Analysis of the Localization of Schizosaccharomyces pombe Glucan Synthases in the Presence of the Antifungal Agent Caspofungin.

In recent years, invasive fungal infections have emerged as a common source of infections in immunosuppressed patients. All fungal cells are surrounded by a cell wall that is essential for cell integrity and survival. It prevents cell death and lysis resulting from high internal turgor pressure. Since the cell wall is not present in animal cells, it is an ideal target for selective invasive fungal infection treatments. The antifungal family known as echinocandins, which specifically inhibit the synthesis of the cell wall ß(13)glucan, has been established as an alternative treatment for mycoses. To explore the mechanism of action of these antifungals, we analyzed the cell morphology and glucan synthases localization in Schizosaccharomyces pombe cells during the initial times of growth in the presence of the echinocandin drug caspofungin. S. pombe are rod-shaped cells that grow at the poles and divide by a central division septum. The cell wall and septum are formed by different glucans, which are synthesized by four essential glucan synthases: Bgs1, Bgs3, Bgs4, and Ags1. Thus, S. pombe is not only a perfect model for studying the synthesis of the fungal ß(1-3)glucan, but also it is ideal for examining the mechanisms of action and resistance of cell wall antifungals. Herein, we examined the cells in a drug susceptibility test in the presence of either lethal or sublethal concentrations of caspofungin, finding that exposure to the drug for long periods at high concentrations (>10 µg/mL) induced cell growth arrest and the formation of rounded, swollen, and dead cells, whereas low concentrations (<10 µg/mL) permitted cell growth with a mild effect on cell morphology. Interestingly, short-term treatments with either high or low concentrations of the drug induced effects contrary to those observed in the susceptibility tests. Thus, low drug concentrations induced a cell death phenotype that was not observed at high drug concentrations, which caused transient fungistatic cell growth arrest. After 3 h, high concentrations of the drug caused the following: (i) a decrease in the GFP-Bgs1 fluorescence level; (ii) altered locations of Bgs3, Bgs4, and Ags1; and (iii) a simultaneous accumulation of cells with calcofluor-stained incomplete septa, which at longer times resulted in septation uncoupling from plasma membrane ingression. The incomplete septa revealed with calcofluor were found to be complete when observed via the membrane-associated GFP-Bgs or Ags1-GFP. Finally, we found that the accumulation of incomplete septa depended on Pmk1, the last kinase of the cell wall integrity pathway.

X-ray computed tomography evaluations of additive manufactured multimaterial composites.

Additive Manufacturing (AM) often produces complex engineered structures by precisely distributing materials in a layer-by-layer fashion. Multimaterial AM is a particularly flexible technique able to combine a range of hard and soft materials to produce designed composites. Critically, the design of AM multimaterial structures requires the development of precise three-dimensional (3D) computed aided design (CAD) files. While such digital design is heavily used, techniques able to validate the physically manufactured composite against the digital design from which it is generated are lacking for AM, especially as any evaluations must be able to distinguish material variation across the 3D space. Nowadays, there is a growing interest in volumetric tools that can provide topological information hidden by the surface of shaped materials. So far, technologies such as Optical microscopy (OM), Scanning Electron Microscopy (SEM), and Coordinate Measuring Machine (CMM) have paved the way into the metrology field to measure the external geometry of physical objects. Currently, alongside conventional metrology tools, X-ray computed tomography (XCT) is emerging to measure the subsurface of the objects but maintaining the integrity of the probed samples. Thereby, the volumetric nature of the XCT investigations and its associated imaging techniques, ensure 3D quantitative measurements comparable to the output data from 2D metrology tools, but above all, supply the missing subsurface description for an exhaustive metrology study. The reward associated with XCT applied to multimaterial AM is a map reflecting the fabricated distribution of materials following CAD, with the benefits of better understanding the mechanical interplay within phases, hence, describing the hidden processes as well as the changes in phases due to a range of mechanical or chemical phenomena. In this study, a nondestructive approach using X-ray computed tomography (XCT) is used to fully evaluate the 3D distribution of multimaterials from an AM process. Specifically, two diverse hard and soft materials are alternatively produced in the form of a fibre embedded in a matrix via ink-jet printing. XCT coupled with imaging evaluation were able to distinguish between the differing materials and, importantly, to demonstrate a reduction in the expected fabricated volumes when compared to the respective CAD designs. LAY DESCRIPTION: Additive Manufacturing (AM) has recently become important in producing complex engineered structures. Using 3D CAD files and/or reconstructed data sets from imaging, hard and soft materials are manufactured independently or in combination, according to geometrical features and shapes in the input data. However, the evaluation of the resultant manufactured parts in comparison with the original 3D drawing is currently lacking. In this sense, X-ray computed tomography (XCT) provides an important metrology tool for mono and multimaterial AM. In this work a volumetric metrology investigation is proposed using higher resolution XCT to provide 3D information comparable to that of the 3D CAD drawings. A commercial high-resolution multijetting material printer (ProJet 5500X, 3D Systems, USA) is used to manufacture single fibre composites, through a complementary deposition of photo sensible polymers. Hard and soft plastics are produced using a UV curable step, resulting in materials of similar attenuation under an X-ray probe. A critical aim of the evaluations is the potential for XCT to distinguish between different UV curable 3D printing materials.

Analysis and application of a suite of recombinant endo-ß(1,3)-D-glucanases for studying fungal cell walls.

BACKGROUND: The fungal cell wall is an essential and robust external structure that protects the cell from the environment. It is mainly composed of polysaccharides with different functions, some of which are necessary for cell integrity. Thus, the process of fractionation and analysis of cell wall polysaccharides is useful for studying the function and relevance of each polysaccharide, as well as for developing a variety of practical and commercial applications. This method can be used to study the mechanisms that regulate cell morphogenesis and integrity, giving rise to information that could be applied in the design of new antifungal drugs. Nonetheless, for this method to be reliable, the availability of trustworthy commercial recombinant cell wall degrading enzymes with non-contaminating activities is vital. RESULTS: Here we examined the efficiency and reproducibility of 12 recombinant endo-ß(1,3)-D-glucanases for specifically degrading the cell wall ß(1,3)-D-glucan by using a fast and reliable protocol of fractionation and analysis of the fission yeast cell wall. This protocol combines enzymatic and chemical degradation to fractionate the cell wall into the four main polymers: galactomannoproteins, α-glucan, ß(1,3)-D-glucan and ß(1,6)-D-glucan. We found that the GH16 endo-ß(1,3)-D-glucanase PfLam16A from Pyrococcus furiosus was able to completely and reproducibly degrade ß(1,3)-D-glucan without causing the release of other polymers. The cell wall degradation caused by PfLam16A was similar to that of Quantazyme, a recombinant endo-ß(1,3)-D-glucanase no longer commercially available. Moreover, other recombinant ß(1,3)-D-glucanases caused either incomplete or excessive degradation, suggesting deficient access to the substrate or release of other polysaccharides. CONCLUSIONS: The discovery of a reliable and efficient recombinant endo-ß(1,3)-D-glucanase, capable of replacing the previously mentioned enzyme, will be useful for carrying out studies requiring the digestion of the fungal cell wall ß(1,3)-D-glucan. This new commercial endo-ß(1,3)-D-glucanase will allow the study of the cell wall composition under different conditions, along the cell cycle, in response to environmental changes or in cell wall mutants. Furthermore, this enzyme will also be greatly valuable for other practical and commercial applications such as genome research, chromosomes extraction, cell transformation, protoplast formation, cell fusion, cell disruption, industrial processes and studies of new antifungals that specifically target cell wall synthesis.

Synthesis of novel pyrazolo[3,4-b]quinolinebisphosphonic acids and an unexpected intramolecular cyclization and phosphonylation reaction.

Novel pyrazolo[3,4-b]quinoline α-ketophosphonic and hydroxymethylenebisphosphonic acid compounds were synthesized using different methodologies, starting from 2-chloro-3-formylquinoline 1. New phosphonic acid compounds were obtained as N-1 derivatives with a side chain with 1 or 3 (n = 1 or 3) methylene groups. All phosphonic acid compounds and their corresponding ester and carboxylic acid precursors were fully characterized, and their structures elucidated by spectroscopic data, using NMR techniques and infrared and high-resolution mass spectroscopy. During the process to obtain the N-1 substituted derivative with two methylene groups (n = 2) in the side chain, an unexpected addition-cyclization cascade reaction was observed, involving the phosphonylation of an aromatic ring and the formation of a new six-member lactam ring to afford a tetracyclic ring system. This was an unexpected result since other pyrazolo[3,4-b]quinoline derivatives and all corresponding pyrazolo[3,4-b]pyridine derivatives already prepared, under similar experimental conditions, did not undergo this reaction. This domino reaction occurs with different phosphite reagents but only affords the six-member ring. The spectroscopic data allowed the identification of the new synthesized tetracyclic compounds and the X-ray diffraction data of compound 11 enabled the confirmation of the proposed structures.

The ancient claudin Dni2 facilitates yeast cell fusion by compartmentalizing Dni1 into a membrane subdomain.

Dni1 and Dni2 facilitate cell fusion during mating. Here, we show that these proteins are interdependent for their localization in a plasma membrane subdomain, which we have termed the mating fusion domain. Dni1 compartmentation in the domain is required for cell fusion. The contribution of actin, sterol-dependent membrane organization, and Dni2 to this compartmentation was analysed, and the results showed that Dni2 plays the most relevant role in the process. In turn, the Dni2 exit from the endoplasmic reticulum depends on Dni1. These proteins share the presence of a cysteine motif in their first extracellular loop related to the claudin GLWxxC(8-10 aa)C signature motif. Structure-function analyses show that mutating each Dni1 conserved cysteine has mild effects, and that only simultaneous elimination of several cysteines leads to a mating defect. On the contrary, eliminating each single cysteine and the C-terminal tail in Dni2 abrogates Dni1 compartmentation and cell fusion. Sequence alignments show that claudin trans-membrane helixes bear small-XXX-small motifs at conserved positions. The fourth Dni2 trans-membrane helix tends to form homo-oligomers in Escherichia plasma membrane, and two concatenated small-XXX-small motifs are required for efficient oligomerization and for Dni2 export from the yeast endoplasmic reticulum. Together, our results strongly suggest that Dni2 is an ancient claudin that blocks Dni1 diffusion from the intercellular region where two plasma membranes are in close proximity, and that this function is required for Dni1 to facilitate cell fusion.

Genetic diversity and differentiation patterns in Micromeria from the Canary Islands are congruent with multiple colonization dynamics and the establishment of species syngameons.

BACKGROUND: Especially on islands closer to the mainland, such as the Canary Islands, different lineages that originated by multiple colonization events could have merged by hybridization, which then could have promoted radiation events (Herben et al., J Ecol 93: 572-575, 2005; Saunders and Gibson, J Ecol 93: 649-652, 2005; Caujapé-Castells, Jesters, red queens, boomerangs and surfers: a molecular outlook on the diversity of the Canarian endemic flora, 2011). This is an alternative to the scenario where evolution is mostly driven by drift (Silvertown, J Ecol 92: 168-173, 2004; Silvertown et al., J Ecol 93: 653-657, 2005). In the former case hybridization should be reflected in the genetic structure and diversity patterns of island species. In the present work we investigate Micromeria from the Canary Islands by extensively studying their phylogeographic pattern based on 15 microsatellite loci and 945 samples. These results are interpreted according to the hypotheses outlined above. RESULTS: Genetic structure assessment allowed us to genetically differentiate most Micromeria species and supported their current classification. We found that populations on younger islands were significantly more genetically diverse and less differentiated than those on older islands. Moreover, we found that genetic distance on younger islands was in accordance with an isolation-by-distance pattern, while on the older islands this was not the case. We also found evidence of introgression among species and islands. CONCLUSIONS: These results are congruent with a scenario of multiple colonizations during the expansion onto new islands. Hybridization contributes to the grouping of multiple lineages into highly diverse populations. Thus, in our case, islands receive several colonization events from different sources, which are combined into sink populations. This mechanism is in accordance with the surfing syngameon hypothesis. Contrary to the surfing syngameon current form, our results may reflect a slightly different effect: hybridization might always be related to colonization within the archipelago as well, making initial genetic diversity to be high to begin with. Thus the emergence of new islands promotes multiple colonization events, contributing to the establishment of hybrid swarms that may enhance adaptive ability and radiation events. With time, population sizes grow and niches start to fill. Consequently, gene-flow is not as effective at maintaining the species syngameon, which allows genetic differentiation and reproductive isolation to be established between species. This process contributes to an even further decrease in gene-flow between species.

The AP-2 complex is required for proper temporal and spatial dynamics of endocytic patches in fission yeast.

In metazoans the AP-2 complex has a well-defined role in clathrin-mediated endocytosis. By contrast, its direct role in endocytosis in unicellular eukaryotes has been questioned. Here, we report co- immunoprecipitation between the fission yeast AP-2 component Apl3p and clathrin, as well as the genetic interactions between apl3Δ and clc1 and sla2Δ/end4Δ mutants. Furthermore, a double clc1 apl3Δ mutant was found to be defective in FM4-64 uptake. In an otherwise wild-type strain, apl3Δ cells exhibit altered dynamics of the endocytic sites, with a heterogeneous and extended lifetime of early and late markers at the patches. Additionally, around 50% of the endocytic patches exhibit abnormal spatial dynamics, with immobile patches and patches that bounce backwards to the cell surface, showing a pervasive effect of the absence of AP-2. These alterations in the endocytic machinery result in abnormal cell wall synthesis and morphogenesis. Our results complement those found in budding yeast and confirm that a direct role of AP-2 in endocytosis has been conserved throughout evolution.

Interpersonal sensitivity and functioning impairment in youth at ultra-high risk for psychosis.

A personality trait that often elicits poor and uneasy interpersonal relationships is interpersonal sensitivity. The aim of the present study was to explore the relationship between interpersonal sensitivity and psychosocial functioning in individuals at ultra-high risk for psychosis as compared to help-seeking individuals who screened negative for an ultra-high risk of psychosis. A total sample of 147 adolescents and young adult who were help seeking for emerging mental health problems participated in the study. The sample was divided into two groups: 39 individuals who met criteria for an ultra-high-risk mental state (UHR), and 108 (NS). The whole sample completed the Interpersonal Sensitivity Measure (IPSM) and the Global Functioning: Social and Role Scale (GF:SS; GF:RS). Mediation analysis was used to explore whether attenuated negative symptoms mediated the relationship between interpersonal sensitivity and social functioning. Individuals with UHR state showed higher IPSM scores and lower GF:SS and GF:RS scores than NS participants. A statistically negative significant correlation between two IPSM subscales (Interpersonal Awareness and Timidity) and GF:SS was found in both groups. Our results also suggest that the relationship between the aforementioned aspects of interpersonal sensitivity and social functioning was not mediated by negative prodromal symptoms. This study suggests that some aspects of interpersonal sensitivity were associated with low level of social functioning. Assessing and treating interpersonal sensitivity may be a promising therapeutic target to improve social functioning in young help-seeking individuals.

Fission yeast Bgs1 glucan synthase participates in the control of growth polarity and membrane traffic.

Rod-shaped fission yeast grows through cell wall expansion at poles and septum, synthesized by essential glucan synthases. Bgs1 synthesizes the linear ß(1,3)glucan of primary septum at cytokinesis. Linear ß(1,3)glucan is also present in the wall poles, suggesting additional Bgs1 roles in growth polarity. Our study reveals an essential collaboration between Bgs1 and Tea1-Tea4, but not other polarity factors, in controlling growth polarity. Simultaneous absence of Bgs1 function and Tea1-Tea4 causes complete loss of growth polarity, spread of other glucan synthases, and spherical cell formation, indicating this defect is specifically due to linear ß(1,3)glucan absence. Furthermore, linear ß(1,3)glucan absence induces actin patches delocalization and sterols spread, which are ultimately responsible for the growth polarity loss without Tea1-Tea4. This suggests strong similarities in Bgs1 functions controlling actin structures during cytokinesis and polarized growth. Collectively, our findings unveil that cell wall ß(1,3)glucan regulates polarized growth, like the equivalent extracellular matrix in neuronal cells.

The FN3 and BRCT motifs in the exomer component Chs5p define a conserved module that is necessary and sufficient for its function.

Chs5p is a component of the exomer, a coat complex required to transport the chitin synthase Chs3p from the trans-Golgi network to the plasma membrane. The Chs5p N-terminal region exhibits fibronectin type III (FN3) and BRCT domains. FN3 domains are present in proteins that mediate adhesion processes, whereas BRCT domains are involved in DNA repair. Several fungi--including Schizosaccharomyces pombe, which has no detectable amounts of chitin--have proteins similar to Chs5p. Here we show that the FN3 and BRCT motifs in Chs5p behave as a module that is necessary and sufficient for Chs5p localization and for cargo delivery. The N-terminal regions of S. cerevisiae Chs5p and S. pombe Cfr1p are interchangeable in terms of Golgi localization, but not in terms of exomer assembly, showing that the conserved function of this module is protein retention in this organelle and that the interaction between the exomer components is organism-specific.

Patients with initial nodal involvement due to breast cancer who have received neoadjuvant chemotherapy: Combined sentinel node-radioguided surgery of the pathological node.

OBJECTIVE: To study the feasibility and usefulness of ultrasound-guided pre-chemotherapy marking of pathologic lymph node followed by sentinel lymph node biopsy (SLNB)-pathologic node radioguided biopsy (ROLL) combined technique, in axillary involvement breast cancer patients undergoing neoadjuvant chemotherapy (NACT). MATERIAL AND METHODS: Prospective diagnostic study of 30 patients with breast cancer and cN1 axillary staging with NACT indication. Before NACT, the biopsied node was marked with a clip (MBN). After NACT an ultrasound was performed and in case of good response a SLNB (99mTc-nanocolloids) plus targeted axillary dissection MBN ROLL biopsy (99mTc-albumin macroaggregates) was performed. Axillary lymph node dissection (ALND) was performed if SLNB and/or MBN were positive for tumor cells. The localization-removal rate of the sentinel lymph node (SLN) and MBN were evaluated. False-negative rate (FNR) and positive predictive value (PPV) of SLNB alone were also evaluated. RESULTS: Thirty patients were included in the study. SLN could be detected in all patients while MBN was successfully removed in 27 (90%). The SLN coincided with MBN in 15 patients (50%). In 12 patients SLNB was negative while MBN positive, leading to a FNR of 44.4% for SLNB alone. We found a PPV of 37% for the SLNB. In 5 patients (18.5%) both SLNB and MBN were negative, avoiding ALND. CONCLUSIONS: SLNB-MBN radioguided biopsy ROLL combined technique is a useful and accessible procedure for accurate axillary restaging after NACT, avoiding the high rate of FNR of SLNB alone in this group of patients and avoiding a great number of ALND.

Natural products targeting the synthesis of ß(1,3)-D-glucan and chitin of the fungal cell wall. Existing drugs and recent findings.

BACKGROUND: During the last three decades systemic fungal infections associated to immunosuppressive therapies have become a serious healthcare problem. Clinical development of new antifungals is an urgent requirement. Since fungal but not mammalian cells are encased in a carbohydrate-containing cell wall, which is required for the growth and viability of fungi, the inhibition of cell wall synthesizing machinery, such as ß(1,3)-D-glucan synthases (GS) and chitin synthases (CS) that catalyze the synthesis of ß(1-3)-D-glucan and chitin, respectively, represent an ideal mode of action of antifungal agents. Although the echinocandins anidulafungin, caspofungin and micafungin are clinically well-established GS inhibitors for the treatment of invasive fungal infections, much effort must still be made to identify inhibitors of other enzymes and processes involved in the synthesis of the fungal cell wall. PURPOSE: Since natural products (NPs) have been the source of several antifungals in clinical use and also have provided important scaffolds for the development of semisynthetic analogues, this review was devoted to investigate the advances made to date in the discovery of NPs from plants that showed capacity of inhibiting cell wall synthesis targets. The chemical characterization, specific target, discovery process, along with the stage of development are provided here. METHODS: An extensive systematic search for NPs against the cell wall was performed considering all the articles published until the end of 2020 through the following scientific databases: NCBI PubMed, Scopus and Google Scholar and using the combination of the terms "natural antifungals" and "plant extracts" with "fungal cell wall". RESULTS: The first part of this review introduces the state of the art of the structure and biosynthesis of the fungal cell wall and considers exclusively those naturally produced GS antifungals that have given rise to both existing semisynthetic approved drugs and those derivatives currently in clinical trials. According to their chemical structure, natural GS inhibitors can be classified as 1) cyclic lipopeptides, 2) glycolipids and 3) acidic terpenoids. We also included nikkomycins and polyoxins, NPs that inhibit the CS, which have traditionally been considered good candidates for antifungal drug development but have finally been discarded after enduring unsuccessful clinical trials. Finally, the review focuses in the most recent findings about the growing field of plant-derived molecules and extracts that exhibit activity against the fungal cell wall. Thus, this search yielded sixteen articles, nine of which deal with pure compounds and seven with plant extracts or fractions with proven activity against the fungal cell wall. Regarding the mechanism of action, seven (44%) produced GS inhibition while five (31%) inhibited CS. Some of them (56%) interfered with other components of the cell wall. Most of the analyzed articles refer to tests carried out in vitro and therefore are in early stages of development. CONCLUSION: This report delivers an overview about both existing natural antifungals targeting GS and CS activities and their mechanisms of action. It also presents recent discoveries on natural products that may be used as starting points for the development of potential selective and non-toxic antifungal drugs.

Dispersal ability, habitat characteristics, and sea-surface circulation shape population structure of Cingula trifasciata (Gastropoda: Rissoidae) in the remote Azores Archipelago.

BACKGROUND: In the marine realm, dispersal ability is among the major factors shaping the distribution of species. In the Northeast Atlantic Ocean, the Azores Archipelago is home to a multitude of marine invertebrates which, despite their dispersal limitations, maintain gene flow among distant populations, with complex evolutionary and biogeographic implications. The mechanisms and factors underlying the population dynamics and genetic structure of non-planktotrophic gastropods within the Azores Archipelago and related mainland populations are still poorly understood. The rissoid Cingula trifasciata is herewith studied to clarify its population structure in the Northeast Atlantic Ocean and factors shaping it, with a special focus in intra-archipelagic dynamics. RESULTS: Coupling microsatellite genotyping by amplicon sequencing (SSR-GBAS) and mitochondrial datasets, our results suggest the differentiation between insular and continental populations of Cingula trifasciata, supporting previously raised classification issues and detecting potential cryptic diversity. The finding of connectivity between widely separated populations was startling. In unique ways, dispersal ability, habitat type, and small-scale oceanographic currents appear to be the key drivers of C. trifasciata's population structure in the remote Azores Archipelago. Dispersal as non-planktotrophic larvae is unlikely, but its small-size adults easily engage in rafting. Although the typical habitat of C. trifasciata, with low hydrodynamics, reduces the likelihood of rafting, individuals inhabiting algal mats are more prone to dispersal. Sea-surface circulation might create dispersal pathways for rafts, even between widely separated populations/islands. CONCLUSIONS: Our results show that gene flow of a marine non-planktotrophic gastropod within a remote archipelago can reveal unanticipated patterns, such that the understanding of life in such areas is far from well-understood. We expect this work to be the starting of the application of SSR-GBAS in other non-model marine invertebrates, providing insights on their population dynamics at distinct geographical scales and on hidden diversity. How transversal is the role played by the complex interaction between functional traits, ecological features, and sea-surface circulation in the population structure of marine invertebrates can be further addressed by expanding this approach to more taxa.

Echinocandin Drugs Induce Differential Effects in Cytokinesis Progression and Cell Integrity.

Fission yeast contains three essential ß(1,3)-D-glucan synthases (GSs), Bgs1, Bgs3, and Bgs4, with non-overlapping roles in cell integrity and morphogenesis. Only the bgs4+ mutants pbr1-8 and pbr1-6 exhibit resistance to GS inhibitors, even in the presence of the wild-type (WT) sequences of bgs1+ and bgs3+. Thus, Bgs1 and Bgs3 functions seem to be unaffected by those GS inhibitors. To learn more about echinocandins' mechanism of action and resistance, cytokinesis progression and cell death were examined by time-lapse fluorescence microscopy in WT and pbr1-8 cells at the start of treatment with sublethal and lethal concentrations of anidulafungin, caspofungin, and micafungin. In WT, sublethal concentrations of the three drugs caused abundant cell death that was either suppressed (anidulafungin and micafungin) or greatly reduced (caspofungin) in pbr1-8 cells. Interestingly, the lethal concentrations induced differential phenotypes depending on the echinocandin used. Anidulafungin and caspofungin were mostly fungistatic, heavily impairing cytokinesis progression in both WT and pbr1-8. As with sublethal concentrations, lethal concentrations of micafungin were primarily fungicidal in WT cells, causing cell lysis without impairing cytokinesis. The lytic phenotype was suppressed again in pbr1-8 cells. Our results suggest that micafungin always exerts its fungicidal effect by solely inhibiting Bgs4. In contrast, lethal concentrations of anidulafungin and caspofungin cause an early cytokinesis arrest, probably by the combined inhibition of several GSs.

[Concept of gender, mainstreaming gender and equal opportunities]. / Concepto género, mainstreaming igualdad oportunidades.

INTRODUCTION: The concept of gender is related to the concepts of socially constructed masculinity and femininity EXPOSURE: Gender Studies that take into account this concept is useful for all disciplines of both health and social, arise in the eighteenth century and incorporated in Spain in the 80s of the twentieth century Agrarian societies with patriarchy changed the social status of women making them dependent. The industrial revolution brought wage employment for women that did not result in positive consequences for overall health or your mental health. CONCLUSIONS: The gender mainstreaming boost since 1985 trying to build a future strategy for the advancement of women, the term for the first time at the Third UN World Conference on Women--Nairobi, 1985--where the government's commitment to acquire implementation of the forward-looking strategies for the advancement of women, which include all the organizations under the UN. Will be in Beijing 1995--Fourth World Conference on Women United Nations--where it was found that equality for women was still limited and not fully protected against discrimination. Policies for gender equality since the United Nations, European Union and Spain have been happening since 1975. Spain has been important in the creation of the Women's Institute in 1983 and the four equal opportunity plans that have been happening since 1988 (1988-1990, 1993-1995, 1997-2000, 2003-2006).

Activation of mGlu2/3 metabotropic glutamate receptors negatively regulates the stimulation of inositol phospholipid hydrolysis mediated by 5-hydroxytryptamine2A serotonin receptors in the frontal cortex of living mice.

The interaction between 5-hydroxytryptamine(2A) (5-HT(2A)) serotonin receptors and metabotropic glutamate (mGlu) 2/3 receptors underlies the antipsychotic activity of mGlu2/3 receptor agonists in experimental animals and humans. The molecular nature of this interaction is only partially known. We here report for the first time that pharmacological activation of mGlu2/3 receptors attenuates the stimulation of polyphosphoinositide (PI) hydrolysis mediated by 5-HT(2A) receptors in the frontal cortex of living mice. Mice were injected intracerebroventricularly with [myo-(3)H]inositol and treated with drugs 1 h after a pretreatment with lithium, which blocks the conversion of inositol monophosphate into free inositol. Systemic injection of the mGlu2/3 receptor agonist (-)-2-oxa-4-aminocyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268) inhibited the stimulation of PI hydrolysis induced by the hallucinogenic 5-HT(2A) receptor agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) without affecting the stimulation by mGlu1/5 or muscarinic receptors. The action of LY379268 was prevented by the preferential mGlu2/3 receptor antagonist (2S,1'S,2'S)-2-(9-xanthylmethyl)-2-(2'-carboxycyclopropyl)glycine (LY341495). N-(4'-cyano-biphenyl-3-yl)-N-(3-pyridinylmethyl)-ethanesulfonamide hydrochloride (LY566332), a selective mGlu2 receptor enhancer, also reduced DOI-stimulated PI hydrolysis when combined with subthreshold doses of LY379268. Systemic LY379268 inhibited DOI-stimulated PI hydrolysis in mice lacking either mGlu2 or mGlu3 receptors but was inactive in double mGlu2/mGlu3 receptor knockout mice, suggesting that both mGlu2 and mGlu3 receptors interact with 5-HT(2A) receptors. Surprisingly, contrasting results were obtained in cortical slice preparations, where LY379268 amplified both DOI- and 3,5-dihydroxyphenylglycine-stimulated PI hydrolysis. Amplification was abrogated by the mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl)pyridine, suggesting that experiments in brain slices are biased by an additional component of receptor-stimulated PI hydrolysis. This highlights the importance of in vivo models for the study of the interaction between 5-HT(2A) and mGlu2/3 receptors.

The Nf2 tumor suppressor, merlin, functions in Rac-dependent signaling.

Mutations in the neurofibromatosis type II (NF2) tumor suppressor predispose humans and mice to tumor development. The study of Nf2+/- mice has demonstrated an additional effect of Nf2 loss on tumor metastasis. The NF2-encoded protein, merlin, belongs to the ERM (ezrin, radixin, and moesin) family of cytoskeleton:membrane linkers. However, the molecular basis for the tumor- and metastasis- suppressing activity of merlin is unknown. We have now placed merlin in a signaling pathway downstream of the small GTPase Rac. Expression of activated Rac induces phosphorylation and decreased association of merlin with the cytoskeleton. Furthermore, merlin overexpression inhibits Rac-induced signaling in a phosphorylation-dependent manner. Finally, Nf2-/- cells exhibit characteristics of cells expressing activated alleles of Rac. These studies provide insight into the normal cellular function of merlin and how Nf2 mutation contributes to tumor initiation and progression.

Synthesis and evaluation of rifabutin analogs against Mycobacterium avium and H(37)Rv, MDR and NRP Mycobacterium tuberculosis.

Clinical utility of rifabutin 1 (RBT), a potent antibiotic used in multidrug regimens for tuberculosis (TB) as well as for infections caused by Mycobacterium avium complex (MAC), has been hampered due to dose-limiting toxicity. RBT analogs 2-11 were synthesized and evaluated against M. avium 1581 and Mycobacterium tuberculosis susceptible and resistant strains in vitro. A selection of candidates were also assayed against non-replicating persistent (NRP) M. tuberculosis. Subsequent in vivo studies with the best preclinical candidate drugs 5 and 8, in a model of progressive pulmonary tuberculosis of Balb/C mice infected either with H(37)Rv drug-sensible strain or with multidrug resistant (MDR) clinical isolates, resistant to all primary antibiotics including rifampicin, were performed. The results disclosed here suggest that 5 and 8 have potential for clinical application.

The fungal cell wall as a target for the development of new antifungal therapies.

In the past three decades invasive mycoses have globally emerged as a persistent source of healthcare-associated infections. The cell wall surrounding the fungal cell opposes the turgor pressure that otherwise could produce cell lysis. Thus, the cell wall is essential for maintaining fungal cell shape and integrity. Given that this structure is absent in host mammalian cells, it stands as an important target when developing selective compounds for the treatment of fungal infections. Consequently, treatment with echinocandins, a family of antifungal agents that specifically inhibits the biosynthesis of cell wall (1-3)ß-D-glucan, has been established as an alternative and effective antifungal therapy. However, the existence of many pathogenic fungi resistant to single or multiple antifungal families, together with the limited arsenal of available antifungal compounds, critically affects the effectiveness of treatments against these life-threatening infections. Thus, new antifungal therapies are required. Here we review the fungal cell wall and its relevance in biotechnology as a target for the development of new antifungal compounds, disclosing the most promising cell wall inhibitors that are currently in experimental or clinical development for the treatment of some invasive mycoses.

Morphological and Mechanical Biomimetic Bone Structures.

Cortical bone is an example of a mineralized tissue containing a compositional distribution of hard and soft phases in 3-dimensional space for mechanical function. X-ray computed tomography (XCT) is able to describe this compositional and morphological complexity but methods to provide a physical output with comparable mechanical function is lacking. A workflow is presented here to establish a method of using high contrast XCT to establish a virtual model of cortical bone that is manufactured using a multiple material capable 3D printer. Resultant 3D printed structures were produced based on more and less remodelled bone designs exhibiting a range of secondary osteon density. Variation in resultant mechanical properties of the 3D printed composite structures for each bone design was achieved using a combination of material components and reasonable prediction of elastic modulus provided using a Hashin-Shtrikman approach. The ability to 3D print composite structures using high contrast XCT to distinguish between compositional phases in a biological structure promises improved anatomical models as well as next-generation mechano-mimetic implants.