Successful production of human epidermal growth factor in tobacco chloroplasts in a biologically active conformation.

Human epidermal growth factor (hEGF) is an important therapeutic compound with multiple applications particularly in pharmaceutical industry. Human EGF has already been expressed in different expression systems, however, the production of hEGF with bioactivity in chloroplasts has not been successful so far. In this study, we expressed a 6 × His-tagged hEGF in tobacco chloroplasts in its native conformation for the potential of large-scale production of hEGF for industrial applications. Several transplastomic plant lines were obtained, which were screened by PCR (polymerase chain reaction) using primers specific to selectable gene aadA, hEGF- and GFP-coding sequences that were included in the chloroplast expression vector. The selected lines were confirmed to be homoplasmic by PCR verification and Southern blot analysis. Immunoblotting assays of homoplasmic lines using antibodies raised against hEGF confirmed the accumulation of hEGF in transplastomic plants and the ELISA results demonstrated the expression levels of hEGF were between 0.124% and 0.165% of the total soluble proteins (TSP), namely, 23.16-25.77 ng/g of the fresh weight. In terms of activity, the data from cell proliferation and elongation assays showed that the tobacco-derived recombinant hEGF was as bioactive as its commercial counterpart. To our knowledge, this is the first report of recombinant production of hEGF with native bioactivity form in the chloroplast stroma. Overall, our results demonstrate the potential of higher plant chloroplasts for the production of a human therapeutic, hEGF, in an active conformation.

Efficient expression of fusion human epidermal growth factor in tobacco chloroplasts.

BACKGROUND: Chloroplast transformation is a robust technology for the expression of recombinant proteins. Various types of pharmaceutical proteins including growth factors have been reported in chloroplasts via chloroplast transformation approach at high expression levels. However, high expression of epidermal growth factor (EGF) in chloroplasts with the technology is still unavailable. RESULTS: The present work explored the high-level expression of recombinant EGF, a protein widely applied in many clinical therapies, in tobacco chloroplasts. In this work, homoplastic transgenic plants expressing fusion protein GFP-EGF, which was composed of GFP and EGF via a linker, were generated. The expression of GFP-EGF was confirmed by the combination of green fluorescent observation and Western blotting. The achieved accumulation of the recombinant fusion GFP-EGF was 10.21 ± 0.27% of total soluble proteins (1.57 ± 0.05 g kg- 1 of fresh leaf). The chloroplast-derived GFP-EGF was capable of increasing the cell viability of the NSLC cell line A549 and enhancing the phosphorylation level of the EGF receptor in the A549 cells. CONCLUSION: The expression of recombinant EGF in tobacco chloroplasts via chloroplast transformation method was achieved at considerable accumulation level. The attempt gives a good example for the application of chloroplast transformation technology in recombinant pharmaceutical protein production.

Hyper-expression of GFP-fused active hFGF21 in tobacco chloroplasts.

Human fibroblast growth factor 21 (hFGF21) is a promising candidate for metabolic diseases. In this study, a tobacco chloroplast transformation vector, pWYP21406, was constructed that consisted of codon-optimized encoding gene hFGF21 fused with GFP at its 5' terminal; it was driven by the promoter of plastid rRNA operon (Prrn) and terminated by the terminator of plastid rps16 gene (Trps16). Spectinomycin-resistant gene (aadA) was the marker and placed in the same cistron between hFGF21 and the terminator Trps16. Transplastomic plants were generated by the biolistic bombardment method and proven to be homoplastic by Southern blotting analysis. The expression of GFP was detected under ultraviolet light and a laser confocal microscope. The expression of GFP-hFGF21 was confirmed by immunoblotting and quantified by enzyme-linked immunosorbnent assay (ELISA). The accumulation of GFP-hFGF21 was confirmed to be 12.44 ± 0.45% of the total soluble protein (i.e., 1.9232 ± 0.0673 g kg-1 of fresh weight). GFP-hFGF21 promoted the proliferation of hepatoma cell line HepG2, inducing the expression of glucose transporter 1 in hepatoma HepG2 cells and improving glucose uptake. These results suggested that a chloroplast expression is a promising approach for the production of bioactive recombinant hFGF21.

Stable and efficient expression of human brain-derived neurotrophic factor in tobacco chloroplasts.

BACKGROUND: Brain-derived neurotrophic factor (BDNF) is an intensively studied neurotrophin that promotes various physiological processes, such as acceleration of cell proliferation and differentiation, and is, therefore widely used in clinical applications. METHODS AND RESULTS: In this study, an expression vector with a codon-optimized hBDNF gene was constructed and transferred into chloroplasts of tobacco by gene-gun. After three or four rounds of selection with optimal spectinomycin concentration, hBDNF was integrated into the chloroplast genome of homoplastomic plants, as confirmed by PCR and Southern hybridization. ELISA indicated that hBDNF fused with GFP represented approximately 15.72% ± 0.33% of total soluble protein in the leaves of transplastomic plants. Moreover, the chloroplast-derived hBDNF displayed biological activity similar to the commercial product. CONCLUSIONS: This is the first case report of hBDNF expression by chloroplast transformation in the plant model, providing an additional pathway for the production of chloroplast-expressed therapeutic proteins.

Characterization of diphenyl phthalate as an agonist for estrogen receptor: an in vitro and in silico study.

Phthalate esters (PAEs) are important pollutants in the environment, which can interfere with the endocrine system by mimicking estrogen. However, limited information is available on modulating the estrogen receptor (ER) of five PAEs including di (2-ethylhexyl) phthalate (DEHP), diisononyl phthalate (DINP), benzyl butyl phthalate (BBP), diphenyl phthalate (DPhP) and dicyclohexyl phthalate (DCHP). This study evaluated the agonistic effects of PAEs on human ER. The cytotoxicity assay showed that there were a significant inhibition of the cell proliferation with treatment of five PAEs. Moreover, DPhP does-dependently enhanced ER-mediated transcriptional activity in the reporter gene assay. The increased expression of estrogen-responsive genes (TFF1, CTSD, and GREB1) was also observed in MCF-7 cells treated with DPhP. The result of molecular docking showed that DPhP tended to bind to the agonist conformation of ER compared with the antagonist conformation of ER, demonstrating its agonist characteristic that has been confirmed in the reporter gene assay. Thus, we found that DPhP may be evaluated as an ER agonist in vitro and it can interfere with the normal function of human ER.

Production of active human FGF21 using tobacco mosaic virus-based transient expression system.

Fibroblast growth factor (FGF) family has a wide range of metabolic processes. FGF21 exerts critical physiological functions in clinical application. This study aimed to explore a convenient and highly efficient approach for rhFGF21 expression using TMV-TES. Firstly, the vector pTTEV-GFP was constructed, followed by optimisation of the expression parameters in Nicotiana benthamiana. Then, the rhFGF21 encoding gene harbouring vector pTTEV-rhFGF21 was constructed. Agrobacterium-mediated vacuum infiltration was performed with the optimised parameters and the expression of rhFGF21 was confirmed by the immunoblotting analysis. ELISA revealed that the protein accumulation of rhFGF21 accounts for 0.11% of total soluble proteins. The biological activity was evaluated and the results suggested that tobacco-expressed rhFGF21 could stimulate the glucose uptake in swiss 3T3-L1 adipocytes, which was similar to the activity of commercial products, suggesting its native biological activity. Therefore, using TMV-TES to express rhFGF21 will be a feasible approach for the mass production of rhFGF21.

A Rapid Pipeline for Pollen- and Anther-Specific Gene Discovery Based on Transcriptome Profiling Analysis of Maize Tissues.

Recently, crop breeders have widely adopted a new biotechnology-based process, termed Seed Production Technology (SPT), to produce hybrid varieties. The SPT does not produce nuclear male-sterile lines, and instead utilizes transgenic SPT maintainer lines to pollinate male-sterile plants for propagation of nuclear-recessive male-sterile lines. A late-stage pollen-specific promoter is an essential component of the pollen-inactivating cassette used by the SPT maintainers. While a number of plant pollen-specific promoters have been reported so far, their usefulness in SPT has remained limited. To increase the repertoire of pollen-specific promoters for the maize community, we conducted a comprehensive comparative analysis of transcriptome profiles of mature pollen and mature anthers against other tissue types. We found that maize pollen has much less expressed genes (>1 FPKM) than other tissue types, but the pollen grain has a large set of distinct genes, called pollen-specific genes, which are exclusively or much higher (100 folds) expressed in pollen than other tissue types. Utilizing transcript abundance and correlation coefficient analysis, 1215 mature pollen-specific (MPS) genes and 1009 mature anther-specific (MAS) genes were identified in B73 transcriptome. These two gene sets had similar GO term and KEGG pathway enrichment patterns, indicating that their members share similar functions in the maize reproductive process. Of the genes, 623 were shared between the two sets, called mature anther- and pollen-specific (MAPS) genes, which represent the late-stage pollen-specific genes of the maize genome. Functional annotation analysis of MAPS showed that 447 MAPS genes (71.7% of MAPS) belonged to genes encoding pollen allergen protein. Their 2-kb promoters were analyzed for cis-element enrichment and six well-known pollen-specific cis-elements (AGAAA, TCCACCA, TGTGGTT, [TA]AAAG, AAATGA, and TTTCT) were found highly enriched in the promoters of MAPS. Interestingly, JA-responsive cis-element GCC box (GCCGCC) and ABA-responsive cis-element-coupling element1 (ABRE-CE1, CCACC) were also found enriched in the MAPS promoters, indicating that JA and ABA signaling likely regulate pollen-specific MAPS expression. This study describes a robust and straightforward pipeline to discover pollen-specific promotes from publicly available data while providing maize breeders and the maize industry a number of late-stage (mature) pollen-specific promoters for use in SPT for hybrid breeding and seed production.

Engineering microalgae through chloroplast transformation to produce high-value industrial products.

The last few years have seen an ever-increasing interest in the exploitation of microalgae as an alternative platform to produce high-value products such as biofuels, industrial enzymes, therapeutic proteins, including antibodies, hormones, and vaccines. Due to some unique attractive features, engineering of the chloroplast genome provides a promising platform for the production of high-value targets because it allows manipulation of metabolic processes in ways that would be impossible, or at least prohibitively difficult through traditional approaches. Since its initial demonstration in 1988 in Chlamydomonas reinhardtii, genetic tools have been developed, which have made it possible to produce high-value molecules in different species. However, the commercial application of microalgae as production platform is hindered by many factors like poor biomass, low product yields, and costly downstream processing methodologies. In this review, we discuss the potential of microalgae to use as an alternative production platform for high-value targets using chloroplast transformation technology.

Stable plastid transformation of rice, a monocot cereal crop.

Rice is one of the most important cereal crops and its biotechnology has been pursued to meet the food demand of ever-growing global population. Rice plastid transformation has been a great challenge to achieve homoplastomic plants due to its low efficiency of regeneration. In this experiment, Japonica rice line 19 was chosen to be the receptor for plastid transformation. A vector harboring smGFP gene was constructed and transferred into rice plastid genome by bombardment. The resistant callus was obtained after long-lasting multiple selections and proved to be in homoplastomic status by molecular testing. The plantlet was regenerated from homoplastomic callus and grown to seeding stage. This is the first case so far to achieve the homoplastomic rice and will be helpful to transform plastid genome of monocotyledonous crops with recalcitrant nature.

ZmSTK1 and ZmSTK2, encoding receptor-like cytoplasmic kinase, are involved in maize pollen development with additive effect.

Pollen germination and pollen tube growth are important physiological processes of sexual reproduction of plants and also are involved in signal transduction. Our previous study reveals that ZmSTK1 and ZmSTK2 are two receptor-like cytoplasmic kinases (RLCK) homologs in Zea mays as members of receptor-like protein kinase (RLK) subfamily, sharing 86% identity at the amino acid level. Here, we report that ZmSTK1 and ZmSTK2, expressed at late stages of pollen development, regulate maize pollen development with additive effect. ZmSTK1 or ZmSTK2 mutation exhibited severe pollen transmission deficiency, which thus influenced pollen fertility. Moreover, the kinase domains of ZmSTKs were cross-interacted with C-terminus of enolases detected by co-immunoprecipitation (Co-IP) and yeast two-hybrid system (Y2H), respectively. Further, the detective ZmSTK1 or ZmSTK2 was associated with decreased activity of enolases and also reduced downstream metabolite contents, which enolases are involved in glycolytic pathway, such as phosphoenolpyruvate (PEP), pyruvate, ADP/ATP, starch, glucose, sucrose and fructose. This study reveals that ZmSTK1 and ZmSTK2 regulate maize pollen development and indirectly participate in glycolytic pathway.

Isolation and characterization of a novel pollen-specific promoter in maize (Zea mays L.).

ZmSTK2_USP, located on the long arm of chromosome 4, belongs to the serine/threonine kinase gene in maize. The sequence analysis of 2100 bp upstream from the start codon ATG has shown that it contains cis-element motifs and two types of anther/pollen-specific promoter elements (GTGA and AGAAA), suggesting that it is the pollen-specific promoter. To investigate the function of ZmSTK2_USP promoter, the GUS gene fusion system was employed. In proZmSTK2_USP-GUS genetically modified plants, GUS activity was detected in mature pollen grains and pollen tubes but not found in other floral and vegetative tissues. These results show that proZmSTK2_USP is the pollen-specific promoter and drives pollen-specific activity during the middle stage of pollen development until pollen maturation.

Production of Bioactive Recombinant Bovine Chymosin in Tobacco Plants.

Chymosin (also known as rennin) plays an essential role in the coagulation of milk in the cheese industry. Chymosin is traditionally extracted from the rumen of calves and is of high cost. Here, we present an alternative method to producing bovine chymosin from transgenic tobacco plants. The CYM gene, which encodes a preprochymosin from bovine, was introduced into the tobacco nuclear genome under control of the viral 35S cauliflower mosaic promoter. The integration and transcription of the foreign gene were confirmed with Southern blotting and reverse transcription PCR (RT-PCR) analyses, respectively. Immunoblotting analyses were performed to demonstrate expression of chymosin, and the expression level was quantified by enzyme-linked immunosorbent assay (ELISA). The results indicated recombinant bovine chymosin was successfully expressed at an average level of 83.5 ng/g fresh weight, which is 0.52% of the total soluble protein. The tobacco-derived chymosin exhibited similar native milk coagulation bioactivity as the commercial product extracted from bovine rumen.

Stable Expression of Basic Fibroblast Growth Factor in Chloroplasts of Tobacco.

Basic fibroblast growth factor (bFGF) is a multifunctional factor in acceleration of cell proliferation, differentiation and transference, and therefore widely used in clinical applications. In this study, expression vector pWX-Nt03 harboring a codon-optimized bFGF gene was constructed and introduced into the tobacco chloroplasts by particle bombardment. After four rounds of selection, bFGF was proved to integrate into the chloroplast genome of regenerated plants and two of four transgenic plants were confirmed to be homoplastomic by PCR and Southern hybridization. ELISA assay indicated that bFGF represented approximately 0.1% of total soluble protein in the leaves of transplastomic tobacco plants. This is the first report of bFGF expression via chloroplast transformation in model plant, providing an additional option for the production of chloroplast-produced therapeutic proteins.

Chloroplast-expressed MSI-99 in tobacco improves disease resistance and displays inhibitory effect against rice blast fungus.

Rice blast is a major destructive fungal disease that poses a serious threat to rice production and the improvement of blast resistance is critical to rice breeding. The antimicrobial peptide MSI-99 has been suggested as an antimicrobial peptide conferring resistance to bacterial and fungal diseases. Here, a vector harboring the MSI-99 gene was constructed and introduced into the tobacco chloroplast genome via particle bombardment. Transformed plants were obtained and verified to be homoplastomic by PCR and Southern hybridization. In planta assays demonstrated that the transgenic tobacco plants displayed an enhanced resistance to the fungal disease. The evaluation of the antimicrobial activity revealed that the crude protein extracts from the transgenic plants manifested an antimicrobial activity against E. coli, even after incubation at 120 °C for 20 min, indicating significant heat stability of MSI-99. More importantly, the MSI-99-containing protein extracts were firstly proved in vitro and in vivo to display significant suppressive effects on two rice blast isolates. These findings provide a strong basis for the development of new biopesticides to combat rice blast.

Studying Salt-Induced Shifts in Gene Expression Patterns of Glucosinolate Transporters and Glucosinolate Accumulation in Two Contrasting Brassica Species.

Brassica crops are well known for the accumulation of glucosinolates-secondary metabolites crucial for plants' adaptation to various stresses. Glucosinolates also functioning as defence compounds pose challenges to food quality due to their goitrogenic properties. Their disruption leaves plants susceptible to insect pests and diseases. Hence, a targeted reduction in seed glucosinolate content is of paramount importance to increase food acceptance. GLUCOSINOLATE TRANSPORTERS (GTRs) present a promising avenue for selectively reducing glucosinolate concentrations in seeds while preserving biosynthesis elsewhere. In this study, 54 putative GTR protein sequences found in Brassica were retrieved, employing Arabidopsis GTR1 and GTR2 templates. Comprehensive bioinformatics analyses, encompassing gene structure organization, domain analysis, motif assessments, promoter analysis, and cis-regulatory elements, affirmed the existence of transporter domains and stress-related regulatory elements. Phylogenetic analysis revealed patterns of conservation and divergence across species. Glucosinolates have been shown to increase under stress conditions, indicating a potential role in stress response. To elucidate the role of GTRs in glucosinolate transportation under NaCl stress in two distinct Brassica species, B. juncea and B. napus, plants were subjected to 0, 100, or 200 mM NaCl. Based on the literature, key GTR genes were chosen and their expression across various plant parts was assessed. Both species displayed divergent trends in their biochemical profiles as well as glucosinolate contents under elevated salt stress conditions. Statistical modelling identified significant contributors to glucosinolate variations, guiding the development of targeted breeding strategies for low-glucosinolate varieties. Notably, GTR2A2 exhibited pronounced expressions in stems, contributing approximately 52% to glucosinolate content variance, while GTR2B1/C2 displayed significant expression in flowers. Additionally, GTR2A1 and GTR1A2/B1 demonstrated noteworthy expression in roots. This study enhances our understanding of glucosinolate regulation under stress conditions, offering avenues to improve Brassica crop quality and resilience.

Mesenchymal Stem Cell-Derived Exosomes Attenuate Murine Cytomegalovirus-Infected Pneumonia via NF-κB/NLRP3 Signaling Pathway.

Reactivation and infection with cytomegalovirus (CMV) are frequently observed in recipients of solid organ transplants, bone marrow transplants, and individuals with HIV infection. This presents an increasing risk of allograft rejection, opportunistic infection, graft failure, and patient mortality. Among immunocompromised hosts, interstitial pneumonia is the most critical clinical manifestation of CMV infection. Recent studies have demonstrated the potential therapeutic benefits of exosomes derived from mesenchymal stem cells (MSC-exos) in preclinical models of acute lung injury, including pneumonia, ARDS, and sepsis. However, the role of MSC-exos in the pathogenesis of infectious viral diseases, such as CMV pneumonia, remains unclear. In a mouse model of murine CMV-induced pneumonia, we observed that intravenous administration of mouse MSC (mMSC)-exos reduced lung damage, decreased the hyperinflammatory response, and shifted macrophage polarization from the M1 to the M2 phenotype. Treatment with mMSC-exos also significantly reduced the infiltration of inflammatory cells and pulmonary fibrosis. Furthermore, in vitro studies revealed that mMSC-exos reversed the hyperinflammatory phenotype of bone marrow-derived macrophages infected with murine CMV. Mechanistically, mMSC-exos treatment decreased activation of the NF-κB/NLRP3 signaling pathway both in vivo and in vitro. In summary, our findings indicate that mMSC-exo treatment is effective in severe CMV pneumonia by reducing lung inflammation and fibrosis through the NF-κB/NLRP3 signaling pathway, thus providing promising therapeutic potential for clinical CMV infection.

The determinants of COVID-19 vaccine uptake among migrants from 109 countries residing in China: A cross-sectional study.

Background: The present study aimed to investigate the prevalence of COVID-19 vaccine uptake among foreign migrants in China and to explore the determinants of their vaccine uptake behavior. Methods: From June to October 2021, we used convenience and snowball sampling to recruit a sample of 764 participants from five cities in which the overwhelming majority of foreign migrants in China live. The chi-square (χ2) tests were used to examine vaccination distribution according to demographic characteristics. Multivariate logistic regression models visualized by forest plot were used to investigate the associations between significant determinants and vaccine uptake. Results: Overall, the prevalence of vaccination rate was 72.9% [95% confidence interval (CI): 69.9-76.0%]. Migrants whose social participation was very active [adjusted odds ratio (AOR): 2.95, 95% CI: 1.36-6.50, P = 0.007] or had perceived COVID-19 progression prevention by the vaccine (AOR: 1.74, 95% CI: 1.01-3.02, P = 0.012) had higher odds of vaccination compared to those whose social participation was inactive or who did not have this perception. Migrants who perceived the vaccine uptake process as complex (AOR: 0.47, 95% CI: 0.27-0.80, P = 0.016) or were unsure of their physical suitability for the vaccine (AOR: 0.40, 95% CI: 0.24-0.68, P < 0.001) had lower odds of vaccination compared to those who did not have these perceptions. Furthermore, migrants from emerging and developing Asian countries (AOR: 2.32, 95% CI: 1.07-5.21, P = 0.04) and the Middle East and Central Asia (AOR: 2.19, 95% CI: 1.07-4.50, P = 0.03) had higher odds of vaccination than those from major advanced economies (G7) countries, while migrants from other advanced economic countries (OR: 0.27, 95% CI: 0.11-0.63, P = 0.003) had lower odds of vaccination than those from G7 countries. Conclusion: It may be beneficial to promote vaccine uptake among migrants by ensuring effective community engagement, simplifying the appointment and uptake process, and advocating the benefits and target populations of the COVID-19 vaccine.

Transformation of alfalfa chloroplasts and expression of green fluorescent protein in a forage crop.

The ability to transform chloroplasts in multiple species is important for improving agricultural traits. Chloroplast transformation of alfalfa (Medicago sativa L.), a useful forage plant with high market value, was achieved using a vector carrying aadA and gfp genes being introduced into the chloroplasts of alfalfa via particle bombardment using leaves and calli as explants. Resistant somatic embryos were generated and developed into plantlets from explants. The transformation efficiency was 1.3% for callus explants and 2.7% for leaf explants. PCR and Southern blotting analyses revealed that the foreign genes were integrated into the transformed chloroplast genome. The occurrence of GFP was further confirmed by fluorescence microscopy. Expression of foreign genes in alfalfa chloroplasts is therefore possible, and provides a novel means for genetic improvement of agronomically important traits and production of value-added proteins.

20(S)-Ginsenoside Rg3 Inhibits Lung Cancer Cell Proliferation by Targeting EGFR-Mediated Ras/Raf/MEK/ERK Pathway.

Lung cancer is the leading cause of cancer death in the world and classified into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). As tyrosine kinase inhibitors (TKIs), several triterpenoid saponins can target to epidermal growth factor receptor (EGFR), a widely used molecular therapeutic target, to exhibit remarkable anti-proliferative activities in cancer cells. As one of triterpenoid saponins, 20([Formula: see text])-ginsenoside Rg3 [20([Formula: see text])-Rg3] was confirmed to be an EGFR-TKI in this work. According to the quantitative real-time reverse transcription-PCR (qRT-PCR) and immunoblotting analysis, 20([Formula: see text])-Rg3 was certified to play a key role on EGFR/Ras/Raf/MEK/ERK signal pathway regulation. Our data demonstrated that 20([Formula: see text])-Rg3 might block the cell cycle at the G0/G1 phase by downregulating CDK2, Cyclin A2, and Cyclin E1. Molecular docking suggested that the combination of both hydrophobic and hydrogen-bonding interactions may help stabilizing the 20([Formula: see text])-Rg3-EGFR binding. Furthermore, their binding stability was assessed by molecular dynamics simulation. Taken together, these data provide the evidence that 20([Formula: see text])-Rg3 could prohibit A549 cell proliferation, probably by arresting the cell cycle at the G0/G1 phase via the EGFR/Ras/Raf/MEK/ERK pathway.