Validation of an ergonomic assessment method using Kinect data in real workplace conditions.

Evaluating potential musculoskeletal disorders risks in real workstations is challenging as the environment is cluttered, which makes it difficult to accurately assess workers' postures. Being marker-free and calibration-free, Microsoft Kinect is a promising device although it may be sensitive to occlusions. We propose and evaluate a RULA ergonomic assessment in real work conditions using recently published occlusion-resistant Kinect skeleton data correction. First, we compared postures estimated with this method to ground-truth data, in standardized laboratory conditions. Second, we compared RULA scores to those provided by two professional experts, in a non-laboratory cluttered workplace condition. The results show that the corrected Kinect data can provide more accurate RULA grand scores, even under sub-optimal conditions induced by the workplace environment. This study opens new perspectives in musculoskeletal risk assessment as it provides the ergonomists with 30 Hz continuous information that could be analyzed offline and in a real-time framework.

Leishmania infection modulates beta-1 integrin activation and alters the kinetics of monocyte spreading over fibronectin.

Contact with Leishmania leads to a decreases in mononuclear phagocyte adherence to connective tissue. In this work, we studied the early stages of bond formation between VLA4 and fibronectin, measured the kinetics of membrane alignment and the monocyte cytoplasm spreading area over a fibronectin-coated surface, and studied the expression of high affinity integrin epitope in uninfected and Leishmania-infected human monocytes. Our results show that the initial VLA4-mediated interaction of Leishmania-infected monocyte with a fibronectin-coated surface is preserved, however, the later stage, leukocyte spreading over the substrate is abrogated in Leishmania-infected cells. The median of spreading area was 72 [55-89] µm(2) for uninfected and 41 [34-51] µm(2) for Leishmania-infected monocyte. This cytoplasm spread was inhibited using an anti-VLA4 blocking antibody. After the initial contact with the fibronectrin-coated surface, uninfected monocyte quickly spread the cytoplasm at a 15 µm(2) s(-1) ratio whilst Leishmania-infected monocytes only made small contacts at a 5.5 µm(2) s(-1) ratio. The expression of high affinity epitope by VLA4 (from 39 ± 21% to 14 ± 3%); and LFA1 (from 37 ± 32% to 18 ± 16%) molecules was reduced in Leishmania-infected monocytes. These changes in phagocyte function may be important for parasite dissemination and distribution of lesions in leishmaniasis.

Use of TIRF to Monitor T-Lymphocyte Membrane Dynamics with Submicrometer and Subsecond Resolution.

A key step of adaptive immune responses is the T lymphocyte capacity to detect the presence of foreign antigens on specialized cells with high speed and specificity during contacts lasting a few minutes. Much evidence suggests that there is a deep link between the lifetime of molecular interactions between T cell receptors and ligands and T cell activation, but the precise mechanisms of bond formation and dissociation remain incompletely understood. Previous experiments done with interference reflection microscopy/reflection interference contrast microscopy disclosed transverse motions with several nanometer average amplitude of micrometer size membrane zones. More recently, total internal reflection fluorescence microscopy was used to show that the initial interaction between primary T lymphocytes and model surfaces involved the tip of microvilli (typically 0.2 µm2 area) generating apparent contacts of a few seconds that allowed cells to detect ligands of their membrane receptors. Here we show that these microvilli displayed minimal lateral displacements but quantitative fluorescence measurement suggested the occurrence of spontaneous transverse fluctuations of order of 67 nm amplitude during 1-s observation periods. This may play a major role in membrane receptor engagement and ensuing signal generation.

T lymphocytes need less than 3 min to discriminate between peptide MHCs with similar TCR-binding parameters.

T lymphocytes need to detect rare cognate foreign peptides among numerous foreign and self-peptides. This discrimination seems to be based on the kinetics of TCRs binding to their peptide-MHC (pMHC) ligands, but there is little direct information on the minimum time required for processing elementary signaling events and deciding to initiate activation. Here, we used interference reflection microscopy to study the early interaction between transfected human Jurkat T cells expressing the 1G4 TCR and surfaces coated with five different pMHC ligands of 1G4. The pMHC concentration required for inducing 50% maximal IFN-γ production by T cells, and 1G4-pMHC dissociation rates measured in soluble phase or on surface-bound molecules, displayed six- to sevenfold variation among pMHCs. When T cells were dropped onto pMHC-coated surfaces, rapid spreading occurred after a 2-min lag. The initial spreading rate measured during the first 45 s, and the contact area, were strongly dependent on the encountered TCR ligand. However, the lag duration did not significantly depend on encountered ligand. In addition, spreading appeared to be an all-or-none process, and the fraction of spreading cells was tightly correlated to the spreading rate and spreading area. Thus, T cells can discriminate between fairly similar TCR ligands within 2 min.

Pose estimation with a Kinect for ergonomic studies: evaluation of the accuracy using a virtual mannequin.

Analyzing human poses with a Kinect is a promising method to evaluate potentials risks of musculoskeletal disorders at workstations. In ecological situations, complex 3D poses and constraints imposed by the environment make it difficult to obtain reliable kinematic information. Thus, being able to predict the potential accuracy of the measurement for such complex 3D poses and sensor placements is challenging in classical experimental setups. To tackle this problem, we propose a new evaluation method based on a virtual mannequin. In this study, we apply this method to the evaluation of joint positions (shoulder, elbow, and wrist), joint angles (shoulder and elbow), and the corresponding RULA (a popular ergonomics assessment grid) upper-limb score for a large set of poses and sensor placements. Thanks to this evaluation method, more than 500,000 configurations have been automatically tested, which would be almost impossible to evaluate with classical protocols. The results show that the kinematic information obtained by the Kinect software is generally accurate enough to fill in ergonomic assessment grids. However inaccuracy strongly increases for some specific poses and sensor positions. Using this evaluation method enabled us to report configurations that could lead to these high inaccuracies. As a supplementary material, we provide a software tool to help designers to evaluate the expected accuracy of this sensor for a set of upper-limb configurations. Results obtained with the virtual mannequin are in accordance with those obtained from a real subject for a limited set of poses and sensor placements.

Human CalDAG-GEFI gene (RASGRP2) mutation affects platelet function and causes severe bleeding.

The nature of an inherited platelet disorder was investigated in three siblings affected by severe bleeding. Using whole-exome sequencing, we identified the culprit mutation (cG742T) in the RAS guanyl-releasing protein-2 (RASGRP2) gene coding for calcium- and DAG-regulated guanine exchange factor-1 (CalDAG-GEFI). Platelets from individuals carrying the mutation present a reduced ability to activate Rap1 and to perform proper αIIbß3 integrin inside-out signaling. Expression of CalDAG-GEFI mutant in HEK293T cells abolished Rap1 activation upon stimulation. Nevertheless, the PKC- and ADP-dependent pathways allow residual platelet activation in the absence of functional CalDAG-GEFI. The mutation impairs the platelet's ability to form thrombi under flow and spread normally as a consequence of reduced Rac1 GTP-binding. Functional deficiencies were confined to platelets and megakaryocytes with no leukocyte alteration. This contrasts with the phenotype seen in type III leukocyte adhesion deficiency caused by the absence of kindlin-3. Heterozygous did not suffer from bleeding and have normal platelet aggregation; however, their platelets mimicked homozygous ones by failing to undergo normal adhesion under flow and spreading. Rescue experiments on cultured patient megakaryocytes corrected the functional deficiency after transfection with wild-type RASGRP2. Remarkably, the presence of a single normal allele is sufficient to prevent bleeding, making CalDAG-GEFI a novel and potentially safe therapeutic target to prevent thrombosis.

T lymphocytes sense antigens within seconds and make a decision within one minute.

Adaptive immune responses are triggered by the rapid and sensitive detection of MHC-bound peptides by TCRs. The kinetics of early TCR/APC contacts are incompletely known. In this study, we used total internal reflection fluorescence microscopy to image human T cell membranes near model surfaces: contact was mediated by mobile protrusions of <0.4 µm diameter. The mean lifetime of contacts with a neutral surface was 8.6 s. Adhesive interactions increased mean contact time to 27.6 s. Additional presence of TCR ligands dramatically decreased contact to 13.7 s, thus evidencing TCR-mediated triggering of a pulling motion within seconds after ligand encounter. After an interaction typically involving 30-40 contacts formed during a 1-min observation period, TCR stimulation triggered a rapid and active cell spreading. Pulling events and cell spreading were mimicked by pharmacological phospholipase Cγ1 activation, and they were prevented by phospholipase Cγ1 inhibition. These results provide a quantitative basis for elucidating the earliest cell response to the detection of foreign Ags.

Biophysical description of multiple events contributing blood leukocyte arrest on endothelium.

Blood leukocytes have a remarkable capacity to bind to and stop on specific blood vessel areas. Many studies have disclosed a key role of integrin structural changes following the interaction of rolling leukocytes with surface-bound chemoattractants. However, the functional significance of structural data and mechanisms of cell arrest are incompletely understood. Recent experiments revealed the unexpected complexity of several key steps of cell-surface interaction: (i) ligand-receptor binding requires a minimum amount of time to proceed and this is influenced by forces. (ii) Also, molecular interactions at interfaces are not fully accounted for by the interaction properties of soluble molecules. (iii) Cell arrest depends on nanoscale topography and mechanical properties of the cell membrane, and these properties are highly dynamic. Here, we summarize these results and we discuss their relevance to recent functional studies of integrin-receptor association in cells from a patient with type III leukocyte adhesion deficiency. It is concluded that an accurate understanding of all physical events listed in this review is needed to unravel the precise role of the multiple molecules and biochemical pathway involved in arrest triggering.

A novel leukocyte adhesion deficiency III variant: kindlin-3 deficiency results in integrin- and nonintegrin-related defects in different steps of leukocyte adhesion.

Leukocyte adhesion deficiency type III is a recently described condition involving a Glanzmann-type bleeding syndrome and leukocyte adhesion deficiency. This was ascribed to a defect of the FERMT3 gene resulting in abnormal expression of kindlin-3, a protein expressed in hematopoietic cells with a major role in the regulation of integrin activation. In this article, we describe a patient with a new mutation of FERMT3 and lack of kindlin-3 expression in platelets and leukocytes. We assayed quantitatively the first steps of kindlin-3-defective leukocyte adhesion, namely, initial bond formation, bond strengthening, and early spreading. Initial bond formation was readily stimulated with neutrophils stimulated by fMLF, and neutrophils and lymphocytes stimulated by a phorbol ester or Mn(2+). In contrast, attachment strengthening was defective in the patient's lymphocytes treated with PMA or Mn(2+), or fMLF-stimulated neutrophils. However, attachment strengthening was normal in patient's neutrophils treated with phorbol ester or Mn(2+). In addition, the patient's T lymphocytes displayed defective integrin-mediated spreading and a moderate but significant decrease of spreading on anti-CD3-coated surfaces. Patient's neutrophils displayed a drastic alteration of integrin-mediated spreading after fMLF or PMA stimulation, whereas signaling-independent Mn(2+) allowed significant spreading. In conclusion, the consequences of kindlin-3 deficiency on ß(2) integrin function depend on both cell type and the stimulus used for integrin activation. Our results suggest looking for a possible kindlin-3 involvement in membrane dynamical event independent of integrin-mediated adhesion.

p8 expression controls pancreatic cancer cell migration, invasion, adhesion, and tumorigenesis.

p8 is a stress gene whose activity is necessary for tumor development and progression. The acquisition of invasive properties by transformed cells is a key event in tumor development. In order to establish whether p8 is involved or not in this phenomenon, we assessed the capacity of p8 at influencing cell adhesion, migration, invasion, and tumorigenesis of pancreatic cancer cells. p8 expression was knocked down by a small interfering RNA (siRNA) in pancreatic cancer-derived Panc-1 and MiaPaCa-2 cells and subsequent changes in cell adhesion, migration, invasion, and tumorigenesis were assessed. Influence of p8 silencing on gene expression was analyzed using cDNA microarrays. The influence of inhibiting CDC42, one of the genes most over-expressed in p8-silenced cells, on the changes observed in p8-silenced cells was also evaluated. Finally, the tumorigenic capacities of Panc-1 cells transfected with control siRNA or p8 siRNA were compared by assessing their ability to form colonies in soft agar and to grow as xenografts in nude mice. Knocking-down p8 in pancreatic cancer cells in vitro decreased migration and invasion while increasing cell adhesion; over-expression produced the opposite effect. Knocking down CDC42 reversed almost completely the effects of silencing p8 in vitro. Finally, cells transfected with p8 siRNA were almost unable to form colonies in soft agar. In addition, p8-deficient Panc-1 cells did not develop tumors when injected subcutaneously in nude mice. In conclusion, p8 expression controls pancreatic cancer cell migration, invasion and adhesion, three processes required for metastasis, at least in part, through CDC42, a major regulator of cytoskeleton organization.

A new method for rapid detection of T lymphocyte decision to proliferate after encountering activating surfaces.

A critical step of the adaptive response is the detection of foreign peptides on antigen presenting cells by T lymphocytes. It is a major challenge for a T lymphocyte to detect the presence of a few tens of cognate ligands or less on the membrane of a cell exposing millions of protein molecules. Detection is followed by the cell decision to undergo full or partial activation or even to start an inhibitory program. While the measurement of cell proliferation or cytokine synthesis is accepted as a reliable means of monitoring T lymphocyte activation, this requires hours or days to complete, which is a significant drawback to relate decision to particular signaling events or to assess lymphocyte reactivity in patients. Here we show that the contact area formed between T lymphocytes and potentially activating surfaces is exquisitely correlated to the proliferative response measured with the standard CFSE technique. Correlation is even better than the Erk activation that was reported as a digital reporter of cell activation. The simple and accurate method of assessing lymphocyte-to-surface contact extension that we describe might be very useful both to monitor lymphocyte reactivity for clinical purposes and to identify early steps of lymphocyte activation.

Do membrane undulations help cells probe the world?

Cells sense physical properties of their environment including substratum rigidity, roughness, and topography of recognition sites. The cell surface displays continuous deformations of nanometer-scale amplitude and Hz frequency. Recent results support the hypothesis that these surface undulations constitute a powerful strategy for the rapid acquisition of environmental cues: transient contact with surroundings generates forces of piconewton intensity as a result of rapid formation and dissociation of intermolecular bonds. The combination of binding and steric forces is expected to drive conformational changes and lateral reorganization of membrane biomolecules, thus generating signaling cascades. We propose that spontaneous membrane mobility shapes the initial information generated by cell-to-surface contacts, and thereby biases later consequences of these interactions.

Biomolecule association rates do not provide a complete description of bond formation.

The efficiency of many cell-surface receptors is dependent on the rate of binding soluble or surface-attached ligands. Much effort was exerted to measure association rates between soluble molecules (three-dimensional k(on)) and, more recently, between surface-attached molecules (two-dimensional [2D] k(on)). According to a generally accepted assumption, the probability of bond formation between receptors and ligands is proportional to the first power of encounter duration. Here we provide new experimental evidence and review published data demonstrating that this simple assumption is not always warranted. Using as a model system the (2D) interaction between ICAM-1-coated surfaces and flowing microspheres coated with specific anti-ICAM-1 antibodies, we show that the probability of bond formation may scale as a power of encounter duration that is significantly higher than 1. Further, we show that experimental data may be accounted for by modeling ligand-receptor interaction as a displacement along a single path of a rough energy landscape. Under a wide range of conditions, the probability that an encounter of duration t resulted in bond formation varied as erfc[(t(0)/t)(1/2)], where t(0) was on the order of 10 ms. We conclude that the minimum contact time for bond formation may be a useful parameter to describe a ligand-receptor interaction, in addition to conventional association rates.

NADPH oxidase 1 controls the persistence of directed cell migration by a Rho-dependent switch of alpha2/alpha3 integrins.

NADPH oxidase 1 (Nox1) is expressed mainly in colon epithelial cells and produces superoxide ions as a primary function. We showed that Nox1 knockdown inhibits directional persistence of migration on collagen I. This paper dissects the mechanism by which Nox1 affects the direction of colonic epithelial cell migration in a two-dimensional model. Transient activation of Nox1 during cell spreading on collagen 1 temporarily inactivated RhoA and led to efficient exportation of alpha2beta1 integrin to the cell surface, which supported persistent directed migration. Nox1 knockdown led to a loss of directional migration which takes place through a RhoA-dependent alpha2/alpha3 integrin switch. Transient RhoA overactivation upon Nox1 inhibition led to transient cytoskeletal reorganization and increased cell-matrix contact associated with a stable increase in alpha3 integrin cell surface expression. Blocking of alpha3 integrin completely reversed the loss of directional persistence of migration. In this model, Nox1 would represent a switch between random and directional migration through RhoA-dependent integrin cell surface expression modulation.

ArgBP2-dependent signaling regulates pancreatic cell migration, adhesion, and tumorigenicity.

The poor prognosis of pancreatic cancer is due to rapid locoregional invasion, the early development of metastases, and the limited efficacy of current therapies. To date, none of the identified oncogenes and suppressors involved in this disease have led to efficient treatments. Here, we describe that the scaffold protein ArgBP2 is repressed during oncogenic transformation of the pancreas. We could show, using a pancreatic cancer cell line model, that this repression of ArgBP2 participates in the progression of this disease. Interestingly, in vitro analyses revealed that the antitumoral potential of ArgBP2 is linked to the control of cell adhesion and migration rather than to the regulation of cell proliferation or sensitivity to apoptosis. Moreover, we could detail part of the molecular mechanism responsible by identifying new ArgBP2-interacting proteins, and show that this function is partly achieved by the control of a WAVE/PTP-PEST/c-Abl signaling complex. These findings point to a new mechanism of pancreatic cancer progression leading to invasion and metastasis and suggest that the ArgBP2 signaling pathway could represent a new target for cancer therapy.

How cells tiptoe on adhesive surfaces before sticking.

Cell membranes are studded with protrusions that were thoroughly analyzed with electron microscopy. However, the nanometer-scale three-dimensional motions generated by cell membranes to fit the topography of foreign surfaces and initiate adhesion remain poorly understood. Here, we describe the dynamics of surface deformations displayed by monocytic cells bumping against fibronectin-coated surfaces. We observed membrane undulations with typically 5 nm amplitude and 5-10 s lifetime. Cell membranes behaved as independent units of micrometer size. Cells detected the presence of foreign surfaces at 50 nm separation, resulting in time-dependent amplification of membrane undulations. Molecular contact then ensued with apparent cell-membrane separation of 30-40 nm, and this distance steadily decreased during the following tens of seconds. Contact maturation was associated with in-plane egress of bulky molecules and robust membrane fluctuations. Thus, membrane undulations may be the major determinant of cell sensitivity to substrate topography, outcome of interaction, and initial kinetics of contact extension.

How Cells feel their environment: a focus on early dynamic events.

It is now well demonstrated that cell adhesion to a foreign surface strongly influences prominent functions such as survival, proliferation, differentiation, migration or mediator release. Thus, a current challenge of major practical and theoretical interest is to understand how cells process and integrate environmental cues to determine future behaviour. The purpose of this review is to summarize some pieces of information that might serve this task. Three sequential points are discussed. First, selected examples are presented to illustrate the influence of substratum chemistry, topography and mechanical properties on nearly all aspects of cell behaviour observed during the days following adhesion. Second, we review reported evidence that long term cell behaviour is highly dependent on the alterations of cell shape and cytoskeletal organization that are often initiated during the minutes to hours following adhesion. Third, we review recently obtained information on cell membrane roughness and dynamics, as well as kinetics and mechanics of molecular interactions. This knowledge is required to understand the influence of substratum structure on cell signaling during the first minute following contact, before the appearance of detectable structural changes. It is suggested that unraveling the earliest phenomena following cell-to-substratum encounter might provide a tractable way of better understanding subsequent events.

Studying Molecular Interactions at the Single Bond Level with a Laminar Flow Chamber.

During the last decade, many investigators developed new methodologies allowing to study ligand-receptor interactions with unprecedented accuracy, up to the single bond level. Reported results include information on bond mechanical properties, association behaviour of surface-attached molecules, and dissection of energy landscapes and reaction pathways. The purpose of the present review is to discuss the potential and limitations of laminar flow chambers operated at low shear rates. This includes a brief review of basic principles, practical tips and problems associated with data interpretation. It is concluded that flow chambers are ideally suited to analyze weak interactions between a number of biomolecules, including the main families of adhesion receptors such as selectins, integrins, cadherins and members of the immunoglobulin superfamily. The sensitivity of the method is limited by the quality of surfaces and efficiency of the studied ligand-receptor couple rather than the hardware. Analyzing interactions with a resolution of a piconewton and a few milliseconds shows that ligand-receptor complexes may experience a number of intermediate binding states, making it necessary to examine the definition of association and dissociation rates. Finally, it is emphasized that association rates measured on surface-bound molecules are highly dependent on parameters unrelated to binding surfaces.

What is the biological relevance of the specific bond properties revealed by single-molecule studies?

During the last decade, many authors took advantage of new methodologies based on atomic force microscopy (AFM), biomembrane force probes (BFPs), laminar flow chambers or optical traps to study at the single-molecule level the formation and dissociation of bonds between receptors and ligands attached to surfaces. Experiments provided a wealth of data revealing the complexity of bond response to mechanical forces and the dependence of bond rupture on bond history. These results supported the existence of multiple binding states and/or reaction pathways. Also, single bond studies allowed us to monitor attachments mediated by a few bonds. The aim of this review is to discuss the impact of this new information on our understanding of biological molecules and phenomena. The following points are discussed: (i) which parameters do we need to know in order to predict the behaviour of an encounter between receptors and ligands, (ii) which information is actually yielded by single-molecule studies and (iii) is it possible to relate this information to molecular structure?