Qiaojun Fang

A histone fold TAF octamer within the yeast TFIID transcriptional coactivator

Gene activity in a eukaryotic cell is regulated by accessory factors to RNA polymerase II, which include the general transcription factor complex TFIID, composed of TBP and TBP-associated factors (TAFs). Three TAFs that contain histone fold motifs (yTAF17, yTAF60 and yTAF61) are critical for transcriptional regulation in the yeast Saccharomyces cerevisiae and are found in both TFIID and SAGA, a multicomponent histone acetyltransferase transcriptional coactivator. Although these three TAFs were proposed to assemble into a pseudooctamer complex, we find instead that yTAF17, yTAF60 and yTAF61 form a specific TAF octamer complex with a fourth TAF found in TFIID, yTAF48. We have reconstituted this complex in vitro and established that it is an octamer containing two copies each of the four components. Point mutations within the histone folds disrupt the octamer in vitro, and temperature-sensitive mutations in the histone folds can be specifically suppressed by overexpressing the other TAF octamer components in vivo. Our results indicate that the TAF octamer is similar both in stoichiometry and histone fold interactions to the histone octamer component of chromatin.

Brain-specific Proteins Decline in the Cerebrospinal Fluid of Humans with Huntington Disease

We integrated five sets of proteomics data profiling the constituents of cerebrospinal fluid (CSF) derived from Huntington disease (HD)-affected and -unaffected individuals with genomics data profiling various human and mouse tissues, including the human HD brain. Based on an integrated analysis, we found that brain-specific proteins are 1.8 times more likely to be observed in CSF than in plasma, that brain-specific proteins tend to decrease in HD CSF compared with unaffected CSF, and that 81% of brain-specific proteins have quantitative changes concordant with transcriptional changes identified in different regions of HD brain. The proteins found to increase in HD CSF tend to be liver-associated. These protein changes are consistent with neurodegeneration, microgliosis, and astrocytosis known to occur in HD. We also discuss concordance between laboratories and find that ratios of individual proteins can vary greatly, but the overall trends with respect to brain or liver specificity were consistent. Concordance is highest between the two laboratories observing the largest numbers of proteins.

A consistent set of statistical potentials for quantifying local side‐chain and backbone interactions

The frequencies of occurrence of atom arrangements in high‐resolution protein structures provide some of the most accurate quantitative measures of interaction energies in proteins. In this report we extend our development of a consistent set of statistical potentials for quantifying local interactions between side‐chains and the polypeptide backbone, as well as nearby side‐chains. Starting with ϕ/ψ/χ1 propensities that select for optimal interactions of the 20 amino acid side‐chains with the 2 flanking peptide bonds, the following 3 new terms are added: (1) a distance‐dependent interaction between the side‐chain at i and the carbonyl oxygens and amide protons of the peptide units at i ± 2, i ± 3, and i ± 4; (2) a distance‐dependent interaction between the side‐chain at position i and side‐chains at positions i + 1 through i + 4; and (3) an orientation‐dependent interaction between the side‐chain at position i and side‐chains at i + 1 through i + 4. The relative strengths of these 4 pseudo free energy terms are estimated by the average information content of each scoring matrix and by assessing their performance in a simple fragment threading test. They vary from −0.4–−0.5 kcal/mole per residue for ϕ/ψ/χ1 propensities to a range of −0.15–−0.6 kcal/mole per residue for each of the other 3 terms. The combined energy function, containing no interactions between atoms more than 4 residues apart, identifies the correct structural fragment for randomly selected 15 mers over 40% of the time, after searching through 232,000 alternative conformations. For 14 out of 20 sets of all‐atom Rosetta decoys analyzed, the native structure has a combined score lower than any of the 1700–1900 decoy conformations. The ability of this energy function to detect energetically important details of local structure is demonstrated by its power to distinguish high‐resolution crystal structures from NMR solution structures. Proteins 2005.

An Evaluation of Blood Compatibility of Silver Nanoparticles.

Silver nanoparticles (AgNPs) have tremendous potentials in medical devices due to their excellent antimicrobial properties. Blood compatibility should be investigated for AgNPs due to the potential blood contact. However, so far, most studies are not systematic and have not provided insights into the mechanisms for blood compatibility of AgNPs. In this study, we have investigated the blood biological effects, including hemolysis, lymphocyte proliferation, platelet aggregation, coagulation and complement activation, of 20 nm AgNPs with two different surface coatings (polyvinyl pyrrolidone and citrate). Our results have revealed AgNPs could elicit hemolysis and severely impact the proliferation and viability of lymphocytes at all investigated concentrations (10, 20, 40 μg/mL). Nevertheless, AgNPs didn’t show any effect on platelet aggregation, coagulation process, or complement activation at up to ~40 μg/mL. Proteomic analysis on AgNPs plasma proteins corona has revealed that acidic and small molecular weight blood plasma proteins were preferentially adsorbed onto AgNPs, and these include some important proteins relevant to hemostasis, coagulation, platelet, complement activation and immune responses. The predicted biological effects of AgNPs by proteomic analysis are mostly consistent with our experimental data since there were few C3 components on AgNPs and more negative than positive factors involving platelet aggregation and thrombosis.

Prediction of protein structure by emphasizing local side-chain/backbone interactions in ensembles of turn fragments.

The prediction strategy used in the CASP5 experiment was premised on the assumption that local side‐chain/backbone interactions are the principal determinants of protein structure at low resolution. Our implementation of this assumption made extensive use of a scoring function based on the propensities of the 20 amino acids for 137 different sub‐regions of the Ramachandran plot, allowing estimation of the quality of fit between a sequence segment and a known conformation. New folds were predicted in three steps: prediction of secondary structure, threading to isolate fragments of protein structures corresponding to one turn plus flanking helices/strands, and recombination of overlapping fragments. The most important step in this fragment ensemble approach, the isolation of turn fragments, employed 2 to 6 sequence homologues when available, with clustering of the best scoring fragments to recover the most common turn arrangement. Recombinants formed between 3 to 8 turn fragments, with cross‐overs confined to helix/strand segments, were selected for compactness plus low energy as estimated by empirical amino acid pair potentials, and the most common overall topology identified by visual inspection. Because significant amounts of steric overlap were permitted during the recombination step, the final model was manually adjusted to reduce overlap and to enhance protein‐like structural features. Even though only one or two models were submitted per target, for several targets the correct chain topology was predicted for fragment lengths up to 100 amino acids. Proteins 2003;53:486–490.

Software Platform for Rapidly Creating Computational Tools for Mass Spectrometry-Based Proteomics

We describe and demonstrate the proteomics computational toolkit provided in the open-source msInspect software distribution. The toolkit includes modules written in Java and in the R statistical programming language to aid the rapid development of proteomics software applications. It contains tools for processing and manipulating standard MS data files, including signal processing of LC-MS data and parsing of MS/MS search results, as well as for modeling proteomics data structures, creating charts, and other common tasks. We present this toolkits capability to rapidly develop new computational tools by presenting an example application, Qurate, a graphical tool for manually curating isotopically labeled peptide quantitative events.

Abraxane, the Nanoparticle Formulation of Paclitaxel Can Induce Drug Resistance by Up-Regulation of P-gp.

P-glycoprotein (P-gp) can actively pump paclitaxel (PTX) out of cells and induces drug resistance. Abraxane, a nanoparticle (NP) formulation of PTX, has multiple clinical advantages over the single molecule form. However, it is still unclear whether Abraxane overcomes the common small molecule drug resistance problem mediated by P-gp. Here we were able to establish an Abraxane-resistant cell line from the lung adenocarcinoma cell line A549. We compared the transcriptome of A549/Abr resistant cell line to that of its parental cell line using RNA-Seq technology. Several pathways were found to be up or down regulated. Specifically, the most significantly up-regulated gene was ABCB1, which translates into P-glycoprotein. We verified the overexpression of P-glycoprotein and confirmed its function by reversing the drug resistance with P-gp inhibitor Verapamil. The results suggest that efflux pathway plays an important role in the Abraxane-resistant cell line we established. However, the relevance of this P-gp mediated Abraxane resistance in tumors of lung cancer patients remains unknown.

Temperature-dependent structural stability and optical properties of ultrathin Hf–Al–O films grown by facing-target reactive sputtering

The structural stability and optical properties of ultrathin HfAlOx films grown by facing-target reactive sputtering, depending on the postannealing temperature, have been determined via x-ray photoelectron spectroscopy and spectroscopic ellipsometry (SE). By virtue of the chemical shifts of Hf4f, Al2p, and Si2p core-level spectra, it has been found that the structural stability of HfAlOx∕Si system sustains up to 800°C. However, the breaking of the Hf–Al–O bond and the phase separation take place drastically at the annealing temperature of 900°C. In particular, the information of an interfacial Si–O–Si bond as the dominant reaction during the postannealing treatment has been observed, confirmed by Fourier transform infrared spectroscopy. Analysis by SE, based on the Tauc-Lorentz model, has indicated that increase in the refractive index and reduction in thickness has been observed as a function of annealing temperature, originating from the annealing-induced higher packing density. The change of the compl...

Bimodal Imprint Chips for Peptide Screening: Integration of High-Throughput Sequencing by MS and Affinity Analyses by Surface Plasmon Resonance Imaging

Peptide probes and drugs have widespread applications in disease diagnostics and therapy. The demand for peptides ligands with high affinity and high specificity toward various targets has surged in the biomedical field in recent years. The traditional peptide screening procedure involves selection, sequencing, and characterization steps, and each step is manual and tedious. Herein, we developed a bimodal imprint microarray system to embrace the whole peptide screening process. Silver-sputtered silicon chip fabricated with microwell array can trap and pattern the candidate peptide beads in a one-well-one-bead manner. Peptides on beads were photocleaved in situ. A portion of the peptide in each well was transferred to a gold-coated chip to print the peptide array for high-throughput affinity analyses by surface plasmon resonance imaging (SPRi), and the peptide left in the silver-sputtered chip was ready for in situ single bead sequencing by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Using the bimodal imprint chip system, affinity peptides toward AHA were efficiently screened out from the 7 × 10(4) peptide library. The method provides a solution for high efficiency peptide screening.

Rapid Screening of Peptide Probes through In Situ Single-Bead Sequencing Microarray

Peptide ligands as targeting probes for in vivo imaging and drug delivery have attracted great interest in the biomedical community. However, high affinity and specificity screening of large peptide libraries remains a tedious process. Here, we report a continuous-flow microfluidic method for one-bead-one-compound (OBOC) combinatorial peptide library screening. We screened a library with 2 × 10(5) peptide beads within 4 h and discovered 140 noncanonical peptide hits targeting the tumor marker, aminopeptidase N (APN). Using the Clustal algorithm, we identified the conserved sequence Tyr-XX-Tyr in the N terminal. We demonstrated that the novel sequence YVEYHLC peptides have both nanomolar affinity and high specificity for APN in ex vivo and in vivo models. We envision that the successful demonstration of this integrated novel nanotechnology for peptide screening and identification open a new avenue for rapid discovery of new peptide-based reagents for disease diagnostics and therapeutics.