Phenotypic, genotypic and proteomic variations between poor and robust colonizing Campylobacter jejuni strains.

Campylobacter jejuni is one of the major causes of bacterial gastrointestinal disease in humans worldwide. This foodborne pathogen colonizes the intestinal tracts of chickens, and consumption of chicken and poultry products is identified as a common route of transmission. We analyzed two C. jejuni strains after oral challenge with 105 CFU/ml of C. jejuni per chick; one strain was a robust colonizer (A74/C) and the other a poor colonizer (A74/O). We also found extensive phenotypic differences in growth rate, biofilm production, and in vitro adherence, invasion, intracellular survival, and transcytosis. Strains A74/C and A74/O were genotypically similar with respect to their whole genome alignment, core genome, and ribosomal MLST, MLST, flaA, porA, and PFGE typing. The global proteomes of the two congenic strains were quantitatively analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and 618 and 453 proteins were identified from A74/C and A74/O isolates, respectively. Cluster of Orthologous Groups (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that carbon metabolism and motility proteins were distinctively overexpressed in strain A74/C. The robust colonizer also exhibited a unique proteome profile characterized by significantly increased expression of proteins linked to adhesion, invasion, chemotaxis, energy, protein synthesis, heat shock proteins, iron regulation, two-component regulatory systems, and multidrug efflux pump. Our study underlines phenotypic, genotypic, and proteomic variations of the poor and robust colonizing C. jejuni strains, suggesting that several factors may contribute to mediating the different colonization potentials of the isogenic isolates.

Trends in the prevalence of cognitive impairment at old age in China, 2002-2018.

INTRODUCTION: China has the world's largest number of older adults with cognitive impairment (CI). We aimed to examine secular trends in the prevalence of CI in China from 2002 to 2018. METHODS: Generalized estimating equations (GEE) was used to assess changes in CI trend in 44,154 individuals (72,027 observations) aged 65 to 105 years old. RESULTS: The prevalence of CI increased from 2002 to 2008 and then decreased until 2018. The age-standardized prevalence increased from 25.7% in 2002, 26.1% in 2005, to 28.2% in 2008, then decreased to 26.0% in 2011, 25.3% in 2014, and 24.9% in 2018. Females and those ≥ 80 years old had greater CI prevalence. DISCUSSION: The prevalence of CI showed an inverted U shape from early 2000s to late 2010s with a peak in 2008. Follow-up studies are needed to confirm the decreasing trend after 2008 and examine the contributing factors and underlying mechanisms of this trend. HIGHLIGHTS: Generalized estimating equations (GEE) were used to assess trends of changes in cognitive impairment (CI). CI prevalence in China increased from 2002 to 2008 and then decreased until 2018. Females and those ≥ 80 years old had greater CI prevalence. Stroke, diabetes, and cigarette smoking were risk factors for CI.

Current Practices in LC-MS Untargeted Metabolomics: A Scoping Review on the Use of Pooled Quality Control Samples.

Untargeted metabolomics is an analytical approach with numerous applications serving as an effective metabolic phenotyping platform to characterize small molecules within a biological system. Data quality can be challenging to evaluate and demonstrate in metabolomics experiments. This has driven the use of pooled quality control (QC) samples for monitoring and, if necessary, correcting for analytical variance introduced during sample preparation and data acquisition stages. Described herein is a scoping literature review detailing the use of pooled QC samples in published untargeted liquid chromatography-mass spectrometry (LC-MS) based metabolomics studies. A literature query was performed, the list of papers was filtered, and suitable articles were randomly sampled. In total, 109 papers were each reviewed by at least five reviewers, answering predefined questions surrounding the use of pooled quality control samples. The results of the review indicate that use of pooled QC samples has been relatively widely adopted by the metabolomics community and that it is used at a similar frequency across biological taxa and sample types in both small- and large-scale studies. However, while many studies generated and analyzed pooled QC samples, relatively few reported the use of pooled QC samples to improve data quality. This demonstrates a clear opportunity for the field to more frequently utilize pooled QC samples for quality reporting, feature filtering, analytical drift correction, and metabolite annotation. Additionally, our survey approach enabled us to assess the ambiguity in the reporting of the methods used to describe the generation and use of pooled QC samples. This analysis indicates that many details of the QC framework are missing or unclear, limiting the reader's ability to determine which QC steps have been taken. Collectively, these results capture the current state of pooled QC sample usage and highlight existing strengths and deficiencies as they are applied in untargeted LC-MS metabolomics.

Quality assurance and quality control reporting in untargeted metabolic phenotyping: mQACC recommendations for analytical quality management.

BACKGROUND: Demonstrating that the data produced in metabolic phenotyping investigations (metabolomics/metabonomics) is of good quality is increasingly seen as a key factor in gaining acceptance for the results of such studies. The use of established quality control (QC) protocols, including appropriate QC samples, is an important and evolving aspect of this process. However, inadequate or incorrect reporting of the QA/QC procedures followed in the study may lead to misinterpretation or overemphasis of the findings and prevent future metanalysis of the body of work. OBJECTIVE: The aim of this guidance is to provide researchers with a framework that encourages them to describe quality assessment and quality control procedures and outcomes in mass spectrometry and nuclear magnetic resonance spectroscopy-based methods in untargeted metabolomics, with a focus on reporting on QC samples in sufficient detail for them to be understood, trusted and replicated. There is no intent to be proscriptive with regard to analytical best practices; rather, guidance for reporting QA/QC procedures is suggested. A template that can be completed as studies progress to ensure that relevant data is collected, and further documents, are provided as on-line resources. KEY REPORTING PRACTICES: Multiple topics should be considered when reporting QA/QC protocols and outcomes for metabolic phenotyping data. Coverage should include the role(s), sources, types, preparation and uses of the QC materials and samples generally employed in the generation of metabolomic data. Details such as sample matrices and sample preparation, the use of test mixtures and system suitability tests, blanks and technique-specific factors are considered and methods for reporting are discussed, including the importance of reporting the acceptance criteria for the QCs. To this end, the reporting of the QC samples and results are considered at two levels of detail: "minimal" and "best reporting practice" levels.

Factors influencing career success of clinical nurses in northwestern China based on Kaleidoscope Career Model: Structural equation model.

AIM: To explore the relationships among self-efficacy, information literacy, social support and career success of clinical nurses and identify factors influencing clinical nurses' career success in northwestern China. BACKGROUND: Understanding the influencing factors of career success is important for the professional development of nurses and the improvement of clinical nursing quality. Many influencing factors of career success have been identified, but there is no large-scale research on the relationships among self-efficacy, information literacy, social support and career success of clinical nurses based on Kaleidoscope Career Model. Studies examining the association of the four factors remain limited. METHODS: A total of 3011 clinical nurses from 30 hospitals in northwestern China were selected in the cross-sectional survey, and the response rate was 94.71%. The clinical nurses completed the online self-report questionnaires including self-efficacy, information literacy, social support rating scale and career success scale. The data were analysed by SPSS23.0 statistical software using t test, analysis of variance, Pearson's correlation and multiple linear regression. Structural equation model (SEM) was used to analyse the influencing factors of career success using Mplus 8.3. RESULTS: The career success of clinical nurses in northwestern China was at a medium level. The linear multivariate regression analysis showed that self-efficacy (ß = .513), social support (ß = .230), information support (ß = .106), information consciousness (ß = -.097), information knowledge (ß = .067), information ethics (ß = -.053), hospital grade (ß = .118), marital status (ß = -.071) and age (ß = -.037) entered regression equation of clinical nurses' career success (all P < .05). SEM results showed that the career success was negatively correlated with demographic characteristics and positively correlated with social support and self-efficacy. CONCLUSION: Demographic characteristics, self-efficacy, social support and information literacy are the influencing factors of nurses' career success, which should be considered in the process of promoting nurses' career success. IMPLICATIONS FOR NURSING MANAGEMENT: Nursing managers need to acknowledge the significance of nurses' career success both for the realization of their own value and for the improvement of clinical nursing quality. They should encourage nurses to enhance self-efficacy and render more social support through incentive policies and foster nurses' information literacy through information technology training so as to improve their career success.

Tuning the UV/Vis Absorption Spectra of Electrochromic Small Molecular Radicals Through Bridge Modulation.

Studies on the optical properties of donor-bridge-acceptor (DBA) materials in their radical anion state are rare but important. Such investigations can help to extend the application of DBA materials to opto-electrochemical devices and no longer limit them to optical physics research. In this work, a series of new DBA materials, TACzs, for overcoming this limitation are reported. All TACzs show strong intramolecular charge transfer (ICT) in their photoexcited states, leading to noticeable solvatochromism. Besides, the electronic structures of their radical anions show great variability, displaying different absorption spectra and diverse colors. Moreover, the potential application of TACzs as electrochromic and electro-fluorochromic materials are discussed. This work demonstrates that manipulating the π bridge between the donor and acceptor in the DBA system is an effective pathway not only to tailor the ICT properties of materials in their neutral state, but also to tune the absorption characteristics of their radical anion state, which makes them very promising for applications in electroluminescent and electrochemical devices.

Stability of the Human Plasma Proteome to Pre-analytical Variability as Assessed by an Aptamer-Based Approach.

Variable processing and storage of whole blood and/or plasma are potential confounders in biomarker development and clinical assays. The goal of the study was to investigate how pre-analytical variables impact the human plasma proteome. Whole blood obtained from 16 apparently healthy individuals was collected in six EDTA tubes and processed randomly under six pre-analytical variable conditions including blood storage at 0 °C or RT for 6 h (B6h0C or B6hRT) before processing to plasma, plasma storage at 4 °C or RT for 24 h (P24h4C or P24hRT), low centrifugal force at 1300 × g, (Low×g), and immediate processing to plasma under 2500 × g (control) followed by plasma storage at -80 °C. An aptamer-based proteomic assay was performed to identify significantly changed proteins (fold change ≥1.2, P < 0.05, and false discovery rate < 0.05) relative to the control from a total of 1305 proteins assayed. Pre-analytical conditions Low×g and B6h0C resulted in the most plasma proteome changes with 200 and 148 proteins significantly changed, respectively. Only 36 proteins were changed under B6hRT. Conditions P24h4C and P24hRT yielded changes of 28 and 75 proteins, respectively. The complement system was activated in vitro under the conditions B6hRT, P24h4C, and P24hRT. The results suggest that particular pre-analytical variables should be controlled for clinical measurement of specific biomarkers.

An Integrated Analysis of Metabolites, Peptides, and Inflammation Biomarkers for Assessment of Preanalytical Variability of Human Plasma.

Discrepancies in blood sample collection and processing could have a significant impact on levels of metabolites, peptides, and protein biomarkers of inflammation in the blood; thus, sample quality control is critical for successful biomarker identification and validation. In this study, we analyzed the effects of several preanalytical processing conditions, including different storage times and temperatures for blood or plasma samples and different centrifugation forces on the levels of metabolites, peptides, and inflammation biomarkers in human plasma samples using ethylenediaminetetraacetic acid (EDTA) as an anticoagulant. Temperature was found to be the major factor for metabolite variation, and both time and temperature were identified as major factors for peptide variation. For inflammation biomarkers, temperature played different roles depending on the sample type (blood or plasma). Low temperature affected inflammation biomarkers in blood, while room temperature impacted inflammation biomarkers in plasma.

Transforming Growth Factor-ß (TGF-ß) Directly Activates the JAK1-STAT3 Axis to Induce Hepatic Fibrosis in Coordination with the SMAD Pathway.

Transforming growth factor-ß (TGF-ß) signals through both SMAD and non-SMAD pathways to elicit a wide array of biological effects. Existing data have shown the association and coordination between STATs and SMADs in mediating TGF-ß functions in hepatic cells, but it is not clear how STATs are activated under these circumstances. Here, we report that JAK1 is a constitutive TGFßRI binding protein and is absolutely required for phosphorylation of STATs in a SMAD-independent manner within minutes of TGF-ß stimulation. Following the activation of SMADs, TGF-ß also induces a second phase of STAT phosphorylation that requires SMADs, de novo protein synthesis, and contribution from JAK1. Our global gene expression profiling indicates that the non-SMAD JAK1/STAT pathway is essential for the expression of a subset of TGF-ß target genes in hepatic stellate cells, and the cooperation between the JAK1-STAT3 and SMAD pathways is critical to the roles of TGF-ß in liver fibrosis.

Apoptosis of enterocytes and nitration of junctional complex proteins promote alcohol-induced gut leakiness and liver injury.

BACKGROUND & AIMS: Binge alcohol exposure causes gut leakiness, contributing to increased endotoxemia and inflammatory liver injury, although the molecular mechanisms are still elusive. This study was aimed at investigating the roles of apoptosis of enterocytes and nitration followed by degradation of intestinal tight junction (TJ) and adherens junction (AJ) proteins in binge alcohol-induced gut leakiness. METHODS: The levels of intestinal (ileum) junctional complex proteins, oxidative stress markers and apoptosis-related proteins in rodents, T84 colonic cells and autopsied human ileums were determined by immunoblot, immunoprecipitation, immunofluorescence, and mass-spectral analyses. RESULTS: Binge alcohol exposure caused apoptosis of gut enterocytes with elevated serum endotoxin and liver injury. The levels of intestinal CYP2E1, iNOS, nitrated proteins and apoptosis-related marker proteins were significantly elevated in binge alcohol-exposed rodents. Differential, quantitative mass-spectral analyses of the TJ-enriched fractions of intestinal epithelial layers revealed that several TJ, AJ and desmosome proteins were decreased in binge alcohol-exposed rats compared to controls. Consistently, the levels of TJ proteins (claudin-1, claudin-4, occludin and zonula occludens-1), AJ proteins (ß-catenin and E-cadherin) and desmosome plakoglobin were very low in binge alcohol-exposed rats, wild-type mice, and autopsied human ileums but not in Cyp2e1-null mice. Additionally, pretreatment with specific inhibitors of CYP2E1 and iNOS prevented disorganization and/or degradation of TJ proteins in alcohol-exposed T84 colonic cells. Furthermore, immunoprecipitation followed by immunoblot confirmed that intestinal TJ and AJ proteins were nitrated and degraded via ubiquitin-dependent proteolysis, resulting in their decreased levels. CONCLUSIONS: These results demonstrated for the first time the critical roles of CYP2E1, apoptosis of enterocytes, and nitration followed by ubiquitin-dependent proteolytic degradation of the junctional complex proteins, in promoting binge alcohol-induced gut leakiness and endotoxemia, contributing to inflammatory liver disease. LAY SUMMARY: Binge alcohol exposure causes gut leakiness, contributing to increased endotoxemia and inflammatory liver injury. Our results demonstrated for the first time the critical roles of apoptosis of enterocytes and nitration followed by ubiquitin-dependent proteolytic degradation of the junctional complex proteins in promoting this gut leakiness and endotoxemia. These results provide insight into the molecular mechanisms of alcohol-induced inflammatory liver disease.

Identification of a novel Wnt5a-CK1ɛ-Dvl2-Plk1-mediated primary cilia disassembly pathway.

Non-motile primary cilium is an antenna-like structure whose defect is associated with a wide range of pathologies, including developmental disorders and cancer. Although mechanisms regulating cilia assembly have been extensively studied, how cilia disassembly is regulated remains poorly understood. Here, we report unexpected roles of Dishevelled 2 (Dvl2) and interphase polo-like kinase 1 (Plk1) in primary cilia disassembly. We demonstrated that Dvl2 is phosphorylated at S143 and T224 in a manner that requires both non-canonical Wnt5a ligand and casein kinase 1 epsilon (CK1ɛ), and that this event is critical to interact with Plk1 in early stages of the cell cycle. The resulting Dvl2-Plk1 complex mediated Wnt5a-CK1ɛ-Dvl2-dependent primary cilia disassembly by stabilizing the HEF1 scaffold and activating its associated Aurora-A (AurA), a kinase crucially required for primary cilia disassembly. Thus, via the formation of the Dvl2-Plk1 complex, Plk1 plays an unanticipated role in primary cilia disassembly by linking Wnt5a-induced biochemical steps to HEF1/AurA-dependent cilia disassembly. This study may provide new insights into the mechanism underlying ciliary disassembly processes and various cilia-related disorders.

Early transcriptional changes in cardiac mitochondria during chronic doxorubicin exposure and mitigation by dexrazoxane in mice.

Identification of early biomarkers of cardiotoxicity could help initiate means to ameliorate the cardiotoxic actions of clinically useful drugs such as doxorubicin (DOX). Since DOX has been shown to target mitochondria, transcriptional levels of mitochondria-related genes were evaluated to identify early candidate biomarkers in hearts of male B6C3F1 mice given a weekly intravenous dose of 3mg/kg DOX or saline (SAL) for 2, 3, 4, 6, or 8 weeks (6, 9, 12, 18, or 24 mg/kg cumulative DOX doses, respectively). Also, a group of mice was pretreated (intraperitoneally) with the cardio-protectant, dexrazoxane (DXZ; 60 mg/kg) 30 min before each weekly dose of DOX or SAL. At necropsy a week after the last dose, increased plasma concentrations of cardiac troponin T (cTnT) were detected at 18 and 24 mg/kg cumulative DOX doses, whereas myocardial alterations were observed only at the 24 mg/kg dose. Of 1019 genes interrogated, 185, 109, 140, 184, and 451 genes were differentially expressed at 6, 9, 12, 18, and 24 mg/kg cumulative DOX doses, respectively, compared to concurrent SAL-treated controls. Of these, expression of 61 genes associated with energy metabolism and apoptosis was significantly altered before and after occurrence of myocardial injury, suggesting these as early genomics markers of cardiotoxicity. Much of these DOX-induced transcriptional changes were attenuated by pretreatment of mice with DXZ. Also, DXZ treatment significantly reduced plasma cTnT concentration and completely ameliorated cardiac alterations induced by 24 mg/kg cumulative DOX. This information on early transcriptional changes during DOX treatment may be useful in designing cardioprotective strategies targeting mitochondria.

Early metabolomics changes in heart and plasma during chronic doxorubicin treatment in B6C3F1 mice.

The present study aimed to identify molecular markers of early stages of cardiotoxicity induced by a potent chemotherapeutic agent, doxorubicin (DOX). Male B6C3F1 mice were dosed with 3 mg kg(-1) DOX or saline via tail vein weekly for 2, 3, 4, 6 or 8 weeks (cumulative DOX doses of 6, 9, 12, 18 or 24 mg kg(-1) , respectively) and euthanized a week after the last dose. Mass spectrometry-based and nuclear magnetic resonance spectrometry-based metabolic profiling were employed to identify initial biomarkers of cardiotoxicity before myocardial injury and cardiac pathology, which were not noted until after the 18 and 24 mg kg(-1) cumulative doses, respectively. After a cumulative dose of 6 mg kg(-1) , 18 amino acids and four biogenic amines (acetylornithine, kynurenine, putrescine and serotonin) were significantly increased in cardiac tissue; 16 amino acids and two biogenic amines (acetylornithine and hydroxyproline) were significantly altered in plasma. In addition, 16 acylcarnitines were significantly increased in plasma and five were significantly decreased in cardiac tissue compared to saline-treated controls. Plasma lactate and succinate, involved in the Krebs cycle, were significantly altered after a cumulative dose of 6 mg kg(-1) . A few metabolites remained altered at higher cumulative DOX doses, which could partly indicate a transition from injury processes at 2 weeks to repair processes with additional injury happening concurrently before myocardial injury at 8 weeks. These altered metabolic profiles in mouse heart and plasma during the initial stages of injury progression due to DOX treatment may suggest these metabolites as candidate early biomarkers of cardiotoxicity. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Integrated microRNA, mRNA, and protein expression profiling reveals microRNA regulatory networks in rat kidney treated with a carcinogenic dose of aristolochic acid.

BACKGROUND: Aristolochic Acid (AA), a natural component of Aristolochia plants that is found in a variety of herbal remedies and health supplements, is classified as a Group 1 carcinogen by the International Agency for Research on Cancer. Given that microRNAs (miRNAs) are involved in cancer initiation and progression and their role remains unknown in AA-induced carcinogenesis, we examined genome-wide AA-induced dysregulation of miRNAs as well as the regulation of miRNAs on their target gene expression in rat kidney. RESULTS: We treated rats with 10 mg/kg AA and vehicle control for 12 weeks and eight kidney samples (4 for the treatment and 4 for the control) were used for examining miRNA and mRNA expression by deep sequencing, and protein expression by proteomics. AA treatment resulted in significant differential expression of miRNAs, mRNAs and proteins as measured by both principal component analysis (PCA) and hierarchical clustering analysis (HCA). Specially, 63 miRNAs (adjusted p value < 0.05 and fold change > 1.5), 6,794 mRNAs (adjusted p value < 0.05 and fold change > 2.0), and 800 proteins (fold change > 2.0) were significantly altered by AA treatment. The expression of 6 selected miRNAs was validated by quantitative real-time PCR analysis. Ingenuity Pathways Analysis (IPA) showed that cancer is the top network and disease associated with those dysregulated miRNAs. To further investigate the influence of miRNAs on kidney mRNA and protein expression, we combined proteomic and transcriptomic data in conjunction with miRNA target selection as confirmed and reported in miRTarBase. In addition to translational repression and transcriptional destabilization, we also found that miRNAs and their target genes were expressed in the same direction at levels of transcription (169) or translation (227). Furthermore, we identified that up-regulation of 13 oncogenic miRNAs was associated with translational activation of 45 out of 54 cancer-related targets. CONCLUSIONS: Our findings suggest that dysregulated miRNA expression plays an important role in AA-induced carcinogenesis in rat kidney, and that the integrated approach of multiple profiling provides a new insight into a post-transcriptional regulation of miRNAs on their target repression and activation in a genome-wide scale.

Distinct roles of GCN5/PCAF-mediated H3K9ac and CBP/p300-mediated H3K18/27ac in nuclear receptor transactivation.

Histone acetyltransferases (HATs) GCN5 and PCAF (GCN5/PCAF) and CBP and p300 (CBP/p300) are transcription co-activators. However, how these two distinct families of HATs regulate gene activation remains unclear. Here, we show deletion of GCN5/PCAF in cells specifically and dramatically reduces acetylation on histone H3K9 (H3K9ac) while deletion of CBP/p300 specifically and dramatically reduces acetylations on H3K18 and H3K27 (H3K18/27ac). A ligand for nuclear receptor (NR) PPARδ induces sequential enrichment of H3K18/27ac, RNA polymerase II (Pol II) and H3K9ac on PPARδ target gene Angptl4 promoter, which correlates with a robust Angptl4 expression. Inhibiting transcription elongation blocks ligand-induced H3K9ac, but not H3K18/27ac, on the Angptl4 promoter. Finally, we show GCN5/PCAF and GCN5/PCAF-mediated H3K9ac correlate with, but are surprisingly dispensable for, NR target gene activation. In contrast, CBP/p300 and their HAT activities are essential for ligand-induced Pol II recruitment on, and activation of, NR target genes. These results highlight the substrate and site specificities of HATs in cells, demonstrate the distinct roles of GCN5/PCAF- and CBP/p300-mediated histone acetylations in gene activation, and suggest an important role of CBP/p300-mediated H3K18/27ac in NR-dependent transcription.

The Shanghai Aging Study: study design, baseline characteristics, and prevalence of dementia.

BACKGROUND: To establish a prospective cohort to enumerate the prevalence, incidence and risk factors for dementia and mild cognitive impairment (MCI) among residents aged ≥60 in an urban community of Shanghai, China. METHODS: Participants received clinical evaluations including physical measurements, demographic and lifestyle questionnaires, physical and neurologic examinations, and neuropsychological testing. Urine and blood samples were collected, aliquoted, and stored. DNA was extracted for Apolipoprotein (APOE) genotyping. Diagnoses of dementia and MCI were made using standard criteria via consensus diagnosis. RESULTS: Among 3,141 participants aged ≥60, 1,438 (45.8%) were men. The average age of participants was 72.3 years (SD 8.1), and they had an average of 11.6 years (SD 4.4) of education. The most common chronic disease of participants was hypertension (56.4%). The frequencies of APOE-​ε2, ε3 and ε4 were 7.9, 82.7 and 9.4%, respectively. We diagnosed 156 (5.0%, 95% CI 4.3-5.8%) participants with dementia. The prevalence rates of Alzheimer's disease and vascular dementia were 3.6% (95% CI 3.0-4.3%) and 0.8% (95% CI 0.5-1.1%). CONCLUSIONS: The Shanghai Aging Study is the first prospective community-based cohort study of cognitive impairment in China, with a comparable study design, procedures, and diagnostic criteria for dementia and MCI to most previous cohort studies in developed countries.

Regulation of androgen receptor activity by tyrosine phosphorylation.

The androgen receptor (AR) is essential for the growth of prostate cancer cells. Here, we report that tyrosine phosphorylation of AR is induced by growth factors and elevated in hormone-refractory prostate tumors. Mutation of the major tyrosine phosphorylation site in AR significantly inhibits the growth of prostate cancer cells under androgen-depleted conditions. The Src tyrosine kinase appears to be responsible for phosphorylating AR, and there is a positive correlation of AR tyrosine phosphorylation with Src tyrosine kinase activity in human prostate tumors. Our data collectively suggest that growth factors and their downstream tyrosine kinases, which are elevated during hormone-ablation therapy, can induce tyrosine phosphorylation of AR and such modification may be important for prostate tumor growth under androgen-depleted conditions.

Regulation of microtubule-based microtubule nucleation by mammalian polo-like kinase 1.

Bipolar spindle formation is pivotal for accurate segregation of mitotic chromosomes during cell division. A growing body of evidence suggests that, in addition to centrosome- and chromatin-based microtubule (MT) nucleation, MT-based MT nucleation plays an important role for proper bipolar spindle formation in various eukaryotic organisms. Although a recently discovered Augmin complex appears to play a central role in this event, how Augmin is regulated remains unknown. Here we provide evidence that a mammalian polo-like kinase 1 (Plk1) localizes to mitotic spindles and promotes MT-based MT nucleation by directly regulating Augmin. Mechanistically, we demonstrated that Cdc2-dependent phosphorylation on a γ-tubulin ring complex (γ-TuRC) recruitment protein, Nedd1/GCP-WD, at the previously uncharacterized S460 residue induces the Nedd1-Plk1 interaction. This step appeared to be critical to allow Plk1 to phosphorylate the Hice1 subunit of the Augmin complex to promote the Augmin-MT interaction and MT-based MT nucleation from within the spindle. Loss of either the Nedd1 S460 function or the Plk1-dependent Hice1 phosphorylation impaired both the Augmin-MT interaction and γ-tubulin recruitment to the spindles, thus resulting in improper bipolar spindle formation that ultimately leads to mitotic arrest and apoptotic cell death. Thus, via the formation of the Nedd1-Plk1 complex and subsequent Augmin phosphorylation, Plk1 regulates spindle MT-based MT nucleation to accomplish normal bipolar spindle formation and mitotic progression.

Identification of Proteomic Biomarkers of Acetaminophen-Induced Hepatotoxicity Using Stable Isotope Labeling.

Quantitative proteomics approaches based on stable isotopic labeling and mass spectrometry have been widely applied to disease research, drug target discovery, biomarker identification, and systems biology. One of the notable stable isotopic labeling approaches is trypsin-catalyzed 18O/16O labeling, which has its own advantages of low sample consumption, simple labeling procedure, cost-effectiveness, and absence of chemical reactions that potentially generate by-products. In this chapter, a protocol for 18O/16O labeling-based quantitative proteomics approach is described with an application to the identification of proteomic biomarkers of acetaminophen (APAP)-induced hepatotoxicity in rats. The protocol involves first the extraction of proteins from liver tissues of control and APAP-treated rats and digestion into peptides by trypsin. After cleaning of the peptides by solid-phase extraction, equal amounts of peptides from the APAP treatment and the control groups are then subject to trypsin-catalyzed 18O/16O labeling. The labeled peptides are combined and fractionated by off-line strong cation exchange liquid chromatography (SCXLC), and each fraction is then analyzed by nanoflow reversed-phase LC coupled online with tandem mass spectrometry (RPLC-MS/MS) for identification and quantification of differential protein expression between APAP-treated rats and controls. The protocol is applicable to quantitative proteomic analysis for a variety of biological samples.

Preharvest spraying of phenylalanine activates the sucrose and respiratory metabolism in muskmelon wounds during healing.

Phenylalanine (Phe) accelerates fruit wound healing by activating phenylpropanoid metabolism. However, whether Phe affects sucrose and respiratory metabolism in fruit during wound healing remains unknown. In this research, we found that preharvest Phe spray promoted sucrose degradation and increased glucose and fructose levels by activating acid invertase (AI), neutral invertase (NI), sucrose synthase (SS) and sucrose phosphate synthase (SPS) on harvested muskmelons. The spray also activated hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK), malate dehydrogenase (MDH), succinate dehydrogenase (SDH) and glucose-6-phosphate dehydrogenase (G6PDH). In addition, the spray improved energy and reducing power levels in the fruit. Taken together, preharvest Phe spray can provide carbon skeleton, energy and reducing power for wound healing by activating the sucrose metabolism, Embden-Meyerhof-Parnas (EMP) pathway, tricarboxylic acid (TCA) cycle and pentose phosphate (PPP) pathway in muskmelon wounds during healing, which is expected to be developed as a new strategy to accelerate fruit wound healing.