Profiling surface proteins on individual exosomes using a proximity barcoding assay

Exosomes have been implicated in numerous biological processes, and they may serve as important disease markers. Surface proteins on exosomes carry information about their tissues of origin. Because of the heterogeneity of exosomes it is desirable to investigate them individually, but this has so far remained impractical. Here, we demonstrate a proximity-dependent barcoding assay to profile surface proteins of individual exosomes using antibody-DNA conjugates and next-generation sequencing. We first validate the method using artificial streptavidin-oligonucleotide complexes, followed by analysis of the variable composition of surface proteins on individual exosomes, derived from human body fluids or cell culture media. Exosomes from different sources are characterized by the presence of specific combinations of surface proteins and their abundance, allowing exosomes to be separately quantified in mixed samples to serve as markers for tissue-specific engagement in disease

Sensitive detection of Aβ protofibrils by proximity ligation - relevance for Alzheimer's disease

<p>Abstract</p> <p>Background</p> <p>Protein aggregation plays important roles in several neurodegenerative disorders. For instance, insoluble aggregates of phosphorylated tau and of Aβ peptides are cornerstones in the pathology of Alzheimer's disease. Soluble protein aggregates are therefore potential diagnostic and prognostic biomarkers for their cognate disorders. Detection of the aggregated species requires sensitive tools that efficiently discriminate them from monomers of the same proteins. Here we have established a proximity ligation assay (PLA) for specific and sensitive detection of Aβ protofibrils via simultaneous recognition of three identical determinants present in the aggregates. PLA is a versatile technology in which the requirement for multiple target recognitions is combined with the ability to translate signals from detected target molecules to amplifiable DNA strands, providing very high specificity and sensitivity.</p> <p>Results</p> <p>For specific detection of Aβ protofibrils we have used a monoclonal antibody, mAb158, selective for Aβ protofibrils in a modified PLA, where the same monoclonal antibody was used for the three classes of affinity reagents required in the assay. These reagents were used for detection of soluble Aβ aggregates in solid-phase reactions, allowing detection of just 0.1 pg/ml Aβ protofibrils, and with a dynamic range greater than six orders of magnitude. Compared to a sandwich ELISA setup of the same antibody the PLA increases the sensitivity of the Aβ protofibril detection by up to 25-fold. The assay was used to measure soluble Aβ aggregates in brain homogenates from mice transgenic for a human allele predisposing to Aβ aggregation.</p> <p>Conclusions</p> <p>The proximity ligation assay is a versatile analytical technology for proteins, which can provide highly sensitive and specific detection of Aβ aggregates - and by implication other protein aggregates of relevance in Alzheimer's disease and other neurodegenerative disorders.</p

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

Co-operative recombination mechanisms promoting gene clustering and lateral transfer of antibacterial drug resistance

Transposons of the Mu superfamily are widespread and have been shown to play an important role in the dissemination of antibiotic resistance among microorganisms. One of these elements, Tn5090/Tn402 is the basal vehicle of the type 1 integrons in which mobile resistance gene cassettes are inserted to form clusters and operons. The transposon was shown to preferentially target recombination sites of the serine family of recombinases that occur in many plasmids and transposons. Mutation analysis revealed that DNA-binding of the targeting factor, a serine recombinase, is essential for efficient transposition, while the recombination activity is not required. Truncated elements were frequently observed and in one instance borne on a composite transposon flanked by IS6100. This new transposon, Tn5089, has allowed the translocation of the integron to small mobilizable IncQ-plasmids that lack the targeting factor and thus are incompetent for insertion of Tn5090/Tn402. Another small replicon, by contrast targeting-positive, was completely sequenced. The transposon Tn5090/Tn402 carries arrayed transposase-binding sites at the ends, which are supposed to arrange the transposase TniA in the appropriate geometry in a recombinationally active complex with DNA. Footprinting showed that transposase, TniA, binds to four 19 bp repeats on one end and to two 19 bp repeats on the other end. Site-specific resolution of Tn5090/Tn402 co-integrates was analysed in an in vitro system. The res site was found to be composed of three unusually organized subsites and expression of TniC was shown to be autoregulated by TniC acting as repressor due to an overlap of the res site with the promoter. The data presented show several aspects of cooperation between transposition and site-specific recombination. This cooperation has enriched genes and combinations of genes that mediate resistance to antibiotic drugs and promotes lateral transfer of these genes. The organization of sites and subsites in the DNA is a subtle genetic code for the formation of the molecule complexes controlling these genetic events

Plasma Protein Profiling Reveal Osteoprotegerin as a Marker of Prognostic Impact for Colorectal Cancer

BACKGROUND: Due to difficulties in predicting recurrences in colorectal cancer stages II and III, reliable prognostic biomarkers could be a breakthrough for individualized treatment and follow-up. OBJECTIVE: To find potential prognostic protein biomarkers in colorectal cancer, using the proximity extension assays. METHODS: A panel of 92 oncology-related proteins was analyzed with proximity extension assays, in plasma from a cohort of 261 colorectal cancer patients with stage II-IV. The survival analyses were corrected for disease stage and age, and the recurrence analyses were corrected for disease stage. The significance threshold was adjusted for multiple comparisons. RESULTS: The plasma proteins expression levels had a greater prognostic relevance in disease stage III colorectal cancer than in disease stage II, and for overall survival than for time to recurrence. Osteoprotegerin was the only biomarker candidate in the protein panel that had a statistical significant association with overall survival (P = .00029). None of the proteins were statistically significantly associated with time to recurrence. CONCLUSIONS: Of the 92 analyzed plasma proteins, osteoprotegerin showed the strongest prognostic impact in patients with colorectal cancer, and therefore osteoprotegerin is a potential predictive marker, and it also could be a target for treatments

Generation of ssDNA aptamers as diagnostic tool for Newcastle avian virus

Aptamers are short single-stranded DNA (ssDNA), RNA or synthetic XNA molecules, which are used as a class of affinity binders recognizing target molecules with a very high affinity and specificity. The aim of this study was to generate and characterize ssDNA aptamers for the detection of Newcastle disease virus (NDV). These aptamers were selected using systematic evolution of ligands by exponential enrichment (SELEX) in combination with quantitative high-throughput DNA sequencing. After three rounds of selections, a highly enriched ssDNA pool was sequenced, and the results were analyzed using FASTAptamer Toolkit. Sequencing reads were sorted by copy numbers and clustered into groups, according to their sequence homology. Top aptameric sequences were used to develop a sandwich enzymatic linked aptamer assay (ELAA) for rapid and sensitive detection of NDV in farm samples. The selected aptamers have an affinity within the nanomolar range, and a high specificity with no cross-reactivity towards other avian viruses. Following optimization of the sandwich ELAA method, the results demonstrated that both selected aptamers Apt_NDV01 and Apt_NDV03 with dissociation constant values of 31 nM and 78.1 nM, respectively, showed the highest specificity and affinity for NDV detection. The ELAA results were verified by quantitative real-time PCR, demonstrating strong concordance, and showing outstanding accuracy for detection of NDV in field sample. In summary, combination of SELEX with high-throughput DNA sequencing allowed rapid screening and selection of aptamers. The selected aptamers allowed recognition of NDV with high affinities. This is the first report that uses a validated sandwich ELAA for rapid and specific detection of NDV in poultry samples

Tyrosine phosphorylation profiling via in situ proximity ligation assay

Background: Tyrosine phosphorylation (pTyr) is an important cancer relevant posttranslational modification since it regulates protein activity and cellular localization. By controlling cell growth and differentiation it plays an important role in tumor development. This paper describes a novel approach for detection and visualization of a panel of pTyr proteins in tumors using in situ proximity ligation assay. Methods: K562 leukemia cells were treated with tyrosine kinase and/or phosphatase inhibitors to induce differences in pTyr levels and mimic cells with different malignant properties. Cells were then probed with one antibody against the pTyr modification and another probe against the detected protein, resulting in a detectable fluorescent signal once the probes were in proximity. Results: Total and protein specific pTyr levels on ABL, SHC, ERK2 and PI3K proteins were detected and samples of control and treated cells were distinguished at the pTyr level using this novel approach. Promising results were also detected for formalin fixed and paraffin embedded cells in the micro array format. Conclusions: This application of in situ proximity ligation assay is valuable in order to study the pTyr modification of a panel of proteins in large data sets to validate mass spectrometric data and to be combined with tissue microarrays. The approach offers new opportunities to reveal the pTyr signatures in cells of different malignant properties that can be used as biomarker of disease in the future

Mitochondrial Respiration-Dependent ANT2-UCP2 Interaction

Adenine nucleotide translocases (ANTs) and uncoupling proteins (UCPs) are known to facilitate proton leak across the inner mitochondrial membrane. However, it remains to be unravelled whether UCP2/3 contribute to significant amount of proton leak in vivo. Reports are indicative of UCP2 dependent proton-coupled efflux of C4 metabolites from the mitochondrial matrix. Previous studies have suggested that UCP2/3 knockdown (KD) contributes to increased ANT-dependent proton leak. Here we investigated the hypothesis that interaction exists between the UCP2 and ANT2 proteins, and that such interaction is regulated by the cellular metabolic demand. Protein-protein interaction was evaluated using reciprocal co-immunoprecipitation and in situ proximity ligation assay. KD of ANT2 and UCP2 was performed by siRNA in human embryonic kidney cells 293A (HEK293A) cells. Mitochondrial and cellular respiration was measured by high-resolution respirometry. ANT2-UCP2 interaction was demonstrated, and this was dependent on cellular metabolism. Inhibition of ATP synthase promoted ANT2-UCP2 interaction whereas high cellular respiration, induced by adding the mitochondrial uncoupler FCCP, prevented interaction. UCP2 KD contributed to increased carboxyatractyloside (CATR) sensitive proton leak, whereas ANT2 and UCP2 double KD reduced CATR sensitive proton leak, compared to UCP2 KD. Furthermore, proton leak was reduced in double KD compared to UCP2 KD. In conclusion, our results show that there is an interaction between ANT2-UCP2, which appears to be dynamically regulated by mitochondrial respiratory activity. This may have implications in the regulation of mitochondrial efficiency or cellular substrate utilization as increased activity of UCP2 may promote a switch from glucose to fatty acid metabolism