Identification of the human Lewisa carbohydrate motif in a secretory peroxidase from a plant cell suspension culture (Vaccinium myrtillus L.)

This paper reports for the first time the presence of the human Lewisa type determinant in glycoproteins secreted by plant cells. A single glycopeptide was identified in the tryptic hydrolysis of the peroxidase VMPxCl from Vaccinium myrtillus L. by HPLC/ESI-MS. The oligosaccharide structures were elucidated by ESI-MS-MS and by methylation analysis before and after removal of fucose by mild acid hydrolysis.info:eu-repo/semantics/publishedVersio

PIG-1 MELK-dependent phosphorylation of nonmuscle myosin II promotes apoptosis through CES-1 Snail partitioning

The mechanism(s) through which mammalian kinase MELK promotes tumorigenesis is not understood. We find that theC.elegansorthologue of MELK, PIG-1, promotes apoptosis by partitioning an anti-apoptotic factor. TheC.elegansNSM neuroblast divides to produce a larger cell that differentiates into a neuron and a smaller cell that dies. We find that in this context, PIG-1 is required for partitioning of CES-1 Snail, a transcriptional repressor of the pro-apoptotic geneegl-1BH3-only.pig-1MELK is controlled by both aces-1Snail- andpar-4LKB1-dependent pathway, and may act through phosphorylation and cortical enrichment of nonmuscle myosin II prior to neuroblast division. We propose thatpig-1MELK-induced local contractility of the actomyosin network plays a conserved role in the acquisition of the apoptotic fate. Our work also uncovers an auto-regulatory loop through whichces-1Snail controls its own activity through the formation of a gradient of CES-1 Snail protein. Author summary Apoptosis is critical for the elimination of 'unwanted' cells. What distinguishes wanted from unwanted cells in developing animals is poorly understood. We report that in theC.elegansNSM neuroblast lineage, the level of CES-1, a Snail-family member and transcriptional repressor of the pro-apoptotic geneegl-1, contributes to this process. In addition, we demonstrate thatC.elegansPIG-1, the orthologue of mammalian proto-oncoprotein MELK, plays a critical role in controlling CES-1(Snail)levels. Specifically, during NSM neuroblast division, PIG-1(MELK)controls partitioning of CES-1(Snail)into one but not the other daughter cell thereby promoting the making of one wanted and one unwanted cell. Furthermore, we present evidence that PIG-1(MELK)acts prior to NSM neuroblast division by locally activating the actomyosin network

Sialoglycoproteins and N-Glycans from Secreted Exosomes of Ovarian Carcinoma Cells

Exosomes consist of vesicles that are secreted by several human cells, including tumor cells and neurons, and they are found in several biological fluids. Exosomes have characteristic protein and lipid composition, however, the results concerning glycoprotein composition and glycosylation are scarce. Here, protein glycosylation of exosomes from ovarian carcinoma SKOV3 cells has been studied by lectin blotting, NP-HPLC analysis of 2-aminobenzamide labeled glycans and mass spectrometry. An abundant sialoglycoprotein was found enriched in exosomes and it was identified by peptide mass fingerprinting and immunoblot as the galectin-3-binding protein (LGALS3BP). Exosomes were found to contain predominantly complex glycans of the di-, tri-, and tetraantennary type with or without proximal fucose and also high mannose glycans. Diantennary glycans containing bisecting N-acetylglucosamine were also detected. This work provides detailed information about glycoprotein and N-glycan composition of exosomes from ovarian cancer cells, furthermore it opens novel perspectives to further explore the functional role of glycans in the biology of exosomes.EU Joint Programme JPND/0003/2011, FCT grant: Pest-OE/EQB/LA0004/2011, FCT PhD fellowship

A Mathematical Model for the Dynamics and Synchronization of Cows

We formulate a mathematical model for daily activities of a cow (eating, lying down, and standing) in terms of a piecewise affine dynamical system. We analyze the properties of this bovine dynamical system representing the single animal and develop an exact integrative form as a discrete-time mapping. We then couple multiple cow "oscillators" together to study synchrony and cooperation in cattle herds. We comment on the relevant biology and discuss extensions of our model. With this abstract approach, we not only investigate equations with interesting dynamics but also develop interesting biological predictions. In particular, our model illustrates that it is possible for cows to synchronize \emph{less} when the coupling is increased.Comment: to appear in Physica

MitoSegNet: Easy-to-use Deep Learning Segmentation for Analyzing Mitochondrial Morphology

While the analysis of mitochondrial morphology has emerged as a key tool in the study of mitochondrial function, efficient quantification of mitochondrial microscopy images presents a challenging task and bottleneck for statistically robust conclusions. Here, we present Mitochondrial Segmentation Network (MitoSegNet), a pretrained deep learning segmentation model that enables researchers to easily exploit the power of deep learning for the quantification of mitochondrial morphology. We tested the performance of MitoSegNet against three feature-based segmentation algorithms and the machine-learning segmentation tool Ilastik. MitoSegNet outperformed all other methods in both pixelwise and morphological segmentation accuracy. We successfully applied MitoSegNet to unseen fluorescence microscopy images of mitoGFP expressing mitochondria in wild-type and catp-6ATP13A2 mutant C. elegans adults. Additionally, MitoSegNet was capable of accurately segmenting mitochondria in HeLa cells treated with fragmentation inducing reagents. We provide MitoSegNet in a toolbox for Windows and Linux operating systems that combines segmentation with morphological analysis

NLRC5 promotes transcription of BTN3A1-3 genes and Vγ9Vδ2 T cell-mediated killing

BTN3A molecules-BTN3A1 in particular-emerged as important mediators of Vγ9Vδ2 T cell activation by phosphoantigens. These metabolites can originate from infections, e.g. with Mycobacterium tuberculosis, or by alterations in cellular metabolism. Despite the growing interest in the BTN3A genes and their high expression in immune cells and various cancers, little is known about their transcriptional regulation. Here we show that these genes are induced by NLRC5, a regulator of MHC class I gene transcription, through an atypical regulatory motif found in their promoters. Accordingly, a robust correlation between NLRC5 and BTN3A gene expression was found in healthy, in M. tuberculosis-infected donors' blood cells, and in primary tumors. Moreover, forcing NLRC5 expression promoted Vγ9Vδ2 T-cell-mediated killing of tumor cells in a BTN3A-dependent manner. Altogether, these findings indicate that NLRC5 regulates the expression of BTN3A genes and hence open opportunities to modulate antimicrobial and anticancer immunity

The permanently chaperone-active small heat shock protein Hsp17 from Caenorhabditis elegans exhibits topological separation of its N-terminal regions

Small Heat shock proteins (sHsps) are a family of molecular chaperones that bind nonnative proteins in an ATP-independent manner. Caenorhabditis elegans encodes 16 different sHsps, among them Hsp17, which is evolutionarily distinct from other sHsps in the nematode. The structure and mechanism of Hsp17 and how these may differ from other sHsps remain unclear. Here, we find that Hsp17 has a distinct expression pattern, structural organization, and chaperone function. Consistent with its presence under nonstress conditions, and in contrast to many other sHsps, we determined that Hsp17 is a mono-disperse, permanently active chaperone in vitro, which interacts with hundreds of different C. elegans proteins under physiological conditions. Additionally, our cryo-EM structure of Hsp17 reveals that in the 24-mer complex, 12 N-terminal regions are involved in its chaperone function. These flexible regions are located on the outside of the spherical oligomer, whereas the other 12 N-terminal regions are engaged in stabilizing interactions in its interior. This allows the same region in Hsp17 to perform different functions depending on the topological context. Taken together, our results reveal structural and functional features that further define the structural basis of permanently active sHsps

Carbohydrate structures of a human tissue plasminogen activator variant expressed in recombinant Chinese hamster ovary cells

AbstractThe carbohydrate structures of a genetically engineered human tissue plasminogen activator variant bearing a single N-glycosylation site at Asn 448 are reported. After isolation of the tryptic glycopeptide and liberation of the N-linked carbohydrates by polypeptide:N-glycosidase F, 6 major oligosaccharide fractions were separated by HPLC on NH2-bonded phase. Their structures were determined by compositional and methylation analyses combined with fast atom bombardment mass spectrometry. Seventy percent of the carbohydrates were of the biantennary complex type with fucose at the proximal GlcNAc and zero, one or two α2-3 linked NeuAc. The remainder were triantennary structures with one, two or three NeuAc