Metabolic Changes Associated With Cardiomyocyte Dedifferentiation Enable Adult Mammalian Cardiac Regeneration.

BACKGROUND: Cardiac regeneration after injury is limited by the low proliferative capacity of adult mammalian cardiomyocytes (CMs). However, certain animals readily regenerate lost myocardium through a process involving dedifferentiation, which unlocks their proliferative capacities. METHODS: We bred mice with inducible, CM-specific expression of the Yamanaka factors, enabling adult CM reprogramming and dedifferentiation in vivo. RESULTS: Two days after induction, adult CMs presented a dedifferentiated phenotype and increased proliferation in vivo. Microarray analysis revealed that upregulation of ketogenesis was central to this process. Adeno-associated virus-driven HMGCS2 overexpression induced ketogenesis in adult CMs and recapitulated CM dedifferentiation and proliferation observed during partial reprogramming. This same phenomenon was found to occur after myocardial infarction, specifically in the border zone tissue, and HMGCS2 knockout mice showed impaired cardiac function and response to injury. Finally, we showed that exogenous HMGCS2 rescues cardiac function after ischemic injury. CONCLUSIONS: Our data demonstrate the importance of HMGCS2-induced ketogenesis as a means to regulate metabolic response to CM injury, thus allowing cell dedifferentiation and proliferation as a regenerative response.

Signaling pathway of globo-series glycosphingolipids and ß1,3-galactosyltransferase V (ß3GalT5) in breast cancer.

The globo-series glycosphingolipids (GSLs) SSEA3, SSEA4, and Globo-H specifically expressed on cancer cells are found to correlate with tumor progression and metastasis, but the functional roles of these GSLs and the key enzyme ß1,3-galactosyltransferase V (ß3GalT5) that converts Gb4 to SSEA3 remain largely unclear. Here we show that the expression of ß3GalT5 significantly correlates with tumor progression and poor survival in patients, and the globo-series GSLs in breast cancer cells form a complex in membrane lipid raft with caveolin-1 (CAV1) and focal adhesion kinase (FAK) which then interact with AKT and receptor-interacting protein kinase (RIP), respectively. Knockdown of ß3GalT5 disrupts the complex and induces apoptosis through dissociation of RIP from the complex to interact with the Fas death domain (FADD) and trigger the Fas-dependent pathway. This finding provides a link between SSEA3/SSEA4/Globo-H and the FAK/CAV1/AKT/RIP complex in tumor progression and apoptosis and suggests a direction for the treatment of breast cancer, as demonstrated by the combined use of antibodies against Globo-H and SSEA4.

Loss of Gut Microbiota Alters Immune System Composition and Cripples Postinfarction Cardiac Repair.

BACKGROUND: The impact of gut microbiota on the regulation of host physiology has recently garnered considerable attention, particularly in key areas such as the immune system and metabolism. These areas are also crucial for the pathophysiology of and repair after myocardial infarction (MI). However, the role of the gut microbiota in the context of MI remains to be fully elucidated. METHODS: To investigate the effects of gut microbiota on cardiac repair after MI, C57BL/6J mice were treated with antibiotics 7 days before MI to deplete mouse gut microbiota. Flow cytometry was applied to examine the changes in immune cell composition in the heart. 16S rDNA sequencing was conducted as a readout for changes in gut microbial composition. Short-chain fatty acid (SCFA) species altered after antibiotic treatment were identified by high-performance liquid chromatography. Fecal reconstitution, transplantation of monocytes, or dietary SCFA or Lactobacillus probiotic supplementation was conducted to evaluate the cardioprotective effects of microbiota on the mice after MI. RESULTS: Antibiotic-treated mice displayed drastic, dose-dependent mortality after MI. We observed an association between the gut microbiota depletion and significant reductions in the proportion of myeloid cells and SCFAs, more specifically acetate, butyrate, and propionate. Infiltration of CX3CR1+ monocytes to the peri-infarct zone after MI was also reduced, suggesting impairment of repair after MI. Accordingly, the physiological status and survival of mice were significantly improved after fecal reconstitution, transplantation of monocytes, or dietary SCFA supplementation. MI was associated with a reorganization of the gut microbial community such as a reduction in Lactobacillus. Supplementing antibiotic-treated mice with a Lactobacillus probiotic before MI restored myeloid cell proportions, yielded cardioprotective effects, and shifted the balance of SCFAs toward propionate. CONCLUSIONS: Gut microbiota-derived SCFAs play an important role in maintaining host immune composition and repair capacity after MI. This suggests that manipulation of these elements may provide opportunities to modulate pathological outcome after MI and indeed human health and disease as a whole.

Real-time in vivo imaging of subpopulations of circulating tumor cells using antibody conjugated quantum dots.

INTRODUCTION: The detection of circulating tumor cells (CTCs) is very important for cancer diagnosis. CTCs can travel from primary tumors through the circulation to form secondary tumor colonies via bloodstream extravasation. The number of CTCs has been used as an indicator of cancer progress. However, the population of CTCs is very heterogeneous. It is very challenging to identify CTC subpopulations such as cancer stem cells (CSCs) with high metastatic potential, which are very important for cancer diagnostic management. RESULTS: We report a study of real-time CTC and CSC imaging in the bloodstreams of living animals using multi-photon microscopy and antibody conjugated quantum dots. We have developed a cancer model for noninvasive imaging wherein pancreatic cancer cells expressing fluorescent proteins were subcutaneously injected into the earlobes of mice and then formed solid tumors. When the cancer cells broke away from the solid tumor, CTCs with fluorescent proteins in the bloodstream at different stages of development could be monitored noninvasively in real time. The number of CTCs observed in the blood vessels could be correlated to the tumor size in the first month and reached a maximum value of approximately 100 CTCs/min after 5 weeks of tumor inoculation. To observe CTC subpopulations, conjugated quantum dots were used. It was found that cluster of differentiation (CD)24+ CTCs can move along the blood vessel walls and migrate to peripheral tissues. CD24+ cell accumulation on the solid tumors' sides was observed, which may provide valuable insight for designing new drugs to target cancer subpopulations with high metastatic potential. We also demonstrated that our system is capable of imaging a minor population of cancer stem cells, CD133+ CTCs, which are found in 0.7% of pancreatic cancer cells and 1%-3% of solid tumors in patients. CONCLUSIONS: With the help of quantum dots, CTCs with higher metastatic potential, such as CD24+ and CD133+ CTCs, have been identified in living animals. Using our approach, it may be possible to investigate detailed metastatic mechanism such as tumor cell extravasation to the blood vessels. In addition, the number of observed CTCs in the blood stream could be correlated with tumor stage in the early stage of cancer.

Random and aligned electrospun PLGA nanofibers embedded in microfluidic chips for cancer cell isolation and integration with air foam technology for cell release.

BACKGROUND: Circulating tumor cells (CTCs) comprise the high metastatic potential population of cancer cells in the blood circulation of humans; they have become the established biomarkers for cancer diagnosis, individualized cancer therapy, and cancer development. Technologies for the isolation and recovery of CTCs can be powerful cancer diagnostic tools for liquid biopsies, allowing the identification of malignancies and guiding cancer treatments for precision medicine. METHODS: We have used an electrospinning process to prepare poly(lactic-co-glycolic acid) (PLGA) nanofibrous arrays in random or aligned orientations on glass slips. We then fabricated poly(methyl methacrylate) (PMMA)-based microfluidic chips embedding the PLGA nanofiber arrays and modified their surfaces through sequential coating with using biotin-(PEG)7-amine through EDC/NHS activation, streptavidin (SA), and biotinylated epithelial-cell adhesion-molecule antibody (biotin-anti-EpCAM) to achieve highly efficient CTC capture. When combined with an air foam technology that induced a high shear stress and, thereby, nondestructive release of the captured cells from the PLGA surfaces, the proposed device system operated with a high cell recovery rate. RESULTS: The morphologies and average diameters of the electrospun PLGA nanofibers were characterized using scanning electron microscopy (SEM) and confocal Raman imaging. The surface chemistry of the PLGA nanofibers conjugated with the biotin-(PEG)7-amine was confirmed through time-of-flight secondary ion mass spectrometry (ToF-SIMS) imaging. The chip system was studied for the effects of the surface modification density of biotin-(PEG)7-amine, the flow rates, and the diameters of the PLGA nanofibers on the capture efficiency of EpCAM-positive HCT116 cells from the spiked liquid samples. To assess their CTC capture efficiencies in whole blood samples, the aligned and random PLGA nanofiber arrays were tested for their abilities to capture HCT116 cells, providing cancer cell capture efficiencies of 66 and 80%, respectively. With the continuous injection of air foam into the microfluidic devices, the cell release efficiency on the aligned PLGA fibers was 74% (recovery rate: 49%), while it was 90% (recovery rate: 73%) on the random PLGA fibers, from tests of 200 spiked cells in 2 mL of whole blood from healthy individuals. Our study suggests that integrated PMMA microfluidic chips embedding random PLGA nanofiber arrays may be suitable devices for the efficient capture and recovery of CTCs from whole blood samples.

Correlational Study of Nonalcoholic Fatty Liver Disease Diagnosed by Ultrasonography with Lipid Profile and Body Mass Index in Adult Nepalese Population.

OBJECTIVE: The purpose of this study was to categorize patients into different grades of nonalcoholic fatty liver disease (NAFLD) by ultrasonography and to compare the findings with their serum lipid profile. MATERIALS AND METHODS: Descriptive, cross-sectional study design was used. One hundred and nine patients without a history of alcohol consumption of age more than 16 years attending general health checkup were selected at Tribhuvan University Teaching Hospital, Maharajganj, Kathmandu, as per the exclusion and inclusion criteria. Ultrasound scanning of the patients was done and their liver size, as well as grading of fatty liver, was done. Data were collected in predesigned pro forma and were analyzed using Statistical Package for the Social Sciences (SPSS) 16.0, IBM (SPSS Inc., Chicago, IL). RESULTS: In this study, the mean age of fatty liver in males was found to be 44.3 years and in females was found to be 51.9 years. 22.9% of patients with NAFLD had increased liver size. Significant association with increasing grades of fatty liver was found with increasing levels of cholesterol (P = 0.028), low-density lipoprotein (LDL) (P = 0.017), liver size (P = 0.001), and body mass index (BMI) (P = 0.045) in patients diagnosed with NAFLD. No significant association with increasing grades of fatty liver was found with increasing levels of triglyceride (P = 0.32) and high-density lipoprotein (P = 0.25). CONCLUSION: Ultrasound is a safe and first-line modality for the evaluation of fatty liver and its grading. Increasing grades of fatty liver had significant association with increasing levels of cholesterol LDL, increasing liver size, and BMI of patients.

Antagonizing SET Augments the Effects of Radiation Therapy in Hepatocellular Carcinoma through Reactivation of PP2A-Mediated Akt Downregulation.

Increasing evidence suggests that SET functions as an oncoprotein and promotes cancer survival and therapeutic resistance. However, whether SET affects radiation therapy (RT)-mediated anticancer effects has not yet been explored. We investigated the impact of SET on RT sensitivity in hepatocellular carcinoma (HCC). Using colony and hepatosphere formation assays, we found that RT-induced proliferative inhibition was critically associated with SET expression. We next tested a novel SET antagonist, N4-(3-ethynylphenyl)-6,7-dimethoxy-N2-(4-phenoxyphenyl) quinazoline-2,4-diamine (EMQA), in combination with RT. We showed that additive use of EMQA significantly enhanced the effects of RT against HCC in vitro and in vivo. Notably, compared with mice receiving either RT or EMQA alone, the growth of PLC5 xenografted tumor in mice receiving RT plus EMQA was significantly reduced without compromising treatment tolerability. Furthermore, we proved that antagonizing SET to restore protein phosphatase 2A-mediated phospho-Akt (p-AKT) downregulation was responsible for the synergism between EMQA and RT. Our data demonstrate a new oncogenic property of SET and provide preclinical evidence that combining a SET antagonist and RT may be effective for treatment of HCC. Further investigation is warranted to validate the clinical relevance of this approach.

Flexible nanopillars to regulate cell adhesion and movement.

Flexible polymer nanopillar substrates were used to systematically demonstrate cell alignment and migration guided by the directional formation of focal adhesions. The polymer nanopillar substrates were constructed to various height specifications to provide an extensive variation of flexibility; a rectangular arrangement created spatial confinement between adjacent nanopillars, providing less spacing in the horizontal and vertical directions. Three polymer nanopillar substrates with the diameter of 400 nm and the heights of 400, 800, and 1200 nm were fabricated. Super-resolution localization imaging and protein pair-distance analysis of vinculin proteins revealed that Chinese hamster ovary (CHO) cells formed mature focal adhesions on 1200 nm high nanopillar substrates by bending adjacent nanopillars to link dot-like adhesions. The spacing confinement of the adjacent nanopillars enhanced the orthogonal directionality of the formation tendency of the mature focal adhesions. The directional formation of the mature focal adhesions also facilitated the organization of actin filaments in the horizontal and vertical directions. Moreover, 78% of the CHO cells were aligned in these two directions, in conformity with the flexibility and nanotopographical cues of the nanopillars. Biased cell migration was observed on the 1200 nm high nanopillar substrates.

Detection of residual rifampicin in urine via fluorescence quenching of gold nanoclusters on paper.

BACKGROUND: Rifampicin or rifampin (R) is a common drug used to treat inactive meningitis, cholestatic pruritus and tuberculosis (TB), and it is generally prescribed for long-term administration under regulated dosages. Constant monitoring of rifampicin is important for controlling the side effects and preventing overdose caused by chronic medication. In this study, we present an easy to use, effective and less costly method for detecting residual rifampicin in urine samples using protein (bovine serum albumin, BSA)-stabilized gold nanoclusters (BSA-Au NCs) adsorbed on a paper substrate in which the concentration of rifampicin in urine can be detected via fluorescence quenching. The intensity of the colorimetric assay performed on the paper-based platforms can be easily captured using a digital camera and subsequently analyzed. RESULTS: The decreased fluorescence intensity of BSA-Au NCs in the presence of rifampicin allows for the sensitive detection of rifampicin in a range from 0.5 to 823 µg/mL. The detection limit for rifampicin was measured as 70 ng/mL. The BSA-Au NCs were immobilized on a wax-printed paper-based platform and used to conduct real-time monitoring of rifampicin in urine. CONCLUSION: We have developed a robust, cost-effective, and portable point-of-care medical diagnostic platform for the detection of rifampicin in urine based on the ability of rifampicin to quench the fluorescence of immobilized BSA-Au NCs on wax-printed papers. The paper-based assay can be further used for the detection of other specific analytes via surface modification of the BSA in BSA-Au NCs and offers a useful tool for monitoring other diseases.

Electrically tunable organic bioelectronics for spatial and temporal manipulation of neuron-like pheochromocytoma (PC-12) cells.

BACKGROUND: Organic bioelectronic devices consisting of alternating poly(3,4-ethylenedioxythiophene) (PEDOT) and reduced graphite oxide (rGO) striped microelectrode arrays were fabricated by lithography technology. It has been demonstrated that the organic bioelectronic devices can be used to spatially and temporally manipulate the location and proliferation of the neuron-like pheochromocytoma cells (PC-12 cells). METHODS: By coating an electrically labile contact repulsion layer of poly(l-lysine-graft-ethylene glycol) (PLL-g-PEG) on the PEDOT electrode, the location and polarity of the PC-12 cells were confined to the rGO electrodes. RESULTS: The outgrowth of spatially confined bipolar neurites was found to align along the direction of the 20µm wide electrode. The location of the PC-12 cells can also be manipulated temporally by applying electrical stimulation during the neurite differentiation of PC-12 cells, allowing the PC-12 cells to cross over the boundary between the PEDOT and the rGO regions and construct neurite networks in an unconfined manner where the contact repulsive coating of PLL-g-PEG was removed. CONCLUSIONS: This adsorption and desorption of the PLL-g-PEG without and with electrical stimulation can be attributed to the tunable surface properties of the PEDOT microelectrodes, whose surface charge can switch from being negative to positive under electrical stimulation. GENERAL SIGNIFICANCE: The electrically tunable organic bioelectronics reported here could potentially be applied to tissue engineering related to the development and regeneration of mammalian nervous systems. The spatial and temporal control in this device would also be used to study the synapse junctions of neuron-neuron contacts in both time and space domains. This article is part of a Special Issue entitled Organic Bioelectronics - Novel Applications in Biomedicine.

3D bioelectronic interface: capturing circulating tumor cells onto conducting polymer-based micro/nanorod arrays with chemical and topographical control.

The three-dimensional (3D) poly(3,4-ethylenedioxythiophene) (PEDOT)-based bioelectronic interfaces (BEIs) with diverse dimensional micro/nanorod array structures, varied surface chemical pro-perties, high electrical conductivity, reversible chemical redox switching, and high optical transparency are used for capturing circulating tumor cells (CTCs). Such 3D PEDOT-based BEIs can function as an efficient clinical diagonstic and therapeutic platform.

Investigation of size-dependent cell adhesion on nanostructured interfaces.

BACKGROUND: Cells explore the surfaces of materials through membrane-bound receptors, such as the integrins, and use them to interact with extracellular matrix molecules adsorbed on the substrate surfaces, resulting in the formation of focal adhesions. With recent advances in nanotechnology, biosensors and bioelectronics are being fabricated with ever decreasing feature sizes. The performances of these devices depend on how cells interact with nanostructures on the device surfaces. However, the behavior of cells on nanostructures is not yet fully understood. Here we present a systematic study of cell-nanostructure interaction using polymeric nanopillars with various diameters. RESULTS: We first checked the viability of cells grown on nanopillars with diameters ranging from 200 nm to 700 nm. It was observed that when cells were cultured on the nanopillars, the apoptosis rate slightly increased as the size of the nanopillar decreased. We then calculated the average size of the focal adhesions and the cell-spreading area for focal adhesions using confocal microscopy. The size of focal adhesions formed on the nanopillars was found to decrease as the size of the nanopillars decreased, resembling the formations of nascent focal complexes. However, when the size of nanopillars decreased to 200 nm, the size of the focal adhesions increased. Further study revealed that cells interacted very strongly with the nanopillars with a diameter of 200 nm and exerted sufficient forces to bend the nanopillars together, resulting in the formation of larger focal adhesions. CONCLUSIONS: We have developed a simple approach to systematically study cell-substrate interactions on physically well-defined substrates using size-tunable polymeric nanopillars. From this study, we conclude that cells can survive on nanostructures with a slight increase in apoptosis rate and that cells interact very strongly with smaller nanostructures. In contrast to previous observations on flat substrates that cells interacted weakly with softer substrates, we observed strong cell-substrate interactions on the softer nanopillars with smaller diameters. Our results indicate that in addition to substrate rigidity, nanostructure dimensions are additional important physical parameters that can be used to regulate behaviour of cells.

Synthesis of tunable and multifunctional Ni-doped near-infrared QDs for cancer cell targeting and cellular sorting.

Here, we report the facile preparation of tunable magnetic Ni-doped near-infrared (NIR) quantum dots (MNIR-QDs) as an efficient probe for targeting, imaging, and cellular sorting applications. We synthesized the MNIR-QDs via a hot colloidal synthesis approach to yield monodisperse and tunable QDs. These hydrophobic QDs were structurally and compositionally characterized and further functionalized with amino-PEG and carboxyl-PEG to improve their biocompatibility. Since QDs are known to be toxic due to the presence of cadmium, we have evaluated the in vitro and in vivo toxicity of our surface-functionalized MNIR-QDs. Our results revealed that surface-functionalized MNIR-QDs did not exhibit significant toxicity at the concentrations used in the experiments and are therefore suitable for biological applications. For further in vitro applications, we covalently linked folic acid to the surface of amino-PEG-coated MNIR-QDs through NHS chemistry to target the folate receptors largely present in the HeLa cells to demonstrate the specific targeting and magnetic behavior of these MNIR-QDs. Improved specificity has been observed with treatment of HeLa cells with the folic acid-linked amino PEG-coated MNIR QDs (FA-PEG-MNIR-QDs) compared to the one without folic acid. Since the synthesized probe has magnetic property, we have also successfully demonstrated sorting between the cells which have taken up the probe with the use of a magnet. Our findings strongly suggest that these functionalized MNIR-QDs can be a potential probe for targeting, cellular sorting, and bioimaging applications.

Development of chitosan oligosaccharide-modified gold nanorods for in vivo targeted delivery and noninvasive imaging by NIR irradiation.

In the present study, we demonstrate the synthesis and applications of multifunctional gold nanorod-based probes for specific targeting and noninvasive imaging based on localized heating generated by gold nanorods after NIR irradiation. The structural design of the probe consists of MUA (11-mercaptoundecanoic acid)-capped gold nanorods covalently linked with low-molecular-weight chitosan oligosaccharide (M(w) ~5000) via carbodiimide (EDC) coupling agent. This surface modification is performed for complete replacement of toxic CTAB (hexadecyltrimethyl-ammonium chloride) and acid-responsive delivery of gold nanorods in acidic environment as known to be present at tumor surrounding areas. The resulting chitosan oligosaccharide-modified gold nanorods (CO-GNRs) were further conjugated with tumor targeting monoclonal antibody against EGFR (epidermal growth factor receptor) to provide localized targeting functionality owing to the overexpression of EGFR in human oral adenosquamous carcinoma cell line CAL 27. Initial in vitro and in vivo toxicity assessments indicated that CO-GNRs did not induce any significant toxicity and are thus suitable for biological applications. Furthermore, selective targeting and accumulation of CO-GNRs were observed in vitro via two-photon luminescence imaging studies in CAL 27, which was also observed through in vivo targeting studies performed via NIR (near-infrared) laser irradiation in CAL 27 xenografts of BALB/c nude mice. Hence, the CO-GNRs that we have developed are biocompatible and nontoxic and can be a potential candidate for in vivo targeted delivery, noninvasive imaging based on localized hyperthermia, and photothermal-related therapies.

Revealing the nanometric structural changes in myocardial infarction models by time-lapse intravital imaging.

In the development of bioinspired nanomaterials for therapeutic applications, it is very important to validate the design of nanomaterials in the disease models. Therefore, it is desirable to visualize the change of the cells in the diseased site at the nanoscale. Heart diseases often start with structural, morphological, and functional alterations of cardiomyocyte components at the subcellular level. Here, we developed straightforward technique for long-term real-time intravital imaging of contracting hearts without the need of cardiac pacing and complex post processing images to understand the subcellular structural and dynamic changes in the myocardial infarction model. A two-photon microscope synchronized with electrocardiogram signals was used for long-term in vivo imaging of a contracting heart with subcellular resolution. We found that the structural and dynamic behaviors of organelles in cardiomyocytes closely correlated with heart function. In the myocardial infarction model, sarcomere shortening decreased from ∼15% (healthy) to ∼8% (diseased) as a result of impaired cardiac function, whereas the distances between sarcomeres increased by 100 nm (from 2.11 to 2.21 µm) in the diastolic state. In addition, T-tubule system regularity analysis revealed that T-tubule structures that were initially highly organized underwent significant remodeling. Morphological remodeling and changes in dynamic activity at the subcellular level are essential to maintain heart function after infarction in a heart disease model.

Design principles of bioinspired interfaces for biomedical applications in therapeutics and imaging.

In the past two decades, we have witnessed rapid developments in nanotechnology, especially in biomedical applications such as drug delivery, biosensing, and bioimaging. The most commonly used nanomaterials in biomedical applications are nanoparticles, which serve as carriers for various therapeutic and contrast reagents. Since nanomaterials are in direct contact with biological samples, biocompatibility is one of the most important issues for the fabrication and synthesis of nanomaterials for biomedical applications. To achieve specific recognition of biomolecules for targeted delivery and biomolecular sensing, it is common practice to engineer the surfaces of nanomaterials with recognition moieties. This mini-review summarizes different approaches for engineering the interfaces of nanomaterials to improve their biocompatibility and specific recognition properties. We also focus on design strategies that mimic biological systems such as cell membranes of red blood cells, leukocytes, platelets, cancer cells, and bacteria.

Exploring the formation of focal adhesions on patterned surfaces using super-resolution imaging.

The formation of focal adhesions on various sizes of fibronectin patterns, ranging from 200 µm to 250 nm, was systematically investigated by total internal reflection fluorescence microscopy and super-resolution imaging. It was found that cells adhered to and spread on these micro/nanopatterns, forming focal adhesions. On a micrometer scale the shape of the focal adhesions was elongated. However, on the nanometer scale, the shape of focal adhesions became dotlike. To further explore the distribution of focal adhesion proteins formed on surfaces, a localization-based super-resolution imaging technique was employed in order to determine the position and density of vinculin proteins. A characteristic distance of 50 nm was found between vinculin molecules in the focal adhesions, which did not depend on the size of the fibronectin nanopatterns. This distance was found to be crucial for the formation of focal adhesions. In addition, the density of vinculin at the focal adhesions formed on the nanopatterns increased as the pattern size decreased. The density of the protein was found to be 425 ± 247, 584 ± 302, and 703 ± 305 proteins µm(-2) on the 600, 400, and 250 nm fibronectin patterns respectively. Whereas 226 ± 77 proteins µm(-2) was measured for the matured focal adhesions on homogeneous fibronectin coated substrates. The increase in vinculin density implies that an increase in mechanical load was applied to the focal adhesions formed on the smaller nanopatterns.

Targeted nuclear delivery using peptide-coated quantum dots.

Core/shell quantum dots (CdSe/Zns) conjugated with various nuclear localization signaling (NLS) peptides, which could facilitate the transportation of quantum dots across the plasma membrane into the nucleus, have been utilized to investigate the uptake mechanism of targeted delivery. Because of their brightness and photostability, it was possible to trace the trajectories of individual quantum dots in living cells using both confocal and total internal reflection microscopes. We found that, when the quantum dots were added to a cell culture, the peptide-coated quantum dots entered the cell nucleus while the uncoated quantum dots remained in the cytoplasm. At 8 nM, most of the peptide coated quantum dots were found in the cytoplasm due to aggregation. However, at a lower concentration (0.08 nM), approximately 25% of the NLS peptide-coated quantum dots entered the cell nucleus. We also found that some quantum dots without NLS coating could also enter the nucleus, suggesting that the size of the quantum dots may play an important role in such a process.

Development of lipid targeting Raman probes for in vivo imaging of Caenorhabditis elegans.

A simple, sensitive, and highly specific lipid targeting Raman probe (Nile red coated silver nanoparticles) has been developed to image living nematode Caenorhabditis elegans (C. elegans). Our idea of imaging lipids in C. elegans is to combine the specificity of the fluorescent dye, Nile red, and the highly enhanced Raman scattering on the silver nanoparticles. Our strategy involves the fabrication of a lipid targeting probe, which is incorporated into the intracellular intestinal granules of C. elegans by incubating these worms in the solution containing Raman probes, resulting in an uptake and subsequent incorporation of these Raman probes into the intestinal granule, thus allowing fast visualization of lipid droplets through a conventional confocal imaging technique.

Localization imaging using blinking quantum dots.

The blinking phenomena of the quantum dots have been utilized in the super-resolution localization microscopy to map out the locations of individual quantum dots on a total internal reflection microscope. Our result indicated that the reconstructed image of quantum dots agreed with the topographic image measured by atomic force microscopy. Because of the superior optical properties of the quantum dots, the high localization resolution can be achieved in the shorter acquisition time with larger detected photon numbers. When the cells were labeled with quantum dots, the sub-cellular structures could be clearly seen in the reconstructed images taken by a commercial microscope without using complicated optical systems, special photo-switchable dye pairs or photo-activated fluorescence proteins.