Dynamic imaging of nascent RNA reveals general principles of transcription dynamics and stochastic splice site selection.

The activities of RNA polymerase and the spliceosome are responsible for the heterogeneity in the abundance and isoform composition of mRNA in human cells. However, the dynamics of these megadalton enzymatic complexes working in concert on endogenous genes have not been described. Here, we establish a quasi-genome-scale platform for observing synthesis and processing kinetics of single nascent RNA molecules in real time. We find that all observed genes show transcriptional bursting. We also observe large kinetic variation in intron removal for single introns in single cells, which is inconsistent with deterministic splice site selection. Transcriptome-wide footprinting of the U2AF complex, nascent RNA profiling, long-read sequencing, and lariat sequencing further reveal widespread stochastic recursive splicing within introns. We propose and validate a unified theoretical model to explain the general features of transcription and pervasive stochastic splice site selection.

Intrinsic Dynamics of a Human Gene Reveal the Basis of Expression Heterogeneity.

Transcriptional regulation in metazoans occurs through long-range genomic contacts between enhancers and promoters, and most genes are transcribed in episodic "bursts" of RNA synthesis. To understand the relationship between these two phenomena and the dynamic regulation of genes in response to upstream signals, we describe the use of live-cell RNA imaging coupled with Hi-C measurements and dissect the endogenous regulation of the estrogen-responsive TFF1 gene. Although TFF1 is highly induced, we observe short active periods and variable inactive periods ranging from minutes to days. The heterogeneity in inactive times gives rise to the widely observed "noise" in human gene expression and explains the distribution of protein levels in human tissue. We derive a mathematical model of regulation that relates transcription, chromosome structure, and the cell's ability to sense changes in estrogen and predicts that hypervariability is largely dynamic and does not reflect a stable biological state.

Multiple models for outbreak decision support in the face of uncertainty.

Policymakers must make management decisions despite incomplete knowledge and conflicting model projections. Little guidance exists for the rapid, representative, and unbiased collection of policy-relevant scientific input from independent modeling teams. Integrating approaches from decision analysis, expert judgment, and model aggregation, we convened multiple modeling teams to evaluate COVID-19 reopening strategies for a mid-sized United States county early in the pandemic. Projections from seventeen distinct models were inconsistent in magnitude but highly consistent in ranking interventions. The 6-mo-ahead aggregate projections were well in line with observed outbreaks in mid-sized US counties. The aggregate results showed that up to half the population could be infected with full workplace reopening, while workplace restrictions reduced median cumulative infections by 82%. Rankings of interventions were consistent across public health objectives, but there was a strong trade-off between public health outcomes and duration of workplace closures, and no win-win intermediate reopening strategies were identified. Between-model variation was high; the aggregate results thus provide valuable risk quantification for decision making. This approach can be applied to the evaluation of management interventions in any setting where models are used to inform decision making. This case study demonstrated the utility of our approach and was one of several multimodel efforts that laid the groundwork for the COVID-19 Scenario Modeling Hub, which has provided multiple rounds of real-time scenario projections for situational awareness and decision making to the Centers for Disease Control and Prevention since December 2020.

mRNA 5' terminal sequences drive 200-fold differences in expression through effects on synthesis, translation and decay.

mRNA regulatory sequences control gene expression at multiple levels including translation initiation and mRNA decay. The 5' terminal sequences of mRNAs have unique regulatory potential because of their proximity to key post-transcriptional regulators. Here we have systematically probed the function of 5' terminal sequences in gene expression in human cells. Using a library of reporter mRNAs initiating with all possible 7-mer sequences at their 5' ends, we find an unexpected impact on transcription that underlies 200-fold differences in mRNA expression. Library sequences that promote high levels of transcription mirrored those found in native mRNAs and define two basic classes with similarities to classic Initiator (Inr) and TCT core promoter motifs. By comparing transcription, translation and decay rates, we identify sequences that are optimized for both efficient transcription and growth-regulated translation and stability, including variants of terminal oligopyrimidine (TOP) motifs. We further show that 5' sequences of endogenous mRNAs are enriched for multi-functional TCT/TOP hybrid sequences. Together, our results reveal how 5' sequences define two general classes of mRNAs with distinct growth-responsive profiles of expression across synthesis, translation and decay.

DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival.

The mTORC1 and mTORC2 pathways regulate cell growth, proliferation, and survival. We identify DEPTOR as an mTOR-interacting protein whose expression is negatively regulated by mTORC1 and mTORC2. Loss of DEPTOR activates S6K1, Akt, and SGK1, promotes cell growth and survival, and activates mTORC1 and mTORC2 kinase activities. DEPTOR overexpression suppresses S6K1 but, by relieving feedback inhibition from mTORC1 to PI3K signaling, activates Akt. Consistent with many human cancers having activated mTORC1 and mTORC2 pathways, DEPTOR expression is low in most cancers. Surprisingly, DEPTOR is highly overexpressed in a subset of multiple myelomas harboring cyclin D1/D3 or c-MAF/MAFB translocations. In these cells, high DEPTOR expression is necessary to maintain PI3K and Akt activation and a reduction in DEPTOR levels leads to apoptosis. Thus, we identify a novel mTOR-interacting protein whose deregulated overexpression in multiple myeloma cells represents a mechanism for activating PI3K/Akt signaling and promoting cell survival.

A mathematical model of SARS-CoV-2 immunity predicts paxlovid rebound.

Nirmatrelvir/ritonavir (Paxlovid), an oral antiviral medication targeting SARS-CoV-2, remains an important treatment for COVID-19. Initial studies of nirmatrelvir/ritonavir were performed in SARS-CoV-2 unvaccinated patients without prior confirmed SARS-CoV-2 infection; however, most individuals have now either been vaccinated and/or have experienced SARS-CoV-2 infection. After nirmatrelvir/ritonavir became widely available, reports surfaced of "Paxlovid rebound," a phenomenon in which symptoms (and SARS-CoV-2 test positivity) would initially resolve, but after finishing treatment, symptoms and test positivity would return. We used a previously described parsimonious mathematical model of immunity to SARS-CoV-2 infection to model the effect of nirmatrelvir/ritonavir treatment in unvaccinated and vaccinated patients. Model simulations show that viral rebound after treatment occurs only in vaccinated patients, while unvaccinated (SARS-COV-2 naïve) patients treated with nirmatrelvir/ritonavir do not experience any rebound in viral load. This work suggests that an approach combining parsimonious models of the immune system could be used to gain important insights in the context of emerging pathogens.

Dynamic Imine Bonding Facilitates Mannan Release from a Nanofibrous Peptide Hydrogel.

Recently, there has been increased interest in using mannan as an immunomodulatory bioconjugate. Despite notable immunological and functional differences between the reduced (R-Man) and oxidized (O-Man) forms of mannan, little is known about the impact of mannan oxidation state on its in vivo persistence or its potential controlled release from biomaterials that may improve immunotherapeutic or prophylactic efficacy. Here, we investigate the impact of oxidation state on the in vitro and in vivo release of mannan from a biocompatible and immunostimulatory multidomain peptide hydrogel, K2(SL)6K2 (abbreviated as K2), that has been previously used for the controlled release of protein and small molecule payloads. We observed that O-Man released more slowly from K2 hydrogels in vitro than R-Man. In vivo, the clearance of O-Man from K2 hydrogels was slower than O-Man alone. We attributed the slower release rate to the formation of dynamic imine bonds between reactive aldehyde groups on O-Man and the lysine residues on K2. This imine interaction was also observed to improve K2 + O-Man hydrogel strength and shear recovery without significantly influencing secondary structure or peptide nanofiber formation. There were no observed differences in the in vivo release rates of O-Man loaded in K2, R-Man loaded in K2, and R-Man alone. These data suggest that, after subcutaneous injection, R-Man naturally persists longer in vivo than O-Man and minimally interacts with the peptide hydrogel. These results highlight a potentially critical, but previously unreported, difference in the in vivo behavior of O-Man and R-Man and demonstrate that K2 can be used to normalize the release of O-Man to that of R-Man. Further, since K2 itself is an adjuvant, a combination of O-Man and K2 could be used to enhance the immunostimulatory effects of O-Man for applications such as infectious disease vaccines and cancer immunotherapy.

Stabilization of Synthetic Collagen Triple Helices: Charge Pairs and Covalent Capture.

Collagen mimetic peptides are composed of triple helices. Triple helical formation frequently utilizes charge pair interactions to direct protein assembly. The design of synthetic triple helices is challenging due to the large number of competing species and the overall fragile nature of collagen mimetics. A successfully designed triple helix incorporates both positive and negative criteria to achieve maximum specificity of the supramolecular assembly. Intrahelical charge pair interactions, particularly those involved in lysine-aspartate and lysine-glutamate pairs, have been especially successful both in driving helix specificity and for subsequent stabilization by covalent capture. Despite this progress, the important sequential and geometric relationships of charged residues in a triple helical context have not been fully explored for either supramolecular assembly or covalent capture stabilization. In this study, we compare the eight canonical axial and lateral charge pairs of lysine and arginine with glutamate and aspartate to their noncanonical, reversed charge pairs. These findings are put into the context of collagen triple helical design and synthesis.

Single-Molecule Imaging Reveals a Switch between Spurious and Functional ncRNA Transcription.

Eukaryotic transcription is pervasive, and many of the resulting RNAs are non-coding. It is unknown whether ubiquitous transcription is functional or simply reflects stochastic transcriptional noise. By single-molecule visualization of the dynamic interplay between coding and non-coding transcription at the GAL locus in living yeast cells, we show that antisense GAL10 ncRNA transcription can switch between functional and spurious under different conditions. During galactose induction, GAL10 sense transcription occurs in short stochastic bursts, which are unaffected by transcription of antisense GAL10 ncRNA, even when both are present simultaneously at the same locus. In contrast, when GAL10 is not induced, ncRNA transcription is critical to prevent transcriptional leakage of GAL1 and GAL10. Suppression of ncRNA transcription by strand-specific CRISPR/dCas9 results in transcriptional leakage of the inducer GAL1, leading to a more sensitive transcription activation threshold, an alteration of metabolic switching, and a fitness defect in competition experiments.

Global analysis of LARP1 translation targets reveals tunable and dynamic features of 5' TOP motifs.

Terminal oligopyrimidine (TOP) motifs are sequences at the 5' ends of mRNAs that link their translation to the mTOR Complex 1 (mTORC1) nutrient-sensing signaling pathway. They are commonly regarded as discrete elements that reside on ∼100 mRNAs that mostly encode translation factors. However, the full spectrum of TOP sequences and their prevalence throughout the transcriptome remain unclear, primarily because of uncertainty over the mechanism that detects them. Here, we globally analyzed translation targets of La-related protein 1 (LARP1), an RNA-binding protein and mTORC1 effector that has been shown to repress TOP mRNA translation in a few specific cases. We establish that LARP1 is the primary translation regulator of mRNAs with classical TOP motifs genome-wide, and also that these motifs are extreme instances of a broader continuum of regulatory sequences. We identify the features of TOP sequences that determine their potency and quantify these as a metric that accurately predicts mTORC1/LARP1 regulation called a TOPscore. Analysis of TOPscores across the transcriptomes of 16 mammalian tissues defines a constitutive "core" set of TOP mRNAs, but also identifies tissue-specific TOP mRNAs produced via alternative transcription initiation sites. These results establish the central role of LARP1 in TOP mRNA regulation on a transcriptome scale and show how it connects mTORC1 to a tunable and dynamic program of gene expression that is tailored to specific biological contexts.

Genomic analysis of diet composition finds novel loci and associations with health and lifestyle.

We conducted genome-wide association studies (GWAS) of relative intake from the macronutrients fat, protein, carbohydrates, and sugar in over 235,000 individuals of European ancestries. We identified 21 unique, approximately independent lead SNPs. Fourteen lead SNPs are uniquely associated with one macronutrient at genome-wide significance (P < 5 × 10-8), while five of the 21 lead SNPs reach suggestive significance (P < 1 × 10-5) for at least one other macronutrient. While the phenotypes are genetically correlated, each phenotype carries a partially unique genetic architecture. Relative protein intake exhibits the strongest relationships with poor health, including positive genetic associations with obesity, type 2 diabetes, and heart disease (rg ≈ 0.15-0.5). In contrast, relative carbohydrate and sugar intake have negative genetic correlations with waist circumference, waist-hip ratio, and neighborhood deprivation (|rg| ≈ 0.1-0.3) and positive genetic correlations with physical activity (rg ≈ 0.1 and 0.2). Relative fat intake has no consistent pattern of genetic correlations with poor health but has a negative genetic correlation with educational attainment (rg ≈-0.1). Although our analyses do not allow us to draw causal conclusions, we find no evidence of negative health consequences associated with relative carbohydrate, sugar, or fat intake. However, our results are consistent with the hypothesis that relative protein intake plays a role in the etiology of metabolic dysfunction.

Cation-π Interactions and Their Role in Assembling Collagen Triple Helices.

Cation-π interactions play a significant role in the stabilization of globular proteins. However, their role in collagen triple helices is less well understood and they have rarely been used in de novo designed collagen mimetic systems. In this study, we analyze the stabilizing and destabilizing effects in pairwise amino acid interactions between cationic and aromatic residues in both axial and lateral sequential relationships. Thermal unfolding experiments demonstrated that only axial pairs are stabilizing, while the lateral pairs are uniformly destabilizing. Molecular dynamics simulations show that pairs with an axial relationship can achieve a near-ideal interaction distance, but pairs in a lateral relationship do not. Arginine-π systems were found to be more stabilizing than lysine-π and histidine-π. Arginine-π interactions were then studied in more chemically diverse ABC-type heterotrimeric helices, where arginine-tyrosine pairs were found to form the best helix. This work helps elucidate the role of cation-π interactions in triple helices and illustrates their utility in designing collagen mimetic peptides.

A scoping review of the distribution and frequency of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae in shrimp and salmon.

Antimicrobial-resistant (AMR) bacteria are a threat to public health as they can resist treatment and pass along genetic material that allows other bacteria to become drug-resistant. To assess foodborne AMR risk, the Codex Guidelines for Risk Analysis of Foodborne AMR provide a framework for risk profiles and risk assessments. Several elements of a risk profile may benefit from a scoping review (ScR). To contribute to a larger risk profile structured according to the Codex Guidelines, our objective was to conduct a ScR of the current state of knowledge on the distribution, frequency and concentrations of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae in salmon and shrimp. Articles were identified via a comprehensive search of five bibliographic databases. Two reviewers screened titles and abstracts for relevance and characterised full-text articles with screening forms developed a priori. Sixteen relevant studies were identified. This review found that there is a lack of Canadian data regarding ESBL-producing Enterobacteriaceae in salmon and shrimp. However, ESBL- producing Escherichia coli, Klebsiella pneumoniae and other Enterobacteriaceae have been isolated in multiple regions with a history of exporting seafood to Canada. The literature described herein will support future decision-making on this issue as research/surveillance and subsequent assessments are currently lacking.

Characterisation of burden of illness measures associated with human (Fluoro)quinolone-resistant Campylobacter spp. infections - a scoping review.

Campylobacter spp. are one of the most common causes of bacterial gastroenteritis in Canada and worldwide. Fluoroquinolones are often used to treat complicated human campylobacteriosis and strains of Campylobacter spp. resistant to these drugs are emerging along the food chain. A scoping review was conducted to summarise how human (fluoro)quinolone-resistant (FQR; quinolones including fluoroquinolones) Campylobacter spp. infections are characterised in the literature by describing how burden of illness (BOI) associated with FQR is measured and reported, describing the variability in reporting of study characteristics, and providing a narrative review of literature that compare BOI measures of FQR Campylobacter spp. infections to those with susceptible infections. The review identified 26 studies that yielded many case reports, a lack of recent literature and a lack of Canadian data. Studies reported 26 different BOI measures and the most common were hospitalisation, diarrhoea, fever and duration of illness. There were mixed results as BOI measures reported in literature were inconsistently defined and there were limited comparisons between resistant and susceptible infections. This presents a challenge when attempting to assess the magnitude of the BOI due to FQR Campylobacter spp., highlighting the need for more research in this area.

Phase transitions may explain why SARS-CoV-2 spreads so fast and why new variants are spreading faster.

The novel coronavirus SARS CoV-2 responsible for the COVID-19 pandemic and SARS CoV-1 responsible for the SARS epidemic of 2002-2003 share an ancestor yet evolved to have much different transmissibility and global impact 1. A previously developed thermodynamic model of protein conformations hypothesized that SARS CoV-2 is very close to a new thermodynamic critical point, which makes it highly infectious but also easily displaced by a spike-based vaccine because there is a tradeoff between transmissibility and robustness 2. The model identified a small cluster of four key mutations of SARS CoV-2 that predicts much stronger viral attachment and viral spreading compared to SARS CoV-1. Here we apply the model to the SARS-CoV-2 variants Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1) and Delta (B.1.617.2)3 and predict, using no free parameters, how the new mutations will not diminish the effectiveness of current spike based vaccines and may even further enhance infectiousness by augmenting the binding ability of the virus.

Archaeal Ribosomal Proteins Possess Nuclear Localization Signal-Type Motifs: Implications for the Origin of the Cell Nucleus.

Eukaryotic cells are divided into the nucleus and the cytosol, and, to enter the nucleus, proteins typically possess short signal sequences, known as nuclear localization signals (NLSs). Although NLSs have long been considered as features unique to eukaryotic proteins, we show here that similar or identical protein segments are present in ribosomal proteins from the Archaea. Specifically, the ribosomal proteins uL3, uL15, uL18, and uS12 possess NLS-type motifs that are conserved across all major branches of the Archaea, including the most ancient groups Microarchaeota and Diapherotrites, pointing to the ancient origin of NLS-type motifs in the Archaea. Furthermore, by using fluorescence microscopy, we show that the archaeal NLS-type motifs can functionally substitute eukaryotic NLSs and direct the transport of ribosomal proteins into the nuclei of human cells. Collectively, these findings illustrate that the origin of NLSs preceded the origin of the cell nucleus, suggesting that the initial function of NLSs was not related to intracellular trafficking, but possibly was to improve recognition of nucleic acids by cellular proteins. Overall, our study reveals rare evolutionary intermediates among archaeal cells that can help elucidate the sequence of events that led to the origin of the eukaryotic cell.

Training Spiking Neural Networks in the Strong Coupling Regime.

Recurrent neural networks trained to perform complex tasks can provide insight into the dynamic mechanism that underlies computations performed by cortical circuits. However, due to a large number of unconstrained synaptic connections, the recurrent connectivity that emerges from network training may not be biologically plausible. Therefore, it remains unknown if and how biological neural circuits implement dynamic mechanisms proposed by the models. To narrow this gap, we developed a training scheme that, in addition to achieving learning goals, respects the structural and dynamic properties of a standard cortical circuit model: strongly coupled excitatory-inhibitory spiking neural networks. By preserving the strong mean excitatory and inhibitory coupling of initial networks, we found that most of trained synapses obeyed Dale's law without additional constraints, exhibited large trial-to-trial spiking variability, and operated in inhibition-stabilized regime. We derived analytical estimates on how training and network parameters constrained the changes in mean synaptic strength during training. Our results demonstrate that training recurrent neural networks subject to strong coupling constraints can result in connectivity structure and dynamic regime relevant to cortical circuits.

A mathematical model for persistent post-CSD vasoconstriction.

Cortical spreading depression (CSD) is the propagation of a relatively slow wave in cortical brain tissue that is linked to a number of pathological conditions such as stroke and migraine. Most of the existing literature investigates the dynamics of short term phenomena such as the depolarization and repolarization of membrane potentials or large ion shifts. Here, we focus on the clinically-relevant hour-long state of neurovascular malfunction in the wake of CSDs. This dysfunctional state involves widespread vasoconstriction and a general disruption of neurovascular coupling. We demonstrate, using a mathematical model, that dissolution of calcium that has aggregated within the mitochondria of vascular smooth muscle cells can drive an hour-long disruption. We model the rate of calcium clearance as well as the dynamical implications on overall blood flow. Based on reaction stoichiometry, we quantify a possible impact of calcium phosphate dissolution on the maintenance of F0F1-ATP synthase activity.

Controversies around the function of LARP1.

The RNA-binding protein LARP1 has generated interest in recent years for its role in the mTOR signalling cascade and its regulation of terminal oligopyrimidine (TOP) mRNA translation. Paradoxically, some scientists have shown that LARP1 represses TOP translation while others that LARP1 activates it. Here, we present opinions from four leading scientists in the field to discuss these and other contradictory findings.

Loss of protein synthesis quality control in host-restricted organisms.

Intracellular organisms, such as obligate parasites and endosymbionts, typically possess small genomes due to continuous genome decay caused by an environment with alleviated natural selection. Previously, a few species with highly reduced genomes, including the intracellular pathogens Mycoplasma and Microsporidia, have been shown to carry degenerated editing domains in aminoacyl-tRNA synthetases. These defects in the protein synthesis machinery cause inaccurate translation of the genetic code, resulting in significant statistical errors in protein sequences that are thought to help parasites to escape immune response of a host. In this study we analyzed 10,423 complete bacterial genomes to assess conservation of the editing domains in tRNA synthetases, including LeuRS, IleRS, ValRS, ThrRS, AlaRS, and PheRS. We found that, while the editing domains remain intact in free-living species, they are degenerated in the overwhelming majority of host-restricted bacteria. Our work illustrates that massive genome erosion triggered by an intracellular lifestyle eradicates one of the most fundamental components of a living cell: the system responsible for proofreading of amino acid selection for protein synthesis. This finding suggests that inaccurate translation of the genetic code might be a general phenomenon among intercellular organisms with reduced genomes.