Miracle-dz/UniFAR-FTUs huggingface dataset:
Activated Cyclin-Dependent Kinase 5 Promotes Microglial Phagocytosis of Fibrillar β-Amyloid by Up-regulating Lipoprotein Lipase Expression*

Amyloid plaques are crucial for the pathogenesis of Alzheimer disease (AD). Phagocytosis of fibrillar β-amyloid (Aβ) by activated microglia is essential for Aβ clearance in Alzheimer disease. However, the mechanism underlying Aβ clearance in the microglia remains unclear. In this study, we performed stable isotope labeling of amino acids in cultured cells for quantitative proteomics analysis to determine the changes in protein expression in BV2 microglia treated with or without Aβ. Among 2742 proteins identified, six were significantly up-regulated and seven were down-regulated by Aβ treatment. Bioinformatic analysis revealed strong over-representation of membrane proteins, including lipoprotein lipase (LPL), among proteins regulated by the Aβ stimulus. We verified that LPL expression increased at both mRNA and protein levels in response to Aβ treatment in BV2 microglia and primary microglial cells. Silencing of LPL reduced microglial phagocytosis of Aβ, but did not affect degradation of internalized Aβ. Importantly, we found that enhanced cyclin-dependent kinase 5 (CDK5) activity by increasing p35-to-p25 conversion contributed to LPL up-regulation and promoted Aβ phagocytosis in microglia, whereas inhibition of CDK5 reduced LPL expression and Aβ internalization. Furthermore, Aβ plaques was increased with reducing p25 and LPL level in APP/PS1 mouse brains, suggesting that CDK5/p25 signaling plays a crucial role in microglial phagocytosis of Aβ. In summary, our findings reveal a potential role of the CDK5/p25-LPL signaling pathway in Aβ phagocytosis by microglia and provide a new insight into the molecular pathogenesis of Alzheimer disease.
A versatile approach for the site-specific modification of recombinant antibodies using a combination of enzyme-mediated bioconjugation and click chemistry.

A unique two-step modular system for site-specific antibody modification and conjugation is reported. The first step of this approach uses enzymatic bioconjugation with the transpeptidase Sortase A for incorporation of strained cyclooctyne functional groups. The second step of this modular approach involves the azide-alkyne cycloaddition click reaction. The versatility of the two-step approach has been exemplified by the selective incorporation of fluorescent dyes and a positron-emitting copper-64 radiotracer for fluorescence and positron-emission tomography imaging of activated platelets, platelet aggregates, and thrombi, respectively. This flexible and versatile approach could be readily adapted to incorporate a large array of tailor-made functional groups using reliable click chemistry whilst preserving the activity of the antibody or other sensitive biological macromolecules.
Quantitative Proteomic Analysis Reveals Effects of Epidermal Growth Factor Receptor (EGFR) on Invasion-promoting Proteins Secreted by Glioblastoma Cells*

Glioblastoma multiforme is a highly invasive and aggressive brain tumor with an invariably poor prognosis. The overexpression of epidermal growth factor receptor (EGFR) is a primary influencer of invasion and proliferation in tumor cells and the constitutively active EGFRvIII mutant, found in 30–65% of Glioblastoma multiforme, confers more aggressive invasion. To better understand how EGFR contributes to tumor aggressiveness, we investigated the effect of EGFR on the secreted levels of 65 rationally selected proteins involved in invasion. We employed selected reaction monitoring targeted mass spectrometry using stable isotope labeled internal peptide standards to quantity proteins in the secretome from five GBM (U87) isogenic cell lines in which EGFR, EGFRvIII, and/or PTEN were expressed. Our results show that cell lines with EGFR overexpression and constitutive EGFRvIII expression differ remarkably in the expression profiles for both secreted and intracellular signaling proteins, and alterations in EGFR signaling result in reproducible changes in concentrations of secreted proteins. Furthermore, the EGFRvIII-expressing mutant cell line secretes the majority of the selected invasion-promoting proteins at higher levels than other cell lines tested. Additionally, the intracellular and extracellular protein measurements indicate elevated oxidative stress in the EGFRvIII-expressing cell line. In conclusion, the results of our study demonstrate that EGFR signaling has a significant effect on the levels of secreted invasion-promoting proteins, likely contributing to the aggressiveness of Glioblastoma multiforme. Further characterization of these proteins may provide candidates for new therapeutic strategies and targets as well as biomarkers for this aggressive disease.
Identification of Candidate Substrates for the Golgi Tul1 E3 Ligase Using Quantitative diGly Proteomics in Yeast*

Maintenance of protein homeostasis is essential for cellular survival. Central to this regulation are mechanisms of protein quality control in which misfolded proteins are recognized and degraded by the ubiquitin-proteasome system. One well-studied protein quality control pathway requires endoplasmic reticulum (ER)-resident, multi-subunit E3 ubiquitin ligases that function in ER-associated degradation. Using fission yeast, our lab identified the Golgi Dsc E3 ligase as required for proteolytic activation of fungal sterol regulatory element-binding protein transcription factors. The Dsc E3 ligase contains five integral membrane subunits and structurally resembles ER-associated degradation E3 ligases. Saccharomyces cerevisiae codes for homologs of Dsc E3 ligase subunits, including the Dsc1 E3 ligase homolog Tul1 that functions in Golgi protein quality control. Interestingly, S. cerevisiae lacks sterol regulatory element-binding protein homologs, indicating that novel Tul1 E3 ligase substrates exist. Here, we show that the S. cerevisiae Tul1 E3 ligase consists of Tul1, Dsc2, Dsc3, and Ubx3 and define Tul1 complex architecture. Tul1 E3 ligase function required each subunit as judged by vacuolar sorting of the artificial substrate Pep12D. Genetic studies demonstrated that Tul1 E3 ligase was required in cells lacking the multivesicular body pathway and under conditions of ubiquitin depletion. To identify candidate substrates, we performed quantitative diGly proteomics using stable isotope labeling by amino acids in cell culture to survey ubiquitylation in wild-type and tul1Δ cells. We identified 3116 non-redundant ubiquitylation sites, including 10 sites in candidate substrates. Quantitative proteomics found 4.5% of quantified proteins (53/1172) to be differentially expressed in tul1Δ cells. Correcting the diGly dataset for these differences increased the number of Tul1-dependent ubiquitylation sites. Together, our data demonstrate that the Tul1 E3 ligase functions in protein homeostasis under non-stress conditions and support a role in protein quality control. This quantitative diGly proteomics methodology will serve as a robust platform for screening for stress conditions that require Tul1 E3 ligase activity.
Novel, Single-Dose Microsphere Formulation of Azithromycin Versus Levofloxacin for the Treatment of Acute Exacerbation of Chronic Bronchitis

Abstract: A novel microsphere formulation of azithromycin enables administration as a single, 2.0-g oral dose, while maintaining tolerability. This multicenter, randomized, double-blind, double-dummy study compared azithromycin microspheres with levofloxacin (500 mg/d for 7 days) in the treatment of acute exacerbations of chronic bronchitis. Subjects had Anthonisen Type 1 exacerbations, were ≥50 years old, and had a ≥20 pack-year smoking history. The primary end point was clinical response at the test of cure visit (Days 14-21). The secondary end point was bacterial response at the test of cure visit in subjects with a baseline pathogen. Clinical cure rates in the clinical per protocol population were 93.6% (206/220) for azithromycin microspheres and 92.7% (202/218) for levofloxacin. Clinical cure rates in subjects with Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis infection were 95% (19/20), 94.7% (18/19), and 92% (23/25), respectively, for azithromycin microspheres, and 100% (26/26), 90.5% (19/21), and 81.3% (13/16), respectively, for levofloxacin. Both treatments were well tolerated. A single dose of azithromycin microspheres was as effective as a 7-day course of levofloxacin in the treatment of acute exacerbations of chronic bronchitis and was well tolerated.
Comparison of the Cytotoxicities and Wound Healing Effects of Hyaluronan, Carbomer, and Alginate on Skin Cells In Vitro

OBJECTIVE: To compare the cytotoxicities and efficacy of hyaluronan (HA), carbomer, and sodium alginate on repairing thermal-injured cells and promoting cell migration. DESIGN: The 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetra-zoliumromide method was used to evaluate the cytotoxicities of HA, carbomer, and sodium alginate on L929 mouse fibroblasts and their repairing ability to thermal-injured HaCaT keratinocytes. A scratch test was used to observe the effects of the 3 materials on cell migration. RESULTS: Hyaluronan with different molecular weights were nontoxic, even at the concentration of 0.5%, whereas carbomer and sodium alginate showed mild or moderate cytotoxicities when their concentrations were higher than 0.1%. Cell viability and cell density of the thermal-injured keratinocytes treated with HA (600, 1070, and 1500 kDa) were increased significantly compared with that of model control (P < .05), whereas carbomer aggravated cell injury, and sodium alginate had no obvious repairing ability. Hyaluronan promoted cell migration significantly with higher cell density in the scratch area, compared with the control after culture for 48 hours; both carbomer and sodium alginate inhibited the cell migration, and carbomer altered the cell morphology completely. CONCLUSIONS: Hyaluronan can repair cell injury and promote cell migration and proliferation. It also has good biocompatibility. As a new type of hydrogel matrix, HA is superior to carbomer and sodium alginate if it is used in wound caring preparations.
Vardenafil: efficacy, tolerability and future directions

Vardenafil is a short-acting PDE5 inhibitor used in the treatment of male erectile dysfunction. It is the most potent and specific of the three commercially available PDE5 inhbitors, which may decrease incidence of side effects, although this has not been demonstrated clinically. Although its efficacy in the treatment of erectile dysfunction has been demonstrated in several studies, recent and continuing studies have shown vardenafil's promise as an effective agent in penile rehabilitation, decreasing lower urinary tract symptoms resulting from benign prostatic hyperplasia and management of pulmonary hypertension.
Nonhealing Ulcers in Patients with Tophaceous Gout: A Systematic Review.

13. Patients with poorly controlled hyperuricemia a. should wear loose-fitting slippers to decrease the risk of tophi. b. are usually diabetic. c. are at increased risk of tophaceous gout. 14. The study by Patel et al revealed that prior to treatment of ulcers from tophaceous gout a. no patients had formed granulation tissue. b. all patients had both diabetes and hypertension. c. patients had positive joint fluid bacterial cultures during attacks of gout.
Cross-linking immunoprecipitation-MS (xIP-MS): Topological Analysis of Chromatin-associated Protein Complexes Using Single Affinity Purification*

In recent years, cross-linking mass spectrometry has proven to be a robust and effective method of interrogating macromolecular protein complex topologies at peptide resolution. Traditionally, cross-linking mass spectrometry workflows have utilized homogenous complexes obtained through time-limiting reconstitution, tandem affinity purification, and conventional chromatography workflows. Here, we present cross-linking immunoprecipitation-MS (xIP-MS), a simple, rapid, and efficient method for structurally probing chromatin-associated protein complexes using small volumes of mammalian whole cell lysates, single affinity purification, and on-bead cross-linking followed by LC-MS/MS analysis. We first benchmarked xIP-MS using the structurally well-characterized phosphoribosyl pyrophosphate synthetase complex. We then applied xIP-MS to the chromatin-associated cohesin (SMC1A/3), XRCC5/6 (Ku70/86), and MCM complexes, and we provide novel structural and biological insights into their architectures and molecular function. Of note, we use xIP-MS to perform topological studies under cell cycle perturbations, showing that the xIP-MS protocol is sufficiently straightforward and efficient to allow comparative cross-linking experiments. This work, therefore, demonstrates that xIP-MS is a robust, flexible, and widely applicable methodology for interrogating chromatin-associated protein complex architectures.
Comparative Phosphoproteomics Analysis of VEGF and Angiopoietin-1 Signaling Reveals ZO-1 as a Critical Regulator of Endothelial Cell Proliferation*

VEGF and angiopoietin-1 (Ang-1) are essential factors to promote angiogenesis through regulation of a plethora of signaling events in endothelial cells (ECs). Although pathways activated by VEGF and Ang-1 are being established, the unique signaling nodes conferring specific responses to each factor remain poorly defined. Thus, we conducted a large-scale comparative phosphoproteomic analysis of signaling pathways activated by VEGF and Ang-1 in ECs using mass spectrometry. Analysis of VEGF and Ang-1 networks of regulated phosphoproteins revealed that the junctional proteins ZO-1, ZO-2, JUP and p120-catenin are part of a cluster of proteins phosphorylated following VEGF stimulation that are linked to MAPK1 activation. Down-regulation of these junctional proteins led to MAPK1 activation and accordingly, increased proliferation of ECs stimulated specifically by VEGF, but not by Ang-1. We identified ZO-1 as the central regulator of this effect and showed that modulation of cellular ZO-1 levels is necessary for EC proliferation during vascular development of the mouse postnatal retina. In conclusion, we uncovered ZO-1 as part of a signaling node activated by VEGF, but not Ang-1, that specifically modulates EC proliferation during angiogenesis.
Fragment-Based Discovery of a Dual pan-RET/VEGFR2 Kinase Inhibitor Optimized for Single-Agent Polypharmacology.

Oncogenic conversion of the RET (rearranged during transfection) tyrosine kinase is associated with several cancers. A fragment-based chemical screen led to the identification of a novel RET inhibitor, Pz-1. Modeling and kinetic analysis identified Pz-1 as a type II tyrosine kinase inhibitor that is able to bind the "DFG-out" conformation of the kinase. Importantly, from a single-agent polypharmacology standpoint, Pz-1 was shown to be active on VEGFR2, which can block the blood supply required for RET-stimulated growth. In cell-based assays, 1.0 nM of Pz-1 strongly inhibited phosphorylation of all tested RET oncoproteins. At 1.0 mg kg(-1) day(-1) per os, Pz-1 abrogated the formation of tumors induced by RET-mutant fibroblasts and blocked the phosphorylation of both RET and VEGFR2 in tumor tissue. Pz-1 featured no detectable toxicity at concentrations of up to 100.0 mg kg(-1), which indicates a large therapeutic window. This study validates the effectiveness and usefulness of a medicinal chemistry/polypharmacology approach to obtain an inhibitor capable of targeting multiple oncogenic pathways.
Comparison of the Proteome of Adult and Cord Erythroid Cells, and Changes in the Proteome Following Reticulocyte Maturation*

Cord blood stem cells are an attractive starting source for the production of red blood cells in vitro for therapy because of additional expansion potential compared with adult peripheral blood progenitors and cord blood banks usually being more representative of national populations than blood donors. Consequently, it is important to establish how similar cord RBCs are to adult cells. In this study, we used multiplex tandem mass tag labeling combined with nano-LC-MS/MS to compare the proteome of adult and cord RBCs and reticulocytes. 2838 unique proteins were identified, providing the most comprehensive compendium of RBC proteins to date. Using stringent criteria, 1674 proteins were quantified, and only a small number differed in amount between adult and cord RBC. We focused on proteins critical for RBC function. Of these, only the expected differences in globin subunits, along with higher levels of carbonic anhydrase 1 and 2 and aquaporin-1 in adult RBCs would be expected to have a phenotypic effect since they are associated with the differences in gaseous exchange between adults and neonates. Since the RBC and reticulocyte samples used were autologous, we catalogue the change in proteome following reticulocyte maturation. The majority of proteins (>60% of the 1671 quantified) reduced in abundance between 2- and 100-fold following maturation. However, ∼5% were at a higher level in RBCs, localized almost exclusively to cell membranes, in keeping with the known clearance of intracellular recycling pools during reticulocyte maturation. Overall, these data suggest that, with respect to the proteome, there is no barrier to the use of cord progenitors for the in vitro generation of RBCs for transfusion to adults other than the expression of fetal, not adult, hemoglobin.
Uncoupling phosphate deficiency from its major effects on growth and transcriptome via PHO1 expression in Arabidopsis.

Inorganic phosphate (Pi) is one of the most limiting nutrients for plant growth in both natural and agricultural contexts. Pi-deficiency leads to a strong decrease in shoot growth, and triggers extensive changes at the developmental, biochemical and gene expression levels that are presumably aimed at improving the acquisition of this nutrient and sustaining growth. The Arabidopsis thaliana PHO1 gene has previously been shown to participate in the transport of Pi from roots to shoots, and the null pho1 mutant has all the hallmarks associated with shoot Pi deficiency. We show here that A. thaliana plants with a reduced expression of PHO1 in roots have shoot growth similar to Pi-sufficient plants, despite leaves being strongly Pi deficient. Furthermore, the gene expression profile normally triggered by Pi deficiency is suppressed in plants with low PHO1 expression. At comparable levels of shoot Pi supply, the wild type reduces shoot growth but maintains adequate shoot vacuolar Pi content, whereas the PHO1 underexpressor maintains maximal growth with strongly depleted Pi reserves. Expression of the Oryza sativa (rice) PHO1 ortholog in the pho1 null mutant also leads to plants that maintain normal growth and suppression of the Pi-deficiency response, despite the low shoot Pi. These data show that it is possible to unlink low shoot Pi content with the responses normally associated with Pi deficiency through the modulation of PHO1 expression or activity. These data also show that reduced shoot growth is not a direct consequence of Pi deficiency, but is more likely to be a result of extensive gene expression reprogramming triggered by Pi deficiency.
N-Acetylcysteine attenuates acute lung injury induced by fat embolism*

Objectives: Fat embolism syndrome is a clinical issue in subjects with long-bone fracture. It may lead to acute lung injury. The mechanisms and therapeutic regimen remain unclear. The present study was designed to investigate the pathologic and biochemical changes after fat embolization in isolated rat lungs, and to test the effects of posttreatment with N-acetylcysteine (NAC). Design: Prospective, randomized, controlled animal study. Setting: University research laboratory. Subjects: A total of 36 perfused lungs isolated from Sprague-Dawley rats. Interventions: The isolated lungs were randomly assigned to receive physiologic saline solution (vehicle group), fat embolism (FE group), or FE with NAC posttreatment (FE + NAC group). There were 12 isolated lungs in each group. FE was produced by introduction of corn oil micelles. NAC at a dose 150 mg/kg was given 10 mins after FE. Measurements and Main Results: The extent of acute lung injury was evaluated by lung weight change, protein concentration in bronchoalveolar lavage, and exhaled nitric oxide. We also measured the pulmonary arterial pressure and capillary filtration coefficient and determined the nitrate/nitrite, methylguanidine, tumor necrosis factor-&agr;, and interleukin-1&bgr; in lung perfusate. Histopathologic changes of the lung were examined and quantified. The levels of neutrophil elastase and myeloperoxidase were determined. The expression of inducible nitric oxide synthase was detected. FE caused acute lung injury as evidenced by the lung weight changes, increases in exhaled nitric oxide and protein concentration in bronchoalveolar lavage, pulmonary hypertension, increased capillary filtration coefficient, and lung pathology. The insult also increased nitrate/nitrite, methylguanidine, tumor necrosis factor-&agr;, and interleukin-1&bgr; in lung perfusate, increased neutrophil elastase and myeloperoxidase levels, and upregulated inducible nitric oxide synthase expression. Posttreatment with NAC abrogated these changes induced by FE. Conclusion: FE caused acute lung injury and associated biochemical changes. Posttreatment with NAC was effective to alleviate the pathologic and biochemical changes caused by FE.
A Simple Light Isotope Metabolic Labeling (SLIM-labeling) Strategy: A Powerful Tool to Address the Dynamics of Proteome Variations In Vivo*

Many quantitative proteomics strategies rely on in vivo metabolic incorporation of amino acids with modified stable isotope profiles into proteins. These methods give rise to multiple ions for each peptide, with possible distortion of the isotopolog distribution, making the overall analytical process complex. We validated an alternative strategy, simple light isotope metabolic labeling (SLIM-labeling), which alleviates many of these problems. SLIM-labeling is based on the in vivo reduction of the isotopic composition of proteins using metabolic precursors with a unique light isotope composition to label all amino acids. This brings a new dimension to in-depth, high resolution MS-based quantitative proteomics. Here, we describe a 12C-based SLIM-labeling strategy using U-[12C]-glucose as the metabolic precursor of all amino acids in the pathogenic yeast Candida albicans. Monoisotopic ion intensity increased exponentially following 12C enrichment, substantially improving peptide identification scores and protein sequence coverage in bottom-up analyses. Multiplexing samples of 12C composition varying from natural abundance (98.93%) to 100% makes it possible to address relative quantification issues, keeping all the critical information for each peptide within a single isotopolog cluster. We applied this method to measure, for the first time, protein turnover at the proteome scale in Candida albicans and its modulation by inhibitors of the proteasome and vacuolar protein degradation systems.