Translocation Dynamics Research Articles

Polymer translocation through a nanopore assisted by an environment of active rods

We use a combination of computer simulations and iso-flux tension propagation (IFTP) theory to investigate translocation dynamics of a flexible linear polymer through a nanopore into an environment composed of repulsive active rods in 2D. We demonstrate that the rod activity induces a crowding effect on the polymer, leading to a time-dependent net force that facilitates translocation into the active environment. Incorporating this force into the IFTP theory for pore-driven translocation allows us to characterise translocation dynamics in detail and derive a scaling form for the average translocation time as $\tilde{\tau} \sim \tilde{L}_{\textrm{r}}^{\nu} / \tilde{F}_{\textrm{SP}} $, where $\tilde{L}_{\textrm{r}}$ and $\tilde{F}_{\textrm{SP}}$ are the rod length and self-propelling force acting on the rods, respectively, and $\nu$ is the Flory exponent.

Boron: More Than an Essential Element for Land Plants?

Although boron (B) is an element that has long been assumed to be an essential plant micronutrient, this assumption has been recently questioned. Cumulative evidence has demonstrated that the players associated with B uptake and translocation by plant roots include a sophisticated set of proteins used to cope with B levels in the soil solution. Here, we summarize compelling evidence supporting the essential role of B in mediating plant developmental programs. Overall, most plant species studied to date have exhibited specific B transporters with tight genetic coordination in response to B levels in the soil. These transporters can uptake B from the soil, which is a highly uncommon occurrence for toxic elements. Moreover, the current tools available to determine B levels cannot precisely determine B translocation dynamics. We posit that B plays a key role in plant metabolic activities. Its importance in the regulation of development of the root and shoot meristem is associated with plant developmental phase transitions, which are crucial processes in the completion of their life cycle. We provide further evidence that plants need to acquire sufficient amounts of B while protecting themselves from its toxic effects. Thus, the development of in vitro and in vivo approaches is required to accurately determine B levels, and subsequently, to define unambiguously the function of B in terrestrial plants.

Internet-mediated Islamist semiotics: policing memes and the R4BIA pizza effect

ABSTRACT This article explores the semiotics and trans-local dynamics of the decision by a Mancunian pizzeria to brand itself using what is widely regarded as a characteristically Islamist internet meme (the so-called ‘R4bia Sign’). The article explores the genesis, genealogy, and development of a salient religio-political symbol as a means to interrogate specific models of cultural production and reproduction. It focuses in particular on the character of meme-making as a mode of political participation and as part of dissident groups’ repertoires of action. In the process, it problematizes contemporary state policy in relation to policing internet-centred discourse among minority and dissident groups. It is particularly critical of indiscriminate interventions which obscure dynamic, reciprocal, and contextual transformations of meaning associated with ostensibly fixed symbols.

Inertial focusing and zeta potential measurements of single-nanoparticles using octet-nanochannels.

Capture-to-translocation dynamics control is an important issue for single-particle and -molecule analyses by resistive pulse waveforms. Here, we report on regulated motions for accurate zeta-potential assessments of single nanoscale objects passing through an octet-nanochannel. We observed ionic spike signals consisting of eight consecutive sub-pulses signifying the ion blockage at the eight sensing zones in series upon electrophoretic translocation of individual nanoparticles. We find an exponential decrease to saturation of the channel-to-channel translocation duration as a nanobead moves forward, reflecting the more restricted radial motion degrees of freedom via inertial effects at the downstream side of the octet channel. This finding enabled a protocol for single-nanoparticle zeta potential estimation impervious to the uncertainty stemming from the stochastic nature of the translocation dynamics. The multi-channel approach presented in this study may be used as a useful tool for analyzing particles and molecules of variable sizes.

Effects of active crowder size and activity-crowding coupling on polymer translocation.

Polymer translocation in complex environments is crucially important to many biological processes in life. In the present work, we adopted two-dimensional Langevin dynamics simulations to study the forced and unbiased polymer translocation dynamics in active and crowded media. The translocation time and probability are analyzed in terms of active force Fa, volume fraction φ and also the crowder size. The non-trivial active crowder size effect and activity-crowding coupling effect as well as the novel mechanism of unbiased translocation between two active environments with different active particle sizes are clarified. Firstly, for forced translocation, we reveal an intriguing non-monotonic dependence of the translocation time on the crowder size in the case of large activity. In particular, crowders of intermediate size similar to the polymer segment are proven to be the most favorable for translocation. Moreover, a facilitation-inhibition crossover of the translocation time with increasing volume fraction is observed, indicating a crucial activity-crowding coupling effect. Secondly, for unbiased translocation driven by different active crowder sizes, the translocation probability demonstrates a novel turnover phenomenon, implying the appearance of an opposite directional preference as the active force exceeds a critical value. The translocation time in both directions decreases monotonically with the active force. The asymmetric activity effect together with the entropic driving scenario provides a reasonable picture for the peculiar behavior observed in unbiased translocation.

IPSC-Derived Microglia for Modeling Human-Specific DAMP and PAMP Responses in the Context of Alzheimer's Disease.

Neuroinflammation in Alzheimer’s disease (AD) has been the focus for identifying targetable pathways for drug development. The role of amyloid beta (Aβ), a prototype of damage-associated molecular patterns (DAMPs), has been implicated in triggering an inflammatory response. As alpha7 nicotinic acetylcholine receptor (α7 nAChR) binds Aβ with high affinity, α7 nAChR may play a role in Aβ-induced neuroinflammation. The conundrum of how α7 nAChR as the mediator of the cholinergic anti-inflammatory response may trigger an inflammatory response has not been resolved. CHRFAM7A, the uniquely human fusion gene between ULK4 and CHRNA7, is a negative regulator of α7 nAChR ionotropic function. To provide the human context, isogenic induced pluripotent stem cell (iPSC) lines were developed from CHRFAM7A null and carrier individuals by genome-editing the null line using TALENs to knock-in CHRFAM7A. In iPSC-derived microglia-like cells, CHRFAM7A mitigated Aβ uptake through the α7 nAChR. Despite the lower Aβ uptake, the presence of CHRFAM7A was associated with an innate immune response that was characterized by NF-κB activation and NF-κB target transcription (TNFA, IL6, and IL1B). LPS, a prototype PAMP, induced a heightened immune response in CHRFAM7A carriers. CHRFAM7A modified the dynamics of NF-κB translocation by prolonging its nuclear presence. CHRFAM7A modified the α7 nAChR metabotropic function, resulting in a human-specific innate immune response. This iPSC model provided an opportunity to elucidate the mechanism and establish high throughput screens.

High Mobility Group Box-1 and Blood-Brain Barrier Disruption.

Increasing evidence suggests that inflammatory responses are involved in the progression of brain injuries induced by a diverse range of insults, including ischemia, hemorrhage, trauma, epilepsy, and degenerative diseases. During the processes of inflammation, disruption of the blood–brain barrier (BBB) may play a critical role in the enhancement of inflammatory responses and may initiate brain damage because the BBB constitutes an interface between the brain parenchyma and the bloodstream containing blood cells and plasma. The BBB has a distinct structure compared with those in peripheral tissues: it is composed of vascular endothelial cells with tight junctions, numerous pericytes surrounding endothelial cells, astrocytic endfeet, and a basement membrane structure. Under physiological conditions, the BBB should function as an important element in the neurovascular unit (NVU). High mobility group box-1 (HMGB1), a nonhistone nuclear protein, is ubiquitously expressed in almost all kinds of cells. HMGB1 plays important roles in the maintenance of chromatin structure, the regulation of transcription activity, and DNA repair in nuclei. On the other hand, HMGB1 is considered to be a representative damage-associated molecular pattern (DAMP) because it is translocated and released extracellularly from different types of brain cells, including neurons and glia, contributing to the pathophysiology of many diseases in the central nervous system (CNS). The regulation of HMGB1 release or the neutralization of extracellular HMGB1 produces beneficial effects on brain injuries induced by ischemia, hemorrhage, trauma, epilepsy, and Alzheimer’s amyloidpathy in animal models and is associated with improvement of the neurological symptoms. In the present review, we focus on the dynamics of HMGB1 translocation in different disease conditions in the CNS and discuss the functional roles of extracellular HMGB1 in BBB disruption and brain inflammation. There might be common as well as distinct inflammatory processes for each CNS disease. This review will provide novel insights toward an improved understanding of a common pathophysiological process of CNS diseases, namely, BBB disruption mediated by HMGB1. It is proposed that HMGB1 might be an excellent target for the treatment of CNS diseases with BBB disruption.

Multiscale simulations of charge and size separation of nanoparticles with a solid-state nanoporous membrane.

Nanoporous membranes provide an attractive approach for rapid filtering of nanoparticles at high-throughput volume, a goal useful to many fields of science and technology. Creating a device to readily separate different particles would require an extensive knowledge of particle-nanopore interactions and particle translocation dynamics. To this end, we use a multiscale model for the separation of nanoparticles by combining microscopic Brownian dynamics simulations to simulate the motion of spherical nanoparticles of various sizes and charges in a system with nanopores in an electrically biased membrane with a macroscopic filtration model accounting for bulk diffusion of nanoparticles and membrane surface pore density. We find that, in general, the separation of differently sized particles is easier to accomplish than of differently charged particles. The separation by charge can be better performed in systems with low pore density and/or smaller filtration chambers when electric nanopore-particle interactions are significant. The results from these simple cases can be used to gain insight in the more complex dynamics of separating, for example, globular proteins.

Pulling a folded polymer through a nanopore

We investigate the translocation dynamics of a folded linear polymer which is pulled through a nanopore by an external force. To this end, we generalize the iso-flux tension propagation theory for end-pulled polymer translocation to include the case of two segments of the folded polymer traversing simultaneously trough the pore. Our theory is extensively benchmarked with corresponding molecular dynamics (MD) simulations. The translocation process for a folded polymer can be divided into two main stages. In the first stage, both branches are traversing the pore and their dynamics is coupled. If the branches are not of equal length, there is a second stage where translocation of the shorter branch has been completed. Using the assumption of equal monomer flux of both branches confirmed by MD simulations, we analytically derive the equations of motion for both branches and characterize the translocation dynamics in detail from the average waiting time and its scaling form.

Effects of AG1 and AG2 QTLs on Nonstructural Carbohydrate and Seed Management Options for Rice Seedling Growth and Establishment under Flooding Stress

Rice (Oryza sativa) plants acquired excess photosynthates in the form of nonstructural carbohydrates (NSCs) in their stems and grain. Despite keen interest in rice NSC, the dynamics of NSC accumulation, translocation and re-accumulation have not yet been well investigated. AG1 and AG2 QTLs associated with flooding tolerance through catalyzing starch into soluble sugar in germinating seeds. Here we conducted three experiments, greenhouse and field to lay the groundwork for large-scale diversity studies on grain NSC and some agronomic traits under direct-seeded rice (DSR) system, using elite lines incorporating AG1, AG2 and AG1-AG2 QTLs into the popular varieties PSB Rc82 and Ciherang-Sub1 along with the donors Kho Hlan On (AG1) and Ma-Zhan Red (AG2). In germinating seedlings, soluble sugars increased, while starch concentration decreased gradually especially in the tolerant checks and AG1-AG2 introgression lines under flooded soil. Soluble sugar accumulation in stem started to increase from the vegetative stage and peaked at the panicle initiation stage then gradually decreased towards the maturity stage. But Sub1-AG lines had higher sugar and starch concentrations at different growth stages than other genotypes in wet season 2016 and dry season 2017. Plant survival rate was positively correlated with the stem NSC at the early vegetative stage (21 days after sowing), and stem NSC was positively associated with plant height at different growth stages. Among the tested seeding rate, the most suitable seeding rate, 4g/m2 with shallow burial depth (0.5cm), resulted in better seedling establishment, relatively higher seedling vigor index and higher leaf area index under flooding in DSR system. Introgression of AG1-AG2 QTLs had no any negative impact on nonstructural carbohydrate, germination rate, and growth and biomass production.

Optogenetic Control Reveals Differential Promoter Interpretation of Transcription Factor Nuclear Translocation Dynamics.

SUMMARYGene expression is thought to be affected not only by the concentration of transcription factors (TFs) but also the dynamics of their nuclear translocation. Testing this hypothesis requires direct control of TF dynamics. Here, we engineer CLASP, an optogenetic tool for rapid and tunable translocation of a TF of interest. Using CLASP fused to Crz1, we observe that, for the same integrated concentration of nuclear TF over time, changing input dynamics changes target gene expression: pulsatile inputs yield higher expression than continuous inputs, or vice versa, depending on the target gene. Computational modeling reveals that a dose-response saturating at low TF input can yield higher gene expression for pulsatile versus continuous input, and that multi-state promoter activation can yield the opposite behavior. Our integrated tool development and modeling approach characterize promoter responses to Crz1 nuclear translocation dynamics, extracting quantitative features that may help explain the differential expression of target genes.

First Responders Shape a Prompt and Sharp NF-κB-Mediated Transcriptional Response to TNF-α.

SummaryNuclear factor (NF)-κB controls the transcriptional response to inflammatory signals by translocating into the nucleus, but we lack a single-cell characterization of the resulting transcription dynamics. Here we show that upon tumor necrosis factor (TNF)-α transcription of NF-κB target genes is heterogeneous in individual cells but results in an average nascent transcription profile that is prompt (i.e., occurs almost immediately) and sharp (i.e., increases and decreases rapidly) compared with NF-κB nuclear localization. Using an NF-κB-controlled MS2 reporter we show that the single-cell nascent transcription is more heterogeneous than NF-κB translocation dynamics, with a fraction of synchronized “first responders” that shape the average transcriptional profile and are more prone to respond to multiple TNF-α stimulations. A mathematical model combining NF-κB-mediated gene activation and a gene refractory state is able to reproduce these features. Our work shows how the expression of target genes induced by transcriptional activators can be heterogeneous across single cells and yet time resolved on average.

Effectors, chaperones, and harpins of the Type III secretion system in the fire blight pathogen Erwinia amylovora: a review

Erwinia amylovora is a plant pathogenic bacterium that causes the disease fire blight in a wide range of Rosaceous plants including apples and pears. This pathogen utilizes the hypersensitive response and pathogenicity (hrp)-type III secretion system (T3SS), an essential pathogenicity factor of E. amylovora, to deliver proteins from bacteria to plant apoplasts or the cytoplasm to regulate host immune responses and physiology. Proteins secreted by the E. amylovora T3SS include five effectors: DspA/E, Eop1, Eop3, Eop4 (AvrRpt2Ea), and HopPtoCEa, two harpins: HrpN and HrpW, and other proteins such as Eop2, HrpJ, and HrpK. Homologs of these T3SS-secreted proteins have been characterized in plant pathogens like Pseudomonas syringae, Xanthomonas spp., Ralstonia spp., plant symbionts like Rhizobium spp. and animal pathogens such as Yersinia spp., implicating a central role of these effector proteins in manipulating bacterial interactions with diverse host species. Translocation of effectors is crucial for T3SS-mediated disease development in hosts. It is thought to be mediated by cognate chaperones, but the translocation of E. amylovora DspA/E was recently shown to be regulated by several different chaperones as well as a previously reported harpin, HrpN, in addition to its cognate chaperone DspB/F. This suggests that the dynamics of effector translocation are more complicated than previously expected in E. amylovora. In this review, we summarize the current knowledge of E. amylovora T3SS effectors, chaperones, and harpins together with their homologs from other plant-associated bacterial species. We also discuss the roles of T3SS-secreted proteins in pathogenicity and fitness via exploring their localization, activity, and direct or indirect targets in host plants. Finally, we provide future perspectives on studying effector biology and the T3SS in E. amylovora and how this fundamental knowledge provides potential applications in preventing fire blight.

Ciliary proteins specify the cell inflammatory response by tuning NFκB signalling, independently of primary cilia.

ABSTRACTComplex inflammatory signalling cascades define the response to tissue injury but also control development and homeostasis, limiting the potential for these pathways to be targeted therapeutically. Primary cilia are subcellular regulators of cellular signalling, controlling how signalling is organized, encoded and, in some instances, driving or influencing pathogenesis. Our previous research revealed that disruption of ciliary intraflagellar transport (IFT), altered the cell response to IL-1β, supporting a putative link emerging between cilia and inflammation. Here, we show that IFT88 depletion affects specific cytokine-regulated behaviours, changing cytosolic NFκB translocation dynamics but leaving MAPK signalling unaffected. RNA-seq analysis indicates that IFT88 regulates one third of the genome-wide targets, including the pro-inflammatory genes Nos2, Il6 and Tnf. Through microscopy, we find altered NFκB dynamics are independent of assembly of a ciliary axoneme. Indeed, depletion of IFT88 inhibits inflammatory responses in the non-ciliated macrophage. We propose that ciliary proteins, including IFT88, KIF3A, TTBK2 and NPHP4, act outside of the ciliary axoneme to tune cytoplasmic NFκB signalling and specify the downstream cell response. This is thus a non-canonical function for ciliary proteins in shaping cellular inflammation.This article has an associated First Person interview with the first author of the paper.

Pulling a DNA molecule through a nanopore embedded in an anionic membrane: tension propagation coupled to electrostatics

We consider the influence of electrostatic forces on driven translocation dynamics of a flexible polyelectrolyte being pulled through a nanopore by an external force on the head monomer. To this end, we augment the iso-flux tension propagation theory with electrostatics for a negatively charged biopolymer pulled through a nanopore embedded in a similarly charged anionic membrane. We show that in the realistic case of a single-stranded DNA molecule, dilute salt conditions characterized by weak charge screening, and a negatively charged membrane, the translocation dynamics is unexpectedly accelerated despite the presence of large repulsive electrostatic interactions between the polymer coil on the cis side and the charged membrane. This is due to the rapid release of the electrostatic potential energy of the coil during translocation, leading to an effectively attractive force that assists end-driven translocation. The speedup results in non-monotonic polymer length and membrane charge dependence of the exponent α characterizing the translocation time of the polymer with length N0. In the regime of long polymers N0 ≳ 500, the translocation exponent exceeds its upper limit α = 2 previously observed for the same system without electrostatic interactions.

Organic carbon storage potential in deep agricultural soil layers: Evidence from long-term experiments in northeast Italy

The significant contribution to the global carbon budget made by soil organic carbon (SOC) inspired the “4 per 1000” initiative. It promotes agricultural practices aimed at raising SOC stocks 0.4 % annually over 20 years. However, the response of topsoil and deep soil profiles to agroecosystem changes is largely unknown at present. Our work aims to quantify deep SOC accumulations in stabilized croplands and grasslands of northeast Italy and identify the best management practices to increase those stores. Soil profiles were collected to 70 and 90 cm depths from three well-established long-term experiments. A total of 1242 soil samples were analyzed for SOC concentrations as a function of soil type, soil management practice, and cropping system. SOC stocks were quantified using the equivalent soil mass method. Results show that SOC stocks averaged 23.2 Mg ha−1 in sandy Arenosol, 59.3 Mg ha−1 in silty loam Cambisol, 111.6 Mg ha−1 in clay loam Gleysol, and 383.5 Mg ha−1 in peaty Histosol. Substantial SOC stocks were found in the subsoil beneath the tilled layer, ranging between 59 % and 74 % in sandy and clay loam soils. Among the considered managements, the SOC accumulation rate of permanent meadow topsoil was higher than croplands (0.299 Mg ha−1 yr−1), which fell to 0.256 Mg ha−1 yr−1 when estimated along the full soil profile. In contrast, organic carbon added through manures and residues, coupled with minimum tillage practices, led to increased average SOC stock rates in the topsoil (0.205 Mg ha−1 yr−1) and throughout the full soil profile (0.386 Mg ha−1 yr−1), suggesting that some translocation dynamics occurred. The long-term adoption of permanent meadow, along with manure or residue addition under minimum tillage, made it possible to achieve “4 per 1000” goals to great depths in naturally poor-SOC sandy and silty loam soils. In SOC-rich soil, only in the topsoil layer achieved this.

Investigating the Effect of Charged Components on Translocation of DNA Molecules in Track-Etched Nanopores

To understand and improve the translocation dynamics of nanopore based sensors, we studied theoretically the translocation of DNA molecules and compared them with experimental results. In our experimental studies, we used conically shaped track-etched polymer (i.e., PET) membrane and built our model based on its physical properties. Simulations were conducted by varying the surface charges of the nanopore and the particle, particle (i.e., DNA) position and applied potential to examine the translocational behavior of DNA and its effect on current-pulse shape. Our results showed that variations in surface and DNA charge density cause conductive distortions indicating the interaction between the surface and the DNA. It also confirmed that the DNA charge density does not remain in its native form in the solution as well as the pore charge during the translocation.

Translocation of links through a pore: effects of link complexity and size

We have used Langevin dynamics to simulate the forced translocation of linked polymer rings through a narrow pore. For fixed size (i.e. fixed number of monomers) the translocation time depends on the link type and on whether the rings are knotted or unknotted. For links with two unknotted rings the crossings between the rings can slow down the translocation and are responsible for a delay as the crossings pass through the pore. The results fall on a set of relatively smooth curves for different link families with the translocation time not always increasing with crossing number within the same family. When one ring is knotted the results depend on whether the link is prime or composite and, for the composite case, they depend on whether the knotted or unknotted ring enters the pore first. We find a similar situation for 3-component links where the results depend on whether the link is prime or composite. These results contribute to our understanding of how the entanglement complexity between filaments impacts their translocation dynamics and should be useful for extending nanopore-sensing techniques to probe the topological properties of these systems.

Dynamics of Co-translational Membrane Protein Integration and Translocation via the Sec Translocon.

An important aspect of cellular function is the correct targeting and delivery of newly synthesized proteins. Central to this task is the machinery of the Sec translocon, a transmembrane channel that is involved in both the translocation of nascent proteins across cell membranes and the integration of proteins into the membrane. Considerable experimental and computational effort has focused on the Sec translocon and its role in nascent protein biosynthesis, including the correct folding and expression of integral membrane proteins. However, the use of molecular simulation methods to explore Sec-facilitated protein biosynthesis is hindered by the large system sizes and long (i.e., minute) time scales involved. In this work, we describe the development and application of a coarse-grained simulation approach that addresses these challenges and allows for direct comparison with both in vivo and in vitro experiments. The method reproduces a wide range of experimental observations, providing new insights into the underlying molecular mechanisms, predictions for new experiments, and a strategy for the rational enhancement of membrane protein expression levels.

Translocation of Heterogeneous Flexible Polymers Assisted by Binding Particles

A polymer chain usually contains two or more types of monomeric species from the perspective of polymer chemistry, which poses a challenge to the understanding of structure-property relationships. It is of course true in the field of polymer translocation. In the present work, I investigate the translocation dynamics of heterogeneous flexible polymers composed of two types of monomers labeled A and B through a nanopore assisted by binding particles (BPs) by using the coarse-grained Langevin dynamics simulations in two-dimensional domains. Specifically, multiblock copolymers with different block lengths and monomeric components are considered. I critically examine how the translocation dynamics responds to the variations in the block length and the monomeric content. Interestingly, it is found that the periodic structure of a multiblock copolymer causes an obvious fingerprint feature in the residence time of individual monomers in which the number of peaks is exactly equal to the number of blocks. These findings provide a basic understanding about the sequence-dynamics relationship for the BPs-assisted translocation of heterogeneous flexible polymers.