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The question whether and which non-human peptides or proteins are present in human milk was raised many decades ago. However, due to cross-reactivity or nonspecific antibody recognition, the accuracy of detection by immunochemical methods has been a concern. Additionally, the relative low-abundance of non-human peptides/proteins in the complex milk sample makes them a challenging target to detect. Here, by deep proteome profiling we detected several non-human peptides, which could be grouped as non-human proteins. We next estimated their concentration in human milk by combining data-dependent shotgun proteomics and parallel reaction monitoring. First, we fractionated human milk at the protein level and were able to detect 1577 human proteins. Additionally, we identified 109 non-human peptides, of which 71 were grouped in to 36 non-human proteins. In the next step, we targeted 37 non-human peptides and 9 of them could be repeatedly quantified in human milk samples. Peptides/proteins originating from bovine milk products were the dominant non-human proteins observed, notably bovine caseins (α-S1-, α-S2-, β-, κ-caseins) and β-lactoglobulin. The method we present here can be expanded to investigate more about non-human peptides and proteins in human milk and have a better understanding of how human milk plays a role in allergy prevention.

The increased speed and sensitivity in mass spectrometry-based proteomics has encouraged its use in biomedical research in recent years. Large-scale detection of proteins in cells, tissues and whole organisms yields highly-complex quantitative data, the analysis of which poses significant challenges. Standardized proteomic workflows are necessary to ensure automated, sharable and reproducible proteomics analysis. Likewise, standardized data processing workflows are also essential for the overall reproducibility of results. To this purpose, we developed PaDuA, a Python package optimized for the processing and analysis of (phospho)proteomics data. PaDuA provides a collection of tools which can be used to build scripted workflows within Jupyter Notebooks, to facilitate bioinformatics analysis by both end-users and developers.

Background The Cambridge Pulmonary Hypertension Outcome Review (CAMPHOR) is the first disease-specific instrument for pulmonary arterial hypertension (PAH) to assess patient-perceived symptoms, activity limitations and quality of life. To be able to use this questionnaire in the Netherlands, the aim of the study was to translate and validate this instrument for the Dutch-speaking population. Methods First the CAMPHOR was translated into Dutch (by means of a bilingual and a lay panel) and field-tested by means of cognitive debriefing interviews with ten PAH patients. For psychometric evaluation, 80 patients with PAH or chronic thromboembolic pulmonary hypertension (CTEPH) were asked to complete the CAMPHOR twice over a two-week period. To test for construct validity, participants also completed the Nottingham Health Profile (NHP). Results The Dutch version of the CAMPHOR showed high internal consistency for all scales (Cronbach’s alpha 0.89–0.91) and excellent reproducibility over two weeks (reliability coefficients 0.87–0.91). Concurrent validity showed that the CAMPHOR scales correlated as expected with the NHP scales. The CAMPHOR was able to distinguish between patient groups based on self-reported general health status, disease severity and NYHA classification demonstrating evidence of known group validity. The CAMPHOR activity limitations scale correlated moderately with the distance walked during the 6‑minute walk test (r = −0.47, p

On 12 May 2018, Iraqis went to the polls to vote for their next parliament, prime minister and cabinet. The elections delivered a surprise, with Muqtada al-Sadr’s Saairun Coalition winning the most seats of any bloc, although not enough to form a government alone. The results also carried a number of clear messages about the state of Iraqi politics, not least the low turnout and the decision of many Iraqis to boycott the elections, reflecting a general malaise and disillusion with the current political leadership and bloated bureaucracy. The revelations of electoral fraud and the very low turnout (of 44.5 percent) indicate that it will be difficult for the political class to overcome its reputation for maintaining the status quo – and for the citizenry to accept it. This report is the second in a series of three produced by the Institute of Regional and International Studies (IRIS) as the outcome of a project examining the mobilisation strategies and results of the 2018 Iraqi elections. This project forms part of the Conflict Research Programme, funded by the UK Department for International Development.

The major treatment effect of prednisone on FVC is reached within 2 to 3 weeks in newly treated sarcoidosis patientshttp://ow.ly/3A3E30h5ZuT

It has now been over a year since the liberation of Mosul by Iraqi government forces in July 2017. The aftermath of this successful military campaign clearly represents a window of opportunity where a sudden reduction in politically motivated violence offers the Government of Iraq and its allies the opportunity to deal with long-term underlying drivers of instability that have repeatedly given rise to organised violence since 2003. However, as the examples of 2003 and 2007, along with the rapid reconstitution of ISIS after 2017 indicate, if these underlying drivers are not properly identified and mediated through accurately targeted policy interventions, then a return to the levels of organised violence that have dominated Iraq for the majority of the last fifteen years is likely. This paper is an attempt to identify those underlying drivers of conflict using the Conflict Research Programme’s four key analytical concepts – moral populism, the political marketplace, public authority and ‘civicness’.

Background Fibrocytes are implicated in Idiopathic Pulmonary Fibrosis (IPF) pathogenesis and increased proportions in the circulation are associated with poor prognosis. Upon tissue injury, fibrocytes migrate to the affected organ. In IPF patients, circulating fibrocytes are increased especially during exacerbations, however fibrocytes in the lungs have not been examined. Therefore, we sought to evaluate if fibrocytes can be detected in IPF lungs and we compare percentages and phenotypic characteristics of lung fibrocytes with circulating fibrocytes in IPF. Methods First we optimized flow cytometric detection circulating fibrocytes using a unique combination of intra- and extra-cellular markers to establish a solid gating strategy. Next we analyzed lung fibrocytes in single cell suspensions of explanted IPF and control lungs and compared characteristics and numbers with circulating fibrocytes of IPF. Results Using a gating strategy for both circulating and lung fibrocytes, which excludes potentially contaminating cell populations (e.g. neutrophils and different leukocyte subsets), we show that patients with IPF have increased proportions of fibrocytes, not only in the circulation, but also in explanted end-stage IPF lungs. These lung fibrocytes have increased surface expression of HLA-DR, increased intracellular collagen-1 expression, and also altered forward and side scatter characteristics compared with their circulating counterparts. Conclusions These findings demonstrate that lung fibrocytes in IPF patients can be quantified and characterized by flow cytometry. Lung fibrocytes have different characteristics than circulating fibrocytes and represent an intermediate cell population between circulating fibrocytes and lung fibroblast. Therefore, more insight in their phenotype might lead to specific therapeutic targeting in fibrotic lung diseases. Electronic supplementary material The online version of this article (10.1186/s12931-018-0798-8) contains supplementary material, which is available to authorized users.

Sort strategy for CD45+/Col-1+ cells. (A) The conventional strategy with the CD45+/Col-1+ cells in red. (B) Position of the same fibrocytes when employing a gating strategy based on additional extracellular markers. The sort strategy is based on extracellular markers and with some modifications previously published [16] (of note: for our research question we did not exclude SSChi cells). CD45+ cells were analyzed for HLA-DR expression and lineage markers to exclude B-cells (CD19), NK cells (CD56) and T-cells (CD3). Lineage negative cells were plotted as CD14 versus CD16 to create a distinct group of cells enriched for CD45+/Col-1+ cells (red box). (PDF 75 kb)

Correction to: Neth Heart J 2016 https://doi.org/10.1007/s12471-016-0849-z Unfortunately the original version of this article contained Electronic Supplementary Material which should not have been published with the article due to copyright reasons. The original version has been updated and the ESM has been removed.

Characteristics of patients and the healthy subjects used for experiments. Data are mean ± standard deviation, unless indicated otherwise. a 1 patient with chronic thromboembolic PH and 4 patients with PH secondary to an auto-immune disease. b 6 patients with IPF, 3 patients with UIP pattern secondary to extrinsic allergic alveolitis, 2 patients with an non-specific interstitial pneumonia and 1 patient with anti-synthetase syndrome. c Mean FVC/FEV1 ratio = 0.71 (2 out of 9 had an obstructive pulmonary function test, both classified as GOLD A). d Assessed by right heart catheterization or suspected with echocardiography. IPF = idiopathic pulmonary fibrosis, IPAH = idiopathic pulmonary arterial hypertension, FVC = forced vital capacity, TLCO = diffusing capacity for carbon monoxide, PAP = pulmonary arterial pressure, RAP = right atrium pressure, Svo2 = mixed venous saturation, PDE5i = phosphodiesterase type 5 inhibitor, ERA = endothelin receptor antagonist, Pros-A = prostacyclin agonist (DOCX 22 kb)

Complete overview collagen-1 and CD15 staining on CD45+/Col-1+ cells and controls. Immunocytochemical images of cultured fibroblasts, cultured fibrocytes, sorted classical monocytes and sorted CD45+/Col-1+ cells. Indicated cells were stained with Collagen-1 or isotype control (rabbit IgG) and CD15. As a control for CD15 we used a buffy coat, nicely showing positive granulocytes next to negative lymphocytes. Magnification for all images was 100Ă . (PDF 1282 kb)

Circulating fibrocyte numbers in patients with IPF and idiopathic pulmonary hypertension (IPAH). (A) Absolute numbers of circulating fibrocytes per ml blood in frozen PBMC of HC, patients with IPF and patients with IPAH. ** PÂ

Correlation circulating CD45+/Col-1+ fibrocytes and granulocytes. For this experiment we analyzed paired total white blood cells (open dots) and PBMCs (black dots) on the same day as blood withdrawal of 9 patients (4 IPF patients and 5 PH patients). Correlation coefficients were calculated using Spearmanâ s rank method. (PDF 102 kb)

SummaryAlthough mass-spectrometry-based screens enable thousands of protein phosphorylation sites to be monitored simultaneously, they often do not cover important regulatory sites. Here, we hypothesized that this is due to the fact that nearly all large-scale phosphoproteome studies are initiated by trypsin digestion. We tested this hypothesis using multiple proteases for protein digestion prior to Ti4+-IMAC-based enrichment. This approach increases the size of the detectable phosphoproteome substantially and confirms the considerable tryptic bias in public repositories. We define and make available a less biased human phosphopeptide atlas of 37,771 unique phosphopeptides, correlating to 18,430 unique phosphosites, of which fewer than 1/3 were identified in more than one protease data set. We demonstrate that each protein phosphorylation site can be linked to a preferred protease, enhancing its detection by mass spectrometry (MS). For specific sites, this approach increases their detectability by more than 1,000-fold.

We present a detailed investigation of the dynamics of impurity-trapped excitons in CaF2:Yb2+using transient photoluminescence enhancement induced via a two-frequency, sequential excitation process employing a UV optical parametric amplifier (OPA) synchronized to an infrared free electron laser (FEL). The temporal behaviour is well approximated by a multi-level rate equation model as relaxation between excited states of the exciton as well as a small contribution from local lattice heating by the FEL which becomes evident due to the 40 cm−1splitting of the exciton excited states giving rise to the transient photoluminescence enhancement itself.

The field of biological research is increasingly trying to understand the complex processes that are too small to observe by eye, even when using a microscope. The real action is taking place at the molecular level; therefore, the only way to investigate this is to use molecular tools. Modern techniques allow studying all proteins in a sample, and the prevailing tool is mass spectrometry based proteomics. As a high-throughput technique, computational analysis is indispensable. In this thesis, we describe several aspects of the treatment of proteomics data using in-house developed tools, and the analysis of several biological experiments. In a first example to illustrate how we used mass spectrometry to investigate biological problems, we investigated the evolution of tyrosine phosphorylation, which possibly arose simultaneously with multicellularity. To investigate this, the placozoan Trichoplax adhaerens was analysed, which is arguably the most primitive animal. We observed several tyrosine kinases in the genome sequence, and phosphoproteomics analysis revealed an extraordinary amount of tyrosine phosphorylation events compared to modern animals. In a second example, we simultaneously analysed RNA, DNA and proteins in a study of a single organ, the rat liver. We compared two different rat strains, the Brown Norwegian (BN) rat, and a variant exhibiting spontaneous hypertension (SHR). Through these measurements we found a differentially regulated rat homolog of a gene that is linked to hypertension in human, CYP17A1, both at the RNA and at the proteome level. The DNA sequence showed a polymorphism in the gene promoter, indicating that a single point mutation may be responsible for the mis-regulation of this gene and protein.

Prednisone is used as first-line therapy for pulmonary sarcoidosis. What dosing strategy has the best balance between effect and side-effects is largely unknown. We analyzed change in forced vital capacity (FVC) and weight during different prednisone doses used in daily practice for treatment naïve pulmonary sarcoidosis patients.Multilevel models were used to describe FVC and weight change over time. Correlations were calculated using linear regression models.Fifty-four patients were included. FVC changed over time (p 0.001), with an average increase of 9.6% predicted (95% CI: 7.2 to 12.1) at 12 months. Weight changed significantly over time (p 0.001), with an average increase of 4.3 kg (95% CI: 3.0 to 5.6) at 12 months. Although FVC and weight changed significantly over time, there was little correlation between prednisone dose and FVC change, while weight increase correlated significantly with cumulative prednisone dose at 24 months. In patients treated with a high cumulative prednisone dose, baseline FVC was on average lower (p = 0.001) compared to low dose treated patients, while no significant differences were observed in need for second/third-line therapy or number of exacerbations. A strategy leading to a low cumulative dose at 12 months was defined by rapid dose tapering to 10 mg/day within 3.5 months.These results suggest that prednisone therapy aimed at improving or preserving FVC in newly- treated pulmonary sarcoidosis can often be reduced in dose, using a treatment regimen that is characterized by early dose tapering.

A key step in shotgun proteomics is the digestion of proteins into peptides amenable for mass spectrometry. Tryptic peptides can be readily sequenced and identified by collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD) because the fragmentation rules are well-understood. Here, we investigate LysargiNase, a perfect trypsin mirror protease, because it cleaves equally specific at arginine and lysine residues, albeit at the N-terminal end. LysargiNase peptides are therefore practically tryptic-like in length and sequence except that following ESI, the two protons are now both positioned at the N-terminus. Here, we compare side-by-side the chromatographic separation properties, gas-phase fragmentation characteristics, and (phospho)proteome sequence coverage of tryptic (i.e., (X)nK/R) and LysargiNase (i.e., K/R(X)n) peptides using primarily electron-transfer dissociation (ETD) and, for comparison, HCD. We find that tryptic and LysargiNase peptides fragment nearly as mirror images. For LysargiNase predominantly N-terminal peptide ions (c-ions (ETD) and b-ions (HCD)) are formed, whereas for trypsin, C-terminal fragment ions dominate (z-ions (ETD) and y-ions (HCD)) in a homologous mixture of complementary ions. Especially during ETD, LysargiNase peptides fragment into low-complexity but information-rich sequence ladders. Trypsin and LysargiNase chart distinct parts of the proteome, and therefore, the combined use of these enzymes will benefit a more in-depth and reliable analysis of (phospho)proteomes.

A key step in shotgun proteomics is the digestion of proteins into peptides amenable for mass spectrometry. Tryptic peptides can be readily sequenced and identified by collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD) because the fragmentation rules are well-understood. Here, we investigate LysargiNase, a perfect trypsin mirror protease, because it cleaves equally specific at arginine and lysine residues, albeit at the N-terminal end. LysargiNase peptides are therefore practically tryptic-like in length and sequence except that following ESI, the two protons are now both positioned at the N-terminus. Here, we compare side-by-side the chromatographic separation properties, gas-phase fragmentation characteristics, and (phospho)proteome sequence coverage of tryptic (i.e., (X)

Posterieure corticale atrofie (PCA) is een neurodegeneratieve aandoening waarbij visuele problemen op de voorgrond staan. De meest voorkomende onderliggende oorzaak van PCA is de ziekte van Alzheimer. Er is geen curatieve therapie voor PCA; visuele ondersteuning bij PCA kan echter een uitkomst bieden. In deze casus beschrijven we een patient met PCA die ondersteuning krijgt bij een revalidatiecentrum dat zich richt op patienten met visuele problematiek. Daarnaast geven we een overzicht van de huidige behandelmethoden en mogelijkheden bij visuele problematiek in het kader van PCA. Passende ondersteuning bij visuele klachten en psychische factoren van een patient met PCA en diens mantelzorger is belangrijk bij het zo veel mogelijk zelfstandig kunnen blijven functioneren van een patient en het voorkomen of verminderen van psychische klachten van patient en mantelzorger.

Diseases at the molecular level are complex and patient dependent, necessitating development of strategies that enable precision treatment to optimize clinical outcomes. Organoid technology has recently been shown to have the potential to recapitulate the in vivo characteristics of the original individual's tissue in a three-dimensional in vitro culture system. Here, we present a quantitative mass-spectrometry-based proteomic analysis and a comparative transcriptomic analysis of human colorectal tumor and healthy organoids derived, in parallel, from seven patients. Although gene and protein signatures can be derived to distinguish the tumor organoid population from healthy organoids, our data clearly reveal that each patient possesses a distinct organoid signature at the proteomic level. We demonstrate that a personalized patient-specific organoid proteome profile can be related to the diagnosis of a patient and with future development contribute to the generation of personalized therapies.

Mass spectrometry (MS)-based proteomics workflows can crudely be classified into two distinct regimes, targeting either relatively small peptides (i.e., 0.7 kDa < Mw < 3.0 kDa) or small to medium sized intact proteins (i.e., 10 kDa < Mw < 30 kDa), respectively, termed bottom-up and top-down proteomics. Recently, a niche has started to be explored covering the analysis of middle-range peptides (i.e., 3.0 kDa < Mw < 10 kDa), aptly termed middle-down proteomics. Although middle-down proteomics can follow, in principle, a modular workflow similar to that of bottom-up proteomics, we hypothesized that each of these modules would benefit from targeted optimization to improve its overall performance in the analysis of middle-range sized peptides. Hence, to generate middle-range sized peptides from cellular lysates, we explored the use of the proteases Asp-N and Glu-C and a nonenzymatic acid induced cleavage. To increase the depth of the proteome, a strong cation exchange (SCX) separation, carefully tuned to improve the separation of longer peptides, combined with reversed phase-liquid chromatography (RP-LC) using columns packed with material possessing a larger pore size, was used. Finally, after evaluating the combination of potentially beneficial MS settings, we also assessed the peptide fragmentation techniques, including higher-energy collision dissociation (HCD), electron-transfer dissociation (ETD), and electron-transfer combined with higher-energy collision dissociation (EThcD), for characterization of middle-range sized peptides. These combined improvements clearly improve the detection and sequence coverage of middle-range peptides and should guide researchers to explore further how middle-down proteomics may lead to an improved proteome coverage, beneficial for, among other things, the enhanced analysis of (co-occurring) post-translational modifications.

Pulmonary fibrosis greatly impacts patients and their partners. Unmet needs of patients are increasingly acknowledged; the needs of partners often remain unnoticed. Little is known about the best way to educate patients and partners. We investigated pulmonary fibrosis patients' and partners' perspectives and preferences in care, and the differences in these between the Netherlands and Germany. Additionally, we evaluated whether interactive interviewing could be a novel education method in this population. Patients and partners were interviewed during pulmonary fibrosis patient information meetings. In the Netherlands, voting boxes were used and results were projected directly. In Germany, questionnaires were used. In the Netherlands, 278 patients and partners participated; in Germany, 51. Many participants experienced anxiety. Almost all experienced misunderstanding, because people do not know what pulmonary fibrosis is. All expressed a need for information, psychological support and care for partners. Use of the interactive voting system was found to be pleasant (70%) and informative (94%). This study improves the knowledge of care needs of patients with pulmonary fibrosis and their partners. There were no major differences between the Netherlands and Germany. Interactive interviewing could be an attractive method to acquire insights into the needs and preferences of patients and partners, while providing them with information at the same time., The needs of patients with pulmonary fibrosis and partners, and the value of interactive interviewing http://ow.ly/M02t307jdEh