Role of small colony variants in persistence of Pseudomonas aeruginosa infections in cystic fibrosis lungs

Jacob G Malone1,21John Innes Centre, Norwich, UK; 2School of Biological Sciences, University of East Anglia, Norwich, UKAbstract: Pseudomonas aeruginosa is an opportunistic pathogen that predominates during the later stages of cystic fibrosis (CF) lung infections. Over many years of chronic lung colonization, P. aeruginosa undergoes extensive adaptation to the lung environment, evolving both toward a persistent, low virulence state and simultaneously diversifying to produce a number of phenotypically distinct morphs. These lung-adapted P. aeruginosa strains include the small colony variants (SCVs), small, autoaggregative isolates that show enhanced biofilm formation, strong attachment to surfaces, and increased production of exopolysaccharides. Their appearance in the sputum of CF patients correlates with increased resistance to antibiotics, poor lung function, and prolonged persistence of infection, increasing their relevance as a subject for clinical investigation. The evolution of SCVs in the CF lung is associated with overproduction of the ubiquitous bacterial signaling molecule cyclic-di-GMP, with increased cyclic-di-GMP levels shown to be responsible for the SCV phenotype in a number of different CF lung isolates. Here, we review the current state of research in clinical P. aeruginosa SCVs. We will discuss the phenotypic characteristics underpinning the SCV morphotype, the clinical implications of lung colonization with SCVs, and the molecular basis and clinical evolution of the SCV phenotype in the CF lung environment.Keywords: small colony variants, cystic fibrosis, cyclic-di-GMP, Pseudomonas aeruginosa, RsmA, antibiotic

Magnetic separation of peripheral nerve-resident cells underscores key molecular features of human Schwann cells and fibroblasts: an immunochemical and transcriptomics approach

Nerve-derived human Schwann cell (SC) cultures are irreplaceable models for basic and translational research but their use can be limited due to the risk of fibroblast overgrowth. Fibroblasts are an ill-defined population consisting of highly proliferative cells that, contrary to human SCs, do not undergo senescence in culture. We initiated this study by performing an exhaustive immunological and functional characterization of adult nerve-derived human SCs and fibroblasts to reveal their properties and optimize a protocol of magnetic-activated cell sorting (MACS) to separate them effectively both as viable and biologically competent cells. We next used immunofluorescence microscopy imaging, flow cytometry analysis and next generation RNA sequencing (RNA-seq) to unambiguously characterize the post-MACS cell products. High resolution transcriptome profiling revealed the identity of key lineage-specific transcripts and the clearly distinct neural crest and mesenchymal origin of human SCs and fibroblasts, respectively. Our analysis underscored a progenitor- or stem cell-like molecular phenotype in SCs and fibroblasts and the heterogeneity of the fibroblast populations. In addition, pathway analysis of RNA-seq data highlighted putative bidirectional networks of fibroblast-to-SC signaling that predict a complementary, yet seemingly independent contribution of SCs and fibroblasts to nerve regeneration. In sum, combining MACS with immunochemical and transcriptomics approaches provides an ideal workflow to exhaustively assess the identity, the stage of differentiation and functional features of highly purified cells from human peripheral nerve tissues.Fil: Peng, Kaiwen. Indiana University. School of Medicine; Estados Unidos. Nanfang Hospital; ChinaFil: Sant, David. University of Utah; Estados Unidos. Miami University. School of Medicine; Estados UnidosFil: Andersen, Natalia Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina. Miami University. School of Medicine; Estados UnidosFil: Silvera, Risset. Miami University. School of Medicine; Estados UnidosFil: Camarena, Vladimir. Miami University. School of Medicine; Estados UnidosFil: Piñero, Gonzalo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Miami University. School of Medicine; Estados UnidosFil: Graham, Regina. Miami University. School of Medicine; Estados UnidosFil: Khan, Aisha. Miami University. School of Medicine; Estados UnidosFil: Xu, Xiao Ming. Indiana University. School of Medicine; Estados UnidosFil: Wang, Gaofeng. Miami University. School of Medicine; Estados UnidosFil: Monje, Paula. Indiana University. School of Medicine; Estados Unidos. Miami University. School of Medicine; Estados Unido

Fundamental Principles in Bacterial Physiology - History, Recent progress, and the Future with Focus on Cell Size Control: A Review

Bacterial physiology is a branch of biology that aims to understand overarching principles of cellular reproduction. Many important issues in bacterial physiology are inherently quantitative, and major contributors to the field have often brought together tools and ways of thinking from multiple disciplines. This article presents a comprehensive overview of major ideas and approaches developed since the early 20th century for anyone who is interested in the fundamental problems in bacterial physiology. This article is divided into two parts. In the first part (Sections 1 to 3), we review the first `golden era' of bacterial physiology from the 1940s to early 1970s and provide a complete list of major references from that period. In the second part (Sections 4 to 7), we explain how the pioneering work from the first golden era has influenced various rediscoveries of general quantitative principles and significant further development in modern bacterial physiology. Specifically, Section 4 presents the history and current progress of the `adder' principle of cell size homeostasis. Section 5 discusses the implications of coarse-graining the cellular protein composition, and how the coarse-grained proteome `sectors' re-balance under different growth conditions. Section 6 focuses on physiological invariants, and explains how they are the key to understanding the coordination between growth and the cell cycle underlying cell size control in steady-state growth. Section 7 overviews how the temporal organization of all the internal processes enables balanced growth. In the final Section 8, we conclude by discussing the remaining challenges for the future in the field.Comment: Published in Reports on Progress in Physics. (https://doi.org/10.1088/1361-6633/aaa628) 96 pages, 48 figures, 7 boxes, 715 reference

Apoptosis in Haematopoietic progenitors

Introduction: Intracranial pressure (ICP) monitoring is a cornerstone of care for patients with severe traumatic brain injury (TBI). The primary goal of ICP treatment is to preserve brain oxygenation, and since brain oxygenation is usually not measured, the control of ICP is used as a surrogate marker. However studies indicating that cerebral hypoxia/ischemia may occur in the face of adequate ICP and cerebral perfusion pressure (CPP) suggest that the interaction between ICP and brain oxygenation is poorly understood and warrants further investigation. This is of particular importance in the context of children in whom the interpretation of relationships between intracranial factors is even more complex due to changing physiological norms with age. To date little scientific data exists in children and treatment threshold values are often extrapolated from adult guidelines. This study aims to better understand the relationship between ICP and brain oxygenation measured as brain tissue oxygen tension (PbtO2) in a large paediatric cohort suffering from severe TBI. Specifically analysis 1) investigated ICP and PbtO2 profiles over time following TBI, 2) examined the relationship between ICP and PbtO2 from time-linked paired observations, 3) explored various critical thresholds for ICP and PbtO2, and 4) interrogated digital data trends depicting the relationship between ICP and PbtO2. The level of agreement between hourly recorded and high frequency electronic data for ICP and PbtO2 was also evaluated. Method: Paired ICP and PbtO2 data from 75 children with severe TBI were tested with correlation and regression. Additional analyses controlled for mean arterial pressure (MAP), arterial partial pressure of oxygen (PaO2), CPP, arterial partial pressure of carbon dioxide (PaCO2) and haemoglobin (Hb) using multivariate logistic regression analysis and general estimating equations. Various thresholds for ICP were examined; these included age-related thresholds to account for the potential influence of age. Receiver-operating curves (ROCs) were used to graphically demonstrate the relationships between various thresholds of ICP and various definitions of low PbtO2. These were constructed for pooled and individual patient data. Interrogation of electronically recorded data allowed for case illustrations examining the relationship between ICP and PbtO2 at selected time points. Hourly and electronic data were compared using Bland and Altman plots and by contrasting the frequency of ICP and PbtO2 perturbations recorded with each system. 5 Result: Analyses using over 8300 hours of paired observations revealed a weak relationship between ICP and PbtO2, with an initially positive but weak slope (r = 0.05) that trended downwards only at higher values of ICP. Controlling for inter-individual differences, as well as MAP, CPP, PaO2, PaCO2 and Hb did not strengthen this association. This poor relationship was further reflected in the examination of threshold ICP values with ROCs, no singular critical ICP threshold for compromised brain oxygenation was discernible. Using age-based thresholds did not improve this relationship and individual patient ROCs demonstrated inter-individual heterogeneity in the relationship between ICP and PbtO2. However, it was clear that in individual patients ICP did exhibit a strong negative relationship with PbtO2 at particular time points, but various different relationships between the 2 variables were also demonstrated. A high level of agreement was found between hourly and electronic data. Conclusion: These results suggest that the relationship between ICP and PbtO2 is highly complex. Although the relationship in individual children at specific time points may be strong, pooled data for the entire cohort of patients, and even for individual patients, suggest only a weak relationship. This is likely because several other factors affect PbtO2 outside of ICP, and some factors affect both independently of each other. These results suggest that more study should be directed at optimising ICP thresholds for treatment in children. The use of complimentary monitoring modalities may assist in this task. Depending on the adequacy of measures of brain perfusion, metabolism or oxygenation, it is possible that targeting a range of ICP values in individual patients may be appropriate; however this would require detailed investigation

Stochastic Effects in Quorum Sensing

[cat] En aquesta tesi, estudiem els efectes de la estocàsticitat en la aparició del comportament col·lectiu en poblacions de bacteris que comuniquen per quorum sensing (QS). Ens centrem en el interruptor genètic com a paradigma dels processos de decisió cel·lulars tant en sistemes de bacteris naturals com sintètics. El nostre mètode es basa en la modelització matemàtica i en les simulacions estocàstiques, tant a nivell d'una cèl·lula individual com a nivell d'una població de cèl·lules. A nivell d'una cèl·lula individual, mostrem que el soroll afavoreix l'estabilitat del fenotip de l'estat ``baix'' de l'interruptor genètic autoactivador i que la regió de biestabilitat s'estén quan creix la intensitat de les fluctuacions, un efecte que hem anomenat estabilització estocàstica. A nivell d'una població de cèl·lules, mostrem que el procés de difusió del mecanisme de QS modifica les fluctuacions i la dinàmica de la molècula autoinductora dins de la cèl·lula i interactua amb el soroll en la expressió genètica. En el sistema canònic de QS LuxR/LuxI, mostrem que el soroll en la expressió genètica de LuxR és el principal factor que controla la variabilitat transitòria de l'activació del QS. El soroll intrínsec disminueix la precisió de la coordinació de la població i modifica la dinàmica de la transició de QS. A més, presentem un model d'una població d'interruptors genètics de toggle switch que comuniquen per l'intercanvi de dos senyals difusius de QS. Mostrem que l'increment de la velocitat de difusió, que augmenta la força de l'acoblament entre les cèl·lules, porta a una transició de fase: va des d'una fase desordenada on les cèl·lules salten de manera aleatòria entre els dos estats de l'interruptor, fins a una fase ordenada amb totes les cèl·lules bloquejades en el mateix estat estable. La mateixa transició s'ha trobat en una població de cèl·lules que creixen exponencialment en un volum tancat, amb totes les cèl·lules entrant en l'estat ordenat quan arriben a una mida crítica del sistema. Els nostres resultats suggereixen un nou mecanisme per la decisió cel·lular col·lectiva basat en el fenomen de la transició de fase.[eng] Stochastic fluctuations, or noise, are ubiquitous in biological systems and play an important role in many cellular processes. Experimental evidences have shown that noise affects the reliability of cell coordination in populations of communicating cells. In this thesis, we study the effects of stochasticity in the emergence of collective behavior in populations of bacteria communicating by QS. We focus on the genetic switch as a paradigm of cellular decision making in both natural and synthetic bacterial systems. Our approach is based on mathematical modeling and stochastic simulations, both at the level of the single cell and at the level of the cell population. We focus on four main topics. In the first topic, we analyze the interplay between intracellular noise and the diffusion process of the QS signaling mechanism. We build a model describing the expression of the signaling molecule and its diffusion in a population of cells, focusing on the situation of very low constitutive expression rate. We show that varying the diffusion rate produces a repertoire of dynamics for the signaling molecule. Our results reveal the contribution of intrinsic noise and transcriptional noise (mRNA copy number fluctuations) in the fluctuations of the signaling molecule. We observe that the total noise exhibits a maximum as a function of the diffusion rate, in contrast to previous studies. Thus, the QS communication mechanism modifies the fluctuations of the signaling molecule inside the cell and interacts with the gene expression noise. In the second topic, we study the effects of gene expression noise on the precision of the population coordination in the QS activation of the LuxR/LuxI system. We analyze the response and dynamics of a population of cells to different levels of autoinducer. Our results show that gene expression noise in LuxR is the main factor that controls the transient variability of the QS activation. This study sheds light on the relation between the single cell stochastic dynamics and the collective behavior in a population of communicating cells. In the third topic, we analyze the effects of intrinsic noise in an autoactivating switch in an isolated single cell. We show that noise promotes the stability of the low-state phenotype of the switch and that the bistable region is extended when increasing the intensity of the fluctuations, an effect that we call stochastic stabilization. Our results show that intrinsic noise modifies the epigenetic landscape as well as the switching rate, which results in complex behavior of the stochastic switching dynamics when varying the intensity of noise. Thus, at the level of a single cell, intrinsic noise contributes to the cell-to-cell variability of the genetic switch and can modify its stable states and its dynamics. In the fourth topic, we build a model of a population of toggle switches communicating by the exchange of two diffusible QS signals. We show that increasing the diffusion rate, which increases the coupling strength between the cells, leads to a phase transition from an unordered phase where the cells randomly flip between the two states of the switch, to an ordered phase with all the cells locked into the same stable state. The same transition is found in a population of cells growing exponentially in a closed volume. Moreover, the response of the cells to a varying external signal exhibits a hysteresis loop. We show that the cell-cell coupling enhances the sensitivity of the population response to the external signal and suggest that this new mechanism could be used to increase the robustness and sensitivity of biosensors. Our results suggest a new mechanism for collective cell decision making based on the phenomenon of phase transition

Suitability of GRK Antibodies for Individual Detection and Quantification of GRK Isoforms in Western Blots

G protein-coupled receptors (GPCRs) are regulated by GPCR kinases (GRKs) which phosphorylate intracellular domains of the active receptor. This results in the recruitment of arrestins, leading to desensitization and internalization of the GPCR. Aside from acting on GPCRs, GRKs regulate a variety of membrane, cytosolic, and nuclear proteins not only via phosphorylation but also by acting as scaffolding partners. GRKs’ versatility is also reflected by their diverse roles in pathological conditions such as cancer, malaria, Parkinson’s-, cardiovascular-, and metabolic disease. Reliable tools to study GRKs are the key to specify their role in complex cellular signaling networks. Thus, we examined the specificity of eight commercially available antibodies targeting the four ubiquitously expressed GRKs (GRK2, GRK3, GRK5, and GRK6) in Western blot analysis. We identified one antibody that did not recognize its antigen, as well as antibodies that showed unspecific signals or cross-reactivity. Hence, we strongly recommend testing any antibody with exogenously expressed proteins to clearly confirm identity of the obtained Western blot results. Utilizing the most-suitable antibodies, we established the Western blot-based, cost-effective simple tag-guided analysis of relative protein abundance (STARPA). This method allows comparison of protein levels obtained by immunoblotting with different antibodies. Furthermore, we applied STARPA to determine GRK protein levels in nine commonly used cell lines, revealing differential isoform expression

Nitrogen efficiency of dairy cattle : from protein evaluation to ammonia emission

Diet optimization contributes considerably to increased nitrogen efficiency of dairy cattle, resulting in reduced nitrogen losses. This thesis focuses on three themes: the potential advances in protein evaluation systems for ruminants, the relationship between dairy cow diet and ammonia emission and the opportunities to monitor ammonia emission from dairy cow barns by application of milk urea content as a practical indicator. Overall, the present work shows that farm management can be aimed at increased nitrogen efficiency of dairy cattle and reduced ammonia emission without compromising other sustainability objectives such as the integral ecological footprint, animal health and farm profitability. </p