Micropatterned biopolymer 3D scaffold for static and dynamic culture of human fibroblasts

During in vivo tissue regeneration, cell behavior is highly influenced by the surrounding environment. Thus, the choice of scaffold material and its microstructure is one of the fundamental steps for a successful in vitro culture. An efficacious method for scaffold fabrication should prove its versatility and the possibility of controlling micro- and nanostructure. In this paper, hyaluronic acid 3D scaffolds were developed through lamination of micropatterned membranes, fabricated after optimization of a soft-lithography method. The scaffold presented here is characterized by a homogeneous hexagonal lattice with porosity of 69%, specific surface area of 287 cm-1, and permeability of 18.9 microm2. The control over the geometry was achieved with an accuracy of 20 mum. This technique allowed not only fabrication of planar 3D scaffolds but also production of thin wall tubular constructs. Mechanical tests, performed on dry tubular scaffolds, show high rupture tensile strength. This construct could be promising not only as engineered vascular grafts but also for regeneration of skin, urethra, and intestinal walls. The biocompatibility of a 3D planar scaffold was tested by seeding human fibroblasts. The cells were cultured in both static and dynamic conditions, in a perfusion bioreactor at different flow rates. Microscope analysis and MTT test showed cell proliferation and viability and a uniform cell distribution likely due to an appropriate lattice structure.     

Insect pest management in the age of synthetic biology

Arthropod crop pests are responsible for 20% of global annual crop losses, a figure predicted to increase in a changing climate where the ranges of numerous species are projected to expand. At the same time, many insect species are beneficial, acting as pollinators and predators of pest species. For thousands of years, humans have used increasingly sophisticated chemical formulations to control insect pests but, as the scale of agriculture expanded to meet the needs of the global population, concerns about the negative impacts of agricultural practices on biodiversity have grown. While biological solutions, such as biological control agents and pheromones, have previously had relatively minor roles in pest management, biotechnology has opened the door to numerous new approaches for controlling insect pests. In this review, we look at how advances in synthetic biology and biotechnology are providing new options for pest control. We discuss emerging technologies for engineering resistant crops and insect populations and examine advances in biomanufacturing that are enabling the production of new products for pest control.     

Removal of adult cyathostomins alters faecal microbiota and promotes an inflammatory phenotype in horses

The interactions between parasitic helminths and gut microbiota are considered to be an important, although as yet incompletely understood, factor in the regulation of immunity, inflammation and a range of diseases. Infection with intestinal helminths is ubiquitous in grazing horses, with cyathostomins (about 50 species of which are recorded) predominating. Consequences of infection include both chronic effects, and an acute inflammatory syndrome, acute larval cyathostominosis, which sometimes follows removal of adult helminths by administration of anthelmintic drugs. The presence of cyathostomins as a resident helminth population of the equine gut (the “helminthome”) provides an opportunity to investigate the effect helminth infection, and its perturbation, has on both the immune system and bacterial microbiome of the gut, as well as to determine the specific mechanisms of pathophysiology involved in equine acute larval cyathostominosis. We studied changes in the faecal microbiota of two groups of horses following treatment with anthelmintics (fenbendazole or moxidectin). We found decreases in both alpha diversity and beta diversity of the faecal microbiota at Day 7 post-treatment, which were reversed by Day 14. These changes were accompanied by increases in inflammatory biomarkers. The general pattern of faecal microbiota detected was similar to that seen in the relatively few equine gut microbiome studies reported to date. We conclude that interplay between resident cyathostomin populations and the bacterial microbiota of the equine large intestine is important in maintaining homeostasis and that disturbance of this ecology can lead to gut dysbiosis and play a role in the aetiology of inflammatory conditions in the horse, including acute larval cyathostominosis.     

Common and specific responses to iron and phosphorus deficiencies in roots of apple tree (Malus × domestica)

Plant Molecular Biology - Iron and phosphorus are abundant elements in soils but poorly available for plant nutrition. The availability of these two nutrients represents a major constraint for...     

Proline-specific aminopeptidase P prevents replication-associated genome instability

Genotoxic stress during DNA replication constitutes a serious threat to genome integrity and causes human diseases. Defects at different steps of DNA metabolism are known to induce replication stress, but the contribution of other aspects of cellular metabolism is less understood. We show that aminopeptidase P (APP1), a metalloprotease involved in the catabolism of peptides containing proline residues near their N-terminus, prevents replication-associated genome instability. Functional analysis of C. elegans mutants lacking APP-1 demonstrates that germ cells display replication defects including reduced proliferation, cell cycle arrest, and accumulation of mitotic DSBs. Despite these defects, app-1 mutants are competent in repairing DSBs induced by gamma irradiation, as well as SPO-11-dependent DSBs that initiate meiotic recombination. Moreover, in the absence of SPO-11, spontaneous DSBs arising in app-1 mutants are repaired as inter-homologue crossover events during meiosis, confirming that APP-1 is not required for homologous recombination. Thus, APP-1 prevents replication stress without having an apparent role in DSB repair. Depletion of APP1 (XPNPEP1) also causes DSB accumulation in mitotically-proliferating human cells, suggesting that APP1’s role in genome stability is evolutionarily conserved. Our findings uncover an unexpected role for APP1 in genome stability, suggesting functional connections between aminopeptidase-mediated protein catabolism and DNA replication.     

Comparative study of the O(2), CO(2) and temperature effect on respiration between "Conference" pear cell protoplasts in suspension and intact pears

The influence of the O(2) and CO(2) concentration and the temperature on the O(2) uptake rate of cool-stored intact pears and pear cell protoplasts in suspension was compared. Protocols to isolate pear cell protoplasts from pear tissue and two methods to measure protoplast respiration have been developed. Modified Michaelis-Menten kinetics were applied to describe the effect of the O(2) and the CO(2) concentration on the O(2) uptake rate and temperature dependence was analysed with an Arrhenius equation. Both systems were described with a non-competitive type of CO(2) inhibition. Due to the inclusion of gas diffusion properties, the Michaelis-Menten constant for intact pears (2.5 mM) was significantly larger than the one for protoplasts in suspension (3 microM), which was in turn larger than the Michaelis-Menten constant obtained in mitochondrial respiration measurements described in the literature. It was calculated that only 3.6% of the total diffusion effect absorbed in the Michaelis-Menten constant for intact pears, could be attributed to intracellular gas diffusion. The number of cells per volume of tissue was counted microscopically to establish a relationship between the pear cell protoplast and intact pear O(2) uptake rate. A remarkable similarity was observed: values of 61.8 nmol kg(-1) s(-1) for protoplasts and 87.1 nmol kg(-1) s(-1) for intact pears were obtained. Also, the inhibitory effect of CO(2) on the respiration rate was almost identical for protoplasts and intact pears, suggesting that protoplast suspensions are useful for the study of other aspects of the respiration metabolism.     

Nitric oxide and superoxide impair human placental amino acid uptake and increase Na+ permeability: implications for fetal growth

Based on evidence that thiol and tyrosine reagents inhibit some amino acid transporters, we tested the hypothesis that NO- and O2- -derived free radicals would impair nutrient uptake by the human placenta. Syncytiotrophoblast microvillous plasma membrane vesicles (MVM) and placental villous fragments were exposed to the drug SIN-1 in the presence or absence of superoxide dismutase (SOD) and hemoglobin (Hb). The uptake of [3H]arginine, [3H]taurine, and [3H]leucine; [14C]MeAIB; and 22Na was studied in MVM, whereas the uptake of [3H]taurine was examined in villous fragments. Nitrotyrosine formation was assessed by Western blotting and quantified by ELISA. In MVM, SIN-1 caused an inhibition of [3H]arginine, [3H]taurine, and [14C]MeAIB uptake but had no significant effect on equilibrium [3H]leucine uptake. These effects were prevented by SOD or Hb, implying that both NO and O2- radicals were essential. In contrast, 22Na+ uptake was significantly increased, and this effect was prevented by SOD. In villous fragments, SIN-1 impaired Na+-dependent [3H]taurine uptake, with no effect on Na+-independent uptake. Increased nitrotyrosine formation was observed in MVM after SIN-1 treatment. Endogenous NO- and O2- -derived free radicals may alter human placental nutrient transfer in vivo, with implications for fetal growth.     

Cellular mechanisms involved in CO(2) and acid signaling in chemosensitive neurons

An increase in CO₂/H⁺ is a major stimulus for increased ventilation and is sensed by specialized brain stem neurons called central chemosensitive neurons. These neurons appear to be spread among numerous brain stem regions, and neurons from different regions have different levels of chemosensitivity. Early studies implicated changes of pH as playing a role in chemosensitive signaling, most likely by inhibiting a K⁺ channel, depolarizing chemosensitive neurons, and thereby increasing their firing rate. Considerable progress has been made over the past decade in understanding the cellular mechanisms of chemosensitive signaling using reduced preparations. Recent evidence has pointed to an important role of changes of intracellular pH in the response of central chemosensitive neurons to increased CO₂/H⁺ levels. The signaling mechanisms for chemosensitivity may also involve changes of extracellular pH, intracellular Ca²⁺, gap junctions, oxidative stress, glial cells, bicarbonate, CO₂, and neurotransmitters. The normal target for these signals is generally believed to be a K⁺ channel, although it is likely that many K⁺ channels as well as Ca²⁺ channels are involved as targets of chemosensitive signals. The results of studies of cellular signaling in central chemosensitive neurons are compared with results in other CO₂- and/or H⁺-sensitive cells, including peripheral chemoreceptors (carotid body glomus cells), invertebrate central chemoreceptors, avian intrapulmonary chemoreceptors, acid-sensitive taste receptor cells on the tongue, and pain-sensitive nociceptors. A multiple factors model is proposed for central chemosensitive neurons in which multiple signals that affect multiple ion channel targets result in the final neuronal response to changes in CO₂/H⁺. hypercapnia; brain stem; ventilation; peripheral chemoreceptor; glia; gap junction; glomus; channel; calcium; potassium; carbonic anhydrase; taste receptor; nociception     

The lys5 mutations of barley reveal the nature and importance of plastidial ADP-Glc transporters for starch synthesis in cereal endosperm

Much of the ADP-Glc required for starch synthesis in the plastids of cereal endosperm is synthesized in the cytosol and transported across the plastid envelope. To provide information on the nature and role of the plastidial ADP-Glc transporter in barley (Hordeum vulgare), we screened a collection of low-starch mutants for lines with abnormally high levels of ADP-Glc in the developing endosperm. Three independent mutants were discovered, all of which carried mutations at the lys5 locus. Plastids isolated from the lys5 mutants were able to synthesize starch at normal rates from Glc-1-P but not from ADP-Glc, suggesting a specific lesion in the transport of ADP-Glc across the plastid envelope. The major plastidial envelope protein was purified, and its sequence showed it to be homologous to the maize (Zea mays) ADP-Glc transporter BRITTLE1. The gene encoding this protein in barley, Hv.Nst1, was cloned, sequenced, and mapped. Like lys5, Hv.Nst1 lies on chromosome 6(6H), and all three of the lys5 alleles that were examined were shown to carry lesions in Hv.Nst1. Two of the identified mutations in Hv.Nst1 lead to amino acid substitutions in a domain that is conserved in all members of the family of carrier proteins to which Hv.NST1 belongs. This strongly suggests that Hv.Nst1 lies at the Lys5 locus and encodes a plastidial ADP-Glc transporter. The low-starch phenotype of the lys5 mutants shows that the ADP-Glc transporter is required for normal rates of starch synthesis. This work on Hv.NST1, together with the earlier work on BRITTLE1, suggests that homologous transporters are probably present in the endosperm of all cereals.