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This paper presents a prototype of a portable and modular electrical impedance tomography (EIT) system for breast tumor detection. The proposed system uses MATLAB to generate three-dimensional representations of breast tissue. The modular architecture of the system allows for flexible customization and scalability. It consists of several interconnected modules. Each module can be easily replaced or upgraded, facilitating system maintenance and future enhancements. Testing of the prototype has shown promising results in preliminary screening based on experimental studies. Agar models were used for the experimental stage of this project. The 3D representations provide clinicians with valuable information for accurate diagnosis and treatment planning. Further research and refinement of the system is warranted to validate its performance in future clinical trials.

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 β(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 β(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.

Zearalenone (ZON), zearalanone (ZAN) and their phase I metabolites: α-zearalenol (α-ZOL), β-zearalenol (β-ZOL), α-zearalalanol (α-ZAL) and β-zearalalanol (β-ZAL) are compounds with estrogenic activity that are metabolized and distributed by the circulatory system in animals and can access the food chain through meat products from livestock. Furthermore, biomonitoring of zearalenones in biological matrices can provide useful information to directly assess mycotoxin exposure; therefore, their metabolites may be suitable biomarkers. The aim of this study was to determine the presence of ZON, ZAN and their metabolites in alternative biological matrices, such as liver, from three different animals: chicken, pig and lamb, in order to evaluate their exposure. A solid-liquid extraction procedure coupled to a GC-MS/MS analysis was performed. The results showed that 69% of the samples were contaminated with at least one mycotoxin or metabolite at varying levels. The highest value (max. 152.62 ng/g of β-ZOL) observed, and the most contaminated livers (42%), were the chicken liver samples. However, pig liver samples presented a high incidence of ZAN (33%) and lamb liver samples presented a high incidence of α-ZOL (40%). The values indicate that there is exposure to these mycotoxins and, although the values are low (ranged to 0.11-152.6 ng/g for α-ZOL and β-ZOL, respectively), analysis and continuous monitoring are necessary to avoid exceeding the regulatory limits and to control the presence of these mycotoxins in order to protect animal and human health.

Nowadays, the bakery industry includes different bioactive ingredients to enrich the nutritional properties of its products, such as betalains from red beetroot ( Beta vulgaris) . However, cereal products are considered a major route of exposure to many mycotoxins, both individually and in combination, due to their daily consumption, if the cereals used contain these toxins. Only the fraction of the contaminant that is released from the food is bioaccessible and bioavailable to produce toxic effects. Foods with bioactive compounds vary widely in chemical structure and function, and some studies have demonstrated their protective effects against toxics. In this study the bioaccessibility and bioavailability of three legislated mycotoxins (AFB 1 , OTA and ZEN), individual and combined, in two breads, one with wheat flour and the other with wheat flour enriched with 20% Beta vulgaris , were evaluated. Bioaccessibility of these three mycotoxins from wheat bread and red beet bread enriched individually at 100 ng/g was similar between the breads: 16% and 14% for AFB1, 16% and 17% for OTA and 26% and 22% for ZEN, respectively. Whereas, when mycotoxins were co-present these values varied with a decreasing tendency: 9% and 15% for AFB1, 13% and 9% for OTA, 4% and 25% for ZEN in wheat bread and in red beet bread, respectively. These values reveal that the presence of other components and the co-presence of mycotoxins can affect the final bioavailability; however, it is necessary to assess this process with in vivo studies to complete the studies.

Impedance measuring acquisition systems focused on breast tumor detection, as well as image processing techniques for 3D imaging, are reviewed in this paper in order to define potential opportunity areas for future research. The description of reported works using electrical impedance tomography (EIT)-based techniques and methodologies for 3D bioimpedance imaging of breast tissues with tumors is presented. The review is based on searching and analyzing related works reported in the most important research databases and is structured according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) parameters and statements. Nineteen papers reporting breast tumor detection and location using EIT were systematically selected and analyzed in this review. Clinical trials in the experimental stage did not produce results in most of analyzed proposals (about 80%), wherein statistical criteria comparison was not possible, such as specificity, sensitivity and predictive values. A 3D representation of bioimpedance is a potential tool for medical applications in malignant breast tumors detection being capable to estimate an ap-proximate the tumor volume and geometric location, in contrast with a tumor area computing capacity, but not the tumor extension depth, in a 2D representation.

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.

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 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.

Electroelastic materials, as for example, 3M VHB 4910, are attracting attention as actuators or generators in some developments and applications. This is due to their capacity of being deformed when submitted to an electric field. Some models of their actuation are available, but recently, viscoelastic models have been proposed to give an account of the dissipative behaviour of these materials. Their response to an external mechanical or electrical force field implies a relaxation process towards a new state of thermodynamic equilibrium, which can be described by a relaxation time. However, it is well known that viscoelastic and dielectric materials, as for example, polymers, exhibit a distribution of relaxation times instead of a single relaxation time. In the present approach, a continuous distribution of relaxation times is proposed via the introduction of fractional derivatives of the stress and strain, which gives a better account of the material behaviour. The application of fractional derivatives is described and a comparison with former results is made. Then, a double generalisation is carried out: the first one is referred to the viscoelastic or dielectric models and is addressed to obtain a nonsymmetric spectrum of relaxation times, and the second one is the adoption of the more realistic Mooney-Rivlin equation for the stress-strain relationship of the elastomeric material. A modified Mooney-Rivlin model for the free energy density of a hyperelastic material, VHB 4910 has been used based on experimental results of previous authors. This last proposal ensures the appearance of the bifurcation phenomena which is analysed for equibiaxial dead loads; time-dependent bifurcation phenomena are predicted by the extended Mooney-Rivlin equations.

It seems that we are far from controlling COVID-19 pandemics, and, consequently, returning to a fully normal life. Until an effective vaccine is found, safety measures as the use of face masks, social distancing, washing hands regularly, etc., have to be taken. Also, the use of appropriate antivirals in order to alleviate the symptoms, to control the severity of the illness and to prevent the transmission, could be a good option that we study in this work. In this paper, we propose a computational random network model to study the transmission dynamics of COVID-19 in Spain. Once the model has been calibrated and validated, we use it to simulate several scenarios where effective antivirals are available. The results show how the early use of antivirals may significantly reduce the incidence of COVID-19 and may avoid a new collapse of the health system., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 Elsevier Ltd. All rights reserved.)

In fission yeast, cytokinesis requires a contractile actomyosin ring (CR) coupled to membrane and septum ingression. Septation proceeds in two phases. In anaphase B, the septum ingresses slowly. During telophase, the ingression rate increases, and the CR becomes dispensable. Here, we explore the relationship between the CR and septation by analyzing septum ultrastructure, ingression, and septation proteins in cells lacking F-actin. We show that the two phases of septation correlate with septum maturation and the response of cells to F-actin removal. During the first phase, the septum is immature and, following F-actin removal, rapidly loses the Bgs1 glucan synthase from the membrane edge and fails to ingress. During the second phase, the rapidly ingressing mature septum can maintain a Bgs1 ring and septum ingression without F-actin, but ingression becomes Cdc42 and exocyst dependent. Our results provide new insights into fungal cytokinesis and reveal the dual function of CR as an essential landmark for the concentration of Bgs1 and a contractile structure that maintains septum shape and synthesis., (© 2019 Ramos et al.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: The breast dense tissue percentage on digital mammograms is one of the most commonly used markers for breast cancer risk estimation. Geometric features of dense tissue over the breast and the presence of texture structures contained in sliding windows that scan the mammograms may improve the predictive ability when combined with the breast dense tissue percentage., Methods: A case/control study nested within a screening program covering 1563 women with craniocaudal and mediolateral-oblique mammograms (755 controls and the contralateral breast mammograms at the closest screening visit before cancer diagnostic for 808 cases) aging 45 to 70 from Comunitat Valenciana (Spain) was used to extract geometric and texture features. The dense tissue segmentation was performed using DMScan and validated by two experienced radiologists. A model based on Random Forests was trained several times varying the set of variables. A training dataset of 1172 patients was evaluated with a 10-stratified-fold cross-validation scheme. The area under the Receiver Operating Characteristic curve (AUC) was the metric for the predictive ability. The results were assessed by only considering the output after applying the model to the test set, which was composed of the remaining 391 patients., Results: The AUC score obtained by the dense tissue percentage (0.55) was compared to a machine learning-based classifier results. The classifier, apart from the percentage of dense tissue of both views, firstly included global geometric features such as the distance of dense tissue to the pectoral muscle, dense tissue eccentricity or the dense tissue perimeter, obtaining an accuracy of 0.56. By the inclusion of a global feature based on local histograms of oriented gradients, the accuracy of the classifier was significantly improved (0.61). The number of well-classified patients was improved up to 236 when it was 208., Conclusion: Relative geometric features of dense tissue over the breast and histograms of standardized local texture features based on sliding windows scanning the whole breast improve risk prediction beyond the dense tissue percentage adjusted by geometrical variables. Other classifiers could improve the results obtained by the conventional Random Forests used in this study., (Copyright © 2019 Elsevier B.V. All rights reserved.)

It is widely accepted in eukaryotes that the cleavage furrow only initiates after mitosis completion. In fission yeast, cytokinesis requires the synthesis of a septum tightly coupled to cleavage furrow ingression. The current cytokinesis model establishes that simultaneous septation and furrow ingression only initiate after spindle breakage and mitosis exit. Thus, this model considers that although Cdk1 is inactivated at early-anaphase, septation onset requires the long elapsed time until mitosis completion and full activation of the Hippo-like SIN pathway. Here, we studied the precise timing of septation onset regarding mitosis by exploiting both the septum-specific detection with the fluorochrome calcofluor and the high-resolution electron microscopy during anaphase and telophase. Contrarily to the existing model, we found that both septum and cleavage furrow start to ingress at early anaphase B, long before spindle breakage, with a slow ingression rate during anaphase B, and greatly increasing after telophase onset. This shows that mitosis and cleavage furrow ingression are not concatenated but simultaneous events in fission yeast. We found that the timing of septation during early anaphase correlates with the cell size and is regulated by the corresponding levels of SIN Etd1 and Rho1. Cdk1 inactivation was directly required for timely septation in early anaphase. Strikingly the reduced SIN activity present after Cdk1 loss was enough to trigger septation by immediately inducing the medial recruitment of the SIN kinase complex Sid2-Mob1. On the other hand, septation onset did not depend on the SIN asymmetry establishment, which is considered a hallmark for SIN activation. These results recalibrate the timing of key cytokinetic events in fission yeast; and unveil a size-dependent control mechanism that synchronizes simultaneous nuclei separation with septum and cleavage furrow ingression to safeguard the proper chromosome segregation during cell division., Competing Interests: The authors have declared that no competing interests exist.

Fungal cleavage furrow formation during cytokinesis relays in the coordinated contraction of an actomyosin-based ring and the centripetal synthesis of both new plasma membrane and a special wall structure named division septum. Through transmission electron microscopy, the septum exhibits a three-layered structure with a central primary septum, flanked at both sides by the secondary septum. In contrast to the chitinous primary septum present in most of fungi, the fission yeast Schizosaccharomyces pombe does not contain chitin, instead it divides through the formation of a linear β(1,3)glucan-rich primary septum, which has been shown to be specifically stained by the fluorochrome Calcofluor white. Recent findings in S. pombe have revealed the importance of septum synthesis for the steady contraction of the ring during cytokinesis. Therefore, to study the molecular mechanisms that connect the extracellular septum wall with the other components of the cytokinetic machinery located in the plasma membrane and cytoplasm, new experimental approaches are needed. Here we describe the methods developed to image the septum structure by fluorescence microscopy, with a special focus in the analysis of septum progression by the use of time-lapse microscopy.

The task of breast density quantification is becoming increasingly relevant due to its association with breast cancer risk. In this work, a semi-automated and a fully automated tools to assess breast density from full-field digitized mammograms are presented. The first tool is based on a supervised interactive thresholding procedure for segmenting dense from fatty tissue and is used with a twofold goal: for assessing mammographic density (MD) in a more objective and accurate way than via visual-based methods and for labeling the mammograms that are later employed to train the fully automated tool. Although most automated methods rely on supervised approaches based on a global labeling of the mammogram, the proposed method relies on pixel-level labeling, allowing better tissue classification and density measurement on a continuous scale. The fully automated method presented combines a classification scheme based on local features and thresholding operations that improve the performance of the classifier. A dataset of 655 mammograms was used to test the concordance of both approaches in measuring MD. Three expert radiologists measured MD in each of the mammograms using the semi-automated tool (DM-Scan). It was then measured by the fully automated system and the correlation between both methods was computed. The relation between MD and breast cancer was then analyzed using a case-control dataset consisting of 230 mammograms. The Intraclass Correlation Coefficient (ICC) was used to compute reliability among raters and between techniques. The results obtained showed an average ICC=0.922 among raters when using the semi-automated tool, whilst the average correlation between the semi-automated and automated measures was ICC=0.838. In the case-control study, the results obtained showed Odds Ratios (OR) of 1.38 and 1.50 per 10% increase in MD when using the semi-automated and fully automated approaches respectively. It can therefore be concluded that the automated and semi-automated MD assessments present a good correlation. Both the methods also found an association between MD and breast cancer risk, which warrants the proposed tools for breast cancer risk prediction and clinical decision making. A full version of the DM-Scan is freely available., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

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 β(1,3)glucan. We show that Bgs4-derived β(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, β(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 β(1,3)glucan is essential for secondary septum formation and correct primary septum completion. Therefore, our results show that extracellular β(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.

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.

Cytokinesis must be initiated only after chromosomes have been segregated in anaphase and must be terminated once cleavage is completed. We show that the fission yeast protein Etd1 plays a central role in both of these processes. Etd1 activates the guanosine triphosphatase (GTPase) Spg1 to trigger signaling through the septum initiation network (SIN) pathway and onset of cytokinesis. Spg1 is activated in late anaphase when spindle elongation brings spindle pole body (SPB)-localized Spg1 into proximity with its activator Etd1 at cell tips, ensuring that cytokinesis is only initiated when the spindle is fully elongated. Spg1 is active at just one of the two SPBs during cytokinesis. When the actomyosin ring finishes constriction, the SIN triggers disappearance of Etd1 from the half of the cell with active Spg1, which then triggers Spg1 inactivation. Asymmetric activation of Spg1 is crucial for timely inactivation of the SIN. Together, these results suggest a mechanism whereby cell asymmetry is used to monitor cytoplasmic partitioning to turn off cytokinesis signaling.

Cytokinesis is a crucial event in the cell cycle of all living cells. In fungal cells, it requires co-ordinated 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)beta-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 bgs1Delta cells and demonstrated that Bgs1p is responsible and essential for linear (1,3)beta-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)beta-d-glucan and partial primary septa. Conversely, both structures are absent in bgs1Delta cells, where there is no Bgs1p. The septum analysis of bgs1(+)-repressed cells indicates that linear (1,3)beta-d-glucan is necessary but not sufficient for primary septum formation. Linear (1,3)beta-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 pressurised liquid extraction (PLE) method for the analysis of ochratoxin A (OTA) in bread samples is given. Parameters such as solvent, temperature, pressure and time were investigated thoroughly. The optimized PLE conditions were: methanol as extraction solvent, 80 degrees C, 2000 psi and a 5-min cycle. OTA was determined by liquid chromatography coupled with fluorescence detection and confirmed by methyl ester derivatization. Under these conditions OTA recovery is 92.3% with a RSD of 5%. Limits of detection and quantification were 0.02 and 0.06 microg/kg, respectively. The proposed method was applied to 20 bread samples, finding two positive samples with OTA levels below the maximum permitted levels by the European Union.

Schizosaccharomyces pombe contains four putative (1,3)beta-D-glucan synthase (GS) catalytic subunits, Bgs1p-4p. In this work, we cloned bgs4+ and show that Bgs4p is the only subunit found to be a part of the GS enzyme and essential for maintaining cell integrity during cytokinesis and polarized growth. Here we show that bgs4+, cwg1+ (cwg1-1 shows reduced cell-wall beta-glucan and GS catalytic activity) and orb11+ (orb11-59 is defective in cell morphogenesis) are the same gene. bgs4+ is essential for spore germination and 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 to compensate for an excess of cell wall degradation during cytokinesis. Shut-off and overexpression analysis suggest that Bgs4p forms part of a GS catalytic multiprotein complex and that Bgs4p-promoted cell-wall beta-glucan alterations induce compensatory mechanisms from other Bgs subunits and (1,3)alpha-D-glucan synthase. Physiological localization studies showed that Bgs4p localizes to the growing ends, the medial ring and septum, and at each stage 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 the stable maintenance of Bgs4p 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)beta-D-glucan necessary to preserve cell integrity when cell wall synthesis or repair are needed.

The cps5-138 fission yeast mutant shows an abnormal lemon-like morphology at 28 degrees C in minimal medium and a lethal thermosensitive phenotype at 37 degrees C. Cell growth is completely inhibited at 28 degrees C in a Ca2+-free medium, in which the wild type is capable of growing normally. Under these conditions, actin patches become randomly distributed throughout the cell, and defects in septum formation and subsequent cytokinesis appear. The mutant cell is hypersensitive to the cell wall-digesting enzymatic complex Novozym234 even under permissive conditions. The gene SPBC31E1.02c, which complements all the mutant phenotypes described above, was cloned and codes for the Ca2+-ATPase homologue Pmr1p. The gene is not essential under optimal growth conditions but is required under conditions of low Ca2+ (<0.1 mM) or high temperature (>35 degrees C). The green fluorescent protein-tagged Cps5 proteins, which are expressed under physiological conditions (an integrated single copy with its own promoter in the cps5Delta strain), display a localization pattern typical of endoplasmic reticulum proteins. Biochemical analyses show that 1,3-beta-D-glucan synthase activity in the mutant is decreased to nearly half that of the wild type and that the mutant cell wall contains no detectable galactomannan when the cells are exposed to a Ca2+-free medium. The mutant acid phosphatase has an increased electrophoretic mobility, suggesting that incomplete protein glycosylation takes place in the mutant cells. These results indicate that S. pombe Pmr1p is essential for the maintenance of cell wall integrity and cytokinesis, possibly by allowing protein glycosylation and the polarized actin distribution to take place normally. Disruption and complementation analyses suggest that Pmr1p shares its function with a vacuolar Ca2+-ATPase homologue, Pmc1p (SPAPB2B4.04c), to prevent lethal activation of calcineurin for cell growth.

Schizosaccharomyces pombe Bgs1p/Cps1p has been identified as a putative (1,3)beta-D-glucan 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)beta-D-glucan synthesis at cytokinesis. We have studied the in vivo physiological localization of Bgs1p in a bgs1delta 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 that 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 occurs only in the new cell. All these data show that Bgs1p is localized to the areas of polarized cell wall growth and so we propose that it might be involved in synthesizing the lineal (1,3)beta-D-glucan of the primary septum, as well as a similar lineal (1,3)beta-D-glucan when other processes of cell wall growth or repair are needed.