VDJServer: A Cloud-Based Analysis Portal and Data Commons for Immune Repertoire Sequences and Rearrangements.

Background: Recent technological advances in immune repertoire sequencing have created tremendous potential for advancing our understanding of adaptive immune response dynamics in various states of health and disease. Immune repertoire sequencing produces large, highly complex data sets, however, which require specialized methods and software tools for their effective analysis and interpretation.

Results: VDJServer is a cloud-based analysis portal for immune repertoire sequence data that provide access to a suite of tools for a complete analysis workflow, including modules for preprocessing and quality control of sequence reads, V(D)J gene segment assignment, repertoire characterization, and repertoire comparison.

VDJPipe: a pipelined tool for pre-processing immune repertoire sequencing data.

Background: Pre-processing of high-throughput sequencing data for immune repertoire profiling is essential to insure high quality input for downstream analysis. VDJPipe is a flexible, high-performance tool that can perform multiple pre-processing tasks with just a single pass over the data files.

Results: Processing tasks provided by VDJPipe include base composition statistics calculation, read quality statistics calculation, quality filtering, homopolymer filtering, length and nucleotide filtering, paired-read merging, barcode demultiplexing, 5' and 3' PCR primer matching, and duplicate reads collapsing.

VDJML: a file format with tools for capturing the results of inferring immune receptor rearrangements.

Background: The genes that produce antibodies and the immune receptors expressed on lymphocytes are not germline encoded; rather, they are somatically generated in each developing lymphocyte by a process called V(D)J recombination, which assembles specific, independent gene segments into mature composite genes. The full set of composite genes in an individual at a single point in time is referred to as the immune repertoire. V(D)J recombination is the distinguishing feature of adaptive immunity and enables effective immune responses against an essentially infinite array of antigens.

Individual heritable differences result in unique cell lymphocyte receptor repertoires of naïve and antigen-experienced cells.

The adaptive immune system's capability to protect the body requires a highly diverse lymphocyte antigen receptor repertoire. However, the influence of individual genetic and epigenetic differences on these repertoires is not typically measured. By leveraging the unique characteristics of B, CD4(+) T and CD8(+) T-lymphocyte subsets from monozygotic twins, we quantify the impact of heritable factors on both the V(D)J recombination process and on thymic selection.

owlcpp: a C++ library for working with OWL ontologies.

Background: The increasing use of ontologies highlights the need for a library for working with ontologies that is efficient, accessible from various programming languages, and compatible with common computational platforms.

Results: We developed owlcpp, a library for storing and searching RDF triples, parsing RDF/XML documents, converting triples into OWL axioms, and reasoning. The library is written in ISO-compliant C++ to facilitate efficiency, portability, and accessibility from other programming languages.

MSPrecise: A molecular diagnostic test for multiple sclerosis using next generation sequencing.

Background: We have previously demonstrated that cerebrospinal fluid-derived B cells from early relapsing-remitting multiple sclerosis (RRMS) patients that express a VH4 gene accumulate specific replacement mutations. These mutations can be quantified as a score that identifies such patients as having or likely to convert to RRMS. Furthermore, we showed that next generation sequencing is an efficient method for obtaining the sequencing information required by this mutation scoring tool, originally developed using the less clinically viable single-cell Sanger sequencing.

The antibody genetics of multiple sclerosis: comparing next-generation sequencing to sanger sequencing.

We previously identified a distinct mutation pattern in the antibody genes of B cells isolated from cerebrospinal fluid (CSF) that can identify patients who have relapsing-remitting multiple sclerosis (RRMS) and patients with clinically isolated syndromes who will convert to RRMS. This antibody gene signature (AGS) was developed using Sanger sequencing of single B cells. While potentially helpful to patients, Sanger sequencing is not an assay that can be practically deployed in clinical settings.

Haplotype Association Mapping Identifies a Candidate Gene Region in Mice Infected With Staphylococcus aureus.

Exposure to Staphylococcus aureus has a variety of outcomes, from asymptomatic colonization to fatal infection. Strong evidence suggests that host genetics play an important role in susceptibility, but the specific host genetic factors involved are not known. The availability of genome-wide single nucleotide polymorphism (SNP) data for inbred Mus musculus strains means that haplotype association mapping can be used to identify candidate susceptibility genes.

Single-molecule imaging of translational output from individual RNA granules in neurons.

Dendritic RNAs are localized and translated in RNA granules. Here we use single-molecule imaging to count the number of RNA molecules in each granule and to record translation output from each granule using Venus fluorescent protein as a reporter. For RNAs encoding activity-regulated cytoskeletal-associated protein (ARC) or fragile X mental retardation protein (FMRP), translation events are spatially clustered near individual granules, and translational output from individual granules is either sporadic or bursty.

The protease domain increases the translocation stepping efficiency of the hepatitis C virus NS3-4A helicase.

Hepatitis C virus (HCV) NS3 protein has two enzymatic activities of helicase and protease that are essential for viral replication. The helicase separates the strands of DNA and RNA duplexes using the energy from ATP hydrolysis. To understand how ATP hydrolysis is coupled to helicase movement, we measured the single turnover helicase translocation-dissociation kinetics and the pre-steady-state P(i) release kinetics on single-stranded RNA and DNA substrates of different lengths.

Experimental and computational analysis of DNA unwinding and polymerization kinetics.

DNA unwinding and polymerization are complex processes involving many intermediate species in the reactions. Our understanding of these processes is limited because the rates of the reactions or the existence of intermediate species is not apparent without specially designed experimental techniques and data analysis procedures. In this chapter we describe how pre-steady state and single-turnover measurements analyzed by model-based methods can be used for estimating the elementary rate constants.

Model-based global analysis of heterogeneous experimental data using gfit.

Regression analysis is indispensible for quantitative understanding of biological systems and for developing accurate computational models. By applying regression analysis, one can validate models and quantify components of the system, including ones that cannot be observed directly. Global (simultaneous) analysis of all experimental data available for the system produces the most informative results.

Mechanism of cadmium-mediated inhibition of Msh2-Msh6 function in DNA mismatch repair.

The observation that Cadmium (Cd(2+)) inhibits Msh2-Msh6, which is responsible for identifying base pair mismatches and other discrepancies in DNA, has led to the proposal that selective targeting of this protein and consequent suppression of DNA repair or apoptosis promote the carcinogenic effects of the heavy metal toxin. It has been suggested that Cd(2+) binding to specific sites on Msh2-Msh6 blocks its DNA binding and ATPase activities. To investigate the mechanism of inhibition, we measured Cd(2+) binding to Msh2-Msh6, directly and by monitoring changes in protein structure and enzymatic activity.

Mechanism of ATP-driven PCNA clamp loading by S. cerevisiae RFC.

Circular clamps tether polymerases to DNA, serving as essential processivity factors in genome replication, and function in other critical cellular processes as well. Clamp loaders catalyze clamp assembly onto DNA, and the question of how these proteins construct a topological link between a clamp and DNA, especially the mechanism by which ATP is utilized for the task, remains open. Here we describe pre-steady-state analysis of ATP hydrolysis, proliferating cell nuclear antigen (PCNA) clamp opening, and DNA binding by Saccharomyces cerevisiae replication factor C (RFC), and present the first kinetic model of a eukaryotic clamp-loading reaction validated by global data analysis.

Multiplexed RNA trafficking in oligodendrocytes and neurons.

In oligodendrocytes and neurons genetic information is transmitted from the nucleus to dendrites in the form of RNA granules. Here we describe how transport of multiple different RNA molecules in individual granules is analogous to the process of multiplexing in telecommunications. In both cases multiple messages are combined into a composite signal for transmission on a single carrier.

Multiplexed dendritic targeting of alpha calcium calmodulin-dependent protein kinase II, neurogranin, and activity-regulated cytoskeleton-associated protein RNAs by the A2 pathway.

In neurons, many different RNAs are targeted to dendrites where local expression of the encoded proteins mediates synaptic plasticity during learning and memory. It is not known whether each RNA follows a separate trafficking pathway or whether multiple RNAs are targeted to dendrites by the same pathway. Here, we show that RNAs encoding alpha calcium calmodulin-dependent protein kinase II, neurogranin, and activity-regulated cytoskeleton-associated protein are coassembled into the same RNA granules and targeted to dendrites by the same cis/trans-determinants (heterogeneous nuclear ribonucleoprotein [hnRNP] A2 response element and hnRNP A2) that mediate dendritic targeting of myelin basic protein RNA by the A2 pathway in oligodendrocytes.

Significant proportions of nuclear transport proteins with reduced intracellular mobilities resolved by fluorescence correlation spectroscopy.

Nuclear transport requires freely diffusing nuclear transport proteins to facilitate movement of cargo molecules through the nuclear pore. We analyzed dynamic properties of importin alpha, importin beta, Ran and NTF2 in nucleus, cytoplasm and at the nuclear pore of neuroblastoma cells using fluorescence correlation spectroscopy. Mobile components were quantified by global fitting of autocorrelation data from multiple cells.

DNA synthesis provides the driving force to accelerate DNA unwinding by a helicase.

Helicases are molecular motors that use the energy of nucleoside 5'-triphosphate (NTP) hydrolysis to translocate along a nucleic acid strand and catalyse reactions such as DNA unwinding. The ring-shaped helicase of bacteriophage T7 translocates along single-stranded (ss)DNA at a speed of 130 bases per second; however, T7 helicase slows down nearly tenfold when unwinding the strands of duplex DNA. Here, we report that T7 DNA polymerase, which is unable to catalyse strand displacement DNA synthesis by itself, can increase the unwinding rate to 114 base pairs per second, bringing the helicase up to similar speeds compared to its translocation along ssDNA.

Design and implementation of dynamic near-infrared optical tomographic imaging instrumentation for simultaneous dual-breast measurements.

Dynamic near-infrared optical tomographic measurement instrumentation capable of simultaneous bilateral breast imaging, having a capability of four source wavelengths and 32 source-detector fibers for each breast, is described. The system records dynamic optical data simultaneously from both breasts, while verifying proper optical fiber contact with the tissue through implementation of automatic schemes for evaluating data integrity. Factors influencing system complexity and performance are discussed, and experimental measurements are provided to demonstrate the repeatability of the instrumentation.

A Brownian motor mechanism of translocation and strand separation by hepatitis C virus helicase.

Helicases translocate along their nucleic acid substrates using the energy of ATP hydrolysis and by changing conformations of their nucleic acid-binding sites. Our goal is to characterize the conformational changes of hepatitis C virus (HCV) helicase at different stages of ATPase cycle and to determine how they lead to translocation. We have reported that ATP binding reduces HCV helicase affinity for nucleic acid.

The DNA-unwinding mechanism of the ring helicase of bacteriophage T7.

Helicases are motor proteins that use the chemical energy of NTP hydrolysis to drive mechanical processes such as translocation and nucleic acid strand separation. Bacteriophage T7 helicase functions as a hexameric ring to drive the replication complex by separating the DNA strands during genome replication. Our studies show that T7 helicase unwinds DNA with a low processivity, and the results indicate that the low processivity is due to ring opening and helicase dissociating from the DNA during unwinding.

The functional interaction of the hepatitis C virus helicase molecules is responsible for unwinding processivity.

Although helicases participate in virtually every cellular process involving nucleic acids, the details of their mechanism including the role of interaction between the subunits remains unclear. Here we study the unwinding kinetics of the helicase from hepatitis C virus using DNA substrates with a range of tail and duplex lengths. The binding of the helicase to the substrates was characterized by electron microscopy and fluorimetric titrations.

Fluorescence correlation spectroscopy and quantitative cell biology.

Fluorescence correlation spectroscopy (FCS) analyzes fluctuations in fluorescence within a small observation volume. Autocorrelation analysis of FCS fluctuation data can be used to measure concentrations, diffusion properties, and kinetic constants for individual fluorescent molecules. Photon count histogram analysis of fluorescence fluctuation data can be used to study oligomerization of individual fluorescent molecules.

ATP binding modulates the nucleic acid affinity of hepatitis C virus helicase.

The helicase of hepatitis C virus (HCV) unwinds nucleic acid using the energy of ATP hydrolysis. The ATPase cycle is believed to induce protein conformational changes to drive helicase translocation along the length of the nucleic acid. We have investigated the energetics of nucleic acid binding by HCV helicase to understand how the nucleotide ligation state of the helicase dictates the conformation of its nucleic acid binding site.

The +2 NTP binding drives open complex formation in T7 RNA polymerase.

Transcription initiation as catalyzed by T7 RNA polymerase consists primarily of promoter binding, strand separation, nucleotide binding, and synthesis of the first phosphodiester bond. The promoter strand separation process occurs at a very fast rate, but promoter opening is incomplete in the absence of the initiating NTPs. In this paper, we investigate how initiating NTPs affect the kinetics and thermodynamics of open complex formation.