Miracle-dz/UniFAR-FTUs huggingface dataset:
Musite, a Tool for Global Prediction of General and Kinase-specific Phosphorylation Sites*

Reversible protein phosphorylation is one of the most pervasive post-translational modifications, regulating diverse cellular processes in various organisms. High throughput experimental studies using mass spectrometry have identified many phosphorylation sites, primarily from eukaryotes. However, the vast majority of phosphorylation sites remain undiscovered, even in well studied systems. Because mass spectrometry-based experimental approaches for identifying phosphorylation events are costly, time-consuming, and biased toward abundant proteins and proteotypic peptides, in silico prediction of phosphorylation sites is potentially a useful alternative strategy for whole proteome annotation. Because of various limitations, current phosphorylation site prediction tools were not well designed for comprehensive assessment of proteomes. Here, we present a novel software tool, Musite, specifically designed for large scale predictions of both general and kinase-specific phosphorylation sites. We collected phosphoproteomics data in multiple organisms from several reliable sources and used them to train prediction models by a comprehensive machine-learning approach that integrates local sequence similarities to known phosphorylation sites, protein disorder scores, and amino acid frequencies. Application of Musite on several proteomes yielded tens of thousands of phosphorylation site predictions at a high stringency level. Cross-validation tests show that Musite achieves some improvement over existing tools in predicting general phosphorylation sites, and it is at least comparable with those for predicting kinase-specific phosphorylation sites. In Musite V1.0, we have trained general prediction models for six organisms and kinase-specific prediction models for 13 kinases or kinase families. Although the current pretrained models were not correlated with any particular cellular conditions, Musite provides a unique functionality for training customized prediction models (including condition-specific models) from users' own data. In addition, with its easily extensible open source application programming interface, Musite is aimed at being an open platform for community-based development of machine learning-based phosphorylation site prediction applications. Musite is available at http://musite.sourceforge. net/.
The Plk1-dependent Phosphoproteome of the Early Mitotic Spindle*

Polo-like kinases regulate many aspects of mitotic and meiotic progression from yeast to man. In early mitosis, mammalian Polo-like kinase 1 (Plk1) controls centrosome maturation, spindle assembly, and microtubule attachment to kinetochores. However, despite the essential and diverse functions of Plk1, the full range of Plk1 substrates remains to be explored. To investigate the Plk1-dependent phosphoproteome of the human mitotic spindle, we combined stable isotope labeling by amino acids in cell culture with Plk1 inactivation or depletion followed by spindle isolation and mass spectrometry. Our study identified 358 unique Plk1-dependent phosphorylation sites on spindle proteins, including novel substrates, illustrating the complexity of the Plk1-dependent signaling network. Over 100 sites were validated by in vitro phosphorylation of peptide arrays, resulting in a broadening of the Plk1 consensus motif. Collectively, our data provide a rich source of information on Plk1-dependent phosphorylation, Plk1 docking to substrates, the influence of phosphorylation on protein localization, and the functional interaction between Plk1 and Aurora A on the early mitotic spindle.
Upregulation of Polymeric Immunoglobulin Receptor Expression by the Heat‐Inactivated Potential Probiotic Bifidobacterium bifidum OLB6378 in a Mouse Intestinal Explant Model

We determined whether a potential probiotic bacterium, Bifidobacterium bifidum OLB6378 (BB6378), exerts beneficial effects on the mucosal immune system in a mouse intestinal explant model. The addition of heat‐inactivated BB6378 to intestinal explants prepared from embryonic day 18 BALB/c mice increased the expression of polymeric immunoglobulin receptor (pIgR) mRNA by two‐ to fivefold. These effects were observed on ileal and colonic explants but not on jejunal explants, suggesting that the BB6378‐induced pIgR upregulation is site‐specific within the mouse intestine. The upregulation of pIgR protein expression in colonic explants was also detected after 24 h of culture. The results of DNA microarray analysis of ileal and colonic samples indicated that BB6378 increased the gene expression of interleukin (IL)‐1α and IL‐1β, and IL‐1α content in colonic explants was significantly increased after 20 h of culture with BB6378. We then examined the involvement of endogenously induced IL‐1α in pIgR mRNA upregulation by using IL‐1α knockout (KO) mice. Contrary to our expectations, pIgR mRNA expression was equally upregulated by BB6378 in colonic explants from BALB/c and IL‐1α KO mice. Conversely, we examined the involvement of Toll‐like receptors in pIgR mRNA upregulation by using MyD88 KO mice. The upregulation of pIgR was completely suppressed in the explants derived from MyD88 KO mice. Taken together, we conclude that in a mouse intestinal explant model, the heat‐inactivated potential probiotic BB6378 increases intestinal pIgR expression in a site‐specific manner and that the upregulation of pIgR could be explained by a direct microbial effect on the epithelium via Toll‐like receptors.
Differential Impact of Caveolae and Caveolin-1 Scaffolds on The Membrane Raft Proteome*

Caveolae, a class of cholesterol-rich lipid rafts, are smooth invaginations of the plasma membrane whose formation in nonmuscle cells requires caveolin-1 (Cav1). The recent demonstration that Cav1-associated cavin proteins, in particular PTRF/cavin-1, are also required for caveolae formation supports a functional role for Cav1 independently of caveolae. In tumor cells deficient for Golgi β-1,6N-acetylglucosaminyltransferase V (Mgat5), reduced Cav1 expression is associated not with caveolae but with oligomerized Cav1 domains, or scaffolds, that functionally regulate receptor signaling and raft-dependent endocytosis. Using subdiffraction-limit microscopy, we show that Cav1 scaffolds are homogenous subdiffraction-limit sized structures whose size distribution differs from that of Cav1 in caveolae expressing cells. These cell lines displaying differing Cav1/caveolae phenotypes are effective tools for probing the structure and composition of caveolae. Using stable isotope labeling by amino acids in cell culture, we are able to quantitatively distinguish the composition of caveolae from the background of detergent-resistant membrane proteins and show that the presence of caveolae enriches the protein composition of detergent-resistant membrane, including the recruitment of multiple heterotrimeric G-protein subunits. These data were further supported by analysis of immuno-isolated Cav1 domains and of methyl-β-cyclodextrin-disrupted detergent-resistant membrane. Our data show that loss of caveolae results in a dramatic change to the membrane raft proteome and that this change is independent of Cav1 expression. The proteomics data, in combination with subdiffraction-limit microscopy, indicates that noncaveolar Cav1 domains, or scaffolds are structurally and functionally distinct from caveolae and differentially impact on the molecular composition of lipid rafts.
Matrix Metalloproteinases in Thoracic Aortic Aneurysm Disease

The proteolytic enzymes known as matrix metalloproteinases, or MMPs, and their inhabitors, the tissue inhabitors of matrix metalloproteinases, or TIMPs, have recently been implicated in the pathophysiology of both abdominal and thoracic aortic aneurysms. There are over thirty different MMPs identifies. MMP-1 and MMP-9 appear to play a pivotal role in thoracic aortic aneurysms. It appears that MMPs are synthesized locally in the diseased aortic wall. The sites of synthesis appear focally concentrated in the aneurysmal aortic segments only. Tissue levels of MMPs appear indeed to be reflected in serum levels, opening the door for serial clinical monitoring. Pharmacologic therapy directed against MMPs promises to provide an alternative for discouraging growth and rupture of aortic aneurysms.
A histomorghometric study of screw‐shaped and removal torque titanium implants with three different surface topographies

Screw-shaped implants with 3 different surface topographies, evidenced visually as well as numerically with an optical profilometer, were inserted in rabbit bone. After a healing period of 12 weeks, a statistically significant higher removal torque was needed to unscrew screws blasted with 25-microns TiO2 particles and screws blasted with 75-microns particles of AI2O3 compared with screws with a turned surface. The histomorphometric evaluation demonstrated a higher percentage of bone-to-metal contact for implants blasted with 25-microns particles of TiO2 compared with the as-machined implants. A greater surface area of bone in threads was found for the turned screws compared with screws blasted with 25-microns TiO2 particles. In the short-term follow-up, there was a better fixation of implants with an average surface roughness of 0.9-1.3-microns and with a homogeneous surface structure than of implants with an average surface roughness of 0.4 microns and with a clear direction of the surface pattern.
Yersinia pestis Versus Yersinia pseudotuberculosis: Effects on Host Macrophages

Yersinia pestis, the causative agent of plague, is proved to be a recently emerged clone from Y. pseudotuberculosis. However, the diseases they cause and their patterns of transmission are very different. People always focus on the genetic changes between Y. pestis and Y. pseudotuberculosis to reveal their pathogenic differences, and little is known about host defence differences to these two Yersinia. In this study, the effects of Y. pestis and Y. pseudotuberculosis on macrophages were analysed. Cell apoptosis showed significant difference after the macrophages were infected by these two strains, and caspase‐3 activity also demonstrated a similar tendency. Further, macrophage function activities were evaluated. We found during the early infection of Y. pestis, several basic functions of macrophages, including phagocytosis, secretion of cytokine tumour necrosis factor‐α and nitric oxide, macrophage polarity and antigen presenting, were significantly interrupted. In comparison, Y. pseudotuberculosis infection showed lower inhibition on macrophages. Especially, Y. pestis infection might cause macrophage to polarize to M2 macrophages in the early phase, compared with Y. pseudotuberculosis infection, which was different from the common acute infection. These results clearly indicated even in the early stage of infection, different host macrophage defence patterns could help us to understand the obvious virulence differences between Y. pestis and Y. pseudotuberculosis.
Intercellular Variation in Signaling through the TGF-β Pathway and Its Relation to Cell Density and Cell Cycle Phase*

Fundamental open questions in signal transduction remain concerning the sequence and distribution of molecular signaling events among individual cells. In this work, we have characterized the intercellular variability of transforming growth factor β-induced Smad interactions, providing essential information about TGF-β signaling and its dependence on the density of cell populations and the cell cycle phase. By employing the recently developed in situ proximity ligation assay, we investigated the dynamics of interactions and modifications of Smad proteins and their partners under native and physiological conditions. We analyzed the kinetics of assembly of Smad complexes and the influence of cellular environment and relation to mitosis. We report rapid kinetics of formation of Smad complexes, including native Smad2-Smad3-Smad4 trimeric complexes, in a manner influenced by the rate of proteasomal degradation of these proteins, and we found a striking cell to cell variation of signaling complexes. The single-cell analysis of TGF-β signaling in genetically unmodified cells revealed previously unknown aspects of regulation of this pathway, and it provided a basis for analysis of these signaling events to diagnose pathological perturbations in patient samples and to evaluate their susceptibility to drug treatment.
Cells Lacking β-Actin are Genetically Reprogrammed and Maintain Conditional Migratory Capacity*

Vertebrate nonmuscle cells express two actin isoforms: cytoplasmic β- and γ-actin. Because of the presence and localized translation of β-actin at the leading edge, this isoform is generally accepted to specifically generate protrusive forces for cell migration. Recent evidence also implicates β-actin in gene regulation. Cell migration without β-actin has remained unstudied until recently and it is unclear whether other actin isoforms can compensate for this cytoplasmic function and/or for its nuclear role. Primary mouse embryonic fibroblasts lacking β-actin display compensatory expression of other actin isoforms. Consistent with this preservation of polymerization capacity, β-actin knockout cells have unchanged lamellipodial protrusion rates despite a severe migration defect. To solve this paradox we applied quantitative proteomics revealing a broad genetic reprogramming of β-actin knockout cells. This also explains why reintroducing β-actin in knockout cells does not restore the affected cell migration. Pathway analysis suggested increased Rho-ROCK signaling, consistent with observed phenotypic changes. We therefore developed and tested a model explaining the phenotypes in β-actin knockout cells based on increased Rho-ROCK signaling and increased TGFβ production resulting in increased adhesion and contractility in the knockout cells. Inhibiting ROCK or myosin restores migration of β-actin knockout cells indicating that other actins compensate for β-actin in this process. Consequently, isoactins act redundantly in providing propulsive forces for cell migration, but β-actin has a unique nuclear function, regulating expression on transcriptional and post-translational levels, thereby preventing myogenic differentiation.
GASTROINTESTINAL , HEPATOBILIARY , AND PANCREATIC PATHOLOGY Contributions of Hepatocytes and Bile Ductular Cells in Ductular Reactions and Remodeling of the Biliary System after Chronic Liver Injury

CME Accreditation Statement: This activity (“ASIP 2014 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.
Development and Validation of RP-HPLC Method for Azilsartan Medoxomil Potassium Quantitation in Human Plasma by Solid Phase Extraction Procedure

Simple and rapid reverse phase high-performance liquid chromatography (RP-HPLC) method was developed and validated using solid phase extraction (SPE) technique for the determination of Azilsartan Medoxomil Potassium (AMP) in human plasma; detection was carried out by photo diode array detector. Chromatographic separation of the analyte AMP was achieved within 7.5 min by Waters symmetry C18 (4.6 × 250 mm, 5 µm) column, mobile phase was 25 mM ammonium acetate buffer (pH 5.5): acetonitrile 55 : 45 v/v, flow rate was 1.0 mL/min, and the detection was carried out at 254 nm. Calibration curve was linear (r2 > 0.9985) in the range of 1.0–9.0 µg/mL, limit of detection (LOD) and limit of quantitation (LOQ) were 0.150 µg/mL and 0.400 µg/mL, respectively, and intra- and interday deviations were between 1.53–8.41% and 1.78–4.59%, respectively. The overall mean recovery of AMP was 92.35%. No any endogenous constituents were found to interfere at retention time of the analyte. This new RP-HPLC method was successfully validated and may be applied to conduct bioavailability and bioequivalence studies of AMP.
A new era of pressure ulcer accountability in acute care.

PURPOSE To familiarize wound care practitioners with the 5 Million Lives Campaign's pressure ulcer prevention intervention. TARGET AUDIENCE This continuing education activity is intended for physicians and nurses with an interest in wound care. OBJECTIVES After reading this article and taking this test, the reader should be able to: 1. List organizational and national initiatives that address pressure ulcer prevention. 2. Identify characteristics of pressure ulcers. 3. Identify 6 strategies that help prevent pressure ulcers.
Unusual ultra-hydrophilic, porous carbon cuboids for atmospheric-water capture.

There is significant interest in high-performance materials that can directly and efficiently capture water vapor, particularly from air. Herein, we report a class of novel porous carbon cuboids with unusual ultra-hydrophilic properties, over which the synergistic effects between surface heterogeneity and micropore architecture is maximized, leading to the best atmospheric water-capture performance among porous carbons to date, with a water capacity of up to 9.82 mmol g(-1) at P/P0 =0.2 and 25 °C (20% relative humidity or 6000 ppm). Benefiting from properties, such as defined morphology, narrow pore size distribution, and high heterogeneity, this series of functional carbons may serve as model materials for fundamental research on carbon chemistry and the advance of new types of materials for water-vapor capture as well as other applications requiring combined highly hydrophilic surface chemistry, developed hierarchical porosity, and excellent stability.
Screening for protein phosphorylation using nanoscale reactions on microdroplet arrays.

We present a novel and straightforward screening method to detect protein phosphorylations in complex protein mixtures. A proteolytic digest is separated by a conventional nanoscale liquid chromatography (nano-LC) separation and the eluate is immediately compartmentalized into microdroplets, which are spotted on a microarray MALDI plate. Subsequently, the enzyme alkaline phosphatase is applied to every second microarray spot to remove the phosphate groups from phosphorylated peptides, which results in a mass shift of n×-80 Da. The MALDI-MS scan of the microarray is then evaluated by a software algorithm to automatically identify the phosphorylated peptides by exploiting the characteristic chromatographic peak profile induced by the phosphatase treatment. This screening method does not require extensive MS/MS experiments or peak list evaluation and can be easily extended to other enzymatic or chemical reactions.
Estimating the Applicability of Wound Care Randomized Controlled Trials to General Wound-Care Populations by Estimating the Percentage of Individuals Excluded from a Typical Wound-Care Population in Such Trials

OBJECTIVE: To determine the percentage of individuals that would be excluded from wound care randomized controlled trials (RCTs) as a surrogate for applicability to general populations. DESIGN: A representative sample of wound-care RCTs was selected from the literature in the past 10 years. Exclusion criteria from the trials were evaluated, and prevalence values for each excluded condition were obtained from a large wound-care population, as well as from the literature. The percentage of patients excluded on this basis was calculated. SETTING: Seventeen RCTs testing "high-technology" wound-care products were evaluated. PATIENTS: Patients in the trials were treated for ulcers (venous, diabetic foot, and pressure ulcers). MAIN OUTCOME MEASURES: A percentage of patients in the study population were excluded for each RCT. MAIN RESULTS: More than 50% of the study population would have been excluded in 15 of the 17 RCTs. When less clinically relevant exclusion criteria were removed, 14 of 17 RCTs would still have excluded between 25% and 50% of the study population. CONCLUSION: The results raise serious questions regarding the applicability of these RCTs to wound-care populations.
A Classifier Based on Accurate Mass Measurements to Aid Large Scale, Unbiased Glycoproteomics*

Determining which glycan moieties occupy specific N-glycosylation sites is a highly challenging analytical task. Arguably, the most common approach involves LC-MS and LC-MS/MS analysis of glycopeptides generated by proteases with high cleavage site specificity; however, the depth achieved by this approach is modest. Nonglycosylated peptides are a major challenge to glycoproteomics, as they are preferentially selected for data-dependent MS/MS due to higher ionization efficiencies and higher stoichiometric levels in moderately complex samples. With the goal of improving glycopeptide coverage, a mass defect classifier was developed that discriminates between peptides and glycopeptides in complex mixtures based on accurate mass measurements of precursor peaks. By using the classifier, glycopeptides that were not fragmented in an initial data-dependent acquisition run may be targeted in a subsequent analysis without any prior knowledge of the glycan or protein species present in the mixture. Additionally, from probable glycopeptides that were poorly fragmented, tandem mass spectra may be reacquired using optimal glycopeptide settings. We demonstrate high sensitivity (0.892) and specificity (0.947) based on an in silico dataset spanning >100,000 tryptic entries. Comparable results were obtained using chymotryptic species. Further validation using published data and a fractionated tryptic digest of human urinary proteins was performed, yielding a sensitivity of 0.90 and a specificity of 0.93. Lists of glycopeptides may be generated from an initial proteomics experiment, and we show they may be efficiently targeted using the classifier. Considering the growing availability of high accuracy mass analyzers, this approach represents a simple and broadly applicable means of increasing the depth of MS/MS-based glycoproteomic analyses.
Interactome Analysis Reveals that C1QBP (complement component 1, q subcomponent binding protein) Is Associated with Cancer Cell Chemotaxis and Metastasis*

The complement component 1, q subcomponent binding protein (C1QBP/p32/HABP1) is a ubiquitously expressed and multicompartmental cellular protein involved in various biological processes. In order to further understand its biological functions, we conducted proteomics analysis of its interactome in this study. An improved sample preparation and mass spectrometric identification strategy was developed combining high-speed centrifugation, formaldehyde labeling, and two-dimensional reverse-phase liquid chromatography. Using this approach, we identified 187 interacting proteins and constructed a highly connected interacting network for C1QBP. Moreover, we explored the interaction between C1QBP and protein kinase C ζ, a key regulator of cell polarity and migration. The results indicated that C1QBP regulated the activity of protein kinase C ζ and modulated EGF-induced cancer cell chemotaxis. In addition, C1QBP was required for breast cancer metastasis in a severe combined immunodeficiency mouse model. Furthermore, C1QBP was observed to be overexpressed in breast cancer tissues, and its expression level was closely linked with distant metastasis and TNM stages. In summary, C1QBP was identified as a novel regulator of cancer metastasis that may serve as a therapeutic target for breast cancer treatment.