The challenge of unprecedented floods and droughts in risk management.

Risk management has reduced vulnerability to floods and droughts globally1,2, yet their impacts are still increasing3. An improved understanding of the causes of changing impacts is therefore needed, but has been hampered by a lack of empirical data4,5. On the basis of a global dataset of 45 pairs of events that occurred within the same area, we show that risk management generally reduces the impacts of floods and droughts but faces difficulties in reducing the impacts of unprecedented events of a magnitude not previously experienced. If the second event was much more hazardous than the first, its impact was almost always higher. This is because management was not designed to deal with such extreme events: for example, they exceeded the design levels of levees and reservoirs. In two success stories, the impact of the second, more hazardous, event was lower, as a result of improved risk management governance and high investment in integrated management. The observed difficulty of managing unprecedented events is alarming, given that more extreme hydrological events are projected owing to climate change3.

BNT162b2, mRNA-1273, and Sputnik V Vaccines Induce Comparable Immune Responses on a Par With Severe Course of COVID-19.

Vaccines against the severe acute respiratory syndrome coronavirus 2, which have been in urgent need and development since the beginning of 2020, are aimed to induce a prominent immune system response capable of recognizing and fighting future infection. Here we analyzed the levels of IgG antibodies against the receptor-binding domain (RBD) of the viral spike protein after the administration of three types of popular vaccines, BNT162b2, mRNA-1273, or Sputnik V, using the same ELISA assay to compare their effects. An efficient immune response was observed in the majority of cases. The obtained ranges of signal values were wide, presumably reflecting specific features of the immune system of individuals. At the same time, these ranges were comparable among the three studied vaccines. The anti-RBD IgG levels after vaccination were also similar to those in the patients with moderate/severe course of the COVID-19, and significantly higher than in the individuals with asymptomatic or light symptomatic courses of the disease. No significant correlation was observed between the levels of anti-RBD IgG and sex or age of the vaccinated individuals. The signals measured at different time points for several individuals after full Sputnik V vaccination did not have a significant tendency to lower within many weeks. The rate of neutralization of the interaction of the RBD with the ACE2 receptor after vaccination with Sputnik V was on average slightly higher than in patients with a moderate/severe course of COVID-19. The importance of the second dose administration of the two-dose Sputnik V vaccine was confirmed: while several individuals had not developed detectable levels of the anti-RBD IgG antibodies after the first dose of Sputnik V, after the second dose the antibody signal became positive for all tested individuals and raised on average 5.4 fold. Finally, we showed that people previously infected with SARS-CoV-2 developed high levels of antibodies, efficiently neutralizing interaction of RBD with ACE2 after the first dose of Sputnik V, with almost no change after the second dose.

IgG Antibodies Develop to Spike but Not to the Nucleocapsid Viral Protein in Many Asymptomatic and Light COVID-19 Cases.

Since SARS-CoV-2 appeared in late 2019, many studies on the immune response to COVID-19 have been conducted, but the asymptomatic or light symptom cases were somewhat understudied as respective individuals often did not seek medical help. Here, we analyze the production of the IgG antibodies to viral nucleocapsid (N) protein and receptor-binding domain (RBD) of the spike protein and assess the serum neutralization capabilities in a cohort of patients with different levels of disease severity. In half of light or asymptomatic cases the antibodies to the nucleocapsid protein, which serve as the main target in many modern test systems, were not detected. They were detected in all cases of moderate or severe symptoms, and severe lung lesions correlated with respective higher signals. Antibodies to RBD were present in the absolute majority of samples, with levels being sometimes higher in light symptom cases. We thus suggest that the anti-RBD/anti-N antibody ratio may serve as an indicator of the disease severity. Anti-RBD IgG remained detectable after a year or more since the infection, even with a slight tendency to raise over time, and the respective signal correlated with the serum capacity to inhibit the RBD interaction with the ACE-2 receptor.

Expression of SARS-CoV-2 surface glycoprotein fragment 319-640 in E. coli, and its refolding and purification.

Sensitive and specific serology tests are essential for epidemiological and public health studies of COVID-19 and for vaccine efficacy testing. The presence of antibodies to SARS-CoV-2 surface glycoprotein (Spike) and, specifically, its receptor-binding domain (RBD) correlates with inhibition of SARS-CoV-2 binding to the cellular receptor and viral entry into the cells. Serology tests that detect antibodies targeting RBD have high potential to predict COVID-19 immunity and to accurately determine the extent of the vaccine-induced immune response. Cost-effective methods of expression and purification of Spike and its fragments that preserve antigenic properties are essential for development of such tests. Here we describe a method of production of His6-tagged S319-640 fragment containing RBD in E. coli. It includes expression of the fragment, solubilization of inclusion bodies, and on-the-column refolding. The antigenic properties of the resulting product are similar, but not identical to the RBD-containing fragment expressed in human cells.

Dual-Antigen System Allows Elimination of False Positive Results in COVID-19 Serological Testing.

Determining the presence of antibodies in serum is important for epidemiological studies, to be able to confirm whether a person has been infected, predicting risks of them getting sick and spreading the disease. During the ongoing pandemic of COVID-19, a positive serological test result can suggest if it is safe to return to work and re-engage in social activities. Despite a multitude of emerging tests, the quality of respective data often remains ambiguous, yielding a significant fraction of false positive results. The human organism produces polyclonal antibodies specific to multiple viral proteins, so testing simultaneously for multiple antibodies appeared a practical approach for increasing test specificity. We analyzed immune response and testing potential for a spectrum of antigens derived from the spike and nucleocapsid proteins of SARS-CoV-2, developed a dual-antigen testing system in the ELISA format and designed a robust algorithm for data processing. Combining nucleocapsid protein and receptor-binding domain for analysis allowed us to completely eliminate false positive results in the tested cohort (achieving specificity within a 95% confidence interval of 97.2-100%). We also tested samples collected from different households, and demonstrated differences in the immune response of COVID-19 patients and their family members; identifying, in particular, asymptomatic cases showing strong presence of studied antibodies, and cases showing none despite confirmed close contacts with the infected individuals.

NusG controls transcription pausing and RNA polymerase translocation throughout the Bacillus subtilis genome.

Transcription is punctuated by RNA polymerase (RNAP) pausing. These pauses provide time for diverse regulatory events that can modulate gene expression. Transcription elongation factors dramatically affect RNAP pausing in vitro, but the genome-wide role of such factors on pausing has not been examined. Using native elongating transcript sequencing followed by RNase digestion (RNET-seq), we analyzed RNAP pausing in Bacillus subtilis genome-wide and identified an extensive role of NusG in pausing. This universally conserved transcription elongation factor is known as Spt5 in archaeal and eukaryotic organisms. B. subtilis NusG shifts RNAP to the posttranslocation register and induces pausing at 1,600 sites containing a consensus TTNTTT motif in the nontemplate DNA strand within the paused transcription bubble. The TTNTTT motif is necessary but not sufficient for NusG-dependent pausing. Approximately one-fourth of these pause sites were localized to untranslated regions and could participate in posttranscription initiation control of gene expression as was previously shown for tlrB and the trpEDCFBA operon. Most of the remaining pause sites were identified in protein-coding sequences. NusG-dependent pausing was confirmed for all 10 pause sites that we tested in vitro. Putative pause hairpins were identified for 225 of the 342 strongest NusG-dependent pause sites, and some of these hairpins were shown to function in vitro. NusG-dependent pausing in the ribD riboswitch provides time for cotranscriptional binding of flavin mononucleotide, which decreases the concentration required for termination upstream of the ribD coding sequence. Our phylogenetic analysis implicates NusG-dependent pausing as a widespread mechanism in bacteria.

Changing climate both increases and decreases European river floods.

Climate change has led to concerns about increasing river floods resulting from the greater water-holding capacity of a warmer atmosphere1. These concerns are reinforced by evidence of increasing economic losses associated with flooding in many parts of the world, including Europe2. Any changes in river floods would have lasting implications for the design of flood protection measures and flood risk zoning. However, existing studies have been unable to identify a consistent continental-scale climatic-change signal in flood discharge observations in Europe3, because of the limited spatial coverage and number of hydrometric stations. Here we demonstrate clear regional patterns of both increases and decreases in observed river flood discharges in the past five decades in Europe, which are manifestations of a changing climate. Our results-arising from the most complete database of European flooding so far-suggest that: increasing autumn and winter rainfall has resulted in increasing floods in northwestern Europe; decreasing precipitation and increasing evaporation have led to decreasing floods in medium and large catchments in southern Europe; and decreasing snow cover and snowmelt, resulting from warmer temperatures, have led to decreasing floods in eastern Europe. Regional flood discharge trends in Europe range from an increase of about 11 per cent per decade to a decrease of 23 per cent. Notwithstanding the spatial and temporal heterogeneity of the observational record, the flood changes identified here are broadly consistent with climate model projections for the next century4,5, suggesting that climate-driven changes are already happening and supporting calls for the consideration of climate change in flood risk management.

The Role of Pyrophosphorolysis in the Initiation-to-Elongation Transition by E. coli RNA Polymerase.

RNA polymerase can cleave a phosphodiester bond at the 3' end of a nascent RNA in the presence of pyrophosphate producing NTP. Pyrophosphorolysis has been characterized during elongation steps of transcription where its rate is significantly slower than the forward rate of NMP addition. In contrast, we report here that pyrophosphorolysis can occur in a millisecond time scale during the transition of Escherichia coli RNA polymerase from initiation to elongation at the psbA2 promoter. This rapid pyrophosphorolysis occurs during productive RNA synthesis as opposed to abortive RNA synthesis. Dissociation of σ70 or RNA extension beyond nine nucleotides dramatically reduces the rate of pyrophosphorolysis. We argue that the rapid pyrophosphorolysis allows iterative cycles of cleavage and re-synthesis of the 3' phosphodiester bond by the productive complexes in the early stage of transcription. This iterative process may provide an opportunity for the σ70 to dissociate from the RNA exit channel of the enzyme, enabling RNA to extend through the channel.

RNA-DNA and DNA-DNA base-pairing at the upstream edge of the transcription bubble regulate translocation of RNA polymerase and transcription rate.

Translocation of RNA polymerase (RNAP) along DNA may be rate-limiting for transcription elongation. The Brownian ratchet model posits that RNAP rapidly translocates back and forth until the post-translocated state is stabilized by NTP binding. An alternative model suggests that RNAP translocation is slow and poorly reversible. To distinguish between these two models, we take advantage of an observation that pyrophosphorolysis rates directly correlate with the abundance of the pre-translocated fraction. Pyrophosphorolysis by RNAP stabilized in the pre-translocated state by bacteriophage HK022 protein Nun was used as a reference point to determine the pre-translocated fraction in the absence of Nun. The stalled RNAP preferentially occupies the post-translocated state. The forward translocation rate depends, among other factors, on melting of the RNA-DNA base pair at the upstream edge of the transcription bubble. DNA-DNA base pairing immediately upstream from the RNA-DNA hybrid stabilizes the post-translocated state. This mechanism is conserved between E. coli RNAP and S. cerevisiae RNA polymerase II and is partially dependent on the lid domain of the catalytic subunit. Thus, the RNA-DNA hybrid and DNA reannealing at the upstream edge of the transcription bubble emerge as targets for regulation of the transcription elongation rate.

Changing climate shifts timing of European floods.

A warming climate is expected to have an impact on the magnitude and timing of river floods; however, no consistent large-scale climate change signal in observed flood magnitudes has been identified so far. We analyzed the timing of river floods in Europe over the past five decades, using a pan-European database from 4262 observational hydrometric stations, and found clear patterns of change in flood timing. Warmer temperatures have led to earlier spring snowmelt floods throughout northeastern Europe; delayed winter storms associated with polar warming have led to later winter floods around the North Sea and some sectors of the Mediterranean coast; and earlier soil moisture maxima have led to earlier winter floods in western Europe. Our results highlight the existence of a clear climate signal in flood observations at the continental scale.

Cotranscriptional Production of Chemically Modified RNA Nanoparticles.

RNA nanoparticles consisting of multiple RNA strands of different sequences forming various three-dimensional structures emerge as promising carriers of siRNAs, RNA aptamers, and ribozymes. In vitro transcription of a mixture of dsDNA templates encoding all the subunits of the RNA nanoparticle may result in cotranscriptional self-assembly of the nanoparticle. Based on our experience with production of RNA nanorings, RNA nanocubes, and RNA three-way junctions, we propose a strategy for optimization of the cotranscriptional production of chemically modified ribonuclease-resistant RNA nanoparticles.

A Cre Transcription Fidelity Reporter Identifies GreA as a Major RNA Proofreading Factor in Escherichia coli.

We made a coupled genetic reporter that detects rare transcription misincorporation errors to measure RNA polymerase transcription fidelity in Escherichia coli Using this reporter, we demonstrated in vivo that the transcript cleavage factor GreA, but not GreB, is essential for proofreading of a transcription error where a riboA has been misincorporated instead of a riboG. A greA mutant strain had more than a 100-fold increase in transcription errors relative to wild-type or a greB mutant. However, overexpression of GreB in ΔgreA cells reduced the misincorporation errors to wild-type levels, demonstrating that GreB at high concentration could substitute for GreA in RNA proofreading activity in vivo.

Production and characterization of a highly pure RNA polymerase holoenzyme from Mycobacterium tuberculosis.

Recent publications have shown that active RNA polymerase (RNAP) from Mycobacterium tuberculosis (MtbRNAP) can be produced by expressing all four subunits in a single recombinant Escherichia coli strain [1-3]. By reducing the number of plasmids and changing the codon usage of the Mtb genes in the co-expression system published by Banerjee et al. [1], we present a simplified, detailed and reproducible protocol for the purification of recombinant MtbRNAP containing the ω subunit. Moreover, we describe the formation of ternary elongation complexes (TECs) with a short fluorescence-labeled RNA primer and DNA oligonucleotides, suitable for transcription elongation studies. The purification of milligram quantities of the pure and highly active holoenzyme omits ammonium sulfate or polyethylene imine precipitation steps [4] and requires only 5 g of wet cells. Our results indicate that subunit assemblies other than α2ßß'ω·σA can be separated by ion-exchange chromatography on Mono Q column and that assemblies with the wrong RNAP subunit stoichiometry lack transcriptional activity. We show that MtbRNAP TECs can be stalled by NTP substrate deprivation and chased upon the addition of missing NTP(s) without the need of any accessory proteins. Finally, we demonstrate the ability of the purified MtbRNAP to initiate transcription from a promoter and establish that its open promoter complexes are stabilized by the M. tuberculosis protein CarD.

Productive mRNA stem loop-mediated transcriptional slippage: Crucial features in common with intrinsic terminators.

Escherichia coli and yeast DNA-dependent RNA polymerases are shown to mediate efficient nascent transcript stem loop formation-dependent RNA-DNA hybrid realignment. The realignment was discovered on the heteropolymeric sequence T5C5 and yields transcripts lacking a C residue within a corresponding U5C4. The sequence studied is derived from a Roseiflexus insertion sequence (IS) element where the resulting transcriptional slippage is required for transposase synthesis. The stability of the RNA structure, the proximity of the stem loop to the slippage site, the length and composition of the slippage site motif, and the identity of its 3' adjacent nucleotides (nt) are crucial for transcripts lacking a single C. In many respects, the RNA structure requirements for this slippage resemble those for hairpin-dependent transcription termination. In a purified in vitro system, the slippage efficiency ranges from 5% to 75% depending on the concentration ratios of the nucleotides specified by the slippage sequence and the 3' nt context. The only previous proposal of stem loop mediated slippage, which was in Ebola virus expression, was based on incorrect data interpretation. We propose a mechanical slippage model involving the RNAP translocation state as the main motor in slippage directionality and efficiency. It is distinct from previously described models, including the one proposed for paramyxovirus, where following random movement efficiency is mainly dependent on the stability of the new realigned hybrid. In broadening the scope for utilization of transcription slippage for gene expression, the stimulatory structure provides parallels with programmed ribosomal frameshifting at the translation level.

Computational and experimental studies of reassociating RNA/DNA hybrids containing split functionalities.

Recently, we developed a novel technique based on RNA/DNA hybrid reassociation that allows conditional activation of different split functionalities inside diseased cells and in vivo. We further expanded this idea to permit simultaneous activation of multiple different functions in a fully controllable fashion. In this chapter, we discuss some novel computational approaches and experimental techniques aimed at the characterization, design, and production of reassociating RNA/DNA hybrids containing split functionalities. We also briefly describe several experimental techniques that can be used to test these hybrids in vitro and in vivo.

Direct competition assay for transcription fidelity.

Accurate transcription is essential for faithful information flow from DNA to RNA and to the protein. Mechanisms of cognate substrate selection by RNA polymerases are currently elucidated by structural, genetic, and biochemical approaches. Here, we describe a fast and reliable approach to quantitative analyses of transcription fidelity, applicable to analyses of RNA polymerase selectivity against misincorporation, incorporation of dNMPs, and chemically modified rNMP analogues. The method is based on different electrophoretic mobility of RNA oligomers of the same length but differing in sequence.

Triggering of RNA interference with RNA-RNA, RNA-DNA, and DNA-RNA nanoparticles.

Control over cellular delivery of different functionalities and their synchronized activation is a challenging task. We report several RNA and RNA/DNA-based nanoparticles designed to conditionally activate the RNA interference in various human cells. These nanoparticles allow precise control over their formulation, stability in blood serum, and activation of multiple functionalities. Importantly, interferon and pro-inflammatory cytokine activation assays indicate the significantly lower responses for DNA nanoparticles compared to the RNA counterparts, suggesting greater potential of these molecules for therapeutic use.

A genetic assay for transcription errors reveals multilayer control of RNA polymerase II fidelity.

We developed a highly sensitive assay to detect transcription errors in vivo. The assay is based on suppression of a missense mutation in the active site tyrosine in the Cre recombinase. Because Cre acts as tetramer, background from translation errors are negligible. Functional Cre resulting from rare transcription errors that restore the tyrosine codon can be detected by Cre-dependent rearrangement of reporter genes. Hence, transient transcription errors are captured as stable genetic changes. We used this Cre-based reporter to screen for mutations of Saccharomyces cerevisiae RPB1 (RPO21) that increase the level of misincorporation during transcription. The mutations are in three domains of Rpb1, the trigger loop, the bridge helix, and in sites involved in binding to TFIIS. Biochemical characterization demonstrates that these variants have elevated misincorporation, and/or ability to extend mispaired bases, or defects in TFIIS mediated editing.

Coliphage HK022 Nun protein inhibits RNA polymerase translocation.

The Nun protein of coliphage HK022 arrests RNA polymerase (RNAP) in vivo and in vitro at pause sites distal to phage λ N-Utilization (nut) site RNA sequences. We tested the activity of Nun on ternary elongation complexes (TECs) assembled with templates lacking the λ nut sequence. We report that Nun stabilizes both translocation states of RNAP by restricting lateral movement of TEC along the DNA register. When Nun stabilized TEC in a pretranslocated register, immediately after NMP incorporation, it prevented binding of the next NTP and stimulated pyrophosphorolysis of the nascent transcript. In contrast, stabilization of TEC by Nun in a posttranslocated register allowed NTP binding and nucleotidyl transfer but inhibited pyrophosphorolysis and the next round of forward translocation. Nun binding to and action on the TEC requires a 9-bp RNA-DNA hybrid. We observed a Nun-dependent toe print upstream to the TEC. In addition, mutations in the RNAP ß' subunit near the upstream end of the transcription bubble suppress Nun binding and arrest. These results suggest that Nun interacts with RNAP near the 5' edge of the RNA-DNA hybrid. By stabilizing translocation states through restriction of TEC lateral mobility, Nun represents a novel class of transcription arrest factors.

In silico design and enzymatic synthesis of functional RNA nanoparticles.

CONSPECTUS: The use of RNAs as scaffolds for biomedical applications has several advantages compared with other existing nanomaterials. These include (i) programmability, (ii) precise control over folding and self-assembly, (iii) natural functionalities as exemplified by ribozymes, riboswitches, RNAi, editing, splicing, and inherent translation and transcription control mechanisms, (iv) biocompatibility, (v) relatively low immune response, and (vi) relatively low cost and ease of production. We have tapped into several of these properties and functionalities to construct RNA-based functional nanoparticles (RNA NPs). In several cases, the structural core and the functional components of the NPs are inherent in the same construct. This permits control over the spatial disposition of the components, intracellular availability, and precise stoichiometry. To enable the generation of RNA NPs, a pipeline is being developed. On one end, it encompasses the rational design and various computational schemes that promote design of the RNA-based nanoconstructs, ultimately producing a set of sequences consisting of RNA or RNA-DNA hybrids, which can assemble into the designed construct. On the other end of the pipeline is an experimental component, which takes the produced sequences and uses them to initialize and characterize their proper assembly and then test the resulting RNA NPs for their function and delivery in cell culture and animal models. An important aspect of this pipeline is the feedback that constantly occurs between the computational and the experimental parts, which synergizes the refinement of both the algorithmic methodologies and the experimental protocols. The utility of this approach is depicted by the several examples described in this Account (nanocubes, nanorings, and RNA-DNA hybrids). Of particular interest, from the computational viewpoint, is that in most cases, first a three-dimensional representation of the assembly is produced, and only then are algorithms applied to generate the sequences that will assemble into the designated three-dimensional construct. This is opposite to the usual practice of predicting RNA structures from a given sequence, that is, the RNA folding problem. To be considered is the generation of sequences that upon assembly have the proper intra- or interstrand interactions (or both). Of particular interest from the experimental point of view is the determination and characterization of the proper thermodynamic, kinetic, functionality, and delivery protocols. Assembly of RNA NPs from individual single-stranded RNAs can be accomplished by one-pot techniques under the proper thermal and buffer conditions or, potentially more interestingly, by the use of various RNA polymerases that can promote the formation of RNA NPs cotransciptionally from specifically designed DNA templates. Also of importance is the delivery of the RNA NPs to the cells of interest in vitro or in vivo. Nonmodified RNAs rapidly degrade in blood serum and have difficulties crossing biological membranes due to their negative charge. These problems can be overcome by using, for example, polycationic lipid-based carriers. Our work involves the use of bolaamphiphiles, which are amphipathic compounds with positively charged hydrophilic head groups at each end connected by a hydrophobic chain. We have correlated results from molecular dynamics computations with various experiments to understand the characteristics of such delivery agents.