Archana Chugh

Cell‐penetrating peptides: Nanocarrier for macromolecule delivery in living cells

Novel classes and applications of cell‐penetrating peptides (CPPs) are being constantly discovered since they were first identified 2 decades ago. These short cationic peptides (nanomolecules) either by covalent binding or by noncovalent binding can traverse cell membranes and deliver a variety of molecules that are unable to overcome the permeability barrier in their own capacity. The ability of the CPPs to deliver variety of macromolecules, such as oligonucleotides, therapeutic drugs, proteins, and medical imaging agents, by forming nanoparticulate carriers in a range of cells has led them to emerge as a potential tool for both macromolecule delivery application and to gain insight into the fundamentals of mechanism of cellular uptake across the plasma membrane. This review explores the recent advances, challenges, and future prospects in the field of CPP‐mediated cargo delivery in mammalian and plant cells. Studies have been conducted into the peptide chemistry and stability of CPP‐macromolecular complexes. Most of the CPPs have been shown to be nontoxic and do not interfere with the functionality of the macromolecules delivered across the cell membrane. The mechanism of uptake of CPP‐cargo complexes and the uptake of CPPs alone across the plasma membrane remains unresolved. As the world of CPPs is rapidly advancing in both mammalian and plant system, there is a promising future for the various applications of transduction and transfection into intact cells.

Study of uptake of cell penetrating peptides and their cargoes in permeabilized wheat immature embryos

The uptake of five fluorescein labeled cell‐penetrating peptides (Tat, Tat2, mutated‐Tat, peptide vascular endothelial‐cadherin and transportan) was studied in wheat immature embryos. Interestingly, permeabilization treatment of the embryos with toluene/ethanol (1 : 20, v/v with permeabilization buffer) resulted in a remarkably higher uptake of cell‐penetrating peptides, whereas nonpermeabilized embryos failed to show significant cell‐penetrating peptide uptake, as observed under fluorescence microscope and by fluorimetric analysis. Among the cell‐penetrating peptides investigated, Tat monomer (Tat) showed highest fluorescence uptake (4.2‐fold greater) in permeabilized embryos than the nonpermeabilized embryos. On the other hand, mutated‐Tat serving as negative control did not show comparable fluorescence levels even in permeabilized embryos. A glucuronidase histochemical assay revealed that Tat peptides can efficiently deliver functionally active β‐glucuronidase (GUS) enzyme in permeabilized immature embryos. Tat2‐mediated GUS enzyme delivery showed the highest number of embryos with GUS uptake (92.2%) upon permeabilization treatment with toluene/ethanol (1 : 40, v/v with permeabilization buffer) whereas only 51.8% of nonpermeabilized embryos showed Tat2‐mediated GUS uptake. Low temperature, endocytosis and macropinocytosis inhibitors reduced delivery of the Tat2–GUS enzyme cargo complex. The results suggest that more than one mechanism of cell entry is involved simultaneously in cell‐penetrating peptide‐cargo uptake in wheat immature embryos. We also studied Tat2‐plasmid DNA (carrying Act‐1GUS) complex formation by gel retardation assay, DNaseI protection assay and confocal laser microscopy. Permeabilized embryos transfected with Tat2–plasmid DNA complex showed 3.3‐fold higher transient GUS gene expression than the nonpermeabilized embryos. Furthermore, addition of cationic transfecting agent Lipofectamine™ 2000 to the Tat2–plasmid DNA complex resulted in 1.5‐fold higher transient GUS gene expression in the embryos. This is the first report demonstrating translocation of various cell‐penetrating peptides and their potential to deliver macromolecules in wheat immature embryos in the presence of a cell membrane permeabilizing agent.

A global conversation about energy from biomass: the continental conventions of the global sustainable bioenergy project

The global sustainable bioenergy (GSB) project was formed in 2009 with the goal of providing guidance with respect to the feasibility and desirability of sustainable, bioenergy-intensive futures. Stage 1 of this project held conventions with a largely common format on each of the worlds continents, was completed in 2010, and is described in this paper. Attended by over 400 persons, the five continental conventions featured presentations, breakout sessions, and drafting of resolutions that were unanimously passed by attendees. The resolutions highlight the potential of bioenergy to make a large energy supply contribution while honouring other priorities, acknowledge the breadth and complexity of bioenergy applications as well as the need to take a systemic approach, and attest to substantial intra- and inter-continental diversity with respect to needs, opportunities, constraints and current practice relevant to bioenergy. The following interim recommendations based on stage 1 GSB activities are offered: — Realize that it may be more productive, and also more correct, to view the seemingly divergent assessments of bioenergy as answers to two different questions rather than the same question. Viewed in this light, there is considerably more scope for reconciliation than might first be apparent, and it is possible to be informed rather than paralysed by divergent assessments. — Develop established and advanced bioenergy technologies such that each contributes to the others success. That is, support and deploy in the near-term meritorious, established technologies in ways that enhance rather than impede deployment of advanced technologies, and support and deploy advanced technologies in ways that expand rather than contract opportunities for early adopters and investors. — Be clear in formulating policies what mix of objectives are being targeted, measure the results of these policies against these objectives and beware of unintended consequences. — Undertake further exploration of land efficiency levers and visions for multiply-beneficial bioenergy deployment. This should be unconstrained by current practices, since we cannot hope to achieve a sustainable and a secure future by continuing the practices that have led to the unsustainable and insecure present. It should also be approached from a global perspective, based on the best science available, and consider the diverse realities, constraints, needs and opportunities extant in different regions of the world. The future trajectory of the GSB project is also briefly considered.

Regeneration via somatic embryogenesis from leaf basal segments and genetic transformation of bread and emmer wheat by particle bombardment

Direct gene transformation methods such as microprojectile bombardment have been successfully employed for obtaining transgenics in cereals in general and wheat in particular. As success of any transformation strategy depends largely upon the regeneration capability of the target explant, the present investigation employs leaf basal segments to achieve high regeneration response via somatic embryogenesis. Basal segments of 5-day-old seedlings of T. aestivum var. CPAN1676 and T. dicoccum var. DDK1001 were cultured on callusing medium for 3 weeks at 26 ± 1 °C, discontinuous light followed by a culture period of 15 days at 21 ± 1 °C in continuous light. The calli were then transferred to auxin-free medium for regeneration in discontinuous light at 26 ± 1 °C. Regeneration via somatic embryogenesis was observed within 2 weeks in T. aestivum var. CPAN1676 and T. dicoccum var. DDK1001 (68 and 82%, respectively). This embryogenic calli were employed further to obtain hygromycin resistance by particle bombardment in T. aestivum and T. dicoccum. A transformation efficiency of 8.6, 7.5 and 4.9% was obtained in T. aestivum var. CPAN1676, PBW343 and T. dicoccum DDK1001, respectively. Presence of the transgene hptII (hygromycin) in T0 plants was confirmed by Southern hybridization.

Cell-penetrating peptides: From mammalian to plant cells.

Internalization of cell-penetrating peptides, well described in mammalian cell system, has recently been reported in a range of plant cells by three independent groups. Despite fundamental differences between animal cell and plant cell composition, the CPP uptake pattern between the mammalian system and the plant system is very similar. Tat, Tat-2 pVEC and transportan internalisation is concentration dependent and non saturable, enhanced at low temperature (4oC), and receptor independent. The use of CPPs as nanocarrier for macromolecular delivery in plant cells is now achievable and the advances made in mammalian cells greatly enhance our understanding of cell-membrane and CPP-macromolecule complex interaction in plant. The cross membrane nanocarrier ability of CPPs promises new avenues in the field of plant biotechnology.

Membrane-Active Peptides from Marine Organisms—Antimicrobials, Cell-Penetrating Peptides and Peptide Toxins: Applications and Prospects

Abstract Marine organisms are known to be a rich and unique source of bioactive compounds as they are exposed to extreme conditions in the oceans. The present study is an attempt to briefly describe some of the important membrane-active peptides (MAPs) such as antimicrobial peptides (AMPs), cell-penetrating peptides (CPPs) and peptide toxins from marine organisms. Since both AMPs and CPPs play a role in membrane perturbation and exhibit interchangeable role, they can speculatively fall under the broad umbrella of MAPs. The study focuses on the structural and functional characteristics of different classes of marine MAPs. Further, AMPs are considered as a potential remedy to antibiotic resistance acquired by several pathogens. Peptides from marine organisms show novel post-translational modifications such as cysteine knots, halogenation and histidino–alanine bridge that enable these peptides to withstand harsh marine environmental conditions. These unusual modifications of AMPs from marine organisms are expected to increase their half-life in living systems, contributing to their increased bioavailability and stability when administered as drug in in vivo systems. Apart from AMPs, marine toxins with membrane-perturbing properties could be essentially investigated for their cytotoxic effect on various pathogens and their cell-penetrating activity across various mammalian cells. The current review will help in identifying the MAPs from marine organisms with crucial post-translational modifications that can be used as template for designing novel therapeutic agents and drug-delivery vehicles for treatment of human diseases.

Role of traditional knowledge in marine bioprospecting

The “marine world” is endowed with diverse life forms. The life under the oceans is bestowed with a unique gene pool and characteristics owing to extreme conditions such as high salt concentration and temperature variations. The marine biodiversity is an extremely rich resource for the development of a wide array of applications in food, pharmaceuticals, cosmetics. Various forms of traditional knowledge, including traditional medicinal knowledge, have been silently developing over the centuries, with the coastal tribes in nations across the globe. Unfortunately, marine traditional knowledge has been underestimated both commercially and legally. It has still not gained its due importance at the international platform for sustainable use and development. An attempt has been made in the present study to collate information on marine traditional knowledge based medicine. Recent trends of marine bioprospecting by various nations including India have been discussed, followed by the study of legal provisions dealing with marine bioprospecting that aim at conservation and sustainable use of marine biodiversity and associated traditional knowledge. Convention of Biological Diversity, United Nations Convention on the Law of the Seas and World Intellectual Property Organization are the major international legal instruments that discuss the concepts of Prior Informed Consent, access and benefit sharing with regard to biopiracy and provide guidelines and limits for conducting marine scientific research.

Marine antimicrobial peptide tachyplesin as an efficient nanocarrier for macromolecule delivery in plant and mammalian cells

Membrane‐active peptides can be classified as cell‐penetrating peptides and antimicrobial peptides (AMPs) that are known to play interchangeable roles. In this study, this dual behaviour was studied for the marine AMP, tachyplesin. It is a well‐established cyclic peptide known to possess antimicrobial properties and was investigated for its cell‐penetrating property and cargo delivery ability. Because of its derivation from a marine organism as well as cyclic nature, it has been shown to possess higher stability in vitro. In this study, its internalization as a cell‐penetrating peptide was established and characterized in both plant and mammalian systems. It was shown to deliver cargo molecules in both living systems, emerging as an efficient nonviral macromolecule nanocarrier.

Cell Penetrating Peptides as Efficient Nanocarriers for Delivery of Antifungal Compound, Natamycin for the Treatment of Fungal Keratitis

PurposeEnhancing the penetration ability of the antifungal drug natamycin, known to possess poor penetration ability through the corneal epithelium, by complexing with cell penetrating peptides.MethodsThe drug, natamycin was conjugated to a cell penetrating peptide, Tat-dimer (Tat2). The uptake ability of the conjugate in human corneal epithelial cells and its antifungal activity against filamentous fungi, F.solani has been elucidated.ResultsThe cellular penetration ability of natamycin increased upon conjugation with Tat2. The conjugation between natamycin and Tat2 also lead to enhanced solubility of the drug in aqueous medium. The antifungal activity of the conjugate increased two- folds in comparison to unconjugated natamycin against clinical isolates of F.solani.ConclusionThe formation of CPP-natamycin complex is clinically significant as it may enhance the bioavailability of natamycin in corneal tissues and aid in efficient management of fungal keratitis.

Microbial synthetic biology for human therapeutics

The emerging field of synthetic biology holds tremendous potential for developing novel drugs to treat various human conditions. The current study discusses the scope of synthetic biology for human therapeutics via microbial approach. In this context, synthetic biology aims at designing, engineering and building new microbial synthetic cells that do not pre-exist in nature as well as re-engineer existing microbes for synthesis of therapeutic products. It is expected that the construction of novel microbial genetic circuitry for human therapeutics will greatly benefit from the data generated by ‘omics’ approaches and multidisciplinary nature of synthetic biology. Development of novel antimicrobial drugs and vaccines by engineering microbial systems are a promising area of research in the field of synthetic biology for human theragnostics. Expression of plant based medicinal compounds in the microbial system using synthetic biology tools is another avenue dealt in the present study. Additionally, the study suggest that the traditional medicinal knowledge can do value addition for developing novel drugs in the microbial systems using synthetic biology tools. The presented work envisions the success of synthetic biology for human therapeutics via microbial approach in a holistic manner. Keeping this in view, various legal and socio-ethical concerns emerging from the use of synthetic biology via microbial approach such as patenting, biosafety and biosecurity issues have been touched upon in the later sections.