Changes in Holstein cow milk and serum proteins during intramammary infection with three different strains of Staphylococcus aureus

<p>Abstract</p> <p>Background</p> <p><it>Staphylococcus aureus </it>is one of the most prevalent pathogens to cause mastitis in dairy cattle. Intramammary infection of dairy cows with <it>S. aureus </it>is often subclinical, due to the pathogen's ability to evade the innate defense mechanisms, but this can lead to chronic infection. A sub-population of <it>S. aureus</it>, known as small colony variant (SCV), displays atypical phenotypic characteristics, causes persistent infections, and is more resistant to antibiotics than parent strains. Therefore, it was hypothesized that the host immune response will be different for SCV than its parental or typical strains of <it>S. aureus</it>. In this study, the local and systemic immune protein responses to intramammary infection with three strains of <it>S. aureus</it>, including a naturally occurring bovine SCV strain (SCV Heba3231), were characterized. Serum and casein-depleted milk cytokine levels (interleukin-8, interferon-γ, and transforming growth factor-β1), as well as serum haptoglobin concentrations were monitored over time after intramammary infection with each of the three <it>S. aureus </it>strains. Furthermore, comparative proteomics was used to evaluate milk proteome profiles during acute and chronic phases of <it>S. aureus </it>intramammary infection.</p> <p>Results</p> <p>Serum IL-8, IFN-γ, and TGF-β1 responses differed in dairy cows challenged with different strains of <it>S. aureus</it>. Changes in overall serum haptoglobin concentrations were observed for each <it>S. aureus </it>challenge group, but there were no significant differences observed between groups. In casein-depleted milk, strain-specific differences in the host IFN-γ response were observed, but inducible IL-8 and TGF-β1 concentrations were not different between groups. Proteomic analysis of the milk following intramammary infection revealed unique host protein expression profiles that were dependent on the infecting strain as well as phase of infection. Notably, the protein, component-3 of the proteose peptone (CPP3), was differentially expressed between the <it>S. aureus </it>treatment groups, implicating it as a potential antimicrobial peptide involved in host defense against <it>S. aureus </it>intramammary infection.</p> <p>Conclusions</p> <p>Intramammary infection of dairy cattle with <it>S. aureus </it>causes an up-regulation of serum and milk immune-related proteins, and these responses vary depending on the infecting strain.</p

Proteomic Identification and Systematic Verification of Biomarkers for Aggressive Prostate Cancer

Despite prostate cancer’s status as the most common cancer to affect men in the Western world, only an estimated 20% of men will die from it. Due to the lack of effective means of prognostication, many men are unnecessarily biopsied and treated. The early and non-invasive identification of lethal disease would have a profound impact on the current practice of prostate cancer management. To this end, the work presented in this thesis describes efforts to characterize proteins in a prostatic fluid known as expressed prostatic secretions (EPS), and to delineate factors in this fluid that demonstrate a high potential as prognostic biomarkers. Using different mass spectrometry-based platforms, I present both a global view of the EPS proteome, as well as targeted, quantitative measures of promising biomarker candidates in this fluid. In order to generate an archive of soluble and secreted factors present in the proteome of EPS, I used shotgun proteomics to analyze direct-EPS and EPS-urine. This led to the identification of over 2000 proteins that comprise the EPS proteome. The data was integrated with other publicly accessible information, such as gene expression profiles and immunohistochemistry images, to characterize this proteome. Furthermore, a collection of prostate-enriched proteins was identified by comparing the proteomes of EPS-urine and urine, demonstrating the unique protein profile of EPS. Next, comparative proteomic profiling of direct-EPS from organ confined and extracapsular tumors produced a long list of potential biomarker candidates. A targeted assay was systematically developed in order to quantitatively assess the performance of the most promising candidates in cohorts of EPS-urines from a heterogeneous population of prostate cancer patients and controls. Utilizing the assay, a number of candidates were verified to be putative biomarkers for prostate cancer diagnosis and prognosis. This information needs to be further validated and holds promise for improved prostate cancer management.Ph.D.2017-02-12 00:00:0

Novel approaches for the identification of biomarkers of aggressive prostate cancer

Ovjesne dizalice su često sredstvo unutrašnjeg transporta komadog materijala. Razne mogućnosti konstrukcijskih izvedbi omogućuju im prilagodbu prema prostoru u kojem se ugrađuju. U prilog im ide i relativno velika nosivost, koja se može postići pogodnim projektiranjem nosača i vitla, kao i jednostavnost rukovanjem. U ovom radu su opisana postojeća rješenja ovjesnih dizalica, te su dane njihove glavne geometrijske i radne karakteristike, koje su kupcu prilikom kupnje takvog proizvoda najinteresantnije. Nakon toga slijedi proračun i dimenzioniranje nosive konstrukcije za dizalicu koja se giba uzduž, po donjem pojasu nosača koji je u vijčanom spoju s okvirnom konstrukcijom koja je temeljnim vijcima spojena u betonski pod hale. Napravljen je proračun komponenti voznih kolica za pogon i prijenos snage. Naposljetku je izvršena provjera stabilnosti i čvrstoće nosive konstrukcije te čvrstoća zavara. Konstrukcijsko rješenje je dobiveno projektiranjem u AutoCAD-u, te je napravljen glavni sklopni crtež, potom sklopni crteži nosive konstrukcije i voznih kolica, te radionički crteži svih nestandardnih dijelova.Hoist systems are often used as material transporting solution in closed areas. A variery of design makes them adjustable to the geometrical area requests. Their advantage is high capacity, achived by properly designed trolley and support structure, also as ease of use. Here are described existing solutions of electric chain hoists, available on market and their working and dimensional characteristics, which are most interesteting to the purchaser. Further is done calculation for steel structure support for chain hoist, which rides longitudinally on lower belt of steel support, which is connected by bolts to the frame structure connected also by bolts to the concrete area ground. Calculation has been made for all trolley components, including power transmission parts. Finally is done calculation of safety for frame structure, regarding deflection and solidity, also as safety of welds. Design has been done using AutoCAD software and by it has been made main assembly drawing, assembly drawings of steel structure and trolley and at least workshop drawings for all non-standard parts

Targeted proteomics identifies liquid-biopsy signatures for extracapsular prostate cancer.

Biomarkers are rapidly gaining importance in personalized medicine. Although numerous molecular signatures have been developed over the past decade, there is a lack of overlap and many biomarkers fail to validate in independent patient cohorts and hence are not useful for clinical application. For these reasons, identification of novel and robust biomarkers remains a formidable challenge. We combine targeted proteomics with computational biology to discover robust proteomic signatures for prostate cancer. Quantitative proteomics conducted in expressed prostatic secretions from men with extraprostatic and organ-confined prostate cancers identified 133 differentially expressed proteins. Using synthetic peptides, we evaluate them by targeted proteomics in a 74-patient cohort of expressed prostatic secretions in urine. We quantify a panel of 34 candidates in an independent 207-patient cohort. We apply machine-learning approaches to develop clinical predictive models for prostate cancer diagnosis and prognosis. Our results demonstrate that computationally guided proteomics can discover highly accurate non-invasive biomarkers

Proteotranscriptomic Analysis Reveals Stage Specific Changes in the Molecular Landscape of Clear-Cell Renal Cell Carcinoma

<div><p>Renal cell carcinoma comprises 2 to 3% of malignancies in adults with the most prevalent subtype being clear-cell RCC (ccRCC). This type of cancer is well characterized at the genomic and transcriptomic level and is associated with a loss of <i>VHL</i> that results in stabilization of HIF1. The current study focused on evaluating ccRCC stage dependent changes at the proteome level to provide insight into the molecular pathogenesis of ccRCC progression. To accomplish this, label-free proteomics was used to characterize matched tumor and normal-adjacent tissues from 84 patients with stage I to IV ccRCC. Using pooled samples 1551 proteins were identified, of which 290 were differentially abundant, while 783 proteins were identified using individual samples, with 344 being differentially abundant. These 344 differentially abundant proteins were enriched in metabolic pathways and further examination revealed metabolic dysfunction consistent with the Warburg effect. Additionally, the protein data indicated activation of ESRRA and ESRRG, and HIF1A, as well as inhibition of FOXA1, MAPK1 and WISP2. A subset analysis of complementary gene expression array data on 47 pairs of these same tissues indicated similar upstream changes, such as increased HIF1A activation with stage, though ESRRA and ESRRG activation and FOXA1 inhibition were not predicted from the transcriptomic data. The activation of ESRRA and ESRRG implied that HIF2A may also be activated during later stages of ccRCC, which was confirmed in the transcriptional analysis. This combined analysis highlights the importance of HIF1A and HIF2A in developing the ccRCC molecular phenotype as well as the potential involvement of ESRRA and ESRRG in driving these changes. In addition, cofilin-1, profilin-1, nicotinamide N-methyltransferase, and fructose-bisphosphate aldolase A were identified as candidate markers of late stage ccRCC. Utilization of data collected from heterogeneous biological domains strengthened the findings from each domain, demonstrating the complementary nature of such an analysis. Together these results highlight the importance of the VHL/HIF1A/HIF2A axis and provide a foundation and therapeutic targets for future studies. (Data are available via ProteomeXchange with identifier PXD003271 and MassIVE with identifier MSV000079511.)</p></div

Identification of Prostate-Enriched Proteins by In-depth Proteomic Analyses of Expressed Prostatic Secretions in Urine

Urinary expressed prostatic secretion or “EPS-urine” is proximal tissue fluid that is collected after a digital rectal exam (DRE). EPS-urine is a rich source of prostate-derived proteins that can be used for biomarker discovery for prostate cancer (PCa) and other prostatic diseases. We previously conducted a comprehensive proteome analysis of direct expressed prostatic secretions (EPS). In the current study, we defined the proteome of EPS-urine employing Multidimensional Protein Identification Technology (MudPIT) and providing a comprehensive catalogue of this body fluid for future biomarker studies. We identified 1022 unique proteins in a heterogeneous cohort of 11 EPS-urines derived from biopsy negative noncancer diagnoses with some benign prostatic diseases (BPH) and low-grade PCa, representative of secreted prostate and immune system-derived proteins in a urine background. We further applied MudPIT-based proteomics to generate and compare the differential proteome from a subset of pooled urines (pre-DRE) and EPS-urines (post-DRE) from noncancer and PCa patients. The direct proteomic comparison of these highly controlled patient sample pools enabled us to define a list of prostate-enriched proteins detectable in EPS-urine and distinguishable from a complex urine protein background. A combinatorial analysis of both proteomics data sets and systematic integration with publicly available proteomics data of related body fluids, human tissue transcriptomic data, and immunohistochemistry images from the Human Protein Atlas database allowed us to demarcate a robust panel of 49 prostate-derived proteins in EPS-urine. Finally, we validated the expression of seven of these proteins using Western blotting, supporting the likelihood that they originate from the prostate. The definition of these prostatic proteins in EPS-urine samples provides a reference for future investigations for prostatic-disease biomarker studies