Concise, gram-scale synthesis of furo[2,3-b]pyridines with functional handles for chemoselective cross-coupling.

A concise 4-step synthesis of furo[2,3-b]pyridines, with handles in the 3- and 5-positions for palladium mediated cross-coupling reactions, is described. The synthetic route has been optimized, with only one step requiring purification by column chromatography. The route is amenable to scale-up, and was successfully executed on a multi-gram scale. Furopyridines are of growing interest in medicinal chemistry, and this route should enable easy access to the core for structure-activity relationship (SAR) studies.

In Depth Analysis of Kinase Cross Screening Data to Identify CAMKK2 Inhibitory Scaffolds.

The calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) activates CAMK1, CAMK4, AMPK, and AKT, leading to numerous physiological responses. The deregulation of CAMKK2 is linked to several diseases, suggesting the utility of CAMKK2 inhibitors for oncological, metabolic and inflammatory indications. In this work, we demonstrate that STO-609, frequently described as a selective inhibitor for CAMKK2, potently inhibits a significant number of other kinases. Through an analysis of literature and public databases, we have identified other potent CAMKK2 inhibitors and verified their activities in differential scanning fluorimetry and enzyme inhibition assays. These inhibitors are potential starting points for the development of selective CAMKK2 inhibitors and will lead to tools that delineate the roles of this kinase in disease biology.

Effect of noise-induced wavelength fluctuation in tunable diode lasers on narrow-linewidth absorption measurements.

Tunable diode laser absorption spectroscopy is being widely used to make sensors for diagnostic purposes in various engineering applications. Since the wavelength of many diode lasers used in such sensors is sensitive to the driving current, even noise as small as a few µArms in the driving current can cause a wavelength fluctuation of ∼±0.5 pm, which is large enough to interfere with sensitive absorption measurements. Although these fluctuations are small, they can cause significant systematic error in measured absorption spectra in applications where the absorption line probed is narrow, as is the case for low-density hypersonic flows. As an example, at a pressure of 300 Pa and 297 K, the error in the full width at half-maximum was ±6.5% in an absorption spectrum obtained using a system based on a vertical-cavity surface-emitting laser scanned at 10 kHz. This paper analyzes the effect of such systematic errors on measured temperature and velocity and suggests some remedial measures.

1.6 MHz scanning rate direct absorption temperature measurements using a single vertical-cavity surface-emitting laser diode.

This paper presents 1.6 MHz scan rate, non-intrusive, time-resolved temperature measurements of a normal shock reflection from a plane end wall within a shock tube. A vertical-cavity surface-emitting laser (VCSEL) was used to conduct tunable diode laser absorption spectroscopy with water vapor as the probe species. The results are compared with analytical predictions. Temperatures measured with this technique agree within a single-scan standard deviation of ±33 K with calculated temperatures at a VCSEL modulation frequency of 800 kHz, which is sufficiently rapid enough to be used to investigate highly transient shock wave interaction processes.

Multiple receivers in a high-resolution near-infrared heterodyne spectrometer.

The paper describes a modification of a high-resolution heterodyne NIR spectrometer described by Rodin, et al. in Opt. Express22, 13825 (2014), wherein the noise amplitude of the heterodyne signal squared was proportional to the power of the incoherent radiation source (the sun). The addition of a second receiver collecting radiation from the sun into a second single-mode fiber created up to a factor of two increase in detected source power spectrum. The ability to add uncorrelated heterodyne IF signals as Gaussian noise (variance) provides the means by which multiple heterodyne receivers of signal from an incoherent source can increase the detected source power by the same multiple, thus avoiding the limit imposed by the antenna theorem for a single receiver (A. E. Siegman, Appl. Opt.5, 1588 (1966)).

Baseline correction for stray light in log-ratio diode laser absorption measurements.

Log-ratio detection is a convenient technique for making temperature and concentration measurements using sensors based on tunable diode laser absorption spectroscopy. In many practical sensing applications, it is difficult to avoid stray light falling on the signal photodiode of the sensor. This stray light acts as noncommon-mode interference and introduces a systematic error in absorption measurements, which is not removed by baseline subtraction. This paper analyzes the factors that determine this systematic error and also presents a calibration method that can correct for it. This correction method is verified using a simple experiment.

Visualization of jet development in laser-induced plasmas.

Laser-induced plasmas in gases are known to generate gaseous jets in the postplasma gas plume. The gaseous jet typically develops toward the laser source, and the experiments presented here show, for the first time to our knowledge, that, under certain conditions, these jets can develop in the opposite direction or may not form at all. The data suggest that this is related to the ratio between the energy absorbed in the plasma and the threshold breakdown energy, effectively leading to multiple plasma initiation sites in the focal waist.

SGC-CAMKK2-1: A Chemical Probe for CAMKK2.

The serine/threonine protein kinase calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) plays critical roles in a range of biological processes. Despite its importance, only a handful of inhibitors of CAMKK2 have been disclosed. Having a selective small molecule tool to interrogate this kinase will help demonstrate that CAMKK2 inhibition can be therapeutically beneficial. Herein, we disclose SGC-CAMKK2-1, a selective chemical probe that targets CAMKK2.

Refolding of lid subdomain of SARS-CoV-2 nsp14 upon nsp10 interaction releases exonuclease activity.

During RNA replication, coronaviruses require proofreading to maintain the integrity of their large genomes. Nsp14 associates with viral polymerase complex to excise the mismatched nucleotides. Aside from the exonuclease activity, nsp14 methyltransferase domain mediates cap methylation, facilitating translation initiation and protecting viral RNA from recognition by the innate immune sensors. The nsp14 exonuclease activity is modulated by a protein co-factor nsp10. While the nsp10/nsp14 complex structure is available, the mechanistic basis for nsp10-mediated modulation remains unclear in the absence of the nsp14 structure. Here, we provide a crystal structure of nsp14 in an apo-form. Comparative analysis of the apo- and nsp10-bound structures explain the modulatory role of the co-factor protein and reveal the allosteric nsp14 control mechanism essential for drug discovery. Further, the flexibility of the N-terminal lid of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp14 structure presented in this study rationalizes the recently proposed idea of nsp14/nsp10/nsp16 ternary complex.

Photoactivated release of membrane impermeant sulfonates inside cells.

Photouncaging delivers compounds with high spatial and temporal control to induce or inhibit biological processes but the released compounds may diffuse out. We here demonstrate that sulfonate anions can be photocaged so that a membrane impermeable compound can enter cells, be uncaged by photoirradiation and trapped within the cell.

Hinge Binder Scaffold Hopping Identifies Potent Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CAMKK2) Inhibitor Chemotypes.

CAMKK2 is a serine/threonine kinase and an activator of AMPK whose dysregulation is linked with multiple diseases. Unfortunately, STO-609, the tool inhibitor commonly used to probe CAMKK2 signaling, has limitations. To identify promising scaffolds as starting points for the development of high-quality CAMKK2 chemical probes, we utilized a hinge-binding scaffold hopping strategy to design new CAMKK2 inhibitors. Starting from the potent but promiscuous disubstituted 7-azaindole GSK650934, a total of 32 compounds, composed of single-ring, 5,6-, and 6,6-fused heteroaromatic cores, were synthesized. The compound set was specifically designed to probe interactions with the kinase hinge-binding residues. Compared to GSK650394 and STO-609, 13 compounds displayed similar or better CAMKK2 inhibitory potency in vitro, while compounds 13g and 45 had improved selectivity for CAMKK2 across the kinome. Our systematic survey of hinge-binding chemotypes identified several potent and selective inhibitors of CAMKK2 to serve as starting points for medicinal chemistry programs.

Diode-laser-based near-resonantly enhanced flow visualization in shock tunnels.

Two new near-resonantly enhanced flow visualization techniques suitable for hypersonic low-density flows in shock or arc tunnels have been developed using seeded lithium (Li) metal as the refractivity-enhancing species. Two semiconductor lasers, single-longitudinal-mode and multimode, are compared with respect to their suitability as light sources for the technique. Transient wake-flow structures around a cylinder and a model of a planetary entry vehicle are visualized to demonstrate the capabilities of this comparatively inexpensive and simple visualization system. The images show flow features which are undetectable with conventional schlieren, shadowgraph, or interferometry techniques. Furthermore, the effect of density inhomogeneities along the line-of-sight outside the region of interest can be reduced by enhancing the refractivity only in selected parts of the flowfield.

Identification of a Human Airway Epithelial Cell Subpopulation with Altered Biophysical, Molecular, and Metastatic Properties.

Lung cancers are documented to have remarkable intratumoral genetic heterogeneity. However, little is known about the heterogeneity of biophysical properties, such as cell motility, and its relationship to early disease pathogenesis and micrometastatic dissemination. In this study, we identified and selected a subpopulation of highly migratory premalignant airway epithelial cells that were observed to migrate through microscale constrictions at up to 100-fold the rate of the unselected immortalized epithelial cell lines. This enhanced migratory capacity was found to be Rac1-dependent and heritable, as evidenced by maintenance of the phenotype through multiple cell divisions continuing more than 8 weeks after selection. The morphology of this lung epithelial subpopulation was characterized by increased cell protrusion intensity. In a murine model of micrometastatic seeding and pulmonary colonization, the motility-selected premalignant cells exhibit both enhanced survival in short-term assays and enhanced outgrowth of premalignant lesions in longer-term assays, thus overcoming important aspects of "metastatic inefficiency." Overall, our findings indicate that among immortalized premalignant airway epithelial cell lines, subpopulations with heritable motility-related biophysical properties exist, and these may explain micrometastatic seeding occurring early in the pathogenesis of lung cancer. Understanding, targeting, and preventing these critical biophysical traits and their underlying molecular mechanisms may provide a new approach to prevent metastatic behavior. Cancer Prev Res; 10(9); 514-24. ©2017 AACRSee related editorial by Hynds and Janes, p. 491.

Multiparameter mechanical and morphometric screening of cells.

We introduce a label-free method to rapidly phenotype and classify cells purely based on physical properties. We extract 15 biophysical parameters from cells as they deform in a microfluidic stretching flow field via high-speed microscopy and apply machine-learning approaches to discriminate different cell types and states. When employing the full 15 dimensional dataset, the technique robustly classifies individual cells based on their pluripotency, with accuracy above 95%. Rheological and morphological properties of cells while deforming were critical for this classification. We also show the application of this method in accurate classifying cells based on their viability, drug screening and detecting populations of malignant cells in mixed samples. We show that some of the extracted parameters are not linearly independent, and in fact we reach maximum classification accuracy by using only a subset of parameters. However, the informative subsets could vary depending on cell types in the sample. This work shows the utility of an assay purely based on intrinsic biophysical properties of cells to identify changes in cell state. In addition to a label-free alternative to flow cytometry in certain applications, this work, also can provide novel intracellular metrics that would not be feasible with labeled approaches (i.e. flow cytometry).

Piezoresistive nanocomposite as an embedded stress sensor in instrumented knee prosthesis.

We characterize the electrical properties of a biocompatible nanocomposite which will be used as a stress sensing material in an instrumented knee implant. The composite is fabricated from multi-walled carbon nanotubes and ultra high molecular weight polyethylene. Experimental cyclic compression loading shows that the composite's resistance exponentially decreases with increasing compression stress, proving its potential for application as a piezoresistive stress sensing material. An analytical model is built to estimate the optimal depth from the tibio-femoral contact surface at which an embedded stress sensor could achieve the highest stress resolution and lowest distortion energy inside the tibial insert.

Size-selective collection of circulating tumor cells using Vortex technology.

A blood-based, low cost alternative to radiation intensive CT and PET imaging is critically needed for cancer prognosis and management of its treatment. "Liquid biopsies" of circulating tumor cells (CTCs) from a relatively non-invasive blood draw are particularly ideal, as they can be repeated regularly to provide up to date molecular information about the cancer, which would also open up key opportunities for personalized therapies. Beyond solely diagnostic applications, CTCs are also a subject of interest for drug development and cancer research. In this paper, we adapt a technology previously introduced, combining the use of micro-scale vortices and inertial focusing, specifically for the high-purity extraction of CTCs from blood samples. First, we systematically varied parameters including channel dimensions and flow rates to arrive at an optimal device for maximum trapping efficiency and purity. Second, we validated the final device for capture of cancer cell lines in blood, considering several factors, including the effect of blood dilution, red blood cell lysis and cell deformability, while demonstrating cell viability and independence on EpCAM expression. Finally, as a proof-of-concept, CTCs were successfully extracted and enumerated from the blood of patients with breast (N = 4, 25-51 CTCs per 7.5 mL) and lung cancer (N = 8, 23-317 CTCs per 7.5 mL). Importantly, samples were highly pure with limited leukocyte contamination (purity 57-94%). This Vortex approach offers significant advantages over existing technologies, especially in terms of processing time (20 min for 7.5 mL of whole blood), sample concentration (collecting cells in a small volume down to 300 µL), applicability to various cancer types, cell integrity and purity. We anticipate that its simplicity will aid widespread adoption by clinicians and biologists who desire to not only enumerate CTCs, but also uncover new CTC biology, such as unique gene mutations, vesicle secretion and roles in metastatic processes.