iRFP is a real time marker for transformation based assays in high content screening

Anchorage independent growth is one of the hallmarks of oncogenic transformation. Here we show that infrared fluorescent protein (iRFP) based assays allow accurate and unbiased determination of colony formation and anchorage independent growth over time. This protocol is particularly compatible with high throughput systems, in contrast to traditional methods which are often labor-intensive, subjective to bias and do not allow further analysis using the same cells. Transformation in a single layer soft agar assay could be documented as early as 2 to 3 days in a 96 well format, which can be easily combined with standard transfection, infection and compound screening setups to allow for high throughput screening to identify therapeutic targets

Nonlinear interaction of meta-atoms through optical coupling

We propose and experimentally demonstrate a multi-frequency nonlinear coupling mechanism between split-ring resonators. We engineer the coupling between two microwave resonators through optical interaction, whilst suppressing the direct electromagnetic coupling. This allows for a power-dependent interaction between the otherwise independent resonators, opening interesting opportunities to address applications in signal processing, filtering, directional coupling, and electromagnetic compatibility.This work was supported by the Ministry of Education and Science of Russian Federation (Project No. 11.G34.31.0020), Russian Foundation for Basic Research (Projects No. 13-02-00411 and 14-02-31783), Scholarship of the President of the Russian Federation for young scientists and graduate students, and by the Australian Research Council (CUDOS Centre of Excellence CE110001018). A.P.S. acknowledges the support of SPIE Scholarship 2012-2013 and the IEEE MTT-S Undergraduate Scholarship for spring 2013. A.P.S. and D.S.F. are also grateful to the “Dynasty” Foundation (Russia)

The Boundary Conditions for Point Transformed Electromagnetic Invisibility Cloaks

In this paper we study point transformed electromagnetic invisibility cloaks in transformation media that are obtained by transformation from general anisotropic media. We assume that there are several cloaks located in different points in space. Our results apply in particular to the first order invisibility cloaks introduced by Pendry et al. and to the high order invisibility cloaks introduced by Hendi et al. and by Cai et al.. We identify the appropriate {\it cloaking boundary conditions} that the solutions of Maxwell equations have to satisfy at the outside, $\partial K_+$, and at the inside, $\partial K_-$, of the boundary of the cloaked object $K$. Namely, that the tangential components of the electric and the magnetic fields have to vanish at $\partial K_+$ -what is always true- and that the normal components of the curl of the electric and the magnetic fields have to vanish at $\partial K_-$. These results are proven requiring that energy be conserved. In the case of one spherical cloak with a spherically stratified $K$ and a radial current at $\partial K$ we verify by an explicit calculation that our {\it cloaking boundary conditions} are satisfied and that cloaking of active devices holds even if the current is at the boundary of the cloaked object. As we prove our results for media that are obtained by transformation from general anisotropic media, our results apply to the cloaking of objects with active and passive devices contained in general anisotropic media, in particular to objects with active and passive devices contained inside general crystals.Comment: This final, published, version has been edited, comments have been adde

Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET

Direct visualization and light control of several cellular processes is a challenge, owing to the spectral overlap of available genetically encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein, miRFP720, and the fully NIR Forster resonance energy transfer (FRET) pair miRFP670-miRFP720, which together enabled design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use in multiplexed imaging and light control of Rho GTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and concurrently used the LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK; showed that Rac1 activity and GDI binding closely depend on the spatiotemporal coordination between these two molecules; and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.Peer reviewe

Modular construction of mammalian gene circuits using TALE transcriptional repressors

An important goal of synthetic biology is the rational design and predictable implementation of synthetic gene circuits using standardized and interchangeable parts. However, engineering of complex circuits in mammalian cells is currently limited by the availability of well-characterized and orthogonal transcriptional repressors. Here, we introduce a library of 26 reversible transcription activator–like effector repressors (TALERs) that bind newly designed hybrid promoters and exert transcriptional repression through steric hindrance of key transcriptional initiation elements. We demonstrate that using the input-output transfer curves of our TALERs enables accurate prediction of the behavior of modularly assembled TALER cascade and switch circuits. We also show that TALER switches using feedback regulation exhibit improved accuracy for microRNA-based HeLa cancer cell classification versus HEK293 cells. Our TALER library is a valuable toolkit for modular engineering of synthetic circuits, enabling programmable manipulation of mammalian cells and helping elucidate design principles of coupled transcriptional and microRNA-mediated post-transcriptional regulation.National Institutes of Health (U.S.) (Grant 5R01CA155320-04)National Institutes of Health (U.S.) (Grant P50GM098792)National Institutes of Health (U.S.) (Grant 1R01CA173712-01

Tajik Basin: A composite record of sedimentary basin evolution in response to tectonics in the Pamir

Investigation of a >6‐km‐thick succession of Cretaceous to Cenozoic sedimentary rocks in the Tajik Basin reveals that this depocentre consists of three stacked basin systems that are interpreted to reflect different mechanisms of subsidence associated with tectonics in the Pamir Mountains: a Lower to mid‐Cretaceous succession, an Upper Cretaceous–Lower Eocene succession and an Eocene–Neogene succession. The Lower to mid‐Cretaceous succession consists of fluvial deposits that were primarily derived from the Triassic Karakul–Mazar subduction–accretion complex in the northern Pamir. This succession is characterized by a convex‐up (accelerating) subsidence curve, thickens towards the Pamir and is interpreted as a retroarc foreland basin system associated with northward subduction of Tethyan oceanic lithosphere. The Upper Cretaceous to early Eocene succession consists of fine‐grained, marginal marine and sabkha deposits. The succession is characterized by a concave‐up subsidence curve. Regionally extensive limestone beds in the succession are consistent with late stage thermal relaxation and relative sea‐level rise following lithospheric extension, potentially in response to Tethyan slab rollback/foundering. The Upper Cretaceous–early Eocene succession is capped by a middle Eocene to early Oligocene (ca. 50–30 Ma) disconformity, which is interpreted to record the passage of a flexural forebulge. The disconformity is represented by a depositional hiatus, which is 10–30 Myr younger than estimates for the initiation of India–Asia collision and overlaps in age with the start of prograde metamorphism recorded in the Pamir gneiss domes. Overlying the disconformity, a >4‐km‐thick upper Eocene–Neogene succession displays a classic, coarsening upward unroofing sequence characterized by accelerating subsidence, which is interpreted as a retro‐foreland basin associated with crustal thickening of the Pamir during India–Asia collision. Thus, the Tajik Basin provides an example of a long‐lived composite basin in a retrowedge position that displays a sensitivity to plate margin processes. Subsidence, sediment accumulation and basin‐forming mechanisms are influenced by subduction dynamics, including periods of slab‐shallowing and retreat

A practical guide to photoacoustic tomography in the life sciences

The life sciences can benefit greatly from imaging technologies that connect microscopic discoveries with macroscopic observations. One technology uniquely positioned to provide such benefits is photoacoustic tomography (PAT), a sensitive modality for imaging optical absorption contrast over a range of spatial scales at high speed. In PAT, endogenous contrast reveals a tissue's anatomical, functional, metabolic, and histologic properties, and exogenous contrast provides molecular and cellular specificity. The spatial scale of PAT covers organelles, cells, tissues, organs, and small animals. Consequently, PAT is complementary to other imaging modalities in contrast mechanism, penetration, spatial resolution, and temporal resolution. We review the fundamentals of PAT and provide practical guidelines for matching PAT systems with research needs. We also summarize the most promising biomedical applications of PAT, discuss related challenges, and envision PAT's potential to lead to further breakthroughs

An interlaboratory comparison on the characterization of a sub-micrometer polydisperse particle dispersion

The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse particle dispersion composed of five sub-populations of poly(methyl methacrylate) (PMMA) and silica beads. The study included 20 participating laboratories from industry, academia, and government, and a variety of state-of-the-art particle-counting instruments. The received datasets were organized by instrument class to enable comparison of intralaboratory and interlaboratory performance. The main findings included high variability between datasets from different laboratories, with coefficients of variation from 13 % to 189 %. Intralaboratory variability was, on average, 37 % of the interlaboratory variability for an instrument class and particle sub-population. Drop-offs at either end of the size range and poor agreement on maximum counts of particle sub-populations were noted. The mean distributions from an instrument class, however, showed the size-coverage range for that class. The study shows that a poly-disperse sample can be used to assess performance capabilities of an instrument set-up (including hardware, software, and user settings) and provides guidance for the development of polydisperse reference materials.Drug Delivery Technolog

Development and Applications of Fluorogen/Light-Up RNA Aptamer Pairs for RNA Detection and More.

The central role of RNA in living systems made it highly desirable to have noninvasive and sensitive technologies allowing for imaging the synthesis and the location of these molecules in living cells. This need motivated the development of small pro-fluorescent molecules called "fluorogens" that become fluorescent upon binding to genetically encodable RNAs called "light-up aptamers." Yet, the development of these fluorogen/light-up RNA pairs is a long and thorough process starting with the careful design of the fluorogen and pursued by the selection of a specific and efficient synthetic aptamer. This chapter summarizes the main design and the selection strategies used up to now prior to introducing the main pairs. Then, the vast application potential of these molecules for live-cell RNA imaging and other applications is presented and discussed.journal article2020importe