Efficacy and safety of piclidenoson in plaque psoriasis: Results from a randomized phase 3 clinical trial (COMFORT-1).

OBJECTIVE: A3 adenosine receptor (A3AR) is overexpressed in the skin and peripheral blood mononuclear cells of psoriasis patients. We investigated the efficacy/safety of piclidenoson (CF101), an orally bioavailable A3AR agonist that inhibits IL-17 and IL-23 production in keratinocytes, in moderate-to-severe plaque psoriasis. METHODS: The randomized, placebo- and active-controlled, double-blind phase 3 COMFORT-1 trial randomized patients (3:3:3:2) to piclidenoson 2 mg BID, piclidenoson 3 mg BID, apremilast 30 mg BID or placebo. At Week 16, patients in the placebo arm were re-randomized (1:1:1) to piclidenoson 2 mg BID, piclidenoson 3 mg BID or apremilast 30 mg BID. The primary end point was the proportion of patients achieving ≥75% improvement in Psoriasis Area and Severity Index (PASI) from baseline (PASI-75) at Week 16 versus placebo. RESULTS: A total of 529 patients were randomized and received ≥1 dose of study medication (safety population). The efficacy analysis population for the primary end point included 426 patients (piclidenoson 2 mg BID, 127; piclidenoson 3 mg BID, 103; apremilast, 118; placebo, 78). Piclidenoson at 2 and 3 mg BID exhibited similar efficacy. The primary end point was met with the 3 mg BID dose: PASI 75 rate of 9.7% versus 2.6% for piclidenoson versus placebo, p = 0.037. The PASI responses with piclidenoson continued to increase throughout the study period in a linear manner. At week 32, analysis in the per-protocol population showed that a greater proportion of patients in the piclidenoson 3 mg BID arm (51/88, 58.0%) achieved improvement from baseline in Psoriasis Disability Index (PDI) compared to apremilast (59/108, 55.1%), and the test for noninferiority trended towards significance (p = 0.072). The safety/tolerability profile of piclidenoson was excellent and superior to apremilast. CONCLUSIONS: Piclidenoson demonstrated efficacy responses that increased over time alongside a favourable safety profile. These findings support its continued clinical development as a psoriasis treatment (ClinicalTrials.gov identifier: NCT03168256).

Monitoring lineages of growing and dividing bacteria reveals an inducible memory of mar operon expression.

In Gram negative bacteria, the multiple antibiotic resistance or mar operon, is known to control the expression of multi-drug efflux genes that protect bacteria from a wide range of drugs. As many different chemical compounds can induce this operon, identifying the parameters that govern the dynamics of its induction is crucial to better characterize the processes of tolerance and resistance. Most experiments have assumed that the properties of the mar transcriptional network can be inferred from population measurements. However, measurements from an asynchronous population of cells can mask underlying phenotypic variations of single cells. We monitored the activity of the mar promoter in single Escherichia coli cells in linear micro-colonies and established that the response to a steady level of inducer was most heterogeneous within individual colonies for an intermediate value of inducer. Specifically, sub-lineages defined by contiguous daughter-cells exhibited similar promoter activity, whereas activity was greatly variable between different sub-lineages. Specific sub-trees of uniform promoter activity persisted over several generations. Statistical analyses of the lineages suggest that the presence of these sub-trees is the signature of an inducible memory of the promoter state that is transmitted from mother to daughter cells. This single-cell study reveals that the degree of epigenetic inheritance changes as a function of inducer concentration, suggesting that phenotypic inheritance may be an inducible phenotype.

Adaptation dynamics between copy-number and point mutations.

Together, copy-number and point mutations form the basis for most evolutionary novelty, through the process of gene duplication and divergence. While a plethora of genomic data reveals the long-term fate of diverging coding sequences and their cis-regulatory elements, little is known about the early dynamics around the duplication event itself. In microorganisms, selection for increased gene expression often drives the expansion of gene copy-number mutations, which serves as a crude adaptation, prior to divergence through refining point mutations. Using a simple synthetic genetic reporter system that can distinguish between copy-number and point mutations, we study their early and transient adaptive dynamics in real time in Escherichia coli. We find two qualitatively different routes of adaptation, depending on the level of functional improvement needed. In conditions of high gene expression demand, the two mutation types occur as a combination. However, under low gene expression demand, copy-number and point mutations are mutually exclusive; here, owing to their higher frequency, adaptation is dominated by copy-number mutations, in a process we term amplification hindrance. Ultimately, due to high reversal rates and pleiotropic cost, copy-number mutations may not only serve as a crude and transient adaptation, but also constrain sequence divergence over evolutionary time scales.

Type 1 piliated uropathogenic Escherichia coli hijack the host immune response by binding to CD14.

A key attribute of persistent or recurring bacterial infections is the ability of the pathogen to evade the host's immune response. Many Enterobacteriaceae express type 1 pili, a pre-adapted virulence trait, to invade host epithelial cells and establish persistent infections. However, the molecular mechanisms and strategies by which bacteria actively circumvent the immune response of the host remain poorly understood. Here, we identified CD14, the major co-receptor for lipopolysaccharide detection, on mouse dendritic cells (DCs) as a binding partner of FimH, the protein located at the tip of the type 1 pilus of Escherichia coli. The FimH amino acids involved in CD14 binding are highly conserved across pathogenic and non-pathogenic strains. Binding of the pathogenic strain CFT073 to CD14 reduced DC migration by overactivation of integrins and blunted expression of co-stimulatory molecules by overactivating the NFAT (nuclear factor of activated T-cells) pathway, both rate-limiting factors of T cell activation. This response was binary at the single-cell level, but averaged in larger populations exposed to both piliated and non-piliated pathogens, presumably via the exchange of immunomodulatory cytokines. While defining an active molecular mechanism of immune evasion by pathogens, the interaction between FimH and CD14 represents a potential target to interfere with persistent and recurrent infections, such as urinary tract infections or Crohn's disease.

Predicting bacterial promoter function and evolution from random sequences.

Predicting function from sequence is a central problem of biology. Currently, this is possible only locally in a narrow mutational neighborhood around a wildtype sequence rather than globally from any sequence. Using random mutant libraries, we developed a biophysical model that accounts for multiple features of σ70 binding bacterial promoters to predict constitutive gene expression levels from any sequence. We experimentally and theoretically estimated that 10-20% of random sequences lead to expression and ~80% of non-expressing sequences are one mutation away from a functional promoter. The potential for generating expression from random sequences is so pervasive that selection acts against σ70-RNA polymerase binding sites even within inter-genic, promoter-containing regions. This pervasiveness of σ70-binding sites implies that emergence of promoters is not the limiting step in gene regulatory evolution. Ultimately, the inclusion of novel features of promoter function into a mechanistic model enabled not only more accurate predictions of gene expression levels, but also identified that promoters evolve more rapidly than previously thought.

Local genetic context shapes the function of a gene regulatory network.

Gene expression levels are influenced by multiple coexisting molecular mechanisms. Some of these interactions such as those of transcription factors and promoters have been studied extensively. However, predicting phenotypes of gene regulatory networks (GRNs) remains a major challenge. Here, we use a well-defined synthetic GRN to study in Escherichia coli how network phenotypes depend on local genetic context, i.e. the genetic neighborhood of a transcription factor and its relative position. We show that one GRN with fixed topology can display not only quantitatively but also qualitatively different phenotypes, depending solely on the local genetic context of its components. Transcriptional read-through is the main molecular mechanism that places one transcriptional unit (TU) within two separate regulons without the need for complex regulatory sequences. We propose that relative order of individual TUs, with its potential for combinatorial complexity, plays an important role in shaping phenotypes of GRNs.

Immunohistochemical expression of non-collagenous extracellular matrix molecules involved in tertiary dentinogenesis following direct pulp capping: a systematic review.

BACKGROUND: Extracellular matrix molecules (ECMM) expression during tertiary dentinogenesis provides useful information for regenerative applications and efficacy of pulp capping materials. AIM: To identify and review the expression and roles of non-collagenous ECMM after successful direct pulp capping (DPC), following mechanical pulp exposures, via immunohistochemistry (IHC). The study addressed the question of where will successful DPC impact the IHC expression of these molecules. DATA SOURCES: In vivo animal and human original clinical studies reporting on ECMM in relation to different follow-up periods were screened and evaluated via descriptive analysis. The electronic literature search was carried out in three databases (MEDLINE/PubMed, Web of Science, Scopus), followed by manual screening of relevant journals and cross-referencing, up to December 2018. STUDY ELIGIBILITY CRITERIA, PARTICIPANTS, AND INTERVENTIONS: Randomized and non-randomized controlled trials, conducted in humans and animals, were selected. Histological evidence for tertiary dentine formation was a prerequisite for IHC evaluation. STUDY APPRAISAL AND SYNTHESIS METHODS: The methodological quality of the included articles was independently assessed using the Systematic Review Centre for Laboratory animal Experimentation (SYRCLE) and the Cochrane risk of bias tool (RoB 1), respectively. RESULTS: From a total of 1534 identified studies, 18 were included. Thirteen papers evaluated animal subjects and five studies were carried out on humans. In animals and humans, fibronectin and tenascin expressions were detected in pulp and odontoblast-like cells (OLC); dentine sialoprotein was expressed in both soft and newly-formed mineralized tissue. In animals, bone sialoprotein was early expressed, in association with OLC and predentin; the immunoreactivity for dentine sialophosphoprotein and dentine matrix protein-1 was associated with the OLC and dentine bridge; osteopontin was expressed in OLC, predentine and reparative dentine. A considerable heterogeneity was found in the methodologies of the included studies, as well as interspecies variability of results in terms of time. CONCLUSIONS AND IMPLICATIONS OF KEY FINDINGS: Within the limited scientific evidence, all non-collagenous ECMM expressions during tertiary dentinogenesis are active and related to soft and hard tissues. There is a shortage of human studies, and future research directions should focus more on them. PROSPERO Protocol: CRD42019121304.

Evolutionary potential of transcription factors for gene regulatory rewiring.

Gene regulatory networks evolve through rewiring of individual components-that is, through changes in regulatory connections. However, the mechanistic basis of regulatory rewiring is poorly understood. Using a canonical gene regulatory system, we quantify the properties of transcription factors that determine the evolutionary potential for rewiring of regulatory connections: robustness, tunability and evolvability. In vivo repression measurements of two repressors at mutated operator sites reveal their contrasting evolutionary potential: while robustness and evolvability were positively correlated, both were in trade-off with tunability. Epistatic interactions between adjacent operators alleviated this trade-off. A thermodynamic model explains how the differences in robustness, tunability and evolvability arise from biophysical characteristics of repressor-DNA binding. The model also uncovers that the energy matrix, which describes how mutations affect repressor-DNA binding, encodes crucial information about the evolutionary potential of a repressor. The biophysical determinants of evolutionary potential for regulatory rewiring constitute a mechanistic framework for understanding network evolution.

Mytilus spp. as sentinels for monitoring microplastic pollution in Norwegian coastal waters: A qualitative and quantitative study.

Microplastic (MP) contamination is ubiquitous in the environment and many species worldwide have been shown to contain MP. The ecological impact of MP pollution is still unknown, thus there is an urgent need for more knowledge. One key task is to identify species suitable as sentinels for monitoring in key eco-compartments, such as coastal waters. In Norway, mussels (Mytilus spp.) have been monitored for hazardous contaminants through OSPAR since 1981. Norway has the longest coastline in Europe and adding MP to the Norwegian Mussel Watch is therefore important in a European and global context. The present study reports MP data in mussels (332 specimens) collected from multiple sites (n = 15) spanning the whole Norwegian coastline. MPs were detected at all locations, except at one site on the west coast. Among the most surprising findings, mussels from the Barents Sea coastline in the Finnmark region, contained significantly more MPs than mussels from most of the southern part of the country, despite the latter sites being located much closer to major urban areas. Only mussels from a site located very close to Oslo, the capital, contained levels similar to those observed in the remote site in Finnmark. In total an average of 1.5 (±2.3) particles ind-1 and 0.97 (±2.61) particles w.w. g-1 was found. The most common MPs were <1 mm in size, and fibres accounted for 83% of particles identified, although there was inter-site variability. Thirteen different polymeric groups were identified; cellulosic being the most common and black rubbery particles being the second. This study suggests Mytilus spp. are suitable for semi-quantitative and qualitatively monitoring of MPs in coastal waters. However, some uncertainties remain including mussel size as a confounding factor that may influence ingestion, the role of depuration and other fate related processes, and this call for further research.

Leaky resistance and the conditions for the existence of lytic bacteriophage.

In experimental cultures, when bacteria are mixed with lytic (virulent) bacteriophage, bacterial cells resistant to the phage commonly emerge and become the dominant population of bacteria. Following the ascent of resistant mutants, the densities of bacteria in these simple communities become limited by resources rather than the phage. Despite the evolution of resistant hosts, upon which the phage cannot replicate, the lytic phage population is most commonly maintained in an apparently stable state with the resistant bacteria. Several mechanisms have been put forward to account for this result. Here we report the results of population dynamic/evolution experiments with a virulent mutant of phage Lambda, λVIR, and Escherichia coli in serial transfer cultures. We show that, following the ascent of λVIR-resistant bacteria, λVIR is maintained in the majority of cases in maltose-limited minimal media and in all cases in nutrient-rich broth. Using mathematical models and experiments, we show that the dominant mechanism responsible for maintenance of λVIR in these resource-limited populations dominated by resistant E. coli is a high rate of either phenotypic or genetic transition from resistance to susceptibility-a hitherto undemonstrated mechanism we term "leaky resistance." We discuss the implications of leaky resistance to our understanding of the conditions for the maintenance of phage in populations of bacteria-their "existence conditions."

Statistical mechanics for metabolic networks during steady state growth.

Which properties of metabolic networks can be derived solely from stoichiometry? Predictive results have been obtained by flux balance analysis (FBA), by postulating that cells set metabolic fluxes to maximize growth rate. Here we consider a generalization of FBA to single-cell level using maximum entropy modeling, which we extend and test experimentally. Specifically, we define for Escherichia coli metabolism a flux distribution that yields the experimental growth rate: the model, containing FBA as a limit, provides a better match to measured fluxes and it makes a wide range of predictions: on flux variability, regulation, and correlations; on the relative importance of stoichiometry vs. optimization; on scaling relations for growth rate distributions. We validate the latter here with single-cell data at different sub-inhibitory antibiotic concentrations. The model quantifies growth optimization as emerging from the interplay of competitive dynamics in the population and regulation of metabolism at the level of single cells.

Phage-host population dynamics promotes prophage acquisition in bacteria with innate immunity.

Temperate bacteriophages integrate in bacterial genomes as prophages and represent an important source of genetic variation for bacterial evolution, frequently transmitting fitness-augmenting genes such as toxins responsible for virulence of major pathogens. However, only a fraction of bacteriophage infections are lysogenic and lead to prophage acquisition, whereas the majority are lytic and kill the infected bacteria. Unless able to discriminate lytic from lysogenic infections, mechanisms of immunity to bacteriophages are expected to act as a double-edged sword and increase the odds of survival at the cost of depriving bacteria of potentially beneficial prophages. We show that although restriction-modification systems as mechanisms of innate immunity prevent both lytic and lysogenic infections indiscriminately in individual bacteria, they increase the number of prophage-acquiring individuals at the population level. We find that this counterintuitive result is a consequence of phage-host population dynamics, in which restriction-modification systems delay infection onset until bacteria reach densities at which the probability of lysogeny increases. These results underscore the importance of population-level dynamics as a key factor modulating costs and benefits of immunity to temperate bacteriophages.

Lack of cations in flow cytometry buffers affect fluorescence signals by reducing membrane stability and viability of Escherichia coli strains.

Buffers are essential for diluting bacterial cultures for flow cytometry analysis in order to study bacterial physiology and gene expression parameters based on fluorescence signals. Using a variety of constitutively expressed fluorescent proteins in Escherichia coli K-12 strain MG1655, we found strong artifactual changes in fluorescence levels after dilution into the commonly used flow cytometry buffer phosphate-buffered saline (PBS) and two other buffer solutions, Tris-HCl and M9 salts. These changes appeared very rapidly after dilution, and were linked to increased membrane permeability and loss in cell viability. We observed buffer-related effects in several different E. coli strains, K-12, C and W, but not E. coli B, which can be partially explained by differences in lipopolysaccharide (LPS) and outer membrane composition. Supplementing the buffers with divalent cations responsible for outer membrane stability, Mg2+ and Ca2+, preserved fluorescence signals, membrane integrity and viability of E. coli. Thus, stabilizing the bacterial outer membrane is essential for precise and unbiased measurements of fluorescence parameters using flow cytometry.

Effects of mutations in phage restriction sites during escape from restriction-modification.

Restriction-modification systems are widespread genetic elements that protect bacteria from bacteriophage infections by recognizing and cleaving heterologous DNA at short, well-defined sequences called restriction sites. Bioinformatic evidence shows that restriction sites are significantly underrepresented in bacteriophage genomes, presumably because bacteriophages with fewer restriction sites are more likely to escape cleavage by restriction-modification systems. However, how mutations in restriction sites affect the likelihood of bacteriophage escape is unknown. Using the bacteriophage λ and the restriction-modification system EcoRI, we show that while mutation effects at different restriction sites are unequal, they are independent. As a result, the probability of bacteriophage escape increases with each mutated restriction site. Our results experimentally support the role of restriction site avoidance as a response to selection imposed by restriction-modification systems and offer an insight into the events underlying the process of bacteriophage escape.

Shaping bacterial population behavior through computer-interfaced control of individual cells.

Bacteria in groups vary individually, and interact with other bacteria and the environment to produce population-level patterns of gene expression. Investigating such behavior in detail requires measuring and controlling populations at the single-cell level alongside precisely specified interactions and environmental characteristics. Here we present an automated, programmable platform that combines image-based gene expression and growth measurements with on-line optogenetic expression control for hundreds of individual Escherichia coli cells over days, in a dynamically adjustable environment. This integrated platform broadly enables experiments that bridge individual and population behaviors. We demonstrate: (i) population structuring by independent closed-loop control of gene expression in many individual cells, (ii) cell-cell variation control during antibiotic perturbation, (iii) hybrid bio-digital circuits in single cells, and freely specifiable digital communication between individual bacteria. These examples showcase the potential for real-time integration of theoretical models with measurement and control of many individual cells to investigate and engineer microbial population behavior.

Regulatory network structure determines patterns of intermolecular epistasis.

Most phenotypes are determined by molecular systems composed of specifically interacting molecules. However, unlike for individual components, little is known about the distributions of mutational effects of molecular systems as a whole. We ask how the distribution of mutational effects of a transcriptional regulatory system differs from the distributions of its components, by first independently, and then simultaneously, mutating a transcription factor and the associated promoter it represses. We find that the system distribution exhibits increased phenotypic variation compared to individual component distributions - an effect arising from intermolecular epistasis between the transcription factor and its DNA-binding site. In large part, this epistasis can be qualitatively attributed to the structure of the transcriptional regulatory system and could therefore be a common feature in prokaryotes. Counter-intuitively, intermolecular epistasis can alleviate the constraints of individual components, thereby increasing phenotypic variation that selection could act on and facilitating adaptive evolution.

An Effective Feedback Loop between Cell-Cell Contact Duration and Morphogen Signaling Determines Cell Fate.

Cell-cell contact formation constitutes an essential step in evolution, leading to the differentiation of specialized cell types. However, remarkably little is known about whether and how the interplay between contact formation and fate specification affects development. Here, we identify a positive feedback loop between cell-cell contact duration, morphogen signaling, and mesendoderm cell-fate specification during zebrafish gastrulation. We show that long-lasting cell-cell contacts enhance the competence of prechordal plate (ppl) progenitor cells to respond to Nodal signaling, required for ppl cell-fate specification. We further show that Nodal signaling promotes ppl cell-cell contact duration, generating a positive feedback loop between ppl cell-cell contact duration and cell-fate specification. Finally, by combining mathematical modeling and experimentation, we show that this feedback determines whether anterior axial mesendoderm cells become ppl or, instead, turn into endoderm. Thus, the interdependent activities of cell-cell signaling and contact formation control fate diversification within the developing embryo.

Complex chromosomal neighborhood effects determine the adaptive potential of a gene under selection.

How the organization of genes on a chromosome shapes adaptation is essential for understanding evolutionary paths. Here, we investigate how adaptation to rapidly increasing levels of antibiotic depends on the chromosomal neighborhood of a drug-resistance gene inserted at different positions of the Escherichia coli chromosome. Using a dual-fluorescence reporter that allows us to distinguish gene amplifications from other up-mutations, we track in real-time adaptive changes in expression of the drug-resistance gene. We find that the relative contribution of several mutation types differs systematically between loci due to properties of neighboring genes: essentiality, expression, orientation, termination, and presence of duplicates. These properties determine rate and fitness effects of gene amplification, deletions, and mutations compromising transcriptional termination. Thus, the adaptive potential of a gene under selection is a system-property with a complex genetic basis that is specific for each chromosomal locus, and it can be inferred from detailed functional and genomic data.

On the mechanistic nature of epistasis in a canonical cis-regulatory element.

Understanding the relation between genotype and phenotype remains a major challenge. The difficulty of predicting individual mutation effects, and particularly the interactions between them, has prevented the development of a comprehensive theory that links genotypic changes to their phenotypic effects. We show that a general thermodynamic framework for gene regulation, based on a biophysical understanding of protein-DNA binding, accurately predicts the sign of epistasis in a canonical cis-regulatory element consisting of overlapping RNA polymerase and repressor binding sites. Sign and magnitude of individual mutation effects are sufficient to predict the sign of epistasis and its environmental dependence. Thus, the thermodynamic model offers the correct null prediction for epistasis between mutations across DNA-binding sites. Our results indicate that a predictive theory for the effects of cis-regulatory mutations is possible from first principles, as long as the essential molecular mechanisms and the constraints these impose on a biological system are accounted for.