Nano-Imprinted Thin Films of Reactive, Azlactone-Containing Polymers: Combining Methods for the Topographic Patterning of Cell Substrates with Opportunities for Facile Post-Fabrication Chemical Functionalization

Approaches to the fabrication of surfaces that combine methods for the topographic patterning of soft materials with opportunities for facile, post-fabrication chemical functionalization could contribute significantly to advances in biotechnology and a broad range of other areas. Here, we report methods that can be used to introduce well defined nano- and microscale topographic features to thin films of reactive polymers containing azlactone functionality using nanoimprint lithography (NIL). We demonstrate that NIL can be used to imprint topographic patterns into thin films of poly(2-vinyl-4,4- dimethylazlactone) and a copolymer of methyl methacrylate and 2-vinyl- 4,4-dimethylazlactone using silicon masters having patterns of grooves and ridges ranging in width from 400 nm to 2μm, demonstrating the potential f this method to transfer patterns to films of these reactive polymers over a range of feature sizes and densities. We demonstrate further that the azlactone functionality of these polymers survives temperatures and pressures associated with NIL, and that topographically patterned films can be readily functionalized post-fabrication by treatment of surface-accessible azlactone functionality with small molecules and polymers containing primary amines. The results of experiments in which NIH-3T3 cells were seeded onto films imprinted with lined patterns having a pitch of 4 demonstrated that cells attach and proliferate on these azlactone-containing films and that they align in the direction of the imprinted pattern. Finally, we demonstrate that the treatment of these materials with amine-functionalized poly(ethylene glycol) (PEG) can be used to create regions of topographically patterned films that prevent cell adhesion. The results of this study suggest approaches to the functionalization of topographically patterned surfaces with a broad range of chemical functionality (e.g., peptides, proteins, carbohydrates, etc.) of biotechnological interest. The ability to manipulate and define both the physical topography and chemical functionality of these reactive materials could provide opportunities to investigate the combined effects of substrate topography and chemical functionality on cell behavior and may also be useful in a broad range of other applications

Fabrication and Selective Functionalization of Amine-Reactive Polymer Multilayers on Topographically Patterned Microwell Cell Culture Arrays

We report an approach to the fabrication and selective functionalization of amine-reactive polymer multilayers on the surfaces of 3-D polyurethane-based microwell cell culture arrays. Reactive layer-by-layer assembly of multilayers using branched polyethyleneimine (BPEI) and the azlactone- functionalized polymer poly(2-vinyl-4,4′-dimethylazlactone) (PVDMA) yielded film-coated microwell arrays that could be chemically functionalized postfabrication by treatment with different amine-functionalized macromolecules or small molecule primary amines. Treatment of film-coated arrays with the small molecule amine d-glucamine resulted in microwell surfaces that resisted the adhesion and proliferation of mammalian fibroblast cells in vitro. These and other experiments demonstrated that it was possible to functionalize different structural features of these arrays in a spatially resolved manner to create dual-functionalized substrates (e.g., to create arrays having either (i) azlactone-functionalized wells, with regions between the wells functionalized with glucamine or (ii) substrates with spatially resolved regions of two different cationic polymers). In particular, spatial control over glucamine functionalization yielded 3-D substrates that could be used to confine cell attachment and growth to microwells for periods of up to 28 days and support the 3-D culture of arrays of cuboidal cell clusters. These approaches to dual functionalization could prove useful for the long-term culture and maintenance of cell types for which the presentation of specific and chemically well-defined 3-D culture environments is required for control over cell growth, differentiation, and other important behaviors. More generally, our approach provides methods for the straightforward chemical functionalization of otherwise unreactive topographically patterned substrates that could prove to be useful in a range of other fundamental and applied contexts. © 2011 American Chemical Society

\u3ci\u3eIn situ\u3c/i\u3e Synthesis of Oligonucleotide Arrays on Surfaces Coated with Crosslinked Polymer Multilayers

We report an approach to the in situ synthesis of oligonucleotide arrays on surfaces coated with crosslinked polymer multilayers. Our approach makes use of methods for the reactive layer-by-layer assembly of thin, amine-reactive multilayers using branched polyethyleneimine (PEI) and the azlactone-functionalized polymer poly(2-vinyl-4,4′-dimethylazlactone) (PVDMA). Postfabrication treatment of film-coated glass substrates with d-glucamine or 4-amino-1-butanol yielded hydroxyl-functionalized films suitable for the Maskless Array Synthesis (MAS) of oligonucleotide arrays. Glucamine-functionalized films yielded arrays of oligonucleotides with fluorescence intensities and signal-to-noise ratios (after hybridization with fluorescently labeled complementary strands) comparable to those of arrays fabricated on conventional silanized glass substrates. These arrays could be exposed to multiple hybridization/dehybridization cycles with only moderate loss of hybridization density. The versatility of the layer-by-layer approach also permitted synthesis directly on thin sheets of film-coated poly(ethylene terephthalate) (PET) to yield flexible oligonucleotide arrays that could be readily manipulated (e.g., bent) and cut into smaller arrays. To our knowledge, this work presents the first use of polymer multilayers as a substrate for the multistep synthesis of complex molecules. Our results demonstrate that these films are robust and able to withstand the ∼450 individual chemical processing steps associated with MAS (as well as manipulations required to hybridize, image, and dehybridize the arrays) without large-scale cracking, peeling, or delamination of the thin films. The combination of layer-by-layer assembly and MAS provides a means of fabricating functional oligonucleotide arrays on a range of different materials and substrates. This approach may also prove useful for the fabrication of supports for the solid-phase synthesis and screening of other macromolecular or small-molecule agents

Engineering bacteria to solve the Burnt Pancake Problem

<p>Abstract</p> <p>Background</p> <p>We investigated the possibility of executing DNA-based computation in living cells by engineering <it>Escherichia coli </it>to address a classic mathematical puzzle called the Burnt Pancake Problem (BPP). The BPP is solved by sorting a stack of distinct objects (pancakes) into proper order and orientation using the minimum number of manipulations. Each manipulation reverses the order and orientation of one or more adjacent objects in the stack. We have designed a system that uses site-specific DNA recombination to mediate inversions of genetic elements that represent pancakes within plasmid DNA.</p> <p>Results</p> <p>Inversions (or "flips") of the DNA fragment pancakes are driven by the <it>Salmonella typhimurium </it>Hin/<it>hix </it>DNA recombinase system that we reconstituted as a collection of modular genetic elements for use in <it>E. coli</it>. Our system sorts DNA segments by inversions to produce different permutations of a promoter and a tetracycline resistance coding region; <it>E. coli </it>cells become antibiotic resistant when the segments are properly sorted. Hin recombinase can mediate all possible inversion operations on adjacent flippable DNA fragments. Mathematical modeling predicts that the system reaches equilibrium after very few flips, where equal numbers of permutations are randomly sorted and unsorted. Semiquantitative PCR analysis of <it>in vivo </it>flipping suggests that inversion products accumulate on a time scale of hours or days rather than minutes.</p> <p>Conclusion</p> <p>The Hin/<it>hix </it>system is a proof-of-concept demonstration of <it>in vivo </it>computation with the potential to be scaled up to accommodate larger and more challenging problems. Hin/<it>hix </it>may provide a flexible new tool for manipulating transgenic DNA <it>in vivo</it>.</p

Dynamical Boson Stars

The idea of stable, localized bundles of energy has strong appeal as a model for particles. In the 1950s John Wheeler envisioned such bundles as smooth configurations of electromagnetic energy that he called {\em geons}, but none were found. Instead, particle-like solutions were found in the late 1960s with the addition of a scalar field, and these were given the name {\em boson stars}. Since then, boson stars find use in a wide variety of models as sources of dark matter, as black hole mimickers, in simple models of binary systems, and as a tool in finding black holes in higher dimensions with only a single killing vector. We discuss important varieties of boson stars, their dynamic properties, and some of their uses, concentrating on recent efforts.Comment: 79 pages, 25 figures, invited review for Living Reviews in Relativity; major revision in 201

Dynamics of Inflammatory Responses After SARS-CoV-2 Infection by Vaccination Status in the USA: A Prospective Cohort Study

BACKGROUND: Cytokines and chemokines play a critical role in the response to infection and vaccination. We aimed to assess the longitudinal association of COVID-19 vaccination with cytokine and chemokine concentrations and trajectories among people with SARS-CoV-2 infection. METHODS: In this longitudinal, prospective cohort study, blood samples were used from participants enrolled in a multi-centre randomised trial assessing the efficacy of convalescent plasma therapy for ambulatory COVID-19. The trial was conducted in 23 outpatient sites in the USA. In this study, participants (aged ≥18 years) were restricted to those with COVID-19 before vaccination or with breakthrough infections who had blood samples and symptom data collected at screening (pre-transfusion), day 14, and day 90 visits. Associations between COVID-19 vaccination status and concentrations of 21 cytokines and chemokines (measured using multiplexed sandwich immunoassays) were examined using multivariate linear mixed-effects regression models, adjusted for age, sex, BMI, hypertension, diabetes, trial group, and COVID-19 waves (pre-alpha or alpha and delta). FINDINGS: Between June 29, 2020, and Sept 30, 2021, 882 participants recently infected with SARS-CoV-2 were enrolled, of whom 506 (57%) were female and 376 (43%) were male. 688 (78%) of 882 participants were unvaccinated, 55 (6%) were partly vaccinated, and 139 (16%) were fully vaccinated at baseline. After adjusting for confounders, geometric mean concentrations of interleukin (IL)-2RA, IL-7, IL-8, IL-15, IL-29 (interferon-λ), inducible protein-10, monocyte chemoattractant protein-1, and tumour necrosis factor-α were significantly lower among the fully vaccinated group than in the unvaccinated group at screening. On day 90, fully vaccinated participants had approximately 20% lower geometric mean concentrations of IL-7, IL-8, and vascular endothelial growth factor-A than unvaccinated participants. Cytokine and chemokine concentrations decreased over time in the fully and partly vaccinated groups and unvaccinated group. Log INTERPRETATION: Initially and during recovery from symptomatic COVID-19, fully vaccinated participants had lower concentrations of inflammatory markers than unvaccinated participants suggesting vaccination is associated with short-term and long-term reduction in inflammation, which could in part explain the reduced disease severity and mortality in vaccinated individuals. FUNDING: US Department of Defense, National Institutes of Health, Bloomberg Philanthropies, State of Maryland, Mental Wellness Foundation, Moriah Fund, Octapharma, HealthNetwork Foundation, and the Shear Family Foundation

The Materials Research Platform: Defining the Requirements from User Stories

A recent meeting focused on accelerated materials design and discovery examined user requirements for a general, collaborative, integrative, and on-demand materials research platform

Identification of multiple risk loci and regulatory mechanisms influencing susceptibility to multiple myeloma

Genome-wide association studies (GWAS) have transformed our understanding of susceptibility to multiple myeloma (MM), but much of the heritability remains unexplained. We report a new GWAS, a meta-analysis with previous GWAS and a replication series, totalling 9974 MM cases and 247,556 controls of European ancestry. Collectively, these data provide evidence for six new MM risk loci, bringing the total number to 23. Integration of information from gene expression, epigenetic profiling and in situ Hi-C data for the 23 risk loci implicate disruption of developmental transcriptional regulators as a basis of MM susceptibility, compatible with altered B-cell differentiation as a key mechanism. Dysregulation of autophagy/apoptosis and cell cycle signalling feature as recurrently perturbed pathways. Our findings provide further insight

Genome-wide association study of classical Hodgkin lymphoma identifies key regulators of disease susceptibility

Several susceptibility loci for classical Hodgkin lymphoma (cHL) have been reported, however much of the heritable risk is unknown. Here, we perform a meta-analysis of two existing genome-wide association studies (GWAS), a new GWAS, and replication totalling 5,314 cases and 16,749 controls. We identify risk loci for all cHL at 6q22.33 (rs9482849, P=1.52 × 10-8) and for nodular sclerosis HL (NSHL) at 3q28 (rs4459895, P=9.43 × 10-17), 6q23.3 (rs6928977, P=4.62 × 10-55 11), 10p14 (rs3781093, P=9.49 × 10-13), 13q34 (rs112998813, P=4.58 × 10-8) and 16p13.13 (rs34972832, P=2.12 × 10-8). Additionally, independent loci within the HLA region are observed for NSHL (rs9269081, HLA-DPB1*03:01, Val86 in HLA-DRB1) and mixed cellularity HL (rs1633096, rs13196329, Val86 in HLA-DRB1). The new and established risk loci localise to areas of active chromatin and show an over-representation of transcription factor binding for determinants of B-cell development and immune response.In the United Kingdom, Bloodwise (LLR; 10021) provided principal funding for the study. Support from Cancer Research UK (C1298/A8362 supported by the Bobby Moore Fund) and the Lymphoma Research Trust is also acknowledged. A.S. is supported by a clinical fellowship from Cancer Research UK. For the UK-GWAS, sample and data acquisition were supported by Breast Cancer Now, the European Union and the Lymphoma Research Trust. The UK-GWAS made use of control genotyping data generated by the WTCCC. For further information, please visit the publishr's website