Drug Discov Today

RNA interference (RNAi) describes the post-transcriptional silencing of gene expression that occurs in response to the introduction of double-stranded RNA into cells. Application of RNAi in experimental systems has provided a great leap forward in the elucidation of gene function. To facilitate large-scale functional genomics studies using RNAi, several high throughput approaches have been developed based on microarray or microwell assays. Recent establishment of large libraries of RNAi reagents combined with a variety of detection assays further opens the door for genome-wide screens of gene function in mammalian cells. The review provides a comprehensive overview on current RNA interference-based high throughput genomic technologies. The cell array- and microwell plate-based loss-of-function cell assays are presented

Carnot-Caratheodory metric and gauge fluctuation in Noncommutative Geometry

Gauge fields have a natural metric interpretation in terms of horizontal distance. The latest, also called Carnot-Caratheodory or subriemannian distance, is by definition the length of the shortest horizontal path between points, that is to say the shortest path whose tangent vector is everywhere horizontal with respect to the gauge connection. In noncommutative geometry all the metric information is encoded within the Dirac operator D. In the classical case, i.e. commutative, Connes's distance formula allows to extract from D the geodesic distance on a riemannian spin manifold. In the case of a gauge theory with a gauge field A, the geometry of the associated U(n)-vector bundle is described by the covariant Dirac operator D+A. What is the distance encoded within this operator ? It was expected that the noncommutative geometry distance d defined by a covariant Dirac operator was intimately linked to the Carnot-Caratheodory distance dh defined by A. In this paper we precise this link, showing that the equality of d and dh strongly depends on the holonomy of the connection. Quite interestingly we exhibit an elementary example, based on a 2 torus, in which the noncommutative distance has a very simple expression and simultaneously avoids the main drawbacks of the riemannian metric (no discontinuity of the derivative of the distance function at the cut-locus) and of the subriemannian one (memory of the structure of the fiber).Comment: published version with additional figures to make the proof more readable. Typos corrected in this ultimate versio

Low Avidity T Cells Do Not Hinder High Avidity T Cell Responses Against Melanoma

The efficacy of T cells depends on their functional avidity, i. e., the strength of T cell interaction with cells presenting cognate antigen. The overall T cell response is composed of multiple T cell clonotypes, involving different T cell receptors and variable levels of functional avidity. Recently, it has been proposed that the presence of low avidity tumor antigen-specific CD8 T cells hinder their high avidity counterparts to protect from tumor growth. Here we analyzed human cytotoxic CD8 T cells specific for the melanoma antigen Melan-A/MART-1. We found that the presence of low avidity T cells did not result in reduced cytotoxicity of tumor cells, nor reduced cytokine production, by high avidity T cells. In vivo in NSG-HLA-A2 mice, the anti-tumor effect of high avidity T cells was similar in presence or absence of low avidity T cells. These data indicate that low avidity T cells are not hindering anti-tumor T cell responses, a finding that is reassuring because low avidity T cells are an integrated part of natural T cell responses

A novel technique to determine the cell type specific response within an in vitro co-culture model via multi-colour flow cytometry

Determination of the cell type specific response is essential towards understanding the cellular mechanisms associated with disease states as well as assessing cell-based targeting of effective therapeutic agents. Recently, there have been increased calls for advanced in vitro multi-cellular models that provide reliable and valuable tools correlative to in vivo. In this pursuit the ability to assess the cell type specific response is imperative. Herein, we report a novel approach towards resolving each specific cell type of a multi-cellular model representing the human lung epithelial tissue barrier via multi-colour flow cytometry (FACS). We proved via ≤ five-colour FACS that the manipulation of this in vitro model allowed each cell type to be resolved with no impact upon cell viability. Subsequently, four-colour FACS verified the ability to determine the biochemical effect (e.g. oxidative stress) of each specific cell type. This technique will be vital in gaining information upon cellular mechanics when using next-level, multi- cellular in vitro strategies

Investigating the interaction of cellulose nanofibers derived from cotton with a sophisticated 3D human lung cell coculture

Cellulose nanofibers are an attractive component of a broad range of nanomaterials. Their intriguing mechanical properties and low cost, as well as the renewable nature of cellulose make them an appealing alternative to carbon nanotubes (CNTs), which may pose a considerable health risk when inhaled. Little is known, however, concerning the potential toxicity of aerosolized cellulose nanofibers. Using a 3D in vitro triple cell coculture model of the human epithelial airway barrier, it was observed that cellulose nanofibers isolated from cotton (CCN) elicited a significantly (p < 0.05) lower cytotoxicity and (pro-)inflammatory response than multiwalled CNTs (MWCNTs) and crocidolite asbestos fibers (CAFs). Electron tomography analysis also revealed that the intracellular localization of CCNs is different from that of both MWCNTs and CAFs, indicating fundamental differences between each different nanofibre type in their interaction with the human lung cell coculture. Thus, the data shown in the present study highlights that not only the length and stiffness determine the potential detrimental (biological) effects of any nanofiber, but that the material used can significantly affect nanofiber–cell interactions

Integrating silver compounds and nanoparticles into ceria nanocontainers for antimicrobial applications

Silver compounds and nanoparticles (NPs) are gaining increasing interest in medical applications, specifically in the treatment and prevention of biomaterial-related infections. However, the silver release from these materials, resulting in a limited antimicrobial activity, is often difficult to control. In this paper, ceria nanocontainers were synthesized by a template-assisted method and were then used to encapsulate silver nitrate (AgNO₃/CeO₂ nanocontainers). Over the first 30 days, a significant level of silver was released, as determined using inductively coupled plasma optical emission spectroscopy (ICP-OES). A novel type of ceria container containing silver NPs (AgNP/CeO₂ containers) was also developed using two different template removal methods. The presence of AgNPs was confirmed both on the surface and in the interior of the ceria containers by X-ray diffraction (XRD), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Upon removal of the template by calcination, the silver was released over a period exceeding three months (>90 days). However, when the template was removed by dissolution, the silver release was shortened to ≤14 days. The antimicrobial activity of the silver-containing CeO₂ containers was observed and the minimum bactericidal concentration (MBC) was determined using the broth dilution method. Investigation on human cells, using a model epithelial barrier cell type (A549 cells), highlighted that all three samples induced a heightened cytotoxicity leading to cell death when exposed to all containers in their raw form. This was attributed to the surface roughness of the CeO₂ nanocontainers and the kinetics of the silver release from the AgNO₃/CeO₂ and AgNP/CeO₂ nanocontainers. In conclusion, despite the need for further emphasis on their biocompatibility, the concept of the AgNP/CeO₂ nanocontainers offers a potentially alternative long-term antibactericidal strategy for implant materials

A Comparative Study of Different In Vitro Lung Cell Culture Systems to Assess the Most Beneficial Tool for Screening the Potential Adverse Effects of Carbon Nanotubes

To determine the potential inhalatory risk posed by carbon nanotubes (CNTs), a tier-based approach beginning with an in vitro assessment must be adopted. The purpose of this study therefore was to compare 4 commonly used in vitro systems of the human lung (human blood monocyte-derived macrophages [MDM] and monocyte-derived dendritic cells [MDDC], 16HBE14o- epithelial cells, and a sophisticated triple cell co-culture model [TCC-C]) via assessment of the biological impact of different CNTs (single-walled CNTs [SWCNTs] and multiwalled CNTs [MWCNTs]) over 24h. No significant cytotoxicity was observed with any of the cell types tested, although a significant (p < .05), dose-dependent increase in tumor necrosis factor (TNF)-α following SWCNT and MWCNT exposure at concentrations up to 0.02mg/ml to MDM, MDDC, and the TCC-C was found. The concentration of TNF-α released by the MDM and MDDC was significantly higher (p < .05) than the TCC-C. Significant increases (p < .05) in interleukin (IL)-8 were also found for both 16HBE14o- epithelial cells and the TCC-C after SWCNTs and MWCNTs exposure up to 0.02mg/ml. The TCC-C, however, elicited a significantly (p < .05) higher IL-8 release than the epithelial cells. The oxidative potential of both SWCNTs and MWCNTs (0.005-0.02mg/ml) measured by reduced glutathione (GSH) content showed a significant difference (p < .05) between each monoculture and the TCC-C. It was concluded that because only the co-culture system could assess each endpoint adequately, that, in comparison with monoculture systems, multicellular systems that take into consideration important cell type-to-cell type interactions could be used as predictive in vitro screening tools for determining the potential deleterious effects associated with CNT

Non-commutative Quantum Mechanics in Three Dimensions and Rotational Symmetry

We generalize the formulation of non-commutative quantum mechanics to three dimensional non-commutative space. Particular attention is paid to the identification of the quantum Hilbert space in which the physical states of the system are to be represented, the construction of the representation of the rotation group on this space, the deformation of the Leibnitz rule accompanying this representation and the implied necessity of deforming the co-product to restore the rotation symmetry automorphism. This also implies the breaking of rotational invariance on the level of the Schroedinger action and equation as well as the Hamiltonian, even for rotational invariant potentials. For rotational invariant potentials the symmetry breaking results purely from the deformation in the sense that the commutator of the Hamiltonian and angular momentum is proportional to the deformation.Comment: 21 page

Risk assessment of released cellulose nanocrystals – mimicking inhalatory exposure

Cellulose nanocrystals (CNCs) exhibit advantageous chemical and mechanical properties that render them attractive for a wide range of applications. During the life-cycle of CNC containing materials the nanocrystals could be released and become airborne, posing a potential inhalatory exposure risk towards humans. Absent reliable and dose-controlled models that mimic this exposure in situ is a central issue in gaining an insight into the CNC-lung interaction. Here, an Air Liquid Interface Cell Exposure system (ALICE), previously designed for studies of spherical nanoparticles, was used for the first time to establish a realistic physiological exposure test method for inhaled fiber shaped nano-objects; in this case, CNCs isolated from cotton. Applying a microscopy based approach the spatially homogenous deposition of CNCs was demonstrated as a prerequisite of the functioning of the ALICE. Furthermore, reliability and controllability of the system to nebulise high aspect ratio nanomaterials (HARN, e.g. CNCs) was shown. This opens the potential to thoroughly investigate the inhalatory risk of CNCs in vitro using a realistic exposure system

Screening of human gene promoter activities using transfected-cell arrays

Promoters are the best characterized transcriptional regulatory sequences in complex genomes because of their predictable location immediately upstream of transcription start sites. Despite a substantial body of literature describing transcriptional promoters, the identification of true start sites for all human transcripts is far from complete. The same is true of the key structural and functional elements responsible for promoter action in different cell types. In order to identify elements responsible for promoter activity, we applied transfected-cell array technology to functionally evaluate promoters for genes involved in inflammatory bowel disease. Seventy-four promoters were examined by reverse transfection of a promoter-fluorescent reporter constructs into a human embryonic kidney cell line (HEK293T). Sixteen (21.6%) promoters were found to be active in HEK293 T cells. Correlations between promoter activity and endogenous transcript level were calculated, and 75% of active promoters were found to be associated with transcriptional activity of their gene counterparts. These results provide experimental evidence of promoter activity, which may aid in understanding the regulation of gene expression. Moreover, this is the first large-scale functional study of regulatory sequences to use a high-throughput transfected-cell array technique