31 research outputs found
Rare X chromosome abnormalities in systemic lupus erythematosus and Sjögren's syndrome
Objective: Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE) are related by clinical and serologic manifestations as well as genetic risks. Both diseases are more commonly found in women than in men, at a ratio of ~10 to 1. Common X chromosome aneuploidies, 47,XXY and 47,XXX, are enriched among men and women, respectively, in either disease, suggesting a dose effect on the X chromosome. Methods: We examined cohorts of SS and SLE patients by constructing intensity plots of X chromosome single-nucleotide polymorphism alleles, along with determining the karyotype of selected patients. Results: Among ~2,500 women with SLE, we found 3 patients with a triple mosaic, consisting of 45,X/46,XX/47,XXX. Among ~2,100 women with SS, 1 patient had 45,X/46,XX/47,XXX, with a triplication of the distal p arm of the X chromosome in the 47,XXX cells. Neither the triple mosaic nor the partial triplication was found among the controls. In another SS cohort, we found a mother/daughter pair with partial triplication of this same region of the X chromosome. The triple mosaic occurs in ~1 in 25,000–50,000 live female births, while partial triplications are even rarer. Conclusion: Very rare X chromosome abnormalities are present among patients with either SS or SLE and may inform the location of a gene(s) that mediates an X dose effect, as well as critical cell types in which such an effect is operative. © 2017, American College of Rheumatolog
Multiplexed Enzyme Activity-Based Probe Display via Hybridization
Emulsions offer the means to miniaturize and parallelize high-throughput screening but require a robust method to localize activity-based fluorescent probes in each droplet. Multiplexing probes in droplets is impractical, though highly desirable for identifying library members that possess very specific activity. Here, we present multiplexed probe immobilization on library beads for emulsion screening. During library bead preparation, we quantitated ∼106 primers per bead by fluorescence in situ hybridization, however emulsion PCR yielded only ∼103 gene copies per bead. We leveraged the unextended bead-bound primers to hybridize complementary probe-oligonucleotide heteroconjugates to the library beads. The probe-hybridized bead libraries were then used to program emulsion in vitro transcription/translation reactions and analyzed by FACS to perform multiplexed activity-based screening of trypsin and chymotrypsin mutant libraries for novel proteolytic specificity. The approach's modularity should permit a high degree of probe multiplexing and appears extensible to other enzyme classes and library types
Hydrogel-Encapsulated Beads Enable Proximity-Driven Encoded Library Synthesis and Screening
Chemoselective Coupling Preserves the Substrate Integrity of Surface-Immobilized Oligonucleotides for Emulsion PCR-Based Gene Library Construction
Combinatorial
bead libraries figure prominently in next-generation
sequencing and are also important tools for in vitro evolution. The
most common methodology for generating such bead libraries, emulsion
PCR (emPCR), enzymatically extends bead-immobilized oligonucleotide
PCR primers in emulsion droplets containing a single progenitor library
member. Primers are almost always immobilized on beads via noncovalent
biotin–streptavidin binding. Here, we describe covalent bead
functionalization with primers (∼10<sup>6</sup> primers/2.8-μm-diameter
bead) via either azide–alkyne click chemistry or Michael addition.
The primers are viable polymerase substrates (4–7% bead-immobilized
enzymatic extension product yield from one thermal cycle). Carbodiimide-activated
carboxylic acid beads only react with oligonucleotides under conditions
that promote nonspecific interactions (low salt, low pH, no detergent),
comparably immobilizing primers on beads, but yielding no detectable
enzymatic extension product. Click-functionalized beads perform satisfactorily
in emPCR of a site-saturation mutagenesis library, generating monoclonal
templated beads (10<sup>4</sup>–10<sup>5</sup> copies/bead,
1.4-kb amplicons). This simpler, chemical approach to primer immobilization
may spur more economical library preparation for high-throughput sequencing
and enable more complex surface elaboration for in vitro evolution
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Strategies to recover proteins from ocular tissues for proteomics
DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis
The
promise of exploiting combinatorial synthesis for small molecule
discovery remains unfulfilled due primarily to the “structure
elucidation problem”: the back-end mass spectrometric analysis
that significantly restricts one-bead-one-compound (OBOC) library
complexity. The very molecular features that confer binding potency
and specificity, such as stereochemistry, regiochemistry, and scaffold
rigidity, are conspicuously absent from most libraries because isomerism
introduces mass redundancy and diverse scaffolds yield uninterpretable
MS fragmentation. Here we present DNA-encoded solid-phase synthesis
(DESPS), comprising parallel compound synthesis in organic solvent
and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides.
Computational encoding language design yielded 148 thermodynamically
optimized sequences with Hamming string distance ≥ 3 and total
read length <100 bases for facile sequencing. Ligation is efficient
(70% yield), specific, and directional over 6 encoding positions.
A series of isomers served as a testbed for DESPS’s utility
in split-and-pool diversification. Single-bead quantitative PCR detected
9 × 10<sup>4</sup> molecules/bead and sequencing allowed for
elucidation of each compound’s synthetic history. We applied
DESPS to the combinatorial synthesis of a 75 645-member OBOC
library containing scaffold, stereochemical and regiochemical diversity
using mixed-scale resin (160-μm quality control beads and 10-μm
screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19
quality control beads showed excellent agreement (<1 ppt) between
DNA sequence-predicted mass and the observed mass. DESPS synergistically
unites the advantages of solid-phase synthesis and DNA encoding, enabling
single-bead structural elucidation of complex compounds and synthesis
using reactions normally considered incompatible with unprotected
DNA. The widespread availability of inexpensive oligonucleotide synthesis,
enzymes, DNA sequencing, and PCR make implementation of DESPS straightforward,
and may prompt the chemistry community to revisit the synthesis of
more complex and diverse libraries
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Proteomic analyses of songbird (Zebra finch; Taeniopygia guttata) retina
Proteomic analyses of male songbird (Zebra finch; Taeniopygia guttata; ZF) retina were performed resulting in identification of 129 proteins. Comparison of T. guttata retinal proteome with that of chicken found proteins detected in both retinas. Immunohistochemical analyses of T. guttata retinal sections and Western analyses of total retinal protein extract were performed confirming presence of select bona fide retinal proteins. Results demonstrate the utility of one-dimensional gel fractionation for mass spectrometry and will be useful for future proteomic comparison of songbird retina and brain tissues in different behavioral and pharmacological studies
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Proteomic Analyses of Songbird (Zebra Finch; Taeniopygia Guttata) Retina
A cell based screening approach for identifying protein degradation regulators
<p>Cellular transitions are achieved by the concerted actions of regulated degradation pathways. In the case of the cell cycle, ubiquitin mediated degradation ensures unidirectional transition from one phase to another. For instance, turnover of the cell cycle regulator cyclin B1 occurs after metaphase to induce mitotic exit. To better understand pathways controlling cyclin B1 turnover, the N-terminal domain of cyclin B1 was fused to luciferase to generate an N-cyclin B1-luciferase protein that can be used as a reporter for protein turnover. Prior studies demonstrated that cell-based screens using this reporter identified small molecules inhibiting the ubiquitin ligase controlling cyclin B1-turnover. Our group adapted this approach for the G2-M regulator Wee1 where a Wee1-luciferase construct was used to identify selective small molecules inhibiting an upstream kinase that controls Wee1 turnover. In the present study we present a screening approach where cell cycle regulators are fused to luciferase and overexpressed with cDNAs to identify specific regulators of protein turnover. We overexpressed approximately 14,000 cDNAs with the N-cyclin B1-luciferase fusion protein and determined its steady-state level relative to other luciferase fusion proteins. We identified the known APC/C regulator Cdh1 and the F-box protein Fbxl15 as specific modulators of N-cyclin B1-luciferase steady-state levels and turnover. Collectively, our studies suggest that analyzing the steady-state levels of luciferase fusion proteins in parallel facilitates identification of specific regulators of protein turnover.</p