ProteinSeq: High-Performance Proteomic Analyses by Proximity Ligation and Next Generation Sequencing

Despite intense interest, methods that provide enhanced sensitivity and specificity in parallel measurements of candidate protein biomarkers in numerous samples have been lacking. We present herein a multiplex proximity ligation assay with readout via realtime PCR or DNA sequencing (ProteinSeq). We demonstrate improved sensitivity over conventional sandwich assays for simultaneous analysis of sets of 35 proteins in 5 µl of blood plasma. Importantly, we observe a minimal tendency to increased background with multiplexing, compared to a sandwich assay, suggesting that higher levels of multiplexing are possible. We used ProteinSeq to analyze proteins in plasma samples from cardiovascular disease (CVD) patient cohorts and matched controls. Three proteins, namely P-selectin, Cystatin-B and Kallikrein-6, were identified as putative diagnostic biomarkers for CVD. The latter two have not been previously reported in the literature and their potential roles must be validated in larger patient cohorts. We conclude that ProteinSeq is promising for screening large numbers of proteins and samples while the technology can provide a much-needed platform for validation of diagnostic markers in biobank samples and in clinical use

The Adhesion GPCR GPR125 is specifically expressed in the choroid plexus and is upregulated following brain injury

<p>Abstract</p> <p>Background</p> <p>GPR125 belongs to the family of <it>Adhesion </it>G protein-coupled receptors (GPCRs). A single copy of GPR125 was found in many vertebrate genomes. We also identified a <it>Drosophila </it>sequence, DmCG15744, which shares a common ancestor with the entire Group III of <it>Adhesio</it>n GPCRs, and also contains Ig, LRR and HBD domains which were observed in mammalian GPR125.</p> <p>Results</p> <p>We found specific expression of GPR125 in cells of the choroid plexus using <it>in situ </it>hybridization and protein-specific antibodies and combined <it>in situ</it>/immunohistochemistry co-localization using cytokeratin, a marker specific for epithelial cells. Induction of inflammation by LPS did not change GPR125 expression. However, GPR125 expression was transiently increased (almost 2-fold) at 4 h after traumatic brain injury (TBI) followed by a decrease (approximately 4-fold) from 2 days onwards in the choroid plexus as well as increased expression (2-fold) in the hippocampus that was delayed until 1 day after injury.</p> <p>Conclusion</p> <p>These findings suggest that GPR125 plays a functional role in choroidal and hippocampal response to injury.</p

Environmental regulation induced foreign direct investment

The last decade has witnessed a renewed interest in the relationship between environmental regulations and international capital flows. However, empirical studies have so far failed to find conclusive evidence for this so-called pollution haven or race to the bottom effect where foreign direct investment (FDI) is assumed to be attracted to low regulation countries, regions or states. In this paper we present a simple theoretical framework to demonstrate that greater stringency in environmental standards can lead to a strategic increase in capital inflows which we refer to as environmental regulation induced FDI. Our result reveals a possible explanation for the mixed results in the empirical literature and provides an illustration of the conditions under which environmental regulations in the host country can affect the location decision of foreign firms

Threshold effect of foreign direct investment on environmental degradation

The aim of this paper is to investigate the threshold effect of foreign direct investment (FDI) on environmental degradation. In empirical analysis, FDI and environmental degradation are jointly determined under the given threshold variable and other exogenous variables. Using carbon dioxide (CO2) emissions per capita as a proxy for environmental degradation, the results show that increasing FDI worsens CO2 emissions after a threshold level of corruption has been reached. Our results demonstrate that increasing FDI will increase CO2 emissions when the degree of corruptibility is relatively high. The study suggests that further FDI and improved environmental quality are competing rather than compatible objectives in high-corruption countries and are compatible rather than competing objectives in low-corruption countries. Higher trade liberalization in low-corruption countries could contribute to negative environmental consequences because of the increased output or economic activity which results from increased trade. The robustness estimation confirms the evidence that pollution and economic development increase together up to a certain income level, after which the trend reverses.info:eu-repo/semantics/publishedVersio

The Origin of GPCRs: Identification of Mammalian like Rhodopsin, Adhesion, Glutamate and Frizzled GPCRs in Fungi

G protein-coupled receptors (GPCRs) in humans are classified into the five main families named Glutamate, Rhodopsin, Adhesion, Frizzled and Secretin according to the GRAFS classification. Previous results show that these mammalian GRAFS families are well represented in the Metazoan lineages, but they have not been shown to be present in Fungi. Here, we systematically mined 79 fungal genomes and provide the first evidence that four of the five main mammalian families of GPCRs, namely Rhodopsin, Adhesion, Glutamate and Frizzled, are present in Fungi and found 142 novel sequences between them. Significantly, we provide strong evidence that the Rhodopsin family emerged from the cAMP receptor family in an event close to the split of Opisthokonts and not in Placozoa, as earlier assumed. The Rhodopsin family then expanded greatly in Metazoans while the cAMP receptor family is found in 3 invertebrate species and lost in the vertebrates. We estimate that the Adhesion and Frizzled families evolved before the split of Unikonts from a common ancestor of all major eukaryotic lineages. Also, the study highlights that the fungal Adhesion receptors do not have N-terminal domains whereas the fungal Glutamate receptors have a broad repertoire of mammalian-like N-terminal domains. Further, mining of the close unicellular relatives of the Metazoan lineage, Salpingoeca rosetta and Capsaspora owczarzaki, obtained a rich group of both the Adhesion and Glutamate families, which in particular provided insight to the early emergence of the N-terminal domains of the Adhesion family. We identified 619 Fungi specific GPCRs across 79 genomes and revealed that Blastocladiomycota and Chytridiomycota phylum have Metazoan-like GPCRs rather than the GPCRs specific for Fungi. Overall, this study provides the first evidence of the presence of four of the five main GRAFS families in Fungi and clarifies the early evolutionary history of the GPCR superfamily

Gene expression profiling in primary breast cancer distinguishes patients developing local recurrence after breast-conservation surgery, with or without postoperative radiotherapy

Introduction Some patients with breast cancer develop local recurrence after breast-conservation surgery despite postoperative radiotherapy, whereas others remain free of local recurrence even in the absence of radiotherapy. As clinical parameters are insufficient for identifying these two groups of patients, we investigated whether gene expression profiling would add further information. Methods We performed gene expression analysis (oligonucleotide arrays, 26,824 reporters) on 143 patients with lymph node-negative disease and tumor-free margins. A support vector machine was employed to build classifiers using leave-one-out cross-validation. Results Within the estrogen receptor-positive (ER+) subgroup, the gene expression profile clearly distinguished patients with local recurrence after radiotherapy (n = 20) from those without local recurrence (n = 80 with or without radiotherapy). The receiver operating characteristic (ROC) area was 0.91, and 5,237 of 26,824 reporters had a P value of less than 0.001 (false discovery rate = 0.005). This gene expression profile provides substantially added value to conventional clinical markers (for example, age, histological grade, and tumor size) in predicting local recurrence despite radiotherapy. Within the ER- subgroup, a weaker, but still significant, signal was found (ROC area = 0.74). The ROC area for distinguishing patients who develop local recurrence from those who remain local recurrence-free in the absence of radiotherapy was 0.66 (combined ER+/ER-). Conclusion A highly distinct gene expression profile for patients developing local recurrence after breast-conservation surgery despite radiotherapy has been identified. If verified in further studies, this profile might be a most important tool in the decision making for surgery and adjuvant therapy

Dysregulation of Mitochondrial Dynamics and the Muscle Transcriptome in ICU Patients Suffering from Sepsis Induced Multiple Organ Failure

BACKGROUND: Septic patients treated in the intensive care unit (ICU) often develop multiple organ failure including persistent skeletal muscle dysfunction which results in the patient's protracted recovery process. We have demonstrated that muscle mitochondrial enzyme activities are impaired in septic ICU patients impairing cellular energy balance, which will interfere with muscle function and metabolism. Here we use detailed phenotyping and genomics to elucidate mechanisms leading to these impairments and the molecular consequences. METHODOLOGY/PRINCIPAL FINDINGS: Utilising biopsy material from seventeen patients and ten age-matched controls we demonstrate that neither mitochondrial in vivo protein synthesis nor expression of mitochondrial genes are compromised. Indeed, there was partial activation of the mitochondrial biogenesis pathway involving NRF2alpha/GABP and its target genes TFAM, TFB1M and TFB2M yet clearly this failed to maintain mitochondrial function. We therefore utilised transcript profiling and pathway analysis of ICU patient skeletal muscle to generate insight into the molecular defects driving loss of muscle function and metabolic homeostasis. Gene ontology analysis of Affymetrix analysis demonstrated substantial loss of muscle specific genes, a global oxidative stress response related to most probably cytokine signalling, altered insulin related signalling and a substantial overlap between patients and muscle wasting/inflammatory animal models. MicroRNA 21 processing appeared defective suggesting that post-transcriptional protein synthesis regulation is altered by disruption of tissue microRNA expression. Finally, we were able to demonstrate that the phenotype of skeletal muscle in ICU patients is not merely one of inactivity, it appears to be an actively remodelling tissue, influenced by several mediators, all of which may be open to manipulation with the aim to improve clinical outcome. CONCLUSIONS/SIGNIFICANCE: This first combined protein and transcriptome based analysis of human skeletal muscle obtained from septic patients demonstrated that losses of mitochondria and muscle mass are accompanied by sustained protein synthesis (anabolic process) while dysregulation of transcription programmes appears to fail to compensate for increased damage and proteolysis. Our analysis identified both validated and novel clinically tractable targets to manipulate these failing processes and pursuit of these could lead to new potential treatments

Is standard breast-conserving therapy (BCT) in elderly breast cancer patients justified? A prospective measurement of acute toxicity according CTC-classification

<p>Abstract</p> <p>Background</p> <p>Breast conserving therapy (BCT) is an accepted treatment for early-stage breast cancer. This study aimed to measure prospectively acute radiation-related toxicity and to create a comprehensive data base for long-term temporal analyses of 3D conformal adjuvant radiotherapy. The specific aspect of age has been neglected by traditional research. Therefore, the impact of age on acute BCT toxicity should be also specifically adressed.</p> <p>Methods</p> <p>Toxicity was measured in 109 patients at initiation (t1), during radiotherapy (t2-t7), and 6 weeks after treatment completion (t8) using a new topographic module. Organ systems were recorded in 15 scales and scored according to symptom intensity (grade 0-5) based on CTC (Common Toxicity Criteria) -classification. Radiotherapy was virtually CT-based planned and applied with 6-MeV-photons. Mean total dose was 60.1 Gy. Patients were stratified by age in 3 Groups: <50, 50-60, and >60 years.</p> <p>Results</p> <p>Registered toxicity was generally low. Mean overall-grade climbed from 0.29-0.40 (t1-t7), and dropped to 0.23 (t8). Univariate analyses revealed slightly higher toxicity in older (> 60 years) versus young patients (< 50 years) in 2 scales only: breast-symmetry (p = 0.033), and arm function (p = 0.007). However, in the scale "appetite" toxicity was higher in younger (< 50 years) versus older (> 60 years) patients (p = 0.039). Toxicity differences in all other scales were not significant. Between older (> 60 years) and midaged patients (50-60 years) no significant differences in toxicity were found. This was also true for the comparison between young (< 50 years) versus midaged patient groups (50-60 years).</p> <p>Conclusion</p> <p>The treatment concept of BCT for breast cancer is generally well tolerated. The toxicity-measurement with the new topographic module is feasible. Not modified standard treatment for BC should be performed in elderly women.</p

Interactional justice at work is related to sickness absence: a study using repeated measures in the Swedish working population

Background: Research has shown that perceived unfairness contributes to higher rates of sickness absence. While shorter, but more frequent periods of sickness absence might be a possibility for the individual to get relief from high strain, long-term sickness absence might be a sign of more serious health problems. The Uncertainty Management Model suggests that justice is particularly important in times of uncertainty, e.g. perceived job insecurity. The present study investigated the association between interpersonal and informational justice at work with long and frequent sickness absence respectively, under conditions of job insecurity. Methods: Data were derived from the 2010, 2012, and 2014 biennial waves of the Swedish Longitudinal Occupational Survey of Health (SLOSH). The final analytic sample consisted of 19,493 individuals. We applied repeated measures regression analyses through generalized estimating equations (GEE), a method for longitudinal data that simultaneously analyses variables at different time points. We calculated risk of long and frequent sickness absence, respectively in relation to interpersonal and informational justice taking perceptions of job insecurity into account. Results: We found informational and interpersonal justice to be associated with risk of long and frequent sickness absence independently of job insecurity and demographic variables. Results from autoregressive GEE provided some support for a causal relationship between justice perceptions and sickness absence. Contrary to expectations, we found no interaction between justice and job insecurity. Conclusions: Our results underline the need for fair and just treatment of employees irrespective of perceived job insecurity in order to keep the workforce healthy and to minimize lost work days due to sickness absence

Evaluation of changes in postnatal care using the "Parents' Postnatal Sense of Security" instrument and an assessment of the instrument's reliability and validity

<p>Abstract</p> <p>Background</p> <p>A sense of security is important for experiences of parenthood in the early postpartum period. The objectives of this study were to evaluate two models of postnatal care using a questionnaire incorporating the Parents' Postpartum Sense of Security (<it>PPSS</it>) instrument and to test the validity of the <it>PPSS </it>instrument.</p> <p>Methods</p> <p>Postal surveys were sent to 234 mothers who had experienced two different forms of postnatal care (study group and control group) and returned by 86.8%. These two groups of mothers were compared for total scores on the <it>PPSS </it>instrument. Demographic variables and mothers' opinions about care interventions were also compared and these variables were tested for correlations with the total <it>PPSS </it>score. A regression analysis was carried out to assess areas of midwifery care which might affect a sense of security. The internal consistency and concurrent validity of the instrument were tested for the total population.</p> <p>Results</p> <p>there were no significant differences between the groups for scores on the <it>PPSS </it>instrument. A total of three variables predicted 26% of the variability on the <it>PPSS </it>scores for the study group and five variables predicted 37% of the variability in the control group. One variable was common to both: "<it>The midwives on the postnatal ward paid attention to the mother as an individual"</it>. There were significant correlations between the total <it>PPSS </it>scores and scores for postpartum talks and visits to the breastfeeding clinic. There was also a significant correlation between the single question: "<it>I felt secure during the first postpartum week</it>" and the total <it>PPSS </it>score. Tests for internal consistency and concurrent validity were satisfactory.</p> <p>Conclusion</p> <p>The proposed new model of care neither improved nor impaired mothers' feelings of security the week following birth. Being seen as an individual by the midwife who provides postnatal care may be an important variable for mothers' sense of postnatal security. It is possible that postpartum talks may encourage the processing of childbirth experiences in a positive direction. Availability of breastfeeding support may also add to a sense of security postpartum. The <it>PPSS </it>instrument has shown acceptable reliability and validity.</p