Onchocerca lupi (Spirurida: Onchocercidae) is a filarial worm parasitizing domestic carnivores and humans. Adult nematodes usually localize beneath in the sclera or in the ocular retrobulbar of infected animals, whilst microfilariae are found in the skin. Therefore, diagnosis of O. lupi is achieved by microscopic and/or molecular detection of microfilariae from skin biopsy and/or surgical removal of adults from ocular tissues of infected hosts. An urgent non-invasive diagnostic tool for the diagnosis of O. lupi in dog is mandatory. In this study, an immunoproteomic analyses was performed using a combination of immunoblotting and mass spectrometry techniques. Onchocerca lupi major antigen (Ol-MJA) and paramyosin (Ol-PARA) proteins were identified as potential biomarkers for serodiagnosis. Linear epitopes were herein scanned for both proteins using high-density peptide microarray. Sera collected from dog infected with O. lupi and healthy animal controls led to the identification of 11 immunodominant antigenic peptides (n = 7 for Ol-MJA; n = 4 for Ol-PARA). These peptides were validated using sera of dogs uniquely infected with the most important filarioids infesting dogs either zoonotic (Dirofilaria repens, Dirofilaria immitis) or not (Acanthocheilonema reconditum and Cercopithifilaria bainae). Overall, six antigenic peptides, three for Ol-MJA and for Ol-PARA, respectively, were selected as potential antigens for the serological detection of canine O. lupi infection. The molecular and proteomic dataset herein reported should provide a useful resource for studies on O. lupi toward supporting the development of new interventions (drugs, vaccines and diagnostics) against canine onchocercosis.
Nutrient transceptors physically interact with the yeast S6/protein kinase B homolog, Sch9, a TOR kinase target
Zhang, Zhiqiang; Cottignie, Ines; Van Zeebroeck, Griet; Thevelein, Johan M.
Multiple starvation-induced, high-affinity nutrient transporters in yeast function as receptors for activation of the protein kinase A (PKA) pathway upon re-addition of their substrate. We now show that these transceptors may play more extended roles in nutrient regulation. The Gap1 amino acid, Mep2 ammonium, Pho84 phosphate and Sul1 sulfate transceptors physically interact in vitro and in vivo with the PKA-related Sch9 protein kinase, the yeast homolog of mammalian S6 protein kinase and protein kinase B. Sch9 is a phosphorylation target of TOR and well known to affect nutrient-controlled cellular processes, such as growth rate. Mapping with peptide microarrays suggests specific interaction domains in Gap1 for Sch9 binding. Mutagenesis of the major domain affects the upstart of growth upon the addition of L-citrulline to nitrogen-starved cells to different extents but apparently does not affect in vitro binding. It also does not correlate with the drop in L-citrulline uptake capacity or transceptor activation of the PKA target trehalase by the Gap1 mutant forms. Our results reveal a nutrient transceptor–Sch9–TOR axis in which Sch9 accessibility for phosphorylation by TOR may be affected by nutrient transceptor–Sch9 interaction under conditions of nutrient starvation or other environmental challenges.
FUSION PROTEIN CONSTRUCTSCOMPRISING ANTI-C3d ANTIBODY AND FACTOR H
Curtis, Michael Steven; Storek, Michael; Violette, Shelia Marie; Kalled, Susan L.; Fahnoe, Kelly C.; Huang, Cheng Ran; Stark, Ellen Garber; Taylor, Frederick Robbins; Caravella, Justin Andrew; Holers, Vernon Michael; Gambel, Phillip
Jan 2021
Abstract
Provided herein are fusion protein constructs that can bind a complement-associated antigen, comprising a targeting moiety and a complement modulator protein, or a fragment thereof or a variant thereof. The targeting moiety is an antibody or an antigen binding fragment thereof, in some examples. Further provided are methods of using the fusion protein constructs, for example, in treating complement mediation conditions.
SARS-CoV-2 Epitope Mapping on Microarrays Highlights Strong Immune-Response to N Protein Region
A workflow for rapid SARS-CoV-2 epitope discovery on peptide microarrays is herein reported. The process started with a proteome-wide screening of immunoreactivity based on the use of a high-density microarray followed by a refinement and validation phase on a restricted panel of probes using microarrays with tailored peptide immobilization through a click-based strategy. Progressively larger, independent cohorts of Covid-19 positive sera were tested in the refinement processes, leading to the identification of immunodominant regions on SARS-CoV-2 spike (S), nucleocapsid (N) protein and Orf1ab polyprotein. A summary study testing 50 serum samples highlighted an epitope of the N protein (region 155–71) providing good diagnostic performance in discriminating Covid-19 positive vs. healthy individuals. Using this epitope, 92% sensitivity and 100% specificity were reached for IgG detection in Covid-19 samples, and no cross-reactivity with common cold coronaviruses was detected. Likewise, IgM immunoreactivity in samples collected within the first month after symptoms onset showed discrimination ability. Overall, epitope 155–171 from N protein represents a promising candidate for further development and rapid implementation in serological tests.
Marker Sequences for Diagnosing and Stratifying Systemic Sclerosis Patients
Budde, Petra
Jan 2021
Abstract
The present invention relates to methods for identifying markers for systemic sclerosis (also scleroderma; SSc) and to the markers identified with the aid of this method, which can differentiate between SSc and other autoimmune diseases on the one hand and between different SSc subgroups on the other hand. The invention also relates to panels, diagnostic devices and test kits which comprise these markers, and to the use and application thereof, for example for the diagnosis, prognosis and therapy control of SSc. The invention also relates to methods for screening and for validating active substances for use in SSc.
Combinatorial Synthesis of Peptide Arrays onto a Microchip
Beyer, M.; Nesterov, A.; Block, I.; Konig, K.; Felgenhauer, T.; Fernandez, S.; Leibe, K.; Torralba, G.; Hausmann, M.; Trunk, U.; Lindenstruth, V.; Bischoff, F. R.; Stadler, V.; Breitling, F.
Arrays promise to advance biology through parallel screening for binding partners. We show the combinatorial in situ synthesis of 40,000 peptide spots per square centimeter on a microchip. Our variant Merrifield synthesis immobilizes activated amino acids as monomers within particles, which are successively attracted by electric fields generated on each pixel electrode of the chip. With all different amino acids addressed, particles are melted at once to initiate coupling. Repetitive coupling cycles should allow for the translation of whole proteomes into arrays of overlapping peptides that could be used for proteome research and antibody profiling.
