Multidomain chimeric enzymes as a promising alternative for biocatalysts improvement: a minireview.

Searching for new and better biocatalysts is an area of study in constant development. In nature, mechanisms generally occurring in evolution, such as genetic duplication, recombination, and natural selection processes, produce various enzymes with different architectures and properties. The recombination of genes that code proteins produces multidomain chimeric enzymes that contain two or more domains that sometimes enhance their catalytic properties. Protein engineering has mimicked this process to enhance catalytic activity and the global stability of enzymes, searching for new and better biocatalysts. Here, we present and discuss examples from both natural and synthetic multidomain chimeric enzymes and how additional domains heighten their stability and catalytic activity. Moreover, we also describe progress in developing new biocatalysts using synthetic fusion enzymes and revise some methodological strategies to improve their biological fitness.

Leaf structure and water relations of an allotetraploid Mediterranean fern and its diploid parents.

Allopolyploidy is a common speciation mechanism in plants; however, its physiological and ecological consequences in niche partitioning have been scarcely studied. In this sense, leaf traits are good proxies to study the adaptive capacity of allopolyploids and diploid parents to their respective environmental conditions. In the present work, leaf water relations (assessed through pressure-volume curves) and structural and anatomical traits of the allotetraploid fern Oeosporangium tinaei and its diploid parents, Oeosporangium hispanicum and Oeosporangium pteridioides, were studied under controlled conditions in response to a water stress (WS) cycle. O. hispanicum showed the lowest osmotic potential at turgor loss point (πtlp ) and leaf capacitance, together with higher leaf mass per area (LMA), leaf thickness (LT), leaf density (LD), and leaf dry matter content (LDMC), whereas O. pteridioides presented the opposite set of traits (high πtlp and capacitance, and low LMA, LT, LD, and LDMC). O. tinaei showed an intermediate position for most of the studied traits. The responsiveness (osmotic and elastic adjustments) to WS was low, although most of the traits explained the segregation of the three species across a range of drought tolerance according to the rank: O. hispanicum > O. tinaei > O. pteridioides. These trait differences may underlie the niche segregation among coexisting populations of the three species in the Mediterranean basin.

Proteome and Interactome Linked to Metabolism, Genetic Information Processing, and Abiotic Stress in Gametophytes of Two Woodferns.

Ferns and lycophytes have received scant molecular attention in comparison to angiosperms. The advent of high-throughput technologies allowed an advance towards a greater knowledge of their elusive genomes. In this work, proteomic analyses of heart-shaped gametophytes of two ferns were performed: the apomictic Dryopteris affinis ssp. affinis and its sexual relative Dryopteris oreades. In total, a set of 218 proteins shared by these two gametophytes were analyzed using the STRING database, and their proteome associated with metabolism, genetic information processing, and responses to abiotic stress is discussed. Specifically, we report proteins involved in the metabolism of carbohydrates, lipids, and nucleotides, the biosynthesis of amino acids and secondary compounds, energy, oxide-reduction, transcription, translation, protein folding, sorting and degradation, and responses to abiotic stresses. The interactome of this set of proteins represents a total network composed of 218 nodes and 1792 interactions, obtained mostly from databases and text mining. The interactions among the identified proteins of the ferns D. affinis and D. oreades, together with the description of their biological functions, might contribute to a better understanding of the function and development of ferns as well as fill knowledge gaps in plant evolution.

The Shared Proteome of the Apomictic Fern Dryopteris affinis ssp. affinis and Its Sexual Relative Dryopteris oreades.

Ferns are a diverse evolutionary lineage, sister to the seed plants, which is of great ecological importance and has a high biotechnological potential. Fern gametophytes represent one of the simplest autotrophic, multicellular plant forms and show several experimental advantages, including a simple and space-efficient in vitro culture system. However, the molecular basis of fern growth and development has hardly been studied. Here, we report on a proteomic study that identified 417 proteins shared by gametophytes of the apogamous fern Dryopteris affinis ssp. affinis and its sexual relative Dryopteris oreades. Most proteins are predicted to localize to the cytoplasm, the chloroplast, or the nucleus, and are linked to enzymatic, binding, and structural activities. A subset of 145 proteins are involved in growth, reproduction, phytohormone signaling and biosynthesis, and gene expression, including homologs of SHEPHERD (SHD), HEAT SHOCK PROTEIN 90-5 (CR88), TRP4, BOBBER 1 (BOB1), FLAVONE 3'-O-METHYLTRANSFERASE 1 (OMT1), ZEAXANTHIN EPOXIDASE (ABA1), GLUTAMATE DESCARBOXYLASE 1 (GAD), and dsRNA-BINDING DOMAIN-LIKE SUPERFAMILY PROTEIN (HLY1). Nearly 25% of the annotated proteins are associated with responses to biotic and abiotic stimuli. As for biotic stress, the proteins PROTEIN SGT1 HOMOLOG B (SGT1B), SUPPRESSOR OF SA INSENSITIVE2 (SSI2), PHOSPHOLIPASE D ALPHA 1 (PLDALPHA1), SERINE/THREONINE-PROTEIN KINASE SRK2E (OST1), ACYL CARRIER PROTEIN 4 (ACP4), and NONHOST RESISTANCE TO P. S. PHASEOLICOLA1 (GLPK) are worth mentioning. Regarding abiotic stimuli, we found proteins associated with oxidative stress: SUPEROXIDE DISMUTASE[CU-ZN] 1 (CSD1), and GLUTATHIONE S-TRANSFERASE U19 (GSTU19), light intensity SERINE HYDROXYMETHYLTRANSFERASE 1 (SHM1) and UBIQUITIN-CONJUGATING ENZYME E2 35 (UBC35), salt and heavy metal stress included MITOCHONDRIAL PHOSPHATE CARRIER PROTEIN 3 (PHT3;1), as well as drought and thermotolerance: LEA7, DEAD-BOX ATP-DEPENDENT RNA HELICASE 38 (LOS4), and abundant heat-shock proteins and other chaperones. In addition, we identified interactomes using the STRING platform, revealing protein-protein associations obtained from co-expression, co-occurrence, text mining, homology, databases, and experimental datasets. By focusing on ferns, this proteomic study increases our knowledge on plant development and evolution, and may inspire future applications in crop species.

A global phylogeny of Stegnogramma ferns (Thelypteridaceae): generic and sectional revision, historical biogeography and evolution of leaf architecture.

The thelypteroid fern genus Stegnogramma s.l. contains around 18-35 species and has a global, cross-continental distribution ranging from tropical to temperate regions. Several genera and infrageneric sections have been recognized previously in Stegnogramma s.l., but their phylogenetic relationships are still unclear. In this study, we present a global phylogeny of Stegnogramma s.l. with the most comprehensive sampling to date and aim to pinpoint the phylogenetic positions of biogeographically and taxonomically important taxa. Based on the reconstructed historical biogeography and character evolution, we propose a new (infra)generic classification and discuss the diversification of Stegnogramma s.l. in a biogeographical context. New names or combinations are made for 12 (infra)species, including transferring the monotypic species of Craspedosorus to Leptogramma. Finally, we discuss a possible link between leaf architecture and ecological adaptation, and hypothesize that the increase in leaf dissection and free-vein proportion is an adaptive feature to cool climates in Stegnogramma s.l.

Fitness of an allopolyploid rupicolous fern compared with its diploid progenitors: from sporogenesis to sporophyte formation.

PREMISE: When two populations of related cytotypes grow in sympatry, the rarer cytotype tends to be excluded due to a frequency-dependent mating disadvantage. Evolutionary models predict that polyploids, which are typically the rarer cytotype upon first formation, should have higher relative fitness and/or higher selfing rates to establish and then coexist with diploid parents. METHODS: Performance during early recruitment was compared among three co-occurring rupicolous fern species: the allotetraploid Cheilanthes tinaei and its diploid ancestors, C. hispanica and C. maderensis. In culture experiments, fresh spores and samples of soil spore banks were tested for variation among cytotypes in germination, survival, fecundity, and mating system of gametophytes. RESULTS: Compared with its diploid parents, C. tinaei fresh spores had higher abortion percentages, lower dispersal ability as a result of its larger spores, and similar vigor at germination. For gametophytes from soil spore banks, C. tinaei had high survival similar to C. maderensis, but its sex expression resembled that of C. hispanica, with a high proportion of males. Patterns of sporophyte formation by females and bisexuals indicate that the polyploid does not have an increased gametophytic selfing rate. Gametophytes were larger in C. tinaei, but its reproductive success (sporophyte formation) was intermediate relative to diploids. CONCLUSIONS: Our results show no evidence of higher selfing or fitness advantage of the allopolyploid over both diploid parents at any stage of early recruitment. These two unexpected findings suggest that further factors, such as niche differentiation, play a more important role in cytotype coexistence.

Biocompatible elastin-like click gels: design, synthesis and characterization.

Elastin-like recombinamer click gels (ELR-CGs) for biomedical applications, such as drug delivery or tissue engineering, have been developed by taking advantage of the click reaction (CuAAC) in the absence of traditional crosslinking agents. ELRs are functionalized with alkyne and azide groups using conventional chemical techniques to introduce the reactivity required to carry out the 1,3-dipolar cycloaddition under mild biocompatible conditions, with no toxic by-products and in short reaction times. Hydrogels with moduli in the range 1,000-10,000 Pa have been synthesized, characterized, and tested in vitro against several cell types. The cells embedded into ELR-CGs possessed high viability and proliferation rate. The mechanical properties, porosity and swelling of the resulting ELR-CGs can easily be tuned by adjusting the ELR concentration. We also show that it is possible to replicate different patterns on the hydrogel surface, thus allowing the use of this type of hydrogel to improve applications that require cell guidance or even differentiation depending on the surface topography.

Catechol biosynthesis from glucose in Escherichia coli anthranilate-overproducer strains by heterologous expression of anthranilate 1,2-dioxygenase from Pseudomonas aeruginosa PAO1.

BACKGROUND: The aromatic compound catechol is used as a precursor of chemical products having multiple applications. This compound is currently manufactured by chemical synthesis from petroleum-derived raw materials. The capacity to produce catechol is naturally present in several microbial species. This knowledge has been applied to the generation of recombinant Escherichia coli strains that can produce catechol from simple carbon sources. RESULTS: Several strains derived from E. coli W3110 trpD9923, a mutant that overproduces anthranilate, were modified by transforming them with an expression plasmid carrying genes encoding anthranilate 1,2-dioxygenase from Pseudomonas aeruginosa PAO1. The additional expression of genes encoding a feedback inhibition resistant version of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase and transketolase from E. coli, was also evaluated. Generated strains were characterized in complex or minimal medium in shake-flask and fed-batch bioreactor cultures and incubation temperatures ranging from 37 to 28°C. These experiments enabled the identification of culture conditions for the production of 4.47 g/L of catechol with strain W3110 trpD9923, expressing 1,2-dioxygenase, DAHP synthase and transketolase. When considering the amount of glucose consumed, a yield of 16% was calculated, corresponding to 42% of the theoretical maximum as determined by elementary node flux analysis. CONCLUSIONS: This work demonstrates the feasibility of applying metabolic engineering for generating E. coli strains for the production of catechol from glucose via anthranilate. These results are a starting point to further optimize environmentally-compatible production capacity for catechol and derived compounds.

Comparative analysis of mycorrhizal communities associated with Struthiopteris spicant (L.) Weiss across Europe and North America.

Introduction: Ferns constitute the second largest group of vascular plants. Previous studies have shown that the diversity and composition of fern communities are influenced by resource availability and water stress, among other factors. However, little is known about the influence of these environmental factors on their biotic interactions, especially regarding the relationship between mycorrhizal fungi and ferns. The present study compares the mycorrhizal communities associated with 36 populations of Struthiopteris spicant L. Weiss across Europe and North America. This species exhibits a great tolerance to variations in light, nutrient, and pH conditions, and it can survive with and without mycorrhizae. Methods: With the aim of determining which environmental factors impact the composition and abundance of the root-associated fungal communities in this species, we used an ITS-focused metabarcoding approach to identify the mycorrhizal fungi present and analyzed the influence of climatic and edaphic variables at global and regional scales. Results and discussion: We encountered striking differences in the relative abundance of arbuscular mycorrhizal fungi (AMF) between S. spicant populations at both spatial levels. We recorded a total of 902 fungal ASVs, but only 2- 4% of the total fungal diversity was observed in each individual, revealing that each fern had a unique fungal community. Light availability and the interactive action of pH and soil nitrogen concentration showed a positive influence on AMF relative abundance, explaining 89% of the variance. However, environmental factors could only explain 4- 8% of the variability in AMF community composition, indicating that it might be determined by stochastic processes. These results support the hypothesis that ferns may be more independent of mycorrhization than other plant groups and interact with fungi in a more opportunistic manner.

Anti-Sigma Factors in E. coli: Common Regulatory Mechanisms Controlling Sigma Factors Availability.

In bacteria, transcriptional regulation is a key step in cellular gene expression. All bacteria contain a core RNA polymerase that is catalytically competent but requires an additional σ factor for specific promoter recognition and correct transcriptional initiation. The RNAP core is not able to selectively bind to a given σ factor. In contrast, different σ factors have different affinities for the RNAP core. As a consequence, the concentration of alternate σ factors requires strict regulation in order to properly control the delicate interplay among them, which favors the competence for the RNAP core. This control is archived by different σ/anti-σ controlling mechanisms that shape complex regulatory networks and cascades, and enable the response to sudden environmental cues, whose global understanding is a current challenge for systems biology. Although there have been a number of excellent studies on each of these σ/anti-σ post-transcriptional regulatory systems, no comprehensive comparison of these mechanisms in a single model organism has been conducted. Here, we survey all these systems in E. coli dissecting and analyzing their inner workings and highlightin their differences. Then, following an integral approach, we identify their commonalities and outline some of the principles exploited by the cell to effectively and globally reprogram the transcriptional machinery. These principles provide guidelines for developing biological synthetic circuits enabling an efficient and robust response to sudden stimuli.

Ecophysiological Differentiation among Two Resurrection Ferns and Their Allopolyploid Derivative.

Theoretically, the coexistence of diploids and related polyploids is constrained by reproductive and competitive mechanisms. Although niche differentiation can explain the commonly observed co-occurrence of cytotypes, the underlying ecophysiological differentiation among cytotypes has hardly been studied. We compared the leaf functional traits of the allotetraploid resurrection fern Oeosporangium tinaei (HHPP) and its diploid parents, O. hispanicum (HH) and O. pteridioides (PP), coexisting in the same location. Our experimental results showed that all three species can recover physiological status after severe leaf dehydration, which confirms their 'resurrection' ability. However, compared with PP, HH had much higher investment per unit area of light-capturing surface, lower carbon assimilation rate per unit mass for the same midday water potential, higher non-enzymatic antioxidant capacity, higher carbon content, and lower contents of nitrogen, phosphorus, and other macronutrients. These traits allow HH to live in microhabitats with less availability of water and nutrients (rock crevices) and to have a greater capacity for resurrection. The higher assimilation capacity and lower antioxidant capacity of PP explain its more humid and nutrient-rich microhabitats (shallow soils). HHPP traits were mostly intermediate between those of HH and PP, and they allow the allotetraploid to occupy the free niche space left by the diploids.

Novel brain-penetrant inhibitor of G9a methylase blocks Alzheimer's disease proteopathology for precision medication.

Current amyloid beta-targeting approaches for Alzheimer's disease (AD) therapeutics only slow cognitive decline for small numbers of patients. This limited efficacy exists because AD is a multifactorial disease whose pathological mechanism(s) and diagnostic biomarkers are largely unknown. Here we report a new mechanism of AD pathogenesis in which the histone methyltransferase G9a noncanonically regulates translation of a hippocampal proteome that defines the proteopathic nature of AD. Accordingly, we developed a novel brain-penetrant inhibitor of G9a, MS1262, across the blood-brain barrier to block this G9a-regulated, proteopathologic mechanism. Intermittent MS1262 treatment of multiple AD mouse models consistently restored both cognitive and noncognitive functions to healthy levels. Comparison of proteomic/phosphoproteomic analyses of MS1262-treated AD mice with human AD patient data identified multiple pathological brain pathways that elaborate amyloid beta and neurofibrillary tangles as well as blood coagulation, from which biomarkers of early stage of AD including SMOC1 were found to be affected by MS1262 treatment. Notably, these results indicated that MS1262 treatment may reduce or avoid the risk of blood clot burst for brain bleeding or a stroke. This mouse-to-human conservation of G9a-translated AD proteopathology suggests that the global, multifaceted effects of MS1262 in mice could extend to relieve all symptoms of AD patients with minimum side effect. In addition, our mechanistically derived biomarkers can be used for stage-specific AD diagnosis and companion diagnosis of individualized drug effects.

Novel brain-penetrant inhibitor of G9a methylase blocks Alzheimer's disease proteopathology for precision medication.

Current amyloid beta-targeting approaches for Alzheimer's disease (AD) therapeutics only slow cognitive decline for small numbers of patients. This limited efficacy exists because AD is a multifactorial disease whose pathological mechanism(s) and diagnostic biomarkers are largely unknown. Here we report a new mechanism of AD pathogenesis in which the histone methyltransferase G9a noncanonically regulates translation of a hippocampal proteome that defines the proteopathic nature of AD. Accordingly, we developed a novel brain-penetrant inhibitor of G9a, MS1262, across the blood-brain barrier to block this G9a-regulated, proteopathologic mechanism. Intermittent MS1262 treatment of multiple AD mouse models consistently restored both cognitive and noncognitive functions to healthy levels. Comparison of proteomic/phosphoproteomic analyses of MS1262-treated AD mice with human AD patient data identified multiple pathological brain pathways that elaborate amyloid beta and neurofibrillary tangles as well as blood coagulation, from which biomarkers of early stage of AD including SMOC1 were found to be affected by MS1262 treatment. Notably, these results indicated that MS1262 treatment may reduce or avoid the risk of blood clot burst for brain bleeding or a stroke. This mouse-to-human conservation of G9a-translated AD proteopathology suggests that the global, multifaceted effects of MS1262 in mice could extend to relieve all symptoms of AD patients with minimum side effect. In addition, our mechanistically derived biomarkers can be used for stage-specific AD diagnosis and companion diagnosis of individualized drug effects. One-Sentence Summary: A brain-penetrant inhibitor of G9a methylase blocks G9a translational mechanism to reverse Alzheimer's disease related proteome for effective therapy.

Epigenetic regulation in the neurogenic niche of the adult dentate gyrus.

The adult dentate gyrus (DG) of the hippocampal formation is a specialized region of the brain that creates new adult-born neurons from a pool of resident adult neural stem and progenitor cells (aNSPCs) throughout life. These aNSPCs undergo epigenetic and epitranscriptomic regulation, including 3D genome interactions, histone modifications, DNA modifications, noncoding RNA mechanisms, and RNA modifications, to precisely control the neurogenic process. Furthermore, the specialized neurogenic niche also uses epigenetic mechanisms in mature neurons and glial cells to communicate signals to direct the behavior of the aNSPCs. Here, we review recent advances of epigenetic regulation in aNSPCs and their surrounding niche cells within the adult DG.

Hypothalamic modulation of adult hippocampal neurogenesis in mice confers activity-dependent regulation of memory and anxiety-like behavior.

Adult hippocampal neurogenesis plays a critical role in memory and emotion processing, and this process is dynamically regulated by neural circuit activity. However, it remains unknown whether manipulation of neural circuit activity can achieve sufficient neurogenic effects to modulate behavior. Here we report that chronic patterned optogenetic stimulation of supramammillary nucleus (SuM) neurons in the mouse hypothalamus robustly promotes neurogenesis at multiple stages, leading to increased production of neural stem cells and behaviorally relevant adult-born neurons (ABNs) with enhanced maturity. Functionally, selective manipulation of the activity of these SuM-promoted ABNs modulates memory retrieval and anxiety-like behaviors. Furthermore, we show that SuM neurons are highly responsive to environmental novelty (EN) and are required for EN-induced enhancement of neurogenesis. Moreover, SuM is required for ABN activity-dependent behavioral modulation under a novel environment. Our study identifies a key hypothalamic circuit that couples novelty signals to the production and maturation of ABNs, and highlights the activity-dependent contribution of circuit-modified ABNs in behavioral regulation.

The Incorporation of Etanercept into a Porous Tri-Layer Scaffold for Restoring and Repairing Cartilage Tissue.

Cartilage diseases currently affect a high percentage of the world's population. Almost all of these diseases, such as osteoarthritis (OA), cause inflammation of this soft tissue. However, this could be controlled with biomaterials that act as an anti-inflammatory delivery system, capable of dosing these drugs over time in a specific area. The objective of this study was to incorporate etanercept (ETA) into porous three-layer scaffolds to decrease the inflammatory process in this soft tissue. ETA is a blocker of pro-inflammatory cytokines, such as tumour necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). For this reason, the scaffold was built based on natural polymers, including chitosan and type I collagen. The scaffold was grafted next to subchondral bone using hydroxyapatite as filler. One of the biomaterials obtained was also crosslinked to compare its mechanical properties with the non-treated one. Both samples' physicochemical properties were studied with SEM, micro-CT and photoacoustic imaging, and their rheological properties were also compared. The cell viability and proliferation of the human chondrocyte C28/I2 cell line were studied in vitro. An in vitro and in vivo controlled release study was evaluated in both specimens. The ETA anti-inflammatory effect was also studied by in vitro TNF-α and IL-6 production. The crosslinked and non-treated scaffolds had rheological properties suitable for this application. They were non-cytotoxic and favoured the in vitro growth of chondrocytes. The in vitro and in vivo ETA release showed desirable results for a drug delivery system. The TNF-α and IL-6 production assay showed that this drug was effective as an anti-inflammatory agent. In an in vivo OA mice model, safranin-O and fast green staining was carried out. The OA cartilage tissue improved when the scaffold with ETA was grafted in the damaged area. These results demonstrate that this type of biomaterial has high potential for clinical applications in tissue engineering and as a controlled drug delivery system in OA articular cartilage.

[Capacity of the oleaginous yeast Clavispora lusitaniae Hi2 to transform agroindustrial residues into lipids]. / Potencial de la levadura oleaginosa Clavispora lusitaniae Hi2 en la conversión de residuos agroindustriales a lípidos.

BACKGROUND: Single-cell oils obtained from oleaginous microorganisms by using lignocellulosic waste hydrolysates are an alternative for producing biodiesel. AIMS: To isolate a yeast strain able to produce lipids from centrifuged nejayote (CN), hydrolyzed nejayote solids (HNS) and hydrolyzed sugarcane bagasse (HSB). METHODS: In order to identify the yeasts recovered, 26S ribosomal DNA was sequenced. The metabolic profile was assessed by using API20C AUX strips. The nutritional characterization of CN, HNS and HSB was performed by quantifying reducing sugars, total carbohydrates, starch, protein and total nitrogen. The biomass and lipid production ability were evaluated by performing growth kinetics of Clavispora lusitaniae Hi2 in combined culture media. RESULTS: Six oleaginous yeast strains were isolated and identified, selecting C. lusitaniae Hi2 to study its lipids production by using nejayote. The C. lusitaniae Hi2 strain can use glucose, xylose, arabinose, galactose and cellobiose as carbon sources. Cultures of C. lusitaniae Hi2 presented the best biomass (5.6±0.28 g/L) and lipid production (0.99±0.09 g/L) at 20 h of incubation with the CN:HNS media in the 25:75 and 50:50 ratios, respectively. CONCLUSIONS: The use of CN, HNS and HSB for the growth of C. lusitaniae Hi2 is an option to take advantage of these agro-industrial residues and generate compounds of biotechnological interest.

Sexual and Apogamous Species of Woodferns Show Different Protein and Phytohormone Profiles.

The gametophyte of ferns reproduces either by sexual or asexual means. In the latter, apogamy represents a peculiar case of apomixis, in which an embryo is formed from somatic cells. A proteomic and physiological approach was applied to the apogamous fern Dryopteris affinis ssp. affinis and its sexual relative D. oreades. The proteomic analysis compared apogamous vs. female gametophytes, whereas the phytohormone study included, in addition to females, three apogamous stages (filamentous, spatulate, and cordate). The proteomic profiles revealed a total of 879 proteins and, after annotation, different regulation was found in 206 proteins of D. affinis and 166 of its sexual counterpart. The proteins upregulated in D. affinis are mostly associated to protein metabolism (including folding, transport, and proteolysis), ribosome biogenesis, gene expression and translation, while in the sexual counterpart, they account largely for starch and sucrose metabolism, generation of energy and photosynthesis. Likewise, ultra-performance liquid chromatography-tandem spectrometry (UHPLC-MS/MS) was used to assess the levels of indol-3-acetic acid (IAA); the cytokinins: 6-benzylaminopurine (BA), trans-Zeatine (Z), trans-Zeatin riboside (ZR), dyhidrozeatine (DHZ), dyhidrozeatin riboside (DHZR), isopentenyl adenine (iP), isopentenyl adenosine (iPR), abscisic acid (ABA), the gibberellins GA3 and GA4, salicylic acid (SA), and the brassinosteroids: brassinolide (BL) and castasterone (CS). IAA, the cytokinins Z, ZR, iPR, the gibberellin GA4, the brassinosteoids castasterone, and ABA accumulated more in the sexual gametophyte than in the apogamous one. When comparing the three apogamous stages, BA and SA peaked in filamentous, GA3 and BL in spatulate and DHRZ in cordate gametophytes. The results point to the existence of large metabolic differences between apogamous and sexual gametophytes, and invite to consider the fern gametophyte as a good experimental system to deepen our understanding of plant reproduction.

The Effects of Crosslinking on the Rheology and Cellular Behavior of Polymer-Based 3D-Multilayered Scaffolds for Restoring Articular Cartilage.

Polymer-based tri-layered (bone, intermediate and top layers) scaffolds used for the restoration of articular cartilage were prepared and characterized in this study to emulate the concentration gradient of cartilage. The scaffolds were physically or chemically crosslinked. In order to obtain adequate scaffolds for the intended application, the impact of the type of calcium phosphate used in the bone layer, the polymer used in the intermediate layer and the interlayer crosslinking process were analyzed. The correlation among SEM micrographs, physical-chemical characterization, swelling behavior, rheological measurements and cell studies were examined. Storage moduli at 1 Hz were 0.3-1.7 kPa for physically crosslinked scaffolds, and 4-5 kPa (EDC/NHS system) and 15-20 kPa (glutaraldehyde) for chemically crosslinked scaffolds. Intrinsic viscoelasticity and poroelasticity were considered in discussing the physical mechanism dominating in different time/frequency scales. Cell evaluation showed that all samples are available as alternatives to repair and/or substitute cartilage in articular osteoarthritis.

Microsatellites reveal substantial among-population genetic differentiation and strong inbreeding in the relict fern Dryopteris aemula.

BACKGROUND AND AIMS: A previous study detected no allozyme diversity in Iberian populations of the buckler-fern Dryopteris aemula. The use of a more sensitive marker, such as microsatellites, was thus needed to reveal the genetic diversity, breeding system and spatial genetic structure of this species in natural populations. METHODS: Eight microsatellite loci for D. aemula were developed and their cross-amplification with other ferns was tested. Five polymorphic loci were used to characterize the amount and distribution of genetic diversity of D. aemula in three populations from the Iberian Peninsula and one population from the Azores. KEY RESULTS: Most microsatellite markers developed were transferable to taxa close to D. aemula. Overall genetic variation was low (H(T) = 0.447), but was higher in the Azorean population than in the Iberian populations of this species. Among-population genetic differentiation was high (F(ST) = 0.520). All loci strongly departed from Hardy-Weinberg equilibrium. In the population where genetic structure was studied, no spatial autocorrelation was found in any distance class. CONCLUSIONS: The higher genetic diversity observed in the Azorean population studied suggested a possible refugium in this region from which mainland Europe has been recolonized after the Pleistocene glaciations. High among-population genetic differentiation indicated restricted gene flow (i.e. lack of spore exchange) across the highly fragmented area occupied by D. aemula. The deviations from Hardy-Weinberg equilibrium reflected strong inbreeding in D. aemula, a trait rarely observed in homosporous ferns. The absence of spatial genetic structure indicated effective spore dispersal over short distances. Additionally, the cross-amplification of some D. aemula microsatellites makes them suitable for use in other Dryopteris taxa.