Multiplexed Sub-Cellular Scale Microarrays from direct DNA Nanolithography

The multiplexed, high-throughput fabrication of microarrays is of vital importance for many applications in life sciences, including drug screening, medical diagnostics and cell biology. In single cell investigations, features smaller than 10 μm are needed for functional manipulation of sub-cellular structures. Several top-down methodologies like electron beam lithography and microcontact printing can be employed for indirect surface patterning at this scale, however those approaches often require clean rooms and multiplexing of several different biomolecules on the same surface is limited [1]. To overcome these obstacles, we combined Dip-pen nanolithography (DPN) and DNA-directed immobilization (DDI) of proteins to fabricate cell-compatible functionalized glass surfaces [2]. We optimized ink formulation for ssDNA printing and the produced arrays were then functionalized with epidermal growth factor (EGF) taking advantage of covalent ssDNA-streptavidin conjugates as adaptor molecules. The surface-immobilized EGF was used for recruiting EGFR in the plasma membrane of MCF7 cells. Via this bottom-up structuring approach, we were able to analyse multiple protein-protein interactions simultaneously in individual living cells [3]. To improve the efficiency of multiplexed surface patterning, we developed a prototype of a robust custom plotter based on 2D polymer-pen lithography (2D-PPL) [4]. This device enables rapid fabrication of microarrays at ambient conditions in a multiplexed direct-writing mode. The printing process was carried out by polymeric pyramidal pens onto which multiple (up to 36) ssDNA solutions can be loaded through a microfluidic inkwell device. Subsequent to optimization of ink viscosity and surface tension by glycerol and tween-20, DNA arrays were plotted and used for DDI of EGF-bearing ssDNA-streptavidin conjugates. The resulting microarrays covered areas of about 0.5 cm2, and were capable of recruiting and activating EGF receptors in sub-cellular regions within human MCF7 cells [4]. References [1] G. Arrabito, B. Pignataro. 2012. Solution Processed Micro- and Nano- Bioarrays for Multiplexed Biosensing. Anal. Chem. 84:5450–5462. [2] G. Arrabito et al. 2013. Biochips for Cell Biology by Combined Dip-Pen Nanolithography and DNA-Directed Protein Immobilization. Small. 9:4243-4249. [3] S. Gandor et al. 2013. A Protein-Interaction Array Inside a Living Cell. Angew. Chem. Int. Ed. Engl. 52:4790–4794. [4] G. Arrabito, et al. 2014. Low-cost Plotter Device for Sub-Cellular Scale Microarray Fabrication. Small. DOI: 10.1002/smll.201303390

Expression and Differential Responsiveness of Central Nervous System Glial Cell Populations to the Acute Phase Protein Serum Amyloid A

Acute-phase response is a systemic reaction to environmental/inflammatory insults and involves hepatic production of acute-phase proteins, including serum amyloid A (SAA). Extrahepatically, SAA immunoreactivity is found in axonal myelin sheaths of cortex in Alzheimer's disease and multiple sclerosis (MS), although its cellular origin is unclear. We examined the responses of cultured rat cortical astrocytes, microglia and oligodendrocyte precursor cells (OPCs) to master pro-inflammatory cytokine tumour necrosis factor (TNF)-\u3b1 and lipopolysaccaride (LPS). TNF-\u3b1 time-dependently increased Saa1 (but not Saa3) mRNA expression in purified microglia, enriched astrocytes, and OPCs (as did LPS for microglia and astrocytes). Astrocytes depleted of microglia were markedly less responsive to TNF-\u3b1 and LPS, even after re-addition of microglia. Microglia and enriched astrocytes showed complementary Saa1 expression profiles following TNF-\u3b1 or LPS challenge, being higher in microglia with TNF-\u3b1 and higher in astrocytes with LPS. Recombinant human apo-SAA stimulated production of both inflammatory mediators and its own mRNA in microglia and enriched, but not microglia-depleted astrocytes. Co-ultramicronized palmitoylethanolamide/luteolin, an established anti-inflammatory/neuroprotective agent, reduced Saa1 expression in OPCs subjected to TNF-\u3b1 treatment. These last data, together with past findings suggest that co-ultramicronized palmitoylethanolamide/luteolin may be a novel approach in the treatment of inflammatory demyelinating disorders like MS

Family-Based Treatments for Serious Juvenile Offenders: A Multilevel Meta-Analysis

OBJECTIVE: Researchers have identified several family-based treatments that hold considerable promise in reducing serious juvenile offending; however, these treatments remain underutilized by youth service systems. In the present study, we used meta-analysis to summarize the findings of research on family-based treatments for serious juvenile offenders. METHOD: We conducted a multilevel meta-analysis that modeled dependencies between multiple effect sizes from the same study. The meta-analysis synthesized 324 effect sizes from 28 studies that met inclusion criteria. Potential moderators (e.g., characteristics of samples, treatments, methods, and measures) were entered as fixed effects in the meta-analytic model. RESULTS: Across studies, family-based treatments produced modest, yet long-lasting, treatment effects (mean d = 0.25 for antisocial behavior, 0.24 overall) relative to comparison conditions. Furthermore, certain characteristics moderated the magnitude of treatment effects; for example, measures of substance use showed the largest effects and measures of peer relationships showed the smallest effects. CONCLUSIONS: Policymakers, administrators, and treatment providers may find it useful to consider the effects of family-based treatments for serious juvenile offenders in their selection of treatments for this population. In addition, investigators who seek to develop and study such treatments may wish to consider the current findings in their future research efforts. (PsycINFO Database Recordstatus: publishe