Development of Container Free Sample Exposure for Synchrotron X-ray Footprinting.

The method of X-ray footprinting and mass spectrometry (XFMS) on large protein assemblies and membrane protein samples requires high flux density to overcome the hydroxyl radical scavenging reactions produced by the buffer constituents and the total protein content. Previously, we successfully developed microsecond XFMS using microfluidic capillary flow and a microfocused broadband X-ray source at the Advanced Light Source synchrotron beamlines, but the excessive radiation damage incurred when using capillaries prevented the full usage of a high-flux density beam. Here we present another significant advance for the XFMS method: the instrumentation of a liquid injection jet to deliver container free samples to the X-ray beam. Our preliminary experiments with a liquid jet at a bending magnet X-ray beamline demonstrate the feasibility of the approach and show a significant improvement in the effective dose for both the Alexa fluorescence assay and protein samples compared to conventional capillary flow methods. The combination of precisely controlled high dose delivery, shorter exposure times, and elimination of radiation damage due to capillary effects significantly increases the signal quality of the hydroxyl radical modification products and the dose-response data. This new approach is the first application of container free sample handling for XFMS and opens up the method for even further advances, such as high-quality microsecond time-resolved XFMS studies

An embedded system for the automated generation of labeled plant images to enable machine learning applications in agriculture

A lack of sufficient training data, both in terms of variety and quantity, is often the bottleneck in the development of machine learning (ML) applications in any domain. For agricultural applications, ML-based models designed to perform tasks such as autonomous plant classification will typically be coupled to just one or perhaps a few plant species. As a consequence, each crop-specific task is very likely to require its own specialized training data, and the question of how to serve this need for data now often overshadows the more routine exercise of actually training such models. To tackle this problem, we have developed an embedded robotic system to automatically generate and label large datasets of plant images for ML applications in agriculture. The system can image plants from virtually any angle, thereby ensuring a wide variety of data; and with an imaging rate of up to one image per second, it can produce lableled datasets on the scale of thousands to tens of thousands of images per day. As such, this system offers an important alternative to time- and cost-intensive methods of manual generation and labeling. Furthermore, the use of a uniform background made of blue keying fabric enables additional image processing techniques such as background replacement and plant segmentation. It also helps in the training process, essentially forcing the model to focus on the plant features and eliminating random correlations. To demonstrate the capabilities of our system, we generated a dataset of over 34,000 labeled images, with which we trained an ML-model to distinguish grasses from non-grasses in test data from a variety of sources. We now plan to generate much larger datasets of Canadian crop plants and weeds that will be made publicly available in the hope of further enabling ML applications in the agriculture sector.Comment: 35 pages, 8 figures, Preprint submitted to PLoS On

Proteostasis by STUB1/HSP70 complex controls sensitivity to androgen receptor targeted therapy in advanced prostate cancer.

Protein homeostasis (proteostasis) is a potential mechanism that contributes to cancer cell survival and drug resistance. Constitutively active androgen receptor (AR) variants confer anti-androgen resistance in advanced prostate cancer. However, the role of proteostasis involved in next generation anti-androgen resistance and the mechanisms of AR variant regulation are poorly defined. Here we show that the ubiquitin-proteasome-system (UPS) is suppressed in enzalutamide/abiraterone resistant prostate cancer. AR/AR-V7 proteostasis requires the interaction of E3 ubiquitin ligase STUB1 and HSP70 complex. STUB1 disassociates AR/AR-V7 from HSP70, leading to AR/AR-V7 ubiquitination and degradation. Inhibition of HSP70 significantly inhibits prostate tumor growth and improves enzalutamide/abiraterone treatments through AR/AR-V7 suppression. Clinically, HSP70 expression is upregulated and correlated with AR/AR-V7 levels in high Gleason score prostate tumors. Our results reveal a novel mechanism of anti-androgen resistance via UPS alteration which could be targeted through inhibition of HSP70 to reduce AR-V7 expression and overcome resistance to AR-targeted therapies

A Candidate Young Massive Planet in Orbit around the Classical T Tauri Star CI Tau

The ~2 Myr old classical T Tauri star CI Tau shows periodic variability in its radial velocity (RV) variations measured at infrared (IR) and optical wavelengths. We find that these observations are consistent with a massive planet in a ~9-day period orbit. These results are based on 71 IR RV measurements of this system obtained over 5 years, and on 26 optical RV measurements obtained over 9 years. CI Tau was also observed photometrically in the optical on 34 nights over ~one month in 2012. The optical RV data alone are inadequate to identify an orbital period, likely the result of star spot and activity induced noise for this relatively small dataset. The infrared RV measurements reveal significant periodicity at ~9 days. In addition, the full set of optical and IR RV measurements taken together phase coherently and with equal amplitudes to the ~9 day period. Periodic radial velocity signals can in principle be produced by cool spots, hot spots, and reflection of the stellar spectrum off the inner disk, in addition to resulting from a planetary companion. We have considered each of these and find the planet hypothesis most consistent with the data. The radial velocity amplitude yields an Msin(i) of ~8.1 M_Jup; in conjunction with a 1.3 mm continuum emission measurement of the circumstellar disk inclination from the literature, we find a planet mass of ~11.3 M_Jup, assuming alignment of the planetary orbit with the disk.Comment: 61 pages, 13 figures, accepted for publication in The Astrophysical Journa

Nanoscale integration of single cell biologics discovery processes using optofluidic manipulation and monitoring.

The new and rapid advancement in the complexity of biologics drug discovery has been driven by a deeper understanding of biological systems combined with innovative new therapeutic modalities, paving the way to breakthrough therapies for previously intractable diseases. These exciting times in biomedical innovation require the development of novel technologies to facilitate the sophisticated, multifaceted, high-paced workflows necessary to support modern large molecule drug discovery. A high-level aspiration is a true integration of "lab-on-a-chip" methods that vastly miniaturize cellulmical experiments could transform the speed, cost, and success of multiple workstreams in biologics development. Several microscale bioprocess technologies have been established that incrementally address these needs, yet each is inflexibly designed for a very specific process thus limiting an integrated holistic application. A more fully integrated nanoscale approach that incorporates manipulation, culture, analytics, and traceable digital record keeping of thousands of single cells in a relevant nanoenvironment would be a transformative technology capable of keeping pace with today's rapid and complex drug discovery demands. The recent advent of optical manipulation of cells using light-induced electrokinetics with micro- and nanoscale cell culture is poised to revolutionize both fundamental and applied biological research. In this review, we summarize the current state of the art for optical manipulation techniques and discuss emerging biological applications of this technology. In particular, we focus on promising prospects for drug discovery workflows, including antibody discovery, bioassay development, antibody engineering, and cell line development, which are enabled by the automation and industrialization of an integrated optoelectronic single-cell manipulation and culture platform. Continued development of such platforms will be well positioned to overcome many of the challenges currently associated with fragmented, low-throughput bioprocess workflows in biopharma and life science research

Testing general relativity using golden black-hole binaries

The coalescences of stellar-mass black-hole binaries through their inspiral, merger, and ringdown are among the most promising sources for ground-based gravitational-wave (GW) detectors. If a GW signal is observed with sufficient signal-to-noise ratio, the masses and spins of the black holes can be estimated from just the inspiral part of the signal. Using these estimates of the initial parameters of the binary, the mass and spin of the final black hole can be uniquely predicted making use of general-relativistic numerical simulations. In addition, the mass and spin of the final black hole can be independently estimated from the merger--ringdown part of the signal. If the binary black hole dynamics is correctly described by general relativity (GR), these independent estimates have to be consistent with each other. We present a Bayesian implementation of such a test of general relativity, which allows us to combine the constraints from multiple observations. Using kludge modified GR waveforms, we demonstrate that this test can detect sufficiently large deviations from GR, and outline the expected constraints from upcoming GW observations using the second-generation of ground-based GW detectors.Comment: 5 pages, 2 fig

Quantum plasmonics: Symmetry-dependent plasmon-molecule coupling and quantized photoconductances

We investigate photoinduced electron transport through nanoparticle dimers connected by linear and ringlike molecules. The two model junctions exhibit similar absorption but distinct photoconductance. The linear-chain junction shows a constant conductance that is independent of photon energies. In contrast, the ring molecules show quantized conductance resonances at energies corresponding to the molecular excitations. These results can be understood from the symmetry and quantization of local molecular excitations, and have implications for plasmon-molecule interactions in general

Leucine Zipper-Bearing Kinase Is a Critical Regulator of Astrocyte Reactivity in the Adult Mammalian CNS.

Reactive astrocytes influence post-injury recovery, repair, and pathogenesis of the mammalian CNS. Much of the regulation of astrocyte reactivity, however, remains to be understood. Using genetic loss and gain-of-function analyses in vivo, we show that the conserved MAP3K13 (also known as leucine zipper-bearing kinase [LZK]) promotes astrocyte reactivity and glial scar formation after CNS injury. Inducible LZK gene deletion in astrocytes of adult mice reduced astrogliosis and impaired glial scar formation, resulting in increased lesion size after spinal cord injury. Conversely, LZK overexpression in astrocytes enhanced astrogliosis and reduced lesion size. Remarkably, in the absence of injury, LZK overexpression alone induced widespread astrogliosis in the CNS and upregulated astrogliosis activators pSTAT3 and SOX9. The identification of LZK as a critical cell-intrinsic regulator of astrocyte reactivity expands our understanding of the multicellular response to CNS injury and disease, with broad translational implications for neural repair