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Epigenetic alterations, such as histone acetylation, regulate the signaling outcomes and phenotypic responses of fibroblasts after growth factor stimulation. The bromodomain and extra-terminal domain-containing proteins (Brd) bind to acetylated histone residues, resulting in recruitment of components of the transcriptional machinery and subsequent gene transcription. Given the central importance of fibroblasts in tissue fibrosis, this study sought to determine the role of Brd proteins in human lung fibroblasts (LFs) after growth factor stimulation and in the murine bleomycin model of lung fibrosis. Using small interfering RNA against human Brd2 and Brd4 and pharmacologic Brd inhibitors, this study found that Brd2 and Brd4 are essential in mediating the phenotypic responses of LFs downstream of multiple growth factor pathways. Growth factor stimulation of LFs causes increased histone acetylation, association of Brd4 with growth factor-responsive genes, and enhanced transcription of these genes that could be attenuated with pharmacologic Brd inhibitors. Of note, lung fibrosis induced after intratracheal bleomycin challenge in mice could be prevented by pretreatment of animals with pharmacologic inhibitors of Brd proteins. This study is the first demonstration of a role for Brd2 and Brd4 proteins in mediating the responses of LFs after growth factor stimulation and in driving the induction of lung fibrosis in mice in response to bleomycin challenge.

Glucokinase (GK) activation as a potential strategy to treat type 2 diabetes (T2D) is well recognized. Compound 1, a glucokinase activator (GKA) lead that we have previously disclosed, caused reversible hepatic lipidosis in repeat-dose toxicology studies. We hypothesized that the hepatic lipidosis was due to the structure-based toxicity and later established that it was due to the formation of a thiourea metabolite, 2. Subsequent SAR studies of 1 led to the identification of a pyrazine-based lead analogue 3, lacking the thiazole moiety. In vivo metabolite identification studies, followed by the independent synthesis and profiling of the cyclopentyl keto- and hydroxyl- metabolites of 3, led to the selection of piragliatin, 4, as the clinical lead. Piragliatin was found to lower pre- and postprandial glucose levels, improve the insulin secretory profile, increase β-cell sensitivity to glucose, and decrease hepatic glucose output in patients with T2D.

Ionization matrix effects are one of the most difficult issues in LC–MS bioanalysis that are without a good and universal solution. Most people in the field are aware of it, but some are not sure how to deal with it. Many laboratories do not routinely assess matrix effects in method validation or substitute it with a different experiment that only indirectly and partially test matrix effects. Others, when matrix effects are mentioned, immediately link them with phospholipids and try to use means to remove them from the samples (e.g., SPE), without understanding if the phospholipids can really impact the analytes. In this article, key issues related to matrix effects will be examined, and methods to address matrix effect problems will be evaluated for effectiveness and practicality.

High quality, ultra-fast bioanalytical LC/MS/MS methods were developed using short columns packed with fused-core particles and high (1.0-3.0 mL/min) flow rates. For more than two years, at flow rates up to 3.0 mL/min, using 0.33 min non-ballistic gradients, these methods were shown to provide comparable or better performance than slower assays for accuracy, precision, sensitivity, specificity, and ruggedness, and met all criteria required by the bioanalytical regulatory guidance.

The preclinical model of bleomycin-induced lung fibrosis, used to investigate mechanisms related to idiopathic pulmonary fibrosis (IPF), has incorrectly predicted efficacy for several candidate compounds suggesting that it may be of limited value. As an attempt to improve the predictive nature of this model, integrative bioinformatic approaches were used to compare molecular alterations in the lungs of bleomycin-treated mice and patients with IPF. Using gene set enrichment analysis we show for the first time that genes differentially expressed during the fibrotic phase of the single challenge bleomycin model were significantly enriched in the expression profiles of IPF patients. The genes that contributed most to the enrichment were largely involved in mitosis, growth factor, and matrix signaling. Interestingly, these same mitotic processes were increased in the expression profiles of fibroblasts isolated from rapidly progressing, but not slowly progressing, IPF patients relative to control subjects. The data also indicated that TGFβ was not the sole mediator responsible for the changes observed in this model since the ALK-5 inhibitor SB525334 effectively attenuated some but not all of the fibrosis associated with this model. Although some would suggest that repetitive bleomycin injuries may more effectively model IPF-like changes, our data do not support this conclusion. Together, these data highlight that a single bleomycin instillation effectively replicates several of the specific pathogenic molecular changes associated with IPF, and may be best used as a model for patients with active disease.

The ultrahigh-resolution capabilities of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) have been demonstrated for accurate mass measurements of complex hydrocarbon mixtures. Ultrahigh resolution is achievable over a wide mass range by the use of stored waveform inverse Fourier transform (SWIFT) ejection to eliminate space-charge effects. The mass measurement stability under different ionization conditions eliminates the need of mass reference standards during acquisition of sample spectra, thereby increasing sensitivity of the accurate mass measurements under low-voltage electron ionization conditions. 27 refs., 4 figs., 2 tabs.

It is desirable to be able to predict the precision of a repeated measurement, based on the result of a single measurement and knowledge of the noise level in the original (time-domain) data. We have previously shown that precision in determination of time-domain signal parameters (initial magnitude, frequency, and exponential damping constant for a noisy exponentially damped sinusoidal signal) from least-squares fit to a frequency-domain FFT spectrum is directly proportional to frequency-domain peak height-to-noise ratio and to the square root of the number of data points per peak width, with a proportionality constant which depends on the spectral type (absorption mode, magnitude mode) and peak shape (e.g., Lorentzian, Gaussian, etc.). In this paper, we show that precision in determination of those same parameters by least-squares fit to the time-domain signal itself is similarly related to time-domain initial amplitude-to-noise ratio and the square root of the number of data points per damping period. In addition, we show that although magnitude-mode spectral noise is well described by a Rayleigh distribution in the signal-free baseline segments of the spectrum, noise in the vicinity of a magnitude-mode spectral peak is more accurately described by a normal (Gaussian) distribution. We then proceed to show that determination of spectral parameters from a time-domain data set is more precise by a factor of √2 than estimates based on the FT absorption-mode spectrum. We further show that padding of the N-point time-domain data set by another N zeroes before FFT improves frequency-domain absorption-mode precision by the same √2 factor. Additional zero-filling does not improve spectral precision. Zero-filling has no effect on precision of spectral parameters determined from time-domain data, and variable effects on parameters determined from fits to magnitude-mode spectra. The above theoretical predictions are supported by analysis of simulated noisy data.

Laser desprption/ionization combined with pulsed (time-of-flight or Fourier transform ion cyclotron resonance) mass spectrometric detection is a powerful technique for analysis of involatile compounds and mixtures. Such experiments were originally conducted with pulsed CO2 lasers. Although a pulsed CO2 laser can be operated in single-shot mode, Nd: YAG lasers perform best with multiple flashes for warm-up before the final Q-switch output light pulse, thus creating the need to synchronize the desired final laser-output pulse with the event sequence for mass spectrometric analysis. In this paper, we describe a new and simple interface (both optical and electronic components) between a Continuum (formerly Quantel) Model YG 660A Nd: YAG laser and an Extrel FTMS-2000 mass spectrometer. The optics are modified from a prior pulsed CO2 laser interface from Extrel. Synchronization betweem th Nd: YAG laser and the mass spectrometer event sequence is achieved by means of a simple timing circuit that uses an inexpensive pulsing device and is triggered by pulses generated directly from the Extrel 1280 data system and cell controller, in contrast to the only prior published method that required an auxiliary microcomputer. The present interface method is highly flexible, and makes possible complex sequence events involving laser pulses for e.g.: desorption/ionization of solids; photoionization of gaseous neutrals; and photodissociation and photodetachment of gaseous ions.

The area under a correctly phased absorption-mode spectral peak is a direct measure of the number of oscillators (ions, spins, molecules) in Fourier transform spectrometry (ion cyclotron resonance, magnetic resonance, interferometry absorbance). However, phase correction can prove difficult when (as in broad-band Fourier transform ion cyclotron resonance (FT/ICR] detection is considerably time-delayed after excitation. In the absence of noise, Huang, Rempel, and Gross showed that a "complex area" method yields the correct absorption-mode peak area, for an unphased noiseless spectrum. In this paper, we show that the number of oscillators may also be obtained from a least-squares fit to a magnitude-mode (i.e., phase-independent) spectrum. In the presence of noise and in the absence of peak overlap, the magnitude-mode method offers precision superior to that based on magnitude-mode peak height, "complex area", or even direct digital integration of a correctly phased absorption-mode peak, as demonstrated by both theoretical derivation and experimental FT/ICR results. The present method thus appears to offer the best available determination of the relative abundances of ions of different mass-to-charge ratio in FT/ICR mass spectrometry.