Perspectives on new opportunities for nano-enabled strategies for gene delivery to plants using nanoporous materials.

MAIN CONCLUSION: Engineered nanocarriers have great potential to deliver different genetic cargos to plant cells and increase the efficiency of plant genetic engineering. Genetic engineering has improved the quality and quantity of crops by introducing desired DNA sequences into the plant genome. Traditional transformation strategies face constraints such as low transformation efficiency, damage to plant tissues, and genotype dependency. Smart nanovehicle-based delivery is a newly emerged method for direct DNA delivery to plant genomes. The basis of this new approach of plant genetic transformation, nanomaterial-mediated gene delivery, is the appropriate protection of transferred DNA from the nucleases present in the cell cytoplasm through the nanocarriers. The conjugation of desired nucleic acids with engineered nanocarriers can solve the problem of genetic manipulation in some valuable recalcitrant plant genotypes. Combining nano-enabled genetic transformation with the new and powerful technique of targeted genome editing, CRISPR (clustered regularly interspaced short palindromic repeats), can create new protocols for efficient improvement of desired plants. Silica-based nanoporous materials, especially mesoporous silica nanoparticles (MSNs), are currently regarded as exciting nanoscale platforms for genetic engineering as they possess several useful properties including ordered and porous structure, biocompatibility, biodegradability, and surface chemistry. These specific features have made MSNs promising candidates for the design of smart, controlled, and targeted delivery systems in agricultural sciences. In the present review, we discuss the usability, challenges, and opportunities for possible application of nano-enabled biomolecule transformation as part of innovative approaches for target delivery of genes of interest into plants.

T-type calcium channels drive the proliferation of androgen-receptor negative prostate cancer cells.

BACKGROUND: Androgen deprivation therapy (ADT) is the treatment of choice for metastatic prostate cancer (PCa). After an initial response to ADT, PCa cells can generate castration resistant (CRPC) or neuroendocrine (NEPC) malignancies, which are incurable. T-type calcium channels (TTCCs) are emerging as promising therapeutic targets for several cancers, but their role in PCa progression has never been investigated. METHODS: To examine the role of TTCCs in PCa, we analyzed their expression level, copy number variants (CNV) and prognostic significance using clinical datasets (Oncomine and cBioPortal). We then evaluated TTCC expression in a panel of PCa cell lines and measured the effect of their inhibition on cell proliferation and survival using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and caspase assays. RESULTS: TTCCs were upregulated in PCas harboring androgen receptor (AR) mutations; CNV rate was positively associated with PCa progression. Higher expression of one TTCC isoform (CACNA1G) predicted poorer postoperative prognosis in early stage PCa samples. Pharmacological or small interfering RNA (siRNA)-based inhibition of TTCCs caused a decrease in PC-3 cell survival and proliferation. CONCLUSIONS: Our results show that TTCCs are overexpressed in advanced forms of PCa and correlate with a poorer prognosis. TTCC inhibition reduces cell proliferation and survival, suggesting that there may be possible value in the therapeutic targeting of TTCCs in advanced PCa.

Current status and biotechnological advances in genetic engineering of ornamental plants.

Cut flower markets are developing in many countries as the international demand for cut flowers is rapidly growing. Developing new varieties with modified characteristics is an important aim in floriculture. Production of transgenic ornamental plants can shorten the time required in the conventional breeding of a cultivar. Biotechnology tools in combination with conventional breeding methods have been used by cut flower breeders to change flower color, plant architecture, post-harvest traits, and disease resistance. In this review, we describe advances in genetic engineering that have led to the development of new cut flower varieties.

Methods for genetic transformation in Dendrobium.

KEY MESSAGE: The genetic transformation of Dendrobium orchids will allow for the introduction of novel colours, altered architecture and valuable traits such as abiotic and biotic stress tolerance. The orchid genus Dendrobium contains species that have both ornamental value and medicinal importance. There is thus interest in producing cultivars that have increased resistance to pests, novel horticultural characteristics such as novel flower colours, improved productivity, longer flower spikes, or longer post-harvest shelf-life. Tissue culture is used to establish clonal plants while in vitro flowering allows for the production of flowers or floral parts within a sterile environment, expanding the selection of explants that can be used for tissue culture or genetic transformation. The latter is potentially the most effective, rapid and practical way to introduce new agronomic traits into Dendrobium. Most (69.4 %) Dendrobium genetic transformation studies have used particle bombardment (biolistics) while 64 % have employed some form of Agrobacterium-mediated transformation. A singe study has explored ovary injection, but no studies exist on floral dip transformation. While most of these studies have involved the use of selector or reporter genes, there are now a handful of studies that have introduced genes for horticulturally important traits.

Enhancement of phosphate absorption by garden plants by genetic engineering: a new tool for phytoremediation.

Although phosphorus is an essential factor for proper plant growth in natural environments, an excess of phosphate in water sources causes serious pollution. In this paper we describe transgenic plants which hyperaccumulate inorganic phosphate (Pi) and which may be used to reduce environmental water pollution by phytoremediation. AtPHR1, a transcription factor for a key regulator of the Pi starvation response in Arabidopsis thaliana, was overexpressed in the ornamental garden plants Torenia, Petunia, and Verbena. The transgenic plants showed hyperaccumulation of Pi in leaves and accelerated Pi absorption rates from hydroponic solutions. Large-scale hydroponic experiments indicated that the enhanced ability to absorb Pi in transgenic torenia (AtPHR1) was comparable to water hyacinth a plant that though is used for phytoremediation causes overgrowth problems.

Expression of flavonoid 3',5'-hydroxylase and acetolactate synthase genes in transgenic carnation: assessing the safety of a nonfood plant.

For 16 years, genetically modified flowers of carnation ( Dianthus caryophyllus ) have been sold to the floristry industry. The transgenic carnation carries a herbicide tolerance gene (a mutant gene encoding acetolactate synthase (ALS)) and has been modified to produce delphinidin-based anthocyanins in flowers, which conventionally bred carnation cannot produce. The modified flower color has been achieved by introduction of a gene encoding flavonoid 3',5'-hydroxylase (F3'5'H). Transgenic carnation flowers are produced in South America and are primarily distributed to North America, Europe, and Japan. Although a nonfood crop, the release of the genetically modified carnation varieties required an environmental risk impact assessment and an assessment of the potential for any increased risk of harm to human or animal health compared to conventionally bred carnation. The results of the health safety assessment and the experimental studies that accompanied them are described in this review. The conclusion from the assessments has been that the release of genetically modified carnation varieties which express F3'5'H and ALS genes and which accumulate delphinidin-based anthocyanins do not pose an increased risk of harm to human or animal health.

Accuracy of transnasal cannulation and dilation of the maxillary ostium in cadavers with intact uncinates.

BACKGROUND: Transnasal cannulation of the natural ostium in patients with an intact uncinate process is complicated by the lack of direct visualization of the ostium. Accuracy of transnasal dilation of the maxillary ostium was evaluated for a malleable-tipped balloon device that was bent to specific angles for avoiding the fontanelle during cannulation. METHODS: Transnasal cannulation and dilation of 42 cadaver maxillary sinus ostia was attempted by 6 surgeons including 3 with very limited clinical experience using the study device. All physicians received procedure training including the technique to shape the balloon device into the recommended 135° maxillary configuration. Tissue dissection was prohibited. Canine fossa trephination and transantral endoscopy were used to evaluate cannulation and dilation outcomes. Physician operators were blinded to transantral images and results were documented by two observers. RESULTS: Appropriate transnasal cannulation and dilation of natural maxillary sinus ostia occurred in 92.9% (39/42) of attempts. Two failures emanated from procedural deviations. In one deviation, the bend angle was changed to 90° and the device tip did not cannulate the ostium. In the second, the device was passed through a preexisting hole in the uncinate and cannulated the natural ostium. A third failure occurred when the device was passed through the fontanelle creating a false lumen. CONCLUSION: Using recommended procedural techniques and a malleable-tipped balloon device, newly trained and experienced physicians alike can perform uncinate-preserving transnasal cannulation and dilation of the maxillary ostium with a high rate of success.

Genetic modification; the development of transgenic ornamental plant varieties.

Plant transformation technology (hereafter abbreviated to GM, or genetic modification) has been used to develop many varieties of crop plants, but only a few varieties of ornamental plants. This disparity in the rate and extent of commercialisation, which has been noted for more than a decade, is not because there are no useful traits that can be engineered into ornamentals, is not due to market potential and is not due to a lack of research and development activity. The GM ornamental varieties which have been released commercially have been accepted in the marketplace. In this article, progress in the development of transgenic ornamentals is reviewed and traits useful to both consumers and producers are identified. In considering possible factors limiting the release of genetically modified ornamental products it is concluded that the most significant barrier to market is the difficulty of managing, and the high cost of obtaining, regulatory approval.

Genetic modification in floriculture.

Micro-propagation, embryo rescue, mutagenesis via chemical or irradiation means and in vitro inter-specific hybridisation methods have been used by breeders in the floriculture industry for many years. In the past 20 years these enabling technologies have been supplemented by genetic modification methods. Though many genes of potential utility to the floricultural industry have been identified, and much has been learnt of the genetic factors and molecular mechanisms underlying phenotypes of great importance to the industry, there are only flower colour modified varieties of carnation and rose in the marketplace. To a large extent this is due to unique financial barriers to market entry for genetically modified varieties of flower crops, including use of technology fees and costs of regulatory approval.

Quantifying pain relief following administration of a novel formulation of paracetamol (acetaminophen).

This article describes how a model-based analysis was used to aid development of a novel formulation technology. Paracetamol (acetaminophen) was used as the motivating example with 4 different formulations (2 developmental and 2 commercial) compared using stochastic (Monte Carlo) pharmacokinetic (PK)-pharmacodynamic (PD) simulations to explore potential differences in pharmacodynamic outcomes. PK models were developed from data collected during an intensively sampled, 4-arm crossover trial in 25 fasted healthy subjects, administered 1 g of paracetamol in 4 different formulations. The PK models were linked to a previously published PD model that quantified pain relief over time following tonsillectomy. The number needed to treat (NNT) was the primary numeric used to compare effectiveness. The developmental formulations were likely to produce faster and greater analgesia with an NNT (compared with placebo) to reduce pain by 50% over a 45-minute interval post dose of 2.75 and 2.88 compared with 4.31 and 3.2 for the commercial products. Over the course of 1 hour, all formulations were comparable. The stochastic simulations provided support that the novel formulation technology was likely to provide a clinically meaningful advantage and should be developed further.

CHR-2797: an antiproliferative aminopeptidase inhibitor that leads to amino acid deprivation in human leukemic cells.

CHR-2797 is a novel metalloenzyme inhibitor that is converted into a pharmacologically active acid product (CHR-79888) inside cells. CHR-79888 is a potent inhibitor of a number of intracellular aminopeptidases, including leucine aminopeptidase. CHR-2797 exerts antiproliferative effects against a range of tumor cell lines in vitro and in vivo and shows selectivity for transformed over nontransformed cells. Its antiproliferative effects are at least 300 times more potent than the prototypical aminopeptidase inhibitor, bestatin. However, the mechanism by which inhibition of these enzymes leads to proliferative changes is not understood. Gene expression microarrays were used to profile changes in mRNA expression levels in the human promyelocytic leukemia cell line HL-60 treated with CHR-2797. This analysis showed that CHR-2797 treatment induced a transcriptional response indicative of amino acid depletion, the amino acid deprivation response, which involves up-regulation of amino acid synthetic genes, transporters, and tRNA synthetases. These changes were confirmed in other leukemic cell lines sensitive to the antiproliferative effects of CHR-2797. Furthermore, CHR-2797 treatment inhibited phosphorylation of mTOR substrates and reduced protein synthesis in HL-60 cells, both also indicative of amino acid depletion. Treatment with CHR-2797 led to an increase in the concentration of intracellular small peptides, the substrates of aminopeptidases. It is suggested that aminopeptidase inhibitors, such as CHR-2797 and bestatin, deplete sensitive tumor cells of amino acids by blocking protein recycling, and this generates an antiproliferative effect. CHR-2797 is orally bioavailable and currently undergoing phase II clinical investigation in the treatment of myeloid leukemia.

Immunohistochemical localization of interleukin-10 in human oral and pharyngeal carcinomas.

OBJECTIVES/HYPOTHESIS: Interleukin-10 (IL-10) is an immunosuppressive cytokine with numerous, well-described effects on the human cellular and humoral immune response. The oncogenic potential of IL-10 has been previously investigated in bronchogenic carcinoma, nasopharyngeal carcinoma, Waldeyer's ring carcinoma, and serum supernatants of patients with squamous cell carcinoma of the head and neck (SCCHN). The purpose of the study was to determine the prevalence and cellular localization of IL-10 in human SC-CHN. STUDY DESIGN: Immunohistochemistry of archival tissues. METHODS: Paraffin-embedded archival tissues were retrospectively obtained from 98 patients with oral and pharyngeal squamous cell carcinoma. Using a standard immunohistochemical technique, these specimens were stained with a polyclonal antibody to IL-10. RESULTS: Using these methods, we found specific localization of antigenic IL-10 to individual tumor cells in 65% of tumors studied. Intensity of staining was significantly, but inversely, related to tumor grade and N stage; there also existed a significant staining predisposition for oral cavity lesions when samples from this site were compared with tissues derived from elsewhere in the pharynx. Furthermore, IL-10 was not localized to normal epithelial keratinocytes or inflammatory cells at the level of sensitivity achieved by the immunohistochemical methods used in the study. CONCLUSIONS: The findings demonstrate that IL-10 can be specifically localized to human oral and pharyngeal cancer cells. These data also suggest an inverse association for both tumor grade and N stage with specific tumor marker staining. Future studies should investigate the role of this cytokine in the pathogenesis of human SCCHN.

Antibacterial activities and characterization of novel inhibitors of LpxC.

Lipid A is the hydrophobic anchor of lipopolysaccharide (LPS) and forms the major lipid component of the outer monolayer of the outer membrane of gram-negative bacteria. Lipid A is required for bacterial growth and virulence, and inhibition of its biosynthesis is lethal to bacteria. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a metalloenzyme that catalyzes the second step in the biosynthesis of lipid A. Inhibitors of LpxC have previously been shown to have antibiotic activities. We have screened a metalloenzyme inhibitor library for antibacterial activities against an Escherichia coli strain with reduced LpxC activity. From this screen, a series of sulfonamide derivatives of the alpha-(R)-amino hydroxamic acids, exemplified by BB-78484 and BB-78485, have been identified as having potent inhibitory activities against LpxC in an in vitro assay. Leads from this series showed gram-negative selective activities against members of the Enterobacteriaceae, Serratia marcescens, Morganella morganii, Haemophilus influenzae, Moraxella catarrhalis, and Burkholderia cepacia. BB-78484 was bactericidal against E. coli, achieving 3-log killing in 4 h at a concentration 4 times above the MIC, as would be predicted for an inhibitor of lipid A biosynthesis. E. coli mutants with decreased susceptibility to BB-78484 were selected. Analysis of these mutants revealed that resistance arose as a consequence of mutations in the fabZ or lpxC genes. These data confirm the antibacterial target of BB-78484 and BB-78485 and validate LpxC as a target for gram-negative selective antibacterials.