Engineering Camelina sativa Seeds as a Green Bioreactor for the Production of Affordable Human Pro-insulin that Demonstrates Anti-diabetic Efficacy in Rats.

The current production of recombinant insulin via fermenter-based platforms (Escherichia coli and yeast) could not fulfill its fast-growing commercial demands, thus leading to a great interest in its sustainable large-scale production at low cost using a plant-based system. In the present study, Agrobacterium tumefaciens-mediated nuclear stable genetic transformation of an industrial oilseed crop, Camelina sativa, to express pro-insulin (with three furin endoprotease cleavage sites) fused with cholera toxin B subunit (CTB) in their seeds was successfully achieved for the first time. The bar gene was used as a selectable marker for selecting transformants and producing herbicide-resistant camelina plants. The transformation process involved the infiltration of camelina inflorescences (at flower buds with partially opened flowers) with A. tumefaciens and harvesting the seeds (T0) at maturity. The T0 seeds were raised into the putative T1 plants sprayed with Basta herbicide (0.03%, v/v), and the survived green transformed plants tested positive for pro-insulin and bar genes. A transformation frequency of 6.96% was obtained. The integration and copy number of the pro-insulin transgene and its expression at RNA and protein levels were confirmed in T1 plants using Southern hybridization, semi-quantitative Reverse Transcriptase-Polymerase Chain Reaction (sqPCR), and quantitative real-time Time PCR (qPCR) and western blot analysis, respectively. Enzyme-linked immunosorbent Assay (ELISA) quantified the amount of expressed pro-insulin protein, and its anti-diabetic efficacy was validated in diabetic rats on oral feeding. Transgenic plants integrated the pro-insulin gene into their genomes and produced a maximum of 197 µg/100 mg of pro-insulin (0.804% of TSP) that had anti-diabetic efficacy in rats.

Synthesis of plant-based, self-adjuvanted, dual antigen specific to Mycobacterium tuberculosis as a novel tuberculosis subunit vaccine that elicits immunogenicity in rabbit.

OBJECTIVES: The only approved vaccine, Bacillus Calmette Guérin (BCG) used in global tuberculosis (TB) immunization programmes has been very effective in childhood TB but not in adult pulmonary and latent TB. Moreover, the emergence of multi-drug resistance-TB cases demands either to increase efficiency of BCG or replace it with the one with improved efficacy. RESULTS: A novel combination of two most effective secreted protein antigens specific for Mycobacterium tuberculosis (Mtb), ESAT-6 and MPT-64 (but not present in BCG strains) fused with a cholera toxin B subunit (CTB) and tagged with 6xHis was expressed for the first time in Escherichia coli as well as in transgenic cucumber plants developed using Agrobacterium tumefaciens-mediated transformation. The recombinant fusion protein (His6x.CTB-ESAT6-MPT64) expressed in E. coli was purified by a single-step affinity chromatography and used to produce polyclonal antibodies in rabbit. The transgenic cucumber lines were confirmed by polymerase chain reaction (PCR), Southern blot hybridization, reverse transcriptase PCR (RT-PCR), real-time PCR (qRT-PCR) and expression of recombinant fusion protein by western blot analysis and its quantification by enzyme-linked immunosorbent assay (ELISA). A maximum value of the fusion protein, 478 ng.g-1 (0.030% of the total soluble protein) was obtained in a transgenic cucumber line. Rabbit immunized orally showed a significant increase in serum IgG levels against the fusion protein as compared to the non-immunized rabbit. CONCLUSIONS: Stable expression of Mtb antigens with CTB in edible cucumber plants (whose fruits are eaten raw) in sufficient amount possibly would facilitate development of a safe, affordable and orally delivered self-adjuvanted, novel dual antigen based subunit vaccine against TB.

Production of Bovine Rotavirus VP6 Subunit Vaccine in a Transgenic Fodder Crop, Egyptian Clover (Berseem, Trifolium alexandrinum) that Elicits Immune Responses in Rabbit.

Group A rotavirus causes acute gastroenteritis in young ones of animals worldwide and is responsible for a high rate of their morbidity and mortality leading to huge economic losses. Developing affordable and safer vaccine on large scale is imperative to reach cattle population worldwide for the long-term control of diarrhea. Rotavirus middle capsid protein layer, VP6, is the most immunogenic and highly conserved protein that induces immune responses against rotavirus. In the present study, bovine group A rotavirus VP6 protein has been expressed for the first time in a highly nutritious and palatable forage crop, Trifolium alexandrinum, using Agrobacterium tumefaciens-mediated stable nuclear transformation. Transgenic nature of the shoots regenerated from cotyledon explants and rooted on hygromycin-containing medium was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, reverse transcription-PCR (RT-PCR) and quantitative real-time PCR (qPCR), and protein expression and quantification by Western blot and enzyme-linked immune-sorbent assay (ELISA), respectively. The transformation efficiency of 2.10% was obtained. The highest amount of VP6 protein produced in a transgenic line was 402 ng/g fresh weights (0.03% of total soluble protein). Oral feeding of transgenic leafy shoots expressing VP6 protein stimulated systemic immunity by inducing significantly higher titers of anti-VP6 serum IgG antibodies in rabbit to reduce rotavirus infection. These transgenic fodder plants offer safer vaccine produced on large scale at low cost with reduced regulatory issues to improve livestock's health and wealth. These plants would be used as alternative to the current live attenuated vaccines to protect young calves against rotavirus infection.

Current advances and future prospects in production of recombinant insulin and other proteins to treat diabetes mellitus.

Diabetes mellitus is the most prevalent deadly disease caused by the destruction and dysfunction of pancreatic ß cells that consequentially increased blood glucose levels. The management of this disease via external administration of insulin/insulin analogs has been difficult and challenging due to their limited production and accessibility at affordable prices. The conventional insulin production platforms (Escherichia coli, Saccharomyces cerevisiae and mammalian cell lines) with limited scalability and high upstream process costs have not been successful in meeting the rapidly increasing insulin demands. However, plants have been used as safe, scalable, environmentally friendly and cost-effective high capacity production platforms for recombinant orally delivered insulin. Recent technological advances in genome engineering and editing technologies for adequate insulin and insulin analogs production, renewable cellular sources of insulin through transplantation of islets or insulin-producing cells and reprogramming or differentiation of non ß cells into ß-like cells, used either alone or in combination, for diabetes containment are reviewed here along with their future prospects.

Tissue Culture- and Selection-Independent Agrobacterium tumefaciens-Mediated Transformation of a Recalcitrant Grain Legume, Cowpea (Vigna unguiculata L. Walp).

A simple and generally fast Agrobacterium-mediated transformation system with no tissue culture and selection steps has been developed for the first time in a recalcitrant food legume, cowpea. The approach involves wounding of 1-day-old germinated seeds with a needle or sonication either alone or in combination of vacuum infiltration with A. tumefaciens EH105 (pCAMBIA2301) carrying a ß-glucuronidase (GUS) gene (uidA) and a neomycin phosphotransferase (nptII) gene for stable transformation. Sonicated and vacuum infiltrated seedlings showed the highest transient GUS activity in 90% of the explants. The sprouted co-cultured seeds directly established in soil and without selection were allowed to develop into plants which on maturity produced T0 seeds. The presence of the alien genes, nptII and uidA in T0 plants and their integration into the genome of T1 plants were confirmed by polymerase chain reaction (PCR) and Southern blot analyses, respectively. The transgenes were inherited in the subsequent T2 generation in a Mendelian fashion and their expression was confirmed by semi-quantitative PCR. The transformation frequency of 1.90% was obtained with sonication followed by vacuum infiltration with Agrobacterium. This approach provides favorable circumstances for the rapid meristem transformation and likely makes translational research ease in an important recalcitrant food legume, cowpea.

Transgenic cowpea plants expressing Bacillus thuringiensis Cry2Aa insecticidal protein imparts resistance to Maruca vitrata legume pod borer.

KEY MESSAGE: Fertile independent transgenic cowpea lines expressing the BtCry2Aa toxin with increased resistance to the most devastating lepidopteran insect pest, Maruca pod borer has been developed for the first time. Cowpea is a staple legume important for food and nutritional security in sub-Saharan Africa and Asia, where its production is limited by the key pest, legume pod borer (Maruca vitrata). Cowpea varieties resistant to M. vitrata are not known, hence, development of Maruca pod borer resistance cowpea through genetic engineering is a promising approach to improve its production. In the present study, transgenic cowpea plants expressing Bacillus thuringiensis Cry2Aa insecticidal protein were developed for the first time using Agrobacterium tumefaciens-mediated transformation of cotyledonary explants. T0 plants recovered from Agrobacterium cocultured explants on medium containing 120 mgl-1 of kanamycin were identified on the basis of the presence of transgenes by PCR, their integration into genome by Southern hybridization and expression of their transcripts by semi quantitative PCR (sqRT-PCR) and quantitative Real-time-PCR (qRT-PCR) and protein by Western blot analysis. The transformation efficiency obtained was 3.47% with 11 independent T0 transgenic lines. The bioefficacy of Cry2Aa protein expressed in randomly selected four T0 plant's leaves and pods was evaluated by feeding Maruca pod borer demonstrated a significant lower damage and a high level of Maruca mortality (more than 90%) for all these Bt lines. The inheritance of transgenes from T0 to T1 progeny plants was demonstrated by PCR analysis. The transgenic plants generated in this study can be used in cowpea breeding program for durable and sustainable legume pod borer resistance.

Biotechnological interventions for the sustainable management of a global pest, whitefly (Bemisia tabaci).

Whiteflies (Bemisia tabaci) are polyphagous invasive hemipteran insects that cause serious losses of important crops by directly feeding on phloem sap and transmitting pathogenic viruses. These insects have emerged as a major threat to global agriculture and food security. Chemically synthesized insecticides are currently the only option to control whiteflies, but the ability of whiteflies to evolve resistance against insecticides has made the management of these insects very difficult. Natural host-plant resistance against whiteflies identified in some crop plants has not been exploited to a great extent. Genetic engineering approaches, such as transgenics and RNA interference (RNAi), are potentially useful for the control of whiteflies. Transgenic plants harboring insecticidal toxins/lectins developed via nuclear or chloroplast transformation are a promising vehicle for whitefly control. Double-stranded RNAs (dsRNAs) of several insect genes, delivered either through microinjection into the insect body cavity or orally via an artificial diet and transiently or stably expressed in transgenic plants, have controlled whiteflies in model plants and in some crops at the laboratory level, but not at the field level. In this review, we highlight the merits and demerits of each delivery method along with strategies for sustained delivery of dsRNAs via fungal entomopathogen/endosymbiont or nontransgenic RNAi approaches, foliar sprays, root absorption or nanocarriers as well as the factors affecting efficient RNAi and their biosafety issues. Genome sequencing and transcriptome studies of whitefly species are facilitating the selection of appropriate genes for RNAi and gene-editing technology for the efficient and resilient management of whiteflies and their transmitted viruses.

Fabrication of an improved amperometric creatinine biosensor based on enzymes nanoparticles bound to Au electrode.

An improved amperometric creatinine biosensor was fabricated that dependent on covalent immobilisation of nanoparticles of creatininase (CANPs), creatinase (CINPs) and sarcosine oxidase (SOxNPs) onto gold electrode (AuE). The CANPs/CINPs/SOxNPs/AuE was characterised by scanning electron microscopy and cyclic voltammetry at various stages. The working electrode exhibited optimal response within 2 s at a potential of 0.6 V, against Ag/AgCl, pH 6.5 and 30 °C. A linear relationship was observed between creatinine concentration range, 0.1-200µM and biosensor response i.e. current in mA, under optimum conditions. Biosensor offered a low detection limit of 0.1 µM with long storage stability. Analytical recoveries of added creatinine in blood sera at 0.5 mM and at 1.0 mM concentrations, were 92.0% and 79.20% respectively. The precision i.e. within and between-batch coefficients of variation were 2.04% and 3.06% respectively. There was a good correlation (R2 = 0.99) between level of creatinine in sera, as calculated by the colorimetric method and present electrode. The CANPs/CINPs/SOxNPs/Au electrode was reused 200 times during the period of 180 days, with just 10% loss in its initial activity, while being stored at 4 °C, when not in use.HighlightsPrepared and characterised creatininase (CA), creatinase (CI) sarcosine oxidase (SOx) nanoparticles and immobilised them onto gold electrode (AuE) for fabrication of an improved amperometric creatinine biosensor.The biosensor displayed a limit of detection (LOD) of 0.1 µM with a linear working range of 0.1 µM-200 µM.The biosensor was evaluated and applied to measure elevated creatinine levels in sera from whom suffering from kidney and muscular disorders.The working electrode retained 90% of its initial activity, while being stored dry at 4 ˚C for 180 days.

Tuberculosis: Current Status, Diagnosis, Treatment and Development of Novel Vaccines.

Tuberculosis (TB) is an infectious disease that mainly affects the lungs and spreads to other organs of the body through the haematogenous route. It is one of the ten major causes of mortality worldwide. India has the highest incidence of new- and multidrug-resistant (MDR) - TB cases in the world. Bacille Calmette-Guerin (BCG) is the vaccine commonly available against TB. BCG does offer some protection against serious forms of TB in childhood but its protective effect wanes with age. Many new innovative strategies are being trailed for the development of effective and potent vaccines like mucosal- and epitope-based vaccines, which may replace BCG or boost BCG responses. The use of nanotechnology for diagnosis and treatment of TB is also in the pipeline along with many other vaccines, which are under clinical trials. Further, in-silico models were developed for finding new drug targets and designing drugs against Mycobacterium tuberculosis (Mtb). These models offer the benefit of computational experiments which are easy, inexpensive and give quick results. This review will focus on the available treatments and new approaches to develop potent vaccines for the treatment of TB.

Biosensing methods for determination of creatinine: A review.

Creatinine is a metabolic product of creatine phosphate in muscles, which provides energy to muscle tissues. Creatinine has been considered as indicator of renal function specifically after dialysis, thyroid malfunction and muscle damage. The normal level of creatinine in the serum and its excretion through urine in apparently healthy individuals is 45-140 µM and 0.8-2.0 gm/day respectively. The level of creatinine reaches >1000 µM in serum during renal, thyroid and kidney dysfunction or muscle disorder. A number of conventional methods such as colorimetric, spectrophotometric and chromatographic are available for determination of creatinine. Besides the advantages of being highly sensitive and selective, these methods have some drawbacks like time-consuming, requirement of sample pre-treatment, high cost instrumental set-up and skilled persons to operate. The sensors/biosensors overcome these drawbacks, as these are fast, easy, cost effective and highly sensitive. This review article describes the classification, operating principles, merits and demerits of various creatinine sensors/biosensors, specifically nanomaterials based biosensors. Creatinine biosensors work optimally within 2-900 s, potential range 0.1-1.0 V, pH range 4.0-10.0, temperature range 25-35 °C and had linear range, 0.004-30000 µM for creatinine with the detection limit between 0.01.01 µM and 520 µM. These biosensors measured creatinine level in sera and urine samples and had storage stability between 4 and 390 days, while being stored dry at 4 °C. The future perspective for further improvement and commercialization of creatinine biosensors are discussed.

Construction and application of amperometric sarcosine biosensor based on SOxNPs/AuE for determination of prostate cancer.

An improved amperometric sarcosine biosensor was constructed based on covalent immobilization of sarcosine oxidase nanoparticles (SOxNPs) onto gold electrode (AuE). The SOxNPs/AuE was characterized by scanning electron microscopy (SEM), fourier transform infrared (FTIR) spectroscopy and electrochemical impedance spectroscopy (EIS) at different stages of its construction. The biosensor worked optimally within 2 s at a potential of 1.0 V, against Ag/AgCl, pH 6.5 and 35 °C. A linear relationship was observed between sarcosine concentration range, 0.1-100 µM and the biosensor response i.e. current in mA under optimum conditions. The biosensor offered a low detection limit of 0.01 µM and gratifying storage stability. The SOxNPs/AuE was unaffected by a number of serum substances at their physiological concentrations. The biosensor measured sarcosine level in sera collected from persons suffering from prostate cancer (mean13.5 µM, n = 8), which was significantly higher (p < 0.01) than those in apparently healthy persons (mean 2.2 µM, n = 8). The SOxNPs/Au electrode was reused 300- times during the span of 180 days, with only 10% loss in its initial activity while being stored dry at 4 °C.

An improved amperometric creatinine biosensor based on nanoparticles of creatininase, creatinase and sarcosine oxidase.

An improved amperometric biosensor for detection of creatinine was developed based on immobilization of nanoparticles (NPs) of creatininase (CA), creatinase (CI), and sarcosine oxidase (SOx) onto glassy carbon (GC) electrode. Transmission electron microscopy (TEM) and fourier transform infrared spectroscopy (FTIR) were employed for characterization of enzyme nanoparticles (ENPs). The GC electrode was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectra (EIS) at different stages of its amendment. The biosensor showed optimum response within 2s at pH 6.0 in 0.1 M sodium phosphate buffer and 25 °C, when operated at 1.0 V against Ag/AgCl. Biosensor exhibited wider linear range from 0.01 µM to 12 µM with a limit of detection (LOD) of 0.01 µM. The analytical recoveries of added creatinine in sera were 97.97 ± 0.1% for 0.1 mM and 98.76 ± 0.2% for 0.15 mM, within and between batch coefficients of variation (CV) were 2.06% and 3.09% respectively. A good correlation (R2 = 0.99) was observed between sera creatinine values obtained by standard enzymic colorimetric method and the present biosensor. This biosensor measured creatinine level in sera of apparently healthy subjects and persons suffering from renal and muscular dysfunction. The ENPs electrode lost 10% of its initial activity within 240 days of its regular uses, when stored at 4 °C.

Progress and challenges in improving the nutritional quality of rice (Oryza sativa L.).

Rice is a staple food for more than 3 billion people in more than 100 countries of the world but ironically it is deficient in many bioavailable vitamins, minerals, essential amino- and fatty-acids and phytochemicals that prevent chronic diseases like type 2 diabetes, heart disease, cancers, and obesity. To enhance the nutritional and other quality aspects of rice, a better understanding of the regulation of the processes involved in the synthesis, uptake, transport, and metabolism of macro-(starch, seed storage protein and lipid) and micronutrients (vitamins, minerals and phytochemicals) is required. With the publication of high quality genomic sequence of rice, significant progress has been made in identification, isolation, and characterization of novel genes and their regulation for the nutritional and quality enhancement of rice. During the last decade, numerous efforts have been made to refine the nutritional and other quality traits either by using the traditional breeding with high through put technologies such as marker assisted selection and breeding, or by adopting the transgenic approach. A significant improvement in vitamins (A, folate, and E), mineral (iron), essential amino acid (lysine), and flavonoids levels has been achieved in the edible part of rice, i.e., endosperm (biofortification) to meet the daily dietary allowance. However, studies on bioavailability and allergenicity on biofortified rice are still required. Despite the numerous efforts, the commercialization of biofortified rice has not yet been achieved. The present review summarizes the progress and challenges of genetic engineering and/or metabolic engineering technologies to improve rice grain quality, and presents the future prospects in developing nutrient dense rice to save the everincreasing population, that depends solely on rice as the staple food, from widespread nutritional deficiencies.

Development of an efficient in vitro plant regeneration system amenable to Agrobacterium- mediated transformation of a recalcitrant grain legume blackgram (Vigna mungo L. Hepper).

An efficient, rapid and direct multiple shoot regeneration system amenable to Agrobacterium-mediated transformation from primary leaf with intact petiole of blackgram (Vigna mungo) is established for the first time. The effect of the explant type and its age, type and concentration of cytokinin and auxin either alone or in combination and genotype on multiple shoot regeneration efficiency and frequency was optimized. The primary leaf explants with petiole excised from 4-day-old seedlings directly developed multiple shoots (an average of 10 shoots/ explant) from the cut ends of the petiole in 95 % of the cultures on MSB (MS salts and B5 vitamins) medium containing 1.0 µM 6-benzylaminopurine. Elongated (2-3 cm) shoots were rooted on MSB medium with 2.5 µM indole-butyric acid and resulted plantlets were hardened and established in soil, where they resumed growth and reached maturity with normal seed set. The regenerated plants were morphologically similar to seed-raised plants and required 8 weeks time from initiation of culture to establish them in soil. The regeneration competent cells present at the cut ends of petiole are fully exposed and are, thus, easily accessible to Agrobacterium, making this plant regeneration protocol amenable for the production of transgenic plants. The protocol was further successfully used to develop fertile transgenic plants of blackgram using Agrobacterium tumefaciens strain EHA 105 carrying a binary vector pCAMBIA2301 that contains a neomycin phosphotransferase gene (nptII) and a ß-glucuronidase (GUS) gene (uidA) interrupted with an intron. The presence and integration of transgenes in putative T0 plants were confirmed by polymerase chain reaction (PCR) and Southern blot hybridization, respectively. The transgenes were inherited in Mendelian fashion in T1 progeny and a transformation frequency of 1.3 % was obtained. This protocol can be effectively used for transferring new traits in blackgram and other legumes for their quantitative and qualitative improvements.

Sesame (Sesamum indicum L.).

Sesame (Sesamum indicum L.) is an important oilseed crop grown in India, China, Korea, Russia, Turkey, Mexico, South America, and several countries of Africa. Sesame seeds are rich in oil, proteins, unsaturated fatty acids, vitamins, minerals, and folic acid. Nearly 70% of the world's sesame is processed into oil and meal, while the remainder is channeled to food and confectionery industries. Production of sesame is limited by several fungal diseases, water logging, salinity, and shattering of seed capsules during harvest. Introgression of useful genes from wild species into cultigens by conventional breeding has not been successful due to postfertilization barriers. The only alternative for the improvement of S. indicum is to transfer genes from other sources through genetic transformation techniques. Here, we describe a simple, fast, and reproducible method for the Agrobacterium-mediated genetic transformation of S. indicum which may be employed for the transfer of desirable traits into this economically important oilseed crop.

Four Hour Temporal Relation of 5-HTP and L-DOPA Induces Inhibitory Responses in Recrudescing Gonad of Indian Palm Squirrel (Funambulus pennantii).

Daily injections of L-dihydroxyphenylalanine (L-DOPA, dopamine precursor) given 4 h after 5-hydroxytryptophan (5-HTP, serotonin precursor) induced inhibitory responses in recrudescing gonad (in the first week of December) of Indian palm squirrel, a seasonally breeding subtropical animal. Other temporal relations (L-DOPA given at 0, 8, 12, 16, and 20 h after 5-HTP administration) did not show any effect on the recrudescing gonad. This inhibitory effect of 4 h was evident under short day length (6 : 18) group but was masked by the increasing day length of nature (NDL, late December onwards) and increased photoperiod of long day group (16 : 8). It is apparent that seasonal testicular recrudescence of Indian palm squirrel during short day length by 4 h relation of 5-HTP and L-DOPA is not a pharmacological effect but actually is an alteration of seasonality in this annually breeding mammal. It seems that endogenous mechanism controlling seasonal testicular recrudescence of Indian palm squirrel is reset by timed daily injections of these neurotransmitter drugs. It is suggested that in spite of different environmental factors (photoperiod, humidity, etc.) used by different species to time their annual reproduction, basic mechanism of seasonality appears to be the same, that is, the temporal synergism of neurotransmitter activity.