Age structure landscapes emerge from the equilibrium between aging and rejuvenation in bacterial populations.

The physiological asymmetry between daughters of a mother bacterium is produced by the inheritance of either old poles, carrying non-genetic damage, or newly synthesized poles. However, as bacteria display long-term growth stability leading to physiological immortality, there is controversy on whether asymmetry corresponds to aging. Here we show that deterministic age structure landscapes emerge from physiologically immortal bacterial lineages. Through single-cell microscopy and microfluidic techniques, we demonstrate that aging and rejuvenating bacterial lineages reach two distinct states of growth equilibria. These equilibria display stabilizing properties, which we quantified according to the compensatory trajectories of continuous lineages throughout generations. Finally, we show that the physiological asymmetry between aging and rejuvenating lineages produces complex age structure landscapes, resulting in a deterministic phenotypic heterogeneity that is neither an artifact of starvation nor a product of extrinsic damage. These findings indicate that physiological immortality and cellular aging can both be manifested in single celled organisms

The HSV-1 Latency-Associated Transcript Functions to Repress Latent Phase Lytic Gene Expression and Suppress Virus Reactivation from Latently Infected Neurons

open access articleHerpes simplex virus 1 (HSV-1) establishes life-long latent infection within sensory neurons, during which viral lytic gene expression is silenced. The only highly expressed viral gene product during latent infection is the latency-associated transcript (LAT), a non-protein coding RNA that has been strongly implicated in the epigenetic regulation of HSV-1 gene expression. We have investigated LAT-mediated control of latent gene expression using chromatin immunoprecipitation analyses and LAT-negative viruses engineered to express firefly luciferase or β-galactosidase from a heterologous lytic promoter. Whilst we were unable to determine a significant effect of LAT expression upon heterochromatin enrichment on latent HSV-1 genomes, we show that reporter gene expression from latent HSV-1 genomes occurs at a greater frequency in the absence of LAT. Furthermore, using luciferase reporter viruses we have observed that HSV-1 gene expression decreases during long-term latent infection, with a most marked effect during LAT-negative virus infection. Finally, using a fluorescent mouse model of infection to isolate and culture single latently infected neurons, we also show that reactivation occurs at a greater frequency from cultures harbouring LAT-negative HSV-1. Together, our data suggest that the HSV-1 LAT RNA represses HSV-1 gene expression in small populations of neurons within the mouse TG, a phenomenon that directly impacts upon the frequency of reactivation and the maintenance of the transcriptionally active latent reservoir

Magnetic behaviour of multisegmented FeCoCu/Cu electrodeposited nanowires

Understanding the magnetic behaviour of multisegmented nanowires (NWs) is a major key for the application of such structures in future devices. In this work, magnetic/non-magnetic arrays of FeCoCu/Cu multilayered NWs electrodeposited in nanoporous alumina templates are studied. Contrarily to most reports on multilayered NWs, the magnetic layer thickness was kept constant (30 nm) and only the non-magnetic layer thickness was changed (0 to 80 nm). This allowed us to tune the interwire and intrawire interactions between the magnetic layers in the NW array creating a three-dimensional (3D) magnetic system without the need to change the template characteristics. Magnetic hysteresis loops, measured with the applied field parallel and perpendicular to the NWs' long axis, showed the effect of the non-magnetic Cu layer on the overall magnetic properties of the NW arrays. In particular, introducing Cu layers along the magnetic NW axis creates domain wall nucleation sites that facilitate the magnetization reversal of the wires, as seen by the decrease in the parallel coercivity and the reduction of the perpendicular saturation field. By further increasing the Cu layer thickness, the interactions between the magnetic segments, both along the NW axis and of neighbouring NWs, decrease, thus rising again the parallel coercivity and the perpendicular saturation field. This work shows how one can easily tune the parallel and perpendicular magnetic properties of a 3D magnetic layer system by adjusting the non-magnetic layer thickness

Distinguishing nanowire and nanotube formation by the deposition current transients

AbstractHigh aspect ratio Ni nanowires (NWs) and nanotubes (NTs) were electrodeposited inside ordered arrays of self-assembled pores (approximately 50 nm in diameter and approximately 50 μm in length) in anodic alumina templates by a potentiostatic method. The current transients monitored during each process allowed us to distinguish between NW and NT formation. The depositions were long enough for the deposited metal to reach the top of the template and form a continuous Ni film. The overfilling process was found to occur in two steps when depositing NWs and in a single step in the case of NTs. A comparative study of the morphological, structural, and magnetic properties of the Ni NWs and NTs was performed using scanning electron microscopy, X-ray diffraction, and vibrating sample magnetometry, respectively.M. P. Proença and C. T. Sousa are thankful to FCT for the doctoral and postdoctoral grants SFRH/BD/43440/2008 and SFRH/BPD/82010/2011, respectively. J. Ventura acknowledges the financial support through FSE/POPH. M Vázquez thanks the Spanish Ministry of Economia y Competitividad, MEC, under project MAT2010-20798-C05-01. J. P. Araújo also thanks the Fundação Gulbenkian for its financial support within the ‘Programa Gulbenkian de Estímulo à Investigação Científica’. The authors acknowledge the funding from FCT through the Associated Laboratory - IN and project PTDC/FIS/105416/2008.Peer Reviewe

IRF-5-dependent signaling restricts Orthobunyavirus dissemination to the central nervous system

ABSTRACT Interferon (IFN)-regulatory factor 5 (IRF-5) is a transcription factor that induces inflammatory responses after engagement and signaling by pattern recognition receptors. To define the role of IRF-5 during bunyavirus infection, we evaluated Oropouche virus (OROV) and La Crosse virus (LACV) pathogenesis and immune responses in primary cells and in mice with gene deletions in Irf3 , Irf5 , and Irf7 or in Irf5 alone. Deletion of Irf3 , Irf5 , and Irf7 together resulted in uncontrolled viral replication in the liver and spleen, hypercytokinemia, extensive liver injury, and an early-death phenotype. Remarkably, deletion of Irf5 alone resulted in meningoencephalitis and death on a more protracted timeline, 1 to 2 weeks after initial OROV or LACV infection. The clinical signs in OROV-infected Irf5 −/− mice were associated with abundant viral antigen and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells in several regions of the brain. Circulating dendritic cell (DC) subsets in Irf5 −/− mice had higher levels of OROV RNA in vivo yet produced lower levels of type I IFN than wild-type (WT) cells. This result was supported by data obtained in vitro , since a deficiency of IRF-5 resulted in enhanced OROV infection and diminished type I IFN production in bone marrow-derived DCs. Collectively, these results indicate a key role for IRF-5 in modulating the host antiviral response in peripheral organs that controls bunyavirus neuroinvasion in mice. IMPORTANCE Oropouche virus (OROV) and La Crosse virus (LACV) are orthobunyaviruses that are transmitted by insects and cause meningitis and encephalitis in subsets of individuals in the Americas. Recently, we demonstrated that components of the type I interferon (IFN) induction pathway, particularly the regulatory transcription factors IRF-3 and IRF-7, have key protective roles during OROV infection. However, the lethality in Irf3 −/− Irf7 −/− (DKO) mice infected with OROV was not as rapid or complete as observed in Ifnar −/− mice, indicating that other transcriptional factors associated with an IFN response contribute to antiviral immunity against OROV. Here, we evaluated bunyavirus replication, tissue tropism, and cytokine production in primary cells and mice lacking IRF-5. We demonstrate an important role for IRF-5 in preventing neuroinvasion and the ensuing encephalitis caused by OROV and LACV

A link between aging and persistence

Despite the various strategies that microorganisms have evolved to resist antibiotics, survival to drug treatments can be driven by subpopulations of susceptible bacteria in a transient state of dormancy. This phenotype, known as bacterial persistence, arises due to a natural and ubiquitous heterogeneity of growth states in bacterial populations. Nonetheless, the unifying mechanism of persistence remains unknown, with several pathways being able to trigger the phenotype. Here, we show that asymmetric damage partitioning, a form of cellular aging, produces the underlying phenotypic heterogeneity upon which persistence is triggered. Using single-cell microscopy and microfluidic devices, we demonstrate that deterministic asymmetry in exponential phase populations leads to a state of growth stability, which prevents the spontaneous formation of persisters. However, as populations approach stationary phase, aging bacteria—those inheriting more damage upon division—exhibit a sharper growth rate decline, increased probability of growth arrest, and higher persistence rates. These results indicate that persistence triggers are biased by bacterial asymmetry, thus acting upon the deterministic heterogeneity produced by cellular aging. This work suggests unifying mechanisms for persistence and offers new perspectives on the treatment of recalcitrant infections

Progressive decline in old pole gene expression signal enhances phenotypic heterogeneity in bacteria

Cell growth and gene expression are heterogeneous processes at the single-cell level, leading to the emergence of multiple physiological states within bacterial populations. Aging is a known deterministic driver of growth asymmetry; however, its role in gene expression heterogeneity remains elusive. Here, we show that aging mother cells undergo a progressive decline in old pole activity, generating asymmetry in protein partitioning, gene expression, and cell morphology. We demonstrate that mother cells, when compared to their daughters, exhibit lower product inheritance and gene expression rates independently of promoter dynamics. The declining activity of maternal old poles generates gene expression gradients that manifest as mother-daughter asymmetry upon division, showing that asymmetry is progressively built over time within the maternal intracellular environment. Moreover, old pole aging correlates with a gradual increase in cell length, leading to morphological asymmetry. These findings provide further evidence for aging as a mechanism to enhance phenotypic heterogeneity in bacterial populations, with possible consequences for stress response and survival

EURL ECVAM Workshop on New Generation of Physiologically-Based Kinetic Models in Risk Assessment

The European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) Strategy Document on Toxicokinetics (TK) outlines strategies to enable prediction of systemic toxicity by applying new approach methodologies (NAM). The central feature of the strategy focuses on using physiologically-based kinetic (PBK) modelling to integrate data generated by in vitro and in silico methods for absorption, distribution, metabolism, and excretion (ADME) in humans for predicting whole-body TK behaviour, for environmental chemicals, drugs, nano-materials, and mixtures. In order to facilitate acceptance and use of this new generation of PBK models, which do not rely on animal/human in vivo data in the regulatory domain, experts were invited by EURL ECVAM to (i) identify current challenges in the application of PBK modelling to support regulatory decision making; (ii) discuss challenges in constructing models with no in vivo kinetic data and opportunities for estimating parameter values using in vitro and in silico methods; (iii) present the challenges in assessing model credibility relying on non-animal data and address strengths, uncertainties and limitations in such an approach; (iv) establish a good kinetic modelling practice workflow to serve as the foundation for guidance on the generation and use of in vitro and in silico data to construct PBK models designed to support regulatory decision making. To gauge the current state of PBK applications, experts were asked upfront of the workshop to fill a short survey. In the workshop, using presentations and discussions, the experts elaborated on the importance of being transparent about the model construct, assumptions, and applications to support assessment of model credibility. The experts offered several recommendations to address commonly perceived limitations of parameterization and evaluation of PBK models developed using non-animal data and its use in risk assessment, these include: (i) develop a decision tree for model construction; (ii) set up a task force for independent model peer review; (iii) establish a scoring system for model evaluation; (iv) attract additional funding to develop accessible modelling software.; (v) improve and facilitate communication between scientists (model developers, data provider) and risk assessors/regulators; and (vi) organise specific training for end users. The experts also acknowledged the critical need for developing a guidance document on building, characterising, reporting and documenting PBK models using non-animal data. This document would also need to include guidance on interpreting the model analysis for various risk assessment purposes, such as incorporating PBK models in integrated strategy approaches and integrating them with in vitro toxicity testing and adverse outcome pathways. This proposed guidance document will promote the development of PBK models using in vitro and silico data and facilitate the regulatory acceptance of PBK models for assessing safety of chemicals

Tuning the magnetic properties of multisegmented Ni/Cu electrodeposited nanowires with controllable Ni lengths

The fabrication of segmented Ni/Cu nanowires (NWs), with tunable structural and magnetic properties, is reported. A potentiostatic electrodeposition method with a single electrolytic bath has been used to fabricate multisegmented Ni/Cu NWs inside a highly hexagonally ordered anodic nanoporous alumina membrane, with diameters of 50 nm and Ni segment lengths (L-Ni) tuned from 10 nm up to 140 nm. The x-ray diffraction results evidenced a strong dependence of the Ni NWs crystallographic face-centered-cubic (fcc) texture along the [220] direction on the aspect ratio of the NWs. The magnetic behavior of the multisegmented Ni/Cu NW arrays, as a function of the magnetic field and temperature, is also studied and correlated with their structural and morphological properties. Micromagnetic simulations, together with the experimental results, showed a dominant antiferromagnetic coupling between Ni segments along the wire length for small low aspect-ratio magnetic segments. When increasing the Ni segments' length, the magnetic interactions between these along the wire became stronger, favouring a ferromagnetic coupling. The Curie temperature of the NWs was also found to strongly depend on the Ni magnetic segment length. Particularly the Curie temperature was found to be reduced 75 K for the 20 nm Ni segments, following the finite-size scaling relation with xi(0) = 8.1 angstrom and gamma = 0.48. These results emphasize the advantages of using a template assisted method to electrodeposit multilayer NWs, as it allows an easy tailor of the respective morphological, chemical, structural and magnetic properties