CRISPR/Cas9 Initiated Transgenic Silkworms as a Natural Spinner of Spider Silk.

Using transgenic silkworms with their natural spinning apparatus has proven to be a promising way to spin spider silk-like fibers. The challenges are incorporating native-size spider silk proteins and achieving an inheritable transgenic silkworm strain. In this study, a CRISPR/Cas9 initiated fixed-point strategy was used to successfully incorporate spider silk protein genes into the Bombyx mori genome. Native-size spider silk genes (up to 10 kb) were inserted into an intron of the fibroin heavy or light chain (FibH or FibL) ensuring that any sequence changes induced by the CRISPR/Cas9 would not impact protein production. The resulting fibers are as strong as native spider silks (1.2 GPa tensile strength). The transgenic silkworms have been tracked for several generations with normal inheritance of the transgenes. This strategy demonstrates the feasibility of using silkworms as a natural spider silk spinner for industrial production of high-performance fibers.

Characterization of physicochemical properties of ivy nanoparticles for cosmetic application.

BACKGROUND: Naturally occurring nanoparticles isolated from English ivy (Hedera helix) have previously been proposed as an alternative to metallic nanoparticles as sunscreen fillers due to their effective UV extinction property, low toxicity and potential biodegradability. METHODS: This study focused on analyzing the physicochemical properties of the ivy nanoparticles, specifically, those parameters which are crucial for use as sunscreen fillers, such as pH, temperature, and UV irradiation. The visual transparency and cytotoxicity of ivy nanoparticles were also investigated comparing them with other metal oxide nanoparticles. RESULTS: Results from this study demonstrated that, after treatment at 100°C, there was a clear increase in the UV extinction spectra of the ivy nanoparticles caused by the partial decomposition. In addition, the UVA extinction spectra of the ivy nanoparticles gradually reduced slightly with the decrease of pH values in solvents. Prolonged UV irradiation indicated that the influence of UV light on the stability of the ivy nanoparticle was limited and time-independent. Compared to TiO2 and ZnO nanoparticles, ivy nanoparticles showed better visual transparency. Methylthiazol tetrazolium assay demonstrated that ivy nanoparticles exhibited lower cytotoxicity than the other two types of nanoparticles. Results also suggested that protein played an important role in modulating the three-dimensional structure of the ivy nanoparticles. CONCLUSIONS: Based on the results from this study it can be concluded that the ivy nanoparticles are able to maintain their UV protective capability at wide range of temperature and pH values, further demonstrating their potential as an alternative to replace currently available metal oxide nanoparticles in sunscreen applications.

Bio-synthesis of gold nanoparticles using English ivy (Hedera helix).

Gold nanoparticles (AuNPs) have drawn significant interest in recent years due to unique properties that make them advantageous in biomedical applications, including drug delivery and tissue engineering. In this paper, we have developed multiple methods for the synthesis of AuNPs using English ivy as the substrate. In the first method, we have used actively growing English ivy shoots to develop a sustainable system for the production of ivy nanoparticles. The second method was developed using the extract from the adventitious roots of English ivy. The nanoparticles formed using both methods were compared to determine the size distribution, morphology, and chemical structure of the nanoparticles. Characterization of the AuNPs was conducted using ultraviolet-visible (UV-Vis) spectroscopy, dynamic light scattering (DLS), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDS). In addition to the structural differences between the AuNPs formed from the different methods, details of the methods in terms of yield, duration, and speed of AuNP formation are also discussed. Further, this paper will show that AuNPs formed using both methods demonstrated efficient uptake in mammalian cells, which provides the potential for biomedical applications. The two methods developed through this research for eco-friendly synthesis of AuNPs present an alternative to traditional chemical synthesis methods.

Evolutionary game theoretic strategy for optimal drug delivery to influence selection pressure in treatment of HIV-1.

Cytotoxic T-lymphocyte (CTL) escape mutation is associated with long-term behaviors of human immunodeficiency virus type 1 (HIV-1). Recent studies indicate heterogeneous behaviors of reversible and conservative mutants while the selection pressure changes. The purpose of this study is to optimize the selection pressure to minimize the long-term virus load. The results can be used to assist in delivery of highly loaded cognate peptide-pulsed dendritic cells (DC) into lymph nodes that could change the selection pressure. This mechanism may be employed for controlled drug delivery. A mathematical model is proposed in this paper to describe the evolutionary dynamics involving viruses and T cells. We formulate the optimization problem into the framework of evolutionary game theory, and solve for the optimal control of the selection pressure as a neighborhood invader strategy. The strategy dynamics can be obtained to evolve the immune system to the best controlled state. The study may shed light on optimal design of HIV-1 therapy based on optimization of adaptive CTL immune response.

Naturally occurring nanoparticles from English ivy: an alternative to metal-based nanoparticles for UV protection.

BACKGROUND: Over the last decade safety concerns have arisen about the use of metal-based nanoparticles in the cosmetics field. Metal-based nanoparticles have been linked to both environmental and animal toxicity in a variety of studies. Perhaps the greatest concern involves the large amounts of TiO2 nanoparticles that are used in commercial sunscreens. As an alternative to using these potentially hazardous metal-based nanoparticles, we have isolated organic nanoparticles from English ivy (Hedera helix). In this study, ivy nanoparticles were evaluated for their potential use in sunscreens based on four criteria: 1) ability to absorb and scatter ultraviolet light, 2) toxicity to mammalian cells, 3) biodegradability, and 4) potential for diffusion through skin. RESULTS: Purified ivy nanoparticles were first tested for their UV protective effects using a standard spectrophotometric assay. Next the cell toxicity of the ivy nanoparticles was compared to TiO2 nanoparticles using HeLa cells. The biodegradability of these nanoparticles was also determined through several digestion techniques. Finally, a mathematical model was developed to determine the potential for ivy nanoparticles to penetrate through human skin. The results indicated that the ivy nanoparticles were more efficient in blocking UV light, less toxic to mammalian cells, easily biodegradable, and had a limited potential to penetrate through human skin. When compared to TiO2 nanoparticles, the ivy nanoparticles showed decreased cell toxicity, and were easily degradable, indicating that they provided a safer alternative to these nanoparticles. CONCLUSIONS: With the data collected from this study, we have demonstrated the great potential of ivy nanoparticles as a sunscreen protective agent, and their increased safety over commonly used metal oxide nanoparticles.

Nanofibers and nanoparticles from the insect-capturing adhesive of the Sundew (Drosera) for cell attachment.

BACKGROUND: The search for naturally occurring nanocomposites with diverse properties for tissue engineering has been a major interest for biomaterial research. In this study, we investigated a nanofiber and nanoparticle based nanocomposite secreted from an insect-capturing plant, the Sundew, for cell attachment. The adhesive nanocomposite has demonstrated high biocompatibility and is ready to be used with minimal preparation. RESULTS: Atomic force microscopy (AFM) conducted on the adhesive from three species of Sundew found that a network of nanofibers and nanoparticles with various sizes existed independent of the coated surface. AFM and light microscopy confirmed that the pattern of nanofibers corresponded to Alcian Blue staining for polysaccharide. Transmission electron microscopy identified a low abundance of nanoparticles in different pattern form AFM observations. In addition, energy-dispersive X-ray spectroscopy revealed the presence of Ca, Mg, and Cl, common components of biological salts. Study of the material properties of the adhesive yielded high viscoelasticity from the liquid adhesive, with reduced elasticity observed in the dried adhesive. The ability of PC12 neuron-like cells to attach and grow on the network of nanofibers created from the dried adhesive demonstrated the potential of this network to be used in tissue engineering, and other biomedical applications. CONCLUSIONS: This discovery demonstrates how a naturally occurring nanofiber and nanoparticle based nanocomposite from the adhesive of Sundew can be used for tissue engineering, and opens the possibility for further examination of natural plant adhesives for biomedical applications.

Tea nanoparticles for immunostimulation and chemo-drug delivery in cancer treatment.

Many health benefits have been associated with tea consumption. In an effort to elucidate the source of these health benefits, numerous phytochemicals have been extracted from tea infusions, some of which have demonstrated promise as clinical therapeutics for cancer therapy. Considering the advantageous properties of organic nanoparticles, the purpose of this study is to develop a method for isolating nanoparticles from tea leaves, and explore potential biomedical applications for these nanoparticles. First, an infusion-dialysis procedure for isolating tea nanoparticles (TNPs) from green tea infusions is developed. Second, atomic force microscopy and scanning electron microscopy reveal that the TNPs are spherical with diameters of 100-300 nm. Third, electrophoretic light scattering is used to determine that the TNPs have a zeta potential of -26.52 mV at pH 7.0. Finally, chemical analysis demonstrates that (-) Epigallocatechin gallate, caffeine, and theobromine are not found in the TNPs. Interestingly, the TNPs do enhance the in vitro secretion of cytokines IL-6, TNF-alpha, and G-CSF, as well as the chemokines RANTES, IP-10, MDC from mouse macrophages RAW264.7, indicating an immunostimulatory effect. As a nanocarrier, the TNPs are able to form complexes with doxorubicin (DOX) and have the potential for applications in drug delivery. Further the DOX-loaded TNPs increase the cellular DOX uptake, compared to free DOX, leading to higher cytotoxicity in the A549 human lung cancer and MCF-7 breast cancer cells. More importantly, the DOX-loaded TNPs significantly increase the DOX uptake and cytotoxicity in MCF-7/ADR multidrug resistant breast cancer cells. In this work, an infusion-dialysis procedure is developed for isolation of the TNPs from green tea, and the potential of these nanoparticles as a multifunctional nanocarrier for cancer therapy in vitro is explored.

Isolation and chemical analysis of nanoparticles from English ivy (Hedera helix L.).

Bio-inspiration for novel adhesive development has drawn increasing interest in recent years with the discovery of the nanoscale morphology of the gecko footpad and mussel adhesive proteins. Similar to these animal systems, it was discovered that English ivy (Hedera helix L.) secretes a high strength adhesive containing uniform nanoparticles. Recent studies have demonstrated that the ivy nanoparticles not only contribute to the high strength of this adhesive, but also have ultraviolet (UV) protective abilities, making them ideal for sunscreen and cosmetic fillers, and may be used as nanocarriers for drug delivery. To make these applications a reality, the chemical nature of the ivy nanoparticles must be elucidated. In the current work, a method was developed to harvest bulk ivy nanoparticles from an adventitious root culture system, and the chemical composition of the nanoparticles was analysed. UV/visible spectroscopy, inductively coupled plasma mass spectrometry, Fourier transform infrared spectroscopy and electrophoresis were used in this study to identify the chemical nature of the ivy nanoparticles. Based on this analysis, we conclude that the ivy nanoparticles are proteinaceous.

Extraction of organic nanoparticles from plants.

Despite the wealth of studies related to the isolation and synthesis of metallic nanoparticles, few studies have been conducted to extract nanoparticles from plants. Nanoparticles isolated from plants have been demonstrated to have unique properties for biomedical and industrial applications. In this chapter, we report a method to extract nanoparticles from plants. The proposed method consists of multiple purification steps, including pre-filtration, dialysis, and size exclusion chromatography.

Evaluation of the nanofibrillar structure of Dioscorea opposite extract for cell attachment.

Dioscorea opposite has been widely used in traditional herbal medicine in the Far East, ameliorating symptoms ranging from abdominal swelling to pain. Previous studies have focused on understanding the chemical components that lead to the medicinal effects of the extract. In this study, we examined the nanostructures formed by the soluble and insoluble parts of the sticky excretion from the mucilaginous rhizome of Dioscorea opposite and evaluated their cellular response. Using atomic force microscopy, we found that the soluble extract of the excretion had the capacity to form a nanofibrillar scaffold composed of uniform ∼10 nm nanofibers with a typical pore size of ∼40 nm, while the insoluble extract formed some nanofibers without specific structure. Cellular response to the two types of nanostructures was tested by seeding with HeLa and MC3T3 cells. The observations suggested that the nanofibrillar scaffold formed from the soluble extract provided an excellent platform for HeLa cell attachment and growth and to a lesser degree for MC3T3 cells, while nanofibers from the insoluble extract displayed no cell attachment and growth. Further analysis by direct incubation of the soluble extract with growing cells indicated that components from the extract preferentially bound to HeLa cells, but not to MC3T3 cells, which might help explain the observed preference of HeLa cells on the nanofibrillar scaffold. The nanofibrillar scaffold created from the Dioscorea opposite extract and its ability to sustain the attachment of specific cell types demonstrate the potential for this natural nanomaterial in tissue engineering applications.

Ultraviolet Extinction and Visible Transparency by Ivy Nanoparticles.

Though much research has been conducted for nanoparticles, naturally occurring nanoparticles have not yet been well explored for their diverse properties and potential applications. This paper reports the optical absorption and scattering properties of nanoparticles secreted by English ivy. Both experimental and theoretical studies have been conducted. Strong ultraviolet extinction and excellent visible transparency are observed, compared to the inorganic TiO(2) and ZnO nanoparticles at similar concentrations. The contributions of absorption and scattering to the total extinction are quantified by simulation of the Mie scattering theory.

A mathematical model on the closing and opening mechanism for venus flytrap.

This paper investigates the opening and closing mechanism for the Venus Flytrap (Dionaea muscipula). A mathematical model has been proposed to explain how the flytrap transitions between open, semi-closed, and closed states. The model accounts for the charge accumulation of action potentials, which generated by mechanical stimulation of the sensitive trigger hairs on the lobes of the flytrap. Though many studies have been reported for the Venus flytrap opening and closing mechanism, this paper attempts to explain the mechanism from nonlinear dynamics and control perspective.

Immunodominance analysis through interactions of CD8+ T cells and DCs in lymph nodes.

Immunodominance is a common phenomenon observed in multiple epitopes immune systems. Previous studies hypothesize that the competition among CD8+ T cell responses against different epitopes can be used to explain immunodominance. This paper proposes a mathematical model that describes the dynamics of CD8+ T cells primed by antigen-presenting dendritic cells (DCs) in the lymph nodes, and shows that the overall avidity of the interactions between peptide-specific T cells and cognate antigen-bearing DCs may determine the immunodominance. The model suggests the probability that a peptide-specific T cell be immunodominant is proportional to (1) the cognate T cell receptor (TCR) affinity, (2) the number of complexes of cognate peptide and major histocompatibility complex (pMHC) per DC, and (3) the half-life of cognate peptide-specific pMHC. The model predicts a threshold density of pMHC complexes for T cell activation. These observations from the mathematical model are consistent with experimental studies in the open literature. For DC-based vaccine design, the model suggests a strategy of immunotherapy based on the injection of cognate antigen-pulsed DCs.

Identification of nanofibers in the Chinese herbal medicine: Yunnan Baiyao.

Yunnan Baiyao is a traditional Chinese herbal medicine that has been used to treat wounds for over 100 years. Here, we use Atomic Force Microscopy (AFM) to determine nano-scale structures of the Yunnan Baiyao. AFM images revealed uniform nanofibers present in relatively high abundance in a solution of this medicine. Fibers were typically 25.1 nm in diameter and ranged in length from 86-726 nm due to processing. Due to the unique adhesive and structural properties of nanofibers, we concluded that these fibers may play a role in platelet aggregation, leading to clotting, and the sealing of wounds.