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Deep Learning Technology: Sebastian Arnold, Betty van Aken, Paul Grundmann, Felix A. Gers and Alexander Löser. Learning Contextualized Document Representations for Healthcare Answer Retrieval. The Web Conference 2020 (WWW'20)
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An exploration on Drynaria species, highly prescribed in bone fracture therapy, successfully isolated flavonoid constituents that induce osteoblast proliferation. Previous studies on Acrostichum aureum Limme failed to show its anti-bacterial activities contrary to its traditional claims in wound management. However, patriscabratine 257 was isolated from the defatted methanol extract of whole plant of A. aureum, and subsequent testing showed it possessed anti-cancer activity in gastric cells and this supprted the traditional use of the plant in peptic ulcer therapy. A decoction from the epiphyte Ficus deltoida has been used to treat diabetes. A study on the hot aqueous extract of this plant revealed anti-hyperglycemic activity by stimulating insulin secretion up to seven-fold. Furthermore, its activity mechanism was related to both the K+ATP-dependant and -non-dependant insulin secretion pathway. However, further studies are required to identify the constituents responsible for the anti-hyperglycaemic activity.
The Indigenous people of Paraguay have used Catasetum barbatum Lindley to topically treat inflammation. Four bioactive compounds were isolated from this species and 2,7-dihydroxy-3,4,8-trimethoxyphenanthrene (confusarin) 595 showed the highest anti-inflammatory activity. The study also revealed the compound to be a non-competitive inhibitor of the H1-receptor.
From the polypodiaceae family, the rhizome of Phymatodes scolopendria (burm.) Ching has been used to treat respiratory disorders. A bioassay-guided phytochemical study on Phymatodes scolopendria (Burm. f.) Pic. Serm. isolated 1,2-benzopyrone (coumarin) 209 as a bronchodilator.
Essential oils (EOs) are defined as volatile secondary metabolites of plants that give the plant a distinctive smell, taste, or both. EOs are produced by more than 17,500 species of plants from many angiosperm families, e.g., Lamiaceae, Rutaceae, Myrtaceae, Zingiberaceae, and Asteraceae, but only about 300 of them are commercialized. Compounds included in the EOs are synthesized in the cytoplasm and plastids of plant cells through the pathways of malonic acid, mevalonic acid, and methyl-d-erythritol-4-phosphate (MEP). They are produced and stored in complex secretory structures, such as glands, secretory cavities, and resin conduits, and are present as drops of liquid in the leaves, stems, flowers and fruits, bark, and roots of plants. Despite containing two or three main components at a level of 20–70%, EOs are very complex mixtures of mainly terpenes, terpenoids, and phenylpropanoids. They may also contain many other compounds, such as fatty acids, oxides, and sulfur derivatives.
EOs are usually obtained as a result of hydrodistillation, steam distillation, dry distillation, or the mechanical cold pressing of plants. At the laboratory scale, the classical method is based on the use of the Clevenger steam distillation apparatus, discovered in 1928. Due to several disadvantages (i.e., placement of valve, fragility), this apparatus was modified by Jakub Deryng in 1951 and it is widely used in Central European countries. Modifications of the simultaneous distillation-extraction (SDE) equipment were described in the manuscript of Arora et al.. The effectiveness of these modifications was described in detail by Baj et al.. At the laboratory scale, modern methods also include processes supported by microwaves and extraction in supercritical fluids. EOs can also be isolated using fermentation, crushing, extraction, or hydrolysis. However, depending on the chosen method, the chemical composition of the obtained EO can unfortunately be different.
Humans have used EOs for thousands of years, not only as ingredients of perfumes or as seasonings for the aromatization of food, but also in folk medicine, because of their many different biological properties, including antimicrobial properties. The antimicrobial qualities are essential in managing the rapidly growing issue of drug-resistant microorganisms. In 2016, about 6 million people died globally due to infections of the upper respiratory tract, tuberculosis, or diarrheal diseases. At the same time, the number of strains of microorganisms resistant to existing antibiotics is constantly increasing. Patients with infections caused by drug-resistant bacteria are, thus, exposed to an increased risk of worse clinical results and even death. Such patients also consume more healthcare resources than patients infected with non-resistant strains of the same bacteria. According to the WHO report on drug resistance, the most serious problems include the resistance of Klebsiella pneumoniae to third-generation cephalosporins and carbapenem, Escherichia coli to third-generation cephalosporins and fluoroquinolone, Staphylococcus aureus to methicillin, Streptococcus pneumoniae to penicillin, and Salmonella sp. to fluoroquinolones. Among the fungal infections, the most common problem is candidiasis caused mainly by Candida albicans and less often by C. glabrata and C. parapsilosis, with more than 20 species of Candida that can cause human infection. Other examples of common fungal infections are aspergillosis, histoplasmosis, and skin mycosis (commonly known as ringworm).
In food production, inhibiting the growth of microorganisms through the use of socially acceptable preservatives is a serious problem. Society’s reluctance to use antibiotics and synthetic preservatives, such as benzoic acid, sorbic acid, lactic acid, propionic acid, acetic acid, and its derivatives, parabens or inorganic sulfites, nitrites, and nitrates, necessitates finding alternative solutions. This may be an application for EOs, especially since chemical preservatives cannot eliminate several pathogenic bacteria, such as Listeria monocytogenes, in food products or delay the growth of spoilage microorganisms. In addition, natural products are inherently better tolerated in the human body, usually with fewer side effects.
Contrary to common opinion, limited EOs possess demonstrated potential as antimicrobial agents. It should be emphasized that although the antimicrobial activity is well established, the real effect is significantly weaker compared to synthetic compounds (including antibiotics). Gram-positive bacteria seem to be much more susceptible to essential oil than Gram-negative organisms. According to available data, the activity is usually correlated with phenolic, aromatic, or alcohol groups. Due to their high volatility, the effective time of action is limited, and features such as encapsulation could be changed. On the other hand, low toxicity level, as well as their natural origin, makes them an attractive alternative in both the food as well as in cosmetic industries. Several practical application could be implemented in these industries. In summary, it should be underlined that the use of EO in microbial stabilization is possible, but all cases must be individually examined.
To evaluate the functional effect of niclosamide on bacterial blight, we first examined its effect on the growth of Xoo bacteria using three different strains, PXO99, 10208, and K3a, and Xanthomonas axonopodis pv. glycines (Xag). The results showed that growth of the Xoo strains was completely inhibited by 5 μg/ml niclosamide, whereas Xag growth was inhibited by 15 μg/ml niclosamide (Fig. 1a). Xag is a pathogen that causes bacterial leaf pustule disease in soybean45. We also examined the effect of niclosamide on the growth of the fungal pathogen Magnaporthe oryzae. Niclosamide did exert an inhibitory effect on the growth of 12 M. oryzae strains, although the inhibition was considerably weaker than that against Xoo (Fig. 1b).
We also tested the effects of parthenolide, a sesquiterpene lactone with anti-tumor activity, on the growth of the Xoo strains PXO99 and 10208. Parthenolide failed to inhibit the growth of either Xoo strain at a concentration of 5 μg/ml, although it slightly inhibited bacterial growth at 50 μg/ml (Fig. 1c). We also tested the effect of niclosamide on the growth of three E. coli strains, Top10, Rosseta2, and DH10b, which served as Gram-negative control bacteria. At lower concentrations, niclosamide had no effect on the growth of these E. coli strains, although it slightly inhibited growth at 50 μg/ml (Fig. 1d).
To determine the minimum inhibitory concentration (MIC) of niclosamide on PXO99 and 10208 growth, we tested concentrations ranging from 0–5 μg/ml. Both Xoo strains could grow in the presence of 4 μg/ml niclosamide, but not 5 μg/ml niclosamide (Fig. 1e; left panel). We further narrowed the MIC of niclosamide from 4–5 μg/ml. As shown in Fig. 1e, the growth of both PXO99 and 10208 was completely inhibited by 4.2 μg/ml niclosamide (Fig. 1e; right panel), indicating that 4.2 μg/ml niclosamide is the MIC for both Xoo strains.
Research on epiphytes that have been used in infectious disease therapy include in wound healing, dysentery, and skin infections. A study on the methanol extract of Adiantum caudatum L., Mant showed anti-fungal activity against common fungi found in wounds (Aspergilus and Candida species), including Aspergillus flavus, A. spinulosus, A. nidulans, and Candida albicans, with minimum inhibitory concentration (MIC) values of 15.6, 15.6, 31.2, and 3.9 µg/mL, respectively. Gallic acid was one of the bioactive constituents. The methanol extract of Ficus natalensis Hochst (a semi-epiphytic plant) showed anti-malarial activity against Plasmodium falciparum, with an half maximal inhibitory concentration (IC50) value of 41.7 µg/mL, and weak bactericidal activity against Staphylococcus aureus, with an MIC value of 99 µg/mL. These results became preliminary data for confirming its traditional uses as malarial fever therapy and wound healing. Phytochemical studies on Pyrrosia sheareri (Bak.) Ching successfully isolated several compounds and were subjected to anti-oxidant testing. While this was not in line with the plant’s ethnomedical uses for dysentery therapy, one of the isolated constituents was protocateuchic acid, which is known to possess anti-bacterial activity. It implies that the traditional uses of the epiphyte was for bacillary dysentery therapy.
All the isolates showed no hemolytic activity and none of the isolated Enterococcus strains harbored virulence factors genes (data not shown).
The antibiotic resistance of the LAB isolates against 15 tested antibiotics were shown in Table 4. In view of the results, all the five LAB exhibited the capacity to resist the impact of cotrimoxazole and tetracycline. On the other hand, they were all susceptible to chloramphenicol and amoxicillin. These strains were characterized by multiple resistances to at least three antibiotics. The E. faecium M6-29 strain was resistant to eleven antibiotics.
Compared with control group, no significant difference was observed in BWG of mice treated with various isolates on days 7 and 14 (data not shown). The percentage of BWG of the mice treated with L. salivarius M2-71 or E. faecium M6-29 was significantly higher (P < 0.05) than that of the control mice on day 21 (Figure 3). In addition, no significant difference in organ index among the different groups was found, including spleen (Figure 4A), liver (Figure 4B), or kidney (Figure 4C).
In sub-Saharan Africa, little information is available on the epidemiology of STEC in humans, food and animals, and current knowledge of STEC sources needs to be improved. Data on the occurrence and the characteristics of STEC in African camels is limited. This study provides evidence for the fecal carriage of STEC among dromedary camels located on a ranch in Laikipia Kenya. The restriction to one farm in Kenya is a limitation of this study and may have led to bias associated with camels from one herd only. In order to obtain a wider picture, analyses of feces from camels on other farms and other regions in Kenya would be required. For this investigation, a total of 163 fecal swabs were obtained. Thereof, 53 (32.5%) tested positive for stx by real-time PCR. STEC was recovered from 20 of the stx positive samples, amounting to 12.3% of all fecal samples. Overall, fecal carriage of STEC was confirmed for 14.5% (17 of a total of 117) of the female camels and 6.7% (3 of 45) of the male camels (Table 2).
The serotypes and virulence genotypes of the STEC isolates are summarized in Table 2. The most frequent serotypes included nine (45% of the isolates) STEC O156:H25 isolates and three (15%) O43:H2 isolates. The remaining strains occurred as single serotypes or were O non-typeable (Table 2). STEC O156:H25 has been described as a persistent colonizer of the bovine gut and has also been linked to human infection in Germany and Switzerland (Table 1). By contrast, STEC O43:H2 is a rare serotype, but has been detected in beef in West Africa.
Further, various stx subtypes were detected among the isolates, including stx2a which is associated with severe disease in STEC infected humans. The stx2a gene was found in a total of 11 (55%) of the isolates, including the nine STEC O156:H25 strains, and the STEC O8:H49 and STEC Ont:H7 isolates, respectively (Table 2). The intimin gene eae was detected in nine (45%) isolates, all belonging to serotype O156:H25 (Table 2). While eae is one of the most prominent virulence factors contributing to pathogenesis, many STEC feature other virulence genes that may enhance their pathogenic potential, such as enterohaemorrhagic E. coli haemolysin ehx. In this study, ehx was detected by PCR in fourteen (70%) of the isolates (Table 2).
Furthermore, we used WGS to provide additional information on the virulence of the analysed STEC. Table 2 shows the presence or absence of a selection of virulence genes that predict severe disease in humans, including espA, tccP, nleA, nleB and tir (de Boer et al.,). Other virulence factors included in Table 2 are iss, which encodes an increased serum survival protein and is associated with non-bloody diarrhoea as well as extra-intestinal pathogenic infections, subAB, which is an emerging pathogenic factor among eae negative human pathogenic STEC isolates, and astA encoding a heat-stable enterotoxin EAST1 that is found mostly in enterohaemorrhagic and enteroaggregative E. coli. The complete ARIES analysis reports on the virulotypes of the 20 sequenced strains are available upon request.
Taken together, espA, tccP, nleA, nleB and tir were detected exclusively among the nine STEC O156:H25 isolates, while iss was identified in 16 (80%), and subAB in four (20%) of the isolates, respectively (Table 2). A minority of two strains (10%) possessed astA (Table 2). Although all STEC can conceivably cause diarrhoea, the risk and severity of infection is linked to the presence of virulence factors, in particular stx2a and eae. Our results show that although 45% of the STEC isolated from camel feces are not highly pathogenic according to their virulence factor profiles, the majority (55%) is associated with stx2a, and STEC O156:H25 isolates in particular, exhibit a virulotype that suggests a potential health risk.
Using the Pasteur scheme, four different sequence types (ST) were identified among 14 isolates (Table 2). Using the Warwick scheme, 11 different STs were identified (Table 2). Of the nine STEC O156:H25 isolates, eight grouped in the sequence type ST300 and ST5334, which differ by one nucleotide in the gyrB gene, indicating close phylogenetic relationship of the isolates. Similarly, the three STEC O43:H2 isolates were assigned to ST937 and ST6275 which differ by one nucleotide in fumC. Two STEC Ont:H20 possessed ST5759. All other isolates belonged to singly occurring STs (Table 2). Accordingly, cgMLST using Ridom SeqSphere+ software divided the strains into distinct clusters that comprised the STEC O156:H25 isolates, the STEC O43:H2 isolates, and the Ont:H20 isolates, respectively, while the remaining strains did not reveal close phylogenetic relationship (Fig. 1).
Finally, all STEC isolates remained fully susceptible to all antimicrobials tested (data not shown).
Common bean, Phaseolus vulgaris, represents a great source of nutrition for millions of people and is the second most important legume crop, after soybean. It is the target of multiple pests and diseases causing substantial losses. For example, on susceptible bean cultivars, bean rust, caused by Uromyces appendiculatus, may cause yield reduction from 18 to 100% with favorable environmental conditions, such as high moisture and temperature between 17 and 27°C. Among the 5 different stages of the bean rust life cycle, basidia, pycnia, aecia, uredinia, and telia, the most devastating on bean is the uredinial stage. The latent period between the germination of an urediniospore and the formation of a sporulating pustule can be as short as 7 days. Signs of infection by Uromyces appendiculatus include the presence of uredinia or spore-producing pustules on the surface of the leaf. The identification of fungal proteins from quiescent and germinating uredospores enhanced the understanding of the infection process of this fungus.
Based upon mapping and quantitative trait loci (QTL) analysis, several genes involved in Colletotrichum lindemuthianum (Co; anthracnose)resistance and other resistance genes for bean common mosaic virus (BCMV), bean golden yellow mosaic virus (BGYMV), common bacterial blight, and bean rust are clustered. The large number of resistance (R) genes for bean rust may correlate with the high pathogen population diversity; with 90 different races identified. The locus Ur-3 confers resistance to 44 out of the 89 U. appendiculatus races present in the USA. Besides the Ur-3 locus, a number of other R genes were identified in bean; such as locus Ur-4 for race 49, locus Ur-11 epistatic to Ur-4 for race 67 or locus Ur-13 mapped to the linkage group B8. To date, no large scale transcriptomic analysis of bean rust infection has been performed to better understand the mechanism of resistance. All of these Ur genes are effective against one specific rust strain, following the gene-for-gene resistance theory. Consequently, gene pyramiding was used to produce cultivars carrying multiple resistance genes. Unfortunately, such resistance may prove to be effective in the field for only a short time due to the adaptation of the fungus to overcome plant defenses. Consequently, unraveling and understanding the mechanisms downstream of these R genes is a key research goal to circumvent the adaptation of the fungus to plant resistance.
We investigated the Phaseolus vulgaris-Uromyces appendiculatus pathosystem at a transcriptional level for a better understanding of the plant response to fungal infection. In this study, we developed a subtractive suppressive hybridization (SSH) library made from the common bean cultivar Early Gallatin that exhibits susceptibility to U. appendiculatus race 41(virulent strain) but resistance to U. appendiculatus race 49 (avirulent strain). The resistance to U. appendiculatus is conferred by the presence of the Ur-4 gene in this cultivar that leads to a hypersensitive response (HR) in presence of the pathogen race 49. This cDNA bean library was enriched in expressed sequence tags (ESTs) that are potentially up-regulated by the compatible and incompatible interactions. More than 20,000 clones from the SSH library were sequenced and assembled into contigs. A total of 10,221 P. vulgaris sequences and 360 U. appendiculatus sequences were added to the NCBI database, significantly increasing the number of ESTs available for common bean. The regulation of 90 genes was confirmed by quantitative real time polymerase chain reaction (qRT-PCR) revealing 3 main expression patterns and highlighting gene regulation that occurs downstream of R protein activation.
The strains were tested for their antimicrobial activities against different bacterial pathogens and the results were shown in Table 3. Among the five LAB isolates, L. salivarius M2-71 exhibited the strongest antimicrobial activity (inhibition zone diameters > 17 mm) toward all of the six indicator bacteria.
The review below (Table 3) describes a selection of the clinical trials and experimental studies using ethnopharmacologically accepted models that have verified the traditional and therefore ethnoveterinary use of the plants described in the results section. In the few cases in which clinical trials have not yet been carried out, the range of therapeutically important and relevant biological properties of the plant is provided. Recent research has indicated that Betonica and Stachys may be separate genera or subgenera and this should be taken into consideration when reviewing the pharmacological literature on betony.
Half of the leaves of 80-day-old rice plants were covered by polythene bags and the remaining systemic leaves were sprayed with 8 μg/ml niclosamide. After treatment, the bag-covered leaves were harvested after incubation for the indicated time periods. Niclosamide was extracted from 0.5 g of each sample using absolute MeOH, and the niclosamide concentration was determined by HPLC separation and fluorescence detection. The relative niclosamide concentration in each sample was compared with the 100 ppb niclosamide standard. Niclosamide levels were expressed as the mean value plus/minus the standard deviation.
The data from our study suggest that dromedary camels are a reservoir of human pathogenic STEC. While none of the fecal samples contained STEC O157:H7 or other non-O157 STEC serogroups frequently associated with severe clinical outcomes such as bloody diarrhoea, HC, or HUS, the majority of the isolates harboured stx2a, and the STEC O156:H25 isolates harboured eae, ehx, and other virulence factors often associated with disease in humans. Their occurrence in fecal samples from camels may represent a threat to humans, especially to those who live in long-term close contact with camels and consume raw camel milk.
Plant pests and pathogens have an important role in global food crops causing significant economic losses in the agricultural industry and threatening food security [,,]. Yam (Dioscorea spp.) is one of the most important staple food crops worldwide and plays a major role in food security and income generation for more than 60 million people in West Africa, with this region contributing over 95% of the world's total yam production. Yams are generally propagated vegetatively through their tubers, which facilitates the spread and accumulation of pathogens, particularly viruses. To date, several virus species belonging to different genera (Potyvirus, Badnavirus, Cucumovirus, Aureusvirus, Potexvirus, Macluravirus, and Carlavirus) [,,,,,] have been reported and characterized in yams. These viral infections restrict the international exchange of yam germplasm and have a significant impact on tuber yields and quality. For example, reports from the Ivory Coast and western Nigeria have described average annual yield losses of 30–50% due to virus infections. Additional constraints to increase yam production and productivity are the unavailability and associated high costs of high-quality virus-free (termed ‘clean’) seed yams and the absence of a formal seed yam certification system.
Infections by potyviruses (genus Potyvirus; family Potyviridae) cause the most economically important diseases of yams and are widespread across the numerous yam growing regions worldwide. The best described potyvirus infecting yam is Yam mosaic virus (YMV), known to infect several species of yam, particularly the most widely cultivated D. rotundata, D. cayenensis and D. alata, while the second most described yam potyvirus, Yam mild mosaic virus (YMMV) is more commonly found on D. alata.
Historic data suggest a strong influence of human activity on the dissemination of viruses through trade and transportation of infected plant material [1,,,]. Applying full phytosanitary surveillance in plant quarantine and certification facilities is unrealistic due to high costs associated with increasing inspection rates. Therefore, there is an urgent need to develop improved detection methods for yam viruses to help make timely decisions on the health status of yam planting material. Several serological and PCR-based methods have been developed and applied for the detection of YMV and YMMV. Some considerations must be taken into account when choosing the detection method, such as sensitivity, specificity, cost and time to obtain results. Although PCR-based assays are often preferred for their sensitivity and specificity, they require specific technical expertise and sophisticated equipment. In addition, PCR-based methods usually require the extraction of high-quality DNA/RNA from the sample material, which is time-consuming, generally involves hazardous chemicals and cannot be done in the field.
An isothermal amplification method called recombinase polymerase amplification (RPA), overcomes the disadvantages of PCR-based assays as it reduces the need for expensive apparatus to control reaction temperature as well as providing rapid and reliable results with sensitivity and specificity comparable to conventional PCR assays. RPA has been successfully used in the detection of several animal [,,], human and plant [,,] pathogens. Recently, we developed a sensitive and robust reverse transcription-recombinase polymerase amplification (RT-RPA) assay for the specific detection of YMV. To develop further this promising diagnostic method and bring it closer to a format suitable for on-site detection of the two most important yam potyviruses, the time-consuming RNA purification step needs to be removed. In this study, we report a RT-RPA method for the detection of YMV and YMMV directly from the crude extract of infected plant material using a simple and inexpensive extraction method. Yam and potyviruses form an excellent combination as a general working model of wide applicability to other plant virus systems as: (1) potyviruses comprise the largest genus of plant RNA viruses causing significant losses in different crops worldwide and (2) yams represent particularly recalcitrant leaf tissue that contain high levels of PCR-inhibitory compounds such as polyphenols and polysaccharides, and hence the technique should be suitable for application to a diverse range of plant species.
The method developed in this study, termed ‘Direct RT-RPA’, thus has the potential to be adapted to any recalcitrant plant species and be used to obtain rapid responses in certification laboratories, reducing costs by minimising quarantine time. In addition, this method will specifically strengthen current efforts in West Africa to multiply and deliver ‘clean’ certified yam planting material to smallholder farmers and thereby improve food and income security.
No additional information is available for this paper.
Gossypol and β-sitosterol were separated from acetone mixture extracted from cottonseed oil sludge and tested for its antiviral effect against TMV in greenhouse, and RSV and SRBSDV in the field trials. As a result, its antiviral effect is better than the commercial agent Ningnanmycin in all three types of viruses including TMV, RSV and SRBSDV, and all infected plant species, indicating that this acetone extract is effectively against a broad spectrum of plant viruses in various plant species including both dicots (tobacco) and monocots (rice). Gossypol and β-sitosterol were seperated from the acetone extract and tested active against TMV. Environmental toxicology showed that this new acetone extract is environmentally friendly and no phytotoxic activity was found in the treated plants.
Gossypol (GOS), a polyphenolic compound isolated from cotton seeds, has been applied as a male contraceptive drug for many years, and also has several new clinical applications including antiviral, antimalarial, and antitumor effects [12–14]. Gossypol, and its derivatives and analogs have been reported as effective agents against human immunodeficiency virus tipe 1 (HIV-1) [15–17], which is proved to be an HIV-1 reverse transcriptase inhibitor by targeting the non-nucleoside inhibitor binding pocket of reverse transcroptase. An et al. also found that the alanine-(-) gossypol derivative, an effective HIV-1 entry inhibitor, can bind to the gp41 hydrophobic pocket and block the formation of the cell fusion-activated gp41 core. In this study, gossypol was found to exhibit effective anti-TMV activity with 54.4% curative effect at 500 μg ml-1.
β-sitosterol, the dominant phytosterol with chemical structures similar to that of cholesterol, has been reported to have several medical uses such as hypocholesterolemic activity [18, 19], anti-inflammatory activity, induction of apoptosis [21, 22], immunomodulatory activity and anti-oxidant effect [24, 25]. In addition, β-sitosterol isolated from aerial parts of Cissus sicyoides showed antibacterial activity against Bacillus subtilis with minimal inhibitory concentration (MIC) of 50 μg mL-1. Peres et al. analyzed chemical compositions and antimicrobial activity of Croton urucurana, and found. Hex/DCM fraction containing β-sitosterol exhibited the highest inhibitory effect against Staphylococcus aureus (0.8 mg mL-1). Gebre-Mariam et al. revealed that extracts containing β-sitosterol from Euclea schimperi showed antiviral activity against coxsackievirus B3 (CVB3), influenza A virus and herpes simplex virus type 1 Kupka (HSV-1). Alphaamyrin, uvaol, ursolic acid and its lactone, and β-sitosterol were detected in the leaves of Euclea schimperi. But there are no relevant reports on antiviral activity of ouabain, amyrin, uvaol, and β-sitosterol. Lin et al. found that β-sitosterol, one of the five major compounds of the Isatis indigotica root, inhibited cleavage activities inhibited cleavage activities of the SARS coronavirus 3C-like protease in cell-free and cell-based assays. In this study, we confirmed that β-sitosterol has antiviral properties in a certain degree, although not better than that of gossypol, with 45.6% curative effect at 500 μg mL-1 against TMV.
When compared with its individual components (gossypol and β-sitosterol), the extract mixture resulted in the best curative effect (71.2%). In the form of complex mixture, the bioactive compounds may simultaneously function as antiviral activity, dependently or independently with each other, thus reinforcing the inhibiton effect against viruses. Another advantage of mixture of antiviral agents has been discussed by previous researches. One important challenge for any antiviral drugs is the development of resistance by various viruses. In this case, mixture of antiviral compounds is more efficient and practical than the single ones. Because a virus that has developed resistance to a particular drug may not be resistant to other antiviral compounds, which have the potential to possess similar, if not identical, antiviral activities.
Natural-product-based antiviral agents have the ability to decompose rapidly, thereby reducing their risk to the environment [32, 33]. In this study, no toxicity was found in tobacco or rice plants. And this acetone extract was slightly toxic to Japanese quail, Daphnia magna, Scenedesmus obliquus and Bombyx mori, and moderately toxic to fish and bees. This environment friendly acetone extract holds potential promise for commecial application in the future. Firstly, from economic considerations, the starting material cottonseed is widely available in China and the cost may be very low. Secondly, cottoseed oil sludge rather than cottonseed oil was served as the raw material, which is the precipitation and enrichment of antiviral effective compounds, so that maximum efforts have been made to improve the concentration of active compounds. Most importantly, the in vivo and field trials demonstrated that this acetone extract was broad-spectrum antiviral, not only against different plant virus (TMV, RSV, SRBSDV), but also more effective in different host varieties from different geographic distributions in China, compared with the commecial agent Ningnanmycin.
In summary, in this study, we firstly discovered acetone extract from cottonseed oil sludge as novel antiviral agent against plant viruses such as TMV, RSV and SRBSDV. Also to our knowledge this is the first report on the application of gossypol and β-sitosterol to plant virus control. Further study will be focused on the antiviral mechanism of these compounds against plant viruses. Hopefully, our results will provide some useful insights for the future virus control strategies.
For the study of phytotoxic activity, the acetone extract was diluted to 500 μg mL-1, sprayed on the leaves of the tested tobacco and rice, and regularly observed. Environmental toxicology was carried out by Institute of Plant Protection (IPP), Chinese Academy of Agricultural Sciences (CAAS) from 2012 to 2013.
More than half of the world’s population relies on rice as a staple source of nourishment. However, over the past decades we have witnessed a serious decline in the overall rice production in several parts of the world due to the unsolicited attack by the several pathogens like fungi and bacteria causing severe diseases, and consequently poses a severe threat to global food security (Niño-Liu et al., 2006; Normile, 2008; Tilman et al., 2017). Xanthomonas oryzae pv. oryzae (Xoo) is a Gram negative rod-shaped bacterium liable for causing the most devastating bacterial leaf blight disease in rice, which causes annual yield losses of 10–50% and even 100% under severe conditions in many rice growing countries (Niño-Liu et al., 2006; Mansfield et al., 2012; Zhang and Wang, 2013). Wounds or hydathodes are the main entry sites for Xoo where it multiplies in the epitheme, and subsequently enters into the xylem vessels where it starts active multiplication, leading to the initiation of leaf wilting (a typical symptom of the blight disease) (Köplin et al., 1992; Ray et al., 2000).
Xoo is well known to produce a wide varieties of virulence factors, including exopolysaccharides, extracellular enzymes, iron-chelating siderophores, and type III secretion-dependent effectors (Ray et al., 2000; Jha et al., 2007; Liang et al., 2016). The production of large amounts of an exopolysaccharide called xanthan is one of the most important virulence factors of this species. Xanthan is a heteropolysaccharide with a cellulose-like backbone and trisaccharide side-chains of two mannose and one glucuronate residues that are attached to every second glucose moiety of the main chain (Jansson et al., 1975; Köplin et al., 1992). The synthesis of xanthan is regulated by the gumBCDEFGHIJKLM genes, located in a single gene cluster of 12 kb which is expressed as an operon from a promoter upstream of the first gene, gumB (Katzen et al., 1998; Vojnov et al., 1998, 2001). As Xoo is a Gram negative bacterium, it also produces a large amount of lipopolysaccharides (LPSs) in its outer membrane. LPSs are crucial molecules for the viability of Gram negative bacteria and in several phases of host–bacterium interaction such as symbiosis, virulence and tolerance (Silipo et al., 2010; Di Lorenzo et al., 2016). Many studies have demonstrated the role of LPSs in inducing the basal plant defenses thereby entitling LPS molecule as a microbe-associated molecular pattern (MAMP) (Di Lorenzo et al., 2016; Meir et al., 2017). Many plant-pathogenic bacteria use quorum sensing (cell–cell communication) to regulate the expression of factors contributing to virulence (Musthafa et al., 2013; Barel et al., 2015). The quorum sensing (QS), increases the virulence of many species of Xanthomonas via the regulation of motility, chemotaxis, stress responses, biofilm dispersal and the synthesis of extracellular enzymes and EPS (LaSarre and Federle, 2013; Shi et al., 2016; Barel et al., 2015).
To control the bacterial leaf blight disease in rice, Bismerthiazole and streptomycin are utilized as the major bactericides, but recent studies have shown that Xoo has developed high resistance against these chemicals (Xu et al., 2010; Zhu et al., 2013; Shi et al., 2015; Yu et al., 2016). Hence, the quest for innovative antibacterial agents remains a major challenge, and such chemical or biological agents are seriously required to control the disease inflicted damages. Over 1 billion people are infected with intestinal nematodes, and many millions are infected with filarial nematodes, flukes, and tapeworms. Niclosamide has been successfully used as an orally bioavailable chlorinated salicylanilide, with anthelmintic and potential antineoplastic activity (Imperi et al., 2013). Niclosamide is a very famous potent drug to cure gastrointestinal tapeworm infections in both humans and animals since 1960s (Weinbach and Garbus, 1969; Kim et al., 2016b). An extensive study in the animal system shows that “niclosamide has effective antiviral activity against the severe acute respiratory syndrome virus, anti-anthrax toxin properties, and anti-neoplastic activity. Niclosamide strongly induces LC3-positive autophagosomes, inhibits the Wnt/ Frizzled pathway, suppresses the autonomous notch-signaling pathway, and inhibits mTOR signaling. Niclosamide uncouples mitochondrial oxidative phosphorylation and thereby slows cell growth” (Weinbach and Garbus, 1969; Wu et al., 2004; Chen et al., 2009; Zhu et al., 2009; Wang et al., 2009; Osada et al., 2011; Fonseca et al., 2012). These findings clearly demonstrate that niclosamide has various curative effects on humans and animals. While searching for a master regulator that effectively functions to protect both animals and plants from infectious diseases recently Kim et al. (2016b) showed that “niclosamide can also inhibit the growth of bacterial plant pathogen Xoo, and it can move long distances from the site of local application to distant rice tissues. Niclosamide also increased the levels of salicylate and induced the expression of defense-related genes thereby suppressing the Xoo-induced leaf wilting. Interestingly, niclosamide had no detrimental effects on vegetative/reproductive growth and yield of rice suggesting that niclosamide can be effectively used to block bacterial leaf blight in rice without any negative effects.” However, in their study the underlying mechanism of action of niclosamide on Xanthomonas oryzae pv. oryzae remained unexplored. This intrigued us to further explore and establish the molecular mechanisms by which niclosamide inhibits Xoo’s bacterial growth.
Wheat yellow mosaic is one of the most devastating soil-borne diseases of winter wheat (Triticum aestivum L.). It was first reported in Japan in the 1920s and China in the 1960s, and then spread continually in Japan and China. According to the statistical data, the disease area was more than 666,700 hectares in the 1990s, the yield loss was estimated to range between 20-40% and could be up to 70-80% during a serious year, even 100%.
Wheat yellow mosaic virus (WYMV), the causal agent of wheat yellow mosaic, belongs to the genus Bymovirus within the family Potyviridae. It is a soil-borne pathogen and is transmitted by the fungus-like organism Polymyxa graminis. The genome of WYMV is comprised of two (+) single-stranded RNAs, RNA1 encodes for coat protein (CP) and six others: P3, 7 K, nuclear inclusion protein a (NIa), nuclear inclusion protein b (NIb), cytoplasmic inclusion protein (CI), 14 K; RNA2 encodes for a polyprotein that contains 28-kDa and 72-kDa proteins.
Concerning virus detection, several methods are used commonly to detect WYMV. ELISA is a reliable method for detecting WYMV and suitable for high-throughput samples; RT-PCR is the most conventional method to detect RNA virus and western blotting detects the target protein for further confirmation. However, the sensitivity of ELISA might not be sufficiently high to detect low concentrations of WYMV, and virus-specific antiserum is required. WYMV can serologically cross-react with wheat spindle streak mosaic virus, and RT-PCR is not perfect either.
Novel nucleic acid amplification methods, loop-mediated isothermal amplification (LAMP) for DNA and RT-LAMP for RNA, have been developed. The high specificity and sensitivity, rapid execution, performance under isothermal condition, time-saving, easy observation of by-products, and low cost make RT-LAMP unrivaled among diagnostic techniques. It is easy and simple to perform only with four appropriate primers, a reverse transcriptase for RNA template, a DNA polymerase and a water bath or heat block for reaction. Therefore, in recent years, many pathogenic viruses have been detected by these methods, including human, animal and plant viruses.
In the present study, the RT-LAMP method was used successfully for detection of WYMV for the first time. This method could result in more accurate diagnosis for monitoring WYMV.
Ten buchu leaves (Barosma betulina or Barosma crenulata), or uva-ursi leaves (Arctostaphylos uva-ursi), were fed to horses after races as a kidney tonic. For minor bladder infections powdered uva-ursi aerial parts and chopped or powdered leaves of dandelions (Taraxacum officinale), were mixed and fed every day until the horse's legs were no longer swollen, or the horse was no longer straining to urinate (usually one to three days). Either fresh or dried parsley (Petroselinum crispum) was added to the feed once a day or more often until the urine cleared up. Dandelion aerial parts were fed ad lib.
Drug resistance in pathogenic microbes is an emerging challenge in crop production and human health care. Multiresistant bacteria have many strategies that threaten the health of animals and plants, such as beta-lactamases that are enzymes synthesized by bacteria to break host resistance. This situation has been provoked by the recent attention paid to identifying new antimicrobial genes against emerging bacterial resistance. Therefore, scientists have been trying to discover novel biocontrol agents to overcome this problem. Antimicrobial peptides (AMPs) have received a considerable amount of attention because of their significant activity against fungi, bacteria, parasites, viruses, inflammation, and tumor cells. It is generally believed that AMPs are short peptides (20–50 amino acids) with low molecular weight and mostly linear cationic α-helices, with effective activity against a broad spectrum of pathogens. AMPs can be isolated from a variety of organisms such as bacteria, fungi, insects, plants, animals, and humans. As a new class of antibiotic with low tendency to induce resistance, high antimicrobial activity, and good selectivity, AMPs have the potential to replace some traditional antibiotics in the future. The Gram-positive bacterial cell envelope is composed of an outer cell wall (a thick peptidoglycan layer and a polysaccharide coat) and an inner cytoplasmic membrane. To achieve their bactericidal activities, AMPs interact with the cell wall or cytomembrane, resulting in membrane interruption and cell lysis. In most of these cases, AMPs are reported to cause disruptions in cell wall or cell membrane integrity, perforation, deformation, and increased water ion and molecular flow across the membrane, which ultimately causes microbial death.
In biotechnology, Bacillus subtilis has been considered an effective tool to study high-level expression of foreign proteins. Additionally, it is a generally regarded as safe (GRAS) organism that does not produce endotoxins. Because of its relatively simple cell structure, high growth rate, short fermentation time, and high capacity to secrete proteins directly into the extracellular medium, it has long been successfully used for the expression of many protein products, including some industrial enzymes (proteases, lipases, and amylases).
Isatis indigotica belongs to the Brassicaceae family and is a biennial herb that has been used as a traditional medicine to cure wounds in Europe and China for centuries. Different compounds isolated from I. indigotica leaves have exerted anti-inflammatory and anti-allergic activities. Extracts of I. indigotica hairy root cultures showed antioxidant activities. Alkaloids isolated from I. indigotica exhibited inhibitory activities against two different types of ureases (Jack bean and Bacillus pasteurii ureases) and significant antifungal activity against Aspergillus niger, Candida albicans, Trichophyton schoenleinii, T. simii, and Macrophomina phaseolina.
To date, most of the research related to AMPs has been focused on extraction, separation, purification, and synthesis of AMPs, as well as some exploration of resistance mechanisms. For the isolation of candidate AMPs, there are two main methods. One method is based on stepwise separation and detection of proteins or polypeptides after isolation from organisms directly. Because the number of AMPs in microbes is usually limited, extensive losses can occur during the isolation and purification procedures. The second method is to synthesize AMPs artificially. This method can improve AMP efficacy and range of performance.
Two factors contribute to a bottleneck in plant resistance breeding—the scarcity of plant resistance genes and the fact that resistance genes are easily overcome by pathogens. To overcome the scarcity of plant resistance genes, we established an antimicrobial gene isolation method using a B. subtilis expression system. To avoid the propensity for resistance genes to be overcome by pathogens, we designed a model in which pathogen-independent, nonself-recognition triggered, and heterosis-based fresh resistance can be generated in F1 hybrids. To the best of our knowledge, I. indigotica has been less well documented for its antimicrobial potential. In the current study, novel AMPs were identified from I. indigotica using the B. subtilis expression system, and further studies were performed to explore their antibacterial and antifungal activities. To determine the basis of the host–pathogen interaction, the potential for these AMPs to control plant diseases and their mechanisms were also investigated in this study.
Some reports have indicated that subcutaneous injections of plant-derived proteins could induce an immunogenic response to plant-specific glycans. Topical applications of plant-derived glycoproteins in humans, however, have not resulted in any adverse effects and therefore represent a potential approach for PMF-based products. Topical application of a recombinant plant secretory antibody prevented oral Streptococcal colonization for at least four months in humans. Topical application of soybean-derived monoclonal antibodies (mAbs) readily diffused in human cervical mucus and prevented herpes simplex virus 2 (HSV-2) infection. Tan et al. (2014) expressed human acidic fibroblast growth factor 1 (FGF-1) in the medicinal plant Salvia miltiorrhiza. The product combined the medicinal function of both FGF-1 and bioactive compounds within the medicinal plant. Topical application of extracts obtained from the transgenic medicinal plant significantly stimulated fibroblast cells, promoted blood vessel formation, and accelerated the healing process of burn wounds in mice. This is an example of how PMF can be used to combine the therapeutic function of a recombinant protein and the inherent properties of a medicinal plant. Topical application of a plant extract would significantly reduce the cost of purification and downstream processing. In general, topical application is safer than oral consumption or injection, which would help to address concerns about public safety.
A primary objective of PMF is to reduce the cost of producing novel therapeutic proteins. Using PMF to create a vegetable, seed or fruit health supplement could be a practical alternative to using PMF to develop a processed pharmaceutical drug. Guan et al. (2014) expressed lumbrokinase, an anti-thrombotic enzyme from earthworm, in sunflower kernels. Mice and rats that were fed the transgenic kernels exhibited a strong degradation of blood clots. Unlike a vaccine or a therapeutic protein, lumbrokinase has been widely sold and used as a health supplement to dissolve blood clots and maintain healthy cardiovascular function in people. This makes lumbrokinase a good candidate for PMF since, in general, health supplements do not need a medical prescription and have less regulations for commercialization.
Rice viral diseases are major threats to rice production and have been distributed worldwide across regions depending on rice cultivation. Two of the most prevalent rice viruses are RSV and RBSDV, which were transmitted by a small brown planthopper (SBPH, Laodelphax striatellus Fallen) [2–4]. When infected with RSV at the seedling stage, normally, rice plants grow poorly and often develop folded and twisted leaves, with the central leaves yellowing and withering; and plant growth may terminate and ultimately the plant will die [5–7]. In China, rice stripe is very serious, especially in Jiangsu province, where about 0.6 M ha per year of rice were infected by RSV during the period of 2000 to 2003, increasing to 1 M ha in 2004. In heavily infected fields, rice yield is reduced by 30–50 %, and in some of the most severely infected fields, no harvest is possible. In RBSDV infected rice always develops stunted stems, dark green, twisted leaves, and white waxy swellings along veins on the abaxial surface of the leaves [3, 9, 10]. The disease caused severely damage on rice in most parts of eastern China with due to widespread release of susceptible cultivars. Since the infection damage was very severe in China and Southeast Asia, the understanding of the responses of rice to viral infection, especially gene expression analysis, is very important for developing strategies for disease control [8, 11].
The widely used method to measure transcript abundance is RT-qPCR compared to reverse transcription-polymerase chain reaction (RT-PCR) and northern blot [12–14]. Besides being a powerful tool, RT-qPCR suffers from certain pitfalls, most important being the normalization with a reference gene [15–17]. In recent years, the reference genes, such as those encoding actin (Actin), tubulin (TUB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S rRNA, are often separately chosen for the normalization in RT-qPCR because of their constant expression levels in living organisms. Nevertheless, different studies sometimes proved different or even opposing results of these reference genes, and it was demonstrated that the transcript levels of these genes actually vary under different experimental conditions [18–21]. For example, different expression levels normalized by a different reference gene could be approximately 100 folds. Furthermore, Myzus persicae’s actin and GAPDH protein were found to interaction with Beet western yellows virus in vitro and some A.pisumwere’s genes (Actin and GAPDH) considered to be potentially related to the transmission of Peaenation mosaic virus and Soybean dwarf virus. Thus, it is important and necessary to select suitable reference gene(s) for different experimental paradigms, particularly in RSV and RBSDV infection conditions which those appropriate internal reference(s) were not identified [25–27].
In this study, we reported the validation of reference genes to identify the most suitable internal control gene(s) for the normalization of RT-qPCR data upon viral infection in rice plants. Using statistical algorithms geNorm and Norm Finder [28, 29], the stability of 14 candidate reference genes (Actin, UBC, 18S rRNA, EF-1α, UBQ 5, GAPDH, α-TUB, β-TUB, eIF-4α, Actin1, UBQ 10, TIP41-like, EXP and Os AOC) was examined and compared. Two best reference genes were identified more stably expressed than traditional ones in RSV- and RBSDV-infected treatments. Our results further indicated that the combination of these two reference genes provides a good starting point for gene expression analysis in rice viral infection plants by RT-qPCR.
The presence of YMV and YMMV was confirmed by RT-PCR using the primer pairs YMV-F/-R and YMMV-F/-R, which amplify a 586 bp and a 249 bp region comprising the coat protein (CP) gene and the 3′ UTR region of the YMV and YMMV genomes, respectively. An assay for detection of the yam actin gene was used as an internal control as described by Silva et al.. RT-PCR amplifications were set up in 20 μL reactions containing either 40 ng RNA or 2 μL of crude extract, 0.2 μM of each primer, 0.25 mM of each dNTP, 2.5 U AMV Reverse Transcriptase (Promega, Southampton, UK), 1 U DreamTaq DNA polymerase and 1x DreamTaq Green buffer (Thermo Scientific, Loughborough, UK) containing 2 mM MgCl2. The following cycle conditions were used: 50 °C for 10 min for reverse transcription, 95 °C for 4.5 min, followed by 30 cycles of 95 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min and one final extension of 72 °C for 5 min. Amplification products were analysed by agarose gel electrophoresis using 1.5% (w/v) agarose gels containing 1x RedSafe nucleic acid stain (iNtRON Biotechnology, Seongnam, South Korea) in 0.5x Tris-Boric acid-EDTA (TBE) buffer.
L. speciosa leaf-derived preparations containing ellagic acid could be useful as an antiviral agent in the prevention or treatment of HRV infection. The antiviral action of ellagic acid may be an indication of at least one of the pharmacological actions of L. speciosa. For the practical use of L. speciosa leaf-derived preparations as novel anti-HRV products to proceed, further research is needed to establish their human safety and whether this activity is exerted in vivo after consumption of L. speciosa leaf-derived products by humans. Historically, a tea from the plant leaves has been used for the treatment of diabetes mellitus in the Philippines
. Rats fed ellagic acid at doses as high as 50 mg/day up to 45 days did not cause any signs of systemic toxicity
. Lastly, detailed tests are needed to understand how to improve anti-HRV potency and stability for eventual commercial development.
The production of plant-derived pharmaceuticals has attracted great interest. Mapp Biopharmaceutical Inc., a company located in San Diego, CA, USA has produced a drug in tobacco leaves called ZMapp, which has been used to combat the 2014 Ebola virus outbreak in Africa. As of October 2014, seven infected patients received an early treatment with ZMapp and fully recovered. Another patient, receiving a late treatment with ZMapp in November 2014, however, succumbed to the disease and died. Additional Ebola patients were unable to receive the treatment due to an insufficient supply of ZMapp. This is unfortunate since it is the only drug to date that has been effectively used to treat patients infected with the Ebola virus, even though it has not been approved by the U.S. Food and Drug Administration (FDA). ZMapp has been subjected to clinical Phase I and 2 trials in 2015, sponsored by the National Institute of Allergy and Infectious Diseases (NIAID) (see Table 1). On 15 September 2015, ZMapp was granted a fast track status by the FDA. Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging disease. Due to the high mortality rate of MERS (above 35%), it caused a public panic in South Korea during May 2015. As of 27 November 2015, MERS-CoV has infected 1618 patients and caused a total of 579 deaths worldwide. Over 26 countries have reported MERS-CoV cases. Currently, no effective drug is available to treat the MERS-CoV virus. Plant Biotechnology Inc. (Hayward, CA, USA) produced an immunoadhesin (DPP4-Fc) in transgenic tobacco. Purified DPP4-Fc exhibits strong binding to MERS-CoV and prevents the virus from infecting lung cells. In June 2015, Plant Biotechnology Inc. received funding from NIAID to support further development and testing of this drug.
The concept of using plants to produce recombinant pharmaceutical proteins, referred to as plant molecular farming (PMF) or pharming (PMP), is not new. Human growth hormone, initially produced in tobacco and sunflower in 1986, was the first-plant-derived recombinant therapeutic protein. Mason et al. later expressed the hepatitis B surface antigen (HBsAg) in transgenic tobacco. This plant-derived antigen was physically and antigenically similar to the HBsAg obtained from human serum and recombinant yeast. The yeast-derived HBsAg is clinically used for HBV vaccination. Since 1994, more than 100 pharmaceutical proteins have been expressed and characterized in plants. By 2011, more than twenty PMF pharmaceuticals were placed in preclinical or clinical trials. Several PMF products have completed Phase 2 trials and one product has been approved by the FDA (Table 1). Although several plant-derived drugs have been commercialized as research and diagnostic reagents (such as tobacco derived aprotinin and rice derived lysozyme from Sigma-Aldrich Company (St. Louis, MO, USA) or received USDA approval as a vaccine additive for use in poultry (Dow Agro Sciences, Indianapolis, IN, USA), the current review mainly focuses on PMF in relation to human pharmaceutical applications. Plants represent a promising system for the production of human pharmaceutical proteins on a large scale, and at a low cost. Many production challenges, however, such as low yield, plant glycosylation, purification and downstream processing hurdles, have limited the development of PMF-based human pharmaceuticals on a clinical scale.
In May 2012, the first PMF-derived enzyme, ELELYSO™ (taliglucerase alfa) (Protalix BioTherapeutics, Karmiel, Israel), was approved for human use by the FDA. ELELYSO™ is based on the use of carrot cells to produce recombinant taliglucerase alfa, which is used in an enzyme replacement therapy to treat adult patients with Gaucher disease. The production and application of ELELYSO™, however, is not representative of other PMF-derived pharmaceuticals for several reasons. Since Gaucher disease is a rare genetic disease, mostly found among Ashkenazi Jews, ELELYSO™ has limited production needs. The FDA also accelerated (fast tracked) the approval process as a treatment for a rare disease. Additionally, the drug is produced in carrot cells using a large bioreactor under very stringent conditions. This process is different from production of other PMF products, which generally use entire leaves, fruits, seeds, or whole plants to produce the recombinant pharmaceutical. The production and approval of ELELYSO™ still represents a major step forward for the whole field of PMF. Many companies have now explored and started product pipelines utilizing plant-expression systems (see Table 2).
Identifying potential genes suitable for PMF and general approaches is becoming more simple and straight forward. Facilitated by the rapid progress in genomics, proteomics, and bioinformatics, a greater number of useful genes are being identified and characterized. Additionally, relatively routine molecular methods have become available for placing the genes of interest into plant expression vectors and transforming them into plants (see Figure 1).
An example of an early proof of concept for PMF is the production of plant-derived edible human vaccines, using leafy plants or fruits. Edible vaccines are an ideal product in concept since the vaccine could be administered to people orally, theoretically without the need of professional health care workers or sterile injections. The tedious and complicated process of purifying and storing the vaccine would also be eliminated since the food product itself would be the vaccine. The plants could also be grown locally, thus negating the cost of long distance transportation and storage. Edible vaccines also avoid the potential risk of infecting patients with a contaminated product since, in general, organisms causing plant diseases do not infect humans. The edible vaccine concept was first proposed by Charles Arntzen and coworkers, after HBsAg (Hepatitis B Virus antigen) was produced in tobacco plants. Mice fed HBsAg-transgenic potatoes exhibited a robust immune response. Uncooked potatoes from transgenic potato plants producing HBsAg were later tested orally in humans. Greater than 60% of the volunteers exhibited strong systemic and mucosal immunity after three doses of potato were consumed. These results demonstrated that plant-edible vaccines could be used in global immunization projects at a low cost. Subsequently, many other vaccine genes were expressed in a variety of crops, including lettuce, banana, and tomato fruits. Several plant vaccines are now in clinical trials that have produced encouraging data. PMF production strategies and challenges, such as biosafety, appropriate expression systems, possible and potential applications, are discussed in the present review with the idea of demonstrating a feasible approach for the potential commercialization of a PMF product.