Publications by authors named "Mohammad Tahir Waheed"

22 Publications

  • Page 1 of 1

Inducible expression of human papillomavirus-16 L1 capsomeres in the plastomes of Nicotiana tabacum: Transplastomic plants develop normal flowers and pollen.

Biotechnol Appl Biochem 2021 Mar 2. Epub 2021 Mar 2.

Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan.

Human papillomavirus type-16 (HPV-16) is the major HPV type involved in causing cervical cancer among women. The disease burden is high in developing and underdeveloped countries. Previously, the constitutive expression of HPV-16 L1 protein led to male sterility in transplastomic tobacco plants. Here, the HPV-16 L1 gene was expressed in chloroplasts of Nicotiana tabacum under the control of an ethanol-inducible promoter, trans-activated by nucleus-derived signal peptide. Plants containing nuclear component were transformed with transformation vector pEXP-T7-L1 by biolistic gun. The transformation and homoplasmic status of transformed plants was verified by polymerase chain reaction and Southern blotting, respectively. Protein was induced by spraying 5% ethanol for 7 consecutive days. The correct folding of L1 protein was confirmed by antigen-capture ELISA using a conformation-specific antibody. The L1 protein accumulated up to 3 μg/g of fresh plant material. The L1 protein was further purified using affinity chromatography. All transplastomic plants developed normal flowers and produced viable seeds upon self-pollination. Pollens also showed completely normal structure under light microscope and scanning electron microscopy. These data confirm the use of the inducible expression as plant-safe approach for expressing transgenes in plants, especially those genes that cause detrimental effects on plant growth and morphology.
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http://dx.doi.org/10.1002/bab.2136DOI Listing
March 2021

Chloroplast genome evolution in the Dracunculus clade (Aroideae, Araceae).

Genomics 2021 Jan 14;113(1 Pt 1):183-192. Epub 2020 Dec 14.

Alpha Genomics Private Limited, Islamabad 45710, Pakistan. Electronic address:

Chloroplast (cp) genomes are considered important for the study of lineage-specific molecular evolution, population genetics, and phylogenetics. Our aim here was to elucidate the molecular evolution in cp genomes of species in the Dracunculus clade (Aroideae, Araceae). We report de novo assembled cp genomes for eight species from eight genera and also retrieved cp genomes of four species from the National Center for Biotechnology Information (NCBI). The cp genomes varied in size from 162,424 bp to 176,835 bp. Large Single Copy (LSC) region ranged in size from 87,141 bp to 95,475 bp; Small Single Copy (SSC) from 14,338 bp to 23,981 bp; and Inverted Repeats (IRa and IRb) from 25,131 bp to 32,708 bp. The expansion in inverted repeats led to duplication of ycf1 genes in four species. The genera showed high similarity in gene content and yielded 113 unique genes (79 protein-coding, 4 rRNA, and 30 tRNA genes). Codon usage, amino acid frequency, RNA editing sites, microsatellites repeats, transition and transversion substitutions, and synonymous and non-synonymous substitutions were also similar across the clade. A previous study reported deletion of ycf1, accD, psbE, trnL-CAA, and trnG-GCC genes in four Amorphophallus species. Our study supports conservative structure of cp genomes in the Dracunculus clade including Amorphophallus species and does not support gene deletion mentioned above. We also report suitable polymorphic loci based on comparative analyses of Dracunculus clade species, which could be useful for phylogenetic inference. Overall, the current study broad our knowledge about the molecular evolution of chloroplast genome in aroids.
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http://dx.doi.org/10.1016/j.ygeno.2020.12.016DOI Listing
January 2021

Plastid genomics of (Solanaceae): insights into molecular evolution, positive selection and the origin of the maternal genome of Aztec tobacco ().

PeerJ 2020 23;8:e9552. Epub 2020 Jul 23.

Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan.

Species of the genus (Solanaceae), commonly referred to as tobacco plants, are often cultivated as non-food crops and garden ornamentals. In addition to the worldwide production of tobacco leaves, they are also used as evolutionary model systems due to their complex development history tangled by polyploidy and hybridization. Here, we assembled the plastid genomes of five tobacco species: and . De novo assembled tobacco plastid genomes had the typical quadripartite structure, consisting of a pair of inverted repeat (IR) regions (25,323-25,369 bp each) separated by a large single-copy (LSC) region (86,510-86,716 bp) and a small single-copy (SSC) region (18,441-18,555 bp). Comparative analyses of plastid genomes with currently available Solanaceae genome sequences showed similar GC and gene content, codon usage, simple sequence and oligonucleotide repeats, RNA editing sites, and substitutions. We identified 20 highly polymorphic regions, mostly belonging to intergenic spacer regions (IGS), which could be suitable for the development of robust and cost-effective markers for inferring the phylogeny of the genus and family Solanaceae. Our comparative plastid genome analysis revealed that the maternal parent of the tetraploid was the common ancestor of and , and the later species is more closely related to . Relaxed molecular clock analyses estimated the speciation event between and appeared 0.56 Ma (HPD 0.65-0.46). Biogeographical analysis supported a south-to-north range expansion and diversification for and related species, where and evolved in North/Central Peru, while developed in Southern Peru and separated from which adapted to the Southern coastal climatic regimes. We further inspected selective pressure on protein-coding genes among tobacco species to determine if this adaptation process affected the evolution of plastid genes. These analyses indicate that four genes involved in different plastid functions, including DNA replication (A) and photosynthesis (B, D and ), came under positive selective pressure as a result of specific environmental conditions. Genetic mutations in these genes might have contributed to better survival and superior adaptations during the evolutionary history of tobacco species.
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http://dx.doi.org/10.7717/peerj.9552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382938PMC
July 2020

Complete Chloroplast Genomes of Anthurium huixtlense and Pothos scandens (Pothoideae, Araceae): Unique Inverted Repeat Expansion and Contraction Affect Rate of Evolution.

J Mol Evol 2020 09 9;88(7):562-574. Epub 2020 Jul 9.

Alpha Genomics Private Limited, Islamabad, 45710, Pakistan.

The subfamily Pothoideae belongs to the ecologically important plant family Araceae. Here, we report the chloroplast genomes of two species of the subfamily Pothoideae: Anthurium huixtlense (size: 163,116 bp) and Pothos scandens (size: 164,719 bp). The chloroplast genome of P. scandens showed unique contraction and expansion of inverted repeats (IRs), thereby increasing the size of the large single-copy region (LSC: 102,956 bp) and decreasing the size of the small single-copy region (SSC: 6779 bp). This led to duplication of many single-copy genes due to transfer to IR regions from the small single-copy (SSC) region, whereas some duplicate genes became single copy due to transfer to large single-copy regions. The rate of evolution of protein-coding genes was affected by the contraction and expansion of IRs; we found higher mutation rates for genes that exist in single-copy regions as compared to those in IRs. We found a 2.3-fold increase of oligonucleotide repeats in P. scandens when compared with A. huixtlense, whereas amino acid frequency and codon usage revealed similarities. The ratio of transition to transversion mutations was 2.26 in P. scandens and 2.12 in A. huixtlense. Transversion mutations mostly translated in non-synonymous substitutions. The phylogenetic inference of the limited species showed the monophyly of the Araceae subfamilies. Our study provides insight into the molecular evolution of chloroplast genomes in the subfamily Pothoideae and family Araceae.
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http://dx.doi.org/10.1007/s00239-020-09958-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7445159PMC
September 2020

Comparison of Chloroplast Genomes among Species of Unisexual and Bisexual Clades of the Monocot Family Araceae.

Plants (Basel) 2020 Jun 11;9(6). Epub 2020 Jun 11.

Alpha Genomics Private Limited, Islamabad 45710, Pakistan.

The chloroplast genome provides insight into the evolution of plant species. We de novo assembled and annotated chloroplast genomes of four genera representing three subfamilies of Araceae: (Lasioideae), , (Zamioculcadoideae), and (Orontioideae), and performed comparative genomics using these chloroplast genomes. The sizes of the chloroplast genomes ranged from 163,770 bp to 169,982 bp. These genomes comprise 113 unique genes, including 79 protein-coding, 4 rRNA, and 30 tRNA genes. Among these genes, 17-18 genes are duplicated in the inverted repeat (IR) regions, comprising 6-7 protein-coding (including trans-splicing gene 12), 4 rRNA, and 7 tRNA genes. The total number of genes ranged between 130 and 131. The A gene was found to be a pseudogene in all four genomes reported here. These genomes exhibited high similarities in codon usage, amino acid frequency, RNA editing sites, and microsatellites. The oligonucleotide repeats and junctions JSB (IRb/SSC) and JSA (SSC/IRa) were highly variable among the genomes. The patterns of IR contraction and expansion were shown to be homoplasious, and therefore unsuitable for phylogenetic analyses. Signatures of positive selection were seen in three genes in including 2, P, and 36. This study is a valuable addition to the evolutionary history of chloroplast genome structure in Araceae.
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http://dx.doi.org/10.3390/plants9060737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355861PMC
June 2020

Expression of ESAT-6 antigen from Mycobacterium tuberculosis in broccoli: An edible plant.

Biotechnol Appl Biochem 2020 Jan 2;67(1):148-157. Epub 2020 Jan 2.

Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan.

Tuberculosis (TB) is one of the major infectious diseases caused by Mycobacterium tuberculosis. The development of an effective and economical vaccine for controlling TB is essential especially for developing countries. Edible plants can serve as biofactories to produce vaccine antigens. In this study, 6 kDa early secretory antigenic target (ESAT-6) of M. tuberculosis was expressed in Brassica oleracea var. italica via Agrobacterium-mediated transformation to facilitate oral delivery of antigen. ESAT-6 gene was cloned using Gateway® cloning strategy. Transformation and presence of transgene was confirmed through PCR. Expression level of transgene was calculated via quantitative real-time PCR (qRT-PCR) and the maximum integrated transgene number was two. Maximum amount of total soluble fraction of ESAT-6 was evaluated by immunoblotting, estimated to accumulate up to 0.5% of total soluble protein. The recombinant ESAT-6 protein was further purified and detected using silver staining and Western blotting. ESAT-6 protein induced humoral immune response in mice immunized orally and subcutaneously. The expression of M. tuberculosis antigen in edible plants could aid in the development of cost-effective and oral delivery of an antigen-based subunit vaccine against TB. To the best our knowledge, it is the first report of expression of a vaccine antigen in broccoli.
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http://dx.doi.org/10.1002/bab.1867DOI Listing
January 2020

Optimization of cell suspension culture of transformed and untransformed lettuce for the enhanced production of secondary metabolites and their pharmaceutical evaluation.

3 Biotech 2019 Sep 22;9(9):339. Epub 2019 Aug 22.

2Department of Biochemistry, Quaid-I-Azam University, Islamabad, 45320 Pakistan.

In vitro suspension culture techniques are cost effective for large-scale production of secondary metabolites. In the present study, firstly, suspension cultures of untransformed were prepared using different hormonal combinations and were subjected to different pH, temperature and salt concentrations. Maximum biomass was obtained for suspensions supplemented with 1.5 mg/L BAP and 0.1 mg/L NAA, at pH 5.8, temperature 28 °C and 0 mM NaCl concentration. Using these parameters, suspensions were produced for and -transformed lines of . All the transgenic lines showed prominent increase in fresh weight (FW) and dry weight (DW) with maximum values for 2 line producing 169.8 mg/mL FW and 25.3 mg/mL DW. The exudates of transformed and untransformed plants were tested for the antioxidant activity and in vivo assays on rats. Maximum phenolic content (261 μg/mL) and flavonoid content (637.6 μg/mL) were obtained for 1 transgenic line. Total antioxidant capacity was found maximum (1451.7 μg/mL) for untransformed lettuce, whereas 1 showed maximum total reducing power activity (637.6 μg/mL). In DPPH assay, maximum activity (104.7 μg/mL) was shown by 3 line. In rats analgesic assay, maximum activity (74.9%) was shown by 2. Line 1 showed maximum anti-inflammatory activity (69.2%) and maximum antidepressant activity (minimum immobility time of 55 s). Maximum anticoagulant activity was observed for 2 with maximum clotting time of 130 s. The present study could help in using lettuce suspension culture as platform for the enhanced production of important metabolites.
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http://dx.doi.org/10.1007/s13205-019-1870-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6704210PMC
September 2019

Characterization of Withania somnifera chloroplast genome and its comparison with other selected species of Solanaceae.

Genomics 2020 03 27;112(2):1522-1530. Epub 2019 Aug 27.

Department of Biochemistry, Quaid-I-Azam University, 45320 Islamabad, Pakistan. Electronic address:

Withania somnifera (L) Dunal, a wonder herb of family Solanaceae, has multiple medicinal properties. Here, we reported the chloroplast genome sequence of Withania somnifera (154,386 bp) which comprises of a large single copy region (85,688 bp), and a small single copy region (18,464 bp), separated by a pair of large inverted repeats (25,117 bp). The chloroplast genome has 132 genes including 86 protein-coding, 37 tRNAs and 8 rRNAs. Comparison of chloroplast genomes of Withania somnifera with four other Solanaceae species revealed similarities in genomic features, including structure, nucleotide content, codon usage, RNA editing sites, simple sequence repeats (SSRs), oligonucleotide repeats, and tandem repeats. We identified 147 simple sequence repeats in protein-coding, and 229 in non-protein-coding regions. We observed numerous post-transcriptional substitutions of Serine to Leucine, specifically at the second nucleotide position of the codon. Maximum likelihood and maximum parsimony tree reconstructed displayed Withania somnifera a sister taxon of Physalis peruviana.
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http://dx.doi.org/10.1016/j.ygeno.2019.08.024DOI Listing
March 2020

Chloroplast-based inducible expression of ESAT-6 antigen for development of a plant-based vaccine against tuberculosis.

J Biotechnol 2019 Nov 24;305:1-10. Epub 2019 Aug 24.

Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, 45320, Islamabad, Pakistan. Electronic address:

Mycobacterium tuberculosis causes tuberculosis in humans. The major disease burden of tuberculosis lies in developing countries. Lack of an effective vaccine for adults is one of the major hurdles for controlling this deadly disease. In the present study, 6 kDa early secretory antigenic target (ESAT-6) of M. tuberculosis was inducibly expressed in chloroplasts of Nicotiana tabacum. The expression of ESAT-6 in chloroplasts was controlled by T7 promoter that was activated by nuclear-generated signal peptide. Tobacco plants, containing nuclear component, were transformed via biolistic bombardment with pEXP-T7-ESAT-6 obtained by Gateway® cloning. Transformation and homoplasmic status of transplastomic plants was confirmed by polymerase chain reaction and Southern blotting. Plants were induced for protein expression by spraying with 5% ethanol for 1 day, 3 days, 7 days and 10 days. ESAT-6 protein was detected by immunoblot analysis and maximum protein was obtained for 10 days induced plants that was estimated to accumulate up to 1.2% of total soluble fraction of protein. Transplastomic plants showed completely normal morphology. Transplastomic and untransformed plants became slightly chlorotic upon prolonged exposure to ethanol until 10 days. Taken together, this data could help in the development of an antigen-based subunit vaccine against tuberculosis.
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http://dx.doi.org/10.1016/j.jbiotec.2019.08.016DOI Listing
November 2019

Disease Status of Afghan Refugees and Migrants in Pakistan.

Front Public Health 2019 3;7:185. Epub 2019 Jul 3.

Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan.

World is facing the largest refugee crisis of its time due to continuously outgoing wars, conflicts and natural disasters. One of the important aspects of refugees and migrants is health. Till date, no comprehensive data was available related to health status of Afghan refugees and internally displaced persons (IDPs) in Pakistan. Here, we present health status for Afghan refugees for last seven years and for IDPs for 2-4 years. For Afghan refugees the data was provided by Commissionerate Afghan Refugee (CAR), Pakistan, whereas data for IDPs was collected from hospitals and Basic health units (BHUs) of different districts of Khyber Pakhtunkhwa namely Peshawar, Dera Ismail Khan and Bannu. Highest number of Afghan refugee's deaths occurred due to cardiovascular problems. Most prevalent reported infections were respiratory tract infections (48.05%). Skin diseases and Diarrhea collectively affected 21.08% of Afghan refugees. Overall, disease burden was more in females than males in Afghan refugee's population. To the best of our knowledge, this is the first comprehensive report on health and disease status of Afghan refugees and IDPs in Pakistan.
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http://dx.doi.org/10.3389/fpubh.2019.00185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6616124PMC
July 2019

Chloroplast genome of Hibiscus rosa-sinensis (Malvaceae): Comparative analyses and identification of mutational hotspots.

Genomics 2020 01 15;112(1):581-591. Epub 2019 Apr 15.

Department of Biochemistry, Quaid-i-Azam University, Islamabad, Pakistan. Electronic address:

Previous studies to resolve phylogenetic and taxonomic discrepancies of Hibiscus remained inconclusive. Here, we report chloroplast genome sequence of Hibiscus rosa-sinensis. Hibiscus rosa-sinensis chloroplast genome was 160,951 bp, comprising of large single copy (89,509 bp) and small single copy (20,246 bp) regions, separated by IRa and IRb (25,598 bp each). The genome contained 130 genes including 85 protein-coding genes, 37 transfer RNAs and 8 ribosomal RNAs. Comparative analyses of chloroplast genomes revealed similar structure among 12 species within family Malvaceae. Evolutionary rates of 77 protein-coding genes showed 95% similarities. Analyses of codon usage, amino acid frequency, putative RNA editing sites, and repeats showed a great extent of similarities between Hibiscus rosa-sinensis and Hibiscus syriacus. We identified 30 mutational hotpots including psbZ-trnG, trnK-rps16, trnD-trnY, trnW-trnP, rpl33-rps18, petG-trnW, trnS-trnG, trnH-psbA, atpB-rbcL, and rpl32-trnL that might be used as polymorphic and robust markers to resolve phylogenetic discrepancies in genus Hibiscus.
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http://dx.doi.org/10.1016/j.ygeno.2019.04.010DOI Listing
January 2020

CRISPR/Cas9-Mediated Immunity in Plants Against Pathogens.

Curr Issues Mol Biol 2018 7;26:55-64. Epub 2017 Sep 7.

Department of Horticulture, Faculty of Agriculture and Natural Sciences, Abant Izzet Baysal University, Bolu, Turkey.

Global crop production is highly threatened due to pathogen invasion. The huge quantity of pesticides application, although harmful to the environment and human health, is carried out to prevent the crop losses worldwide, every year. Therefore, understanding the molecular mechanisms of pathogenicity and plant resistance against pathogen is important. The resistance against pathogens is regulated by three important phytohormones viz. salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). Here we review possible role of CRISPR technology to understand the plant pathogenicity by mutating genes responsible for pathogen invasion or up-regulating the phytohormones genes or resistant genes. Thus hormone biosynthesis genes, receptor and feeding genes of pathogens could be important targets for modifications using CRISPR/Cas9 following multiplexing tool box strategy in order to edit multiple genes simultaneously to produce super plants. Here we put forward our idea thatthe genes would be either mutated in case of plant receptor protein targets of pathogens or up-regulation of resistant genes or hormone biosynthesis genes will be better choice for resistance against pathogens.
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http://dx.doi.org/10.21775/cimb.026.055DOI Listing
August 2018

Transformation of Lettuce with rol ABC Genes: Extracts Show Enhanced Antioxidant, Analgesic, Anti-Inflammatory, Antidepressant, and Anticoagulant Activities in Rats.

Appl Biochem Biotechnol 2017 Mar 12;181(3):1179-1198. Epub 2016 Oct 12.

Department of Biochemistry, Quaid-i-Azam University, Islamabad, 45320, Pakistan.

Lettuce is an edible crop that is well known for dietary and antioxidant benefits. The present study was conducted to investigate the effects of rol ABC genes on antioxidant and medicinal potential of lettuce by Agrobacterium-mediated transformation. Transgene integration and expression was confirmed through PCR and real-time RT-PCR, respectively. The transformed plants showed 91-102 % increase in total phenolic contents and 53-65 % increase in total flavonoid contents compared to untransformed plants. Total antioxidant capacity and total reducing power increased up to 112 and 133 % in transformed plants, respectively. Results of DPPH assay showed maximum 51 % increase, and lipid peroxidation assay exhibited 20 % increase in antioxidant activity of transformed plants compared to controls. Different in vivo assays were carried out in rats. The transgenic plants showed up to 80 % inhibition in both hot plate analgesic assay and carrageenan-induced hind paw edema test, while untransformed plants showed only 45 % inhibition. Antidepressant and anticoagulant potential of transformed plants was also significantly enhanced compared to untransformed plants. Taken together, the present work highlights the use of rol genes to enhance the secondary metabolite production in lettuce and improve its analgesic, anti-inflammatory, antidepressant, and anticoagulatory properties.
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http://dx.doi.org/10.1007/s12010-016-2277-3DOI Listing
March 2017

Effect of Rol Genes on Polyphenols Biosynthesis in Artemisia annua and Their Effect on Antioxidant and Cytotoxic Potential of the Plant.

Appl Biochem Biotechnol 2016 Aug 16;179(8):1456-68. Epub 2016 Apr 16.

Department of Biochemistry, Quaid-i-Azam University Islamabad, Islamabad, Pakistan.

Flavonoids are famous for their antioxidant capacity and redox potential. They can combat with cell aging, lipid peroxidation, and cancer. In the present study, Artemisia annua hybrid (Hyb8001r) was subjected to qualitative and quantitative analysis of flavonoids through HPLC. Rol genes transgenics of A. annua were also evaluated for an increase in their flavonoid content along with an increase in antioxidant and cytotoxic potential. This was also correlated with the expression level of flavonoids biosynthetic pathway genes as determined by real-time qPCR. Phenylalanine ammonia-lyase and chalcone synthase genes were found to be significantly more highly expressed in rol B (four to sixfold) and rol C transgenics (3.8-5.5-fold) than the wild-type plant. Flavonoids detected in the wild-type A. annua through HPLC include rutin (0.31 mg/g DW), quercetin (0.01 mg/g DW), isoquercetin (0.107 mg/g DW) and caffeic acid (0.03 mg/g DW). Transgenics of the rol B gene showed up to threefold increase in rutin and caffeic acid, sixfold increase in isoquercetin, and fourfold increase in quercetin. Whereas, in the case of transgenics of rol C gene, threefold increase in rutin and quercetin, 5 fold increase in isoquercetin, and 2.6-fold increase in caffeic acid was followed. Total phenolics and flavonoids content was also found to be increased in rol B (1.5-fold) and rol C (1.4-fold) transgenics as compared to the wild-type plant along with increased free radical scavenging activity. Similarly, the cytotoxic potential of rol gene transgenics against MCF7, HeLA, and HePG2 cancer cell lines was found to be significantly enhanced than the wild-type plant of A. annua. Current findings support the fact that rol genes can alter the secondary metabolism and phytochemical level of the plant. They increased the flavonoids content of A. annua by altering the expression level of flavonoids biosynthetic pathway genes. Increased flavonoid content also enhanced the antioxidant and cytotoxic potential of the plant.
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http://dx.doi.org/10.1007/s12010-016-2077-9DOI Listing
August 2016

Need of cost-effective vaccines in developing countries: What plant biotechnology can offer?

Springerplus 2016 22;5:65. Epub 2016 Jan 22.

Department of Applied Plant Sciences and Plant Biotechnology, University of Natural Resources and Applied Life Sciences, Konrad Lorenz Straße 24, 3430 Tulln an der Donau, Austria ; AIT Austrian Institute of Technology GmbH, Donau-City-Straße 1, 1220 Vienna, Austria.

To treat current infectious diseases, different therapies are used that include drugs or vaccines or both. Currently, the world is facing an increasing problem of drug resistance from many pathogenic microorganisms. In majority of cases, when vaccines are used, formulations consist of live attenuated microorganisms. This poses an additional risk of infection in immunocompromised patients and people suffering from malnutrition in developing countries. Therefore, there is need to improve drug therapy as well as to develop next generation vaccines, in particular against infectious diseases with highest mortality rates. For patients in developing countries, costs related to treatments are one of the major hurdles to reduce the disease burden. In many cases, use of prophylactic vaccines can help to control the incidence of infectious diseases. In the present review, we describe some infectious diseases with high impact on health of people in low and middle income countries. We discuss the prospects of plants as alternative platform for the development of next-generation subunit vaccines that can be a cost-effective source for mass immunization of people in developing countries.
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http://dx.doi.org/10.1186/s40064-016-1713-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4722051PMC
February 2016

Transformation of Lactuca sativa L. with rol C gene results in increased antioxidant potential and enhanced analgesic, anti-inflammatory and antidepressant activities in vivo.

3 Biotech 2016 Dec 5;6(2):215. Epub 2016 Oct 5.

Department of Biochemistry, Quaid-i-Azam University, Islamabad, 45320, Pakistan.

Lettuce is an important edible crop which possesses various medicinal properties. In this study Lactuca sativa L. (cv Grand Rapids) was transformed by Agrobacterium-mediated transformation with rol C gene. Transgene integration and expression was confirmed through PCR and semiquantitative RT-PCR. The transformed extracts were evaluated for their in vitro antioxidant and in vivo analgesic, anti-inflammatory and antidepressant activities in rats. The transformed plants showed 53-98 % increase in total phenolic and 45-58 % increase in total flavonoid contents compared with untransformed plants. Results of total reducing power and total antioxidant capacity exhibited 90-118 and 61-75 % increase in transformed plants, respectively. In contrast to control, DPPH, lipid peroxidation and DNA protection assay showed up to 37, 20 and 50 % enhancement in transformed plants, respectively. The extracts showed similar but significant enhancement behavior in hot plate analgesic and carrageenan-induced hind paw edema test. The transformed extracts showed 72.1 and 78.5 % increase for analgesic and anti-inflammatory activities, respectively. The transformants of rol C gene exhibited prominent antidepressant activity with 64-73 % increase compared with untransformed plants. In conclusion, the present work suggests that transformation with rol C gene can be used to generate lettuce with enhanced medicinally important properties, such as antioxidant, analgesic, anti-inflammatory and antidepressant potential.
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http://dx.doi.org/10.1007/s13205-016-0533-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5052223PMC
December 2016

Expression of HPV-16 L1 capsomeres with glutathione-S-transferase as a fusion protein in tobacco plastids: an approach for a capsomere-based HPV vaccine.

Hum Vaccin Immunother 2014 19;10(10):2975-82. Epub 2014 Nov 19.

a Department of Biotechnology ; Quaid-i-Azam University ; Islamabad , Pakistan.

Human Papillomavirus (HPV) is the main cause of cervical cancer, which is the second most severe cancer of women worldwide, particularly in developing countries. Although vaccines against HPV infection are commercially available, they are neither affordable nor accessible to women in low income countries e.g. Africa. Thus, alternative cost-effective vaccine production approaches need to be developed. This study uses tobacco plants to express pentameric capsomeres of HPV that have been reported to generate elevated immune responses against HPV. A modified HPV-16 L1 (L1_2xCysM) protein has been expressed as a fusion protein with glutathione-S-transferase (GST) in tobacco chloroplasts following biolistic transformation. In total 7 transplastomic lines with healthy phenotypes were generated. Site specific integration of the GST-L1_2xCysM and aadA genes was confirmed by PCR. Southern blot analysis verified homogenous transformation of all transplastomic lines. Antigen capture ELISA with the conformation-specific antibody Ritti01, showed protein expression as well as the retention of immunogenic epitopes of L1 protein. In their morphology, GST-L1 expressing tobacco plants were identical to wild type plants and yielded fertile flowers. Taken together, these data enrich knowledge for future development of cost-effective plant-made vaccines against HPV.
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http://dx.doi.org/10.4161/21645515.2014.970973DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5443053PMC
September 2015

Agrobacterium-mediated transformation of tomato with rolB gene results in enhancement of fruit quality and foliar resistance against fungal pathogens.

PLoS One 2014 9;9(5):e96979. Epub 2014 May 9.

Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan.

Tomato (Solanum lycopersicum L.) is the second most important cultivated crop next to potato, worldwide. Tomato serves as an important source of antioxidants in human diet. Alternaria solani and Fusarium oxysporum cause early blight and vascular wilt of tomato, respectively, resulting in severe crop losses. The foremost objective of the present study was to generate transgenic tomato plants with rolB gene and evaluate its effect on plant morphology, nutritional contents, yield and resistance against fungal infection. Tomato cv. Rio Grande was transformed via Agrobacterium tumefaciens harbouring rolB gene of Agrobacterium rhizogenes. rolB. Biochemical analyses showed considerable improvement in nutritional quality of transgenic tomato fruits as indicated by 62% increase in lycopene content, 225% in ascorbic acid content, 58% in total phenolics and 26% in free radical scavenging activity. Furthermore, rolB gene significantly improved the defence response of leaves of transgenic plants against two pathogenic fungal strains A. solani and F. oxysporum. Contrarily, transformed plants exhibited altered morphology and reduced fruit yield. In conclusion, rolB gene from A. rhizogenes can be used to generate transgenic tomato with increased nutritional contents of fruits as well as improved foliar tolerance against fungal pathogens.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0096979PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4016209PMC
January 2015

A novel chloroplast transformation vector compatible with the Gateway(®) recombination cloning technology.

Transgenic Res 2013 Dec 29;22(6):1273-8. Epub 2013 Jun 29.

Department of Crop Sciences, University of Natural Resources and Life Sciences, Vienna, Konrad Lorenz Street 24, 3430, Tulln, Austria.

To analyze the suitability of Gateway(®) vectors for transformation of chloroplasts, we converted a standard plastid transformation vector into a Gateway(®) destination vector containing the necessary recombination sites attR1 and attR2. Insertion of the green fluorescent protein (GFP) coding sequence with associated T7g10 ribosome binding site into this destination vector created the expression vector for transformation of tobacco chloroplasts with the biolistic method. Correct integration of the transgene into the plastid genome was verified by PCR and the homoplasmic nature of the transformed plants was confirmed by Southern Blot analysis. Expression of the GFP reporter protein was monitored by confocal laser scanning microscopy (CLSM) and quantification by western blot analysis showed a GFP accumulation level of 3% total soluble protein (TSP). The presented results clearly demonstrate that the Gateway(®) recombination sites are compatible with all steps of plastid transformation, from generation of transplastomic plants to expression of GFP. This is the first report of a plastid transformation vector made by the Gateway(®) recombinant cloning technology, which proves the suitability of this system for use in chloroplasts.
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http://dx.doi.org/10.1007/s11248-013-9726-3DOI Listing
December 2013

How can plant genetic engineering contribute to cost-effective fish vaccine development for promoting sustainable aquaculture?

Plant Mol Biol 2013 Sep 1;83(1-2):33-40. Epub 2013 Jun 1.

Bioforsk, Norwegian Institute for Agricultural and Environmental Research, Ås, Norway.

Aquaculture, the fastest growing food-producing sector, now accounts for nearly 50 % of the world's food fish (FAO in The state of world fisheries and aquaculture. FAO, Rome, 2010). The global aquaculture production of food fish reached 62.7 million tonnes in 2011 and is continuously increasing with an estimated production of food fish of 66.5 million tonnes in 2012 (a 9.4 % increase in 1 year, FAO, www.fao.org/fishery/topic/16140 ). Aquaculture is not only important for sustainable protein-based food fish production but also for the aquaculture industry and economy worldwide. Disease prevention is the key issue to maintain a sustainable development of aquaculture. Widespread use of antibiotics in aquaculture has led to the development of antibiotic-resistant bacteria and the accumulation of antibiotics in the environment, resulting in water and soil pollution. Thus, vaccination is the most effective and environmentally-friendly approach to combat diseases in aquaculture to manage fish health. Furthermore, when compared to >760 vaccines against human diseases, there are only about 30 fish vaccines commercially available, suggesting the urgent need for development and cost-effective production of fish vaccines for managing fish health, especially in the fast growing fish farming in Asia where profit is minimal and therefore given high priority. Plant genetic engineering has made significant contributions to production of biotech crops for food, feed, valuable recombinant proteins etc. in the past three decades. The use of plants for vaccine production offers several advantages such as low cost, safety and easy scaling up. To date a large number of plant-derived vaccines, antibodies and therapeutic proteins have been produced for human health, of which a few have been made commercially available. However, the development of animal vaccines in plants, especially fish vaccines by genetic engineering, has not yet been addressed. Therefore, there is a need to exploit plant biotechnology for cost effective fish vaccine development in plants, in particular, edible crops for oral fish vaccines. This review provides insight into (1) the current status of fish vaccine and vaccination in aquaculture, (2) plant biotechnology and edible crops for fish vaccines for oral administration, (3) regulatory constraints and (4) conclusions and future perspectives.
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http://dx.doi.org/10.1007/s11103-013-0081-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3755229PMC
September 2013

Plant-derived vaccines: an approach for affordable vaccines against cervical cancer.

Hum Vaccin Immunother 2012 Mar 13;8(3):403-6. Epub 2012 Feb 13.

Department of Applied Plant Sciences and Applied Plant Biotechnology, University of Natural Resources and Applied Life Sciences (BOKU), Vienna, Austria.

Several types of human papillomavirus (HPV) are causatively associated with cervical cancer, which is the second most common cancer in women worldwide. HPV-16 and 18 are among the high risk types and responsible for HPV infection in more than 70% of the cases. The majority of cervical cancer cases occur in developing countries. Currently available HPV vaccines are expensive and probably unaffordable for most women in low and middle income countries. Therefore, there is a need to develop cost-effective vaccines for these countries. Due to many advantages, plants offer an attractive platform for the development of affordable vaccines. These include low cost of production, scalability, low health risks and the potential ability to be used as unprocessed or partially processed material. Among several techniques, chloroplast transformation is of eminent interest for the production of vaccines because of high yield of foreign protein and lack of transgene transmission through pollen. In this commentary, we focus on the most relevant aspects of plant-derived vaccines that are decisive for the future development of cost-effective HPV vaccines.
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http://dx.doi.org/10.4161/hv.18568DOI Listing
March 2012

New areas of plant-made pharmaceuticals.

Expert Rev Vaccines 2011 Feb;10(2):151-3

Department of Applied Plant Sciences and Plant Biotechnology, University of Natural Resources and Applied Life Sciences, Gregor-Mendel-Strasse 33, 1180 Vienna, Austria.

On October 15 2010 the meeting 'Recombinant Pharmaceutical Manufacturing from Plants - The Future of Molecular Farming' hosted by EuroScicon was held at BioPark Hertfordshire, Welwyn Garden city, UK. The scientific program of this very eventful meeting was wide ranging and covered diverse aspects of biopharming. The highlights presented included: safety issues in biopharming; coexpression of multiple proteins; steps towards vaccine generation; and engineering of secondary metabolites and medicinal plants. This article summarizes the stimulating scientific presentations and fruitful panel discussions that subsequently arose during and after this event.
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http://dx.doi.org/10.1586/erv.10.166DOI Listing
February 2011