The Darroch and Frost analysis was conducted nearly 20 years ago, and the interviews were limited to women practicing vaginal intercourse. To our knowledge, a more recent study linking likelihood of product use and price sensitivity has not been conducted, or at least not reported, to include other populations of potential microbicide users such as heterosexual couples practicing anal sex or gay men practicing unprotected rectal intercourse. Nevertheless, the 1999 study established an initial price point and price sensitivity for potential users of microbicides in the USA. Griffithsin has a broader spectrum of antiviral activity than HIV-specific PrEP agents, including activity against HSV-2 and HCV, which are co-transmitted with HIV-1 . Hence, Griffithsin might command a higher price due to its broader antiviral activity and its potential to obviate prevention and treatment costs for co-transmitted viruses. In the USA, the cost of the oral PrEP drug Truvada ranges from $1,300 to over $1,700 per month for the uninsured, but treatment is typically covered by insurance with user co-payments of $80–$150 per month. So even if a Griffithsin-containing microbicide sold for $5 per application , a user of 2 packs per month would pay $100 for the microbicide, which is in the range of PrEP, with the potential added benefit of controlling co-transmitted viruses. Consumers in wealthier economies might be receptive to microbicides costing $1–2 or even more per dose; however, consumers in lesser-developed economies might find $1–2/dose to be prohibitive. Hence, absent subsidies, there exists a continuing need to lower COGS for APIs such as Griffithsin. We can conclude that a COGS of <$0.40/dose of Griffithsin DS as determined in this study, and an estimated user cost of $1– 2/dose, mobile vertical grow racks might enable at least some simpler formulations of the drug to be economically marketed.
For more complex formulations and delivery systems, or for higher doses of the drug, lower COGS for bulk Griffithsin would be desirable.The environmental assessment of the plant-based production of Griffithsin indicates low impact, particularly if the plant nutrient solutions are recycled in a hydroponic system and if waste streams containing TMV are treated in a bio-waste heat or chemical treatment process. The assessment method used, although semi-quantitative, utilizes mass input and output stream data generated by SuperPro, along with independent assessment of compound toxicity and/or environmental impact , and allows comparison between alternative production strategies, process configurations or chemical components used in the manufacturing process. Our low environmental impact assessment for plant-based manufacturing should compare favorably with fermentation based approaches to producing Griffithsin. In the latter, the complexities of purification suggest less efficient utilization of materials and higher disposal volumes, although a side-by-side environmental analysis between the two platforms was not conducted in this study.Upstream, Griffithsin expression rates were based on empirical findings using TMV whole virion as the expression vector, which can achieve typically 0.5–1.0 g Griffithsin/kg plant biomass . An average pilot-scale expression rate of0.52 g/kg was used in our model . Although this expression level is quite good for TMV, higher Griffithsin expression levels can be achieved with different technology. For example, Nomad Bioscience GmbH has achieved Griffithsin expression in N. benthamiana exceeding 2.5 g Griffithsin/kg FW biomass using NomadicTM agrobacterial vectors applied to plants either through vacuum infiltration or agrospray , albeit these results were obtained in small-scale studies. For example, even with the same recovery efficiency of 70% assumed in the current model, the output of Griffithsin at the higher expression level would be 1.75 g API/kg plant material, instead of the current 0.37 g/kg; this represents more than 4.7- times the modeled output of protein per kg biomass.
Under such conditions, the costliest parts of the current process, namely biomass production and upstream procedures, would be lowered by the reduced biomass needs to produce the required 20 kg/year of API. Although a full analysis of the cost of agrobacterial inoculation for Griffithsin production needs to be conducted, we know from similar analyses that economics can be favorably impacted by higher expression efficiencies. We can therefore envision that by using a more efficient induction process the per-dose production cost could be less than the current $0.32. Still other gene expression methods can be considered, including using transgenic plants expressing Griffithsin either in constitutive or inducible systems , which could also lead to higher API accumulation in host plant biomass and potentially lower COGS . Increasing expression yield upstream might shift costs to downstream operations to handle process streams with higher concentrations of API. Definition of the comparative cost benefits of these improvements relative to the current process modeled awaits a subsequent evaluation. From a process standpoint, improvements in the efficiency of lighting technologies and/or incorporating solar panels would reduce upstream utilities costs, one of the major contributors to the upstream operating costs. Improving hydroponic nutrient utilization through recycling and minimizing runoff in the simulation model will reduce raw material costs as well as aqueous waste disposal costs, thereby reducing the COGS. In the downstream portion of the process consumables play a major role, particularly dead-end filters and plate-and frame filters; if these could be replaced with tangential flow filtration systems that utilize reusable, cleanable ceramic filters, downstream operating costs could be further reduced. At the time of this writing, such systems were being considered and their impact on Griffithsin COGS will be the subject of a future analysis. Nitrogen is a vital macro-nutrient for plant growth and development. Plants have evolved a range of mechanisms to adapt to imbalanced nitrogen conditions. In agricultural systems, high-yield of crops relies on application of nitrogen fertilizers. But a large part of nitrogen deposited in the soil can’t be absorbed by plants and is lost to the environment, resulting in severe environmental and ecological pollution. Improving the nitrogen use effciency of crops is the key to solve these problems. Studying the genes and mechanisms involved in regulating nitrogen uptake and assimilation can be a prerequisite for improving NUE of crops, therefore it is of great importance for sustaining agriculture.
Nitrate and ammonium are the main nitrogen forms used by plants and most crops, like maize and wheat, take up nitrate as the major nitrogen source. In addition to its nutrient role, nitrate acts also as a signaling molecule for plants. It regulates the expression levels of many genes, including genes directly involved in nitrate assimilation, namely NIAs, NiR, and some NRTs. It is also involved in many adaptive responses of plants, such as root development and architecture, seed dormancy, fowering time, circadian system, leaf development, stomatal movements, and auxin transportation. Nitrate is taken up into plants by nitrate transporters and high afinity and low afinity nitrate uptake systems have been identified. Four gene families have been identified that encode nitrate transporters in Arabidopsis: NRT1/PTR , NRT2 , CLC , and SLAC1/SLAH. Among these families, NRT1/PTR belongs to the low afinity transport system, and NRT2 belongs to the high afinity transport system. NRT1.1 , which belongs to NRT/PTR family, functions in nitrate uptake as both high afinity and low afinity transporter.In addition to the nitrate transport systems, genes involved in nitrate signaling have also been identified. Most of these genes were found to function in root architecture or primary nitrate responses. Te genes functioning in root architecture include the ANR1, the first molecular component isolated by classic molecular genetics approach, is a MADS box transcription factor and positively regulates lateral root branching under sufficient nitrate condition. miR393/AFB3 and NAC4 have been demonstrated to regulate the root system architecture in nitrate signaling using systems approach. Te split-root assays indicated that TCP20 was involved in systemic nitrate signaling for root foraging. Recently, TCP20 was found to regulate root meristem growth under nitrogen starvation and to interact with NLP6&7. HHO1 and HRS1 are two nitrate-responsive transcription factors isolated by genome-wide analyses. They function in the repression of primary root growth under both phosphate starvation and nitrate supply conditions. During last several years,vertical garden growing the nitrate regulatory factors involved in the primary nitrate response have been identified. NRT1.1, in addition to its transport function, was identified to work as a nitrate sensor. Te study on the crystal structure of NRT1.1 has demonstrated that Tr101 phosphorylation is essential for nitrate transport rate and provides further insights into its transport mechanisms. CIPK8 and CIPK23 which belong to CBL-interacting protein kinase family are important players in responding to primary nitrate. CIPK8 works positively while CIPK23 functions negatively in nitrate regulation. Te expression of both CIPK8 and CIPK23 is regulated by NRT1.1. Recently, NRG2 which is an essential nitrate regulatory gene was isolated by forward genetics screen. NRG2 acts as a positive nitrate regulatory factor and modulates NRT1.1 expression and can interact with NLP7. Additionally, several transcription factors were identified to be involved in primary nitrate response, for example, NLP6, NLP7, LBD37/38/39, TGA1, TGA4, and SPL9. NLP7 is NIN-like protein and acts as an important nitrate positive regulator. NLP7 was isolated by reverse genetics strategy and the nlp7 mutants exhibit a nitrogen-starved phenotype. Te nitrate condition can affect the NLP7′s nuclear retention. Previous studies have demonstrated that the nitrate response cis-element NRE can be bound by NLPs and contain a DNA-binding domain RWP-PK and protein-protein interaction domains typeI/II Phox and Ben1p. ChIP-chip assays showed that NLP7 could bind 851 genes containing NRT1.1, NRT2.1, LBD37/38. In addition, over expression of NLP7 can increase plant biomass, nitrogen uptake, total nitrogen content, and expression levels of genes involved in nitrogen assimilation and signaling.
Moreover, NLP7 can control plant root growth under both N-limited and N-rich conditions. NLP6 also functions positively in nitrate regulation, is retained in the nucleus in nitrate-treated plants and can activate the expression of nitrate-responsive genes. LBD37/38/39 are negative regulators in nitrate signaling. Tey are involved in primary nitrate response and can affect nitrogen status, growth, and nitrogen-dependent shoot branching. TGA1, TGA4, and SPL9 were isolated by systems approach. TGA1 and TGA4 belong to bZIP transcription factor family and TGA1 can bind to the promoters of NRT2.1 and NRT2.2. SPL9 is demonstrated to be a nitrate regulatory hub. Although these nitrate regulatory genes have been identified, our understanding of the nitrate regulatory gene network is still incomplete. For example, both NLP7 and NRT1.1 play essential roles in regulating nitrate signaling and ChIP-chip assay showed that NLP7 might bind NRT1.1, however, their relationship and underlining mechanism remain unclear. In this paper, we investigated the relationship between NRT1.1 and NLP7 in nitrate regulation. Our analyses reveal that NLP7 acts as a positive regulatory factor upstream of NRT1.1 when NH4 + is present and modulates the nitrate signaling function of NRT1.1. NLP7 might function in another pathway to regulate nitrate signaling independent of NRT1.1. In addition, transcriptome data showed that four GO terms related to nitrogen were regulated by NRT1.1 as well as NLP7 in nitrate signaling, providing more evidence to support our above conclusion. Furthermore, the ChIP and EMSA assays indicated that NLP7 could bind to specific regions of the NRT1.1 promoter. Our findings not only further elucidate the relationship between NRT1.1 and NLP7, but also provide insights into the network of the nitrate regulatory genes.To study the relationship between NLP7 and NRT1.1, the expression levels of NRT1.1 was detected firstly under potassium nitrate and ammonium nitrate conditions. Figure 1a showed that the transcript levels of NRT1.1 in the nlp7 mutants were not notably changed under potassium nitrate condition, but was significantly decreased in mutant plants under ammonium nitrate condition . This indicates that the expression levels of NRT1.1 can be modulated by NLP7 in the presence of NH4 +. In order to test if NLP7 is regulated by NRT1.1, we tested NLP7 expression in chl1-5 and chl1-13 mutants in potassium nitrate and ammonium nitrate mediums. Te expression of NLP7 was not changed in the nrt1.1 mutants . This result indicates that NRT1.1 may not regulate the expression of NLP7. We also tested the NRT1.1 expression response to nitrate in WT and the nlp7 mutants. qPCR results showed that the induction of NRT1.1 by nitrate was notably decreased in the nlp7 mutants, indicating that NLP7 affects the response of NRT1.1 to nitrate .To elucidate the relationship between NLP7 and NRT1.1, the single mutants: nlp7-4 and chl1-13 which contain the nitrate-responsive NRP-YFP transgene, both of which were isolated by our mutant screens described previously were crossed to obtain the double mutant chl1-13 nlp7-4.