Search

Serological testing to evaluate antigen-specific antibodies in plasma is generally performed by rapid lateral flow test strips that lack quantitative results or by high complexity immunoassays that are time- and labor-intensive but provide semi-quantitative results. Here, we describe a novel application of biolayer interferometry for the rapid detection of antigen-specific antibody levels in plasma samples, and demonstrate its utility for quantification of SARS-CoV-2 antibodies. Our biolayer interferometry immunosorbent assay (BLI-ISA) utilizes single-use biosensors in an automated “dip-and-read” format, providing real-time optical measurements of antigen loading, plasma antibody binding, and antibody isotype detection. Complete semi-quantitative results are obtained in less than 20 min. BLI-ISA meets or exceeds the performance of high complexity methods such as Enzyme-Linked Immunosorbent Assay (ELISA) and Chemiluminescent Immunoassay. Importantly, our method can be immediately implemented on existing BLI platforms for urgent COVID-19 studies, such as serosurveillance and the evaluation of vaccine candidates. In a broader sense, BLI-ISA can be developed as a novel diagnostic platform to evaluate antibodies and other biomolecules in clinical specimens.

The biosynthetic potential of marine-sediment-derived Gram-negative bacteria is poorly understood. Sampling of California near-shore marine environments afforded isolation of numerous Gram-negative bacteria in the Proteobacteria and Bacteriodetes phyla, which were grown in the laboratory to provide extracts whose metabolites were identified by comparative analyses of LC-mass spectrometry and MSn data. Overall, we developed an assemblage of seven bacterial strains grown in five different media types designed to coax out unique secondary metabolite production as a function of varying culture conditions. The changes in metabolite production patterns were tracked using the GNPS MS2 fragmentation pattern analysis tool. A variety of nitrogen-rich metabolites were visualized from the different strains grown in different media, and strikingly, all of the strains examined produced the same new, proton-atom-deficient compound, 1-methyl-4-methylthio-β-carboline (1), C13H12N2S. Scaleup liquid culture of Achromobacter spanius (order: Burkholderiales; class: Betaproteobacteria) provided material for the final structure elucidation. The methods successfully combined in this work should stimulate future studies of molecules from marine-derived Gram-negative bacteria.

An evaluation of Indonesian plants to identify compounds with immune modulating activity revealed that the methanolic extract of an Alphonsea javanica Scheff specimen possessed selective anti-inflammatory activity in a nuclear factor-kappa B (NF-κB) luciferase and MTT assay using transfected macrophage immune (Raw264.7) cells. A high-throughput LC/MS-ELSD based library approach of the extract in combination with the NF-κB and MTT assays revealed the styryl lactone (+)-altholactone (2) was responsible for the activity. Compound 2, its acetylated derivate (+)-3-O-acetylaltholactone (3), and the major compound of this class, (+)-goniothalmin (1), were further evaluated to determine their anti-inflammatory potential in the NF-κB assay. Concentration-response studies of 1-3 indicated that only 2 possessed NF-κB based anti-inflammatory activity. Compound 2 reduced the LPS-induced NO production, phosphorylation of IκBα, and the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) using Western blot analysis. Further studies using qPCR indicated 2 reduced the expression of eight pro-inflammatory cytokines/enzymes (0.8-5.0μM) which included: COX-2, iNOS, IP-10, IL-1β, MCP-1, GCS-F, IL-6 and IFN-β. These results indicated that 2 displays broad spectrum immune modulating activity by functioning as an anti-inflammatory agent against LPS-induced NF-κB signaling. Conversely the selective cytotoxicity and in vivo anti-tumor and anti-inflammatory activity previously reported for 1 do not appear to arise from a mechanism that is linked to the NF-κB immune mediated pathway.

Opioid therapeutics are excellent analgesics, whose utility is compromised by dependence. Morphine (1) and its clinically relevant derivatives such as OxyContin (2), Vicodin (3), and Dilaudid (4) are “biased” agonists at the μ opioid receptor (OR), wherein they engage G protein signaling but poorly engage β-arrestin and the endocytic machinery. In contrast, endorphins, the endogenous peptide agonists for ORs, are potent analgesics, show reduced liability for tolerance and dependence, and engage both G protein and β-arrestin pathways as “balanced” agonists. We set out to determine if marine-derived alkaloids could serve as novel OR agonist chemotypes with a signaling profile distinct from morphine and more similar to the endorphins. Screening of 96 sponge-derived extracts followed by LC-MS-based purification to pinpoint the active compounds and subsequent evaluation of a mini library of related alkaloids identified two structural classes that modulate the ORs. These included the following: aaptamine (10), 9-demethyl aaptamine (11), demethyl (oxy)–aaptamine (12) with activity at the δ-OR (EC50: 5.1, 4.1, 2.3 μM, respectively) and fascaplysin (17), and 10-bromo fascaplysin (18) with activity at the μ-OR (EC50: 6.3, 4.2 μM respectively). An in vivo evaluation of 10 using δ-KO mice indicated its previously reported antidepressant-like effects are dependent on the δ-OR. Importantly, 17 functioned as a balanced agonist promoting both G protein signaling and β-arrestin recruitment along with receptor endocytosis similar to the endorphins. Collectively these results demonstrate the burgeoning potential for marine natural products to serve as novel lead compounds for therapeutic targets in neuroscience research.