Foliar-applied iron and zinc nanoparticles improved plant growth, phenolic compounds, essential oil yield, and rosmarinic acid production of lemon balm (Melissa officinalis L.).

Metallic nanoparticles (NPs) have been highlighted to improve plant growth and development in the recent years. Although positive effects of some NPs have been reported on medicinal plants, the knowledge for stimulations application of iron (Fe) and zinc (Zn) NPs is not available. Hence, the present work aimed to discover the effects of Fe NPs at 10, 20, and 30 mg L-1 and Zn NPs at 60 and 120 mg L-1 on growth, water content, photosynthesis pigments, phenolic content, essential oil (EO) quality, and rosmarinic acid (RA) production of lemon balm (Melissa officinalis L.). The results showed that Fe NPs at 20 and 30 mg L-1 and Zn NPs at 120 mg L-1 significantly improved biochemical attributes. Compared with control plants, the interaction of Fe NPs at 30 mg-1 and Zn NPs at 120 mg L-1 led to noticeable increases in shoot weight (72%), root weight (92%), chlorophyll (Chl) a (74%), Chl b (47%), RA (66%), proline (81%), glycine betaine (GB, 231%), protein (286%), relative water content (8%), EO yield (217%), total phenolic content (63%), and total flavonoid content (57%). Heat map analysis revealed that protein, GB, EO yield, shoot weight, root weight, and proline had the maximum changes upon Fe NPs. Totally, the present study recommended the stimulations application of Fe NPs at 20-30 mg L-1 and Zn NPs at 120 mg L-1 to reach the optimum growth and secondary metabolites of lemon balm.

The oncogenic role of NOTCH1 as biomarker in oral squamous cell carcinoma and oral lichen planus.

Background: Oral squamous cell carcinoma (OSCC) is the most common type of oral cancer with heterogeneous molecular pathogenesis. Oral lichen planus (OLP) is demonstrated potentially can transfer to OSCC malignant lesions. Unfortunately, there are no definitive prognostic and predictive biomarkers for the clinical management of OSCC patients. The present research is the first study that compared an oral premalignant lesion such as OLP to malignant lesions like OSCC for NOTCH1 expression levels to better understand its oncogenic or tumor suppressive role. Materials and Methods: In this cross-sectional study, mRNA expression of NOTCH1 was evaluated by quantitative polymerase chain reaction in 65 tissue-embedded Paraffin-Block samples, including 32 OSCC and 33 OLP. Furthermore, we collected demographic information and pathological data, including tumor stage and grade. The association between NOTCH1 and GAPDH gene expressions was determined by Chi-squared, Spearman, and Mann-Whitney tests. A P < 0.05 was considered statistically significant for all statistical analyses. Results: Comparison of OSCC and OLP groups showed a statistically significant difference between the quantitative expression of the NOTCH1 gene (P < 0.001). Qualitative gene expression was divided into low expression and high expression. Both study groups demonstrated a statistically significant gene expression difference (P < 0.001). There was a statistically significant difference between age and NOTCH1 expression in the OLP group (P = 0.036). There was no correlation between NOTCH1 expression and age, gender, tumor grade, and stage. Conclusion: Since the OSCC is a malignant lesion and the OLP showed the possible nature of malignancy transformation, we can consider the NOTCH1 as a biomarker for the assessment of the tumorigenesis process with a definition of a standard threshold for potentially malignant lesions and malignant OSCC tumors.

Evaluating the efficiency of CHEF and CMV promoter with IRES and Furin/2A linker sequences for monoclonal antibody expression in CHO cells.

In recent years, monoclonal antibodies (mAbs) have been developed as powerful therapeutic and diagnostic agents and Chinese hamster ovary (CHO) cells have emerged as the dominant host for the recombinant expression of these proteins. A critical step in recombinant expression is the utilization of strong promoters, such as the Chinese Hamster Elongation Factor-1α (CHEF-1) promoter. To compare the strengths of CHEF with cytomegalovirus (CMV) promoter for mAb expression in CHO cells, four bicistronic vectors bearing either internal ribosome entry site (IRES) or Furin/2A (F2A) sequences were designed. The efficiency of these promoters was evaluated by measuring level of expressed antibody in stable cell pools. Our results indicated that CHEF promoter-based expression of mAbs was 2.5 fold higher than CMV-based expression in F2A-mediated vectors. However, this difference was less significant in IRES-mediated mAb expressing cells. Studying the stability of the F2A expression system in the course of 18 weeks, we observed that the cells having CHEF promoter maintained their antibody expression at higher level than those transfected with CMV promoter. Further analyses showed that both IRES-mediated vectors, expressed mAbs with correct size, whereas in antibodies expressed via F2A system heterogeneity of light chains were detected due to incomplete furin cleavage. Our findings indicated that the CHEF promoter is a viable alternative to CMV promoter-based expression in F2A-mediated vectors by providing both higher expression and level of stability.

Monoclonal antibodies expression improvement in CHO cells by PiggyBac transposition regarding vectors ratios and design.

Establishing stable Chinese Hamster Ovary (CHO) cells producing monoclonal antibodies (mAbs) usually pass through the random integration of vectors to the cell genome, which is sensitive to gene silencing. One approach to overcome this issue is to target a highly transcribed region in the genome. Transposons are useful devices to target active parts of genomes, and PiggyBac (PB) transposon can be considered as a good option. In the present study, three PB transposon donor vectors containing both heavy and light chains were constructed, one contained independent expression cassettes while the others utilized either an Internal Ribosome Entry Site (IRES) or 2A element to express mAb. Conventional cell pools were created by transferring donor vectors into the CHO cells, whereas transposon-based cells were generated by transfecting the cells with donor vectors with a companion of a transposase-encoding helper vector, with 1:2.5 helper/donor vectors ratio. To evaluate the influence of helper/donor vectors ratio on expression, the second transposon-based cell pools were generated with 1:5 helper/donor ratio. Expression levels in the transposon-based cells were two to five -folds more than those created by conventional method except for the IRES-mediated ones, in which the observed difference increased more than 100-fold. The results were dependent on both donor vector design and vectors ratios.

PhiC31 integrase can improve the efficiency of different construct designs for monoclonal antibody expression in CHO cells.

OBJECTIVES: Several types of expression vectors have been used for recombinant protein expression in Chinese hamster ovary cells (CHO) which usually result in variable and unstable levels of expression. METHODS AND RESULTS: In this study, we have compared the mAb0014 expression level of single ORF/IRES vector and dual ORF vector in the presence and absence of phiC31 integrase targeting system. Both expression vectors contain an elongation factor 1α (EF1α) promoter upstream of LC and harboring an attB site. CHO-S cells were co-transfected with single ORF/IRES or dual ORF vectors along with a phiC31 integrase expression vector which can catalyze recombination between attB site and pseudo-attP sites presented in the mammalian genome. Our results demonstrated that dual ORF vector in the presence of phiC31 integrase expression vectors (+FC31 2P) generated more recombinant antibody in comparison to its negative control (-FC31 2P). Moreover, both of +FC31 2P and -FC31 2P cell pools yield higher recombinant protein in comparison to single ORF/IRES vector (FC31 IRES) cell pools. Stability of expression in phiC31 co-transfected cell pools (+FC31 2P and +FC31 IRES) had no considerable changes. CONCLUSIONS: Our results indicated that the dual ORF vector using integrase can support the generation of cell lines with stable transgene expression at an elevated mAb relative to single ORF/IRES vector.

Evaluating the expression profile and stability of different UCOE containing vector combinations in mAb-producing CHO cells.

BACKGROUND: As the demand for monoclonal antibodies (mAb) increases, more efficient expression methods are required for their manufacturing process. Transcriptional gene silencing is a common phenomenon in recombinant cell lines which leads to expression reduction and instability. There are reports on improved antibody expression in ubiquitous chromatin opening element (UCOE) containing both heavy and light chain gene constructs. Here we investigate the impact of having these elements as part of the light chain, heavy chain or both genes during cell line development. In this regard, non-UCOE and UCOE vectors were constructed and stable Chinese hamster ovary (CHO) cell pools were generated by different vector combinations. RESULTS: Expression analysis revealed that all UCOE cell pools had higher antibody yields compared to non-UCOE cells, Moreover the most optimal expression was obtained by cells containing just the UCOE on heavy chain. In terms of stability, it was shown that the high level of expression was kept consistence for more than four months in these cells whereas the expression titers were reduced in the other UCOE pools. CONCLUSIONS: In conclusion, UCOE significantly enhanced the level and stability of antibody expression and the use of this element with heavy chain provided more stable cell lines with higher production level.

Growth inhibition of human breast cancer cells and down-regulation of ODC1 and ADA genes by Nepeta binaloudensis

ABSTRACT Nepeta binaloudensis Jamzad, Lamiaceae, is a rare medicinal plant endemic to Iran. In spite of many studies about the chemical constituents and antibacterial effects of this species, no report has been provided about its cytotoxic and anticancer activities. In this study we have evaluated the effects of EtOH 70%, hexane and aqueous extracts of N. binaloudensis on the cell proliferation and n-hexane extract on the expression of adenosine deaminase and ornithine decarboxylase 1 genes in breast cancer cell lines (MCF-7, MDA-MB-231) compared to non-cancer line (MCF-10A). The cell lines were subjected to increasing doses of the extracts ranging from 10 to 320 µg/ml. Cell viability was quantified by MTS assay. Expression of adenosine deaminase and ornithine decarboxylase 1 genes was analyzed by real time PCR. N. binaloudensis inhibited the growth of malignant cells in a time and dose-dependent manner. Among extracts of N. binaloudensis, the hexane extract was found to be more toxic compared to other extracts. Results showed a marked decrease in the expression of ornithine decarboxylase 1 and adenosine deaminase genes in cancer cell lines. At 60 µg/ml concentration of N. binaloudensis hexane extract ornithine decarboxylase 1 and adenosine deaminase mRNA expression were reduced 4.9 fold and 3.5 fold in MCF-7 cell line and 3.6 fold and 2.6 fold in MDA-MB-231 cell line compared to control, respectively. The result of our study highlights the potential influences of N. binaloudensis hexane extract on ornithine decarboxylase 1 and adenosine deaminase genes expression in breast cancer cells and its relation to inhibition of cancer cell growth.

Evaluating the efficiency of phiC31 integrase-mediated monoclonal antibody expression in CHO cells.

Traditional methods to generate CHO cell lines rely on random integration(s) of the gene of interest and result in unpredictable and unstable protein expression. In comparison, site-specific recombination methods increase the recombinant protein expression by inserting transgene at a locus with specific expression features. PhiC31 serine integrase, catalyze unidirectional integration that occurs at higher frequency in comparison with the reversible integration carried out by recombinases such as Cre. In this study, using different ratios of phiC31 serine integrase, we evaluated the phiC31 mediated gene integration for expression of a humanized IgG1 antibody (mAb0014) in CHO-S cells. Light chain (LC) and heavy chain (HC) genes were expressed in one operon under EF1α promoter and linked by internal ribosome entry site (IRES) element. The clonal selection was carried out by limiting dilution. Targeted integration approach increased recombinant protein yield and stability in cell pools. The productivity of targeted cell pools was about 4 mg/L and about 40 µg/L in the control cell pool. The number of integrated transgenes was about 19 fold higher than the control cells pools. Our results confirmed that the phiC31 integrase leads to mAb expression in more than 90% of colonies. The productivity of the PhiC31 integrated cell pools was stable for three months in the absence of selection as compared with conventional transfection methods. Hence, utilizing PhiC31 integrase can increase protein titer and decrease the required time for protein expression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1570-1576, 2016.

Antibacterial and Antifungal Activity of Holothuria leucospilota Isolated From Persian Gulf and Oman Sea.

BACKGROUND: Emergence of antimicrobial resistance toward a number of conventional antibiotics has triggered the search for antimicrobial agents from a variety of sources including the marine environment. OBJECTIVES: The aim of this study was to evaluate the antimicrobial potential of Holothuria leucospilota from Qeshm and Kharg Islands against some selected bacteria and fungi. MATERIALS AND METHODS: In this investigation, sea cucumbers from two coastal cities of Persian Gulf were collected in March and May 2011 and identified by the scale method according to the food and agriculture organization of the United Nations. Antibacterial activity of hydroalcoholic extracts of the body wall, cuvierian organs and coelomic fluid, methanol, chloroform, and n-hexane extracts of the body wall were evaluated by the spot test. In addition, their antifungal activity was assessed by the broth dilution method. RESULTS: The displayed effect was microbiostatic at concentrations of 1000 and 2000 µg/mL rather than microbicidal. The highest activity of hydroalcoholic extracts was exhibited by body wall, cuvierian organs and coelomic fluid against Escherichia coli, Salmonella typhi, Staphylococcus aureus and Pseudomonas aeruginosa; Aspergillus niger, A. fumigatus, A. flavus and A. brasilensis. However, none of the methanol, chloroform and n-haxane extracts showed appreciable effects against Shigella dysenteriae, Proteus vulgaris, Bacillus cereus, S. epidermidis and Candida albicans. Moreover, cuvierian organs did not possess any antifungal potential. CONCLUSIONS: Our data indicated that water-methanol extracts from the body wall of H. leucospilota possess antibacterial and antifungal activity. However, additional and in-depth studies are required to isolate and identify the active component(s).