Late-Stage Reshaping of Phage-Displayed Libraries to Macrocyclic and Bicyclic Landscapes using a Multipurpose Linchpin.

Genetically encoded libraries (GEL) are increasingly being used for the discovery of ligands for "undruggable" targets that cannot be addressed with small molecules. Foundational GEL platforms like phage-, yeast-, ribosome-, and mRNA-display have enabled the display of libraries composed of 20 natural amino acids (20AA). Unnatural amino acids (UAA) and chemical post-translational modification (cPTM) expanded GEL beyond the 20AA space to yield unnatural linear, cyclic, and bicyclic peptides.

The liquid lectin array detects compositional glycocalyx differences using multivalent DNA-encoded lectins on phage.

Selective detection of disease-associated changes in the glycocalyx is an emerging field in modern targeted therapies. Detecting minor glycan changes on the cell surface is a challenge exacerbated by the lack of correspondence between cellular DNA/RNA and glycan structures. We demonstrate that multivalent displays of lectins on DNA-barcoded phages-liquid lectin array (LiLA)-detect subtle differences in density of glycans on cells.

Measuring carbohydrate recognition profile of lectins on live cells using liquid glycan array (LiGA).

Glycans constitute a significant fraction of biomolecular diversity on cellular surfaces across all kingdoms of life. As the structure of glycans is not directly encoded by the organism's DNA, it is impossible to use high-throughput DNA technologies to study the role of cellular glycosylation or to understand how glycocalyx is recognized by glycan-binding proteins (GBPs). To address this gap, we recently described a liquid glycan array (LiGA) platform that allows profiling of glycan-GBP interactions on the surface of live cells in vitro and in vivo using next-generation sequencing.

DNA-Encoded Multivalent Display of Chemically Modified Protein Tetramers on Phage: Synthesis and Applications.

Phage display links the phenotype of displayed polypeptides with the DNA sequence in the phage genome and offers a universal method for the discovery of proteins with novel properties. However, the display of large multisubunit proteins on phages remains a challenge. A majority of protein display systems are based on monovalent phagemid constructs, but methods for the robust display of multiple copies of large proteins are scarce.

Colloid chemistry pitfall for flow cytometric enumeration of viruses in water.

Flow cytomtery (FCM) has become a standard approach to enumerate viruses in water research. However, the nature of the fluorescent signal in flow cytometric analysis of water samples and the mechanism of its formation, have not been addressed for bacteriophages expected in wastewaters. Here we assess the behaviour of fluorescent DNA-staining dyes in aqueous solutions, as well as sensitivity and accuracy of FCM for enumeration of DNA-stained model bacteriophages λ, P1, and T4.

A lab-on-chip for malaria diagnosis and surveillance.

Background: Access to timely and accurate diagnostic tests has a significant impact in the management of diseases of global concern such as malaria. While molecular diagnostics satisfy this need effectively in developed countries, barriers in technology, reagent storage, cost and expertise have hampered the introduction of these methods in developing countries. In this study a simple, lab-on-chip PCR diagnostic was created for malaria that overcomes these challenges.

A novel method for sample delivery and testing of whole blood: gel strip PCR for point of care (POC) molecular diagnostics.

Testing of whole blood in miniaturized PCR is compromised by the opaque nature of whole blood that leads to physical masking of a fluorescent signal. We demonstrate a method to perform real-time PCR with whole blood that avoids interference from the opacity of whole blood.

A miniaturized and integrated gel post platform for multiparameter PCR detection of herpes simplex viruses from raw genital swabs.

Herpes simplex virus (HSV) is one of the most prevalent viruses, with acute and recurrent infections in humans. The current gold standard for the diagnosis of HSV is viral culture which takes 2-14 days and has low sensitivity. In contrast, DNA amplification by polymerase chain reaction (PCR) can be performed within 1-2 h.

In-gel technology for PCR genotyping and pathogen detection.

This work describes the use of polyacrylamide gel and PCR reagents photopolymerized in a mold to create an array of semisolid posts that serve as reaction vessels for parallel PCR amplification of an externally added template. DNA amplification occurred in a cylindrical, self-standing 9 × 9 array of gel posts each less than 1 μL in volume. Photopolymerization of the gel with an intercalating dye added prior to polymerization permitted acquisition of real-time PCR data and melting curve analysis data without the need for any type of post-PCR staining procedures.

Strategies for enhancing the speed and integration of microchip genetic amplification.

In this work, we explore the use of methods that allow a significant acceleration of genetic analysis within microchips fabricated from low thermal conductivity materials such as glass or polymers. Although these materials are highly suitable for integrating a number of genetic analysis techniques onto lab-on-a-chip devices, their low thermal conductivity limits the rate at which heat can be transferred and hence lowers the speed of thermal cycling. However, short thermal cycling times are the key to bringing PCR to clinical point-of-care applications.

Microfluidic platform for single nucleotide polymorphism genotyping of the thiopurine S-methyltransferase gene to evaluate risk for adverse drug events.

Prospective clinical pharmacogenetic testing of the thiopurine S-methyltransferase gene remains to be realized despite the large body of evidence demonstrating clinical benefit for the patient and cost effectiveness for health care systems. We describe an entirely microchip-based method to genotype for common single nucleotide polymorphisms in the thiopurine S-methyltransferase gene that lead to serious adverse drug reactions for patients undergoing thiopurine therapy. Restriction fragment length polymorphism and allele-specific polymerase chain reaction have been adapted to a microfluidic chip-based polymerase chain reaction and capillary electrophoresis platform to genotype the common *2, *3A, and *3C functional alleles.

Microfluidic chips for detecting the t(4;14) translocation and monitoring disease during treatment using reverse transcriptase-polymerase chain reaction analysis of IgH-MMSET hybrid transcripts.

Diagnosis platforms incorporating low-cost microfluidic chips enable sensitive, rapid, and accurate genetic analysis that could facilitate customized therapies tailored to match the vulnerabilities of any types of cancer. Using ex vivo cancer cells, we have detected the unique molecular signature and a chromosomal translocation in multiple myeloma. Multiple myeloma is characterized by IgH rearrangements and translocations that enable unequivocal identification of malignant cells, detected here with integrated microfluidic chips incorporating genetic amplification via reverse transcriptase-polymerase chain reaction and capillary electrophoresis.

CD4 T cells play major effector role and CD8 T cells initiating role in spontaneous autoimmune myocarditis of HLA-DQ8 transgenic IAb knockout nonobese diabetic mice.

In humans, spontaneous autoimmune attack against cardiomyocytes often leads to idiopathic dilated cardiomyopathy (IDCM) and life-threatening heart failure. HLA-DQ8 transgenic IAb knockout NOD mice (NOD.DQ8/Ab(0); DQA1*0301, DQB1*0302) develop spontaneous anticardiomyocyte autoimmunity with pathology very similar to human IDCM, but why the heart is targeted is unknown.

Genetic analysis of completely sequenced disease-associated MHC haplotypes identifies shuffling of segments in recent human history.

The major histocompatibility complex (MHC) is recognised as one of the most important genetic regions in relation to common human disease. Advancement in identification of MHC genes that confer susceptibility to disease requires greater knowledge of sequence variation across the complex. Highly duplicated and polymorphic regions of the human genome such as the MHC are, however, somewhat refractory to some whole-genome analysis methods.

Sensitive detection using microfluidics technology of single cell PCR products from high and low abundance IgH VDJ templates in multiple myeloma.

Human cancer is inherently heterogeneous, so the ability to monitor individual cancer cells at every clinic visit would be a valuable tool. This work describes the first step towards developing handheld and automated devices for molecular and phenotypic analysis of cancer cells. Here, we show that use of capillary electrophoresis to detect PCR product amplified from either transcripts (high abundance template) or genomic DNA (low abundance template) encoding clonotypic immunoglobulin heavy chain VDJ of plasma cells from patients with multiple myeloma.

Complete MHC haplotype sequencing for common disease gene mapping.

The future systematic mapping of variants that confer susceptibility to common diseases requires the construction of a fully informative polymorphism map. Ideally, every base pair of the genome would be sequenced in many individuals. Here, we report 4.