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[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‑β(1,3)‑d‑glucanases for specifically degrading the cell wall β(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, β(1,3)‑d‑glucan and β(1,6)‑d‑glucan. We found that the GH16 endo‑β(1,3)‑d‑glucanase PfLam16A from Pyrococcus furiosus was able to completely and reproducibly degrade β(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‑β(1,3)‑d‑glucanase no longer commercially available. Moreover, other recombinant β(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‑β(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 β(1,3)‑d‑glucan. This new commercial endo‑β(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 othern 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, This work was supported by grants PGC2018-098924-B-I00 (Spanish Ministry of Science and Innovation, MICINN , Spain; and the European Regional Developmental Fund, FEDER, EU), and CSI150P20 and “Escalera de Excelencia” CLU-2017-03 (Regional Government of Castile and Leon, JCYL, Spain; and the European Regional Developmental Fund, FEDER, EU).

Fission yeast contains three essential β(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., This research was funded by grants PGC2018-098924-B-I00 (the Spanish Ministry of Science and Innovation, MICINN, Spain; the European Regional Developmental Fund, FEDER, EU), and CSI150P20 and “Escalera de Excelencia” CLU-2017-03 (Regional Government of Castile and Leon, JCYL, Spain; and the European Regional Developmental Fund, FEDER, EU).

[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 β(1,3)-D-glucan synthases (GS) and chitin synthases (CS) that catalyze the synthesis of β(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., J.C.G.C., M.A.C. and J.C.R. acknowledge Ministerio de Ciencia e Innovación (MICINN; PGC2018-098924-B-I00) and Junta de Castilla y León/FEDER (CSI150P20 and "Escalera de Excelencia" CLU-2017-03), Spain, for funds. L.A.S. acknowledges Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) PICT2016-1833 and Universidad Nacional de Rosario (UNR) 1BIO571 for funds. E.B. acknowledges Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) for a postdoctoral fellowship. L.A.S. is member of the CONICET Researcher career. L.A.S. and E.B. belong to the teaching staff of Pharmacognosy area.

The cell wall is a structure external to the plasma membrane that is essential for the survival of the fungi. This polysaccharidic structure confers resistance to the cell internal turgor pressure and protection against mechanical injury. The fungal wall is also responsible for the shape of these organisms due to different structural polysaccharides, such as β-(1,3)-glucan, which form fibers and confer rigidity to the cell wall. These polysaccharides are not present in animal cells and therefore they constitute excellent targets for antifungal chemotherapies. Cell wall damage leads to the activation of MAPK signaling pathways, which respond to the damage by activating the repair of the wall and the maintenance of the cell integrity. Fission yeast Schizosaccharomyces pombe is a model organism for the study morphogenesis, cell wall, and how different inputs might regulate this structure. We present here a short overview of the fission yeast wall composition and provide information about the main biosynthetic activities that assemble this cell wall. Additionally, we comment the recent advances in the knowledge of the cell wall functions and discuss the role of the cell integrity MAPK signaling pathway in the regulation of fission yeast wall., This work was supported by grants BIO2015-69958-P and BFU2017-82423-P from MINECO, Spain and CSI068P17 from Junta de Castilla y León.

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)β-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., We thank Emma Keck for language revision. J.C.G.C acknowledges support from a Postdoctoral Contract granted by the University of Salamanca, Spain. This publication was supported by the grants BIO2015-69958-P from the Ministry of Science, Innovation and Universities (MICIU) (http://www.ciencia.gob.es/) and grants CSI068P17 and Escalera de Excelencia CLU-2017-03 from the Regional Government of Castile and Leon (JCyL/FEDER) and the European Regional Development Fund (ERDF) (https://www.jcyl.es/).

Background: Phytolacca tetramera is an endemic plant from Argentina that is currently at serious risk because its environment is subjected to a high anthropic impact. A previous study has shown that berry extracts obtained from this plant display antifungal activity against multiple human-pathogenic fungi when tested with a non-standardized method. Further evidences of the antifungal properties of other parts of the plant and studies of mechanism of antifungal action of the antifungal chemically characterized extracts are required. Purpose: This study aimed to gain further evidence of the antifungal activity of P. tetramera berry, leaf and root extracts in order to find the most active extract to be developed as an Herbal Medicinal Antifungal Product. The medicinal usefulness of P. tetramera extracts as antifungal agents will serve as an important support to create concience and carry out actions tending to the preservation of this threatened species and its environment. Materials and methods: Chemical analysis of all P. tetramera extracts, including quantitation of selected markers, was performed through UHPLC-ESI-MS/MS and UPLC-ESI-MS techniques according to the European Medicines Agency (EMA). The antifungal activity of the quantified extracts was tested with the standardized CLSI microbroth dilution method against Candida spp. Antifungal mechanisms of the most active extract were studied by examination of morphological changes by phase-contrast and fluorescence microscopies and both, cellular and enzymatic assays targeting either the fungal membrane or the cell wall. Results: The antifungal activity of twelve P. tetramera extracts was tested against Candida albicans and Candida glabrata. The dichloromethane extract from berries (PtDEb) showed the best activity. Phytolaccagenin (PhytG) and phytolaccoside B (PhytB) were selected as the main active markers for the antifungal P. tetramera extracts. The quantitation of these active markers in all extracts showed that PtDEb possessed the highest amount of PhytG and PhytB. Finally, studies on the mechanism of antifungal action showed that the most active PtDEb extract produces morphological changes compatible with a damage of the cell wall and/or the plasma membrane. Cellular and enzymatic assays showed that PtDEb would not damage the fungal cell wall by itself, but would alter the plasma membrane. In agreement, PtDEb was found to bind to ergosterol, the main sterol of the fungal plasma membrane. Conclusion: Studies of the anti-Candida activity of P. tetramera extracts led to the selection of PtDEb as the most suitable extract, confirming the antifungal properties of the threatened species P. tetramera. The new data give a valuable reason for the definitive protection of this sp. and its natural environment thus allowing further studies for the future development of an Herbal Medicinal Antifungal Product., Susana A. Zacchino and Laura Svetaz (L.S.) acknowledge Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), PICT2014-1170 and PICT2016-1833 and National University of Rosario, Argentina (UNR) for funds. J.C.R. acknowledges MINECO (BIO2015-69958-P), Junta de Castilla y León/FEDER (CSI068P17 and >Escalera de Excelencia> CLU-2017-03), Spain, for funds. Estefanía Butassi (E.B.) acknowledges Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) for doctoral and post-doctoral fellowships. L.S. is a member of the CONICET Researcher career; L.S. and E.B. belong to the teaching staff of Pharmacognosy area. Jean-Luc Wolfender and Shuaizhen Zhou (S.Z.) acknowledge Sino Swiss Science and Technology Cooperation Program (SSSTC) IP18-092011 for the funding of the post-doctoral stay of S.Z. The MEDINA authors Francisca Vicente, Caridad Díaz and José Pérez-del Palacio disclosed the receipt of financial support from Fundación MEDINA, a public-private partnership of Merck Sharp & Dohme de España S.A./Universidad de Granada/Junta de Andalucía, Spain.

Background Laparoscopic nephrectomy for living donors is the current procedure of choice. Hand-assisted laparoscopic donor nephrectomy (HALDN) is the variation of this technique currently used in our institution. Though the advantages and disadvantages have been described for this procedure, the graft function compared with open surgery has been shown to be equal. We compared the outcomes of patients undergoing the former standard open donor nephrectomy (ODN) versus the current HALDN technique. Methods In this retrospective, comparative, and analytic study we reviewed our institutional database of renal transplantation procedures from January 2005 to April 2011 for perioperative variables and 1-year follow-up data. Donor renal function was evaluated with serum creatinine concentrations and estimated glomerular filtration rates with the Chronic Kidney Disease–Epidemiology formula. Complications were reported with the Clavien-Dindo classification. Results The 190 consecutive donors included 99 ODN and 91 HALDN, who did not show baseline differences. ODN had a shorter mean operative time (217 ± 57.5 vs 270 ± 60.1 minutes) and shorter warm ischemia time (2.12 ± 1.4 vs 4.62 ± 2.7 minutes). HALDN had less operative blood loss (274.4 ± 198.1 vs 202.99 ± 157.1 mL) and shorter in-hospital stay (5.58 ± 2.2 vs 4.23 ± 1.8 days). There were no significant differences in 30-day surgical complications or transfusion requirements. No graft loss was reported. No difference in renal function was observed between the groups at days 1–2 or months 1, 6, or 12 after nephrectomy. Conclusions Laparoscopic surgery has replaced conventional open surgery for living renal donors. HALDN is a safe and successful procedure compared with ODN. It is now the procedure of choice in our institution.

24 p.-7 fig., In fungal cells cytokinesis requires coordinated closure of a contractile actomyosin ring (CAR) and synthesis of a special cell wall structure known as the division septum. Many CAR proteins have been identified and characterized, but how these molecules interact with the septum synthesis enzymes to form the septum remains unclear. Our genetic study using fission yeast shows that cooperation between the paxillin homolog Pxl1, required for ring integrity, and Bgs1, the enzyme responsible for linear β(1,3)glucan synthesis and primary septum formation, is required for stable anchorage of the CAR to the plasma membrane before septation onset, and for cleavage furrow formation. Thus, lack of Pxl1 in combination with Bgs1 depletion, causes failure of ring contraction and lateral cell wall overgrowth towards the cell lumen without septum formation. We also describe here that Pxl1 concentration at the CAR increases during cytokinesis and that this increase depends on the SH3 domain of the F-BAR protein Cdc15. In consequence, Bgs1 depletion in cells carrying a cdc15ΔSH3 allele causes ring disassembly and septation blockage, as it does in cells lacking Pxl1. On the other hand, the absence of Pxl1 is lethal when Cdc15 function is affected, generating a large sliding of the CAR with deposition of septum wall material along the cell cortex, and suggesting additional functions for both Pxl1 and Cdc15 proteins. In conclusion, our findings indicate that CAR anchorage to the plasma membrane through Cdc15 and Pxl1, and concomitant Bgs1 activity, are necessary for CAR maintenance and septum formation in fission yeast., This work was supported by the Spanish Ministry of Science and Innovation (BFU2010-15641 and BFU2013-39394-P) to PP. JCR was financed by the Spanish Ministry of Science and Innovation (BIO2012-35372), and Junta de Castilla y León, Spain (CSI037U14).

Capítulo 9., Fungi have emerged worldwide as increasingly frequent causes of healthcare-associated infections. Invasive fungal infections can be life-threatening. However, the number of antifungal agents available and their use in therapy is very limited. Recently, a new family of specific fungal cell wall synthesis inhibitors has emerged as an alternative antifungal therapy and is gaining increasing relevance yearly. The cell wall is a multilayer dynamic structure, essential to the integrity and shape of the fungal cell, whose function is to counteract the osmotic forces that could otherwise produce fungal cell lysis. The cell wall is absent in nonfungal cells, therefore representing a useful target in discovering selective drugs for the treatment of fungal infections without causing toxicity in the host. Although fungi exhibit a considerable diversity in their cell wall structure, all present β(1,3)-, β(1,6)- and α(1,3)-glucans, chitin, and mannoproteins as their major cell wall components. Three different cell wall synthesis inhibitors of the lipopeptide family of echinocandins, named caspofungin, micafungin, and anidulafungin, are commercially available and new classes of cell wall synthesis inhibitors are emerging. This review provides an overview of what is so far known about the different classes of cell wall-affecting antifungal agents and their mechanism of action, offering new alternatives with clinical potential.

This article is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license)., Cytokinesis has been extensively studied in different models, but the role of the extracellular cell wall is less understood. Here we studied this process in fission yeast. The essential protein Bgs4 synthesizes the main cell wall ß(1,3)glucan. We show that Bgs4-derived ß(1,3)glucan is required for correct and stable actomyosin ring positioning in the cell middle, before the start of septum formation and anchorage to the cell wall. Consequently, ß(1,3)glucan loss generated ring sliding, oblique positioned rings and septa, misdirected septum synthesis indicative of relaxed rings, and uncoupling between a fast ring and membrane ingression and slow septum synthesis, suggesting that cytokinesis can progress with defective septum pushing and/or ring pulling forces. Moreover, Bgs4-derived ß(1,3)glucan is essential for secondary septum formation and correct primary septum completion. Therefore, our results show that extracellular ß(1,3)glucan is required for cytokinesis to connect the cell wall with the plasma membrane and for contractile ring function, as proposed for the equivalent extracellular matrix in animal cells., We thank the Ramón Areces Foundation for Instituto de Biología Funcional y Genómica financial support. J. Muñoz, M. Ramos, and J.A. Clemente-Ramos acknowledge support by fellowships from Junta de Castilla y León, Consejo Superior de Investigaciones Científicas, and Ministerio de Ciencia e Innovación (MICINN; Spain), respectively; J.C.G. Cortés by a “Juan de la Cierva” contract from MICINN; and I.M. Martins by a fellowship from Fundação para a Ciência e a Tecnologia (Portugal). This work was supported by grants BIO2009-10597, BFU2010-15641, and BIO2012-35372 (MICINN), CSI376A12-2 (Junta de Castilla y León), and OTKA 101323 (Hungarian Scientific Research Fund).

This article is distributed under the terms of a Creative Commons License Attribution–Noncommercial–Share Alike 3.0 Unported license.-- et al., Fungal cytokinesis requires the assembly of a dividing septum wall. In yeast, the septum has to be selectively digested during the critical cell separation process. Fission yeast cell wall α(1-3)glucan is essential, but nothing is known about its localization and function in the cell wall or about cooperation between the α- and β(1-3)glucan synthases Ags1 and Bgs for cell wall and septum assembly. Here, we generate a physiological Ags1-GFP variant and demonstrate a tight colocalization with Bgs1, suggesting a cooperation in the important early steps of septum construction. Moreover, we define the essential functions of α(1-3)glucan in septation and cell separation. We show that α(1-3)glucan is essential for both secondary septum formation and the primary septum structural strength needed to support the physical forces of the cell turgor pressure during cell separation. Consequently, the absence of Ags1 and therefore α(1-3) glucan generates a special and unique side-explosive cell separation due to an instantaneous primary septum tearing caused by the turgor pressure. © 2012 Cortés et al., We thank the Ramón Areces Foundation for IBFG financial support. J.C.G. Cortés acknowledges support from “Juan de la Cierva” program granted by Minsterio de Ciencia e Innovación (MICINN, Spain). This work was supported by grants BIO2009-10597 (MICINN) and CSI038A11-2 (Junta de Castilla y León, Spain).

To explore the opinion of physicians about euthanasia and the treatment of dying patients.A comparative survey.We interviewed 38 family physicians (FP), 38 specialty physicians (SP) and 38 medical students (MS). The survey had 30 items, five of them about experience with terminal patients which were not used for the students.Descriptive statistics and chi 2 or Fisher test to compare proportions between groups.One hundred and two (89%) of the interviewed had a correct concept of euthanasia; 105 (92%) thought that life is holy and untouchable; 29 (25%) agreed there are persons more valuable than others, and four (4%) consider that some should die in certain situations. In relation to patients with brain death, 79 (69%) believed they should not receive futile treatment, but 42 (37%) said they should be attended until cardiac arrest occurred. All agreed with the need of the patients to receive comfort and peace, but only 49/76 (64%) of the physicians and 28 (74%) of the students were in favor of sending dying patients to their home. Nine FP (23%) and 14 SP (36%) stated that in many occasions they lacked elements to solve ethical dilemmas. Thirty six (32%) agreed with the use of passive euthanasia and 21 (18%) with active euthanasia; the latter was more frequent among students. Nine FP (24%) and 13 SP (34%) said they had exceeded therapy sometimes and 23 (61%) of the FP and 19 (50%) of the SP considered they had stopped treatment too early in some cases. We found no differences in regard to euthanasia between physicians and students (chi 2 = 0.32, p = 0.71) nor between the physicians with frequent vs occasional contact with terminal patients (Fisher = 0.13).A third of the physicians agreed with some form of euthanasia but this frequency is smaller than that in other countries.