I am uncertain” vs “it is uncertain”. How linguistic markers of the uncertainty source affect uncertainty communication

Two psychological sources of uncertainty bear implications for judgment and decision-making: external uncertainty is seen as stemming from properties of the world, whereas internal uncertainty is seen as stemming from lack of knowledge. The apparent source of uncertainty can be conveyed through linguistic markers, such as the pronoun of probability phrases (e.g., I am uncertain vs. It is uncertain). Here, we investigated whether and when speakers use different pronoun subjects as such linguistic markers (Exp. 1 and 2) and what hearers infer from them (Exp. 3 and 4). Speakers more often described higher probabilities and knowable outcomes with internal probability phrases. In dialogue, speakers mirrored the source of their conversational partner. Markers of the source had a main effect or interacted with the probability conveyed and speaker expertise to shape the judgments and decisions of hearers. For example, experts voicing an internal probability phrase were judged as more knowledgeable than experts using an external probability phrase whereas the result was the opposite for lay speakers. We discuss how these findings inform our understanding of subjective uncertainty and uncertainty communication theories

Genetic Variants of the DSF Quorum Sensing System in Stenotrophomonas maltophilia Influence Virulence and Resistance Phenotypes Among Genotypically Diverse Clinical Isolates

Altres ajuts: Departament de Salut, Generalitat de Catalunya: SLT002/16/00349The pathogenicity of Stenotrophomonas maltophilia is regulated in part by its quorum sensing (QS) system. The main QS signaling molecule in S. maltophilia is known as diffusible signal factor (DSF), and the rpf gene cluster is responsible for its synthesis and perception. Two cluster variants have been previously described, rpf -1 and rpf -2, which differ basically in the conditions under which DSF is produced. Here, correlations between the rpf variant and antibiotic susceptibility, LPS electrophoretic profiles and virulence-related phenotypes were evaluated for a collection of 78 geographically and genetically diverse clinical strains of S. maltophilia. In general there were associations between previously established genogroups and the genetic variant of the rpf cluster. However, only few genotype-phenotype correlations could be observed. Resistance to the β-lactam antibiotics ceftazidime and ticarcillin was associated with strains carrying the rpf -1 variant, whereas strains of variant rpf -2, particularly those of genogroup C, showed higher resistance levels to colistin. Strains of variant rpf -2 were also significantly more virulent to Galleria mellonella larvae than those of rpf -1, most likely due to an increased ability of rpf -2 strains to form biofilms. A comparative genomic analysis revealed the presence of proteins unique to individual genogroups. In particular, the strains of genogroup C share an operon that encodes for a new virulence determinant in S. maltophilia related to the synthesis of an alternative Flp/Tad pilus. Overall, this study establishes a link between the DSF-based QS system and the virulence and resistance phenotypes in this species, and identifies potential high-risk clones circulating in European hospitals

Genetic variants of the DSF quórum sensing system in Stenotrophomonas maltophilia influence virulence and resistance phenotypes among genotypically diverse clinical isoaltes

The pathogenicity of Stenotrophomonas maltophilia is regulated in part by its quorum sensing (QS) system. The main QS signaling molecule in S. maltophilia is known as diffusible signal factor (DSF), and the rpf gene cluster is responsible for its synthesis and perception. Two cluster variants have been previously described, rpf-1 and rpf-2, which differ basically in the conditions under which DSF is produced. Here, correlations between the rpf variant and antibiotic susceptibility, LPS electrophoretic profiles and virulence-related phenotypes were evaluated for a collection of 78 geographically and genetically diverse clinical strains of S. maltophilia. In general there were associations between previously established genogroups and the genetic variant of the rpf cluster. However, only few genotype-phenotype correlations could be observed. Resistance to the β-lactam antibiotics ceftazidime and ticarcillin was associated with strains carrying the rpf-1 variant, whereas strains of variant rpf-2, particularly those of genogroup C, showed higher resistance levels to colistin. Strains of variant rpf-2 were also significantly more virulent to Galleria mellonella larvae than those of rpf-1, most likely due to an increased ability of rpf-2 strains to form biofilms. A comparative genomic analysis revealed the presence of proteins unique to individual genogroups. In particular, the strains of genogroup C share an operon that encodes for a new virulence determinant in S. maltophilia related to the synthesis of an alternative Flp/Tad pilus. Overall, this study establishes a link between the DSF-based QS system and the virulence and resistance phenotypes in this species, and identifies potential high-risk clones circulating in European hospitals

Synthesis and evaluation of novel furanones as biofilm inhibitors in opportunistic human pathogens

Diseases caused by biofilm-forming pathogens are becoming increasingly prevalent and represent a major threat to human health. This trend has prompted a search for novel inhibitors of microbial biofilms which could, for example, be used to potentiate existing antibiotics. Naturally-occurring, halogenated furanones isolated from marine algae have proven to be effective biofilm inhibitors in several bacterial species. In this work, we report the synthesis of a library of novel furanones and their subsequent evaluation as biofilm inhibitors in several opportunistic human pathogens including S. enterica, S. aureus, E. coli, S. maltophilia, P. aeruginosa and C. albicans. A number of the most potent compounds were subjected to further analysis by confocal laser-scanning microscopy for their effects on P. aeruginosa and C. albicans biofilms individually, in addition to mixed polymicrobial biofilms. Lastly, we investigated the impact of a promising candidate on survival rates in vivo using a Galleria mellonella model

Production of functional inclusion bodies in endotoxin-free Escherichia coli

Escherichia coli is the workhorse for gene cloning and production of soluble recombinant proteins in both biotechnological and biomedical industries. The bacterium is also a good producer of several classes of protein-based self-assembling materials such as inclusion bodies (IBs). Apart from being a relatively pure source of protein for in vitro refolding, IBs are under exploration as functional, protein-releasing materials in regenerative medicine and protein replacement therapies. Endotoxin removal is a critical step for downstream applications of therapeutic proteins. The same holds true for IBs as they are often highly contaminated with cell-wall components of the host cells. Here, we have investigated the production of IBs in a recently developed endotoxin-free E. coli strain. The characterization of IBs revealed this mutant as a very useful cell factory for the production of functional endotoxin-free IBs that are suitable for the use at biological interfaces without inducing endotoxic responses in human immune cell

Detoxifying Escherichia coli for endotoxin-free production of recombinant proteins

Es va publicar un treball amb esmenes a aquest article que es pot consultar a https://ddd.uab.cat/record/185349 (DOI 10.1186/s12934-015-0265-x)Background: lipopolysaccharide (LPS), also referred to as endotoxin, is the major constituent of the outer leaflet of the outer membrane of virtually all Gram-negative bacteria. The lipid A moiety, which anchors the LPS molecule to the outer membrane, acts as a potent agonist for Toll-like receptor 4/myeloid differentiation factor 2-mediated pro-inflammatory activity in mammals and, thus, represents the endotoxic principle of LPS. Recombinant proteins, commonly manufactured in Escherichia coli, are generally contaminated with endotoxin. Removal of bacterial endotoxin from recombinant therapeutic proteins is a challenging and expensive process that has been necessary to ensure the safety of the final product. -Results: as an alternative strategy for common endotoxin removal methods, we have developed a series of E. coli strains that are able to grow and express recombinant proteins with the endotoxin precursor lipid IVA as the only LPS-related molecule in their outer membranes. Lipid IVA does not trigger an endotoxic response in humans typical of bacterial LPS chemotypes. Hence the engineered cells themselves, and the purified proteins expressed within these cells display extremely low endotoxin levels. - Conclusions: this paper describes the preparation and characterization of endotoxin-free E. coli strains, and demonstrates the direct production of recombinant proteins with negligible endotoxin contamination

Improved mini-Tn 7 Delivery Plasmids for Fluorescent Labeling of

Fluorescently labeled bacterial cells have become indispensable for many aspects of microbiological research, including studies on biofilm formation as an important virulence factor of various opportunistic bacteria of environmental origin such as . Using a Tn 7 -based genomic integration system, we report the construction of improved mini-Tn 7 delivery plasmids for labeling of with sfGFP, mCherry, tdTomato and mKate2 by expressing their codon-optimized genes from a strong, constitutive promoter and an optimized ribosomal binding site. Transposition of the mini-Tn 7 transposons into single neutral sites located on average 25 nucleotides downstream of the 3'-end of the conserved glmS gene of different wild-type strains did not have any adverse effects on the fitness of their fluorescently labeled derivatives. This was demonstrated by comparative analyses of growth, resistance profiles against 18 antibiotics of different classes, the ability to form biofilms on abiotic and biotic surfaces, also independent of the fluorescent protein expressed, and virulence in . It is also shown that the mini-Tn 7 elements remained stably integrated in the genome of over a prolonged period of time in the absence of antibiotic selection pressure. Overall, we provide evidence that the new improved mini-Tn 7 delivery plasmids are valuable tools for generating fluorescently labeled strains that are indistinguishable in their properties from their parental wild-type strains. IMPORTANCE The bacterium is an important opportunistic nosocomial pathogen that can cause bacteremia and pneumonia in immunocompromised patients with a high rate of mortality. It is now considered as a clinically relevant and notorious pathogen in cystic fibrosis patients but has also been isolated from lung specimen of healthy donors. The high intrinsic resistance to a wide range of antibiotics complicates treatment and most likely contributes to the increasing incidence of infections worldwide. One important virulence-related trait of is the ability to form biofilms on any surface, which may result in the development of increased transient phenotypic resistance to antimicrobials. The significance of our work is to provide a mini-Tn 7 -based labeling system for to study the mechanisms of biofilm formation or host-pathogen interactions with live bacteria under non-destructive conditions

Structural and functional features of self-assembling protein nanoparticles produced in endotoxin-free Escherichia coli

Background: production of recombinant drugs in process-friendly endotoxin-free bacterial factories targets to a lessened complexity of the purification process combined with minimized biological hazards during product application. The development of nanostructured recombinant materials in innovative nanomedical activities expands such a need beyond plain functional polypeptides to complex protein assemblies. While Escherichia coli has been recently modified for the production of endotoxin-free proteins, no data has been so far recorded regarding how the system performs in the fabrication of smart nanostructured materials. - Results: we have here explored the nanoarchitecture and in vitro and in vivo functionalities of CXCR4-targeted, self-assembling protein nanoparticles intended for intracellular delivery of drugs and imaging agents in colorectal cancer. Interestingly, endotoxin-free materials exhibit a distinguishable architecture and altered size and target cell penetrability than counterparts produced in conventional E. coli strains. These variant nanoparticles show an eventual proper biodistribution and highly specific and exclusive accumulation in tumor upon administration in colorectal cancer mice models, indicating a convenient display and function of the tumor homing peptides and high particle stability under physiological conditions. - Discussion: the observations made here support the emerging endotoxin-free E. coli system as a robust protein material producer but are also indicative of a particular conformational status and organization of either building blocks or oligomers. This appears to be promoted by multifactorial stress-inducing conditions upon engineering of the E. coli cell envelope, which impacts on the protein quality control of the cell factory

Synthesis and evaluation of novel furanones as biofilm inhibitors in opportunistic human pathogens.

Diseases caused by biofilm-forming pathogens are becoming increasingly prevalent and represent a major threat to human health. This trend has prompted a search for novel inhibitors of microbial biofilms which could, for example, be used to potentiate existing antibiotics. Naturally-occurring, halogenated furanones isolated from marine algae have proven to be effective biofilm inhibitors in several bacterial species. In this work, we report the synthesis of a library of novel furanones and their subsequent evaluation as biofilm inhibitors in several opportunistic human pathogens including S. enterica, S. aureus, E. coli, S. maltophilia, P. aeruginosa and C. albicans. A number of the most potent compounds were subjected to further analysis by confocal laser-scanning microscopy for their effects on P. aeruginosa and C. albicans biofilms individually, in addition to mixed polymicrobial biofilms. Lastly, we investigated the impact of a promising candidate on survival rates in vivo using a Galleria mellonella model

Phenotypic and transcriptomic analyses of seven clinical Stenotrophomonas maltophilia isolates identify a small set of shared and commonly regulated genes involved in the biofilm lifestyle

Stenotrophomonas maltophilia is one of the most frequently isolated multidrug-resistant nosocomial opportunistic pathogens. It contributes to disease progression in cystic fibrosis (CF) patients and is frequently isolated from wounds, infected tissues, and catheter surfaces. On these diverse surfaces S. maltophilia lives in single-species or multispecies biofilms. Since very little is known about common processes in biofilms of different S. maltophilia isolates, we analyzed the biofilm profiles of 300 clinical and environmental isolates from Europe of the recently identified main lineages Sgn3, Sgn4, and Sm2 to Sm18. The analysis of the biofilm architecture of 40 clinical isolates revealed the presence of multicellular structures and high phenotypic variability at a strain-specific level. Further, transcriptome analyses of biofilm cells of seven clinical isolates identified a set of 106 shared strongly expressed genes and 33 strain-specifically expressed genes. Surprisingly, the transcriptome profiles of biofilm versus planktonic cells revealed that just 9.43% ± 1.36% of all genes were differentially regulated. This implies that just a small set of shared and commonly regulated genes is involved in the biofilm lifestyle. Strikingly, iron uptake appears to be a key factor involved in this metabolic shift. Further, metabolic analyses implied that S. maltophilia employs a mostly fermentative growth mode under biofilm conditions. The transcriptome data of this study together with the phenotypic and metabolic analyses represent so far the largest data set on S. maltophilia biofilm versus planktonic cells. This study will lay the foundation for the identification of strategies for fighting S. maltophilia biofilms in clinical and industrial settings. IMPORTANCE Microorganisms living in a biofilm are much more tolerant to antibiotics and antimicrobial substances than planktonic cells are. Thus, the treatment of infections caused by microorganisms living in biofilms is extremely difficult. Nosocomial infections (among others) caused by S. maltophilia, particularly lung infection among CF patients, have increased in prevalence in recent years. The intrinsic multidrug resistance of S. maltophilia and the increased tolerance to antimicrobial agents of its biofilm cells make the treatment of S. maltophilia infection difficult. The significance of our research is based on understanding the common mechanisms involved in biofilm formation of different S. maltophilia isolates, understanding the diversity of biofilm architectures among strains of this species, and identifying the differently regulated processes in biofilm versus planktonic cells. These results will lay the foundation for the treatment of S. maltophilia biofilms