Monitoring induced gene expression of single cells in a multilayer microchip

We present a microfluidic system that facilitates long-term measurements of single cell response to external stimuli. The difficulty of addressing cells individually was overcome by using a two-layer microfluidic device. The top layer is designed for trapping and culturing of cells while the bottom layer is employed for supplying chemical compounds that can be transported towards the cells in defined concentrations and temporal sequences. A porous polyester membrane that supports transport and diffusion of compounds from below separates the microchannels of both layers. The performance and potential of the device are demonstrated using human embryonic kidney cells (HEK293) transfected with an inducible gene expression system. Expression of a fluorescent protein (ZsGreen1-DR) is observed while varying the concentration and exposure time of the inducer tetracycline. The study reveals the heterogeneous response of the cells as well as average responses of tens of cells that are analyzed in parallel. The microfluidic platform enables systematic studies under defined conditions and is a valuable tool for general single cell studies to obtain insights into mechanisms and kinetics that are not accessible by conventional macroscopic methods. Figure A two-layer microfluidic device is presented that facilitates measurements of single cell response to external stimul

Preparation of neuronal co-cultures with single cell precision

Microfluidic embodiments of the Campenot chamber have attracted great interest from the neuroscience community. These interconnected co-culture platforms can be used toinvestigate a variety of questions, spanning developmental and functional neurobiology to infection and disease propagation. However, conventional systems require significant cellular inputs (many thousands per compartment), inadequate for studying low abundance cells, such as primary dopaminergic substantia nigra, spiral ganglia and Drosophilia melanogaster neurons, and impractical for high throughput experimentation. The dense cultures are also highly locally entangled, with few outgrowths (<10%) interconnecting the two cultures. In this paper straightforward microfluidic and patterning protocols are described which address these challenges: (i) a microfluidic single neuron arraying method, and (ii) a water masking method for plasma patterning biomaterial coatings to register neurons and promote outgrowth between compartments. Minimalistic neuronal co-cultures were prepared with high-level (>85%) inter-compartment connectivity and can be used for high throughput neurobiology experiments with single cell precisio

Species Abundance Patterns in Complex Evolutionary Dynamics

An analytic theory of species abundance patterns (SAPs) in biological networks is presented. The theory is based on multispecies replicator dynamics equivalent to the Lotka-Volterra equation, with diverse interspecies interactions. Various SAPs observed in nature are derived from a single parameter. The abundance distribution is formed like a widely observed left-skewed lognormal distribution. As the model has a general form, the result can be applied to similar patterns in other complex biological networks, e.g. gene expression.Comment: 4 pages, 3 figures. Physical Review Letters, in pres

Taxonomic decomposition of the latitudinal gradient in species diversity of North American floras

Aim: To test the latitudinal gradient in plant species diversity for self-similarity across taxonomic scales and amongst taxa. Location: North America. Methods: We used species richness data from 245 local vascular plant floras to quantify the slope and shape of the latitudinal gradients in species diversity (LGSD) across all plant species as well as within each family and order. We calculated the contribution of each family and order to the empirical LGSD. Results: We observed the canonical LGSD when all plants were considered with floras at the lowest latitudes having, on average, 451 more species than floras at the highest latitudes. When considering slope alone, most orders and families showed the expected negative slope, but 31.7% of families and 27.7% of orders showed either no significant relationship between latitude and diversity or a reverse LGSD. Latitudinal patterns of family diversity account for at least 14% of this LGSD. Most orders and families did not show the negative slope and concave-down quadratic shape expected by the pattern for all plant species. A majority of families did not make a significant contribution in species to the LGSD with 53% of plant families contributing little to nothing to the overall gradient. Ten families accounted for more than 70% of the gradient. Two families, the Asteraceae and Fabaceae, contributed a third of the LGSD. Main Conclusions: The empirical LGSD we describe here is a consequence of a gradient in the number of families and diversification within relative few plant families. Macroecological studies typically aim to generate models that are general across taxa with the implicit assumption that the models are general within taxa. Our results strongly suggest that models of the latitudinal gradient in plant species richness that rely on environmental covariates (e.g. temperature, energy) are likely not general across plant taxa

Micropatterning neuronal networks

Spatially organised neuronal networks have wide reaching applications, including fundamental research, toxicology testing, pharmaceutical screening and the realisation of neuronal implant interfaces. Despite the large number of methods catalogued in the literature there remains the need to identify a method that delivers high pattern compliance, long-term stability and is widely accessible to neuroscientists. In this comparative study, aminated (polylysine/polyornithine and aminosilanes) and cytophobic (poly(ethylene glycol) (PEG) and methylated) material contrasts were evaluated. Backfilling plasma stencilled PEGylated substrates with polylysine does not produce good material contrasts, whereas polylysine patterned on methylated substrates becomes mobilised by agents in the cell culture media which results in rapid pattern decay. Aminosilanes, polylysine substitutes, are prone to hydrolysis and the chemistries prove challenging to master. Instead, the stable coupling between polylysine and PLL-g-PEG can be exploited: Microcontact printing polylysine onto a PLL-g-PEG coated glass substrate provides a simple means to produce microstructured networks of primary neurons that have superior pattern compliance during long term (>1 month) cultur

Prognostic and predictive value of circulating tumor cells and CXCR4 expression as biomarkers for a CXCR4 peptide antagonist in combination with carboplatin-etoposide in small cell lung cancer: exploratory analysis of a phase II study.

Background Circulating tumor cells (CTCs) and chemokine (C-X-C motif) receptor 4 (CXCR4) expression in CTCs and tumor tissue were evaluated as prognostic or predictive markers of CXCR4 peptide antagonist LY2510924 plus carboplatin-etoposide (CE) versus CE in extensive-stage disease small cell lung cancer (ED-SCLC). Methods This exploratory analysis of a phase II study evaluated CXCR4 expression in baseline tumor tissue and peripheral blood CTCs and in post-treatment CTCs. Optimum cutoff values were determined for CTC counts and CXCR4 expression in tumors and CTCs as predictors of survival outcome. Kaplan-Meier estimates and hazard ratios were used to determine biomarker prognostic and predictive values. Results There was weak positive correlation at baseline between CXCR4 expression in tumor tissue and CTCs. Optimum cutoff values were H-score ≥ 210 for CXCR4+ tumor, ≥7% CTCs with CXCR4 expression (CXCR4+ CTCs), and ≥6 CTCs/7.5 mL blood. Baseline H-score for CXCR4+ tumor was not prognostic of progression-free survival (PFS) or overall survival (OS). Baseline CXCR4+ CTCs ≥7% was prognostic of shorter PFS. CTCs ≥6 at baseline and cycle 2, day 1 were prognostic of shorter PFS and OS. None of the biomarkers at their respective optimum cutoffs was predictive of treatment response of LY2510924 plus CE versus CE. Conclusions In patients with ED-SCLC, baseline CXCR4 expression in tumor tissue was not prognostic of survival or predictive of LY2510924 treatment response. Baseline CXCR4+ CTCs ≥7% was prognostic of shorter PFS. CTC count ≥6 at baseline and after 1 cycle of treatment were prognostic of shorter PFS and OS

The Effects of ARRA Funding on Broadband Availability and Adoption

The American Recovery and Reinvestment Act (ARRA), passed in 2009, provided funds to help the American economy recover from the 2008 economic crisis. More than $7 billion was designated to the advancement of broadband services, and critically, improvement in broadband infrastructure and adoption in underserved areas. This thesis sought to discover if there is a significant causal relationship between the amount of ARRA funding given to a U.S. state and its increase of broadband availability/adoption rates between 2010 (when all ARRA projects were announced), 2013, and 2016 (after all projects were completed). After running various regressions, I found that there is no such relationship between a state’s logarithmic level of ARRA funding and percent increases in either availability or adoption rates. In fact, only one regression displayed any significant causal relationship at all: a negative relationship between funding received and a state’s 2016 adoption rate. The results, explained below, contain lessons for policymakers going forward as society’s dependence on high-speed internet continues to increase

Toward a theory for diversity gradients: the abundance–adaptation hypothesis

The abundance–adaptation hypothesis argues that taxa with more individuals and faster generation times will have more evolutionary ‘experiments’ allowing expansion into, and diversification within, novel habitats. Thus, as older taxa have produced more individuals over time, and smaller taxa have higher population sizes and faster generation times, the Latitudinal Diversity Gradients (LDGs) of these clades should show shallower slopes. We describe the LDGs for archaea, bacteria, fungi, invertebrates and trees from six North American forests. For three focal groups – bacteria, ants, and trees – older taxa had shallower LDG slopes than the more recent, terminal taxa. Across 12 orders of magnitude of body mass, LDG slopes were steeper in larger taxa. The slopes of LDGs vary systematically with body size and clade age, underscoring the non-canonical nature of LDGs. The steepest LDG slopes were found for the largest organisms while the smallest, from bacteria to small litter-soil invertebrates, have shallower- to zero-slope LDGs. If tropical niche conservatism is the failure of clades to adapt to, and diversify in temperate habitats, then the steep LDGs of chordates and plants likely arise from the decreased ability of clades with large individuals to adapt to the multiple challenges of extra-tropical life