External fixator-assisted plating osteosynthesis in a rabbit model of femoral bone defects appears to be a feasible and reproducible surgical technique: preliminary insights from a bone substitute study.

PURPOSE: The aims of this study were to assess the complications associated with the use of an external fixator-assisted plate osteosynthesis technique to stabilize a femoral bone defect in a rabbit model and to evaluate if this technique could avoid the mispositioning and the displacement of the femoral fragments during the surgical procedure. METHODS: A preliminary cadaveric animal study was conducted to develop a new technique of external fixator-assisted plating. Thirty rabbits underwent a surgical procedure consisting in the creation of a femoral bone defect and, subsequently an implantation of a bone substitute through the assistance of a temporary external fixator. The fixator's ability to maintain length and alignment during surgery was documented. All intraoperative complications were prospectively collected. RESULTS: No complications related to the use of the temporary external fixator were reported. The technique successfully prevented mispositioning and dislocation during plating in all the rabbits. CONCLUSION: In a rabbit animal model, the use of an external fixator-assisted plate osteosynthesis technique appears to be feasible and effective in avoiding misposition and rotation of femoral fragments when performing osteotomy and plating to create a mid-diaphyseal femoral defect.

The Iron Man of the Renaissance: the contribution of Girolamo Fabrizi d'Acquapendente.

PURPOSE: To highlight the most relevant contributions of the Italian Renaissance surgeon Girolamo Fabrizi d'Acquapendente in the field of orthopaedics and traumatology. METHODS: An extensive research on the life and achievements of Girolamo Fabrizi was conducted on University Libraries as well as on electronic databases like PubMed. RESULTS: Girolamo Fabrizi d'Acquapendente is known for his embryology and physiology studies, particularly on sensory organs and blood circulation. He founded the world's first permanent anatomical theater established at the University of Padua and inaugurated in 1595. His most notable publications include surgical and definitely orthopedics works such as "De fracturis" (On fractures) and "De luxationibus" (On joint displacement). He outlined some principles of treatment that are still valid nowadays such as anatomical reduction and stabilization of the fracture, that were applied using the equipment available at that time. He described and illustrated maneuvers and instruments, such as pulleys, winches, splint, and bandages. He further depicted the famous "Oplomochlion" ("the armored man"), which is actually a collection of all of the braces used at the time to correct congenital and post-traumatic deformities. CONCLUSION: Bracing and prosthetic replacements have accompanied medical history throughout the centuries, from Ancient Egypt to the present, but it was the ingenuity of Renaissance surgeons that pushed biomedical technology to new heights: Girolamo Fabrizi d'Acquapendente was one of the most illustrious contributors to these great achievements.

Minimally Manipulated Mesenchymal Stem Cells for the Treatment of Knee Osteoarthritis: A Systematic Review of Clinical Evidence.

BACKGROUND: The use of laboratory-expanded mesenchymal stem cells (MSCs) is subject to several restrictions, resulting in "minimal manipulation" methods becoming the current most popular strategy to increase the use of MSCs in an orthopaedic practice. The aim of the present systematic review is to assess the clinical applications of "minimally" manipulated MSCs, either as bone marrow aspirate concentrate (BMAC) or as stromal vascular fraction (SVF), in the treatment of knee osteoarthritis (OA). METHODS: A systematic review of three databases (PubMed, ScienceDirect, and Google Scholar) was performed using the following keywords: "Knee Osteoarthritis" with "(Bone marrow aspirate) OR (bone marrow concentrate)" or with "(adipose-derived mesenchymal stem cells) OR (adipose derived stromal cells) OR (stromal vascular fraction) OR (SVF)" as either keywords or MeSH terms. The reference lists of all retrieved articles were further reviewed for identification of potentially relevant studies. RESULTS: Twenty-three papers were included in the final analysis (10 on BMAC and 13 on SVF). Of these, only 4 were randomized controlled trials (RCTs). Bias risk evaluation, performed using a modified Coleman score, revealed an overall poor quality of the studies. In terms of clinical application, despite the apparent safety of minimally manipulated MSCs and the short-term positive clinical outcomes associated with their use, clinicians reported different preparation and administration methods, ranging from single intra-articular injections to intraosseous applications to administration in combination with other surgical procedures. CONCLUSIONS: The available literature is undermined by both the lack of high-quality studies and the varied clinical settings and different protocols reported in the few RCTs presently published. This prevents any recommendation on the use of either product in a clinical practice. Nevertheless, the use of minimally manipulated MSCs (in the form of BMAC or SVF) has been shown to be safe and have some short-term beneficial effects.

Combined toxic effects and DNA damage to two plant species exposed to binary metal mixtures (Cd/Pb).

Acute and long-term (3-, 10- and 56-day exposure) laboratory toxicity tests were carried out to assess the individual and combined toxic effects of cadmium (Cd) and lead (Pb) in Brassica oleracea and Trifolium repens. In addition to morphological parameters, this work also used comet assay to address endpoints in relation to genotoxicity. Bioaccumulation was measured to demonstrate the influence of the mixture on the concentrations of each metal in the plant. The statistical method reported by Ince et al. (1999) was used to evaluate the types of interaction between Cd and Pb in each treatment and concerning their combined effect. This study concludes that the combined effects of binary metal combinations of Cd/Pb on morphological parameters are most often additive, sometimes antagonistic and more rarely synergistic, thus extending the findings of previous publications on this subject. DNA damage analysis revealed concentration- and time-dependent interactions. Synergistic effects of mixed metals (more breaks than individually applied metals) are observed in T. repens after a short exposure. Antagonistic effects are statistically significant after 10 days-exposure, suggesting competition between metals. At 56 days, the rate of DNA damage observed in plants exposed to the Cd/Pb mixture was similar to that measured in plants exposed to lead only and was significantly lower than the rate of DNA damage induced by Cd. This supports the idea that there may be competition between metals and also strengthens the hypothesis that long-term reparation mechanisms may be implemented. Cd/Pb co-exposure does not significantly influence the bioaccumulation of each metal. It is nevertheless important to note that a statistically significant 'interaction' is not necessarily biologically relevant and should therefore be considered with caution when assessing heavy metals combined effects.

Combined effect of Cd and Pb spiked field soils on bioaccumulation, DNA damage, and peroxidase activities in Trifolium repens.

The present study was designed to investigate the combined effects of Cd and Pb on accumulation and genotoxic potential in white clover (Trifolium repens). For this purpose, T. repens was exposed to contaminated soils (2.5-20 mg kg(-1) cadmium (Cd), 250-2000 mg kg(-1) lead (Pb) and a mixture of these two heavy metals) for 3, 10 and 56 days. The resulting bioaccumulation of Cd and Pb, DNA damage (comet assay) and peroxidase activities (APOX and GPOX) were determined. The exposure time is a determinant factor in experiments designed to measure the influence of heavy metal contamination. The accumulation of Cd or Pb resulting from exposure to the two-metal mixture does not appear to depend significantly on whether the white clover is exposed to soil containing one heavy metal or both. However, when T. repens is exposed to a Cd/Pb mixture, the percentage of DNA damage is lower than when the plant is exposed to monometallic Cd. DNA damage is close to that observed in the case of monometallic Pb exposure. Peroxidase activity cannot be associated with DNA damage under these experimental conditions.

Antioxidant defense gene analysis in Brassica oleracea and Trifolium repens exposed to Cd and/or Pb.

This study focused on the expression analysis of antioxidant defense genes in Brassica oleracea and in Trifolium repens. Plants were exposed for 3, 10, and 56 days in microcosms to a field-collected suburban soil spiked by low concentrations of cadmium and/or lead. In both species, metal accumulations and expression levels of genes encoding proteins involved and/or related to antioxidant defense systems (glutathione transferases, peroxidases, catalases, metallothioneins) were quantified in leaves in order to better understand the detoxification processes involved following exposure to metals. It appeared that strongest gene expression variations in T. repens were observed when plants are exposed to Cd (metallothionein and ascorbate peroxidase upregulations) whereas strongest variations in B. oleracea were observed in case of Cd/Pb co-exposures (metallothionein, glutathione transferase, and peroxidase upregulations). Results also suggest that there is a benefit to use complementary species in order to better apprehend the biological effects in ecotoxicology.

Antioxidant responses of Annelids, Brassicaceae and Fabaceae to pollutants: a review.

Pollutants, such as Metal Trace Elements (MTEs) and organic compounds (polycyclic aromatic hydrocarbons, pesticides), can impact DNA structure of living organisms and thus generate damage. For instance, cadmium is a well-known genotoxic and mechanisms explaining its clastogenicity are mainly indirect: inhibition of DNA repair mechanisms and/or induction of Reactive Oxygen Species (ROS). Animal or vegetal cells use antioxidant defense systems to protect themselves against ROS produced during oxidative stress. Because tolerance of organisms depends, at least partially, on their ability to cope with ROS, the mechanisms of production and management of ROS were investigated a lot in Ecotoxicology as markers of biotic and abiotic stress. This was mainly done through the measurement of enzyme activities The present Review focuses on 3 test species living in close contact with soil that are often used in soil ecotoxicology: the worm Eisenia fetida, and two plant species, Trifolium repens (white clover) and Brassica oleracea (cabbage). E. fetida is a soil-dwelling organism commonly used for biomonitoring. T. repens is a symbiotic plant species which forms root nodule with soil bacteria, while B. oleracea is a non-symbiotic plant. In literature, some oxidative stress enzyme activities have already been measured in those species but such analyses do not allow distinction between individual enzyme involvements in oxidative stress. Gene expression studies would allow this distinction at the transcriptomic level. A literature review and a data search in molecular database were carried out on the basis of keywords in Scopus, in PubMed and in Genbank™ for each species. Molecular data regarding E. fetida were already available in databases, but a lack of data regarding oxidative stress related genes was observed for T. repens and B. oleracea. By exploiting the conservation observed between species and using molecular biology techniques, we partially cloned missing candidates involved in oxidative stress and in metal detoxification in E. fetida, T. repens and B. oleracea.

Biomarker measurements in Trifolium repens and Eisenia fetida to assess the toxicity of soil contaminated with landfill leachate: a microcosm study.

To assess the toxicity of a soil contaminated with landfill leachate, biomarker measurements in two species living in close contact with the soil, i.e. a plant species Trifolium repens and an animal species Eisenia fetida, were conducted. Briefly, both species were studied after simultaneous exposure conducted in microcosms. The organisms were exposed to soil supplemented with pure leachate, leachate diluted to 50%; leachate diluted to 25% and without leachate. After a 10 weeks exposure period, we observed an increase in the Olive Trail Moment in T. repens, compared to the reference, for 50% and pure leachate. The response observed appears to be dose-dependent and linear in our experimental conditions. Addition of the leachate to the reference soil induced an increase in Cd-Metallothionein-coding mRNA quantity in E. fetida. In addition, expression level of another gene implied in detoxification and coding Phytochelatin synthase was significantly induced in worms exposed to the reference soil spiked with the leachate, regardless presence of T. repens. Thus, T. repens and E. fetida can be used in a complementary manner to assess soil quality. Sensitivities of the test species yield sensitive bioassays as both species responded at low doses despite the buffering effect of the soil.

Maternal perinatal undernutrition has long-term consequences on morphology, function and gene expression of the adrenal medulla in the adult male rat.

Epidemiological studies suggest that maternal undernutrition sensitises to the development of chronic adult diseases, such as type 2 diabetes, hypertension and obesity. Although the physiological mechanisms involved in this 'perinatal programming' remain largely unknown, alterations of stress neuroendocrine systems such as the hypothalamic-pituitary-adrenal (HPA) and sympathoadrenal axes might play a crucial role. Despite recent reports showing that maternal perinatal undernutrition disturbs chromaffin cells organisation and activity in male rats at weaning, its long-term effects on adrenal medulla in adult animals are unknown. Using a rat model of maternal perinatal 50% food restriction (FR50) from the second week of gestation until weaning, histochemistry approaches revealed alterations in noradrenergic chromaffin cells aggregation and in cholinergic innervation in the adrenal medulla of 8-month-old FR50 rats. Electron microscopy showed that chromaffin cell granules exhibited ultrastructural changes in FR50 rats. These morphological changes were associated with reduced circulating levels and excretion of catecholamines. By contrast, catecholamine plasma levels were significantly increased after a 16 or 72 h of fasting, indicating that the responsiveness of the sympathoadrenal system to food deprivation was accentuated in FR50 adult rats. Among 384 pituitary adenylate cyclase-activating polypeptide-sensitive genes, we identified 129 genes (33.6%) that were under expressed (ratio < 0.7) in FR50 animals. A large number of these genes are involved in cytoskeleton remodelling and vesicle trafficking. Taken together, our results show that maternal perinatal undernutrition programmes adrenomedullary function and gene expression in adult male rats. Because catecholamines contribute to metabolic homeostasis, as well as arterial blood pressure regulation, the alterations observed in the adrenal medulla of adult male FR50 rats may participate in the programming of chronic adult diseases.

The strong induction of metallothionein gene following cadmium exposure transiently affects the expression of many genes in Eisenia fetida: a trade-off mechanism?

Metal pollution causes disturbances at various levels of biological organization in most species. Important physiological functions could be affected in the exposed individuals and among the main physiological functions, immunity may provide one (or more) effector(s) whose expression can be directly affected by a metal exposure in various macroinvertebrates. Protein expressions were studied in order to test them as molecular biomarkers of metal exposure in Eisenia fetida. Selected effectors were calmodulin, heat shock proteins, superoxide dismutase, catalase, metallothionein, beta-adrenergic receptor kinase, pyruvate carboxylase, transcriptionally controlled tumor protein, protein kinase C, ubiquitin and cyclophilin-A. The level of expression of each gene was analysed in whole organism following exposures to cadmium in soil using real-time PCR. Metallothionein, transcriptionally controlled tumor protein and cyclophilin-A expression were also measured following copper exposures in soil because these genes seemed to be sensitive to copper. This work enabled to distinguish metallothionein and cyclophilin-A among the 15 selected effectors. A strong decrease of the number of transcripts was also detected for most effectors soon after the exposure to cadmium suggesting that a trade-off mechanism occurs.

Cloning and real-time PCR testing of 14 potential biomarkers in Eisenia fetida following cadmium exposure.

Important biological activities could be affected in metal exposed species, and amongthe main physiological functions, immunity may provide one (or more) effector(s) which expression can be directly affected by a metal exposure in various macroinvertebrates. As many proteinic effectors showed a high degree of homology between species, we have developed a PCR approach to characterize partial mRNA sequences of selected effectors in the laboratory model, Eisenia fetida. After cloning, levels of expression of each gene were analyzed following exposures (80 and 800 mg/kg) to cadmium spiked soils using real-time PCR. An implemented approach was allowed to test quickly potential biomarkers in Eisenia fetida. Selected effectors were calmodulin, heat shock proteins, superoxide dismutase, catalase, metallothionein, beta-adrenergic receptor kinase, pyruvate carboxylase, trancriptionally controlled tumor protein, protein kinase C, and ubiquitin. Most of the selected effectors did not show variations of expression level after exposure. Others expressed weak changes of expression as heat shock proteins. At lastfor catalase and metallothionein, early suitable variations of expression were observed.

Involvement of penaeidins in defense reactions of the shrimp Litopenaeus stylirostris to a pathogenic vibrio.

The present study reports for the first time the involvement of an antimicrobial peptide in the defense reactions of a shrimp infected by a pathogenic Vibrio, Vibrio penaeicida. New members of the penaeidin family were characterized in the shrimp Litopenaeus stylirostris by RT-PCR and RACE-PCR from hemocyte total RNAs, and by mass spectrometry detection and immunolocalization of mature peptides in shrimp hemocytes. In infected shrimps, bacteria and penaeidin distribution colocalized in the gills and the lymphoid organ that represented the main infected sites. Moreover, the shrimp immune response to infection involved massive hemocyte recruitment to infection sites where released penaeidin may participate in the isolation and elimination of the bacteria, We show that the ability of the shrimps to circumvent shrimp infections is closely related to a recovery phase based on the hematopoietic process.

A reappraisal of pacemaker timing cycles pertaining to automatic mode switching.

Automatic mode switching algorithms of dual chamber pacemakers require fundamental changes in the operation of pacemaker timing cycles to optimize detection of supraventricular tachyarrhythmias. The timing cycles related to mode switching are basically independent of the algorithm design. Blanking periods (when the sensing amplifier is temporarily disabled) should be optimized to a relatively small fraction of the pacing cycle to enhance atrial sensing and prevent far-field sensing. This review explains the function of the timing cycles pertaining to mode switching and proposes simpler terminology to facilitate the understanding of pacemaker function and electrographic interpretation of complex recordings.

Evidence for an endocannabinoid system in the central nervous system of the leech Hirudo medicinalis.

In invertebrates, like Hydra and sea urchins, evidence for a functional cannabinoid system was described. The partial characterization of a putative CB1 cannabinoid receptor in the leech Hirudo medicinalis led us to investigate the presence of a complete endogenous cannabinoid system in this organism. By using gas chromatography-mass spectrometry, we demonstrate the presence of the endocannabinoids anandamide (N-arachidonoylethanolamine, 21.5+/-0.7 pmol/g) and 2-arachidonoyl-glycerol (147.4+/-42.7 pmol/g), and of the biosynthetic precursor of anandamide, N-arachidonylphosphatidyl-ethanolamine (16.5+/-3.3 pmol/g), in the leech central nervous system (CNS). Anandamide-related molecules such as N-palmitoylethanolamine (32.4+/-1.6 pmol/g) and N-linolenoylethanolamine (5.8 pmol/g) were also detected. We also found an anandamide amidase activity in the leech CNS cytosolic fraction with a maximal activity at pH 7 and little sensitivity to typical fatty acid amide hydrolase (FAAH) inhibitors. Using an antiserum directed against the amidase signature sequence, we focused on the identification and the localization of the leech amidase. Firstly, leech nervous system protein extract was subjected to Western blot analysis, which showed three immunoreactive bands at ca. approximately 42, approximately 46 and approximately 66 kDa. The former and latter bands were very faint and were also detected in whole homogenates from the coelenterate Hydra vulgaris, where the presence of CB1-like receptors, endocannabinoids and a FAAH-like activity was reported previously. Secondly, amidase immunocytochemical detection revealed numerous immunoreactive neurons in the CNS of three species of leeches. In addition, we observed that leech amidase-like immunoreactivity matches to a certain extent with CB1-like immunoreactivity. Finally, we also found that stimulation by anandamide of this receptor leads, as in mammals, to inhibition of cAMP formation, although this effect appeared to be occurring through the previously described anandamide-induced and CB1-mediated activation of nitric oxide release. Taken together, these results suggest the existence of a complete and functional cannabinoid system in leeches.

Original involvement of antimicrobial peptides in mussel innate immunity.

Recently, the existence and extended diversity of antimicrobial peptides has been revealed in two mussel species. These molecules are classified into four groups according to common features of their primary structure: defensins, mytilins, myticins and mytimycin. In Mytilus galloprovincialis, gene structure reveals synthesis as precursors in circulating hemocytes. Synthesised even in absence of challenge, the precursors mature and the peptides are stored in granules as active forms. The different peptides are engaged in the destruction of bacteria inside phagocytes, before being released into hemolymph to participate in systemic responses. Such involvement in anti-infectious responses is unique, and apparently more related to those of mammalian phagocytes than to those of insects.

Ligand-induced internalization of neurotensin in transfected COS-7 cells: differential intracellular trafficking of ligand and receptor.

The neuropeptide neurotensin (NT) is known to be internalized in a receptor-mediated fashion into its target cells. To gain insight into the mechanisms underlying this process, we monitored in parallel the migration of the NT1 neurotensin receptor subtype and a fluorescent analog of NT (fluo-NT) in COS-7 cells transfected with a tagged NT1 construct. Fluo-NT internalization was prevented by hypertonic sucrose, potassium depletion and cytosol acidification, demonstrating that it proceeded via clathrin-coated pits. Within 0-30 minutes, fluo-NT accumulated together with its receptor in Acridine Orange-positive, acidic organelles. These organelles concentrated transferrin and immunostained positively for rab 5A, therefore they were early endosomes. After 30-45 minutes, the ligand and its receptor no longer colocalized. Fluo-NT was first found in rab 7-positive late endosomes and later in a nonacidic juxtanuclear compartment identified as the Trans-Golgi Network (TGN) by virtue of its staining for syntaxin 6. This juxtanuclear compartment also stained positively for rab 7 and for the TGN/pericentriolar recycling endosome marker rab 11, suggesting that the ligand could have been recruited to the TGN from either late or recycling endosomes. By that time, internalized receptors were detected in Lamp-1-immunoreactive lysosomes. These results demonstrate that neurotensin/NT1 receptor complexes follow a recycling cycle that is unique among the G protein-coupled receptors studied to date, and provide the first evidence for the targeting of a nonendogenous protein from endosomes to the TGN.

Differential distribution and defence involvement of antimicrobial peptides in mussel.

In previous papers, we characterised 3 types of 4-kDa, cysteine-rich, cationic antimicrobial peptides: MGDs (for Mytilus galloprovincialis defensins), mytilins and myticins, which are abundant in the mussel hemocytes. In the present work, we revealed a differential distribution of the cells expressing the different genes. In addition, using confocal and electron microscopy, we confirmed that defensins and mytilins were partially located in different sub-types of circulating hemocytes although the peptides can be located in the same cell, and even in the same granule. We also demonstrated that mytilins exert their microbicidal effect within the cells through the process of phagosome-mytilin granule fusion leading to the co-location of ingested bacteria and mytilins.

Therostasin, a novel clotting factor Xa inhibitor from the rhynchobdellid leech, Theromyzon tessulatum.

Therostasin is a potent naturally occurring tight-binding inhibitor of mammalian Factor Xa (K(i), 34 pm), isolated from the rhynchobdellid leech Theromyzon tessulatum. Therostasin is a cysteine-rich protein (8991 Da) consisting of 82 amino acid residues with 16 cysteine residues. Its amino acid sequence has been determined by a combination of techniques, including Edman degradation, enzymatic cleavage, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) on the native and s-beta-pyridylethylated compound. Sequence analysis reveals that it shares no significant homology with other Factor Xa inhibitors except for the putative reactive site. Moreover, it contains a signature pattern for proteins of the endothelin family, potent vasoconstrictors isolated in mammal and snake venom. Therostasin cDNA (825 bp) codes for a polypeptide of 82 amino acid residues preceded by 19 residues, representing a signal peptide sequence. As for the other known inhibitors of Factor Xa, therostasin is expressed and stored in the cells of the leech salivary glands.