Ctdnep1 and Eps8L2 regulate dorsal actin cables for nuclear positioning during cell migration.

Cells actively position their nuclei within the cytoplasm for multiple cellular and physiological functions.1-3 Consequently, nuclear mispositioning is usually associated with cell dysfunction and disease, from muscular disorders to cancer metastasis.4-7 Different cell types position their nuclei away from the leading edge during cell migration.8-11 In migrating fibroblasts, nuclear positioning is driven by an actin retrograde flow originated at the leading edge that drives dorsal actin cables away from the leading edge. The dorsal actin cables connect to the nuclear envelope by the linker of nucleoskeleton and cytoskeleton (LINC) complex on transmembrane actin-associated nuclear (TAN) lines.12-14 Dorsal actin cables are required for the formation of TAN lines. How dorsal actin cables are organized to promote TAN lines formation is unknown. Here, we report a role for Ctdnep1/Dullard, a nuclear envelope phosphatase,15-22 and the actin regulator Eps8L223-25 on nuclear positioning and cell migration. We demonstrate that Ctdnep1 and Eps8L2 directly interact, and this interaction is important for nuclear positioning and cell migration. We also show that Ctdnep1 and Eps8L2 are involved in the formation and thickness of dorsal actin cables required for TAN lines engagement during nuclear movement. We propose that Ctdnep1-Eps8L2 interaction regulates dorsal actin cables for nuclear movement during cell migration.

The role of the cell nucleus in mechanotransduction.

Mechanical forces are known to influence cellular processes with consequences at the cellular and physiological level. The cell nucleus is the largest and stiffest organelle, and it is connected to the cytoskeleton for proper cellular function. The connection between the nucleus and the cytoskeleton is in most cases mediated by the linker of nucleoskeleton and cytoskeleton (LINC) complex. Not surprisingly, the nucleus and the associated cytoskeleton are implicated in multiple mechanotransduction pathways important for cellular activities. Herein, we review recent advances describing how the LINC complex, the nuclear lamina, and nuclear pore complexes are involved in nuclear mechanotransduction. We will also discuss how the perinuclear actin cytoskeleton is important for the regulation of nuclear mechanotransduction. Additionally, we discuss the relevance of nuclear mechanotransduction for cell migration, development, and how nuclear mechanotransduction impairment leads to multiple disorders.

Methods to Measure Perinuclear Actin Dynamics During Nuclear Movement in Migrating Cells.

The nucleus is specifically positioned within a cell in diverse biological contexts. There are multiple connections between the nuclear envelope and the cytoskeleton and these connections are involved in nuclear positioning. During cell polarization prior to cell migration, nuclear envelope proteins bind to the actin cytoskeleton and get organized into linear arrays, known as transmembrane actin-associated nuclear (TAN) lines to move the nucleus away from the leading edge. Here we describe methods to study perinuclear actin dynamics, including measurement of the thickness of actin cables coupled to TAN lines, measurement of the number of perinuclear actin cables, and ablation of perinuclear actin cables. These methods are used to identify mechanisms of nuclear positioning.

Dealing with the nucleus during cell migration.

The position of the nucleus within cells is a key event during cell migration. The movement and positioning of the nucleus strongly impacts cell migration. Notably, the last two years largely contributed to emphasise the dynamicity of the nucleus-cytoskeleton interactions that occur during cell migration. Nuclei are under continuous tension from opposing intracellular forces and its tether to the cytoskeleton can be regulated at different levels. Interestingly, it was showed how nuclear positioning is highly related to cell function. In most migrating cells, including cancer cells, the nucleus can be the rate limiting step of cell migration and is placed away from the leading edge. By contrast, leukocytes position their nucleus close to the lamellipodia at the leading edge, and the nucleus contributes to drilling through the endothelium. Differences in cell migration in 2D versus 3D environments are also evident. The mechanisms and forces at play during nuclear positioning and translocation are clearly affected by the nature of the substrate. As such nuclear positioning during cell migration can vary between cell types and environments. In this review we aim to give an overview of the latest discoveries in the field revealing how nuclear positioning is tightly regulated, not only by intrinsic nuclear properties, such as deformability, nuclear envelope content or nucleus-cytoskeleton connectivity, but also by the microenvironment.

Nuf and Rip11 requirement for polarity determinant recycling during Drosophila development.

A tight relationship between apico-basal polarity and trafficking is essential for epithelial physiology and tissue homeostasis. Recent studies have described how some Rab GTPases, key components of the intracellular traffic machinery, contribute to the establishment of cell polarity in vertebrates. We have demonstrated a novel connection between cell polarity and trafficking: in Drosophila epithelia, the apical determinant aPKC is recycled via Rab11-Nuf-recycling endosomes to maintain cell polarity. Furthermore, the phosphorylation of Nuf by aPKC allows aPKC to control the sub-cellular localization of Nuf and its own membrane accumulation. Here we review these data and show the different contribution of the 2 Drosophila Rab11 adaptor proteins, Nuf and Rip11, to the maintenance of Drosophila embryonic ectoderm polarity.

SnapShot: Nucleo-cytoskeletal Interactions.

The nucleus is connected to the cytoskeleton, and these connections are involved in multiple functions such as nuclear positioning, shape and stiffness, cytoskeleton organization, mechanotransduction, gene expression, chromosome positioning, DNA repair, and cell migration.

Nuclear fallout provides a new link between aPKC and polarized cell trafficking.

BACKGROUND: Cell polarity, essential for cell physiology and tissue coherence, emerges as a consequence of asymmetric localization of protein complexes and directional trafficking of cellular components. Although molecules required in both processes are well known their relationship is still poorly understood. RESULTS: Here we show a molecular link between Nuclear Fallout (Nuf), an adaptor of Rab11-GTPase to the microtubule motor proteins during Recycling Endosome (RE) trafficking, and aPKC, a pivotal kinase in the regulation of cell polarity. We demonstrate that aPKC phosphorylates Nuf modifying its subcellular distribution. Accordingly, in aPKC mutants Nuf and Rab11 accumulate apically indicating altered RE delivery. We show that aPKC localization in the apico-lateral cortex is dynamic. When we block exocytosis, by means of exocyst-sec mutants, aPKC accumulates inside the cells. Moreover, apical aPKC concentration is reduced in nuf mutants, suggesting aPKC levels are maintained by recycling. CONCLUSIONS: We demonstrate that active aPKC interacts with Nuf, phosphorylating it and, as a result, modifying its subcellular distribution. We propose a regulatory loop by which Nuf promotes aPKC apical recycling until sufficient levels of active aPKC are reached. Thus, we provide a novel link between cell polarity regulation and traffic control in epithelia.

Impact of incident comorbidity on functional loss in elderly chronic kidney disease patients undergoing hemodialysis.

The incidence of end stage renal disease in older persons has been increasing progressively over the last 10 years. Improved survival rates with renal replacement therapy are making this increased prevalence even more pronounced. The usual risks of morbidity and requirements for specialized care associated with older people increase dramatically when they have chronic kidney disease (CKD). It has been seen that the majority of patients in hemodialysis units are over the age of 60, and have significant co-morbidities. The relationship between older age, chronic disorders and functional dependence (FD) is well known. Accordingly, nursing care planning must be designed with this in mind. The aim of this study was to assess whether the comorbidity associated with CKD modifies FD in patients on hemodialysis. We undertook a prospective longitudinal cohort study of hemodialysis outpatients in Málaga, Spain, using the Barthel test to establish FD and the Charlson comorbidity index to quantify comorbidity. All health events were analyzed to select those study patients with incident comorbidity, understood as the appearance of a new disease that could modify the Charlson comorbidity index, and determine the change in FD. Multivariate linear regression showed that the best model for predicting functional loss was that which considered comorbidity adjusted for age, particularly when it occurred as a result of hospital admission, as it was shown to have an important predictive value for the onset of a decrease in functional dependency scores in patients with CKD.

Design, synthesis and evaluation of 4,5-di-substituted acridone ligands with high G-quadruplex affinity and selectivity, together with low toxicity to normal cells.

A series of 4,5-di-substituted acridones have been designed and synthesized. Several compounds show high affinity for telomeric G-quadruplex DNA in classical and competition FRET assays, together with low duplex DNA affinity, although they do not show activity in a telomerase assay or evidence of telomere shortening. They have low toxicity against a panel of cancer cell lines and a normal human fibroblast line, and produce potent senescence-based long-term growth arrest in the MCF7 and A549 cancer cell lines.

Targeting human gastrointestinal stromal tumor cells with a quadruplex-binding small molecule.

Most of human gastrointestinal stromal tumors (GIST) are driven by activating mutations in the proto-oncogene KIT, a tyrosine kinase receptor. Clinical treatment with imatinib targets the kinase domain of KIT, but tumor regrowth occurs as a result of the development of resistant mutations in the kinase active site. An alternative small-molecule approach to GIST therapy is described, in which the KIT gene is directly targeted, and thus, kinase resistance may be circumvented. A naphthalene diimide derivative has been used to demonstrate the concept of dual quadruplex targeting. This compound strongly stabilizes both telomeric quadruplex DNA and quadruplex sites in the KIT promoter in vitro. It is shown here that the compound is a potent inducer of growth arrest in a patient-derived GIST cell line at a concentration (approximately 1 microM) that also results in effective inhibition of telomerase activity and almost complete suppression of KIT mRNA and KIT protein expression. Molecular modeling studies with a telomeric quadruplex have been used to rationalize aspects of the experimental quadruplex melting data.

Técnica modificada de Seldinger. Canalización de catéteres venosos centrales a través de catéteres venosos periféricos / Seldinger modified technique canalizing central blood vessel catheters through peripheral blood vessel catheters

Se describe el procedimiento de inserción de catéteres venosos centrales a través de acceso periférico, utilizando la técnica modificada de Seldinger, dado que en el servicio de críticos del Hospital Regional Universitario Carlos Haya de Málaga se atiende un elevado número de pacientes, que necesitan tener canalizado un catéter venoso central para optimizar el tratamiento que reciben. En numerosos casos, no es posible la inserción del catéter venoso central tipo DRUM, por el insuficiente calibre de las venas; entonces realizamos el procedimiento de canalización venosa central utilizando la técnica modificada de Seldinger. Trabajo expuesto en el III Congreso Nacional de Enfermería Sociosanitaria(AU)

The authors describe the procedure to insert central blood vessel catheters through peripheral blood vessel catheters using the Seldinger modified technique since critically ill patients service at the Carlos Haya Regional University Hospital in Malaga cares for a high number of patients who need to have canalized a central blood vessel catheter to maximize the treatment they receive. In many cases it is not possible to insert a DRUM type central blood vessel catheter due to an insufficient blood vessel caliber and then the nursing team at this hospital opts to carry out a procedure to canalize a central blood vessel using the Seldinger modified technique. This report was presented at the III National Social-Sanitary Nursing Congress(AU)

Topology conservation and loop flexibility in quadruplex-drug recognition: crystal structures of inter- and intramolecular telomeric DNA quadruplex-drug complexes.

Knowledge of the biologically relevant topology is critical for the design of drugs targeting quadruplex nucleic acids. We report here crystal structures of a G-quadruplex-selective ligand complexed with two human telomeric DNA quadruplexes. The intramolecular quadruplex sequence d[TAGGG(TTAGGG)(3)] and the bimolecular quadruplex sequence d(TAGGGTTAGGGT) were co-crystallized with a tetra-substituted naphthalene diimide quadruplex-binding ligand. The structures were solved and refined to 2.10- and 2.20-A resolution, respectively, revealing that the quadruplex topology in both structures is unchanged by the addition of the ligands, retaining a parallel-stranded arrangement with external double-chain-reversal propeller loops. The parallel topology results in accessible external 5' and 3' planar G-tetrad surfaces for ligand stacking. This also enables significant ligand-induced conformational changes in several TTA propeller loops to take place such that the loops themselves are able to accommodate bound drug molecules without affecting the parallel quadruplex topology, by stacking on the external TTA connecting loop nucleotides. Ligands are bound into the external TTA loop nucleotides and stack onto G-tetrad surfaces. These crystal structures provide a framework for further ligand development of the naphthalene diimide series to enhance selectivity and affinity.

Tri- and tetra-substituted naphthalene diimides as potent G-quadruplex ligands.

A series of tri- and tetra-substituted naphthalene diimides have been designed and synthesized. Several compounds show exceptional affinity for telomeric G-quadruplex DNA in classical and competition FRET assays and SPR studies. They inhibit telomerase in the TRAP assay, and show potent senescence-based short-term anti-proliferative effects on MCF7 and A549 cancer cell lines, and localize in the nucleus and particularly the nucleolus of MCF7 cells.

[Seldinger modified technique]. / Técnica modificada de Seldinger. Canalización de catéteres venosos centrales a través de catéteres venosos periféricos.

The authors describe the procedure to insert central blood vessel catheters through peripheral blood vessel catheters using the Seldinger modified technique since critically ill patients service at the Carlos Haya Regional University Hospital in Malaga cares for a high number of patients who need to have canalized a central blood vessel catheter to maximize the treatment they receive. In many cases it is not possible to insert a DRUM type central blood vessel catheter due to an insufficient blood vessel caliber and then the nursing team at this hospital opts to carry out a procedure to canalize a central blood vessel using the Seldinger modified technique. This report was presented at the III National Social-Sanitary Nursing Congress.

Synthesis of distamycin A polyamides targeting G-quadruplex DNA.

A number of amide-linked oligopyrroles based on distamycin molecules have been synthesized by solid-state methods, and their interactions with a human intramolecular G-quadruplex have been measured by a melting procedure. Several of these molecules show an enhanced ratio of quadruplex vs. duplex DNA binding compared to distamycin itself, including one with a 2,5-disubstituted pyrrole group. Quadruplex affinity increases with the number of pyrrole groups, and it is suggested that this is consistent with a mixed groove/G-quartet stacking binding mode.

Trisubstituted acridines as G-quadruplex telomere targeting agents. Effects of extensions of the 3,6- and 9-side chains on quadruplex binding, telomerase activity, and cell proliferation.

The synthesis is reported of a group of 3,6,9-trisubstituted acridine compounds as telomeric quadruplex-stabilizing ligands with systematic variations at the 3-, 6-, and 9-positions. A new microwave-assisted methodology has been developed for trisubstituted acridine synthesis. Structure-activity relationships are reported using surface plasmon resonance and a fluorescence melting assay to examine quadruplex binding, together with a telomerase inhibition assay. These reveal relationships between G-quadruplex stabilization and telomerase inhibition and optimal 3,6- and 9-substituent side-chain lengths for maximal activity. Qualitative molecular modeling using molecular dynamics simulations has been undertaken on four quadruplex-DNA complexes. Long-term exposure of MCF7 cancer cells to a subset of the most active compounds, at doses lower than the IC(50) values, showed that one compound produced a marked decrease in population growth, accompanied by senescence, which is consistent with telomere targeting by this agent.

Synthesis and antibody-binding studies of a series of parasite fuco-oligosaccharides.

Complex multifucosylated oligosaccharides are structural elements of glycoprotein and glycolipid subsets of larval, egg, and adult stages of Schistosoma, the parasitic worms that cause schistosomiasis, a serious disease affecting more than 200 million people in the tropics. The fucosylated structures are thought to play an important role in the immunology of schistosomiasis. Defined schistosomal oligosaccharides that enable immunological studies are difficult to obtain from natural sources. Therefore, we have chemically synthesized spacer-linked GlcNAc, Fucalpha1-3GlcNAc, Fucalpha1-2Fucalpha1-3GlcNAc, and Fucalpha1-2Fucalpha1-2Fucalpha1-3GlcNAc. This series of linear oligosaccharides was used to screen a library of anti-schistosome monoclonal antibodies by surface plasmon resonance spectroscopy. Interestingly, the reactive antibodies could be grouped according to their specificity for the different oligosaccharides tested, showing that these oligosaccharides form different immunological entities based on the number and linkage of the fucose residues. Subsequently, the thus defined monoclonal antibodies were used to visualize the expression of the corresponding oligosaccharide epitopes by adult Schistosoma mansoni worms.