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.

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.

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.

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.

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.

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.

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.

Lin28, a major translation reprogramming factor, gains access to YB-1-packaged mRNA through its cold-shock domain.

The RNA-binding protein Lin28 (Lin28a) is an important pluripotency factor that reprograms translation and promotes cancer progression. Although Lin28 blocks let-7 microRNA maturation, Lin28 also binds to a large set of cytoplasmic mRNAs directly. However, how Lin28 regulates the processing of many mRNAs to reprogram global translation remains unknown. We show here, using a structural and cellular approach, a mixing of Lin28 with YB-1 (YBX1) in the presence of mRNA owing to their cold-shock domain, a conserved ß-barrel structure that binds to ssRNA cooperatively. In contrast, the other RNA binding-proteins without cold-shock domains tested, HuR, G3BP-1, FUS and LARP-6, did not mix with YB-1. Given that YB-1 is the core component of dormant mRNPs, a model in which Lin28 gains access to mRNPs through its co-association with YB-1 to mRNA may provide a means for Lin28 to reprogram translation. We anticipate that the translational plasticity provided by mRNPs may contribute to Lin28 functions in development and adaptation of cancer cells to an adverse environment.

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.

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.

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.

Quaking RNA-Binding Proteins Control Early Myofibril Formation by Modulating Tropomyosin.

Skeletal muscle contraction is mediated by myofibrils, complex multi-molecular scaffolds structured into repeated units, the sarcomeres. Myofibril structure and function have been extensively studied, but the molecular processes regulating its formation within the differentiating muscle cell remain largely unknown. Here we show in zebrafish that genetic interference with the Quaking RNA-binding proteins disrupts the initial steps of myofibril assembly without affecting early muscle differentiation. Using RNA sequencing, we demonstrate that Quaking is required for accumulation of the muscle-specific tropomyosin-3 transcript, tpm3.12. Further functional analyses reveal that Tpm3.12 mediates Quaking control of myofibril formation. Moreover, we identified a Quaking-binding site in the 3' UTR of tpm3.12 transcript, which is required in vivo for tpm3.12 accumulation and myofibril formation. Our work uncovers a Quaking/Tpm3 pathway controlling de novo myofibril assembly. This unexpected developmental role for Tpm3 could be at the origin of muscle defects observed in human congenital myopathies associated with tpm3 mutation.

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.

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.

Indirect excitation of ultrafast demagnetization.

Does the excitation of ultrafast magnetization require direct interaction between the photons of the optical pump pulse and the magnetic layer? Here, we demonstrate unambiguously that this is not the case. For this we have studied the magnetization dynamics of a ferromagnetic cobalt/palladium multilayer capped by an IR-opaque aluminum layer. Upon excitation with an intense femtosecond-short IR laser pulse, the film exhibits the classical ultrafast demagnetization phenomenon although only a negligible number of IR photons penetrate the aluminum layer. In comparison with an uncapped cobalt/palladium reference film, the initial demagnetization of the capped film occurs with a delayed onset and at a slower rate. Both observations are qualitatively in line with energy transport from the aluminum layer into the underlying magnetic film by the excited, hot electrons of the aluminum film. Our data thus confirm recent theoretical predictions.

Controlling the Cyanobacterial Clock by Synthetically Rewiring Metabolism.

Circadian clocks are oscillatory systems that allow organisms to anticipate rhythmic changes in the environment. Several studies have shown that circadian clocks are connected to metabolism, but it is not generally clear whether metabolic signaling is one voice among many that influence the clock or whether metabolic cycling is the major clock synchronizer. To address this question in cyanobacteria, we used a synthetic biology approach to make normally autotrophic cells capable of growth on exogenous sugar. This allowed us to manipulate metabolism independently from light and dark. We found that feeding sugar to cultures blocked the clock-resetting effect of a dark pulse. Furthermore, in the absence of light, the clock efficiently synchronizes to metabolic cycles driven by rhythmic feeding. We conclude that metabolic activity, independent of its source, is the primary clock driver in cyanobacteria.

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.

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.

Quantifying selective pressures driving bacterial evolution using lineage analysis.

Organisms use a variety of strategies to adapt to their environments and maximize long-term growth potential, but quantitative characterization of the benefits conferred by the use of such strategies, as well as their impact on the whole population's rate of growth, remains challenging. Here, we use a path-integral framework that describes how selection acts on lineages -i.e. the life-histories of individuals and their ancestors- to demonstrate that lineage-based measurements can be used to quantify the selective pressures acting on a population. We apply this analysis to E. coli bacteria exposed to cyclical treatments of carbenicillin, an antibiotic that interferes with cell-wall synthesis and affects cells in an age-dependent manner. While the extensive characterization of the life-history of thousands of cells is necessary to accurately extract the age-dependent selective pressures caused by carbenicillin, the same measurement can be recapitulated using lineage-based statistics of a single surviving cell. Population-wide evolutionary pressures can be extracted from the properties of the surviving lineages within a population, providing an alternative and efficient procedure to quantify the evolutionary forces acting on a population. Importantly, this approach is not limited to age-dependent selection, and the framework can be generalized to detect signatures of other trait-specific selection using lineage-based measurements. Our results establish a powerful way to study the evolutionary dynamics of life under selection, and may be broadly useful in elucidating selective pressures driving the emergence of antibiotic resistance and the evolution of survival strategies in biological systems.