Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood

Convenient and well-performing protein detection methods for a wide range of targets are in great demand for biomedical research and future diagnostics. Assays without the need for washing steps while still unaffected when analyzing complex biological samples are difficult to develop. Herein, we report a well-characterized nucleic acid proximity-based assay using antibodies, called Proximity Extension Assay (PEA), showing good performance in plasma samples. Target-specific antibody pairs are linked to DNA strands that upon simultaneous binding to the target analyte create a real-time PCR amplicon in a proximity-dependent manner enabled by the action of a DNA polymerase. 3′Exonuclease-capable polymerases were found to be clearly superior in sensitivity over non-3′exonuclease ones. A PEA was set up for IL-8 and GDNF in a user-friendly, homogenous assay displaying femtomolar detection sensitivity, good recovery in human plasma, high specificity and up to 5-log dynamic range in 1 μL samples. Furthermore, we have illustrated the use of a macro-molecular crowding matrix in combination with this homogeneous assay to drive target binding for low-affinity antibodies, thereby improving the sensitivity and increasing affinity reagent availability by lowering assay development dependency on high-affinity antibodies. Assay performance was also confirmed for a multiplex version of PEA

Using adjusted local assortativity with Molecular Pixelation unveils colocalization of membrane proteins with immunological significance

Advances in spatial proteomics and protein colocalization are a driving force in the understanding of cellular mechanisms and their influence on biological processes. New methods in the field of spatial proteomics call for the development of algorithms and open up new avenues of research. The newly introduced Molecular Pixelation (MPX) provides spatial information on surface proteins and their relationship with each other in single cells. This allows for in silico representation of neighborhoods of membrane proteins as graphs. In order to analyze this new data modality, we adapted local assortativity in networks of MPX single-cell graphs and created a method that is able to capture detailed information on the spatial relationships of proteins. The introduced method can evaluate the pairwise colocalization of proteins and access higher-order similarity to investigate the colocalization of multiple proteins at the same time. We evaluated the method using publicly available MPX datasets where T cells were treated with a chemokine to study uropod formation. We demonstrate that adjusted local assortativity detects the effects of the stimuli at both single- and multiple-marker levels, which enhances our understanding of the uropod formation. We also applied our method to treating cancerous B-cell lines using a therapeutic antibody. With the adjusted local assortativity, we recapitulated the effect of rituximab on the polarity of CD20. Our computational method together with MPX improves our understanding of not only the formation of cell polarity and protein colocalization under stimuli but also advancing the overall insight into immune reaction and reorganization of cell surface proteins, which in turn allows the design of novel therapies. We foresee its applicability to other types of biological spatial data when represented as undirected graphs

Bulk hadron production at high rapidities

Recent experimental observations on the `bulk' features of particle production at high (pseudo)rapidities will be reviewed. This kinematic region is of interest mostly because of its relevance to the theoretical description of initial state effects of nuclei at ultra-relativistic energies. Measurements of the charged hadron multiplicity density as well as the pseudorapidity dependence of the elliptic and directed flow exhibit a remarkable scaling property as a function of collision energy. This scaling seems to hold for pions and even photons and J/Psi-s, but is violated for protons. The special role of baryons will be discussed using selected results on nuclear transparency and baryon stopping.Comment: 10 pages, 8 figures. Prepared for the Proceedings of the Quark Matter 2005 Conferenc

Gel-Based Proteomics of Clinical Samples Identifies Potential Serological Biomarkers for Early Detection of Colorectal Cancer

The burden of colorectal cancer (CRC) is considerable—approximately 1.8 million people are diagnosed each year with CRC and of these about half will succumb to the disease. In the case of CRC, there is strong evidence that an early diagnosis leads to a better prognosis, with metastatic CRC having a 5-year survival that is only slightly greater than 10% compared with up to 90% for stage I CRC. Clearly, biomarkers for the early detection of CRC would have a major clinical impact. We implemented a coherent gel-based proteomics biomarker discovery platform for the identification of clinically useful biomarkers for the early detection of CRC. Potential protein biomarkers were identified by a 2D gel-based analysis of a cohort composed of 128 CRC and site-matched normal tissue biopsies. Potential biomarkers were prioritized and assays to quantitatively measure plasma expression of the candidate biomarkers were developed. Those biomarkers that fulfilled the preset criteria for technical validity were validated in a case-control set of plasma samples, including 70 patients with CRC, adenomas, or non-cancer diseases and healthy individuals in each group. We identified 63 consistently upregulated polypeptides (factor of four-fold or more) in our proteomics analysis. We selected 10 out of these 63 upregulated polypeptides, and established assays to measure the concentration of each one of the ten biomarkers in plasma samples. Biomarker levels were analyzed in plasma samples from healthy individuals, individuals with adenomas, CRC patients, and patients with non-cancer diseases and we identified one protein, tropomyosin 3 (Tpm3) that could discriminate CRC at a significant level (p = 0.0146). Our results suggest that at least one of the identified proteins, Tpm3, could be used as a biomarker in the early detection of CRC, and further studies should provide unequivocal evidence for the real-life clinical validity and usefulness of Tpm3

The safety effect of increased pedestrian protection, autonomous emergency braking for pedestrians and bicyclists on passenger cars, and speed management

The overall objective of this paper was to estimate the effect of increased pedestrian protection and speed management to reduce serious injuries among pedestrians and bicyclists. More specifically, the aim was to estimate the injury mitigating effects of the following interventions: AEB with pedestrian and bicyclist detection, Euro NCAP pedestrian test score, Active Bonnet, Traffic calming at pedestrian and bicycle crossings, and additionally, the combined effect of the above-mentioned treatments. The main source of data was the Swedish traffic data acquisition system, where information of road traffic crashes between passenger cars and pedestrians or bicyclists for the period January 2003–December 2022 was obtained. Cars with optional fitment of AEB systems were identified, and the license registration number was used to access individual car equipment lists to identify whether the vehicle was equipped with AEB with pedestrian and/or cyclist detection. Information about traffic calming at pedestrian and bicycle crossings was obtained from the Swedish Transport Administration. The injury metric used was risk of permanent medical impairment (RPMI) of at least one percent and ten percent. RPMI captures the risk of long-term medical impairment based on a diagnosed injury location and Abbreviated Injury Severity score. The relative difference between the mean values of RPMI (mRPMI1% and mRPMI10%+) was calculated and tested using an independent two sample t-test which was conducted for unequal sample sizes and variance. Pedestrian mRPMI10%+ was reduced by 34%–44% in speed zones 10–50 km/h comparing the group struck by cars equipped with AEB with pedestrian detection compared to the group struck by cars without the system. However the reduction was only significant at 90% level. The pattern was similar also for mRPMI1%+. For cyclists, the mRPMI10%+ was reduced by 35% at speed zones 10–50 km/h. For crashes within +/- 20 meters from a pedestrian or bicycle crossing, the AEB system reduced 60% (p = 0.05) of pedestrians mRPMI10%+ at crossings with good safety standard compared to crossings of poor safety standard. The comparison of cars with poor performance (1–9 p) in the NCAP pedestrian test and cars with a high score (28–36 p) showed that pedestrian mRPMI10%+ was reduced by 48% (p < 0.01) across all speed limits, and by 64% including only those aged ≤ 64 years. For bicyclists, a significant reduction of cyclist mRPMI10%+ was found comparing low scoring cars to high scoring cars in 10–30 km/h speed limit (-73%, p = 0.02) and across all speed limits (-36%, p = 0.06). Including only those aged ≤ 64 years, the reduction was 49% (p < 0.01). For the active bonnet, a significant reduction of mRPMI1%+ by 24% was observed but given that the rate of helmet wearing was higher in the group struck by cars with active bonnet, this difference cannot be attributed to an effect of the active bonnet. The STA safety rating of pedestrian and bicycle crossings showed that overall pedestrian mRPMI1%+ was reduced by 15% (p = 0.06), while cyclists mRPMI10%+ was reduced by 32% (p = 0.01) comparing crossings of high safety level to crossings of poor safety level. The analysis of combined interventions showed that the total reduction of pedestrians and cyclists mRPMI10%+ together was 69% (p < 0.01), from 6.4% to 2%. This paper demonstrates that a road environment with adapted infrastructure and speed, combined with passenger car technologies that improve the safety for vulnerable road users, creates significant reductions of serious injuries among pedestrians and bicyclists

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

Multiplex amplification of all coding sequences within 10 cancer genes by Gene-Collector

Herein we present Gene-Collector, a method for multiplex amplification of nucleic acids. The procedure has been employed to successfully amplify the coding sequence of 10 human cancer genes in one assay with uniform abundance of the final products. Amplification is initiated by a multiplex PCR in this case with 170 primer pairs. Each PCR product is then specifically circularized by ligation on a Collector probe capable of juxtapositioning only the perfectly matched cognate primer pairs. Any amplification artifacts typically associated with multiplex PCR derived from the use of many primer pairs such as false amplicons, primer-dimers etc. are not circularized and degraded by exonuclease treatment. Circular DNA molecules are then further enriched by randomly primed rolling circle replication. Amplification was successful for 90% of the targeted amplicons as seen by hybridization to a custom resequencing DNA micro-array. Real-time quantitative PCR revealed that 96% of the amplification products were all within 4-fold of the average abundance. Gene-Collector has utility for numerous applications such as high throughput resequencing, SNP analyses, and pathogen detection

Risk of subsequent invasive breast carcinoma after in situ breast carcinoma in a population covered by national mammographic screening

Sweden was the first country to establish a nationwide breast cancer screening service. We used the Swedish Family-Cancer Database to evaluate the risk of invasive carcinoma after in situ carcinoma of the breast. Risk estimates for contralateral and ipsilateral invasive malignancies following age and histology specific in situ breast carcinomas were calculated using Poisson's regression analysis. The agreement between concordant and discordant morphologies of invasive and in situ breast cancer was measured using the kappa statistic. Women with in situ breast cancer showed a relative risk of 2.03 for contralateral and 3.94 for ipsilateral invasive breast cancer. The risk was higher for in situ carcinomas diagnosed before the age of 50 years and after lobular in situ breast cancers. A comparison of the risks during the past decades suggested that the risk of ipsilateral breast cancer has increased in Sweden but that of contralateral breast cancer has remained unchanged. In situ and the subsequent invasive breast cancers did not seem to share their morphologies

Electromagnetic form factors of the nucleon in a covariant diquark model

We present a simple covariant constituent diquark-quark model for the nucleon. The nucleon is assumed to be composed of a scalar diquark and a quark which interact via a quark exchange. Starting from the Bethe-Salpeter equation, the instantaneous approximation leads to a diquark-quark Salpeter equation. In the Mandelstam formalism, the electromagnetic form factors of the nucleon are calculated for momentum transfers up to q^2 = - 3 \; (\mbox{GeV/c})^2. A remarkable description of the experimental data is obtained. Especially, the model gives nearly the right values for the proton and (negative) neutron charge radii, and a qualitative description of the magnetic form factors.Comment: 17 pages, revtex, 8 figures in additional fil

The G Protein-Coupled Receptor GPR17: Overview and Update

The GPR17 receptor is a G protein-coupled receptor (GPCR) that seems to respond to two unrelated families of endogenous ligands: nucleotide sugars (UDP, UDP-galactose, and UDP-glucose) and cysteinyl leukotrienes (LTD4 , LTC4 , and LTE4 ), with significant affinity at micromolar and nanomolar concentrations, respectively. This receptor has a broad distribution at the level of the central nervous system (CNS) and is found in neurons and in a subset of oligodendrocyte precursor cells (OPCs). Unfortunately, disparate results emerging from different laboratories have resulted in a lack of clarity with regard to the role of GPR17-targeting ligands in OPC differentiation and in myelination. GPR17 is also highly expressed in organs typically undergoing ischemic damage and has various roles in specific phases of adaptations that follow a stroke. Under such conditions, GPR17 plays a crucial role; in fact, its inhibition decreases the progression of ischemic damage. This review summarizes some important features of this receptor that could be a novel therapeutic target for the treatment of demyelinating diseases and for repairing traumatic injury