Control and Manipulation of Pathogens with an Optical Trap for Live Cell Imaging of Intercellular Interactions

The application of live cell imaging allows direct visualization of the dynamic interactions between cells of the immune system. Some preliminary observations challenge long-held beliefs about immune responses to microorganisms; however, the lack of spatial and temporal control between the phagocytic cell and microbe has rendered focused observations into the initial interactions of host response to pathogens difficult. This paper outlines a method that advances live cell imaging by integrating a spinning disk confocal microscope with an optical trap, also known as an optical tweezer, in order to provide exquisite spatial and temporal control of pathogenic organisms and place them in proximity to host cells, as determined by the operator. Polymeric beads and live, pathogenic organisms (Candida albicans and Aspergillus fumigatus) were optically trapped using non-destructive forces and moved adjacent to living cells, which subsequently phagocytosed the trapped particle. High resolution, transmitted light and fluorescence-based movies established the ability to observe early events of phagocytosis in living cells. To demonstrate the broad applicability of this method to immunological studies, anti-CD3 polymeric beads were also trapped and manipulated to form synapses with T cells in vivo, and time-lapse imaging of synapse formation was also obtained. By providing a method to exert fine control of live pathogens with respect to immune cells, cellular interactions can be captured by fluorescence microscopy with minimal perturbation to cells and can yield powerful insight into early responses of innate and adaptive immunity.National Institute of Biomedical Imaging and Bioengineering (U.S.) (grant T32EB006348)Massachusetts General Hospital (Department of Medicine Internal Funds)Center for Computational and Integrative Biology (Development fund)Center for Computational and Integrative Biology (AI062773)Center for Computational and Integrative Biology (grant AI062773)Center for Computational and Integrative Biology (grant DK83756)Center for Computational and Integrative Biology (grant DK 043351)National Institute of Allergy and Infectious Diseases (U.S.)National Institutes of Health (U.S.) (grant AI057999

Multidimensional Atomic Force Microscopy: A Versatile Novel Technology for Nanopharmacology Research

Nanotechnology is giving us a glimpse into a nascent field of nanopharmacology that deals with pharmacological phenomena at molecular scale. This review presents our perspective on the use of scanning probe microscopy techniques with special emphasis to multidimensional atomic force microscopy (m-AFM) to explore this new field with a particular emphasis to define targets, design therapeutics, and track outcomes of molecular-scale pharmacological interactions. The approach will be to first discuss operating principles of m-AFM and provide representative examples of studies to understand human health and disease at the molecular level and then to address different strategies in defining target macromolecules, screening potential drug candidates, developing and characterizing of drug delivery systems, and monitoring target–drug interactions. Finally, we will discuss some future directions including AFM tip-based parallel sensors integrated with other high-throughput technologies which could be a powerful platform for drug discovery

¿Psicología de la Educación o Psicología Escolar? Esa es la cuestión

Este artigo apresenta alguns dados oriundos da tese de doutorado sobre a história do campo de conhecimento e prática da Psicologia em sua relação com a Educação no Brasil. Este estudo foi conduzido baseado no fundamento epistêmico-filosófico do materialismo histórico dialético e na nova história, utilizando fontes bibliográficas históricas e cinco relatos orais de personagens da Psicologia Educacional e Escolar. Os depoimentos e o material das fontes escritas constituíram o corpus documental cuja organização seguiu a metodologia da história oral e historiografia plural. Foi realizada análise descritivo-analítica compreendida em duas etapas: a) análise documental (fontes não orais) e b) construção de indicadores e núcleos de significação dos registros orais. A partir das análises, compôs-se uma periodização da história da Psicologia Educacional e Escolar brasileira por meio de marcos históricos da área. No presente artigo destaca-se a discussão acerca da conceituação e terminologias utilizadas pela Psicologia Educacional e Escolar ao longo do tempo e de como essas mudanças nas nomenclaturas da área refletem questões epistemológicas, ideológicas e políticas

Mutational Analysis of Circulating Tumor Cells Using a Novel Microfluidic Collection Device and qPCR Assay

AbstractCirculating tumor cells (CTCs) provide a readily accessible source of tumor material from patients with cancer. Molecular profiling of these rare cells can lead to insight on disease progression and therapeutic strategies. A critical need exists to isolate CTCs with sufficient quantity and sample integrity to adapt to conventional analytical techniques. We present a microfluidic platform (IsoFlux) that uses flow control and immunomagnetic capture to enhance CTC isolation. A novel cell retrieval mechanism ensures complete transfer of CTCs into the molecular assay. Improved sensitivity to the capture antigen was demonstrated by spike-in experiments for three cell lines of varying levels of antigen expression. We obtained spike-in recovery rates of 74%, 75%, and 85% for MDA-MB-231 (low), PC3 (middle), and SKBR3 (high) cell lines. Recovery using matched enumeration protocols and matched samples (PC3) yielded 90% and 40% recovery for the IsoFlux and CellSearch systems, respectively. In matched prostate cancer samples (N = 22), patients presenting more than four CTCs per blood draw were 95% and 36% using IsoFlux and CellSearch, respectively. An assay for detecting KRAS mutations was described along with data from patients with colorectal cancer, of which 87% presented CTCs above the assay's limit of detection (four CTCs). The CTC KRAS mutant rate was 50%, with 46% of patients displaying a CTC KRAS mutational status that differed from the previously acquired tissue biopsy data. The microfluidic system and mutation assay presented here provide a complete workflow to track oncogene mutational changes longitudinally with high success rates

Characterization of the nanoscale properties of individual amyloid fibrils

We report the detailed mechanical characterization of individual amyloid fibrils by atomic force microscopy and spectroscopy. These self-assembling materials, formed here from the protein insulin, were shown to have a strength of 0.6 ± 0.4 GPa, comparable to that of steel (0.6–1.8 GPa), and a mechanical stiffness, as measured by Young's modulus, of 3.3 ± 0.4 GPa, comparable to that of silk (1–10 GPa). The values of these parameters reveal that the fibrils possess properties that make these structures highly attractive for future technological applications. In addition, analysis of the solution-state growth kinetics indicated a breakage rate constant of 1.7 ± 1.3 × 10(−8) s(−1), which reveals that a fibril 10 μm in length breaks spontaneously on average every 47 min, suggesting that internal fracturing is likely to be of fundamental importance in the proliferation of amyloid fibrils and therefore for understanding the progression of their associated pathogenic disorders