In vitro inflammation and toxicity assessment of pre- and post-incinerated organomodified nanoclays to macrophages using high-throughput screening approaches.

BACKGROUND: Organomodified nanoclays (ONC), two-dimensional montmorillonite with organic coatings, are increasingly used to improve nanocomposite properties. However, little is known about pulmonary health risks along the nanoclay life cycle even with increased evidence of airborne particulate exposures in occupational environments. Recently, oropharyngeal aspiration exposure to pre- and post-incinerated ONC in mice caused low grade, persistent lung inflammation with a pro-fibrotic signaling response with unknown mode(s) of action. We hypothesized that the organic coating presence and incineration status of nanoclays determine the inflammatory cytokine secretary profile and cytotoxic response of macrophages. To test this hypothesis differentiated human macrophages (THP-1) were acutely exposed (0-20 µg/cm2) to pristine, uncoated nanoclay (CloisNa), an ONC (Clois30B), their incinerated byproducts (I-CloisNa and I-Clois30B), and crystalline silica (CS) followed by cytotoxicity and inflammatory endpoints. Macrophages were co-exposed to lipopolysaccharide (LPS) or LPS-free medium to assess the role of priming the NF-κB pathway in macrophage response to nanoclay treatment. Data were compared to inflammatory responses in male C57Bl/6J mice following 30 and 300 µg/mouse aspiration exposure to the same particles. RESULTS: In LPS-free media, CloisNa exposure caused mitochondrial depolarization while Clois30B exposure caused reduced macrophage viability, greater cytotoxicity, and significant damage-associated molecular patterns (IL-1α and ATP) release compared to CloisNa and unexposed controls. LPS priming with low CloisNa doses caused elevated cathepsin B/Caspage-1/IL-1ß release while higher doses resulted in apoptosis. Clois30B exposure caused dose-dependent THP-1 cell pyroptosis evidenced by Cathepsin B and IL-1ß release and Gasdermin D cleavage. Incineration ablated the cytotoxic and inflammatory effects of Clois30B while I-CloisNa still retained some mild inflammatory potential. Comparative analyses suggested that in vitro macrophage cell viability, inflammasome endpoints, and pro-inflammatory cytokine profiles significantly correlated to mouse bronchioalveolar lavage inflammation metrics including inflammatory cell recruitment. CONCLUSIONS: Presence of organic coating and incineration status influenced inflammatory and cytotoxic responses following exposure to human macrophages. Clois30B, with a quaternary ammonium tallow coating, induced a robust cell membrane damage and pyroptosis effect which was eliminated after incineration. Conversely, incinerated nanoclay exposure primarily caused elevated inflammatory cytokine release from THP-1 cells. Collectively, pre-incinerated nanoclay displayed interaction with macrophage membrane components (molecular initiating event), increased pro-inflammatory mediators, and increased inflammatory cell recruitment (two key events) in the lung fibrosis adverse outcome pathway.

Nanobiosensing with graphene and carbon quantum dots: Recent advances.

Graphene and carbon quantum dots (GQDs and CQDs) are relatively new nanomaterials that have demonstrated impact in multiple different fields thanks to their unique quantum properties and excellent biocompatibility. Biosensing, analyte detection and monitoring wherein a key feature is coupled molecular recognition and signal transduction, is one such field that is being greatly advanced by the use of GQDs and CQDs. In this review, recent progress on the development of biotransducers and biosensors enabled by the creative use of GQDs and CQDs is reviewed, with special emphasis on how these materials specifically interface with biomolecules to improve overall analyte detection. This review also introduces nano-enabled biotransducers and different biosensing configurations and strategies, as well as highlights key properties of GQDs and CQDs that are pertinent to functional biotransducer design. Following relevant introductory material, the literature is surveyed with emphasis on work performed over the last 5 years. General comments and suggestions to advance the direction and potential of the field are included throughout the review. The strategic purpose is to inspire and guide future investigations into biosensor design for quality and safety, as well as serve as a primer for developing GQD- and CQD-based biosensors.

Predicting Nanotube Fibrogenicity through Stem Cell-Mediated Fibroblast Focus and Spheroid Formation.

Fibroblast stem cells or stemlike cells (FSCs) are proposed to play a pivotal role in extracellular matrix (ECM) regeneration by serving as a key source of ECM-producing fibroblasts. We developed a mechanism-based in vitro model for fibrogenicity testing of nanomaterials based on their ability to induce FSCs. Using a FSC-enriched fibroblast focus model to mimic in vivo fibrogenic response, we demonstrated a dose-dependent increase in fibroblast focus formation and collagen production by primary lung fibroblasts treated with multiwalled carbon nanotubes (MWCNTs). The focus-forming cells exhibited stem properties as indicated by stem cell markers expression, sphere formation, and ALDH activity assays. Inhibition of ALDH activity diminished the focus and sphere formation as well as collagen production. In vivo animal studies supported the in vitro findings and indicated the potential utility of FSC-based assays as a rapid screening tool for fibrogenicity testing of nanomaterials. This study also unveils a novel mechanism of nanotube-induced fibrogenesis through ALDH-dependent FSC activation.

Mesothelin promotes epithelial-to-mesenchymal transition and tumorigenicity of human lung cancer and mesothelioma cells.

BACKGROUND: Lung cancer and pleural mesothelioma are two of the most deadly forms of cancer. The prognosis of lung cancer and mesothelioma is extremely poor due to limited treatment modalities and lack of understanding of the disease mechanisms. We have identified mesothelin as a potentially unique therapeutic target that as a specific advantage appears nonessential in most cell types. Mesothelin (MSLN), a plasma membrane differentiation antigen, is expressed at a high level in many human solid tumors, including 70% of lung cancer and nearly all mesotheliomas. However, the role of MSLN in the disease process and underlying mechanisms is largely unknown. METHODS: ShRNA knockdown and overexpression of MSLN were performed in human cancer cell lines and corresponding normal cells, respectively. Tumorigenic and metastatic effects of MSLN were examined by tumor sphere formation, migration, and invasion assays in vitro, as well as xenograft tumor assay in vivo. EMT and CSCs were detected by qPCR array, immunoblotting and flow cytometry. RESULTS: MSLN plays a key role in controlling epithelial-to-mesenchymal transition (EMT) and stem properties of human lung cancer and mesothelioma cells that control their tumorigenicity and metastatic potential. Firstly, MSLN was found to be highly upregulated in non-small cell lung cancer (NSCLC) patient tissues and in lung carcinoma and mesothelioma cell lines. Secondly, genetic knockdown of MSLN significantly reduced anchorage-independent cell growth, tumor sphere formation, cell adhesion, migration and invasion in vitro, as well as tumor formation and metastasis in vivo. Thirdly, ectopic overexpression of MSLN induced the malignant phenotype of non-cancerous cells, supporting its role as an oncogene. Finally, mechanistic studies revealed that knockdown of MSLN reversed EMT and attenuated stem cell properties, in addition to inhibiting tumor growth and metastasis. CONCLUSIONS: These results indicate an essential role of MSLN in controlling EMT and stem cell properties of human lung cancer and mesothelioma cells. Since EMT is an important process in tumor progression and metastasis, and MSLN is nonessential in most normal tissue, our findings on MSLN may provide new insights into the disease mechanisms and may aid in the development of novel targeted therapy for lung cancer and mesothelioma.

Toxicity evaluations of nanoclays and thermally degraded byproducts through spectroscopical and microscopical approaches.

BACKGROUND: Montmorillonite is a type of nanoclay that originates from the clay fraction of the soil and is incorporated into polymers to form nanocomposites with enhanced mechanical strength, barrier, and flammability properties used for food packaging, automotive, and medical devices. However, with implementation in such consumer applications, the interaction of montmorillonite-based composites or derived byproducts with biological systems needs to be investigated. METHODS: Herein we examined the potential of Cloisite Na+ (pristine) and Cloisite 30B (organically modified montmorillonite nanoclay) and their thermally degraded byproducts' to induce toxicity in model human lung epithelial cells. The experimental set-up mimicked biological exposure in manufacturing and disposal areas and employed cellular treatments with occupationally relevant doses of nanoclays previously characterized using spectroscopical and microscopical approaches. For nanoclay-cellular interactions and for cellular analyses respectively, biosensorial-based analytical platforms were used, with induced cellular changes being confirmed via live cell counts, viability assays, and cell imaging. RESULTS: Our analysis of byproducts' chemical and physical properties revealed both structural and functional changes. Real-time high throughput analyses of exposed cellular systems confirmed that nanoclay induced significant toxic effects, with Cloisite 30B showing time-dependent decreases in live cell count and cellular viability relative to control and pristine nanoclay, respectively. Byproducts produced less toxic effects; all treatments caused alterations in the cell morphology upon exposure. CONCLUSIONS: Our morphological, behavioral, and viability cellular changes show that nanoclays have the potential to produce toxic effects when used both in manufacturing or disposal environments. GENERAL SIGNIFICANCE: The reported toxicological mechanisms prove the extensibility of a biosensorial-based platform for cellular behavior analysis upon treatment with a variety of nanomaterials.

Role of mesothelin in carbon nanotube-induced carcinogenic transformation of human bronchial epithelial cells.

Carbon nanotubes (CNTs) have been likened to asbestos in terms of morphology and toxicity. CNT exposure can lead to pulmonary fibrosis and promotion of tumorigenesis. However, the mechanisms underlying CNT-induced carcinogenesis are not well defined. Mesothelin (MSLN) is overexpressed in many human tumors, including mesotheliomas and pancreatic and ovarian carcinomas. In this study, the role of MSLN in the carcinogenic transformation of human bronchial epithelial cells chronically exposed to single-walled CNT (BSW) was investigated. MSLN overexpression was found in human lung tumors, lung cancer cell lines, and BSW cells. The functional role of MSLN in the BSW cells was then investigated by using stably transfected MSLN knockdown (BSW shMSLN) cells. MSLN knockdown resulted in significantly decreased invasion, migration, colonies on soft agar, and tumor sphere formation. In vivo, BSW shMSLN cells formed smaller primary tumors and less metastases. The mechanism by which MSLN contributes to these more aggressive behaviors was investigated by using ingenuity pathway analysis, which predicted that increased MSLN could induce cyclin E expression. We found that BSW shMSLN cells had decreased cyclin E, and their proliferation rate was reverted to nearly that of untransformed cells. Cell cycle analysis showed that the BSW shMSLN cells had an increased G2 population and a decreased S phase population, which is consistent with the decreased rate of proliferation. Together, our results indicate a novel role of MSLN in the malignant transformation of bronchial epithelial cells following CNT exposure, suggesting its utility as a potential biomarker and drug target for CNT-induced malignancies.

Combinatorial approaches to evaluate nanodiamond uptake and induced cellular fate.

Nanodiamonds (NDs) are an emerging class of engineered nanomaterials that hold great promise for the next generation of bionanotechnological products to be used for drug and gene delivery, or for bio-imaging and biosensing. Previous studies have shown that upon their cellular uptake, NDs exhibit high biocompatibility in various in vitro and in vivo set-ups. Herein we hypothesized that the increased NDs biocompatibility is a result of minimum membrane perturbations and their reduced ability to induce disruption or damage during cellular translocation. Using multi-scale combinatorial approaches that simulate ND-membrane interactions, we correlated NDs real-time cellular uptake and kinetics with the ND-induced membrane fluctuations to derive energy requirements for the uptake to occur. Our discrete and real-time analyses showed that the majority of NDs internalization occurs within 2 h of cellular exposure, however, with no effects on cellular viability, proliferation or cellular behavior. Furthermore, our simulation analyses using coarse-grained models identified key changes in the energy profile, membrane deformation and recovery time, all functions of the average ND or ND-based agglomerate size. Understanding the mechanisms responsible for ND-cell membrane interactions could possibly advance their implementation in various biomedical applications.

Analysis of affinities between specific biological ligands using atomic force microscopy.

In the cell, protein-ligand recognition involves association and dissociation processes controlled by the affinity of the two binding partners and chemical harvesting of adenosine triphosphate energy. Fundamental knowledge of selected recognition events is currently translated in a synthetic environment for biosensors, immunoassays and diagnosis applications, or for pharmaceutical development. However, in order to advance such fields, one needs to determine the lifetime and binding efficiency of the two partners, as well as the complex energy landscape parameters. We employed contact mode atomic force microscopy to evaluate the association and dissociation events between streptavidin protein and its anti-streptavidin antibody ligand currently used for nucleotide array, ELISA, and flow cytometry applications, just to name a few. Using biotin as the control, our analysis helped characterize and differentiate multi- or single bonds of different strengths as well as associated energy landscapes to determine the protein-ligand structural arrangement at nanointerfaces and how these depend on the specificity of the ligand-recognition reaction. Our results suggest that understanding the importance of the rupture forces between a protein and its ligand could serve as the first step to protect on-off switches for biomedical research applications where specificity and selectivity are foremost sought.

Genotoxicity of multi-walled carbon nanotubes at occupationally relevant doses.

Carbon nanotubes are commercially-important products of nanotechnology; however, their low density and small size makes carbon nanotube respiratory exposures likely during their production or processing. We have previously shown mitotic spindle aberrations in cultured primary and immortalized human airway epithelial cells exposed to single-walled carbon nanotubes (SWCNT). In this study, we examined whether multi-walled carbon nanotubes (MWCNT) cause mitotic spindle damage in cultured cells at doses equivalent to 34 years of exposure at the NIOSH Recommended Exposure Limit (REL). MWCNT induced a dose responsive increase in disrupted centrosomes, abnormal mitotic spindles and aneuploid chromosome number 24 hours after exposure to 0.024, 0.24, 2.4 and 24 µg/cm² MWCNT. Monopolar mitotic spindles comprised 95% of disrupted mitoses. Three-dimensional reconstructions of 0.1 µm optical sections showed carbon nanotubes integrated with microtubules, DNA and within the centrosome structure. Cell cycle analysis demonstrated a greater number of cells in S-phase and fewer cells in the G2 phase in MWCNT-treated compared to diluent control, indicating a G1/S block in the cell cycle. The monopolar phenotype of the disrupted mitotic spindles and the G1/S block in the cell cycle is in sharp contrast to the multi-polar spindle and G2 block in the cell cycle previously observed following exposure to SWCNT. One month following exposure to MWCNT there was a dramatic increase in both size and number of colonies compared to diluent control cultures, indicating a potential to pass the genetic damage to daughter cells. Our results demonstrate significant disruption of the mitotic spindle by MWCNT at occupationally relevant exposure levels.

Effect of fiber length on carbon nanotube-induced fibrogenesis.

Given their extremely small size and light weight, carbon nanotubes (CNTs) can be readily inhaled by human lungs resulting in increased rates of pulmonary disorders, particularly fibrosis. Although the fibrogenic potential of CNTs is well established, there is a lack of consensus regarding the contribution of physicochemical attributes of CNTs on the underlying fibrotic outcome. We designed an experimentally validated in vitro fibroblast culture model aimed at investigating the effect of fiber length on single-walled CNT (SWCNT)-induced pulmonary fibrosis. The fibrogenic response to short and long SWCNTs was assessed via oxidative stress generation, collagen expression and transforming growth factor-beta (TGF-ß) production as potential fibrosis biomarkers. Long SWCNTs were significantly more potent than short SWCNTs in terms of reactive oxygen species (ROS) response, collagen production and TGF-ß release. Furthermore, our finding on the length-dependent in vitro fibrogenic response was validated by the in vivo lung fibrosis outcome, thus supporting the predictive value of the in vitro model. Our results also demonstrated the key role of ROS in SWCNT-induced collagen expression and TGF-ß activation, indicating the potential mechanisms of length-dependent SWCNT-induced fibrosis. Together, our study provides new evidence for the role of fiber length in SWCNT-induced lung fibrosis and offers a rapid cell-based assay for fibrogenicity testing of nanomaterials with the ability to predict pulmonary fibrogenic response in vivo.

Enzyme-based formulations for decontamination: current state and perspectives.

Development of noncorrosive, cost-effective, environmentally benign, and broad-spectrum antimicrobial formulations is necessary for clinical, industrial, and domestic purposes. Many current decontaminating formulations are effective, but they require the use of strong oxidizing agents or organic solvents that have deleterious effects on human health and the surrounding environment. The emergence of antibiotic-resistant pathogens has motivated researchers to develop enzyme-based self-decontaminating formulations as alternatives to such chemical decontamination approaches. Hydrolytic and oxidative enzymes can be used to deactivate pathogens, including bacteria, spores, viruses, and fungi. Laccases, haloperoxidases, and perhydrolases catalyze the generation of biocidal oxidants, such as iodine, bromine, hypohalous acid (e.g., HOCl or HOBr), and peracetic acid. These oxidants have broad-spectrum antimicrobial activity. Due to the multi-pathway action of these oxidants, it has proven extremely difficult for microbes to gain resistance. Thus far, few examples have been reported on enzyme-based antimicrobial formulations. For these reasons, various enzyme-containing antimicrobial formulations are highlighted in this review.

Perhydrolase-nanotube paint composites with sporicidal and antiviral activity.

AcT (perhydrolase) containing paint composites were prepared leading to broad-spectrum decontamination. AcT was immobilized onto multi-walled carbon nanotubes (MWNTs) and then incorporated into latex-based paints to form catalytic coatings. These AcT-based paint composites showed a 6-log reduction in the viability of spores of Bacillus cereus and Bacillus anthracis (Sterne) within 60 min. The paint composites also showed >4-log reduction in the titer of influenza virus (X-31) within 10 min (initially challenged with 10(7) PFU/mL). AcT-based paint composites were also tested using various perhydrolase acyl donor substrates, including propylene glycol diacetate (PGD), glyceryl triacetate, and ethyl acetate, with PGD observed to be the best among the substrates tested for generation of peracetic acid and killing of bacillus spores. The operational stability of paint composites was also studied at different relative humidities and temperatures to simulate real-life operation.

Electric Cell-Substrate Sensing for Real-Time Evaluation of Metal-Organic Framework Toxicological Profiles.

Metal-organic frameworks (MOFs) are hybrids formed through the coordination of metal ions and organic linkers in organic solvents. The implementation of MOFs in biomedical and industrial applications has led to concerns regarding their safety. Herein, the profile of a selected MOF, a zeolitic imidazole framework, was evaluated upon exposure to human lung epithelial cells. The platform for evaluation was a real-time technique (i.e., electric cell-substrate impedance sensing [ECIS]). This study identifies and discusses some of the deleterious effects of the selected MOF on the exposed cells. Furthermore, this study demonstrates the benefits of using the real-time method versus other biochemical assays for comprehensive cell evaluations. The study concludes that observed changes in cell behavior could hint at possible toxicity induced upon exposure to MOFs of different physicochemical characteristics and the dosage of those frameworks being used. By understanding changes in cell behavior, one foresees the ability to improve safe-by-design strategies of MOFs to be used for biomedical applications by specifically tailoring their physicochemical characteristics.

Digitoxin and a synthetic monosaccharide analog inhibit cell viability in lung cancer cells.

Mechanisms of digitoxin-inhibited cell growth and induced apoptosis in human non-small cell lung cancer (NCI-H460) cells remain unclear. Understanding how digitoxin or derivate analogs induce their cytotoxic effect below therapeutically relevant concentrations will help in designing and developing novel, safer and more effective anti-cancer drugs. In this study, NCI-H460 cells were treated with digitoxin and a synthetic analog D6-MA to determine their anti-cancer activity. Different concentrations of digitoxin and D6-MA were used and the subsequent changes in cell morphology, viability, cell cycle, and protein expressions were determined. Digitoxin and D6-MA induced dose-dependent apoptotic morphologic changes in NCI-H460 cells via caspase-9 cleavage, with D6-MA possessing 5-fold greater potency than digitoxin. In comparison, non-tumorigenic immortalized bronchial and small airway epithelial cells displayed significantly less apoptotic sensitivity compared to NCI-H460 cells suggesting that both digitoxin and D6-MA were selective for NSCLC. Furthermore, NCI-H460 cells arrested in G(2)/M phase following digitoxin and D6-MA treatment. Post-treatment evaluation of key G2/M checkpoint regulatory proteins identified down-regulation of cyclin B1/cdc2 complex and survivin. Additionally, Chk1/2 and p53 related proteins experienced down-regulation suggesting a p53-independent cell cycle arrest mechanism. In summary, digitoxin and D6-MA exert anti-cancer effects on NCI-H460 cells through apoptosis or cell cycle arrest, with D6-MA showing at least 5-fold greater potency relative to digitoxin.

Microtubules and Quantum Dots Integration Leads to Conjugates with Applications in Biosensors and Bionanodevices.

This chapter describes compiled methods for the formation and manipulation of microtubule-kinesin-carbon nanodots conjugates in user-defined synthetic environments. Specifically, by using inherited self-assembly and self-recognition properties of tubulin cytoskeletal protein and by interfacing this protein with lab synthesized carbon nanodots, bio-nano hybrid interfaces were formed. Further manipulation of such biohybrids under the mechanical cycle of kinesin 1 ATP-ase molecular motor led to their integration on user-controlled engineered surfaces. Presented methods are foreseen to lead to microtubule-molecular motor-hybrid based assemblies formation with applications ranging from biosensing, to nanoelectronics and single molecule printing, just to name a few.

Hyaluronic Acid Allows Enzyme Immobilization for Applications in Biomedicine.

Enzymes are proteins that control the efficiency and effectiveness of biological reactions and systems, as well as of engineered biomimetic processes. This review highlights current applications of a diverse range of enzymes for biofuel production, plastics, and chemical waste management, as well as for detergent, textile, and food production and preservation industries respectively. Challenges regarding the transposition of enzymes from their natural purpose and environment into synthetic practice are discussed. For example, temperature and pH-induced enzyme fragilities, short shelf life, low-cost efficiency, poor user-controllability, and subsequently insufficient catalytic activity were shown to decrease pertinence and profitability in large-scale production considerations. Enzyme immobilization was shown to improve and expand upon enzyme usage within a profit and impact-oriented commercial world and through enzyme-material and interfaces integration. With particular focus on the growing biomedical market, examples of enzyme immobilization within or onto hyaluronic acid (HA)-based complexes are discussed as a definable way to improve upon and/or make possible the next generation of medical undertakings. As a polysaccharide formed in every living organism, HA has proven beneficial in biomedicine for its high biocompatibility and controllable biodegradability, viscoelasticity, and hydrophilicity. Complexes developed with this molecule have been utilized to selectively deliver drugs to a desired location and at a desired rate, improve the efficiency of tissue regeneration, and serve as a viable platform for biologically accepted sensors. In similar realms of enzyme immobilization, HA's ease in crosslinking allows the molecule to user-controllably enhance the design of a given platform in terms of both chemical and physical characteristics to thus best support successful and sustained enzyme usage. Such examples do not only demonstrate the potential of enzyme-based applications but further, emphasize future market trends and accountability.

Active Nanointerfaces Based on Enzyme Carbonic Anhydrase and Metal-Organic Framework for Carbon Dioxide Reduction.

Carbonic anhydrases are enzymes capable of transforming carbon dioxide into bicarbonate to maintain functionality of biological systems. Synthetic isolation and implementation of carbonic anhydrases into membrane have recently raised hopes for emerging and efficient strategies that could reduce greenhouse emission and the footprint of anthropogenic activities. However, implementation of such enzymes is currently challenged by the resulting membrane's wetting capability, overall membrane performance for gas sensing, adsorption and transformation, and by the low solubility of carbon dioxide in water, the required medium for enzyme functionality. We developed the next generation of enzyme-based interfaces capable to efficiently adsorb and reduce carbon dioxide at room temperature. For this, we integrated carbonic anhydrase with a hydrophilic, user-synthesized metal-organic framework; we showed how the framework's porosity and controlled morphology contribute to viable enzyme binding to create functional surfaces for the adsorption and reduction of carbon dioxide. Our analysis based on electron and atomic microscopy, infrared spectroscopy, and colorimetric assays demonstrated the functionality of such interfaces, while Brunauer-Emmett-Teller analysis and gas chromatography analysis allowed additional evaluation of the efficiency of carbon dioxide adsorption and reduction. Our study is expected to impact the design and development of active interfaces based on enzymes to be used as green approaches for carbon dioxide transformation and mitigation of global anthropogenic activities.

Thin Films of Metal-Organic Framework Interfaces Obtained by Laser Evaporation.

Properties such as large surface area, high pore volume, high chemical and thermal stability, and structural flexibility render zeolitic imidazolate frameworks (ZIFs) well-suited materials for gas separation, chemical sensors, and optical and electrical devices. For such applications, film processing is a prerequisite. Herein, matrix-assisted pulsed laser evaporation (MAPLE) was successfully used as a single-step deposition process to fabricate ZIF-8 films. By correlating laser fluency and controlling the specific transfer of lab-synthesized ZIF-8, films with user-controlled physical and chemical properties were obtained. Films' characteristics were evaluated by scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy, X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and X-ray photoelectron spectroscopy (XPS). The analysis showed that frameworks of ZIF-8 can be deposited successfully and controllably to yield polycrystalline films. The deposited films maintained the integrity of the individual ZIF-8 framework, while undergoing minor crystalline and surface chemistry changes. No significant changes in particle size were observed. Our study demonstrated control over both the MAPLE deposition conditions and the outcome, as well as the suitability of the listed deposition method to create composite architectures that could potentially be used in applications ranging from selective membranes to gas sensors.

A bio-pen for direct writing of single molecules on user-functionalized surfaces.

Advancing ultrahigh resolution (below 10 nm) direct writing technologies could lead to impacts in areas as diverse as disease detection, genetic analysis and nanomanufacturing. Current methods based on electron-beams and photo- or dip-pen nanolithography are laborious and lack flexibility when aiming to create single molecule patterns for application specific integration. We hypothesize that a novel strategy could be developed to allow for writing of parallel and yet individually addressable patterns of single molecules on user-controlled surfaces. The strategy is based on using in vitro self-recognition of tubulin protein to assemble rigid protofilaments of microtubules, with one such microtubule to be subsequently used as a "bio-pen" capable of writing "inks" of single kinesin molecules in user-defined environments. Our results show that single kinesin inks could be written under the energy of adenosine triphosphate hydrolysis and observed by both atomic force and optical microscopy. Upon extending ink functionalities, the integration of soft and hard materials for nanostructure assembly and complex single molecule pattern formation is envisioned.

Potential antitumor activity of digitoxin and user-designed analog administered to human lung cancer cells.

BACKGROUND: Cardiac glycosides (CGs), such as digitoxin, are traditionally used for treatment of congestive heart failure; recently they also gained attention for their anticancer properties. Previous studies showed that digitoxin and a synthetic L-sugar monosaccharide analog treatment decreases cancer cell proliferation, increases apoptosis, and pro-adhesion abilities; however, no reports are available on their potential to alter lung cancer cell cytoskeleton structure and reduce migratory ability. Herein, we investigated the anticancer effects of digitoxin and its analog, digitoxigenin-α-L-rhamnoside (D6MA), to establish whether cytoskeleton reorganization and reduced motility are drug-induced cellular outcomes. METHODS: We treated non-small cell lung carcinoma cells (NSCLCs) with sub-therapeutic, therapeutic, and toxic concentrations of digitoxin and D6MA respectively, followed by both single point and real-time assays to evaluate changes in cellular gene and protein expression, adhesion, elasticity, and migration. RESULTS: Digitoxin and D6MA induced a decrease in matrix metalloproteinases expression via altered focal adhesion signaling and a suppression of the phosphoinositide 3-kinases / protein kinase B pathway which lead to enhanced adhesion, altered elasticity, and reduced motility of NSCLCs. Global gene expression analysis identified dose-dependent changes to nuclear factor kappa-light-chain-enhancer, epithelial tumor, and microtubule dynamics signaling. CONCLUSIONS: Our study demonstrates that digitoxin and D6MA can target antitumor signaling pathways to alter NSCLC cytoskeleton and migratory ability to thus potentially reduce their tumorigenicity. SIGNIFICANCE: Discovering signaling pathways that control cancer's cell phenotype and how such pathways are affected by CG treatment will potentially allow for active usage of synthetic CG analogs as therapeutic agents in advanced lung conditions.