The effect of hydrophobic patterning on micromolding of aqueous-derived silk structures

A novel micromolding approach was developed to process liquid biopolymers with high aqueous solvent contents (\u3e90% water). Specifically silk fibroin was cast into a well-defined scaffold-like structures for potential tissue engineering applications. A method was developed to pattern the hydrophilicity and hydrophobicity of the polydimethylsiloxane (PDMS) mold surfaces. The water based biopolymer solution could then be directly applied to the desired regions on the cast surface. The variations in degree of hydrophilicity and hydrophobicity on the PDMS surfaces were quantified through contact angle measurements and compared to the outcome of the molded silk structures. Through this method free-standing structures (vs. relief surface-patterning) could be fabricated. © 2008 Materials Research Society

The effect of hydrophobic patterning on micromolding of aqueous-derived silk structures

A novel micromolding approach was developed to process liquid biopolymers with high aqueous solvent contents (\u3e90% water). Specifically silk fibroin was cast into a well-defined scaffold-like structures for potential tissue engineering applications. A method was developed to pattern the hydrophilicity and hydrophobicity of the polydimethylsiloxane (PDMS) mold surfaces. The water based biopolymer solution could then be directly applied to the desired regions on the cast surface. The variations in degree of hydrophilicity and hydrophobicity on the PDMS surfaces were quantified through contact angle measurements and compared to the outcome of the molded silk structures. Through this method free-standing structures (vs. relief surface-patterning) could be fabricated. © 2008 Materials Research Society

A New Toolbox for Assessing Single Cells

Unprecedented access to the biology of single cells is now feasible, enabled by recent technological advancements that allow us to manipulate and measure sparse samples and achieve a new level of resolution in space and time. This review focuses on advances in tools to study single cells for specific areas of biology. We examine both mature and nascent techniques to study single cells at the genomics, transcriptomics, and proteomics level. In addition, we provide an overview of tools that are well suited for following biological responses to defined perturbations with single-cell resolution. Techniques to analyze and manipulate single cells through soluble and chemical ligands, the microenvironment, and cell-cell interactions are provided. For each of these topics, we highlight the biological motivation, applications, methods, recent advances, and opportunities for improvement. The toolbox presented in this review can function as a starting point for the design of single-cell experiments. © 2014 by Annual Reviews

The distribution of polysaccharide-specific antibody-secreting and memory B cells against the four polysaccharides.

<p>The number of PS-specific ASCs (A) and MBCs (B) specific for each antigen at days 0, 7 and 28 post-vaccination are depicted. The radius of each circle in the center panel is proportional to the number of PS-specific ASCs or MBCs detected, and the antigen specificity is indicated by the color of each circle. The antigen distribution for all ASCs and MBCs within each vaccine-group are depicted in pie charts. Numbers in the center of the pie charts indicate the total numbers of ASCs or MBCs detected. The distribution of antigen specificity was compared using a 2XN Fisher’s test.</p

Neutralizing antibodies against West Nile virus identified directly from human B cells by single-cell analysis and next generation sequencing

West Nile virus (WNV) infection is an emerging mosquito-borne disease that can lead to severe neurological illness and currently has no available treatment or vaccine. Using microengraving, an integrated single-cell analysis method, we analyzed a cohort of subjects infected with WNV – recently infected and post-convalescent subjects – and efficiently identified four novel WNV neutralizing antibodies. We also assessed the humoral response to WNV on a single-cell and repertoire level by integrating next generation sequencing (NGS) into our analysis. The results from single-cell analysis indicate persistence of WNV-specific memory B cells and antibody-secreting cells in post-convalescent subjects. These cells exhibited class-switched antibody isotypes. Furthermore, the results suggest that the antibody response itself does not predict the clinical severity of the disease (asymptomatic or symptomatic). Using the nucleotide coding sequences for WNV-specific antibodies derived from single cells, we revealed the ontogeny of expanded WNV-specific clones in the repertoires of recently infected subjects through NGS and bioinformatic analysis. This analysis also indicated that the humoral response to WNV did not depend on an anamnestic response, due to an unlikely previous exposure to the virus. The innovative and integrative approach presented here to analyze the evolution of neutralizing antibodies from natural infection on a single-cell and repertoire level can also be applied to vaccine studies, and could potentially aid the development of therapeutic antibodies and our basic understanding of other infectious diseases.National Institutes of Health (U.S.) (U19AI089992)National Institutes of Health (U.S.) (AI091816)National Institutes of Health (U.S.) (R01AI104739)National Institutes of Health (U.S.) (1F32AI112359-01)National Institutes of Health (U.S.) (T15LM07056)Human Immunology Project ConsortiumGillson Longenbaugh Foundatio

The heavy-chain gene-family usage and CDR3 length of polysaccharide-specific memory B cells.

<p>Sequences recovered from PS-specific MBCs were analyzed by IMGT/HighV-QUEST using the rhesus macaque immunoglobulin database. The heavy chain alleles of each B cell were generated by RT-PCR. (A) Heavy-chain gene-family usage at day 0 and 7 after immunization with 23vPS or 13vPnC. The number in the pie chart is the average number of variable sequences recovered from PS-specific MBCs. Pairwise statistical analysis was performed by Fisher’s Exact Test. (B) Distribution of heavy-chain gene-family usage for each PS serotype from PS-specific MBCs at 7 days following immunization with 13vPnC. The number in the pie chart is the average number of PS-specific MBCs against each antigen. Statistical analysis was performed by Fisher’s Exact Test. (C) The average CDR3 amino acid length of PS-specific MBCs at 0 and 7 days after immunization.</p

The distribution of polysaccharide-specific antibody-secreting and memory B cells against the four polysaccharides.

<p>The number of PS-specific ASCs (A) and MBCs (B) specific for each antigen at days 0, 7 and 28 post-vaccination are depicted. The radius of each circle in the center panel is proportional to the number of PS-specific ASCs or MBCs detected, and the antigen specificity is indicated by the color of each circle. The antigen distribution for all ASCs and MBCs within each vaccine-group are depicted in pie charts. Numbers in the center of the pie charts indicate the total numbers of ASCs or MBCs detected. The distribution of antigen specificity was compared using a 2XN Fisher’s test.</p

Longitudinal multiparameter single-cell analysis of macaques immunized with pneumococcal protein-conjugated or unconjugated polysaccharide vaccines reveals distinct antigen specific memory B cell repertoires

<div><p>Background</p><p>The efficacy of protein-conjugated pneumococcal polysaccharide vaccines has been well characterized for children. The level of protection conferred by unconjugated polysaccharide vaccines remains less clear, particularly for elderly individuals who have had prior antigenic experience through immunization with unconjugated polysaccharide vaccines or natural exposure to <i>Streptococcus pneumoniae</i>.</p><p>Methods</p><p>We compared the magnitude, diversity and genetic biases of antigen-specific memory B cells in two groups of adult cynomolgus macaques that were immunized with a 7-valent conjugated vaccine and boosted after five years with either a 13-valent pneumococcal polysaccharide conjugate vaccine (13vPnC) or a 23-valent unconjugated pneumococcal polysaccharide vaccine (23vPS) using microengraving (a single-cell analysis method) and single-cell RT-PCR.</p><p>Results</p><p>Seven days after boosting, the mean frequency of antigen-specific memory B cells was significantly increased in macaques vaccinated with 13vPnC compared to those receiving 23vPS. The 13vPnC-vaccinated macaques also exhibited a more even distribution of antibody specificities to four polysaccharides in the vaccine (PS4, 6B, 14, 23F) that were examined. However, single-cell analysis of the antibody variable region sequences from antigen-specific B cells elicited by unconjugated and conjugated vaccines indicated that both the germline gene segments forming the heavy chains and the average lengths of the Complementary Determining Region 3 (CDR3) were similar.</p><p>Conclusions</p><p>Our results confirm that distinctive differences can manifest between antigen-specific memory B cell repertoires in nonhuman primates immunized with conjugated and unconjugated pneumococcal polysaccharide vaccines. The study also supports the notion that the conjugated vaccines have a favorable profile in terms of both the frequency and breadth of the anamnestic response among antigen-specific memory B cells.</p></div