Background Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor. Methods To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe the optimization of saliva pretreatment protocols to enable analytically sensitive viral detection by RT-LAMP. We optimized the RT-LAMP reaction conditions and implemented high-throughput unbiased methods for assay interpretation. We tested whether saliva pretreatment could also enable viral detection by conventional reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Finally, we validated these assays on clinical samples. Results The optimized saliva pretreatment protocol enabled analytically sensitive extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP or RT-qPCR. In simulated samples, the optimized RT-LAMP assay had a limit of detection of 59 (95% confidence interval: 44-104) particle copies per reaction. We highlighted the flexibility of LAMP assay implementation using three readouts: naked-eye colorimetry, spectrophotometry, and real-time fluorescence. In a set of 30 clinical saliva samples, colorimetric RT-LAMP and RT-qPCR assays performed directly on pretreated saliva samples without RNA extraction had accuracies greater than 90%. Conclusions Rapid and extraction-free detection of SARS-CoV-2 from saliva by colorimetric RT-LAMP is a simple, sensitive, and cost-effective approach with broad potential to expand diagnostic testing for the virus causing COVID-19.

In situ assays of transcription factor (TF) binding are confounded by cellular heterogeneity and represent averaged profiles in complex tissues. Single cell RNA-seq (scRNA-seq) is capable of resolving different cell types based on gene expression profiles, but no technology exists to directly link specific cell types to the binding pattern of TFs in those cell types. Here, we present self-reporting transposons (SRTs) and their use in single cell calling cards (scCC), a novel assay for simultaneously capturing gene expression profiles and mapping TF binding sites in single cells. First, we show how the genomic locations of SRTs can be recovered from mRNA. Next, we demonstrate that SRTs deposited by the piggyBac transposase can be used to map the genome-wide localization of the TFs SP1, through a direct fusion of the two proteins, and BRD4, through its native affinity for piggyBac. We then present the scCC method, which maps SRTs from scRNA-seq libraries, thus enabling concomitant identification of cell types and TF binding sites in those same cells. As a proof-of-concept, we show recovery of cell type-specific BRD4 and SP1 binding sites from cultured cells. Finally, we map Brd4 binding sites in the mouse cortex at single cell resolution, thus establishing a new technique for studying TF biology in situ..

Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold standard diagnostic assays are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment (PPE), instrumentation, and labor. Here we present an approach to overcome these challenges with the development of a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step. We describe our optimizations of the LAMP reaction and saliva pre-treatment protocols that enabled rapid and sensitive detection of < 10 2 viral genomes per reaction in contrived saliva controls. We also observed high performance of this assay on a limited number of clinical saliva samples. While thorough validation on additional clinical samples will be needed before such an assay can be widely used, these preliminary results demonstrate a promising approach to overcome the current bottlenecks limiting widespread testing.

The concept of mutually unbiased bases is studied for N pairs of continuous variables. To find mutually unbiased bases reduces, for specific states related to the Heisenberg-Weyl group, to a problem of symplectic geometry. Given a single pair of continuous variables, three mutually unbiased bases are identified while five such bases are exhibited for two pairs of continuous variables. For N = 2, the golden ratio occurs in the definition of these mutually unbiased bases suggesting the relevance of number theory not only in the finite-dimensional setting. 03.65.Ta Mutually unbiased (MU) bases of Hilbert spaces with finite dimension d (as defined by Eq. (1) below) are a useful tool. If you want to experimentally determine the state of a quantum system, given only a limited supply of identical copies, the optimal strategy is to perform measurements with respect to MU bases [1]. To pass a secret message to a second party, you could use quantum cryptography to establish a shared key, a procedure which relies on MU bases in the space. Sending a physical system carrying a spin through a noisy environment, the effect of the interactions on the state of the spin might be modelled by a specific quantum channel, conveniently described in terms of MU bases [5]. Finally, if you happen to be captured by a mean king, you might be able to meet his challenge by knowing about entangled states and MU bases [6,7]. Many of the ideas which underlie physical concepts defined for discrete variables, that is, in a Hilbert space of finite dimension, survive the transition from spin operators to position and momentum operators. Quantum key distribution [8] and quantum teleportation [9], for example, possess counterparts for continuous variables [10] which act on an infinite-dimensional Hilbert space. It is thus natural to inquire into MU bases for continuous variables which, in fact, naturally occur in Feynman's path integral formulation of quantum mechanics [11]. The properties of MU bases in an infinite-dimensional space might also provide new insights into the existence of complete sets of MU bases in spaces of finite dimension not equal to the power of a prime.Let us recall the definition of MU bases in C d and some of their properties. Two orthonormal basessince each state of one basis gives rise to the same probability distribution when measured with respect to the other basis. The value of the overlap κ is not arbitrary but one derives from (1) that κ ≡ 1/ √ d by using the completeness of the basis B b , say.Schwinger [12] describes how to construct two MU bases from any orthonormal basis of C d . They are found to be the eigenbases of two operatorsÛ andV each shifting cyclically the elements of the other basis. These operators satisfy commutation relations of HeisenbergWeyl type,ÛV = e 2πi/dVÛ , describing finite translations in a discrete phase space [13]. This approach has been generalized in [14], where it is shown that if one finds n unitaries each cyclically shifting the eigenbases of all other unitaries then these n bases ...

Transcription factors (TFs) enact precise regulation of gene expression through site-specific, genome-wide binding. Common methods for TF-occupancy profiling, such as chromatin immunoprecipitation, are limited by requirement of TF-specific antibodies and provide only end-point snapshots of TF binding. Alternatively, TF-tagging techniques, in which a TF is fused to a DNA-modifying enzyme that marks TF-binding events across the genome as they occur, do not require TF-specific antibodies and offer the potential for unique applications, such as recording of TF occupancy over time and cell type specificity through conditional expression of the TF–enzyme fusion. Here, we create a viral toolkit for one such method, calling cards, and demonstrate that these reagents can be delivered to the live mouse brain and used to report TF occupancy. Further, we establish a Cre-dependent calling cards system and, in proof-of-principle experiments, show utility in defining cell type-specific TF profiles and recording and integrating TF-binding events across time. This versatile approach will enable unique studies of TF-mediated gene regulation in live animal models.