The effect of hydration on mechanical anisotropy,topography and fibril organization of the osteonal lamellae

The effect of hydration on the mechanical properties of osteonal bone, in directions parallel and perpendicular to the bone axis, was studied on three length scales: (i) the mineralized fibril level (~100 nm), (ii) the lamellar level (~6 µm); and (iii) the osteon level (up to ~30 µm).We used a number of techniques, namely atomic force microscopy (AFM), nanoindentation and microindentation. The mechanical properties (stiffness, modulus and/or hardness) have been studied under dry and wet conditions. On all three length scales the mechanical properties under dry conditions were found to be higher by 30–50% compared to wet conditions. Also the mechanical anisotropy, represented by the ratio between the properties in directions parallel and perpendicular to the osteon axis (anisotropy ratio, designated here by AnR), surprisingly decreased somewhat upon hydration. AFM imaging of osteonal lamellae revealed a disappearance of the distinctive lamellar structure under wet conditions. Altogether, these results suggest that a change in mineralized fibril orientation takes place upon hydration.     

Nascent multicellular life and the emergence of individuality

The evolution of multicellular organisms from unicellular ancestors involves a shift in the level at which selection operates. It is usual to think about this shift in terms of the emergence of traits that cause heritable differences in reproductive output at the level of nascent collectives. Defining these traits and the causes of their origin lies at the heart of understanding the evolution of multicellular life. In working toward a mechanistic, take-nothing-for-granted account, we begin by recognizing that the standard Lewontin formulation of properties necessary and sufficient for evolution by natural selection does not necessarily encompass Darwinian evolution in primitive collectives where parent-offspring relationships may have been poorly defined. This, we suggest, limits the ability to conceptualize and capture the earliest manifestations of Darwinian properties. By way of solution we propose a relaxed interpretation of Lewontin’s conditions and present these in the form of a set of necessary requirements for evolution by natural selection based upon the establishment of genealogical connections between recurrences of collectives. With emphasis on genealogy – as opposed to reproduction – it is possible to conceive selection acting on collectives prior to any manifestation of heritable variance in fitness. Such possibility draws attention to the evolutionary emergence of traits that strengthen causal relationships between recurrences – traits likely to underpin the emergence of forms of multiplication that establish parent-offspring relationships. Application of this framework to collectives of marginal status, particularly those whose recurrence is not defined by genealogy, makes clear that change at the level of collectives need not arise from selection acting at the higher level. We conclude by outlining applicability of our framework to loosely defined collectives of cells, such as those comprising the slugs of social amoeba and microbes that constitute the human microbiome.     

A molecular timescale of eukaryote evolution and the rise of complex multicellular life

Background  

The pattern and timing of the rise in complex multicellular life during Earth's history has not been established. Great disparity persists between the pattern suggested by the fossil record and that estimated by molecular clocks, especially for plants, animals, fungi, and the deepest branches of the eukaryote tree. Here, we used all available protein sequence data and molecular clock methods to place constraints on the increase in complexity through time.     

Immortality and the base of multicellular life: Lessons from cnidarian stem cells

Cnidarians are phylogenetically basal members of the animal kingdom (>600 million years old). Together with plants they share some remarkable features that cannot be found in higher animals. Cnidarians and plants exhibit an almost unlimited regeneration capacity and immortality. Immortality can be ascribed to the asexual mode of reproduction that requires cells with an unlimited self-renewal capacity. We propose that the basic properties of animal stem cells are tightly linked to this archaic mode of reproduction. The cnidarian stem cells can give rise to a number of differentiated cell types including neuronal and germ cells. The genomes of Hydra and Nematostella, representatives of two major cnidarian classes indicate a surprising complexity of both genomes, which is in the range of vertebrates. Recent work indicates that highly conserved signalling pathways control Hydra stem cell differentiation. Furthermore, the availability of genomic resources and novel technologies provide approaches to analyse these cells in vivo. Studies of stem cells in cnidarians will therefore open important insights into the basic mechanisms of stem cell biology. Their critical phylogenetic position at the base of the metazoan branch in the tree of life makes them an important link in unravelling the common mechanisms of stem cell biology between animals and plants.     

An in vivo microdialysis characterization of the transient changes in the interstitial dialysate concentration of metabolites and cytokines in human skeletal muscle in response to insertion of a microdialysis probe

Skeletal muscle has recently been described as an endocrine organ, capable of releasing cytokines and regulators of metabolism. Microdialysis of the interstitial space of skeletal muscle enables analysis of the release of such cytokines. The purpose of this study was to determine the transient changes in concentration of metabolites and cytokines in human skeletal muscle in a 7 h period following the insertion of a microdialysis probe. In total, sixteen microdialysis catheters were inserted into the vastus lateralis of male participants (age 26.2 ± 1.35 y, height 180.8 ± 3.89 cm, mass 83.9 ± 3.86 kg, BMI 25.7 ± 0.87 kg m⁻², body fat 26.1 ± 3.0%). Serial samples were analyzed by micro-enzymatic and multiplexed immunoassay. Muscle interstitial glucose and lactate levels remained stable throughout, amino acid concentrations stabilized after 2.5 h, however, insertion of a microdialysis catheter induced a 29-fold increase in peak IL-6 (p < 0.001) and 35-fold increase in peak IL-8 concentrations (p < 0.001) above basal levels 6 h post insertion. In contrast to stable amino acid, glucose and lactate concentrations after 2 h, commonly reported markers of tissue homeostasis in in vivo microdialysis, the multi-fold increase in IL-6 and IL-8 following insertion of a microdialysis catheter is indicative of a sustained disturbance of tissue homeostasis.     

Effects of radiation on host-tumor interactions using the multicellular tumor spheroid model

Summary Use of the multicellular tumor spheroid as a tumor model allows separate host or tumor treatment with ionizing radiation and examination of the effects on host-tumor immune interactions. Spheroids of EMT6/Ro, a BALB/c mammary tumor were implanted into the peritoneal cavity of syngeneic immunized mice, recovered, and dissociated into single cells. Cytolytic activity of mature spheroid associated cells and peritoneal cells was resistant to radiation doses as high as 1000 rads when irradiated directly prior to assay. Mice irradiated (200, 400, 700 rads) 24 h prior to spheroid injection had an increased number of tumor cells and decreased number of tumor infiltrating and peritoneal host cells upon spheroid recovery. This was paralleled by an increased colony forming efficiency per spheroid. Cytolytic activity of the spheroid associated cells against radiolabeled EMT6 cells was in many cases decreased with radiation although lysis was the same on a per cell basis. Cytolytic activity by peritoneal cells from these mice increased with dose as measured on a per cell basis. This activity from irradiated animals was carried out by a Thyl⁺ cell.     

Complex life cycles of multicellular eukaryotes: new approaches based on the use of model organisms

A wide variety of life cycles can be found in the different groups of multicellular eukaryotes. Here we provide an overview of this variety, and review some of the theoretical arguments that have been put forward to explain the evolutionary stability of different life cycle strategies. We also describe recent progress in the analysis of the haploid-diploid life cycle of the model angiosperm Arabidopsis thaliana and show how new molecular data are providing a means to test some of the theoretical predictions. Finally, we describe an emerging model organism from the brown algae, Ectocarpus siliculosus, and highlight the potential of this system for the investigation of the mechanisms that regulate complex life cycles.     

Residual stress and strain in the lamellar unit of the porcine aorta: experiment and analysis

The opened-up configuration of the artery wall has long been assumed to be stress-free. This is questionable in a microscopic level. The aortic media is made of concentric layers whose unit is called a lamellar unit, a pair of elastic lamina (EL) and a smooth muscle-rich layer (SML). Recently, we found that the EL was about 2.5 times stiffer than the SML. If the circumferential stress in the in vivo condition is the same between the two layers, residual stress of each layer should be different because the stress-strain relationships differ. Such residual stress is not released fully by radial cutting, but is released in the area close to the cut surface, causing hills and valleys on the surface due to residual stress. To check this hypothesis, we have developed a scanning micro indentation tester, a scaled-up version of the atomic force microscope, and measured the topography and the stiffness distribution of the cut surface. The surface of the section of porcine thoracic aortas shows hill and valley pattern corresponding with their histology. The hills were more than three times stiffer than the valleys, indicating that the hills are the ELs and the valleys the SMLs, and the ELs are compressed and the SMLs stretched in the lamellar unit. A finite element analysis showed that the residual stress in the EL and the SML is much higher than those estimated in the unloaded ring-like segments. Fairly large stress may still reside in the opened-up aortic wall.     

Volume effects on fatigue life of equine cortical bone

Materials, including bone, often fail due to loading in the presence of critical flaws. The relative amount, location, and interaction of these flaws within a stressed volume of material play a role in determining the failure properties of the structure. As materials are generally imperfect, larger volumes of material have higher probabilities of containing a flaw of critical size than do smaller volumes. Thus, larger volumes tend to fail at fewer cycles compared with smaller volumes when fatigue loaded to similar stress levels. A material is said to exhibit a volume effect if its failure properties are dependent on the specimen volume. Volume effects are well documented in brittle ceramics and composites and have been proposed for bone. We hypothesized that (1) smaller volumes of cortical bone have longer fatigue lives than similarly loaded larger volumes and (2) that compared with microstructural features, specimen volume was able to explain comparable amounts of variability in fatigue life. In this investigation, waisted rectangular specimens (n=18) with nominal cross-sections of 3×4 mm and gage lengths of 10.5, 21, or 42 mm, were isolated from the mid-diaphysis of the dorsal region of equine third metacarpal bones. These specimens were subjected to uniaxial load controlled fatigue tests, with an initial strain range of 4000 microstrain. The group having the smallest volume exhibited a trend of greater log fatigue life than the larger volume groups. Each volume group exhibited a significant positive correlation between the logarithm of fatigue life and the cumulative failure probability, indicating that the data follow the two-parameter Weibull distribution. Additionally, log fatigue life was negatively correlated with log volume, supporting the hypothesis that smaller stressed volumes of cortical bone possess longer fatigue lives than similarly tested larger stressed volumes.     

Effects of the peptide mycotoxin destruxin E on insect haemocytes and on dynamics and efficiency of the multicellular immune reaction

Destruxins (DTXs) are cyclic peptide toxins secreted by the entomopathogenic fungus Metarhizium anisopliae var. anisopliae. The effects of DTX E, the most active compound of this family on haemocytes, the immunocompetent insect cells, and on the dynamics and efficacy of the multicellular defense of insect hosts have been investigated. Ultrastructural alterations have been observed in circulating plasmatocytes and granular haemocytes, and in attached haemocytes of Galleria mellonella larvae treated with a toxic dose of DTX E (LC50). These changes appear as a consequence of disturbances induced in the cellular calcium balance. An effect on the cell surface of granulocytes was also noted in cells incubated with the toxin and FITC-Con A, even when the concentration of DTX was as low as 0.005 microg/ml. Morphological studies of haemocytic capsules formed in vivo revealed disturbances of the multicellular defense mechanism after toxin treatment However, an attempt to establish if these changes were significant was unsuccessful. In contrast, comparative assays regarding the effect of toxin treatment on the efficacy of the antifungal effect of encapsulation has given conclusive results. The germination of injected Aspergillus niger spores became slightly but significantly increased, and when the granuloma were incubated the fungus escaped more easily from the haemocytic envelope. These results are discussed in terms of significance of the contribution of DTXs to the fungal infection process. It is suggested that the fungal peptides may intervene during the disease by a true immune-inhibitory effect occurring at doses which do not cause paralysis or any general sign of toxicity (e.g., 0.8 microg/g of body weight).     

Influence of functional unit on the life cycle assessment of traction batteries

Goal, Scope and Background This paper describes the influence of the choice of the functional unit on the results of an environmental assessment of different battery technologies for electric and hybrid vehicles. Battery, hybrid and fuel cell electric vehicles are considered as being environmentally friendly. However, the batteries they use are sometimes said to be environmentally unfriendly. At the current state of technology different battery types can be envisaged: lead-acid, nickel-cadmium, nickel-metal hydride, lithium-ion and sodium-nickel chloride. The environmental impacts described in this paper are based on a life cycle assessment (LCA) approach. One of the first critical stages of LCA is the definition of an appropriate and specific functional unit for electric and hybrid vehicle application. Most of the known LCA studies concerning batteries were performed while choosing different functional units, although this choice can influence the final results. An adequate functional unit, allowing to compare battery technologies in their real life vehicle application should be chosen. The results of the LCA are important as they will be used as a decision support for the end-of-life vehicles directive 2000/53/EC (Official Journal of the European Communities L269/24 2000). As a consequence, a thorough analysis is required to define an appropriate functional unit for the assessment of batteries for electric vehicles. This paper discusses this issue and will mainly focus on traction batteries for electric vehicles. Main Features An overview of the different parameters to be considered in the definition of a functional unit to compare battery technologies for battery electric vehicle application is described and discussed. An LCA study is performed for the most relevant potential functional units. SimaPro 6 is used as a software tool and Eco-indicator 99 as an impact assessment method. The influence of the different selected functional units on the results (Eco-indicator Points) is discussed. The environmental impact of the different electric vehicle battery technologies is described. A sensitivity analysis illustrates the robustness of the obtained results. Results and Discussion Five main parameters are considered in each investigated functional unit: an equal depth of discharge is assumed, a relative number of batteries required during the life of the vehicle is calculated, the energy losses in the battery and the additional vehicle consumption due to the battery mass is included and the same lifetime distance target is taken into account. On the basis of the energy content, battery mass, number of cycles and vehicle autonomy three suitable functional units are defined: ‘battery packs with an identical mass’, ‘battery packs with an identical energy content’ and ‘battery packs with an identical one-charge range’. The results show that the differences in the results between these three functional units are small and imply less variation on the results than the other uncertainties inherent to LCA studies. On the other hand, the results obtained using other, less adequate, functional units can be quite different. Conclusions When performing an LCA study, it’s important to choose an appropriate functional unit. Most of the time, this choice is unambiguous. However, sometimes this choice is more complicated when different correlated parameters have to be considered, as it is the case for traction batteries. When using a realistic functional unit, the result is not influenced significantly by the choice of one out of the three suitable functional units. Additionally, the life cycle assessment allowed concluding that three electric vehicle battery technologies have a comparable environmental impact: lead-acid, nickel-cadmium and nickel-metal hydride. Lithium-ion and sodium-nickel chloride have lower environmental impacts than the three previously cited technologies when used in a typical battery electric vehicle application. Recommendations and Perspectives The article describes the need to consider all relevant parameters for the choice of a functional unit for an electric vehicle battery, as this choice can influence the conclusions. A more standardised method to define the functional unit could avoid these differences and could make it possible to compare the results of different traction battery LCA studies more easily.     

Influence of the lamellar phase unbinding energy on the relative stability of lamellar and inverted cubic phases

Based on curvature energy considerations, nonbilayer phase-forming phospholipids in excess water should form stable bicontinuous inverted cubic (Q_II) phases at temperatures between the lamellar (L_α) and inverted hexagonal (H_II) phase regions. However, the phosphatidylethanolamines (PEs), which are a common class of biomembrane phospholipids, typically display direct L_α/H_II phase transitions and may form intermediate Q_II phases only after the temperature is cycled repeatedly across the L_α/H_II phase transition temperature, T_H, or when the H_II phases are cooled from T > T_H. This raises the question of whether models of inverted phase stability, which are based on curvature energy alone, accurately predict the relative free energy of these phases. Here we demonstrate the important role of a noncurvature energy contribution, the unbinding energy of the L_α phase bilayers, g_u, that serves to stabilize the L_α phase relative to the nonlamellar phases. The planar L_α phase bilayers must separate for a Q_II phase to form and it turns out that the work of their unbinding can be larger than the curvature energy reduction on formation of Q_II phase from L_α at temperatures near the L_α/Q_II transition temperature (T_Q). Using g_u and elastic constant values typical of unsaturated PEs, we show that g_u is sufficient to make T_Q > T_H for the latter lipids. Such systems would display direct L_α → H_II transitions, and a Q_II phase might only form as a metastable phase upon cooling of the H_II phase. The g_u values for methylated PEs and PE/phosphatidylcholine mixtures are significantly smaller than those for PEs and increase T_Q by only a few degrees, consistent with observations of these systems. This influence of g_u also rationalizes the effect of some aqueous solutes to increase the rate of Q_II formation during temperature cycling of lipid dispersions. Finally, the results are relevant to protocols for determining the Gaussian curvature modulus, which substantially affects the energy of intermediates in membrane fusion and fission. Recently, two such methods were proposed based on measuring T_Q and on measuring Q_II phase unit cell dimensions, respectively. In view of the effect of g_u on T_Q that we describe here, the latter method, which does not depend on the value of g_u, is preferable.     

Effects of thioredoxin on established airway remodeling in a chronic antigen exposure asthma model

The development and treatment of asthma remains a subject of considerable interest in the medical community. Previous studies implicate an important role of cytokines in the pathology of asthma. In this current study, we examined whether redox-active protein thioredoxin 1 (TRX1) could prevent airway remodeling in an ovalbumin (OVA)-driven mouse chronic antigen exposure asthma model. Balb/c mice were sensitized and then challenged nine times with OVA (days 19-45). In this protocol, airway remodeling was established by day 34. Administration of recombinant human TRX1 during antigen challenge (days 18-32) significantly inhibited airway remodeling, eosinophilic pulmonary inflammation, airway hyperresponsiveness and resulted in decreased lung expression of eotaxin, macrophage inflammatory protein-1alpha and IL-13. Airway remodeling and eosinophilic pulmonary inflammation was also prevented in chronic OVA-exposed Balb/c human TRX1 transgenic mice. Importantly, TRX1-administration, after the establishment of airway remodeling (days 35-45), resulted in improved airway pathology. Our results suggest TRX1 prevents the development of airway remodeling, and also improves established airway remodeling by inhibiting production of chemokines and Th2 cytokines in the lungs.     

Effects of load carriage and fatigue on gait characteristics

The objective of this study was to determine the main and interactive effects of load carriage and fatigue on gait characteristics. Twelve young male participants were recruited in this study. Fatiguing protocol involved a running exercise, and fatigue was considered to be induced when the participants first gave an RPE rating at or above 17. Gait data were collected when the participants walked on a medical treadmill at their self-selected comfortable speed, both before and right after the fatiguing exercise. Different back-carrying loads (i.e. 0, 7.5, and 15 kg) were applied separately to the participants during the walking trials. Gait variability measures and kinematic measures were used to quantify gait characteristics. The results showed that gait width variability, hip range of motion, and trunk range of motion increased with fatigue and with the application of the heavy load. These findings suggest that both fatigue and load carriage compromise gait. Findings from this study can help better understand how fatigue and load carriage affect gait, and further aid in developing interventions that are able to minimize fall risks especially with the application of fatigue and/or external load.     

Effects of DAP on diaphragm force and fatigue, including fatigue due to neurotransmission failure

Van Lunteren, Erik, and Michelle Moyer. Effects of DAPon diaphragm force and fatigue, including fatigue due toneurotransmission failure. J. Appl.Physiol. 81(5): 2214-2220, 1996.Among theaminopyridines, 3,4-diaminopyridine (DAP) is a more effectiveK⁺ channel blocker than is4-aminopyridine (4-AP), and, furthermore, DAP enhances neuromusculartransmission. Because 4-AP improves muscle contractility, wehypothesized that DAP would also increase force and, in addition,ameliorate fatigue and improve the neurotransmission failure componentof fatigue. Rat diaphragm strips were studied in vitro (37°C). Infield-stimulated muscle, 0.3 mM DAP significantly increased diaphragmtwitch force, prolonged contraction time, and shifted theforce-frequency relationship to the left without altering peak tetanicforce, resulting in increased force at stimulation frequencies 50 Hz.During 20-Hz intermittent stimulation, DAP increased diaphragm peakforce compared with control during a 150-s fatigue run and,furthermore, significantly improved maintenance of intratrain force.The relative contribution of neurotransmission failure to fatigue wasestimated by comparing the force generated by phrenic nerve-stimulatedmuscles with that generated by curare-treated field-stimulated muscles.DAP significantly increased force in nerve-stimulated muscles and, inaddition, reduced the neurotransmission failure contribution todiaphragm fatigue. Thus DAP increases muscle force atlow-to-intermediate stimulation frequencies, improves overall force andintratrain fatigue during 20-Hz intermittent stimulation, and reducesneurotransmission failure.

     

Effects of different mesenchymal stromal cell sources and delivery routes in experimental emphysema

We sought to assess whether the effects of mesenchymal stromal cells (MSC) on lung inflammation and remodeling in experimental emphysema would differ according to MSC source and administration route. Emphysema was induced in C57BL/6 mice by intratracheal (IT) administration of porcine pancreatic elastase (0.1 UI) weekly for 1 month. After the last elastase instillation, saline or MSCs (1×10⁵), isolated from either mouse bone marrow (BM), adipose tissue (AD) or lung tissue (L), were administered intravenously (IV) or IT. After 1 week, mice were euthanized. Regardless of administration route, MSCs from each source yielded: 1) decreased mean linear intercept, neutrophil infiltration, and cell apoptosis; 2) increased elastic fiber content; 3) reduced alveolar epithelial and endothelial cell damage; and 4) decreased keratinocyte-derived chemokine (KC, a mouse analog of interleukin-8) and transforming growth factor-β levels in lung tissue. In contrast with IV, IT MSC administration further reduced alveolar hyperinflation (BM-MSC) and collagen fiber content (BM-MSC and L-MSC). Intravenous administration of BM- and AD-MSCs reduced the number of M1 macrophages and pulmonary hypertension on echocardiography, while increasing vascular endothelial growth factor. Only BM-MSCs (IV > IT) increased the number of M2 macrophages. In conclusion, different MSC sources and administration routes variably reduced elastase-induced lung damage, but IV administration of BM-MSCs resulted in better cardiovascular function and change of the macrophage phenotype from M1 to M2.     

The effects of testing methods on the flexural fatigue life of human cortical bone

A flexural model of four-point bending fatigue that has been experimentally validated for human cortical bone under load control was used to determine how load and displacement control testing affects the fatigue behavior of human cortical bone in three-point and symmetric four-point bending. Under load control, it was predicted that three-point bending produced no significant differences in fatigue life when compared to four-point bending. However, three-point bending produced less stiffness loss with increasing cycles than four-point bending. In four-point bending, displacement control was predicted to produce about one and a half orders of magnitude greater fatigue life when compared to load control. This prediction agrees with experimental observations of equine cannon bone tested in load and displacement control (Gibson et al., 1998). Displacement controlled three-point bending was found to produce approximately a 25% greater fatigue life when compared to load control. The prediction of longer fatigue life under displacement control may have clinical relevance for the repair of damaged bone. The model can also be adapted to other geometric configurations, including modeling of whole long bones, and with appropriate fatigue data, other cortical bone types.     

Effects of highly selective sensory/motor nerve injury on bone metabolism and bone remodeling in rats

Objectives:This work aimed to investigate the mechanism of selective sensory/motor nerve injury in affecting bone metabolism and remodeling.Methods:The selective sensory/motor nerve injury rat model was constructed through posterior rhizotomy (PRG), anterior rhizotomy (ARG), or anterior combined with posterior rhizotomy (APRG) at the L_4-6 sensory/motor nerves on the right side of rats. Sham-operated (SOG) rats served as control. At 8 weeks after surgery, the sciatic nerves, spinal cord segments L₅ and tibial tissues were collected for analysis.Results:the integrity of trabecular bone was damaged, the number of trabecular bone was decreased and the number of osteoclasts were increased in ARG group. ARG activated NF-κβ and PPAR-γ pathways, and inhibited Wnt/β-catenin pathway. ARG group exhibited high turnover bone metabolism. In PRG group, the trabecular bone morphology became thinner, and the number of osteoclasts was increased. NF-κβ pathway was activated and OPG/RANKL ratio was decreased in PRG group. The activated osteoclasts, reduced osteoblasts activity and lower turnover bone metabolism were observed in PRG group. Additionally, the bone metabolism in APRG group was similar to ARG group.Conclusion:The posterior rhizotomy and anterior rhizotomy induced the different degree of osteoporosis in rats, which may attribute to regulate Wnt/β-catenin, NF-κβ and PPAR-γ signalling pathways.