Development of a 3D Tissue‐Engineered Skeletal Muscle and Bone Co‐culture System

In vitro 3D tissue‐engineered (TE) structures have been shown to better represent in vivo tissue morphology and biochemical pathways than monolayer culture, and are less ethically questionable than animal models. However, to create systems with even greater relevance, multiple integrated tissue systems should be recreated in vitro. In the present study, the effects and conditions most suitable for the co‐culture of TE skeletal muscle and bone are investigated. High‐glucose Dulbecco's modified Eagle medium (HG‐DMEM) supplemented with 20% fetal bovine serum followed by HG‐DMEM with 2% horse serum is found to enable proliferation of both C2C12 muscle precursor cells and TE85 human osteosarcoma cells, fusion of C2C12s into myotubes, as well as an upregulation of RUNX2/CBFa1 in TE85s. Myotube formation is also evident within indirect contact monolayer cultures. Finally, in 3D co‐cultures, TE85 collagen/hydroxyapatite constructs have significantly greater expression of RUNX2/CBFa1 and osteocalcin/BGLAP in the presence of collagen‐based C2C12 skeletal muscle constructs; however, fusion within these constructs appears reduced. This work demonstrates the first report of the simultaneous co‐culture and differentiation of 3D TE skeletal muscle and bone, and represents a significant step toward a full in vitro 3D musculoskeletal junction model.     

Biased polymers in the origin of life

We delve into the study of a Markov chain formalism applied to the thermal prebiotic oligomerization of amino acids. We find for the case of only two types of electromagnetic interactions, that the steady state attainment by the Markov chain is a built in mechanism limiting the expected variability in sequences in a population of polymers. Such result may be of importance as it makes more accessible the replication of a minimal chemical machinery compatible with life.     

Ventilatory Chemosensory Drive Is Blunted in the mdx Mouse Model of Duchenne Muscular Dystrophy (DMD)

Duchenne Muscular Dystrophy (DMD) is caused by mutations in the DMD gene resulting in an absence of dystrophin in neurons and muscle. Respiratory failure is the most common cause of mortality and previous studies have largely concentrated on diaphragmatic muscle necrosis and respiratory failure component. Here, we investigated the integrity of respiratory control mechanisms in the mdx mouse model of DMD. Whole body plethysmograph in parallel with phrenic nerve activity recordings revealed a lower respiratory rate and minute ventilation during normoxia and a blunting of the hypoxic ventilatory reflex in response to mild levels of hypoxia together with a poor performance on a hypoxic stress test in mdx mice. Arterial blood gas analysis revealed low PaO₂ and pH and high PaCO₂ in mdx mice. To investigate chemosensory respiratory drive, we analyzed the carotid body by molecular and functional means. Dystrophin mRNA and protein was expressed in normal mice carotid bodies however, they are absent in mdx mice. Functional analysis revealed abnormalities in Dejours test and the early component of the hypercapnic ventilatory reflex in mdx mice. Together, these results demonstrate a malfunction in the peripheral chemosensory drive that would be predicted to contribute to the respiratory failure in mdx mice. These data suggest that investigating and monitoring peripheral chemosensory drive function may be useful for improving the management of DMD patients with respiratory failure.     

Dual effects of nitric oxide on cat carotid body chemoreception.

We studied the effects of nitric oxide (NO) released by NO donors on cat carotid body (CB) chemosensory activity during normoxia and hypoxia. CBs excised from pentobarbital sodium-anaesthetized cats were perfused with Tyrode at 38 degrees C and pH 7.40. The frequency of chemosensory discharges (f(x)) was recorded from the carotid sinus nerve, and changes of NO concentration were measured by a chronoamperometric technique, with NO-selective carbon-fiber microelectrodes inserted in the CB. During steady chemosensory excitation induced by hypoxia, bolus injections of NO (DeltaNO = 0. 5-12 microM), released by S-nitroso-N-acetylpenicillamine (SNAP) and 6-(2-hydroxy-1-methyl-nitrosohydrazino)-N-methyl-1-hexanamine++ + (NOC-9), transiently reduced f(x) in a dose-dependent manner. However, during normoxia, the same concentration of NO (DeltaNO = 0. 5-13 microM) released by the NO donors increased f(x) in a dose-dependent manner. The present results show a dual effect of NO on CB chemoreception that is dependent on the PO(2) levels. During hypoxia, NO is predominantly an inhibitor of chemoreception, whereas, in normoxia, NO increased f(x). The mechanisms by which NO produces chemosensory excitation during normoxia remain to be determined.     

On the origin of life event

On the assumption of a uniform sample space probability hypothesis it is estimated a maximum number of polypeptides (or other kind of polymers) that could be synthesized in the prebiotic Earth. Besides, on the basis of five premises that are postulated as indispensable requirements for the origin of a living system, under the constraints of a protein-nucleic acid chemistry, it is concluded categorically that the origin of life event could not be the result of unbiased polymerization phenomena. On the contrary, biased and specific patterns of polymerization had to be an essential component in this fundamental event.Finally, several theories on the origin of life and complementary concepts like hypercyclic organization and self-organization phenomena in dissipative structures are discussed in the light of the conclusions arrived at in this work.     

Expression of Lex antigen in Schistosoma japonicum and S.haematobium and immune responses to Lex in infected animals: lack of Lex expression in other trematodes and nematodes

Adults of the human parasitic trematode Schistosoma mansoni, which causes hepatosplenic/intestinal complications in humans, synthesize glycoconjugates containing the Lewis x (Lex) Galbeta1-->4(Fucalpha1-- >3)GlcNAcbeta1-->R, but not sialyl Lewis x (sLex), antigen. We now report on our analyses of Lexand sLexexpression in S.haematobium and S.japonicum, which are two other major species of human schistosomes that cause disease, and the possible autoimmunity to these antigens in infected individuals. Antigen expression was evaluated by both ELISA and Western blot analyses of detergent extracts of parasites using monoclonal antibodies. Several high molecular weight glycoproteins in both S. haematobium and S. japonicum contain the Lexantigen, but no sialyl Lexantigen was detected. In addition, sera from humans and rodents infected with S.haematobium and S.japonicum contain antibodies reactive with Lex. These results led us to investigate whether Lexantigens are expressed in other helminths, including the parasitic trematode Fasciola hepatica , the parasitic nematode Dirofilaria immitis (dog heartworm), the ruminant nematode Haemonchus contortus , and the free-living nematode Caenorhabditis elegans . Neither Lexnor sialyl-Lexis detectable in these other helminths. Furthermore, none of the helminths, including schistosomes, express Lea, Leb, Ley, or the H- type 1 antigen. However, several glycoproteins from all helminths analyzed are bound by Lotus tetragonolobus agglutinin , which binds Fucalpha1-->3GlcNAc, and Wisteria floribunda agglutinin, which binds GalNAcbeta1-->4GlcNAc (lacdiNAc or LDN). Thus, schistosomes may be unique among helminths in expressing the Lexantigen, whereas many different helminths may express alpha1,3-fucosylated glycans and the LDN motif.      

Identification of genes required for de novo DNA methylation in Arabidopsis

De novo DNA methylation in Arabidopsis thaliana is catalyzed by the methyltransferase DRM2, a homolog of the mammalian de novo methyltransferase DNMT3. DRM2 is targeted to DNA by small interfering RNAs (siRNAs) in a process known as RNA-directed DNA Methylation (RdDM). While several components of the RdDM pathway are known, a functional understanding of the underlying mechanism is far from complete. We employed both forward and reverse genetic approaches to identify factors involved in de novo methylation. We utilized the FWA transgene, which is methylated and silenced when transformed into wild-type plants, but unmethylated and expressed when transformed into de novo methylation mutants. Expression of FWA is marked by a late-flowering phenotype, which is easily scored in mutant versus wild-type plants. By reverse genetics we discovered the requirement for known RdDM effectors AGO6 and NRPE5a for efficient de novo methylation. A forward genetic approach uncovered alleles of several components of the RdDM pathway, including alleles of clsy1, ktf1 and nrpd/e2, which have not been previously shown to be required for the initial establishment of DNA methylation. Mutations were mapped and genes cloned by both traditional and whole genome sequencing approaches. The methodologies and the mutant alleles discovered will be instrumental in further studies of de novo DNA methylation.Key words: DNA methylation, Arabidopsis, de novo, genetic screen, whole-genome sequencing     

The Conservation and Management of Tunas and Their Relatives: Setting Life History Research Priorities

Scombrids (tunas, bonitos, Spanish mackerels and mackerels) support important fisheries in tropical, subtropical and temperate waters around the world, being one of the most economically- and socially-important marine species globally. Their sustainable exploitation, management and conservation depend on accurate life history information for the development of quantitative fisheries stock assessments, and in the fishery data-poor situations for the identification of vulnerable species. Here, we assemble life history traits (maximum size, growth, longevity, maturity, fecundity, spawning duration and spawning interval) for the 51 species of scombrids globally. We identify major biological gaps in knowledge and prioritize life history research needs in scombrids based on their biological gaps in knowledge, the importance of their fisheries and their current conservation status according to the International Union for Conservation of Nature Red List. We find that the growth and reproductive biology of tunas and mackerel species have been more extensively studied than for Spanish mackerels and bonitos, although there are notable exceptions in all groups. We also reveal that reproductive biology of species, particular fecundity, is the least studied biological aspect in scombrids. We identify two priority groups, including 32 species of scombrids, and several populations of principal market tunas, for which life history research should be prioritized following the species-specific life history gaps identified in this study in the coming decades. By highlighting the important gaps in biological knowledge and providing a priority setting for life history research in scombrid species this study provides guidance for management and conservation and serves as a guide for biologists and resource managers interested in the biology, ecology, and management of scombrid species.     

Effects of marine reserve characteristics on the protection of fish populations: a meta-analysis

Meta-analyses of published data for 19 marine reserves reveal that marine protected areas enhance species richness consistently, but their effect on fish abundance is more variable. Overall, there was a slight (11%) but significant increase in fish species number inside marine reserves, with all reserves sharing a common effect. There was a substantial but non-significant increase in overall fish abundance inside marine reserves compared to adjacent, non-reserve areas. When only species that are the target of fisheries were considered, fish abundance was significantly higher (by 28%) within reserve boundaries. Marine reserves vary significantly in the extent and direction of their response. This variability in relative abundance was not attributable to differences in survey methodology among studies, nor correlated with reserve characteristics such as reserve area, years since protection, latitude nor species diversity. The effectiveness of marine reserves in enhancing fish abundance may be largely related to the intensity of exploitation outside reserve boundaries and to the composition of the fish community within boundaries. It is recommended that studies of marine reserve effectiveness should routinely report fishing intensity, effectiveness of enforcement and habitat characteristics.