Selective lateral muscle activation in moderate medial knee osteoarthritis subjects does not unload medial knee condyle

There is some debate in the literature regarding the role of quadriceps-hamstrings co-contraction in the onset and progression of knee osteoarthritis. Does co-contraction during walking increase knee contact loads, thereby causing knee osteoarthritis, or might it be a compensatory mechanism to unload the medial tibial condyle? We used a detailed musculoskeletal model of the lower limb to test the hypothesis that selective activation of lateral hamstrings and quadriceps, in conjunction with inhibited medial gastrocnemius, can actually reduce the joint contact force on the medial compartment of the knee, independent of changes in kinematics or external forces. “Baseline” joint loads were computed for eight subjects with moderate medial knee osteoarthritis (OA) during level walking, using static optimization to resolve the system of muscle forces for each subject?s scaled model. Holding all external loads and kinematics constant, each subject?s model was then perturbed to represent non-optimal “OA-type” activation based on mean differences detected between electromyograms (EMG) of control and osteoarthritis subjects. Knee joint contact forces were greater for the “OA-type” than the “Baseline” distribution of muscle forces, particularly during early stance. The early-stance increase in medial contact load due to the “OA-type” perturbation could implicate this selective activation strategy as a cause of knee osteoarthritis. However, the largest increase in the contact load was found at the lateral condyle, and the “OA-type” lateral activation strategy did not increase the overall (greater of the first or second) medial peak contact load. While “OA-type” selective activation of lateral muscles does not appear to reduce the medial knee contact load, it could allow subjects to increase knee joint stiffness without any further increase to the peak medial contact load.     

Calcium ionophore A23187 reveals calcium related cellular stress as "I-Bodies": an old actor in a new role

Calcimycin (A23187) is an ionophore widely used in studies related to calcium dynamics in cells, but its fluorometric potential to reveal intracellular physiology has not been explored. Exploiting the microenvironment-induced changes in its fluorescence, we show that a brief exposure of cells to non-toxic concentrations (≤3 μM) of the ionophore results in the characteristic organization of the ionophore forming brightly fluorescent cytoplasmic bodies termed “I-Bodies”, which are closely related to stress linked disturbances/changes in calcium homeostasis. “I-Bodies” appear to be Ca²⁺ rich intracellular sites formed during stress-induced release of intracellular Ca²⁺, causing dysfunction and aggregation of mitochondria, providing scaffold for high density packing of A23187. Formation of “I-Bodies” in cells exposed to ionizing radiation and certain anticancer drugs suggest their potential in revealing alterations in calcium signaling and mitochondrial function during (related to) macromolecular damage-induced cell death. The absence of “I-Bodies” in non-malignant cells and their varying numbers in malignant cells with 5 fold increase in fluorescence imply that they can be potential biomarkers of cancer. Thus, “I-Bodies” are novel indicators of endogenous and induced stress linked to disturbances in calcium homeostasis in cells, with a potential to serve as biomarker of cancer.     

How to win ATP and influence Ca signaling

This brief review discusses recent advances in studies of mitochondrial Ca²⁺ signaling and considers how the relationships between mitochondria and Ca²⁺ responses are shaped in secretory epithelial cells. Perhaps the more precise title of this review could have been “How to win ATP and influence Ca²⁺ signaling in secretory epithelium with emphasis on exocrine secretory cells and specific focus on pancreatic acinar cells”. But “brevity is a virtue” and the authors hope that many of the mechanisms discussed are general and applicable to other tissues and cell types. Among these mechanisms are mitochondrial regulation of Ca²⁺ entry and the role of mitochondria in the formation of localized Ca²⁺ responses. The roles of Ca²⁺ signaling in the physiological adjustment of bioenergetics and in mitochondrial damage are also briefly discussed.     

Recombinase-mediated cassette exchange (RMCE) — A rapidly-expanding toolbox for targeted genomic modifications

Starting in 1991, the advance of Tyr-recombinases Flp and Cre enabled superior strategies for the predictable insertion of transgenes into compatible target sites of mammalian cells. Early approaches suffered from the reversibility of integration routes and the fact that co-introduction of prokaryotic vector parts triggered uncontrolled heterochromatization. Shortcomings of this kind were overcome when Flp-Recombinase Mediated Cassette Exchange entered the field in 1994. RMCE enables enhanced tag-and-exchange strategies by precisely replacing a genomic target cassette by a compatible donor construct. After “gene swapping” the donor cassette is safely locked in, but can nevertheless be re-mobilized in case other compatible donor cassettes are provided (“serial RMCE”). These features considerably expand the options for systematic, stepwise genome modifications. The first decade was dominated by the systematic generation of cell lines for biotechnological purposes. Based on the reproducible expression capacity of the resulting strains, a comprehensive toolbox emerged to serve a multitude of purposes, which constitute the first part of this review. The concept per se did not, however, provide access to high-producer strains able to outcompete industrial multiple-copy cell lines. This fact gave rise to systematic improvements, among these certain accumulative site-specific integration pathways. The exceptional value of RMCE emerged after its entry into the stem cell field, where it started to contribute to the generation of induced pluripotent stem (iPS-) cells and their subsequent differentiation yielding a variety of cell types for diagnostic and therapeutic purposes. This topic firmly relies on the strategies developed in the first decade and can be seen as the major ambition of the present article. In this context an unanticipated, potent property of serial Flp-RMCE setups concerns the potential to re-open loci that have served to establish the iPS status before the site underwent the obligatory silencing process. Other relevant options relate to the introduction of composite Flp-recognition target sites (“heterospecific FRT-doublets”), into the LTRs of lentiviral vectors. These “twin sites” enhance the safety of iPS re-programming and -differentiation as they enable the subsequent quantitative excision of a transgene, leaving behind a single “FRT-twin”. Such a strategy combines the established expression potential of the common retro- and lentiviral systems with options to terminate the process at will. The remaining genomic tag serves to identify and characterize the insertion site with the goal to identify genomic “safe harbors” (GOIs) for re-use. This is enabled by the capacity of “FRT-twins” to accommodate any incoming RMCE-donor cassette with a compatible design.     

Neurodegenerative disease-specific induced pluripotent stem cell research

Neurodegenerative disease-specific induced pluripotent stem cell (iPSC) research contributes to the following 3 areas; “Disease modeling”, “Disease material” and “Disease therapy”.“Disease modeling”, by recapitulating the disease phenotype in vitro, will reveal the pathomechanisms. Neurodegenerative disease-specific iPSC-derived non-neuronal cells harboring disease-causative protein(s), which play critical roles in neurodegeneration including motor neuron degeneration in amyotrophic lateral sclerosis, could be “Disease material”, the target cell(s) for drug screening. These differentiated cells also could be used for “Disease therapy”, an autologous cellular replacement/neuroprotection strategy, for patients with neurodegenerative disease.Further progress in these areas of research can be made for currently incurable neurodegenerative diseases.     

The sense of water in the blowfly Protophormia terraenovae

The gustatory system of the blowfly, Protophormia terraenovae, is a relatively simple biological model for studies on chemosensory input and behavioral output. It appears to have renewed interest as a model for studies on the role of water channels, namely aquaporins or aquaglyceroporins, in water detection. To this end, we investigated the presence of water channels, their role in “water” and “salt” cell responsiveness and the transduction mechanism involved. For the first time our electrophysiological results point to the presence of an aquaglyceroporin in the chemoreceptor membrane of the “water” cell in the blowfly taste chemosensilla whose transduction mechanism ultimately involves an intracellular calcium increase and consequently cell depolarization. This hypothesis is also supported by calcium imaging data following proper stimulation. This mechanism is triggered by “water” cell stimulation with hypotonic solutions and/or solutes such as glycerol which crosses the membrane by way of aquaglyceroporins. Behavioral output indicates that the “sense” of water in blowflies is definitely not dependent on the “water” cell only, but also on the “salt” cell sensitivity. These findings also hypothesize a new role for aquaglyceroporin in spiking cell excitability.     

On the relationship between mouth opening and “broken mirror neurons” in autistic individuals

Electromyographies of the mylohyoid muscle (MH) during the execution of the goal-oriented action “grasping to eat” have been used to determine the time relationship between the opening of the mouth and the beginning of the movement. This has been used to distinguish the behaviour of typical developing (TD) children from that of highly functioning autistic (ASD) individuals. The results of previous studies appeared to provide evidence of a deficit in action chain organization in ASD subjects and prompted the hypothesis of a “broken” mirror neuron system (MNS) for these individuals. Our results show the MH activation timing is not reliable in discriminating between TD and ASD children and the distance between the food and the subject plays a key role on the MH activation timing and cannot be neglected when analysing these type of data. The preliminary investigation on the effects of external perturbations also shows that these might have an effect on the results and further investigations are warranted. It appears that there is not enough evidence to support a link between ASD and a broken mirror network system (MNS), and the experimental results must be carefully interpreted before developing therapeutic or rehabilitative protocols.     

Baroreflex variability and “resetting”: A new perspective

A new framework is proposed for the interpretation of spontaneous cardiac baroreflex sensitivity data and the general concept of baroreflex resetting. The framework is used to explore baroreflex function along two separate lines of inquiry: one following a direct intervention in baroreflex function in individual subjects, another in a group of subjects where baroreflex function may have been compromised by coronary artery disease or aging. It is found that under baseline conditions the baroreflex is in a “free-floating” state in which the gain or “sensitivity” is highly variable, while under orthostatic stress or in the absence of or reduced vagal input the gain is more tightly controlled with an expected decline in sensitivity but a very large decline in the variability of that sensitivity. It is concluded that baroreflex “resetting” is better viewed not simply as a change in baroreflex sensitivity but rather as a change in the “focus” or “attention” of the baroreflex as expressed by an observed decline in the variability of the measured gain. The results do not support the interpretation of baroreflex “resetting” as a departure from or return to a universal “set point” as in homeostasis or open loop models.     

Uncoupled binding and folding of immune signaling-related intrinsically disordered proteins

The classical protein structure-function paradigm has been challenged by the emergence of intrinsically disordered proteins (IDPs), the proteins that do not adopt well-defined three-dimensional structures under physiological conditions. This development was accompanied by the introduction of a “coupled binding and folding” paradigm that suggests folding of IDPs upon binding to their partners. However, our recent studies challenge this general view by revealing a novel, previously unrecognized phenomenon – uncoupled binding and folding. This biologically important mechanism is characteristic of members of a new family of IDPs involved in immune signaling and underlies their unusual properties including: (1) specific homodimerization, (2) the lack of folding upon binding to a well-folded protein, another IDP molecule, or to lipid bilayer membranes, and (3) the “scissors-cut paradox”. The third phenomenon occurs in diverse IDP interactions and suggests that properties of IDP fragments are not necessarily additive in the context of the entire protein. The “no disorder-to-order transition” type of binding is distinct from known IDP interactions and is characterized by an unprecedented observation of the lack of chemical shift and peak intensity changes in multidimensional NMR spectra, a fingerprint of proteins, upon complex formation. Here, I focus on those interactions of IDPs with diverse biological partners where the binding phase driven by electrostatic interactions is not be necessarily followed by the hydrophobic folding phase. I also review new multidisciplinary knowledge about immune signaling-related IDPs and show how it expands our understanding of cell function with multiple applications in biology and medicine.     

Hommes et environnements au Paléolithique supérieur en Ukraine continentale et en Crimée : introduction

Research, conducted under the ANR project “Mammouths”, on “the end of the mammoth steppe: Man/Environment relationship during late Pleniglacial in Eastern Europe”, is the subject of several contributions, a part of them is published in this volume, under the heading “Humans and environments during Upper Paleolithic in mainland Ukraine and Crimea”, in the French journal L’anthropologie.     

Hem-1: putting the "WAVE" into actin polymerization during an immune response

Most active processes by immune cells including adhesion, migration, and phagocytosis require the coordinated polymerization and depolymerization of filamentous actin (F-actin), which is an essential component of the actin cytoskeleton. This review focuses on a newly characterized hematopoietic cell-specific actin regulatory protein called hematopoietic protein-1 [Hem-1, also known as Nck-associated protein 1-like (Nckap1l or Nap1l)]. Hem-1 is a component of the “WAVE [WASP (Wiskott-Aldrich syndrome protein)-family verprolin homologous protein]” complex, which signals downstream of activated Rac to stimulate F-actin polymerization in response to immuno-receptor signaling. Genetic studies in cell lines and in mice suggest that Hem-1 regulates F-actin polymerization in hematopoietic cells, and may be essential for most active processes dependent on reorganization of the actin cytoskeleton in immune cells.     

How does the immune response get started?

An effective adaptive immune response requires the prior induction of the regulatory effector T-helper (eTh). There are two competing models of how this cell is induced to effectors. Under the Associative Recognition of Antigen (ARA) or “two signal” model, the T-helper requires eTh in order to be induced to eTh, an “autocatalytic” process. Under the “costimulation” model eTh are induced by an antigen-unspecific signal derived from an “activated” APC. Under the ARA model the problem of the origin of the primer eTh is posed. A nonself antigen-independent pathway to eTh is proposed as well as an experiment to reveal its existence. In the costimulation framework no primer eTh need be postulated but it lacks a mechanism that, in the absence of ARA, accounts for the self-nonself discrimination and the determination of effector class.     

Cell death pathology: cross-talk with autophagy and its clinical implications

Autophagy is a self-digesting mechanism that cells adopt to respond to stressful stimuli. Morphologically, cells dying by autophagy show multiple cytoplasmic double-membraned vacuoles, and, if prolonged, autophagy can lead to cell death, “autophagic cell death”. Thus, autophagy can act both as a temporary protective mechanism during a brief stressful episode and be a mode of cell death in its own right. In this mini-review we focus on recent knowledge concerning the connection between autophagy and programmed cell death, evaluating their possible implications for therapy in pathologies like cancer and neurodegeneration.     

Towards high-yield production of pharmaceutical proteins with plant cell suspension cultures

“Molecular farming” in plants with significant advantages in cost and safety is touted as a promising platform for the production of complex pharmaceutical proteins. While whole-plant produced biopharmaceuticals account for a significant portion of the preclinical and clinical pipeline, plant cell suspension culture, which integrates the merits of whole-plant systems with those of microbial fermentation, is emerging as a more compliant alternative “factory”. However, low protein productivity remains a major obstacle that limits extensive commercialization of plant cell bioproduction platform. This review highlights the advantages and recent progress in plant cell culture technology and outlines viable strategies at both the biological and process engineering levels for advancing the economic feasibility of plant cell-based protein production. Approaches to overcome and solve the associated challenges of this culture system that include non-mammalian glycosylation and genetic instability will also be discussed.     

Isolation and characterization of a large soluble form of fibronectin that stimulates adhesion, spreading, and alignment of mouse erythroleukemia cells

Fibronectin (FN) is a major component of the extracellular matrix which plays important roles in a variety of cellular processes including cell adhesion, and migration. The soluble cellular form of FN has a monomer molecular weight of approximately 250 kDa, and generally exists as a dimer of 500 kDa. We have isolated a different form of soluble FN from mouse breast cancer cell line SC115 conditioned medium (CM) and purified it to homogeneity as evidenced by both native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate PAGE. It still exhibits a monomeric form of about 250 kDa while its form in the CM is stable and soluble with an apparent tetrameric molecular weight in the range of 800-1000 kDa. This form of FN is a potent cell adhesion factor (AF) that induces adhesion to polystyrene, elongation, spreading, alignment or “track” formation, and migration of mouse erythroleukemia cells. Column fractions homogeneous for AF protein were able to stimulate 10% cell adhesion at concentrations of 23 ng/ml and 1.9 ng/cm². Purified AF induced 50% cell adhesion at 94 ng/ml and 7.5 ng/cm². AF also increased the migration of human aortic smooth muscle and vascular endothelial cells. However, this form of FN differs from other forms as it does not bind tightly to either gelatin or heparin. Studies of this AF should shed light on adhesion of cells to extracellular matrix molecules and on cell migration, both of which are critical in several biological processes such as wound healing, metastasis, matrix formation and structure, and organ development.     

Trials, Travails and Triumphs: An Account of RNA Catalysis in RNase P

Last December marked the 20th anniversary of the Nobel Prize in Chemistry to Sidney Altman and Thomas Cech for their discovery of RNA catalysts in bacterial ribonuclease P (an enzyme catalyzing 5′ maturation of tRNAs) and a self-splicing rRNA of Tetrahymena, respectively. Coinciding with the publication of a treatise on RNase P,¹ this review provides a historical narrative, a brief report on our current knowledge, and a discussion of some research prospects on RNase P.

“…the great thing about science is that you can actually solve a problem. You can take something which is confused, a mess, and not only find a solution, but prove it's the right one.”²

-Sydney Brenner

“In research the front line is almost always in a fog.”³

-Francis Crick

     

Le Magdalénien de la grotte « Grappin » à Arlay (Jura, France) : nouveaux regards

The “Grappin” or “Saint-Vincent's” cave, on the western slope of Jura, East of France, has been explored since 1889. From then to 1960, it has yielded substantial material dated to middle “à navettes” Magdalenian developing during the late Pleniglacial. The study of this settlement, although often mentioned, was never dealt with comprehensively until now. Due to its scientific importance for the middle Magdalenian of western Europe, the site is to be reinvestigated through a global interdisciplinary project entitled “The Tardiglacial and the start of Holocene in the Jura and its margins”. This paper will review our present knowledge of the site, radiocarbon dates and archaeological data. It also focuses on ornaments and engraved mobile art.     

The impact of adhesion on cellular invasion processes in cancer and development

In this paper we consider a simple continuous model to describe cell invasion, incorporating the effects of both cell-cell adhesion and cell-matrix adhesion, along with cell growth and proteolysis by cells of the surrounding extracellular matrix (ECM). We demonstrate that the model is capable of supporting both noninvasive and invasive tumour growth according to the relative strength of cell-cell to cell-matrix adhesion. Specifically, for sufficiently strong cell-matrix adhesion and/or sufficiently weak cell-cell adhesion, degradation of the surrounding ECM accompanied by cell-matrix adhesion pulls the cells into the surrounding ECM. We investigate the criticality of matrix heterogeneity on shaping invasion, demonstrating that a highly heterogeneous ECM can result in a “fingering” of the invasive front, echoing observations in real-life invasion processes ranging from malignant tumour growth to neural crest migration during embryonic development.