A new software tool for carbohydrate microarray data storage, processing, presentation, and reporting

Publisher Copyright: © 2022 The Author(s) 2022. Published by Oxford University Press. This project is supported by Wellcome Trust Biomedical Resource grants (WT099197/Z/12/Z, 108430/Z/15/Z and 218304/Z/19/Z); March of Dimes European Prematurity Research Centre grant 22-FY18-82 and NIH Commons Fund 1U01GM125267-01Glycan microarrays are essential tools in glycobiology and are being widely used for assignment of glycan ligands in diverse glycan recognition systems. We have developed a new software, called Carbohydrate microArray Analysis and Reporting Tool (CarbArrayART), to address the need for a distributable application for glycan microarray data management. The main features of CarbArrayART include: (i) Storage of quantified array data from different array layouts with scan data and array-specific metadata, such as lists of arrayed glycans, array geometry, information on glycan-binding samples, and experimental protocols. (ii) Presentation of microarray data as charts, tables, and heatmaps derived from the average fluorescence intensity values that are calculated based on the imaging scan data and array geometry, as well as filtering and sorting functions according to monosaccharide content and glycan sequences. (iii) Data export for reporting in Word, PDF, and Excel formats, together with metadata that are compliant with the guidelines of MIRAGE (Minimum Information Required for A Glycomics Experiment). CarbArrayART is designed for routine use in recording, storage, and management of any slide-based glycan microarray experiment. In conjunction with the MIRAGE guidelines, CarbArrayART addresses issues that are critical for glycobiology, namely, clarity of data for evaluation of reproducibility and validity.publishersversionpublishe

Synthesis of α-glucans in fission yeast spores is carried out by three α-glucan synthase paralogues, Mok12p, Mok13p and Mok14p

El pdf del artículo es la versión pre-print.-- et al.Fission yeast possesses a family of (1,3)-α-glucan synthase-related genes; one of them, mok1+/ags1+, plays an essential function in morphogenesis during vegetative growth. Here we show that three mok1+ paralogues –mok12+, mok13+ and mok14+– are required for sporulation to succeed, acting at different stages of the spore wall maturation process. Mutation of mok12+ affected the efficiency of spore formation and spore viability. Deletion of mok13+ does not affect spore viability but the spores showed reduced resistance to stress conditions. mok14Δ mutant spores failed to accumulate the amylose-like spore wall-specific polymer. mok12+, mok13+ and mok14+ expression was restricted to sporulating cells and the proteins localized to the spore envelope but with different timing. mok11+ was also induced during the sporulation process although its deletion did not show apparently a sporulation defect. In vegetative cells, β-glucans are more abundant than α-glucans (55% versus 28%). In spores, the situation was the opposite, α-glucans accounted for 46% while β-glucans were approximately 38% of the total polysaccharides. We found at least two types of α-glucan polymers, Mok12p and Mok13p, were involved in the synthesis of the greater part of α-glucan in the spores envelope, a polymer that is mainly digested with α-1,3 glucanase, while Mok14p, homologous to starch synthases, was required for the synthesis of the iodine-reactive polymer that is made of α-1,4 glucose residues.I. García was supported by a fellowship from the Junta de Castilla y León; V. Tajadura and V. Martín were supported from a fellowship granted by the MEC, Spain. This work was supported by Grants BIO2001-1663, CSI7/01 and CSI02C05.Peer Reviewe

Role of Rho GTPases and Rho-GEFs in the regulation of cell shape and integrity in fission yeast

El pdf del artículo es la versión pre-print.The Rho family of GTPases are highly conserved molecular switches that control some of the most fundamental processes of cell biology, including morphogenesis, vesicular transport, cell division and motility. Guanine nucleotide-exchange factors (GEFs) are directly responsible for the activation of Rho-family GTPases in response to extracellular stimuli. In fission yeast, there are seven Dbl-related GEFs and they activate six Rho-type GTPases within a particular spatio-temporal context. The failure to do so might have consequences reflected in aberrant phenotypes and in some cases lead to cell death. In this review, we briefly summarize the role of Rho GTPases and Rho-GEFs in the establishment and maintenance of cell polarity and cell integrity in Schizosaccharomyces pombe. Copyright © 2006 John Wiley & Sons, Ltd.P. Garcia and I. Garcia were supported by a fellowship from the Junta de Castilla y León and V. Tajadura acknowledges support from a fellowship granted by the Ministerio de Educación y Ciencia, Spain. This work was supported by Grant BFU2005-01557 from the Comisión Interministerial de Ciencia y Tecnología, Spain, and CSI02C05 from the Junta de Castilla y León.Peer Reviewe

Rgf1p Is a Specific Rho1-GEF That Coordinates Cell Polarization with Cell Wall Biogenesis in Fission Yeast

Rho1p regulates cell integrity by controlling the actin cytoskeleton and cell wall synthesis. We have identified a new GEF, designated Rgf1p, which specifically regulates Rho1p during polarized growth. The phenotype of rgf1 null cells was very similar to that seen after depletion of Rho1p, 30% of cells being lysed. In addition, rgf1(+) deletion caused hypersensitivity to the antifungal drug Caspofungin and defects in the establishment of bipolar growth. rho1(+), but none of the other GTPases of the Rho-family, suppressed the rgf1Δ phenotypes. Moreover, deletion of rgf1(+) suppressed the severe growth defect in rga1(+) null mutants (a Rho1-GAP, negative regulator). Rgf1p and Rho1p coimmunoprecipitated and overexpression of rgf1(+) specifically increased the GTP-bound Rho1p; it caused changes in cell morphology, and a large increase in β(1,3)-glucan synthase activity. These effects were similar to those elicited when the hyperactive rho1-G15V allele was expressed. A genetic relationship was observed between Rgf1p, Bgs4p (β[1,3]-glucan synthase), and Pck1p (protein kinase C [PKC] homologue); Bgs4p and Pck1p suppressed the hypersensitivity to Caspofungin in rgf1Δ mutants. Rgf1p localized to the growing ends and the septum, where Rho1, Pck1p, and Bgs4p are known to function. Our results suggest that Rgf1p probably activates the Rho functions necessary for coordinating actin deposition with cell wall biosynthesis during bipolar growth, allowing the cells to remodel their wall without risk of rupture

Two E-selectin ligands, BST-2 and LGALS3BP, predict metastasis and poor survival of ER-negative breast cancer

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Identification of RGF1p and RGF3p domains involved in function and subcellular targeting

Resumen del póster presentado a la EMBO Conference on Fission Yeast: Pombe 2013; 7th International Fission Yeast Meeting celebrada en Londres (UK) del 24 al 29 de junio de 2013.In fission yeast, three guanine nucleotide-exchange factors (GEFs) activate Rho1p: Rgf1p, Rgf2p and Rgf3p. Rgf1p localizes to the cell tips in interphase cells and to the division septum in mitotic cells. Rgf1p is not essential for viability, but it does play a role in cell integrity and is also necessary for the establishment of bipolar growth. Rgf3p localizes exclusively to the septum and is essential for maintaining cell integrity during cell separation. The three GEFs contain a RhoGEF domain, which contacts the Rho GTPase to catalyze nucleotide exchange, and an associated Pleckstrin homology (PH) domain, which fine-tunes the exchange process. Rgf1p and Rgf2p also contain a putative Dishevelled, Egl-10 y Pleckstrin (DEP) domain N-terminal to the DH domain and a Citron Homology (CNH) domain at the C-terminus. Rgf3p also contains a CNH domain but lacks a DEP domain.The function of the DEP and the CNH domains is still unknown. We tested the functional relevance of the different domains in Rgf1p and Rgf3p. As expected, the RhoGEF and the PH domains are both essential for function. However, the N-terminus and the DEP domain are dispensable for full function. From these studies we have learnt that the CNH domain is essential for function in both GEFs. Rgf1pΔCNH cells, carrying a deletion in the CNH domain and Rgf1p ΔC45 cells lacking the last 45 aa of the Rgf1p, showed similar phenotypes than the null mutant regarding polarity and cell integrity. In addition, for the Rgf1p ΔC45 construct, the normal localization of Rgf1p at the two tips was disrupted and the signal was mainly monopolar. Our results demonstrate that the CNH domain of the Rgf1p and particularly the C-end (last 45 aa), is required for Rgf1p anchorage to the new growing tip allowing the establishment of bipolar growth. A similar situation was shown for Rgf3p; deletion of the CNH domain in Rgf3p renders the cells unviable and this domain is also required for localization of the protein to the septum.Peer Reviewe

O-linked mucin-type glycosylation in breast cancer

Changes in mucin-type O-linked glycosylation are seen in over 90% of breast cancers where increased sialylation is often observed and a change from branched glycans to linear glycans is often seen. There are many mechanisms involved including increased/altered expression of glycosyltransferases and relocalisation to the endoplasmic reticulum of the enzymes responsible for the addition of the first sugar, N-acetyl-d-galactosamine. It is now becoming clear that these changes can contribute to tumour growth and progression by modulating the micro-environment through glycan-sensing lectins expressed on immune cells, by modulating interactions with tumour surface receptors and by binding to selectins. The understanding of how changes in mucin-type O-linked glycosylation influence tumour growth and progression reveals new potential targets for therapeutic intervention in the treatment of breast cancer.</jats:p