinformatics or in silico-based approaches are the easiest and cheapest ways to identify desired miRNAs. In this study, several miRNAs in Echinophora platyloba were identified, and their potential roles were reported. E. platyloba, which belongs to the Umbelliferae family, is an endemic plant in Iran found in the Kermanshah, Hamedan, and Lorestan provinces; it has important medicinal uses such as cytotoxic activity in breast cancer, treatment of dysmenorrhea, central and peripheral analgesic effects, and hepatoprotective effects on acute acetaminophen-induced liver injuries. To this end, the RNA was extracted from E. platyloba leaf and sent to the Beijing genome institute for RNA sequencing. After quality control, low-quality data was filtered, and de novo assembly was performed. Detection of miRNAs was then performed by miRDeep (v37) and miRBase tools. Accordingly, we identified seven miRNAs from the leaf dataset, and their secondary structures were evaluated. Target genes of the detected miRNAs were identified through the psRNA target website.

Abstract Background Leishmania infantum is the major causative agent of visceral leishmaniasis in Mediterranean regions. Isoenzyme electrophoresis (IE), as a biochemical technique, is applied in the characterization of Leishmania species. The current study attempted to investigate the isoenzyme patterns of logarithmic and stationary promastigotes and axenic amastigotes (amastigote-like) of L. infantum using IE. The antioxidant activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX) was also checked in the aforementioned forms. Method After L. infantum cultivation and obtaining logarithmic and stationary promastigotes, axenic amastigotes were achieved by incubation of stationary promastigotes at 37 °C for 48 h. The lysate samples were prepared and examined for six enzymatic systems including glucose-6-phosphate dehydrogenase (G6PD), nucleoside hydrolase 1 (NH1), malate dehydrogenase (MDH), glucose-phosphate isomerase (GPI), malic enzyme (ME), and phosphoglucomutase (PGM). Additionally, the antioxidant activity of SOD and GPX was measured. Results GPI, MDH, NH1, and G6PD enzymatic systems represented different patterns in logarithmic and stationary promastigotes and axenic amastigotes of L. infantum. PGM and ME showed similar patterns in the aforementioned forms of parasite. The highest level of SOD activity was determined in the axenic amastigote form and GPX activity was not detected in different forms of L. infantum. Conclusion The characterization of leishmanial-isoenzyme patterns and the measurement of antioxidant activity of crucial antioxidant enzymes, including SOD and GPX, might reveal more information in the biology, pathogenicity, and metabolic pathways of Leishmania parasites and consequently drive to designing novel therapeutic strategies in leishmaniasis treatment.

ObjectivesThe research was conducted to study changes in expression pattern of some genes involved in drought tolerance due to the problem of terminal drought stress in rainfed lentil agriculture in Iran.Material and methodsIn order to identify the consensus sequence of the coding sequence (CDS) of selected lentil genes; Aquaporin PIP2, Fructose bisphosphate aldolase, Oxygen-evolving enhancer protein2, WRKY1 and Xyloglucan endotransglucosylase/hydrolase, expressed sequence tag (EST) and reads were identified through similar ortholog sequences in other leguminosae plants. 90, 60 and 30 percent of field capacity were considered as control, medium and severe stress respectively. RNA was extracted from both control and severe drought stress treated plants leaf samples, cDNA was synthesized from RNA samples. Expression profiles of the genes, were determined via real time PCR technique. To represent the effect of drought stress on lentil plant, some morphological, physiological and biochemical characteristics were measured.ResultsCharacteristics like plant height, shoot fresh weight, root fresh weight, shoot dry weight, root dry weight, relative water content, leaf area index, chlorophyll and protein content decreased due to drought stress, leaf trichome density, electrolyte leakage index, proline and carbohydrate content, malondialdehyde and hydrogen peroxide increased in drought stress. Analysis of real time PCR demonstrated expression of AQUAPIP2، FBA and WRKY1 genes were increased and expression of XTH and OEE2 genes were decreased under severe drought stress condition.ConclusionsThe results show that AQUAPIP2, FBA and WRKY1 genes are involved in drought tolerance. Due to higher expression of AQUAPIP2 and WRKY1 genes in the tolerant genotype, reduction of RWC in this genotype is less. Less increase in hydrogen peroxide in the tolerant genotype, partly influenced by higher expression of AquaPIP2 gene and higher activity of antioxidant enzymes, which was influenced by higher expression of AquaPIP2 gene in tolerant genotype than susceptible genotype. The increase in carbohydrate content in both genotypes was partially affected by FBA gene expression.

The chemical constituents of essential oils of Datura stramonium L. and D. innoxia (family Solanaceae) are being reported. In this study, the collected seeds of Ardabil and Urmia were planted in grow bags and placed for three months in a greenhouse of the College of Agriculture, Tarbiat Modares University in Tehran. Under greenhouse conditions, the average air temperature was 25 °C. Air-dried areal parts of D. stramonium and D. innoxia were prepared and their essential oils (EOs) were isolated by hydrodistillation for 3 h in a Clevenger-type apparatus. EOs content and composition were examined with gas chromatography techniques. The EOs yield of D. stramonium and D. innoxia were 0.03% and 0.02% (v/w), respectively. The major compounds in the D. stramonium EOs were Camphor (29.9%), Selin-11-en-4alpha-ol (13.0%), and Borneol (10.4%). Those in the D. innoxia were mostly fatty alkanes, including n-Decane (19.99%), 7-Pentadecyne (11.78%), and Linoleic acid (6.13%). The other important components identified in the EOs of Datura were Dihydrocitronellol, n-Dodecane, Isobornyl acetate, Germacrene D, and n-Tetradecane. Generally, the EOs of the areal parts in D. stramonium showed oxygenated monoterpenes.

Abstract High-temperature stress is widely considered a main plant-growth-limiting factor. The positive effects of 24-epibrassinolide (EBR) as analogs of brassinosteroids (BRs) in modulating abiotic stresses have led this hormone to be referred to as a growth regulator in plants. The current study highlights the influence of EBR on enhancing tolerance to high-temperature and altering the diosgenin content in fenugreek. Different amounts of EBR (4, 8, and 16 μM), harvesting times (6, and 24 h), as well as temperature regimes (23 °C, and 42 °C) were, used as treatments. EBR application under normal temperature and high-temperature stress resulted in decreased malondialdehyde content and electrolyte leakage percentage, while the activity of antioxidant enzymes improved significantly. Exogenous EBR application possibly contributes to activating the nitric oxide, H2O2, and ABA-dependent pathways, enhancing the biosynthesis of abscisic acid and auxin, and regulating the signal transduction pathways, which raises fenugreek tolerance to high-temperature. The SQS (eightfold), SEP (2.8-fold), CAS (11-fold), SMT (17-fold), and SQS (sixfold) expression, considerably increased following EBR application (8 μM) compared to the control. Compared to the control, when the short-term (6 h) high-temperature stress was accompanied by EBR (8 μM), a sixfold increase in diosgenin content was achieved. Our findings highlight the potential role of exogenous 24-epibrassinolide in mitigating the high-temperature stress in fenugreek by stimulating the biosynthesis processes of enzymatic and non-enzymatic antioxidants, chlorophylls, and diosgenin. In conclusion, the current results could be of utmost importance in breeding or biotechnology-based programs of fenugreek and also in the researches related to the engineering of the biosynthesis pathway of diosgenin in this valuable plant.

Abstract Redroot Pigweed (Amaranthus retroflexus L.) is an important weed that is highly competitive with common bean. Photosynthetic pigments, the activity of antioxidant enzymes, the relative expression of a number of antioxidant enzyme and light response genes, were studied in three of common bean cultivars and in V4 and R7 stages under Redroot Pigweed free and infested. The presence of weeds reduced the content of chlorophyll, relative chlorophyll and anthocyanin of common bean leaves. With the increase of weed competition, the expression of antioxidant genes and enzymes increased, which indicates the increase of their activity in order to reduce the amount of reactive oxygen species. Among the studied antioxidant enzymes, the activity of catalase and ascorbate peroxidase produced in the leaves was higher than that of superoxide dismutase. With the increase of weed interference, the expression of phytochrome interacting factor 3 (PIF3) gene as a positive regulator of light signals is increased and the expression of phytochrome rapidly regulated1 (PAR1) gene as a negative regulator is decreased. Chlorophyll a/b-binding protein (CAB1) and auxin-responsive protein IAA8 (IAA8) genes also down-regulated with increasing competition. Along with the decrease of CAB expression in the conditions of competition with weeds, the chlorophyll a, b content also decreased. Correlation between gene expression and physiological traits related to them highlights the prominent role of CWCP in maintaining yield potential.

Abstract To study the effects of foliar application of putrescine (distilled water (0), 0.75, 1.5, and 2.25 mM) and water deficit stress (20%, 40%, 60%, and 80% available soil water depletion (ASWD)) on the physiological, biochemical, and molecular attributes of Salvia officinalis L., a factorial experiment was performed in a completely randomized design with three replications in the growth chamber. The results of Real-Time quantitative polymerase chain reaction (qRT-PCR) analysis showed that putrescine concentration, irrigation regime, and the two-way interaction between irrigation regime and putrescine concentration significantly influenced cineole synthase (CS), sabinene synthase (SS), and bornyl diphosphate synthase (BPPS) relative expression. The highest concentration of 1,8-cineole, camphor, α-thujone, β-thujone, CS, SS, and BPPS were obtained in the irrigation regime of 80% ASWD with the application of 0.75 mM putrescine. There was high correlation between expression levels of the main monoterpenes synthase and the concentration of main monoterpenes. The observed correlation between the two enzyme activities of ascorbate peroxidase (APX) and catalase (CAT) strongly suggests they have coordinated action. On the other hand, the highest peroxidase (PO) and superoxide dismutase (SOD) concentrations were obtained with the application of 0.75 mM putrescine under the irrigation regime of 40% ASWD. Putrescine showed a significant increase in LAI and RWC under water deficit stress. There was an increasing trend in endogenous putrescine when putrescine concentration was increased in all irrigation regimes. Overall, the results suggest that putrescine may act directly as a stress-protecting compound and reduced H2O2 to moderate the capacity of the antioxidative system, maintain the membrane stability, and increase secondary metabolites under water deficit stress.

Abstract Nowadays, with the development and advancement of next-generation sequencing technologies, a new path has been provided for transcriptomic studies. In this study, the transcriptome of Dracocephalum kotschyi Boiss., as an endemic and endangered plant which is contained a large amount of valuable secondary metabolites with antioxidant and anticancer properties, was sequenced. Then functional annotation and gene ontology analysis for 165,597 assembled transcripts were performed, most were associated with the metabolic pathways. This might be because there are various active biochemical pathways in this plant. Furthermore, after comprehensive transcript annotation, the putative genes involved in the main metabolic pathways of D. kotschyi were identified. Then, the biosynthetic pathway of its valuable methoxylated flavones was proposed. Finally, the accumulations of important methoxylated-flavone metabolites in three different tissues were quantified by HPLC. The relative expression of the genes involved in the proposed pathway was investigated by qRT-PCR, which indicated high expression levels in the bud tissue. The present results may lead to the design strategies to preserve the genetic diversity of endangered D. kotschyi plants and apply the new methods for engineering its valuable methoxylated-flavones pathway.

Objective:The relationship between drug resistance and the expression of hexokinase (HK) has been indicated in leishmaniasis. According to the prolonged treatment period in cutaneous leishmaniasis (CL) patients co-infected with Crithidia in Iran, this study aims to investigate the expression of HK in the proteome of Leishmania major and Crithidia using a proteomic approach.Methods:A total of 205 samples were removed from the lesions of patients in Fars province, Iran, for the characterization of L. major and Crithidia using polymerase chain reaction (PCR). After protein extraction, two-dimensional gel electrophoresis was employed for protein separation. Several spots were isolated for HK determination in the proteomes of L. major and Crithidia using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/TOF MS).Results:The PCR results showed 5 positive cases for Crithidia and 96 positive cases for L. major. MALDI TOF/TOF MS indicated HK as a common protein in the proteome of L. major and Crithidia. HK was up-regulated in the Crithidia proteome in comparison with the L. major proteome.Conclusion:Since a relationship between HK expression and drug resistance has been indicated in leishmaniasis, the overexpression of HK in Crithidia might be related to the increased duration of the treatment period in CL patients co-infected with Crithidia.

Background. Parkinson’s disease (PD) has been described in dopamine brain level reductions. Conversely, several studies have shown that Toxoplasma parasite can increase the level of dopamine in an infected host. This study was conducted to assess the serum, cerebral dopamine levels, and downregulation of Parkinson’s disease manifestations in mice with chronic toxoplasmosis. Methods. PD induction was done by oral inoculation of rotenone into BALB/c mice. To induce the chronic infection, cysts of T. gondii Prugniaud strain (genotype II) were injected intraperitoneally into the mice. The rotarod test was used for the evaluation of functional motor disorders in experimental mice. The serum and cerebral dopamine levels of the mice were also measured by using high-performance liquid chromatography (HPLC) on consecutive periods (10 days). Results. Findings of the rotarod test showed the highest and lowest average of running duration belonged to the uninfected mice and PD mice, respectively. Remarkably, the running duration of infected mice with PD was higher than PD mice. As well, the level of serum and cerebral dopamine increased in mice with PD and toxoplasmosis in comparison with PD mice. Conclusion. Increasing the serum and cerebral dopamine levels in mice infected with toxoplasmosis is related to the presence of the parasite. Moreover, the dopamine upregulation due to the infection is effective in the reduction of PD complications.

The complement system exerts crucial functions both in innate immune responses and adaptive humoral immunity. This pivotal system plays a major role dealing with pathogen invasions including protozoan parasites. Different pathogens including parasites have developed sophisticated strategies to defend themselves against complement killing. Some of these strategies include the employment, mimicking or inhibition of host’s complement regulatory proteins, leading to complement evasion. Therefore, parasites are proven to use the manipulation of the complement system to assist them during infection and persistence. Herein, we attempt to study the interaction´s mechanisms of some prominent infectious protozoan parasites including Plasmodium, Toxoplasma, Trypanosoma, and Leishmania dealing with the complement system. Moreover, several crucial proteins that are expressed, recruited or hijacked by parasites and are involved in the modulation of the host´s complement system are selected and their role for efficient complement killing or lysis evasion is discussed. In addition, parasite’s complement regulatory proteins appear as plausible therapeutic and vaccine targets in protozoan parasitic infections. Accordingly, we also suggest some perspectives and insights useful in guiding future investigations.

Paclitaxel® (PC) is one of the most effective and profitable anti-cancer drugs. The most promising sources of this compound are natural materials such as tissue cultures of Taxus species and, more recently, hazelnut (Corylus avellana L.). A large part of the PC biosynthetic pathway in the yew tree and a few steps in the hazelnut have been identified. Since understanding the biosynthetic pathway of plant-based medicinal metabolites is an effective step toward their development and engineering, this paper aimed to identify taxadiene-5α-ol-O-acetyltransferase (TDAT) in hazelnut. TDAT is one of the key genes involved in the third step of the PC biosynthetic pathway. In this study, the TDAT gene was isolated using the nested-PCR method and then characterized. The cotyledon-derived cell mass induced with 150 μM of methyl jasmonate (MeJA) was utilized to isolate RNA and synthesize the first-strand cDNA. The full-length cDNA of TDAT is 1423 bp long and contains a 1302 bp ORF encoding 433 amino acids. The phylogenetic analysis of this gene revealed high homology with its ortholog genes in Quercus suber and Juglans regia. Bioinformatics analyses were used to predict the secondary and tertiary structures of the protein. Due to the lack of signal peptide, protein structure prediction suggested that this protein may operate at the cytoplasm. The homologous superfamily of the T5AT protein, encoded by TDAT, has two domains. The highest and lowest hydrophobicity of amino acids were found in proline 142 and lysine 56, respectively. T5AT protein fragment had 24 hydrophobic regions. The tertiary structure of this protein was designed using Modeler software (V.9.20), and its structure was verified based on the results of the Verify3D (89.46%) and ERRAT (90.3061) programs. The T5AT enzyme belongs to the superfamily of the transferase, and the amino acids histidine 164, cysteine 165, leucine 166, histidine 167, and Aspartic acid 168 resided at its active site. More characteristics of TDAT, which would aid PC engineering programs and maximize its production in hazelnut, were discussed.

Thrombolytic and fibrinolytic therapies are effective treatments to dissolve blood clots in stroke therapy. Thrombolytic drugs activate plasminogen to its cleaved form plasmin, a proteolytic enzyme that breaks the crosslinks between fibrin molecules. The FDA-approved human tissue plasminogen activator Reteplase (rPA) is a non-glycosylated protein produced in E. coli. rPA is a deletion mutant of the wild-type Alteplase that benefits from an extended plasma half-life, reduced fibrin specificity and the ability to better penetrate into blood clots. Different methods have been proposed to improve the production of rPA. Here we show for the first time the transient expression in Nicotiana benthamiana of rPA fused to the immunoglobulin fragment crystallizable (Fc) domain on an IgG1, a strategy commonly used to improve the stability of therapeutic proteins. Despite our success on the expression and purification of dimeric rPA-Fc fusions, protein instability results in high amounts of Fc-derived degradation products. We hypothesize that the "Y"- shape of dimeric Fc fusions cause steric hindrance between protein domains and leads to physical instability. Indeed, mutations of critical residues in the Fc dimerization interface allowed the expression of fully stable rPA monomeric Fc-fusions. The ability of rPA-Fc to convert plasminogen into plasmin was demonstrated by plasminogen zymography and clot lysis assay shows that rPA-Fc is able to dissolve blood clots ex vivo. Finally, we addressed concerns with the plant-specific glycosylation by modulating rPA-Fc glycosylation towards serum-like structures including α2,6-sialylated and α1,6-core fucosylated N-glycans completely devoid of plant core fucose and xylose residues.

Background. Toxoplasma parasite alters the transduction of neurotransmitter signals and leads to changes in the level of brain neurotransmitters including tyrosine and dopamine, so behavior changes can occur in infected hosts. Based on this concept, this study was conducted for evaluation of the tyrosine and dopamine serum levels in infected mice with chronic toxoplasmosis. Materials and Methods. Toxoplasma gondii (Prugniaud strain II) was injected intraperitoneally into BALB/c mice to induce chronic toxoplasmosis. Modified agglutination test (MAT), polymerase chain reaction (PCR), and microscopic methods were conducted to confirm the induction of chronic toxoplasmosis. The infected mouse sera were separated at days 40, 50, 60, 70, and 80 for evaluation of tyrosine and dopamine serum levels using high-performance liquid chromatography (HPLC). Results. Microscopic methods confirmed the formation of the Toxoplasma cysts in mouse tissues. Inducing chronic toxoplasmosis is also confirmed by MAT, PCR, and histological methods. HPLC results indicated a decrease in serum tyrosine level at day 40 in infected mice in comparison to control, and the levels were too low to be measured at other times. However, a significantly high serum dopamine level was observed that gradually increased after parasite inoculation. Conclusions. No detection of tyrosine level in most of the sample groups is probably related to the very low concentration of tyrosine in sera. However, low concentration of tyrosine at day 40 and increase of dopamine in most of the sample groups suggest the production of dopamine from tyrosine due to the presence of Toxoplasma in infected mice.

Toxoplasma gondii is a pathogenic protozoan parasite that infects the nucleated cells of warm-blooded hosts leading to an infectious zoonotic disease known as toxoplasmosis. The infection outcomes might be severe and fatal in patients with immunodeficiency, diabetes, and pregnant women and infants. The One Health approach to toxoplasmosis highlights that the health of humans is closely related to the health of animals and our common environment. The presence of drug resistance and side effects, the further improvement of sensitivity and specificity of serodiagnostic tools and the potentiality of vaccine candidates to induce the host immune response are considered as justifiable reasons for the identification of novel targets for the better management of toxoplasmosis. Thus, the identification of new critical proteins in the proteome of Toxoplasma parasites can also be helpful in designing and test more effective drugs, vaccines, and diagnostic tools. Accordingly, in this study we present important proteins found in the proteome of the life cycle-specific stages of Toxoplasma parasites that are potential diagnostic or vaccine candidates. The current study might help to understand the complexity of these parasites and provide a possible source of strategies and biomolecules that can be further evaluated in the pathobiology of Toxoplasma parasites and for diagnostics and vaccine trials against this disease.

ISSR markers were applied to evaluate the genetic diversity and differentiation of 270 individuals of 27 Iranian C. melo landraces of various varietal groups include vars. inodorous, cantalupensis, reticulatus, ameri, dudaim. Genetic diversity among the studied genotypes obtained by GeneAlex analysis (H = 0.08, I = 0.12, Na = 0.77, PPL = 22.6%). Cluster analysis divided Iranian melon landraces into two main cluster. Non-sweet genotype (dudaim group) was well separated from sweet genotypes (inodorous, ameri, reticulatus, cantalupensis). The most similar genotypes were BANI and TONI (0.95) and the most dissimilar ones were GER and TS (0.58). AMOVA result showed that the percentage of genetic variation among and within Iranian melon is 69% and 31%, respectively. All landraces evaluated based on 10 morphological traits which revealed the diversity of melon varietal groups. Bayesian analysis assigned ten landraces to Pop 1, eight landraces to Pop 2 and nine melon landraces to Pop 3. Bayesian and UPGMA cluster analyses demonstrated the almost related results. Our results indicated that ISSR markers technique alongside polyacrylamide gel analysis could be helpful to discriminate varieties of melon. Keywords: Orthogonal contrasts analysis, Morphological traits, AMOVA, Bayesian analysis, UPGMA cluster analysis

Human Leukocyte Antigen-G (HLA-G), a polymorphic non-classical HLA (HLA-Ib) with immune-regulatory properties in cancers and infectious diseases, presents both membrane-bound and soluble (sHLA-G) isoforms. Polymorphism has implications in host responses to pathogen infections and in pathogenesis. Differential expression patterns of HLA-G/sHLA-G or its polymorphism seem to be related to different pathological conditions, potentially acting as a disease progression biomarker. Pathogen antigens might be involved in the regulation of both membrane-bound and sHLA-G levels and impact immune responses during co-infections. The upregulation of HLA-G in viral and bacterial infections induce tolerance to infection. Recently, sHLA-G was found useful to identify the prognosis of Coronavirus disease 2019 (COVID-19) among patients and it was observed that the high levels of sHLA-G are associated with worse prognosis. The use of pathogens, such as Plasmodium falciparum, as immune modulators for other infections could be extended for the modulation of membrane-bound HLA-G in COVID-19-infected tissues. Overall, such information might open new avenues concerning the effect of some pathogens such as parasites in decreasing the expression level of HLA-G to restrict pathogenesis in some infections or to influence the immune responses after vaccination among others.

Extraction of high quality RNA is a critical step in molecular genetics studies. Hazelnut is one of the most important nuts plants in the world. The presence of the taxol and other taxanes in hazelnut plant necessitates explaining their biosynthesis pathway and identifying the candidate genes. Therefore, an easy and practical method is necessary for RNA extraction from hazelnuts. Hazelnut has high levels of phenolic compounds. High amounts of polyphenolic and polysaccharide compounds in plants could be causing problems in RNA extraction procedures. To avoid these problems, a simple and efficient method can be used based on cetyltrimethylammonium bromide (CTAB) extraction buffer and lithium chloride for extraction of high quality RNA from different parts of hazelnut plant. Using this method, a high-quality RNA sample (light absorbed in the A260/A280 was 2.04

Alliinase is the key enzyme in allicin biosynthesis pathway. In the current study, the identification and sequencing of alliinase genes along with determination of allicin contents were reported for Allium species with a novel report for Iranian endemic species. The presence of different isoforms in the Allium being discovered for the first time. In bulbs tissue, the highest allicin concentration was in Allium sativum, A. umbilicatum, and A. fistolosum (1.185%, 0.367%, and 0.34%, respectively), followed by A. spititatum (0.072%), A. lenkoranicum (0.055%), A. atroviolaseum (0.36%), A. rubellum (0.041%), and A. stamineum (0.007%). The highest allicin content in the leaves and roots were in A. sativum (0.13%), and A. stamineum (0.195%), respectively. The ORFs length ranged from 1416 in A. sativum (iso-alliinase2; ISA2) to 1523 bp in A. sativum (alliinase); the identity with A. sativum (alliinase) varies from 95% to 68% for A. ampeloprasum, and A. sativum (iso-alliinase1, ISA1) respectively. These data suggested that both ISA1 and ISA2 had a high expression in the roots and bulbs compared to A. sativum as the control in all species. Note that ISA1 and ISA2 were not expressed in the leaves. The results showed that isoforms expression patterns among different tissues in Allium species were variable. The presence of various isoforms is a possible explanation for the difference between the species in terms of obtained results, especially the amount of allicin.

Opium poppy (Papaver somniferum L.) is one of the ancient medical crops, which produces several important alkaloids such as morphine, noscapine, sanguinarine and codeine. MicroRNAs are endogenous non-coding RNAs that play important regulatory roles in plant diverse biological processes. Many plant miRNAs are encoded as single transcriptional units, in contrast to animal miRNAs, which are often clustered. Herein, using computational approaches, a total of 22 miRNA precursors were identified, which five of them were located as a clustered in pre-ribosomal RNA. Afterward, the transcript level of the precursor and the mature of clustered miRNAs in two species of the Papaveraceae family, i.e. P. somniferum L. and P. bracteatum L, were quantified by RT-PCR. With respect to obtained results, these clustered miRNAs were expressed differentially in different tissues of these species. Moreover, using target prediction and Gene Ontology (GO)-based on functional classification indicated that these miRNAs might play crucial roles in various biological processes as well as metabolic pathways. In this study, we discovered the clustered miRNA derived from pre-rRNA, which may shed some light on the importance of miRNAs in the plant kingdom.

To estimate the genetic diversity of chickpea germplasm from Iran, a total of 307 landraces from 4 regions including: northern areas (29 from Ardebil, 3 from Qazvin and 5 from Mazanderan provinces), temperate (16 from Kermanshah, 2 from Semnan, 54 from Khorasan and 20 from Kerman provinces), semi-arid (28 from Ghom and 56 from Isfahan provinces) and cold areas (15 from West Azarbayjan, 52 from Tehran and 27 from East Azarbayjan provinces) were analysed using 16 microsatellite loci. The number of alleles per microsatellite locus ranged from 8 to 29, with an average of 19.31 per locus. A high level of genetic diversity in the northern area (He = 0.76), even with a limited number of available landraces (37) compared with the other three regions (84-94), might confirm the northern Persia as part of the chickpea centre of origin. The neighbour-joining tree showed a low relationship between molecular divergence and the geographical grouping of chickpea. Moreover, cluster analyses based on molecular data showed that the northern area was separated clearly from the other three regions, indicating a physical barrier or geographical and environmental differences among these regions. A wide genetic diversity of Iranian chickpea landraces is a critical component for future selection and use of this germplasm for future breeding of chickpea.

IntroductionCommon bean (Phaseolus vulgaris L.) has been considered as an important source of protein and calorie. This plant plays an important role in supplying the human's protein. Bean crop is highly susceptible to weeds due to low growth rate early in the growing season. Therefore, competition with weeds causes a significant reduction in grain yield at the end of the growing season. Weed competition is a kind of non-living stress, which affects plant yield. Weeds and crops are always competing for obtaining nutrients, water, light and space (CO2). Examining the effect of weed interference time during the critical period provides valuable information regarding the physiological outcome of competition between weeds and crops. Red bean cultivars, with different morphological and genetic characteristics, show different responses in the presence of weeds, which may ultimately result in possible difference in yield. Unlimited growth genotypes showed higher competitive ability than those with limited growth genotypes. However, there are contradictory reports about the effect of growth habits on competitive ability. Measurement of growth indices is necessary to study the competitive ability of species during the growth period. The aim of this study was to investigate the response of some morphophysiological traits of three red bean cultivars with two different growth habits to weeds. Materials and MethodsA field experiment was conducted to investigate the morphological and physiological characteristics of three red bean cultivars with two different growth habits (semi erect and erect) under weed competition in the experimental research station at the Faculty of Agriculture, University of Ilam, Iran. The experiment was conducted as factorial based on a randomized complete block design with two factors including: cultivars (Sayad, Derakhshan and line D81083) and weed competition at two levels (weed free and infested during the growing season) with three replications. The traits related to red bean such as plant height, biomass, chlorophyll a, b and total chlorophyll, anthocyanin and carotenoid at the stages V4, R6, R7 and grain yield and yield components were measured at physiological maturation stage. The importance value of weed (IVW) was measured in two stages V4 and R9. Competitive index (CI) was used to evaluate the competitive ability of common bean varieties against weeds. The software of SPSS 22, SAS 9.4 and Excel 2013 were used for test normality, data analysis and drawing charts, respectively. Means comparison was performed by the least significant difference test (LSD). Results and Discussion Convolvulus arvensis had the highest importance value of weed (IVW) index from the beginning of growth until stage V4. At stage R9, the Purtulaca oleracea and Amaranthus retroflaxus had the highest values of IVW by 27.33% and 21.92%, respectively. The latter are dominant species in the western regions of the country. The results showed that cultivar type and weed competition had significant effects on the bean crop height at all studied stages. In addition, the effect of interaction between cultivar and weed competition was significant on leaf area (LA) and height at V4 and R7 stages, respectively. At all stages, traits such as height and leaf area of red bean cultivars were significantly decreased owing to weed competition compared to the weed control. Line D81083 and Sayad had the highest and lowest leaf area in competition with weeds during all stages, respectively. The effect of interaction between cultivar and weed competition was significant on grain yield and number of pods per plant. Line D81083 and Derakhshan cultivars had the highest and lowest seed yield in competition with weeds, respectively. Line D81083 had the most competitive index among the cultivars. There were no significant differences between Sayad and Derakhshan in all studied traits. Weed competition had a significant effect on chlorophyll a, b and total chlorophyll at V4 and R7 stages. Anthocyanin and carotenoids were not affected by the treatments at V4 stage. Overall, chlorophyll a, b and total chlorophyll were significantly increased in the absence of weeds. The effect of differences in anthocyanin value was significant based on the cultivar and weed presence, while carotenoid content was only affected by the cultivar type at R7 stage. ConclusionThe present study showed that use of line D81083 could increase grain yield of red bean up to 2-fold under weed free conditions compared to crop-weed interference conditions. Line D81083 had the highest grain yield, pod per plant, 100-grain weight, and competition index when full-season weed competition occurred.

Background. Recombinant plasminogen activator (r-PA) consists of the Kringle-2 and protease domains of human tissue-type plasminogen. It is used clinically to treat coronary artery thrombosis and acute myocardial infarction. However, the expression and production of reteplase (r-PA) are limited due to its susceptibility to proteolysis during manufacturing processes. Therefore, efforts have been made to address this limitation. Materials and methods. To enhance the conformational stability of r-PA and increase its resistance to proteolysis, we used Gly 6 Ala substitutions in the Kringle-2 domain through in silico. We created an in silico mutant collection with eight structures, incorporating four designated mutations (R103S, G39A, G53A, and G55A). Using MODELLER software and homology modeling, we developed three-dimensional structures for two Kringle-2 and tissue plasminogen activator protease domains, including the wild noncleavable form (R103S) and mutants with all four designated mutations. We assessed protein stability using a dynamic cross-correlation matrix by extracting global properties such as Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF) from trajectory files. Results. The findings revealed that a single glycine--alanine substitution (G39A) enhanced the conformational stability of r-PA, as evidenced by improvements in RMSD, RMSF, radius of gyration, surface accessibility, hydrogen bond formation, eigenvector projection, and density analysis. Conclusion. The conformational stability of r-PA conferred by glycine replacement with alanine may decrease the propensity for proteolysis in protease -- rich environments across various recombinant systems and potentially enhance its production and expression levels. [ABSTRACT FROM AUTHOR]

The chemical constituents of essential oils of Datura stramonium L. and D. innoxia (family Solanaceae) are being reported. In this study, the collected seeds of Ardabil and Urmia were planted in grow bags and placed for three months in a greenhouse of the College of Agriculture, Tarbiat Modares University in Tehran. Under greenhouse conditions, the average air temperature was 25 °C. Air-dried areal parts of D. stramonium and D. innoxia were prepared and their essential oils (EOs) were isolated by hydrodistillation for 3 h in a Clevenger-type apparatus. EOs content and composition were examined with gas chromatography techniques. The EOs yield of D. stramonium and D. innoxia were 0.03% and 0.02% (v/w), respectively. The major compounds in the D. stramonium EOs were Camphor (29.9%), Selin-11-en-4alpha-ol (13.0%), and Borneol (10.4%). Those in the D. innoxia were mostly fatty alkanes, including n-Decane (19.99%), 7-Pentadecyne (11.78%), and Linoleic acid (6.13%). The other important components identified in the EOs of Datura were Dihydrocitronellol, n-Dodecane, Isobornyl acetate, Germacrene D, and n-Tetradecane. Generally, the EOs of the areal parts in D. stramonium showed oxygenated monoterpenes. [ABSTRACT FROM AUTHOR]