New cell wall-affecting antifungal antibiotics

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.Peer reviewe

Bgs1p localization during the life cycle

[EN]Schizosaccharomyces pombe Bgs1p/Cps1p has been identified as a putative (1,3)β-Dglucan synthase (GS) catalytic subunit with a possible function during cytokinesis and polarized growth. To study this possibility, double mutants of cps1-12 and cdc septation mutants, were made. The double mutants displayed several hypersensitive phenotypes and altered actin distribution. Epistasis analysis showed mutations prior to septum synthesis were dominant over cps1-12, while cps1-12 was dominant over the end of septation mutant cdc16-116, suggesting Bgs1p is involved in septum cell-wall (1,3)β-D-glucan synthesis at cytokinesis. We have studied the in vivo physiological localization of Bgs1p in a bgs1Δ strain containing a functional GFP-bgs1+ gene (integrated single copy and expressed under its own promoter). During vegetative growth, Bgs1p always localizes to the growing zones: one or both ends during cell growth, and contractile ring and septum during cytokinesis. Bgs1p localization in cdc septation mutants indicates that Bgs1p needs the medial ring and septation initiation network (SIN) proteins to localize properly with the rest of septation components. Bgs1p localization in the actin mutant cps8-188 shows it depends on actin localization. In addition, Bgs1p remains polarized in the mislocalized growing poles and septa of tea1-1 and tea2-1 mutants. During the meiotic process of the life cycle, Bgs1p localizes to the mating projection, to the cell-to-cell contact zone during cell fusion and to the neck area during zygote formation. Also, Bgs1p localization suggests it collaborates in forespore and spore wall synthesis. During spore germination, Bgs1p localizes first around the spore during isotropic growth, then to the zone of polarized growth and finally, to the medial ring and septum. At the end of spore-cell division, the Bgs1p displacement to the old end only occurs in the new cell. All these data shows Bgs1p is localized to the areas of polarized cell wall growth and according to that, we propose it might be involved in synthesizing the lineal (1,3)β-D-glucan of the primary septum, as well as a similar lineal (1,3)β-D-glucan when other processes of cell wall growth or repair are needed

A Family of Multifunctional Thiamine-Repressible Expression Vectors for Fission Yeast

[EN]A series of thiamine-repressible shuttle vectors has been constructed to allow a more efficient DNA manipulation in Schizosaccharomyces pombe. These high-copy-number vectors with regulatable expression (pJR) are based on the backbone of the pREP-3X, pREP-41X and pREP-81X plasmids. The pJR vectors are all uniform in structure, containing i) sequences for replication (ori) and selection (AmpR) in Escherichia coli, ii) the f1 ori sequence of the phage f1 for packaging of ssDNA, making them suitable for site-directed mutagenesis, and iii) the ars1 sequence for replication in S. pombe. The pJR vectors differ among them in i) the selectable marker (Saccharomyces cerevisiae LEU 2 gene, that complements S. pombe leu1- gene, and S. pombe ura4+ and his3+ genes); ii) the thiaminerepressible nmt1 promoter (3X, 41X and 81X with extremely high, moderate or low transcription efficiency, respectively); and iii) the multiple cloning site (two multiple cloning sites, with twelve restriction sites each). The expression level of the pJR vectors has been analyzed using the β-galactosidase gene as reporter. Three levels of expression for each nmt1 promoter version, with any selectable marker and for either repressed or induced conditions, have been found. The expression is dependent on the distance to the initiation codon, varying from 0.001 to 15 times the activity characterized for the pREP plasmids. Also, the gene expression has been found to be extremely sensitive to the nucleotide sequence prior to the initiation codon, being up to 50 fold higher with an A/T sequence than with a G/C sequence. Finally, the β-galactosidase mRNA levels were found to be similar in each nmt1 series, suggesting a translational effect on gene expression. As a result, any of these eighteen new vectors allow performing gene expression in fission yeast as well as a more versatile cloning, sequencing and mutagenesis directly in the plasmid without the need for subcloning into intermediary vectors

Bgs4 is essential for cytokinesis and cell growth

[EN]Schizosaccharomyces pombe contains four putative (1,3)β-D-glucan synthase (GS) catalytic subunits, Bgs1p to Bgs4p. In this work, we cloned bgs4+ and show that Bgs4p is the only subunit 1) essential for maintaining cell integrity during both cytokinesis and polarized growth, and 2) found to be part of the GS enzyme. Here we show that bgs4+, cwg1+ (cwg1-1 shows reduced cell-wall β-glucan and GS catalytic activity) and orb11+ (orb11-59 is defective in cell morphogenesis) are the same gene. bgs4+ is essential during spore germination. bgs4+ shut-off produces cell lysis at growing poles and mainly at the septum prior to cytokinesis, suggesting that Bgs4p is essential for cell wall growth and for compensating an excess of cell wall degradation during cytokinesis. Shut-off and overexpression analysis suggest that 1) Bgs4p forms part of a GS catalytic multiprotein complex, and 2) Bgs4p-promoted cell-wall β-glucan alterations induce compensatory mechanisms from other Bgs subunits and (1,3)α-D-glucan synthase. Physiological localization studies showed that Bgs4p localizes to the growing ends, the medial ring and septum, and in each process of wall synthesis or remodeling that occurs during sexual differentiation: mating, zygote and spore formation, and spore germination. Bgs4p timing and requirements for proper positioning during cytokinesis and its localization pattern during spore maturation differ from those of Bgs1p. Bgs4p localizes overlapping the contractile ring once Bgs1p is present and a Calcofluor white-stained septum material is detected, suggesting that Bgs4p is involved in a late process of secondary or general septum synthesis. Unlike Bgs1p, Bgs4p needs the medial ring but not the Septation Initiation Network proteins to localize with the other septation components. Furthermore, Bgs4p localization depends on the polarity establishment proteins. Finally, F-actin is necessary for Bgs4p delocalization from and relocalization to the growing regions, but it is not needed for its stable maintenance at the growing sites, poles and septum. All these data show for the first time an essential role for a Bgs subunit in the synthesis of a (1,3)β-D-glucan necessary to preserve cell integrity when cell wall synthesis or repair are needed

The effect of magnetic activity on low-mass stars in eclipsing binaries

In recent years, analyses of eclipsing binary systems have unveiled differences between the observed fundamental properties of low-mass stars and those predicted by stellar structure models. Particularly, radius and effective temperatures computed from models are ~ 5-10% lower and ~ 3-5% higher than observed, respectively. These discrepancies have been attributed to different factors, notably to the high levels of magnetic activity present on these stars. In this paper, we test the effect of magnetic activity both on models and on the observational analysis of eclipsing binaries using a sample of such systems with accurate fundamental properties. Regarding stellar models, we have found that unrealistically high spot coverages need to be assumed to reproduce the observations. Tests considering metallicity effects and missing opacities on models indicate that these are not able to explain the radius discrepancies observed. With respect to the observations, we have tested the effect of several spot distributions on the light curve analysis. Our results show that spots cause systematic deviations on the stellar radii derived from light curve analysis when distributed mainly over the stellar poles. Assuming the existence of polar spots, overall agreement between models and observations is reached when ~ 35% spot coverage is considered on stellar models. Such spot coverage induces a systematic deviation in the radius determination from the light curve analysis of ~ 3% and is also compatible with the modulations observed on the light curves of these systems. Finally, we have found that the effect of activity or rotation on convective transport in partially radiative stars may also contribute to explain the differences seen in some of the systems with shorter orbital periods.Comment: 12 pages, 9 figures, accepted for publication in Ap

Machine learning in critical care: state-of-the-art and a sepsis case study

Background: Like other scientific fields, such as cosmology, high-energy physics, or even the life sciences, medicine and healthcare face the challenge of an extremely quick transformation into data-driven sciences. This challenge entails the daunting task of extracting usable knowledge from these data using algorithmic methods. In the medical context this may for instance realized through the design of medical decision support systems for diagnosis, prognosis and patient management. The intensive care unit (ICU), and by extension the whole area of critical care, is becoming one of the most data-driven clinical environments. Results: The increasing availability of complex and heterogeneous data at the point of patient attention in critical care environments makes the development of fresh approaches to data analysis almost compulsory. Computational Intelligence (CI) and Machine Learning (ML) methods can provide such approaches and have already shown their usefulness in addressing problems in this context. The current study has a dual goal: it is first a review of the state-of-the-art on the use and application of such methods in the field of critical care. Such review is presented from the viewpoint of the different subfields of critical care, but also from the viewpoint of the different available ML and CI techniques. The second goal is presenting a collection of results that illustrate the breath of possibilities opened by ML and CI methods using a single problem, the investigation of septic shock at the ICU. Conclusion: We have presented a structured state-of-the-art that illustrates the broad-ranging ways in which ML and CI methods can make a difference in problems affecting the manifold areas of critical care. The potential of ML and CI has been illustrated in detail through an example concerning the sepsis pathology. The new definitions of sepsis and the relevance of using the systemic inflammatory response syndrome (SIRS) in its diagnosis have been considered. Conditional independence models have been used to address this problem, showing that SIRS depends on both organ dysfunction measured through the Sequential Organ Failure (SOFA) score and the ICU outcome, thus concluding that SIRS should still be considered in the study of the pathophysiology of Sepsis. Current assessment of the risk of dead at the ICU lacks specificity. ML and CI techniques are shown to improve the assessment using both indicators already in place and other clinical variables that are routinely measured. Kernel methods in particular are shown to provide the best performance balance while being amenable to representation through graphical models, which increases their interpretability and, with it, their likelihood to be accepted in medical practice.Peer ReviewedPostprint (published version

Simple sugar intake and hepatocellular carcinoma: epidemiological and mechanistic insight

Sugar intake has dramatically increased during the last few decades. Specifically, there has been a clear trend towards higher consumption of fructose and high fructose corn syrup, which are the most common added sugars in processed food, soft drinks and other sweetened beverages. Although still controversial, this rising trend in simple sugar consumption has been positively associated with weight gain and obesity, insulin resistance and type 2 diabetes mellitus and non-alcoholic fatty liver disease. Interestingly, all of these metabolic alterations have also been related to the development of hepatocellular carcinoma. The purpose of this review is to discuss the evidence coming from epidemiological studies and data from animal models relating the consumption of simple sugars, and specifically fructose, with an increased risk of hepatocellular carcinoma and to gain insight into the putative molecular mechanisms involved

Bgs1p is responsible for primary septum formation

[EN]Cytokinesis is a crucial event in the cell cycle of all living cells. In fungal cells, it requires coordinated contraction of an actomyosin ring and synthesis of both plasmatic membrane and a septum structure that will constitute the new cell wall end. Schizosaccharomyces pombe contains four essential putative (1,3)β-D-glucan synthase catalytic subunits, Bgs1p to Bgs4p. Here we examined the function of Bgs1p in septation by studying the lethal phenotypes of bgs1 + shut-off and bgs1 Δ cells and demonstrated that Bgs1p is responsible and essential for linear (1,3)β-D-glucan and primary septum formation. bgs1 + shut-off generates a more than 300-fold Bgs1p reduction, but the septa still present large amounts of disorganized linear (1,3)β-D-glucan and partial primary septa. Conversely, both structures are absent in bgs1 Δ cells, where there is no Bgs1p. The septum analysis of bgs1+-repressed cells indicates that linear (1,3)β-D-glucan is necessary but not sufficient for primary septum formation. Linear (1,3)β-D-glucan is the polysaccharide that specifically interacts with the fluorochrome Calcofluor white in fission yeast. We also show that in the absence of Bgs1p abnormal septa are formed, but the cells cannot separate and eventually die

A Quotient Basis Kernel for the prediction of mortality in severe sepsis patients

In this paper, we describe a novel kernel for multinomial distributions, namely the Quotient Basis Kernel (QBK), which is based on a suitable reparametrization of the input space through algebraic geometry and statistics. The QBK is used here for data transformation prior to classification in a medical problem concerning the prediction of mortality in patients suffering severe sepsis. This is a common clinical syndrome, often treated at the Intensive Care Unit (ICU) in a time-critical context. Mortality prediction results with Support Vector Machines using QBK compare favorably with those obtained using alternative kernels and standard clinical procedures.Postprint (published version

Critical appraisal of international guidelines for the management of fecal incontinence in adults: is it possible to define what to do in different clinical scenarios?

Faecal incontinence; Treatment algorithm; Guidelines; ReviewIncontinencia fecal; Algoritmo de tratamiento; Guias; RevisiónIncontinència fecal; Algorisme de tractament; Guies; RevisióFecal incontinence (FI) is a complex often multifactorial functional disorder which is associated with a significant impact on patients’ quality of life. There is a broad spectrum of symptoms, and degrees of severity and diverse patient backgrounds. Several treatment algorithms from different professional societies and experts are available in the literature. However, no consensus has been reached on several aspects of FI management. We performed a critical review of the most recently pub- lished guidelines on FI, emphasising the lack of consensus, highlighting specific topics mentioned in each of the guidelines that are not covered in the others and defining the treatment proposed in different clinical scenarios