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We present an optical method that combines confocal microscopy with position modulation to perform axial tracking and topographic imaging of fluorescent surfaces. Using a remote focusing system, the confocal observation volume is oscillated in the axial direction. The resulting modulation of the detected signal is used as a feedback to precisely control the distance to an object of interest. The accuracy of this method is theoretically analyzed and the axial-locking accuracy is experimentally evaluated. Topographic imaging is demonstrated on fluorescently coated beads and fixed cells. This microscope allows for nanometric topography or tracking of dynamic fluorescent surfaces.

Fish growth can be modulated through genetic selection. However, it is not known whether growth regulatory mechanisms modulated by genetic selection can provide information about phenotypic growth variations among families or populations. Following a five-generation breeding program that selected for the absence of early sexual maturity and increased growth in brook charr we aimed to understand how the genetic selection process modifies the growth regulatory pathway of brook charr at the molecular level. To achieve this, we studied the regulation of growth traits at three different levels: 1) between lines-one under selection, the other not, 2) among-families expressing differences in average growth phenotypes, which we termed family performance, and 3) among individuals within families that expressed extreme growth phenotypes, which we termed slow- and fast-growing. At age 1+, individuals from four of the highest performing and four of the lowest performing families in terms of growth were sampled in both the control and selected lines. The gene expression levels of three reference and ten target genes were analyzed by real-time PCR. Results showed that better growth performance (in terms of weight and length at age) in the selected line was associated with an upregulation in the expression of genes involved in the growth hormone (GH)/insulin growth factor-1 (IGF-1) axis, including the igf-1 receptor in pituitary; the gh-1 receptor and igf-1 in liver; and ghr and igf-1r in white muscle. When looking at gene expression within families, family performance and individual phenotypes were associated with upregulations of the leptin receptor and neuropeptid Y-genes related to appetite regulation-in the slower-growing phenotypes. However, other genes related to appetite (ghrelin, somatostatin) or involved in muscle growth (myosin heavy chain, myogenin) were not differentially expressed. This study highlights how transcriptomics may improve our understanding of the roles of different key endocrine steps that regulate physiological performance. Large variations in growth still exist in the selected line, indicating that the full genetic selection potential has not been reached.

Oral tongue squamous cell carcinoma is one of the most prevalent head and neck cancers. During tumor progression, elastin fragments are released in the tumor microenvironment. Among them, we previously identified a nonapeptide, AG-9, that stimulates melanoma progression in vivo in a mouse melanoma model. In the present paper, we studied AG-9 effect on tongue squamous cell carcinoma invasive properties. We demonstrated that AG-9 stimulates cell invasion in vitro in a modified Boyen chamber model. It increases MMP-2 secretion, analyzed by zymography and MT1-MMP expression, studied by Western blot. The stimulatory effect was mediated through Ribosomal Protein SA (RPSA) receptor binding as demonstrated by SiRNA experiments. The green tea-derived polyphenol, (&minus, )-epigallocatechin-3-gallate (EGCG), was previously shown to bind RPSA. Molecular docking experiments were performed to compare the preferred areas of interaction of AG-9 and EGCG with RPSA and suggested overlapping areas. This was confirmed by competition assays. EGCG abolished AG-9-induced invasion, MMP-2 secretion, and MT1-MMP expression.

The nucleoprotein filament (NPF) is the fundamental element of homologous recombination (HR), a major mechanism for the repair of double-strand DNA breaks in the cell. The NPF is made of the damaged DNA strand surrounded by recombinase proteins, and its sensitivity to base-pairing mismatches is a crucial feature that guarantees the fidelity of the repair. The concurrent recombinases are also essential for several steps of HR. In this work, we used torque-sensitive magnetic tweezers to probe and apply mechanical constraints to single nucleoprotein filaments (NPFs). We demonstrated that the NPF undergoes structural transitions from a stretched to a compact state, and we measured the corresponding mechanochemical signatures. Using an active two-state model, we proposed a free-energy landscape for the NPF transition. Using this quantitative model, we explained both how the sensitivity of the NPF to the homology length is regulated by its structural transition and how the cooperativity of Rad51 favors selectivity to relatively long homologous sequences.

We study the orientational order of an immobile fish school. Starting from the second Newton's law, we show that the inertial dynamics of orientations is ruled by an Ornstein-Uhlenbeck process. This process describes the dynamics of alignment between neighboring fish in a shoal, a dynamics already used in the literature for mobile fish schools. Firstly, in a fluid at rest, we calculate the global polarization (i.e. the mean orientation of the fish) which decreases rapidly as a function of the noise. We show that the faster a fish is able to reorient itself, the more the school can afford to reorder itself for important noise values. Secondly, in the prescence of a stream, each fish tends to orient itself and swims against the flow: the so-called rheotaxis. So even in the presence of a flow, it results in an immobile fish school. By adding an individual rheotaxis effect to alignment interaction between fish, we show that in a noisy environment, individual rheotaxis is enhanced by alignment interactions between fish., Comment: 11 pages, 9 figures

Foerster Resonance Energy Transfer (FRET) allows for the visualization of nanometer-scale distances and distance changes. This sensitivity is regularly achieved in single-molecule experiments in vitro but is still challenging in biological materials. Despite many efforts, quantitative FRET in living samples is either restricted to specific instruments or limited by the complexity of the required analysis. With the recent development and expanding utilization of FRET-based biosensors, it becomes essential to allow biologists to produce quantitative results that can directly be compared. Here, we present a new calibration and analysis method allowing for quantitative FRET imaging in living cells with a simple fluorescence microscope. Aside from the spectral crosstalk corrections, two additional correction factors were defined from photophysical equations, describing the relative differences in excitation and detection efficiencies. The calibration is achieved in a single step, which renders the Quantitative Three-Image FRET (QuanTI-FRET) method extremely robust. The only requirement is a sample of known stoichiometry donor:acceptor, which is naturally the case for intramolecular FRET constructs. We show that QuanTI-FRET gives absolute FRET values, independent of the instrument or the expression level. Through the calculation of the stoichiometry, we assess the quality of the data thus making QuanTI-FRET usable confidently by non-specialists., 13 pages, 3 figures

In the Estuary and Gulf of St. Lawrence, northern shrimp (Pandalus borealis) and Greenland halibut (Reinhardtius hippoglossoides) are usually found at depths > 150 m and thus frequently inhabit hypoxic areas (18–50% saturation). The impact of a one-week exposure to different levels of dissolved oxygen (100, 40, 30, and 20% saturation) at 5 °C was evaluated in adult shrimp and juvenile Greenland halibut; the effect of acute exposure to severe hypoxia was also assessed in Greenland halibut. The activities of key enzymes involved in aerobic (citrate synthase [CS], cytochrome c oxidase [COX]) and anaerobic (pyruvate kinase [PK], phosphoenolpyruvate carboxykinase [PEPCK], lactate dehydrogenase [LDH]) pathways, and of enzymes involved in antioxidant defence (superoxide dismutase, glutathione peroxidase [GPx], and catalase [CAT]) were measured. Quantitative real-time PCR analysis was also performed in Greenland halibut. In northern shrimp exposed to chronic hypoxia, muscle CS activity decreased by ~ 40%. Muscle LDH activity was significantly reduced, with a more intense reduction in males. At the same time, hepatopancreas GPx activity increased under hypoxia, and this response was stronger in males. Overall, the results suggest the presence of a threshold above 40% saturation and higher hypoxia tolerance in males. In juvenile Greenland halibut, exposure to chronic hypoxia elicited a more wide-ranging enzymatic response than did acute exposure to severe hypoxia. Under chronic hypoxia, CS activity decreased and PK and LDH activity were respectively 46% and 57% lower than in normoxia. There were no major changes in the activity of antioxidant enzymes, but activity in normoxia was high compared to other fish species. Interestingly, the relative expression of genes coding for muscle COX (severe hypoxia), liver PEPCK (chronic), and CAT (chronic) activities were triggered in hypoxia. The absence of a corresponding change in enzyme activity makes the interpretation of these results difficult, but clearly there was a response at the transcription level. Overall, the results indicate that these two species are particularly well adapted to withstand severe hypoxia. -- Keywords : Aerobic pathwa ; Anaerobic pathway ; Antioxidant defence ; Metabolic capacity ; Gene expression ; Enzyme activity.

Collagen IV is a major component of the dermo-epidermal junction (DEJ). To study expression of collagen IV upon aging in the DEJ and dermal fibroblasts isolated from the same patients. A model of senescent fibroblasts was developed in order to identify biological compounds that might restore the level of collagen IV. Skin fragments of women (30 to 70 years old) were collected. Localisation of collagen IV expression in the DEJ was studied by immunofluorescence. Fibroblast collagen IV expression was studied by real-time PCR, ELISA, and western blotting. Premature senescence was simulated by exposing fibroblasts to subcytotoxic H2O2 concentrations. Collagen IV decreased in the DEJ and fibroblasts relative to age. TGF-β1 treatment significantly increased collagen IV gene and protein expression in fibroblasts and restored expression in the model of senescence. Addition of TGF-β1-neutralizing antibody to fibroblast cultures decreased collagen IV expression. Taken together, the results suggest that the decrease in collagen IV in the DEJ, relative to age, could be due to a decrease in collagen IV expression by senescent dermal fibroblasts and may involve TGF-β1 signalling.

Type XIX collagen is a minor collagen associated with basement membranes. It was isolated for the first time in a human cDNA library from rhabdomyosarcoma and belongs to the FACITs family (Fibril Associated Collagens with Interrupted Triple Helices). Previously, we demonstrated that the NC1 domain of collagen XIX (NC1(XIX)) exerts anti-tumor properties on melanoma cells by inhibiting their migration and invasion. In the present work, we identified for the first time the integrin αvβ3 as a receptor of NC1(XIX). Moreover, we demonstrated that NC1(XIX) inhibits the FAK/PI3K/Akt/mTOR pathway, by decreasing the phosphorylation and activity of the major proteins involved in this pathway. On the other hand, NC1(XIX) induced an increase of GSK3β activity by decreasing its degree of phosphorylation. Treatments targeting this central signaling pathway in the development of melanoma are promising and new molecules should be developed. NC1(XIX) seems to have the potential for the design of new anti-cancer drugs.

Methylmercury (MeHg) is highly immunotoxic and can alter the health status of the harbour seal, Phoca vitulina, from the North Sea. To investigate the mechanism of MeHg-induced toxicity in harbour seal lymphocytes, Concanavalin A (ConA)-stimulated peripheral blood leucocytes were exposed in vitro to sublethal concentrations of MeHgCl (0.2, 1, and 2 µM) for 72 h and then analysed for their viability and ultrastructure. After 72 h of incubation, cells were counted with a propidium iodide staining technique, a metabolic MTS assay was performed, and cells exposed to 1 µM of MeHgCl were observed by transmission electron microscopy (TEM). Alive cell numbers decreased with increased MeHgCl concentrations. In presence of ConA and 1 µM of MeHgCl, TEM images revealed a higher frequency of apoptotic cells. Exposed cells displayed condensation of the chromatin at the nuclear membrane and mitochondrial damages. The results suggest that in vitro MeHgCl-induced apoptosis in harbour seal lymphocytes through a mitochondrial pathway.

Cells are able to sense and react to their physical environment by translating a mechanical cue into an intracellular biochemical signal that triggers biological and mechanical responses. This process, called mechanotransduction, controls essential cellular functions such as proliferation and migration. The cellular response to an external mechanical stimulation has been investigated with various static and dynamic systems, so far limited to global deformations or to local stimulation through discrete substrates. To apply local and dynamic mechanical constraints at the single cell scale through a continuous surface, we have developed and modelled magneto-active substrates made of magnetic micro-pillars embedded in an elastomer. Constrained and unconstrained substrates are analysed to map surface stress resulting from the magnetic actuation of the micro-pillars and the adherent cells. These substrates have a rigidity in the range of cell matrices, and the magnetic micro-pillars generate local forces in the range of cellular forces, both in traction and compression. As an application, we followed the protrusive activity of cells subjected to dynamic stimulations. Our magneto-active substrates thus represent a new tool to study mechanotransduction in single cells, and complement existing techniques by exerting a local and dynamic stimulation, traction and compression, through a continuous soft substrate.

How a nano-searcher finds its nano-target is a general problem in non-equilibrium statistical physics. It becomes vital when the searcher is a damaged DNA fragment trying to find its counterpart on the intact homologous chromosome. If the two copies are paired, that intact homologous sequence serves as a template to reconstitute the damaged DNA sequence, enabling the cell to survive without genetic mutations. To succeed, the search must stop only when the perfect homology is found. The biological process that ensures such a genomic integrity is called Homologous Recombination and is promoted by the Recombinase proteins. In this article, we use torque-sensitive magnetic tweezers to measure the free-energy landscape of the human Recombinase hRad51 protein assembled a DNA fragment. Based on our measurements we model the hRad51/DNA complex as an out-of-equilibrium two-state system and provide a thermodynamical description of Homologous Recombination. With this dynamical two-state model, we suggest a mechanism by which the recombinase proteins discriminate between homologous and a nonhomologous sequences.

Northern shrimp is an important commercial species in the Estuary and Gulf of St. Lawrence. It is usually found at depths > 150 m and thus frequently inhabits hypoxic areas (18–40% saturation) in this region. To evaluate the impact of hypoxia on adult shrimp, males and females were exposed to different levels of dissolved oxygen at two temperatures (5 and 8 °C). Standard and maximal metabolic rates as well as the critical oxygen threshold were measured. In addition, metabolic and antioxidant enzyme activities were measured at 5 °C. Females had a higher critical oxygen threshold than males at both temperatures (15.5 and 22.2 vs. 9.0 and 13.8 at 5 and 8 °C respectively), indicating that they were less tolerant of hypoxia. A decrease in glycolytic and fermentation enzyme activities confirmed this result: in females, severe hypoxia significantly decreased the specific activities of citrate synthase and of enzymes involved in anaerobic biochemical pathways (lactate dehydrogenase, pyruvate kinase, and phosphoenolpyruvate carboxykinase (PEPCK)); in males, only the PEPCK activity decreased significantly while glutathione peroxidase (antioxidant defense) activity increased significantly. In females, severe hypoxia (22% sat.) did not affect the standard metabolic rate but significantly reduced (by ~ 43%) the maximum metabolic rate compared to normoxia. Consequently, aerobic scope was reduced by ~ 58% at 22% sat. compared to normoxia. This suggests that the shrimp's flexibility to respond to metabolic demands, including such activities as vertical migration, foraging, and egg production, could be reduced in hypoxic conditions, especially in females.

The interactions and coordination of biomolecules are crucial for most cellular functions. The observation of protein interactions in live cells may provide a better understanding of the underlying mechanisms. After fluorescent labeling of the interacting partners and live-cell microscopy, the colocalization is generally analyzed by quantitative global methods. Recent studies have addressed questions regarding the individual colocalization of moving biomolecules, usually by using single-particle tracking (SPT) and comparing the fluorescent intensities in both color channels. Here, we introduce a new method that combines SPT and correlation methods to obtain a dynamical 3D colocalization analysis along single trajectories of dual-colored particles. After 3D tracking, the colocalization is computed at each particle’s position via the local 3D image cross correlation of the two detection channels. For every particle analyzed, the output consists of the 3D trajectory, the time-resolved 3D colocalization information, and the fluorescence intensity in both channels. In addition, the cross-correlation analysis shows the 3D relative movement of the two fluorescent labels with an accuracy of 30 nm. We apply this method to the tracking of viral fusion events in live cells and demonstrate its capacity to obtain the time-resolved colocalization status of single particles in dense and noisy environments.

Type XIX collagen is a minor collagen associated with basement membranes in vascular, neuronal, mesenchymal, and epithelial tissues. We demonstrated that the NC1, C-terminal, domain of collagen XIX inhibits the migration capacities of tumor cells and exerts a strong inhibition of tumor growth. Other basement membrane collagens or derived fragments were measured in biological fluids such as blood and urine of patients and appeared to be useful for diagnosis, prognosis, or treatment monitoring. The aim of this study was to develop and validate methods to measure collagen XIX and its fragments in human cell cultures, tissue extracts, and human biological fluids. For that purpose, we developed real-time PCR, Western blot, and competitive enzyme-linked immunosorbent assays. We demonstrated that the methods developed in this paper are specific for collagen XIX. We showed that it is expressed in human cell cultures, tissue extracts, and various biological fluids. These methods may be used in various human tissue extracts and biological fluids such as serum, amniotic fluid, cord blood, and many other fluids. Collagen XIX or its fragments could constitute new biomarkers for human diseases as well as for diagnosis and/or prognosis.

Rationale Neutrophils play an important role in the inflammatory process associated with chronic obstructive pulmonary disease (COPD). Lung-infiltrating neutrophils secrete elastinolytic proteases that participate in elastin breakdown and the formation of elastin peptides (EPs). Objectives We hypothesized that circulating neutrophil-associated immune response may be modulated by EPs during COPD. Methods Neutrophils obtained from patients with either stable or exacerbated COPD and controls were cultured with or without EPs. Cell chemotaxis was analysed by the Boyden method and cytokine expression was analysed by ELISA and real-time reverse transcriptase PCR. Bacterial phagocytosis and killing of ingested bacteria were evaluated after incubation with Pseudomonas aeruginosa. Reactive oxygen species (ROS) measurement and elastin receptor expression were determined by flow cytometry. Results Chemotactic activity of neutrophils from patients with COPD towards the VGVAPG EP was reduced compared with controls. VGVAPG increased proinflammatory cytokine synthesis and bacterial load, but reduced ROS production in neutrophils from controls and from patients with stable COPD. Patients with exacerbated COPD were unresponsive to VGVAPG treatment. These findings were associated with a decreased or almost complete loss of S-Gal elastin receptor expression in neutrophils from patients with stable or exacerbated COPD, respectively. Conclusions The study demonstrates that the response of neutrophils from patients with COPD to VGVAPG varied according to COPD phase and critical level of S-Gal expression. S-Gal downregulation could result from a feedback mechanism induced by high levels of EPs.

HIV (human immunodeficiency virus) diverts the cellular ESCRT (endosomal sorting complex required for transport) machinery to promote virion release from infected cells. The ESCRT consists of four heteromeric complexes (ESCRT-0 to ESCRT-III), which mediate different membrane abscission processes, most importantly formation of intralumenal vesicles at multivesicular bodies. The ATPase VPS4 (vacuolar protein sorting 4) acts at a late stage of ESCRT function, providing energy for ESCRT dissociation. Recruitment of ESCRT by late-domain motifs in the viral Gag polyprotein and a role of ESCRT in HIV release are firmly established, but the order of events, their kinetics and the mechanism of action of individual ESCRT components in HIV budding are unclear at present. Using live-cell imaging, we show late-domain-dependent recruitment of VPS4A to nascent HIV particles at the host cell plasma membrane. Recruitment of VPS4A was transient, resulting in a single or a few bursts of at least two to five VPS4 dodecamers assembling at HIV budding sites. Bursts lasted for ∼35 s and appeared with variable delay before particle release. These results indicate that VPS4A has a direct role in membrane scission leading to HIV-1 release.

International audience; The human recombinase hRad51 is a key protein for the maintenance of genome integrity and for cancer development. Polymerization and depolymerization of hRad51 on duplex DNA were studied here using a new generation of magnetic tweezers, measuring DNA twist in real time with a resolution of 5 degrees . Our results combined with earlier structural information suggest that DNA is somewhat less extended by hRad51 than by RecA (4.5 vs. 5.1 A per base pair) and untwisted by 18.2 degrees per base pair. They also confirm a stoichiometry of 3-4 bp per protein in the hRad51-dsDNA nucleoprotein filament. At odds with earlier claims, we show that after initial deposition of a multimeric nucleus, nucleoprotein filament growth occurs by addition/release of single proteins, involving DNA twisting steps of 65 degrees +/- 5 degrees. Simple numeric simulations show that this mechanism is an efficient way to minimize nucleoprotein filament defects. Nucleoprotein filament growth from a preformed nucleus was observed at hRad51 concentrations down to 10 nM, whereas nucleation was never observed below 100 nM in the same buffer. This behavior can be associated with the different stoichiometries of nucleation and growth. It may be instrumental in vivo to permit efficient continuation of strand exchange by hRad51 alone while requiring additional proteins such as Rad52 for its initiation, thus keeping the latter under the strict control of regulatory pathways.

Basement membrane collagens or derived fragments are measured in biological fluids such as blood and urine of patients and appear to be useful for diagnosis, prognostication, or treatment monitoring as proposed for endostatin, a fragment of collagen XVIII, or tumstatin, a fragment of collagen IV. Tetrastatin, the NC1 alpha 4 collagen IV domain, was previously reported to inhibit tumor growth and angiogenesis. The aim of this study was to develop and validate a method to measure tetrastatin concentrations in human fluids. We developed a competitive enzyme-linked immunosorbent assay (ELISA). It allowed measuring tetrastatin levels in human serum, bronchial aspiration and bronchoalveolar lavage fluids, and lung tissue extracts. The tetrastatin level was significantly higher in tumor tissues than in healthy lung tissues. Tetrastatin competitive ELISA could be useful to quantify tetrastatin in tissues and biological fluids for the diagnosis or prognostication of diseases in which basement membrane metabolism may be altered, especially tumor progression.

Actin filament dynamics govern many key physiological processes from cell motility to tissue morphogenesis. A central feature of actin dynamics is the capacity of filaments to polymerize and depolymerize at their ends in response to cellular conditions. It is currently thought that filament kinetics can be described by a single rate constant for each end. In this study, using direct visualization of single actin filament elongation, we show that actin polymerization kinetics at both filament ends are strongly influenced by the binding of proteins to the lateral filament surface. We also show that the pointed-end has a non-elongating state that dominates the observed filament kinetic asymmetry. Estimates of flexibility as well as effects on fragmentation and growth suggest that the observed kinetic diversity arises from structural alteration. Tuning elongation kinetics by exploiting the malleability of the filament structure may be a ubiquitous mechanism to generate a rich variety of cellular actin dynamics. DOI: http://dx.doi.org/10.7554/eLife.04599.001, eLife digest Actin is one of the most abundant proteins in cells. It forms networks of filaments that provide structural support and generate the forces needed for cell movement, division, and many other processes in cells. Filaments of actin continuously change in length as actin molecules are added or removed at the ends. One end of an actin filament—called the barbed-end—grows much faster than the other, known as the pointed-end. Many other proteins also help the actin filaments to form. Some of these proteins bind to the ends of the filaments, where they directly control the growth of the filaments. Other proteins bind along the length of the filaments, but how these ‘side-binding’ proteins influence the growth of filaments is not clear. In this study, Crevenna et al. used a technique called ‘total internal reflection fluorescence (TIRF) microscopy’ to study how several side-binding proteins affect the growth of actin filaments in an artificial system. The growth of the barbed-ends was strongly influenced by which side-binding protein was interacting with the filament. For example, the barbed-end grew rapidly when a protein called VASP was present but grew more slowly in the presence of the protein α-actinin. Although the growth at the pointed-end was generally slow and sporadic, the side-binding proteins also had noticeable effects. Crevenna et al. found that when the side-binding proteins were present at low levels, filament growth was similar for all proteins studied. It was only when the proteins were present at higher levels that the growth of the actin filaments was altered depending on the specific side-binding protein present. One side-binding protein called α-actinin also altered the shape of the actin filament so that when it was present at high levels, the filaments curved in a particular direction. Together, these results suggest that the growth, structure, and flexibility of actin filaments can be strongly influenced by the various proteins that bind along the length of the filaments. The next challenges are to understand the precise details of how these side-binding proteins are able to alter the growth and shape of actin and investigate how they influence other processes that control the structure of actin networks in cells. DOI: http://dx.doi.org/10.7554/eLife.04599.002

Greenland halibut (Reinhardtius hippoglossoides), especially juveniles, are frequently found in severely hypoxic areas (18%–25% saturation) of the St. Lawrence Estuary. We investigated the tolerance of this species to hypoxia and evaluated the consequences of low oxygen levels on metabolic capacity. At 5 °C, juveniles had a higher critical oxygen threshold than adults (15% versus 11% saturation), indicating that they were less tolerant to hypoxia. Severe hypoxia (19% saturation) did not affect the juveniles' standard metabolic rate but significantly reduced (by 55%) their maximum metabolic rate compared with normoxia. Consequently, the aerobic scope was reduced by 72% in hypoxia compared with normoxia. In juveniles, severe hypoxia increased the duration of digestive processes. The decrease in aerobic scope in hypoxia and the determination of critical oxygen threshold at a saturation level close to actual field dissolved oxygen values strongly suggest that juveniles from the St. Lawrence Estuary are living at the edge of their metabolic capacity. Consequently, the growth and distribution of Greenland halibut could be affected if there are further declines in dissolved oxygen availability.

The NC1 domains from the different α(IV) collagen chains were found to exert anti-tumorigenic and/or anti-angiogenic activities. A limitation to the therapeutic use of these matrikines is the large amount of purified recombinant proteins, in the milligram range in mice that should be administered daily throughout the experimental procedures. In the current study, we developed a new therapeutic approach based on tumstatin (NC1α3(IV)) overexpression in vivo in a mouse melanoma model. Gene electrotransfer of naked plasmid DNA (pDNA) is particularly attractive because of its simplicity, its lack of immune responsiveness and its safety. The pDNA electrotransfer in muscle mediates a substantial gene expression that lasts several months. A pVAX1© vector containing the tumstatin cDNA was injected into the legs of C57BL/6 mice and submitted to electrotranfer. Sera were collected at different times and tumstatin was quantified by ELISA. Tumstatin secretion reached a plateau at day 21 with an expression level of 12 μg/mL. For testing the effects of tumstatin expression on tumor growth in vivo, B16F1 melanoma cells were subcutaneously injected in mice 7 days after empty pVAX1© (Mock) or pVAX1©-tumstatin electrotransfer. Tumstatin expression triggered a large decrease in tumor growth and an increase in mouse survival. This new therapeutic approach seems promising to inhibit tumor progression in vivo.

The technical advances of single particle tracking (SPT) have come with an increasing demand for such techniques to follow single biomolecules as they accomplish their function in living cells. From the single particle trajectory, quantitative mechanistic information can be obtained that are unattainable in classical ensemble methods. Traditionally, SPT is performed in two dimensions (2D) for technical simplicity. However, life occurs in three dimensions (3D) and different methods have been recently developed to track particles in 3D. Now, is the third dimension worth the effort? Here, we tracked fluorescent nanoparticles in living cells using a home-made orbital tracking microscope capable of tracking particles in 3D in real-time with a high spatial resolution1. The nanoparticles were tracked in two different cell types having different 3D aspect ratios: 3D Dictyostelium discoideum and quasi-2D HuH-7 human cells. To be compared, the 3D trajectories and their 2D projections were analyzed with a time-resolved algorithm2 based on a local mean square displacement (MSD) analysis. The distributions and characteristics of the active and passive phases were calculated in both cases. Here, we show that intra-cellular diffusion is not purely isotropic and that 2D trajectories cannot be simply scaled up to 3D. The estimation of the diffusion coefficient was more strongly biased in the HuH-7 cells whereas the active transport analysis in these quasi-2D cells was only barely affected by the projection. Inversely, a third of the active phases in the roundish amoeba were wrongly assigned to passive phases in the 2D analysis revealing the quasi isotropic organization of the cell's cytoskeleton. Hence, for an accurate determination of the diffusion coefficient and characterization of the different dynamic phases, 3D tracking and analysis are required.1Dupont et al. Nanoscale (2011)2Arcizet et al. PRL (2008)

International audience; Three-dimensional tracking of single-walled carbon nanotubes (SWNT) with an orbital tracking microscope is demonstrated. We determine the viscosity regime (above 250 cP) at which the rotational diffusion coefficient can be used for length estimation. We also demonstrate SWNT tracking within live HeLa cells and use these findings to spatially map corral volumes (0.27-1.32 μm(3)), determine an active transport velocity (455 nm/s), and calculate local viscosities (54-179 cP) within the cell. With respect to the future use of SWNTs as sensors in living cells, we conclude that the sensor must change the fluorescence signal by at least 4-13% to allow separation of the sensor signal from fluctuations due to rotation of the SWNT when measuring with a time resolution of 32 ms.

Background NC1 domains from α1, α2, α3 and α6(IV) collagen chains were shown to exert anti-tumor or anti-angiogenic activities, whereas the NC1 domain of the α4(IV) chain did not show such activities so far. Methodology/Principal Findings We demonstrate in the present paper that the NC1 α4(IV) domain exerts a potent anti-tumor activity both in vitro and in an experimental human melanoma model in vivo. The overexpression of NC1 α4(IV) in human UACC-903 melanoma cells strongly inhibited their in vitro proliferative (–38%) and invasive (–52%) properties. MT1-MMP activation was largely decreased and its cellular distribution was modified, resulting in a loss of expression at the migration front associated with a loss of migratory phenotype. In an in vivo xenograft model in athymic nude mice, the subcutaneous injection of NC1 α4(IV)-overexpressing melanoma cells induced significantly smaller tumors (–80% tumor volume) than the Mock cells, due to a strong inhibition of tumor growth. Exogenously added recombinant human NC1 α4(IV) reproduced the inhibitory effects of NC1 α4(IV) overexpression in UACC-903 cells but not in dermal fibroblasts. An anti-αvβ3 integrin blocking antibody inhibited cell adhesion on recombinant human NC1 α4(IV) substratum. The involvement of αvβ3 integrin in mediating NC1 α4(IV) effect was confirmed by surface plasmon resonance (SPR) binding assays showing that recombinant human NC1 α4(IV) binds to αvβ3 integrin (KD = 148±9.54 nM). Conclusion/Significance Collectively, our results demonstrate that the NC1 α4(IV) domain, named tetrastatin, is a new endogenous anti-tumor matrikine.

International audience; We previously demonstrated that the CNYYSNS peptide derived from tumstatin inhibited in vivo tumor progression. The YSNS motif formed a β-turn crucial for biological activity. More recently, a YSNSG cyclopeptide with a constrained β-turn on the YSNS residues was designed. Intraperitoneal administration of the YSNSG cyclopeptide inhibited in vivo melanoma progression more efficiently than the native linear peptide. In the present article, we showed that the YSNSG cyclopeptide also triggered an inhibition of in vivo tumor neovascularization and we further analyzed its in vitroantiangiogenic effect. The YSNSG cyclopeptide did not alter endothelial cell proliferation but inhibited cell migration by 83% in an in vitro wound healing model. The inhibition was mediated by a decrease in active MT1-MMP at the migration front as well as a decrease in u-PA and u-PAR expression. The cyclopeptide also altered β1-integrin distribution in endothelial cell lamellipodia, induced a strong decrease in the phosphorylated focal adhesion kinase (p125FAK), disorganized F-actin stress fibers and decreased the number of lamellipodia, resulting in a non migratory phenotype. Our results confirm the YSNSG cyclopeptide as a potent antitumor agent, through both the inhibition of invasive properties of tumor cells and the antiangiogenic activity.

Gold islands, vapor deposited on silicon and quartz by microsphere lithography patterning, are used to nucleate arrays of ZnO nanorods. ZnO is grown on approximately 0.32 microm2 Au islands by carbothermal reduction in a tube furnace. Scanning electron microscopy (SEM) and energy dispersive atomic X-ray spectroscopy (EDS) confirm that the gold effectively controls the sites of nucleation of ZnO. Atomic force microscopy (AFM) shows that approximately 30 nm diameter nanorods grow horizontally, along the surface. Alloy droplets that are characteristic of the vapor-liquid-solid (VLS) mechanism are observed at the tips of the nanorods. The spatial growth direction of VLS catalyzed ZnO nanorods is along the substrate when they nucleate from gold islands on silicon and quartz. The energy of adhesion of the VLS droplet to the surface can account for the horizontal growth.

Colorectal cancers (CRC) with high level of microsatellite instability (MSI-H) are characterized by lower metastasis propensity and better prognosis than their stable microsatellite (MSS) counterpart. It was hypothesized that the difference in cancer progression might be related to distinct gelatinase-tissue inhibitors of metalloproteinase (TIMPs) balance in MSI-H and MSS sporadic CRC.Levels of gelatinase-A (MMP-2) and -B (MMP-9), TIMP-1 and -2 and membrane-type matrix metallo-proteinase-1 (MT1-MMP) were compared in tumors and normal mucosa from patients with MSI-H and MSS CRC.Active levels of MMP-2 and -9, normalized to normal mucosa, were lower in MSI-H than MSS CRC. There was a trend for higher levels of TIMP-1 and TIMP-2 within MSI-H tumors compared with MSS tumors (p=0.08 and p=0.15, respectively), while TIMP-2 amounts were significantly higher in adjacent normal tissue (p0.001) in patients with MSI-H vs. MSS cancers. There was also a trend for lower MT1-MMP activity in MSI-H than in MSS CRC.Our data suggest that the distinct invasive and metastatic behaviors of MSI-H and MSS CRC may be related to different patterns of gelatinase secretion and regulation.

Kinesin is a dimeric molecular motor, which processively moves along the microtubule in a sequence of 8 nm steps. The stepping dynamics needs to be clarified: there are controversial reports about the existence of substructures in the mechanical step and the step timescale. We present observations of the kinesin steps in the absence of external forces, measured with subnanometre precision and microsecond time resolution using a technique which we have recently introduced and named travelling wave tracking. The data suggest that, at zero load, the 8 nm step occurs in less than 30 its and without any long mechanical substeps.

Single-particle tracking (SPT) is of growing importance in the biophysical community. It is used to investigate processes such as drug and gene delivery, viral uptake, intracellular trafficking or membrane-bound protein mobility. Traditionally, SPT is performed in two dimensions (2D) because of its technical simplicity. However, life occurs in three dimensions (3D) and many methods have been recently developed to track particles in 3D. Now, is the third dimension worth the effort? Here we investigate the differences between the 2D and 3D analyses of intracellular transport with the 3D development of a time-resolved mean square displacement (MSD) analysis introduced previously. The 3D trajectories, and the 2D projections, of fluorescent nanoparticles were obtained with an orbital tracking microscope in two different cell types: in Dictyostelium discoideum ameba and in adherent, more flattened HuH-7 human cells. As expected from the different 3D organization of both cells' cytoskeletons, a third of the active transport was lost upon projection in the ameba whereas the identification of the active phases was barely affected in the HuH-7 cells. In both cell types, we found intracellular diffusion to be anisotropic and the diffusion coefficient values derived from the 2D analysis were therefore biased.

Background It is thought that foamy viruses (FVs) enter host cells via endocytosis because all FV glycoproteins examined display pH-dependent fusion activities. Only the prototype FV (PFV) glycoprotein has also significant fusion activity at neutral pH, suggesting that its uptake mechanism may deviate from other FVs. To gain new insights into the uptake processes of FV in individual live host cells, we developed fluorescently labeled infectious FVs. Results N-terminal tagging of the FV envelope leader peptide domain with a fluorescent protein resulted in efficient incorporation of the fluorescently labeled glycoprotein into secreted virions without interfering with their infectivity. Double-tagged viruses consisting of an eGFP-tagged PFV capsid (Gag-eGFP) and mCherry-tagged Env (Ch-Env) from either PFV or macaque simian FV (SFVmac) were observed during early stages of the infection pathway. PFV Env, but not SFVmac Env, containing particles induced strong syncytia formation on target cells. Both virus types showed trafficking of double-tagged virions towards the cell center. Upon fusion and subsequent capsid release into the cytosol, accumulation of naked capsid proteins was observed within four hours in the perinuclear region, presumably representing the centrosomes. Interestingly, virions harboring fusion-defective glycoproteins still promoted virus attachment and uptake, but failed to show syncytia formation and perinuclear capsid accumulation. Biochemical and initial imaging analysis indicated that productive fusion events occur predominantly within 4–6 h after virus attachment. Non-fused or non-fusogenic viruses are rapidly cleared from the cells by putative lysosomal degradation. Quantitative monitoring of the fraction of individual viruses containing both Env and capsid signals as a function of time demonstrated that PFV virions fused within the first few minutes, whereas fusion of SFVmac virions was less pronounced and observed over the entire 90 minutes measured. Conclusions The characterized double-labeled FVs described here provide new mechanistic insights into FV early entry steps, demonstrating that productive viral fusion occurs early after target cell attachment and uptake. The analysis highlights apparent differences in the uptake pathways of individual FV species. Furthermore, the infectious double-labeled FVs promise to provide important tools for future detailed analyses on individual FV fusion events in real time using advanced imaging techniques.

Single particle tracking (SPT) in biological systems is a quickly growing field. Many new technologies are being developed providing new tracking capabilities, which also lead to higher demands and expectations for SPT. Following a single biomolecule as it performs its function provides quantitative mechanistic information that cannot be obtained in classical ensemble methods. From the 3D trajectory, information is available over the diffusional behavior of the particle and precise position information can also be used to elucidate interactions of the tracked particle with its surroundings. Thus, three-dimensional (3D) SPT is a very valuable tool for investigating cellular processes. This review presents recent progress in 3D SPT, from image-based techniques toward more sophisticated feedback approaches. We focus mainly on the feedback technique known as orbital tracking. We present here a modified version of the original orbital tracking in which the intensities from two z-planes are simultaneously measured allowing a concomitant wide-field imaging. The system can track single particles with a precision down to 5 nm in the x-y plane and 7 nm in the axial direction. The capabilities of the system are demonstrated using single virus tracing to follow the infection pathway of Prototype Foamy Virus in living cells.

Background Mercury is known to bioaccumulate and to magnify in marine mammals, which is a cause of great concern in terms of their general health. In particular, the immune system is known to be susceptible to long-term mercury exposure. The aims of the present study were (1) to determine the mercury level in the blood of free-ranging harbour seals from the North Sea and (2) to examine the link between methylmercury in vitro exposure and immune functions using seal and human mitogen-stimulated peripheral blood mononuclear cells (T-lymphocytes). Methods Total mercury was analysed in the blood of 22 harbour seals. Peripheral blood mononuclear cells were isolated from seals (n = 11) and from humans (n = 9). Stimulated lymphocytes of both species were exposed to functional tests (proliferation, metabolic activity, radioactive precursor incorporation) under increasing doses of methylmercury (0.1 to 10 μM). The expression of cytokines (IL-2, IL-4 and TGF-β) was investigated in seal lymphocytes by RT-PCR and by real time quantitative PCR (n = 5) at methylmercury concentrations of 0.2 and 1 μM. Finally, proteomics analysis was attempted on human lymphocytes (cytoplasmic fraction) in order to identify biochemical pathways of toxicity at concentration of 1 μM (n = 3). Results The results showed that the number of seal lymphocytes, viability, metabolic activity, DNA and RNA synthesis were reduced in vitro, suggesting deleterious effects of methylmercury concentrations naturally encountered in free-ranging seals. Similar results were found for human lymphocytes. Functional tests showed that a 1 μM concentration was the critical concentration above which lymphocyte activity, proliferation and survival were compromised. The expression of IL-2 and TGF-β mRNA was weaker in exposed seal lymphocytes compared to control cells (0.2 and 1 μM). Proteomics showed some variation in the protein expression profile (e.g. vimentin). Conclusion Our results suggest that seal and human PBMCs react in a comparable way to MeHg in vitro exposure with, however, larger inter-individual variations. MeHg could be an additional cofactor in the immunosuppressive pollutant cocktail generally described in the blood of seals and this therefore raises the possibility of additional additive effects in the marine mammal immune system.