In-depth analyses of kinase-dependent tyrosine phosphoproteomes based on metal ion-functionalized soluble nanopolymers.

The ability to obtain in-depth understanding of signaling networks in cells is a key objective of systems biology research. Such ability depends largely on unbiased and reproducible analysis of phosphoproteomes. We present here a novel proteomics tool, polymer-based metal ion affinity capture (PolyMAC), for the highly efficient isolation of phosphopeptides to facilitate comprehensive phosphoproteome analyses. This approach uses polyamidoamine dendrimers multifunctionalized with titanium ions and aldehyde groups to allow the chelation and subsequent isolation of phosphopeptides in a homogeneous environment. Compared with current strategies based on solid phase micro- and nanoparticles, PolyMAC demonstrated outstanding reproducibility, exceptional selectivity, fast chelation times, and high phosphopeptide recovery from complex mixtures. Using the PolyMAC method combined with antibody enrichment, we identified 794 unique sites of tyrosine phosphorylation in malignant breast cancer cells, 514 of which are dependent on the expression of Syk, a protein-tyrosine kinase with unusual properties of a tumor suppressor. The superior sensitivity of PolyMAC allowed us to identify novel components in a variety of major signaling networks, including cell migration and apoptosis. PolyMAC offers a powerful and widely applicable tool for phosphoproteomics and molecular signaling.

Regulation of Syk by phosphorylation on serine in the linker insert.

The Syk protein-tyrosine kinase is phosphorylated on multiple tyrosines after the aggregation of the B cell antigen receptor. However, metabolic labeling experiments indicate that Syk is inducibly phosphorylated to an even greater extent on serine after receptor ligation. A combination of phosphopeptide mapping and mass spectrometric analyses indicates that serine 291 is a major site of phosphorylation. Serine 291 lies within a 23-amino acid insert located within the linker B region that distinguishes Syk from SykB and Zap-70. The phosphorylation of serine-291 by protein kinase C enhances the ability of Syk to couple the antigen receptor to the activation of the transcription factors NFAT and Elk-1. Protein interaction studies indicate a role for the phosphorylated linker insert in promoting an interaction between Syk and the chaperone protein, prohibitin.

One-step extraction of subcellular proteins from eukaryotic cells.

Conventional biochemical analysis mainly focuses on the expression level of cellular proteins from entire cells. However, it has been increasingly acknowledged that the subcellular location of proteins often carries important information. Analysis of subcellular proteins conventionally requires subcellular fractionation which involves two steps: cell lysis to release proteins and high-speed centrifugation to separate the homogenate. Such approach requires bulky and expensive equipment and is not compatible with processing scarce cell samples of limited volume. In this study, we apply microfluidic flow-through electroporation to breach cell membranes and extract cytosolic proteins selectively in a single step. We demonstrate that this approach allows monitoring the translocation of the transcription factor NF-kappaB from the cytosol to the nucleus without the need of subcellular fractionation. Our technique is compatible with the processing of samples of various sizes and provides a simple and universal tool for bioanalytical analysis and spatial proteomics.

Nipple Reconstruction with the Biodesign Nipple Reconstruction Cylinder: A Prospective Clinical Study.

BACKGROUND: Nipple reconstruction is the last stage in cosmetic reconstruction of the breast after mastectomy, but no method produces reliable and consistent aesthetic results. This study examined the use of the Biodesign Nipple Reconstruction Cylinder (NRC) during reconstruction of the nipple after mastectomy. METHODS: Patients with a history of breast cancer and mastectomy desiring nipple reconstruction were invited to participate. After obtaining consent, unilateral or bilateral nipple reconstruction was performed. Skin flaps were raised, the NRC was placed beneath the flaps as a stent, and the site was protected for up to 4 weeks with a nipple shield. Nipple projection was measured for 12 months after surgery. Patient satisfaction was measured and adverse events were recorded. Follow-up examinations were performed at 1 week, and then at 1, 3, 6, and 12 months after surgery. RESULTS: Eighty-two nipple reconstructions were performed in 50 patients. Related postoperative adverse events were minor, but reported in 8 reconstructions (9.8%) representing 7 patients (14.0%). Average projection at 6 and 12 months was 4.1 ± 1.6 mm and 3.8 ± 1.5 mm, respectively, compared with 10.5 ± 2.2 mm 1 week after surgery. Of patients completing the satisfaction questionnaire at 12 months, 70/75 (93.3%) of reconstructions were rated "pleased" or "very pleased" with the overall outcome. Overall, 45/46 (97.8%) patients would recommend nipple reconstruction to other women. CONCLUSIONS: The Biodesign NRC offers a safe alternative to nipple reconstruction, resulting in stable projection and a high level of patient satisfaction for 12 months after placement.

Intracellular targets for a phosphotyrosine peptidomimetic include the mitotic kinesin, MCAK.

SH2 domains are attractive targets for chemotherapeutic agents due to their involvement in the formation of protein-protein interactions critical to many signal transduction cascades. Little is known, however, about how synthetic SH2 domain ligands would influence the growth properties of tumor cells or with which intracellular proteins they would interact due to their highly charged nature and enzymatic lability. In this study, a prodrug delivery strategy was used to introduce an enzymatically stable, phosphotyrosine peptidomimetic into tumor cells. When tested in a human tumor cell panel, the prodrug exhibited a preference for inhibiting the growth of leukemia and lymphoma cells. In these cells, it was largely cytostatic and induced endoreduplication and the appearance of midbodies. Proteomic analyses identified multiple targets that included mitotic centromere-associated kinesin (MCAK). Molecular modeling studies suggested the ATP-binding site on MCAK as the likely site of drug interaction. Consistent with this, ATP inhibited the drug-MCAK interaction and the drug inhibited MCAK ATPase activity. Accordingly, the effects of the prodrug on the assembly of the mitotic spindle and alignment of chromosomes were consistent with the identification of MCAK as an important intracellular target.

[Post-Intensive Care Unit mortality and related prognostic factors in a cohort of critically ill patients with multi-organ dysfunction]. / Mortalidad tras el alta de la Unidad de Cuidados Intensivos y factores pronósticos relacionados en una cohorte de pacientes críticos con disfunción multiorgánica.

BACKGROUND AND OBJECTIVE: To assess the post-Intensive Care Unit (ICU) mortality (in-hospital and one year after hospital discharge) and the associated factors. PATIENTS AND METHOD: Cohort design in medical-surgical patients with multi-organ dysfunction syndrome (MODS) during the first 24h of admission to ICU. We recorded the following data: personal background, functional general situation, general information about admission to ICU, hospital stay and contact by phone after one year of hospital discharge. We registered mortality at the follow-up at anytime. Cox regression was performed to evaluate mortality factors. RESULTS: Five hundred and forty five patients were recruited. During the study period 256 patients (52.9%) died; out of them 29.5% in ICU; 14.8% of 384 patients transferred to the ward died. Of 327 discharged patients, 266 (81.3%) were contacted; 14.3% of those had died. In-hospital death-related factors were age (odds ratio [OR] 1.04; 95% confidence interval [95% CI] 1.02-1.06; P<.01) and a decreased functional general status (OR 1.7; 95% CI 1.1-2.9; P<.05). Post-hospitalisation mortality-related variables were: diminished functional general status (OR 2.42; 95% CI 1.23-4.75; P<.01) and readmission after discharge from hospital (1.45 OR; 95% CI 1.19-1.76; P<.001). CONCLUSIONS: Patients admitted for a medical-surgical MODS presented a mortality of 52.9% within one year. The factors influencing hospital mortality are age and a generally diminished functional status, both being not modifiable factors. After discharge, the decreased general functional status remained central along with the re-hospitalisation.

Akt2 inhibits the activation of NFAT in lymphocytes by modulating calcium release from intracellular stores.

The engagement of antigen receptors on lymphocytes leads to the activation of phospholipase C-γ, the mobilization of intracellular calcium and the activation of the NFAT transcription factor. The coupling of antigen receptors to the activation of NFAT is modulated by numerous cellular effectors including phospho-inositide 3-kinase (PI3K), which is activated following receptor cross-linking. The activation of PI3K has both positive and negative effects on the receptor-mediated activation of NFAT. An increase in the level and activity of Akt2, a target of activated PI3K, potently inhibits the subsequent activation of NFAT. In contrast, an elevation in Akt1 has no effect on signaling. Signaling pathways operating both upstream and downstream of inositol 1,4,5-trisphosphate (IP3)-stimulated calcium release from intracellular stores are unaffected by Akt2. An increase in the level of Akt2 has no significant effect on the initial amplitude, but substantially reduces the duration of calcium mobilization. The ability of Akt2 to inhibit prolonged calcium mobilization is abrogated by the administration of a cell permeable peptide that blocks the interaction between Bcl-2 and the IP3 receptor. Thus, Akt2 is a negative regulator of NFAT activation through its ability to inhibit calcium mobilization from the ER.

Two closely spaced tyrosines regulate NFAT signaling in B cells via Syk association with Vav.

Activated Syk, an essential tyrosine kinase in B cell signaling, interacts with Vav guanine nucleotide exchange factors and regulates Vav activity through tyrosine phosphorylation. The Vav SH2 domain binds Syk linker B by an unusual recognition of two closely spaced Syk tyrosines: Y342 and Y346. The binding affinity is highest when both Y342 and Y346 are phosphorylated. An investigation in B cells of the dependence of Vav phosphorylation and NFAT activation on phosphorylation of Y342 and Y346 finds that cellular response levels match the relative binding affinities of the Vav1 SH2 domain for singly and doubly phosphorylated linker B peptides. This key result suggests that the uncommon recognition determinant of these two closely spaced tyrosines is a limiting factor in signaling. Interestingly, differences in affinities for binding singly and doubly phosphorylated peptides are reflected in the on rate, not the off rate. Such a control mechanism would be highly effective for regulating binding among competing Syk binding partners. The nuclear magnetic resonance (NMR) structure of Vav1 SH2 in complex with a doubly phosphorylated linker B peptide reveals diverse conformations associated with the unusual SH2 recognition of two phosphotyrosines. NMR relaxation indicates compensatory changes in loop fluctuations upon binding, with implications for nonphosphotyrosine interactions of Vav1 SH2.

Mortalidad tras el alta de la Unidad de Cuidados Intensivos y factores pronósticos relacionados en una cohorte de pacientes críticos con disfunción multiorgánica / Post-Intensive Care Unit mortality and related prognostic factors in a cohort of critically ill patients with multi-organ dysfunction

Fundamento y objetivo: Evaluar la mortalidad tras el alta de la Unidad de Cuidados Intensivos –post-UCI– (hospitalaria y seguimiento a un año) y los factores asociados. Pacientes y método: Diseño de cohortes en enfermos medicoquirúrgicos con síndrome de disfunción multiorgánica (SDMO) durante sus primeras 24h de ingreso. Se registraron antecedentes personales, situación basal, datos generales de ingreso en UCI, estancia hospitalaria y datos de supervivencia mediante contacto telefónico al año. Se evaluó la mortalidad en cualquier momento del seguimiento, relizando una regresión de Cox para valorar factores de mortalidad. Resultados: Se reclutaron 545 pacientes. En el total del período de estudio fallecieron 256 pacientes (52,9%); de los ingresados en UCI fallecieron el 29,5%, mientras que de los 384 enfermos que pasaron a planta falleció el 14,8%. De los 327 enfermos dados de alta hospitalaria se ha contactado con el 81,3% (266 pacientes), de los que han fallecido el 14,3%. Los factores relacionados con la muerte hospitalaria fueron la edad (odds ratio [OR] 1,04; intervalo de confianza del 95% [IC 95%] 1,02-1,06; p<0,01) y una situación basal funcional disminuida (OR 1,7; IC 95% 1,1-2,9; p<0,05). Las variables relacionadas con la mortalidad posthospitalaria fueron: una situación basal funcional disminuida (OR 2,42; IC 95% 1,23-4,75; p<0,01) y el reingreso tras el alta hospitalaria (OR 1,45; IC 95% 1,19-1,76; p<0,001). Conclusiones: Los pacientes ingresados por un SDMO medicoquirúrgico presentan una mortalidad al año de seguimiento del 52,9%. Los factores que más influyen en la mortalidad hospitalaria son la edad y una situación funcional basal disminuida, ambos factores no modificables. Tras el alta hospitalaria, la situación funcional basal disminuida sigue siendo transcendental, junto con el reingreso hospitalario (AU)

Background and objective: To assess the post-Intensive Care Unit (ICU) mortality (in-hospital and one year after hospital discharge) and the associated factors. Patients and method: Cohort design in medical-surgical patients with multi-organ dysfunction syndrome (MODS) during the first 24h of admission to ICU. We recorded the following data: personal background, functional general situation, general information about admission to ICU, hospital stay and contact by phone after one year of hospital discharge. We registered mortality at the follow-up at anytime. Cox regression was performed to evaluate mortality factors. Results: Five hundred and forty five patients were recruited. During the study period 256 patients (52.9%) died; out of them 29.5% in ICU; 14.8% of 384 patients transferred to the ward died. Of 327 discharged patients, 266 (81.3%) were contacted; 14.3% of those had died. In-hospital death-related factors were age (odds ratio [OR] 1.04; 95% confidence interval [95% CI] 1.02-1.06; P<.01) and a decreased functional general status (OR 1.7; 95% CI 1.1-2.9; P<.05). Post-hospitalisation mortality-related variables were: diminished functional general status (OR 2.42; 95% CI 1.23-4.75; P<.01) and readmission after discharge from hospital (1.45 OR; 95% CI 1.19-1.76; P<.001). Conclusions: Patients admitted for a medical-surgical MODS presented a mortality of 52.9% within one year. The factors influencing hospital mortality are age and a generally diminished functional status, both being not modifiable factors. After discharge, the decreased general functional status remained central along with the re-hospitalisation (AU)