86 research outputs found
Fluorescence Signal Enhancement in Antibody Microarrays Using Lightguiding Nanowires
Fluorescence-based detection assays play an essential role in the life sciences and medicine. To offer better detection sensitivity and lower limits of detection (LOD), there is a growing need for novel platforms with an improved readout capacity. In this context, substrates containing semiconductor nanowires may offer significant advantages, due to their proven light-emission enhancing, waveguiding properties, and increased surface area. To demonstrate and evaluate the potential of such nanowires in the context of diagnostic assays, we have in this work adopted a well-established single-chain fragment antibody-based assay, based on a protocol previously designed for biomarker detection using planar microarrays, to freestanding, SiO2-coated gallium phosphide nanowires. The assay was used for the detection of protein biomarkers in highly complex human serum at high dilution. The signal quality was quantified and compared with results obtained on conventional flat silicon and plastic substrates used in the established microarray applications. Our results show that using the nanowire-sensor platform in combination with conventional readout methods, improves the signal intensity, contrast, and signal-to-noise by more than one order of magnitude compared to flat surfaces. The results confirm the potential of lightguiding nanowires for signal enhancement and their capacity to improve the LOD of standard diagnostic assays
On-tissue dataset-dependent MALDI-TIMS-MS bioimaging
Trapped ion mobility spectrometry (TIMS) adds an additional separation dimension to mass spectrometry (MS) imaging, however, the lack of fragmentation spectra (MS) impedes confident compound annotation in spatial metabolomics. Here, we describe spatial ion mobility-scheduled exhaustive fragmentation (SIMSEF), a dataset-dependent acquisition strategy that augments TIMS-MS imaging datasets with MS spectra. The fragmentation experiments are systematically distributed across the sample and scheduled for multiple collision energies per precursor ion. Extendable data processing and evaluation workflows are implemented into the open source software MZmine. The workflow and annotation capabilities are demonstrated on rat brain tissue thin sections, measured by matrix-assisted laser desorption/ionisation (MALDI)-TIMS-MS, where SIMSEF enables on-tissue compound annotation through spectral library matching and rule-based lipid annotation within MZmine and maps the (un)known chemical space by molecular networking. The SIMSEF algorithm and data analysis pipelines are open source and modular to provide a community resource
Identification of a serum biomarker signature associated with metastatic prostate cancer
Purpose: Improved early diagnosis and determination of aggressiveness of prostate cancer (PC) is important to select suitable treatment options and to decrease over-treatment. The conventional marker is total prostate specific antigen (PSA) levels in blood, but lacks specificity and ability to accurately discriminate indolent from aggressive disease. Experimental design: In this study, we sought to identify a serum biomarker signature associated with metastatic PC. We measured 157 analytes in 363 serum samples from healthy subjects, patients with non-metastatic PC and patients with metastatic PC, using a recombinant antibody microarray. Results: A signature consisting of 69 proteins differentiating metastatic PC patients from healthy controls was identified. Conclusions and clinical relevance: The clinical value of this biomarker signature requires validation in larger independent patient cohorts before providing a new prospect for detection of metastatic PC
CSF Surfactant Protein Changes in Preterm Infants After Intraventricular Hemorrhage
Introduction: Surfactant proteins (SP) have been shown to be inherent proteins of the
human CNS and are altered during acute and chronic disturbances of CSF circulation.
Aim of the study was to examine the changes of surfactant protein concentrations in CSF
of preterm babies suffering from intraventricular hemorrhage.
Patients and Methods: Consecutive CSF samples of 21 preterm infants with
intraventricular hemorrhages (IVH) and posthemorrhagic hydrocephalus (PHHC) were
collected at primary intervention, after 5–10 days and at time of shunt insertion 50
days after hemorrhage. Samples were analyzed for surfactant proteins A, B, C, and G by
ELISA assays and the results were compared to 35 hydrocephalus patients (HC) without
hemorrhage and 6 newborn control patients.
Results and Discussion: Premature patients with IVH showed a significant elevation
of surfactant proteins SP-A, C, and G compared to HC and control groups: mean
values for the respective groups were SP-A 4.19 vs. 1.08 vs. 0.38 ng/ml. Mean SP-C
3.63 vs. 1.47 vs. 0.48 ng/ml. Mean SP-G 3.86 vs. 0.17 vs. 0.2 ng/ml. SP-A and G
concentrations were slowly falling over time without reaching normal values. SP-C levels
declined faster following neurosurgical interventions and reached levels comparable to
those of hydrocephalus patients without hemorrhage.
Conclusion: Intraventricular hemorrhages of premature infants cause posthemorrhagic
CSF flow disturbance and are associated with highly significant elevations of surfactant
proteins A, C, and G independent of total CSF protein concentrations
Units for Promoter Measurement in Mammalian Cells
The purpose of this RFC is to provide units for the characterization of promoter strength for use in mammalian cells. RMPU is mRNA based and
directly proportional to PoPS, whereas REU is protein based and not proportional to PoPS
Epigenetic dynamics of monocyte-to-macrophage differentiation
Background Monocyte-to-macrophage differentiation involves major biochemical and structural changes. In order to elucidate the role of gene regulatory changes during this process, we used high-throughput sequencing to analyze the complete transcriptome and epigenome of human monocytes that were differentiated in vitro by addition of colony-stimulating factor 1 in serum-free medium. Results Numerous mRNAs and miRNAs were significantly up- or down-regulated. More than 100 discrete DNA regions, most often far away from transcription start sites, were rapidly demethylated by the ten eleven translocation enzymes, became nucleosome-free and gained histone marks indicative of active enhancers. These regions were unique for macrophages and associated with genes involved in the regulation of the actin cytoskeleton, phagocytosis and innate immune response. Conclusions In summary, we have discovered a phagocytic gene network that is repressed by DNA methylation in monocytes and rapidly de-repressed after the onset of macrophage differentiation
Epigenetic adaptations of the masticatory mucosa to periodontal inflammation
Background: In mucosal barrier interfaces, flexible responses of gene expression to long-term environmental
changes allow adaptation and fine-tuning for the balance of host defense and uncontrolled not-resolving inflammation.
Epigenetic modifications of the chromatin confer plasticity to the genetic information and give insight into
how tissues use the genetic information to adapt to environmental factors. The oral mucosa is particularly exposed
to environmental stressors such as a variable microbiota. Likewise, persistent oral inflammation is the most important
intrinsic risk factor for the oral inflammatory disease periodontitis and has strong potential to alter DNA-methylation
patterns. The aim of the current study was to identify epigenetic changes of the oral masticatory mucosa in response
to long-term inflammation that resulted in periodontitis.
Methods and results: Genome-wide CpG methylation of both inflamed and clinically uninflamed solid gingival
tissue biopsies of 60 periodontitis cases was analyzed using the Infinium MethylationEPIC BeadChip. We validated
and performed cell-type deconvolution for infiltrated immune cells using the EpiDish algorithm. Effect sizes of DMPs
in gingival epithelial and fibroblast cells were estimated and adjusted for confounding factors using our recently
developed “intercept-method”. In the current EWAS, we identified various genes that showed significantly different
methylation between periodontitis-inflamed and uninflamed oral mucosa in periodontitis patients. The strongest
differences were observed for genes with roles in wound healing (ROBO2, PTP4A3), cell adhesion (LPXN) and innate
immune response (CCL26, DNAJC1, BPI). Enrichment analyses implied a role of epigenetic changes for vesicle trafficking
gene sets.
Conclusions: Our results imply specific adaptations of the oral mucosa to a persistent inflammatory environment
that involve wound repair, barrier integrity, and innate immune defense
DNA methylation-based classification of sinonasal tumors
The diagnosis of sinonasal tumors is challenging due to a heterogeneous spectrum of various differential diagnoses as well as poorly defined, disputed entities such as sinonasal undifferentiated carcinomas (SNUCs). In this study, we apply a machine learning algorithm based on DNA methylation patterns to classify sinonasal tumors with clinical-grade reliability. We further show that sinonasal tumors with SNUC morphology are not as undifferentiated as their current terminology suggests but rather reassigned to four distinct molecular classes defined by epigenetic, mutational and proteomic profiles. This includes two classes with neuroendocrine differentiation, characterized by IDH2 or SMARCA4/ARID1A mutations with an overall favorable clinical course, one class composed of highly aggressive SMARCB1-deficient carcinomas and another class with tumors that represent potentially previously misclassified adenoid cystic carcinomas. Our findings can aid in improving the diagnostic classification of sinonasal tumors and could help to change the current perception of SNUCs
Complement Factor H-Related Proteins CFHR2 and CFHR5 Represent Novel Ligands for the Infection-Associated CRASP Proteins of Borrelia burgdorferi
Background: One virulence property of Borrelia burgdorferi is its resistance to innate immunity, in particular to complement-mediated killing. Serum-resistant B. burgdorferi express up to five distinct complement regulator-acquiring surface proteins (CRASP) which interact with complement regulator factor H (CFH) and factor H-like protein 1 (FHL1) or factor H-related protein 1 (CFHR1). In the present study we elucidate the role of the infection-associated CRASP-3 and CRASP-5 protein to serve as ligands for additional complement regulatory proteins as well as for complement resistance of B. burgdorferi. Methodology/Principal Findings: To elucidate whether CRASP-5 and CRASP-3 interact with various human proteins, both borrelial proteins were immobilized on magnetic beads. Following incubation with human serum, bound proteins were eluted and separated by Glycine-SDS-PAGE. In addition to CFH and CFHR1, complement regulators CFHR2 and CFHR5 were identified as novel ligands for both borrelial proteins by employing MALDI-TOF. To further assess the contributions of CRASP-3 and CRASP-5 to complement resistance, a serum-sensitive B. garinii strain G1 which lacks all CFH-binding proteins was used as a valuable model for functional analyses. Both CRASPs expressed on the B. garinii outer surface bound CFH as well as CFHR1 and CFHR2 in ELISA. In contrast, live B. garinii bound CFHR1, CFHR2, and CFHR5 and only miniscute amounts of CFH as demonstrated by serum adsorption assays and FACS analyses. Further functional analysis revealed that upon NHS incubation, CRASP-3 or CRASP-5 expressing borreliae were killed by complement. Conclusions/Significance: In the absence of CFH and the presence of CFHR1, CFHR2 and CFHR5, assembly and integration of the membrane attack complex was not efficiently inhibited indicating that CFH in co-operation with CFHR1, CFHR2 and CFHR5 supports complement evasion of B. burgdorferi
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