Pattern formation and polarity sorting of driven actin filaments on lipid membranes

Collective motion of active matter is ubiquitously observed, ranging from propelled colloids to flocks of bird, and often features the formation of complex structures composed of agents moving coherently. However, it remains extremely challenging to predict emergent patterns from the binary interaction between agents, especially as only a limited number of interaction regimes have been experimentally observed so far. Here, we introduce an actin gliding assay coupled to a supported lipid bilayer, whose fluidity forces the interaction between self-propelled filaments to be dominated by steric repulsion. This results in filaments stopping upon binary collisions and eventually aligning nematically. Such a binary interaction rule results at high densities in the emergence of dynamic collectively moving structures including clusters, vortices, and streams of filaments. Despite the microscopic interaction having a nematic symmetry, the emergent structures are found to be polar, with filaments collectively moving in the same direction. This is due to polar biases introduced by the stopping upon collision, both on the individual filaments scale as well as on the scale of collective structures. In this context, positive half-charged topological defects turn out to be a most efficient trapping and polarity sorting conformation.

Collective motion of active matter is ubiquitous, with observations ranging from flocks of birds (1) and schools of fish (2) to propelled colloids (3). The interactions between agents in such systems lead to the formation of complex structures including clusters, swirls, or lanes of agents moving coherently (4). The structure of the emerging patterns strongly depends on both the agents’ shape and their velocity alignment mechanism. A particular case is that of elongated microscopic particles that translate along their major axis in a quasi-two-dimensional environment and only interact upon collision (5, 6). In the context of cytoskeletal systems, gliding actin filaments or microtubules propelled by molecular motors are found to be able to readily crawl over each other and only retain a weak level of alignment upon binary collisions, which eventually leads at high densities to a diverse set of patterns (7). Such resulting patterns are found to be strongly dependent on this weak microscopic alignment interaction, and therefore, even slightly tuning it causes the system to switch between polar and nematic phases, separated by a coexistence regime (8, 9). Observed structures in cytoskeletal systems with weak to moderate interactions include nematic lanes, polar waves, and vortices (10–12). Conversely, pattern formation in systems of elongated bacteria or granular matter is often based on hard interactions with a strong steric component (13–18). In this repulsion-dominated regime, particles are unable to crawl over each other and must stop upon collision. In the limiting case of spherical self-propelled particles, this kind of steric interaction can lead to a stable phase separation between stuck and moving particles, the so-called motility induced phase separation (MIPS) (19). On the other hand, in the case of elongated particles, steric effects can still act as velocity aligning mechanisms. As orientation mismatches are unstable, particles end up aligning and this leads to flocking behavior rather than to phase separation (5, 20–25). This latter case, in which strong steric constraints dominate binary interactions but alignment is still present, is poorly understood, and how modeling has to be extended to account for the emergent collective behavior of elongated, flexible agents with volume exclusion also remains still under debate (26–30). This is partly due to the lack of microscopic experimental systems allowing to explore this regime. Semiflexible cytoskeletal filaments would be the best candidate, but their volume exclusion is usually too weak. However, having them propelled by motors immobilized on a fluid membrane would be a promising route to bridge this experimental gap (31).Here, we enforce a steric repulsion-dominated interaction, leading to alignment between actin filaments by coupling myosin motors to a fluid-supported lipid bilayer. Because of the slippage of the motors on the membrane, the force propelling the filaments is too weak to enable filaments to crawl over each other and thus effectively implements a repulsion-dominated regime, with filaments stopping upon collisions. Eventually, however, because of the thermal fluctuations of their tips, filaments can align and resume motion. The experimental realization of such a microscopic binary interaction, based on volume exclusion, enables us to observe and quantify the resulting pattern formation process in a system of active semiflexible filaments. We then first characterize the interaction at the single filament scale, showing that it leads to nematic alignment. As the filaments’ density is increased, patterns of collective motion emerge, ranging from clusters to thick streams and vortices. Despite the nematic collision rule, we find the emerging structures to be locally polar. The repulsion-dominated interaction indeed introduces a polar bias not only due to the tendency of active filaments or clusters to keep moving together after a polar collision but also by forcing filaments with similar orientation to stop and accumulate when encountering an obstacle. In particular, at high densities, such an interaction leads to the formation of transient local +1/2 topological defects, which act as wedges and, therefore, effectively trap and polarity-sort motile filaments. We interpret this trapping mechanism as an analog of MIPS for elongated self-propelled particles.     

Glass transition and rheological redundancy in F-actin solutions

The unique mechanical performance of animal cells and tissues is attributed mostly to their internal biopolymer meshworks. Its perplexing universality and robustness against structural modifications by drugs and mutations is an enigma in cell biology and provides formidable challenges to materials science. Recent investigations could pinpoint highly universal patterns in the soft glassy rheology and nonlinear elasticity of cells and reconstituted networks. Here, we report observations of a glass transition in semidilute F-actin solutions, which could hold the key to a unified explanation of these phenomena. Combining suitable rheological protocols with high-precision dynamic light scattering, we can establish a remarkable rheological redundancy and trace it back to a highly universal exponential stretching of the single-polymer relaxation spectrum of a "glassy wormlike chain." By exploiting the ensuing generalized time-temperature superposition principle, the time domain accessible to microrheometry can be extended by several orders of magnitude, thus opening promising new metrological opportunities.     

Blood chemistry measurements and D-Dimer levels associated with fatal and nonfatal outcomes in humans infected with Sudan Ebola virus

Blood samples from patients infected with the Sudan species of Ebola virus (EBOV), obtained during an outbreak of disease in Uganda in 2000, were tested for a panel of analytes to evaluate their clinical condition and to compare values obtained for patients with fatal and nonfatal cases and for uninfected (hospitalized control) patients. Liver function tests showed higher levels of aspartate aminotransferase (AST) in blood samples from patients with fatal cases than in samples from patients with nonfatal cases, whereas alanine aminotransferase levels were comparable and only slightly increased in all patients, suggesting that increased blood AST levels are due to a greater degree of injury in tissues other than the liver. Significantly higher levels of amylase, urea nitrogen, and creatinine suggest that acute pancreatitis and renal dysfunction develop in fatal cases, whereas reduced albumin and calcium levels may be linked to these conditions or to liver damage. d-Dimer levels in blood specimens were drastically increased in patients with fatal and nonfatal infections but were 4 times higher in patients with fatal cases than in patients who survived (180,000 vs. 44,000 ng/mL), during the most acute period of the infection (6-8 days after onset). These results indicate that disseminated intravascular coagulation is an early and important component of EBOV disease. This study has identified levels of analytes with prognostic value, which can also be used to target therapeutic interventions, and expands on the findings of prior blood tests conducted on this group of patients.     

Outbreaks of filovirus hemorrhagic fever: time to refocus on the patient

In the 40 years since the recognition of filoviruses as agents of lethal human disease, there have been no specific advances in antiviral therapies or vaccines and few clinical studies on the efficacy of supportive care. On 20 September 2006, experts from 14 countries representing 68 institutions integrally involved in the response to outbreaks of filovirus hemorrhagic fever gathered at the National Microbiology Laboratory of the Public Health Agency of Canada in Winnipeg to discuss possible remedies for this grim situation, in a unique workshop entitled "Marburg and Ebola Hemorrhagic Fever: Feasibility of Prophylaxis and Therapy." A summary of the opportunities for and challenges to improving treatment of filovirus hemorrhagic fevers is presented here.     

Safety studies conducted on high-purity trans-resveratrol in experimental animals

trans-Resveratrol is a naturally occurring polyphenolic compound found in a variety of foods, but predominantly in grapes. Safety studies were conducted on high-purity trans-resveratrol (Resvida™), including skin and eye irritation, dermal sensitization, subchronic and reproductive toxicity, genotoxicity, and absorption, metabolism and excretion. Resvida™ was non-irritating to skin and eyes and non-sensitizing. It was non-mutagenic in a bacterial reverse mutation assay in Salmonella typhimurium and Escherichia coli, but exhibited clastogenic activity in a chromosomal aberration test in human lymphocytes. However, in an in vivo bone marrow micronucleus test in rats, Resvida™ was non-genotoxic. In a 28-day study, Resvida™ caused no adverse effects in rats at 50, 150 and 500 mg/kg bw/day. Similarly, in a 90-day study, Resvida™ did not cause any adverse effects in rats at up to 700 mg/kg bw/day; the highest dose tested. Resvida™ did not induce any adverse reproductive effects in an embryo–fetal toxicity study in rats at a dose of 750 mg/kg bw/day. Also, in vitro and in vivo absorption, metabolism, and excretion studies in Caco-2 cells, rat primary hepatocytes and male and female rats (in vivo) show that Resvida™ is readily absorbed, metabolized and excreted. These studies provide evidence that Resvida™ is well tolerated and non-toxic.     

Structural and biologic characterization of pegylated recombinant IFN-alpha2b.

The type I interferon-alpha (IFN-alpha) family is a family of natural small proteins that have clinically important anti-infective and antitumor activity. We have developed a semisynthetic protein-polymer conjugate of IFN-alpha2b (Intron A) by attaching a 12,000-Da monomethoxypolyethylene glycol (PEG-12000) polymer to the protein. PEG conjugation is thought to increase the serum half-life and thereby prolong patient exposure to IFN-alpha2b without altering the biologic potency to the protein. Matrix-assisted laser desorption ionization/mass spectrometry (MALDI-MS), high-performance size exclusion chromatography (HPSEC), circular dichroism (CD) analysis and tryptic digestion peptide analysis of PEG Intron demonstrated that the IFN-alpha2b protein was approximately 95% monopegylated and that the primary, the secondary, and the tertiary structures were unaltered. Pegylation did not affect the epitope recognition of antibodies used for Intron A quantitation. An extensive analysis of the pegylated positional isomers revealed that approximately 50% of PEG Intron was monopegylated on the His(34) residue of the IFN-alpha2b protein. The highest antiviral activity of the pegylated positional isomers for PEG Intron was associated with the His(34) pegylated isomer. The specific activity for PEG Intron in an antiviral cytopathic protection assay was 28%, relative to Intron A. However, the potency of PEG Intron, defined as bioactivity independent of protein concentration, was comparable to Intron A at both the molecular and cellular levels in a battery of in vitro assays. Equivalent units of PEG Intron and Intron A were indistinguishable for the induction of several key IFN-induced genes, including 2',5'-oligoadenylate synthetase (2',5'-OAS) and protein kinase R (PKR), in Molt 4 cells. The antiviral dose-response curves revealed that there were no significant differences between PEG Intron and Intron A. This demonstrated that the introduction of more IFN-alpha2b protein associated with equivalent unit dosing of PEG Intron did not create any antagonism or agonism in the antiviral assay. In assays for the immune response, PEG Intron and Intron A displayed comparable potency for both natural-killer (NK) and lymphokine-activated killer (LAK) cell cytolytic activity and for the induction of class I major histocompatibility protein. These results demonstrate that PEG Intron maintains an in vitro biologic potency profile for both antiviral and immunotherapeutic activity that is highly comparable to that of Intron A.     

Cancer incidence in a trial of an antiapoptotic agent for Parkinson's disease

We performed a placebo‐controlled trial of CEP‐1347, an inhibitor of neuronal apoptotic cell death, in patients with early Parkinson's disease (PD) to determine whether long‐term therapy would slow disability progression. This also provided an opportunity to monitor cancer incidence in a large cohort of PD patients followed prospectively including periods before and after patients developed disability requiring dopaminergic therapy. This was a multicenter study of 806 patients with early PD, without disability requiring dopaminergic therapy, assigned randomly to placebo or one of three doses of CEP‐1347. Patients were monitored for an average of 1.8 years (1,467 patient‐years) with routine cancer screening evaluations and annual skin examinations by a dermatologist. There was no significant excess of cancers among patients taking CEP‐1347 compared with placebo for any cancer type (all P > 0.1). Nonmelanoma skin cancers were the most common cancer type observed. The incidence of melanomas was 20.9 times that predicted in the general population. Most melanomas occurred in patients who had never taken dopaminergic therapy. We found no evidence that CEP‐1347 affected cancer incidence within 2 years of follow‐up. Melanoma occurrence in our PD patients was greater than predicted compared with the general population and was unrelated to dopaminergic therapy. Clinical surveillance of PD patients for melanoma may be warranted. © 2010 Movement Disorder Society