The experimental power of FR900359 to study Gq-regulated biological processes.

Despite the discovery of heterotrimeric αβγ G proteins ∼25 years ago, their selective perturbation by cell-permeable inhibitors remains a fundamental challenge. Here we report that the plant-derived depsipeptide FR900359 (FR) is ideally suited to this task. Using a multifaceted approach we systematically characterize FR as a selective inhibitor of Gq/11/14 over all other mammalian Gα isoforms and elaborate its molecular mechanism of action. We also use FR to investigate whether inhibition of Gq proteins is an effective post-receptor strategy to target oncogenic signalling, using melanoma as a model system. FR suppresses many of the hallmark features that are central to the malignancy of melanoma cells, thereby providing new opportunities for therapeutic intervention. Just as pertussis toxin is used extensively to probe and inhibit the signalling of Gi/o proteins, we anticipate that FR will at least be its equivalent for investigating the biological relevance of Gq

Denoising Two-Photon Calcium Imaging Data

Two-photon calcium imaging is now an important tool for in vivo imaging of biological systems. By enabling neuronal population imaging with subcellular resolution, this modality offers an approach for gaining a fundamental understanding of brain anatomy and physiology. Proper analysis of calcium imaging data requires denoising, that is separating the signal from complex physiological noise. To analyze two-photon brain imaging data, we present a signal plus colored noise model in which the signal is represented as harmonic regression and the correlated noise is represented as an order autoregressive process. We provide an efficient cyclic descent algorithm to compute approximate maximum likelihood parameter estimates by combing a weighted least-squares procedure with the Burg algorithm. We use Akaike information criterion to guide selection of the harmonic regression and the autoregressive model orders. Our flexible yet parsimonious modeling approach reliably separates stimulus-evoked fluorescence response from background activity and noise, assesses goodness of fit, and estimates confidence intervals and signal-to-noise ratio. This refined separation leads to appreciably enhanced image contrast for individual cells including clear delineation of subcellular details and network activity. The application of our approach to in vivo imaging data recorded in the ferret primary visual cortex demonstrates that our method yields substantially denoised signal estimates. We also provide a general Volterra series framework for deriving this and other signal plus correlated noise models for imaging. This approach to analyzing two-photon calcium imaging data may be readily adapted to other computational biology problems which apply correlated noise models.National Institutes of Health (U.S.) (DP1 OD003646-01)National Institutes of Health (U.S.) (R01EB006385-01)National Institutes of Health (U.S.) (EY07023)National Institutes of Health (U.S.) (EY017098

SQUID gradiometry for magnetocardiography using different noise cancellation techniques

Magnetocardiographic (MCG) measurements in unshielded environment require efficient noise cancellation techniques. We have applied two software gradiometry methods to analyze the time series of signal and reference data recorded outside magnetic shielding with high temperature superconducting quantum interference device (HTS SQUID) based gradiometers. One method uses adaptive frequency dependent gradiometer coefficients determined in the Fourier domain to subtract the reference from the signal data. The other method combines recently developed techniques for nonlinear projection with properties of the wavelet transform to extract noise in state space. The analyzed MCG data sets showed improved signal-to-noise ratios for both methods as compared to the data recorded with the electronic gradiometer. In this way, it is possible to increase the bandwidth from 130 Hz for our electronic gradiometer to 250 Hz without using any additional filtering

Adoptive T Cell Therapy Targeting Different Gene Products Reveals Diverse and Context-Dependent Immune Evasion in Melanoma

Tumor immune escape limits durable responses to T cell therapy. Here, we examined how regulation and function of gene products that provide the target epitopes for CD8(+) T cell anti-tumor immunity influence therapeutic efficacy and resistance. We used a CRISPR-Cas9-based method (CRISPitope) in syngeneic melanoma models to fuse the same model CD8(+) T cell epitope to the C-termini of different endogenous gene products. Targeting melanosomal proteins or oncogenic CDK4(R24C )(Cyclin-dependent kinase 4) by adoptive cell transfer (ACT) of the same epitope-specific CD8(+) T cells revealed diverse genetic and non-genetic immune escape mechanisms. ACT directed against melanosomal proteins, but not CDK4(R24C), promoted melanoma dedifferentiation, and increased myeloid cell infiltration. CDK4(R24C) antigen persistence was associated with an interferon-high and T-cell-rich tumor microenvironment, allowing for immune checkpoint inhibition as salvage therapy. Thus, the choice of target antigen determines the phenotype and immune contexture of recurrent melanomas, with implications to the design of cancer immunotherapies