Modifying the Replication of Geminiviral Vectors Reduces Cell Death and Enhances Expression of Biopharmaceutical Proteins in Nicotiana benthamiana Leaves

Plants are a promising platform to produce biopharmaceutical proteins, however, the toxic nature of some proteins inhibits their accumulation. We previously created a replicating geminiviral expression system based on bean yellow dwarf virus (BeYDV) that enables very high-level production of recombinant proteins. To study the role of replication in this system, we generated vectors that allow separate and controlled expression of BeYDV Rep and RepA proteins. We show that the ratio of Rep and RepA strongly affects the efficiency of replication. Rep, RepA, and vector replication all elicit the plant hypersensitive response, resulting in cell death. We find that a modest reduction in expression of Rep and RepA reduces plant leaf cell death which, despite reducing the accumulation of viral replicons, increases target protein accumulation. A single nucleotide change in the 5′ untranslated region (UTR) reduced Rep/RepA expression, reduced cell death, and enhanced the production of monoclonal antibodies. We also find that replicating vectors achieve optimal expression with lower Agrobacterium concentrations than non-replicating vectors, further reducing cell death. Viral UTRs are also shown to contribute substantially to cell death, while a native plant-derived 5′ UTR does not

Acupuncture for chronic neck pain: a pilot for a randomised controlled trial

Background: Acupuncture is increasingly being used for many conditions including chronic neck pain. However the evidence remains inconclusive, indicating the need for further well-designed research. The aim of this study was to conduct a pilot randomised controlled parallel arm trial, to establish key features required for the design and implementation of a large-scale trial on acupuncture for chronic neck pain. Methods: Patients whose GPs had diagnosed neck pain were recruited from one general practice, and randomised to receive usual GP care only, or acupuncture ( up to 10 treatments over 3 months) as an adjunctive treatment to usual GP care. The primary outcome measure was the Northwick Park Neck Pain Questionnaire (NPQ) at 3 months. The primary analysis was to determine the sample size for the full scale study. Results: Of the 227 patients with neck pain identified from the GP database, 28 (12.3%) consenting patients were eligible to participate in the pilot and 24 (10.5%) were recruited to the trial. Ten patients were randomised to acupuncture, receiving an average of eight treatments from one of four acupuncturists, and 14 were randomised to usual GP care alone. The sample size for the full scale trial was calculated from a clinically meaningful difference of 5% on the NPQ and, from this pilot, an adjusted standard deviation of 15.3%. Assuming 90% power at the 5% significance level, a sample size of 229 would be required in each arm in a large-scale trial when allowing for a loss to follow-up rate of 14%. In order to achieve this sample, one would need to identify patients from databases of GP practices with a total population of 230,000 patients, or approximately 15 GP practices roughly equal in size to the one involved in this study (i.e. 15,694 patients). Conclusion: This pilot study has allowed a number of recommendations to be made to facilitate the design of a large-scale trial, which in turn will help to clarify the existing evidence base on acupuncture for neck pain

Asymmetric function theory

The classical theory of symmetric functions has a central position in algebraic combinatorics, bridging aspects of representation theory, combinatorics, and enumerative geometry. More recently, this theory has been fruitfully extended to the larger ring of quasisymmetric functions, with corresponding applications. Here, we survey recent work extending this theory further to general asymmetric polynomials.Comment: 36 pages, 8 figures, 1 table. Written for the proceedings of the Schubert calculus conference in Guangzhou, Nov. 201

Improvement of the transient expression system for production of recombinant proteins in plants

An efficient and high yielding expression system is required to produce recombinant proteins. Furthermore, the transient expression system can be used to identify the localization of proteins in plant cells. In this study, we demonstrated that combination of a geminiviral replication and a double terminator dramatically enhanced the transient protein expression level in plants. The GFP protein was expressed transiently in lettuce, Nicotiana benthamiana, tomatoes, eggplants, hot peppers, melons, and orchids with agroinfiltration. Compared to a single terminator, a double terminator enhanced the expression level. A heat shock protein terminator combined with an extensin terminator resulted in the highest protein expression. Transiently expressed GFP was confirmed by immunoblot analysis with anti-GFP antibodies. Quantitative analysis revealed that the geminiviral vector with a double terminator resulted in the expression of at least 3.7 mg/g fresh weight of GFP in Nicotiana benthamiana, approximately 2-fold that of the geminiviral vector with a single terminator. These results indicated that combination of the geminiviral replication and a double terminator is a useful tool for transient expression of the gene of interest in plant cells

Perspective Chapter: Natural Adjuvants for Mucosal Vaccines—The Promise of Tomatine as an Inherent Adjuvant in Tomatoes

Certain natural immunostimulatory compounds inherent to plants, animals, and microorganisms, in both terrestrial and aquatic ecosystems, have been reported to enhance the immunogenicity of vaccines by conferring an adjuvant effect and/or possessing potent immunomodulatory properties acting as immunogens themselves. In this chapter, we summarize the current state of vaccine adjuvant development and application, encompassing a range of immunomodulatory compounds that improve protective immune responses and enhance vaccine efficacy. We place special focus on the effectiveness of tomatine, inherent to tomatoes, as a natural immunostimulant. We discuss the adjuvant- and immunomodulatory-properties of tomatine and its advantages in plant-based vaccine production, cost-effectiveness, development, safety profiles, and applications compared to other adjuvants and vaccine delivery systems. This chapter provides a futuristic overview and insights into the promise of tomatine for the development of safer, easily-scalable, sustainable, and more efficient vaccines

Perspective Chapter: The Most Natural Possible Vaccine Administered in the Most Natural Possible Way - Noninvasive over Injectable Vaccine Delivery Routes

This chapter provides perspective on the routes of vaccine administration, comparing invasive and noninvasive delivery methods. We begin with an analysis of the most frequently used routes of administration: invasive, such as traditional needle-based injections (intramuscular and subcutaneous) and noninvasive, including oral and intranasal routes. We discuss recent advancements, for example, aerosols and jet injectors, as well as other novel administration methods for immunization such as improved mucosally-administered vaccines. Finally, we provide an update on how different delivery methods can impact consumer (vaccine recipients) compliance rates and vaccine availability (e.g., cold chain logistics in areas of the world with infrastructure limitations) from the perspectives of both the vaccine provider and the vaccine recipient

Evaluation of a toxoid fusion protein vaccine produced in plants to protect poultry against necrotic enteritis

Background Necrotic enteritis (NE) is caused by type A strains of the bacterium Clostridium perfringens. Total global economic losses to the poultry industry due to NE is estimated to be over two billion dollars annually. Traditionally, NE has been effectively controlled by inclusion of antibiotics in the diet of poultry. However, recent concerns regarding the impact of this practice on increasing antibiotic resistance in human pathogens have led us to consider alternative approaches, such as vaccination, for controlling this disease. NE strains of C. perfringens produce two major toxins, a-toxin and NetB. Immune responses against either toxin can provide partial protection against NE. Methods We have developed a fusion protein combining a non-toxic carboxyl-terminal domain of a-toxin (PlcC) and an attenuated, mutant form of NetB (NetB-W262A) for use as a vaccine antigen to immunize poultry against NE. We utilized a DNA sequence that was codon-optimized for Nicotiana benthamiana to enable high levels of expression. The 6-His tagged PlcC-NetB fusion protein was synthesized in N. benthamiana using a geminiviral replicon transient expression system, purified by metal affinity chromatography, and used to immunize broiler birds. Results Immunized birds produced a strong serum IgY response against both the plant produced PlcC-NetB protein and against bacterially produced His-PlcC and His-NetB. Immunized birds were significantly protected against a subsequent in-feed challenge with virulent C. perfringens when treated with the fusion protein. These results indicate that a plant-produced PlcC-NetB toxoid is a promising vaccine candidate for controlling NE in poultry

Targeted Isolation of Antibodies Directed against Major Sites of SIV Env Vulnerability

The simian immunodeficiency virus (SIV) challenge model of lentiviral infection is often used as a model to human immunodeficiency virus type 1 (HIV-1) for studying vaccine mediated and immune correlates of protection. However, knowledge of the structure of the SIV envelope (Env) glycoprotein is limited, as is knowledge of binding specificity, function and potential efficacy of SIV antibody responses. In this study we describe the use of a competitive probe binding sort strategy as well as scaffolded probes for targeted isolation of SIV Env-specific monoclonal antibodies (mAbs). We isolated nearly 70 SIV-specific mAbs directed against major sites of SIV Env vulnerability analogous to broadly neutralizing antibody (bnAb) targets of HIV-1, namely, the CD4 binding site (CD4bs), CD4-induced (CD4i)-site, peptide epitopes in variable loops 1, 2 and 3 (V1, V2, V3) and potentially glycan targets of SIV Env. The range of SIV mAbs isolated includes those exhibiting varying degrees of neutralization breadth and potency as well as others that demonstrated binding but not neutralization. Several SIV mAbs displayed broad and potent neutralization of a diverse panel of 20 SIV viral isolates with some also neutralizing HIV-27312A. This extensive panel of SIV mAbs will facilitate more effective use of the SIV non-human primate (NHP) model for understanding the variables in development of a HIV vaccine or immunotherapy

Acupuncture, or non-directive counselling versus usual care for the treatment of depression: a pilot study

<p>Abstract</p> <p>Background</p> <p>Depression is one of the most common reasons for consulting in primary care. Acupuncture is a popular complementary therapy choice for depression but its evidence base is poor with more robust high quality trials being required. More than half of depressed patients experience painful symptoms, with severe pain being associated with poor response to antidepressants. Acupuncture may have much to offer as an intervention for depression that also helps alleviate pain. Non-directive counselling is the most widely used psychological approach for depression in NHS settings, and provides a useful pragmatic comparison for acupuncture that would, according to our pre-trial qualitative research, be of high interest to doctors and patients.</p> <p>Methods and design</p> <p>The pilot study uses five arms and involves a pragmatic design. All patients will continue to receive usual care. Four groups of patients will be allocated to acupuncture, or non-directive counselling, in addition to usual GP care. The acupuncture and counselling arms will be further split into two groups to explore different treatment regimens. The primary outcome measure is the BDI II. Potentially eligible patients will be screened for depression using the PHQ-9, which is also a secondary outcome measure. Other secondary measures include the SF 36 bodily pain subscale, the CORE OM, the WBQ-12 and the EQ5D. Health economic data will be collected and measures of therapeutic engagement will be used to compare patient's views of therapists and GPs. The study will employ a fully randomised preference design with collection of data on patient preferences and prior expectations.</p> <p>Discussion</p> <p>This study has been implemented, and data are currently being analysed to inform the design of a full scale trial. Two practical operational issues that impacted on study implementation are discussed. Firstly, the challenge of recruiting depressed patients via GP consultation. Secondly, the problem of poor uptake and high attrition for counselling and acupuncture, which appeared to be associated with poor questionnaire return, and resulted in missing data. These problems may be relevant to other researchers working in the area of depression, or similar illnesses, where patients may lack motivation and energy to engage in research, or attend for treatment.</p> <p>Trial Registration</p> <p>Current Controlled Trials (ISRCTN 59267538)</p

Western Indian Ocean marine and terrestrial records of climate variability: a review and new concepts on land-ocean interactions since AD 1660

We examine the relationship between three tropical and two subtropical western Indian Ocean coral oxygen isotope time series to surface air temperatures (SAT) and rainfall over India, tropical East Africa and southeast Africa. We review established relationships, provide new concepts with regard to distinct rainfall seasons, and mean annual temperatures. Tropical corals are coherent with SAT over western India and East Africa at interannual and multidecadal periodicities. The subtropical corals correlate with Southeast African SAT at periodicities of 16–30 years. The relationship between the coral records and land rainfall is more complex. Running correlations suggest varying strength of interannual teleconnections between the tropical coral oxygen isotope records and rainfall over equatorial East Africa. The relationship with rainfall over India changed in the 1970s. The subtropical oxygen isotope records are coherent with South African rainfall at interdecadal periodicities. Paleoclimatological reconstructions of land rainfall and SAT reveal that the inferred relationships generally hold during the last 350 years. Thus, the Indian Ocean corals prove invaluable for investigating land–ocean interactions during past centuries