Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components.

The recent Zika virus outbreak highlights the need for low-cost diagnostics that can be rapidly developed for distribution and use in pandemic regions. Here, we report a pipeline for the rapid design, assembly, and validation of cell-free, paper-based sensors for the detection of the Zika virus RNA genome. By linking isothermal RNA amplification to toehold switch RNA sensors, we detect clinically relevant concentrations of Zika virus sequences and demonstrate specificity against closely related Dengue virus sequences. When coupled with a novel CRISPR/Cas9-based module, our sensors can discriminate between viral strains with single-base resolution. We successfully demonstrate a simple, field-ready sample-processing workflow and detect Zika virus from the plasma of a viremic macaque. Our freeze-dried biomolecular platform resolves important practical limitations to the deployment of molecular diagnostics in the field and demonstrates how synthetic biology can be used to develop diagnostic tools for confronting global health crises. PAPERCLIP.

Massively parallel CRISPRi assays reveal concealed thermodynamic determinants of dCas12a binding.

The versatility of CRISPR-Cas endonucleases as a tool for biomedical research has led to diverse applications in gene editing, programmable transcriptional control, and nucleic acid detection. Most CRISPR-Cas systems, however, suffer from off-target effects and unpredictable nonspecific binding that negatively impact their reliability and broader applicability. To better evaluate the impact of mismatches on DNA target recognition and binding, we develop a massively parallel CRISPR interference (CRISPRi) assay to measure the binding energy between tens of thousands of CRISPR RNA (crRNA) and target DNA sequences. By developing a general thermodynamic model of CRISPR-Cas binding dynamics, our results unravel a comprehensive map of the energetic landscape of nuclease-dead Cas12a (dCas12a) from Francisella novicida as it inspects and binds to its DNA target. Our results reveal concealed thermodynamic factors affecting dCas12a DNA binding, which should guide the design and optimization of crRNA that limits off-target effects, including the crucial role of an extended protospacer adjacent motif (PAM) sequence and the impact of the specific base composition of crRNA-DNA mismatches. Our generalizable approach should also provide a mechanistic understanding of target recognition and DNA binding when applied to other CRISPR-Cas systems.

Time-resolved study of laser emission in nitrogen gas pumped by two near IR femtosecond laser pulses.

The time profile of a lasing signal at 391.4 nm emitted by a weakly ionized gas of nitrogen molecules at low pressure is measured under double excitation with intense femtosecond laser pulses at 800 nm. An abrupt decrease in emission occurs at the time of arrival of the second pulse. It is explained by a transfer of population from ground to first excited ionic level and by a disruption of coherence, terminating the conditions for lasing in a V-scheme without population inversion.

YB-1, an abundant core mRNA-binding protein, has the capacity to form an RNA nucleoprotein filament: a structural analysis.

The structural rearrangements accompanying mRNA during translation in mammalian cells remain poorly understood. Here, we discovered that YB-1 (YBX1), a major partner of mRNAs in the cytoplasm, forms a linear nucleoprotein filament with mRNA, when part of the YB-1 unstructured C-terminus has been truncated. YB-1 possesses a cold-shock domain (CSD), a remnant of bacterial cold shock proteins that have the ability to stimulate translation under the low temperatures through an RNA chaperone activity. The structure of the nucleoprotein filament indicates that the CSD of YB-1 preserved its chaperone activity also in eukaryotes and shows that mRNA is channeled between consecutive CSDs. The energy benefit needed for the formation of stable nucleoprotein filament relies on an electrostatic zipper mediated by positively charged amino acid residues in the YB-1 C-terminus. Thus, YB-1 displays a structural plasticity to unfold structured mRNAs into extended linear filaments. We anticipate that our findings will shed the light on the scanning of mRNAs by ribosomes during the initiation and elongation steps of mRNA translation.

Quantum erasing of laser emission in N2.

Cavity-free lasing of N2+ induced by a femtosecond laser pulse at 800 nm is nearly totally suppressed by a delayed twin control pulse. We explain this surprising effect within the V-scheme of lasing without population inversion. A fast transfer of population between nitrogen ionic states X2Σg+ and A2Πu, induced by the second pulse, terminates the conditions for amplification in the system. The appearance of short lasing bursts at delays corresponding to revivals of rotational wave packets is explained along the same lines.

Ancient hot and cold genes and chemotherapy resistance emergence.

We use a microfabricated ecology with a doxorubicin gradient and population fragmentation to produce a strong Darwinian selective pressure that drives forward the rapid emergence of doxorubicin resistance in multiple myeloma (MM) cancer cells. RNA sequencing of the resistant cells was used to examine (i) emergence of genes with high de novo substitution densities (i.e., hot genes) and (ii) genes never substituted (i.e., cold genes). The set of cold genes, which were 21% of the genes sequenced, were further winnowed down by examining excess expression levels. Both the most highly substituted genes and the most highly expressed never-substituted genes were biased in age toward the most ancient of genes. This would support the model that cancer represents a revision back to ancient forms of life adapted to high fitness under extreme stress, and suggests that these ancient genes may be targets for cancer therapy.

Memory and fitness optimization of bacteria under fluctuating environments.

Bacteria prudently regulate their metabolic phenotypes by sensing the availability of specific nutrients, expressing the required genes for their metabolism, and repressing them after specific metabolites are depleted. It is unclear, however, how genetic networks maintain and transmit phenotypic states between generations under rapidly fluctuating environments. By subjecting bacteria to fluctuating carbon sources (glucose and lactose) using microfluidics, we discover two types of non-genetic memory in Escherichia coli and analyze their benefits. First, phenotypic memory conferred by transmission of stable intracellular lac proteins dramatically reduces lag phases under cyclical fluctuations with intermediate timescales (1-10 generations). Second, response memory, a hysteretic behavior in which gene expression persists after removal of its external inducer, enhances adaptation when environments fluctuate over short timescales (< 1 generation). Using a mathematical model we analyze the benefits of memory across environmental fluctuation timescales. We show that memory mechanisms provide an important class of survival strategies in biology that improve long-term fitness under fluctuating environments. These results can be used to understand how organisms adapt to fluctuating levels of nutrients, antibiotics, and other environmental stresses.

Costs of Clock-Environment Misalignment in Individual Cyanobacterial Cells.

Circadian rhythms are endogenously generated daily oscillations in physiology that are found in all kingdoms of life. Experimental studies have shown that the fitness of Synechococcus elongatus, a photosynthetic microorganism, is severely affected in non-24-h environments. However, it has been difficult to study the effects of clock-environment mismatch on cellular physiology because such measurements require a precise determination of both clock state and growth rate in the same cell. Here, we designed a microscopy platform that allows us to expose cyanobacterial cells to pulses of light and dark while quantitatively measuring their growth, division rate, and circadian clock state over many days. Our measurements reveal that decreased fitness can result from a catastrophic growth arrest caused by unexpected darkness in a small subset of cells with incorrect clock times corresponding to the subjective morning. We find that the clock generates rhythms in the instantaneous growth rate of the cell, and that the time of darkness vulnerability coincides with the time of most rapid growth. Thus, the clock mediates a fundamental trade-off between growth and starvation tolerance in cycling environments. By measuring the response of the circadian rhythm to dark pulses of varying lengths, we constrain a mathematical model of a population's fitness under arbitrary light/dark schedules. This model predicts that the circadian clock is only advantageous in highly regular cycling environments with frequencies sufficiently close to the natural frequency of the clock.

The transcription factor Vox represses endoderm development by interacting with Casanova and Pou2.

Endoderm and mesoderm are both formed upon activation of Nodal signaling but how endoderm differentiates from mesoderm is still poorly explored. The sox-related gene casanova (sox32) acts downstream of the Nodal signal, is essential for endoderm development and requires the co-factor Pou2 (Pou5f1, Oct3, Oct4) in this process. Conversely, BMP signals have been shown to inhibit endoderm development by an as yet unexplained mechanism. In a search for Casanova regulators in zebrafish, we identified two of its binding partners as the transcription factors Pou2 and Vox, a member of the Vent group of proteins also involved in the patterning of the gastrula. In overexpression studies we show that vox and/or Vent group genes inhibit the capacity of Casanova to induce endoderm, even in the presence of its co-factor Pou2, and that Vox acts as a repressor in this process. We further show that vox, but not other members of the Vent group, is essential for defining the proper endodermal domain size at gastrulation. In this process, vox acts downstream of BMPs. Cell fate analysis further shows that Vox plays a key role downstream of BMP signals in regulating the capacity of Nodal to induce endoderm versus mesoderm by modulating the activity of the Casanova/Pou2 regulatory system.

Cell motility and drug gradients in the emergence of resistance to chemotherapy.

The emergence of resistance to chemotherapy by cancer cells, when combined with metastasis, is the primary driver of mortality in cancer and has proven to be refractory to many efforts. Theory and computer modeling suggest that the rate of emergence of resistance is driven by the strong selective pressure of mutagenic chemotherapy and enhanced by the motility of mutant cells in a chemotherapy gradient to areas of higher drug concentration and lower population competition. To test these models, we constructed a synthetic microecology which superposed a mutagenic doxorubicin gradient across a population of motile, metastatic breast cancer cells (MDA-MB-231). We observed the emergence of MDA-MB-231 cancer cells capable of proliferation at 200 nM doxorubicin in this complex microecology. Individual cell tracking showed both movement of the MDA-MB-231 cancer cells toward higher drug concentrations and proliferation of the cells at the highest doxorubicin concentrations within 72 h, showing the importance of both motility and drug gradients in the emergence of resistance.

Recollision-Induced Superradiance of Ionized Nitrogen Molecules.

We propose a new mechanism to explain the origin of optical gain in the transitions between the excited and ground states of the ionized nitrogen molecule following irradiation of neutral nitrogen molecules with an intense ultrashort laser pulse. An efficient transfer of population to the excited state is achieved via field-induced multiple recollisions. We show that the proposed excitation mechanism must lead to a superradiant emission, a feature that we confirm experimentally.

Fine-tuning of Hh signaling by the RNA-binding protein Quaking to control muscle development.

The development of the different muscles within the somite is a complex process that involves the Hedgehog (Hh) signaling pathway. To specify the proper number of muscle cells and organize them spatially and temporally, the Hh signaling pathway needs to be precisely regulated at different levels, but only a few factors external to the pathway have been described. Here, we report for the first time the role of the STAR family RNA-binding protein Quaking A (QkA) in somite muscle development. We show in zebrafish that the loss of QkA function affects fast muscle fiber maturation as well as Hh-induced muscle derivative specification and/or morphogenesis. Mosaic analysis reveals that fast fiber maturation depends on the activity of QkA in the environment of fast fiber progenitors. We further show that Hh signaling requires QkA activity for muscle development. By an in silico approach, we screened the 3'UTRs of known Hh signaling component mRNAs for the Quaking response element and found the transcription factor Gli2a, a known regulator of muscle fate development. Using destabilized GFP as a reporter, we show that the gli2a mRNA 3'UTR is a functional QkA target. Consistent with this notion, the loss of QkA function rescued slow muscle fibers in yot mutant embryos, which express a dominant-negative Gli2a isoform. Thus, our results reveal a new mechanism to ensure muscle cell fate diversity by fine-tuning of the Hh signaling pathway via RNA-binding proteins.

Frugal day-ahead forecasting of multiple local electricity loads by aggregating adaptive models.

This paper focuses on day-ahead electricity load forecasting for substations of the distribution network in France; therefore, the corresponding problem lies between the instability of a single consumption and the stability of a countrywide total demand. Moreover, this problem requires to forecast the loads of over one thousand substations; consequently, it belongs to the field of multiple time series forecasting. To that end, the paper applies an adaptive methodology that provided excellent results at a national scale; the idea is to combine generalized additive models with state-space representations. However, extending this methodology to the prediction of over a thousand time series raises a computational issue. It is solved by developing a frugal variant that reduces the number of estimated parameters: forecasting models are estimated only for a few time series and transfer learning is achieved by relying on aggregation of experts. This approach yields a reduction of computational needs and their associated emissions. Several variants are built, corresponding to different levels of parameter transfer, to find the best trade-off between accuracy and frugality. The selected method achieves competitive results compared to individual models. Finally, the paper highlights the interpretability of the models, which is important for operational applications.

Overcoming Leak Sensitivity in CRISPRi Circuits Using Antisense RNA Sequestration and Regulatory Feedback.

The controlled binding of the catalytically dead CRISPR nuclease (dCas) to DNA can be used to create complex, programmable transcriptional genetic circuits, a fundamental goal of synthetic biology. This approach, called CRISPR interference (CRISPRi), is advantageous over existing methods because the programmable nature of CRISPR proteins in principle enables the simultaneous regulation of many different targets without crosstalk. However, the performance of dCas-based genetic circuits is limited by both the sensitivity to leaky repression within CRISPRi logic gates and retroactive effects due to a shared pool of dCas proteins. By utilizing antisense RNAs (asRNAs) to sequester gRNA transcripts as well as CRISPRi feedback to self-regulate asRNA production, we demonstrate a mechanism that suppresses unwanted repression by CRISPRi and improves logical gene circuit function in Escherichia coli. This improvement is particularly pronounced during stationary expression when CRISPRi circuits do not achieve the expected regulatory dynamics. Furthermore, the use of dual CRISPRi/asRNA inverters restores the logical performance of layered circuits such as a double inverter. By studying circuit induction at the single-cell level in microfluidic channels, we provide insight into the dynamics of antisense sequestration of gRNA and regulatory feedback on dCas-based repression and derepression. These results demonstrate how CRISPRi inverters can be improved for use in more complex genetic circuitry without sacrificing the programmability and orthogonality of dCas proteins.

A plasmid system with tunable copy number.

Plasmids are one of the most commonly used platforms for genetic engineering and recombinant gene expression in bacteria. The range of available copy numbers for cloning vectors is largely restricted to the handful of Origins of Replication (ORIs) that have been isolated from plasmids found in nature. Here, we introduce two systems that allow for the continuous, finely-tuned control of plasmid copy number between 1 and 800 copies per cell: a plasmid with an anhydrotetracycline-controlled copy number, and a parallelized assay that is used to generate a continuous spectrum of 1194 ColE1-based copy number variants. Using these systems, we investigate the effects of plasmid copy number on cellular growth rates, gene expression, biosynthesis, and genetic circuit performance. We perform single-cell timelapse measurements to characterize plasmid loss, runaway plasmid replication, and quantify the impact of plasmid copy number on the variability of gene expression. Using our assay, we find that each plasmid imposes a 0.063% linear metabolic burden on their hosts, hinting at a simple relationship between metabolic burdens and plasmid DNA synthesis. Our systems enable the precise control of gene expression, and our results highlight the importance of tuning plasmid copy number as a powerful tool for the optimization of synthetic biological systems.

Analytic description and optimization of magneto-optical Kerr setups with photoelastic modulation.

Instruments based on the magneto-optical Kerr effect are routinely used to probe surface magnetic properties. These tools rely on the characterization of the polarization state of reflected light from the sample to collect information on its magnetization. Here, we present a theoretical optimization of common setups based on the magneto-optical Kerr effect. A detection scheme based on a simple analyzer and photodetector and one made from a polarizing beam splitter and balanced photodetectors are considered. The effect of including a photoelastic modulator (PEM) and a lock-in amplifier to detect the signal at harmonics of the modulating frequency is studied. Jones formalism is used to derive general expressions that link the intensity of the measured signal to the magneto-optical Fresnel reflection coefficients for any orientation of the polarizing optical components. Optimal configurations are then defined as those that allow measuring the Kerr rotation and ellipticity while minimizing nonmagnetic contributions from the diagonal Fresnel coefficients in order to improve the signal-to-noise ratio (SNR). The expressions show that with the PEM, setups based on polarizing beam splitters inherently offer a twofold higher signal than commonly used analyzers, and the experimental results confirm that the SNR is improved by more than 150%. Furthermore, we find that while all proposed detection schemes measure Kerr effects, only those with polarizing beam splitters allow measuring the Kerr rotation directly when no modulator is included. This accommodates, for instance, time-resolved measurements at relatively low laser pulse repetition rates. Ultrafast demagnetization measurements are presented as an example of such applications.

Polarity of the CRISPR roadblock to transcription.

CRISPR (clustered regularly interspaced short palindromic repeats) utility relies on a stable Cas effector complex binding to its target site. However, a Cas complex bound to DNA may be removed by motor proteins carrying out host processes and the mechanism governing this removal remains unclear. Intriguingly, during CRISPR interference, RNA polymerase (RNAP) progression is only fully blocked by a bound endonuclease-deficient Cas (dCas) from the protospacer adjacent motif (PAM)-proximal side. By mapping dCas-DNA interactions at high resolution, we discovered that the collapse of the dCas R-loop allows Escherichia coli RNAP read-through from the PAM-distal side for both Sp-dCas9 and As-dCas12a. This finding is not unique to RNAP and holds for the Mfd translocase. This mechanistic understanding allowed us to modulate the dCas R-loop stability by modifying the guide RNAs. This work highlights the importance of the R-loop in dCas-binding stability and provides valuable mechanistic insights for broad applications of CRISPR technology.

Increased energy demand from anabolic-catabolic processes drives ß-lactam antibiotic lethality.

ß-Lactam antibiotics disrupt the assembly of peptidoglycan (PG) within the bacterial cell wall by inhibiting the enzymatic activity of penicillin-binding proteins (PBPs). It was recently shown that ß-lactam treatment initializes a futile cycle of PG synthesis and degradation, highlighting major gaps in our understanding of the lethal effects of PBP inhibition by ß-lactam antibiotics. Here, we assess the downstream metabolic consequences of treatment of Escherichia coli with the ß-lactam mecillinam and show that lethality from PBP2 inhibition is a specific consequence of toxic metabolic shifts induced by energy demand from multiple catabolic and anabolic processes, including accelerated protein synthesis downstream of PG futile cycling. Resource allocation into these processes is coincident with alterations in ATP synthesis and utilization, as well as a broadly dysregulated cellular redox environment. These results indicate that the disruption of normal anabolic-catabolic homeostasis by PBP inhibition is an essential factor for ß-lactam antibiotic lethality.

Anomalous spatial redistribution of competing bacteria under starvation conditions.

Bacterial cells evolved under prolonged stress often have a growth advantage in stationary phase (GASP); we expect GASP cells to maintain a proliferative state and dominate wild-type cells during starvation, especially when nutrients are limited and the medium has been conditioned. However, when we compete GASP mutants against wild-type cells in a chain of microfluidic microhabitat patches (MHPs) with alternating nutrient-rich and nutrient-limited regions, we observe the reverse effect: wild-type cells achieve maximum relative density under nutrient-limited conditions, while GASP cells dominate nutrient-rich regions. We explain this surprising observation in terms of ideal free distributions, where we show that wild-type cells maximize their fitness at high cell density by redistributing themselves to sparsely populated MHPs. At the microscopic level, we describe how biofilm formation also contributes to the population redistribution. We conclude by discussing the implications of these results for social interactions of more complex organisms.

Polarization control of high order harmonics in the EUV photon energy range.

We report the generation of circularly polarized high order harmonics in the extreme ultraviolet range (18-27 nm) from a linearly polarized infrared laser (40 fs, 0.25 TW) focused into a neon filled gas cell. To circularly polarize the initially linearly polarized harmonics we have implemented a four-reflector phase-shifter. Fully circularly polarized radiation has been obtained with an efficiency of a few percents, thus being significantly more efficient than currently demonstrated direct generation of elliptically polarized harmonics. This demonstration opens up new experimental capabilities based on high order harmonics, for example, in biology and materials science. The inherent femtosecond time resolution of high order harmonic generating table top laser sources renders these an ideal tool for the investigation of ultrafast magnetization dynamics now that the magnetic circular dichroism at the absorption M-edges of transition metals can be exploited.