A fully integrated microfluidic platform for highly sensitive analysis of immunochemical parameters.

We present a novel fully integrated centrifugal microfluidic platform for highly sensitive immunoassays in point-of-care settings. The platform consists of a disposable cartridge containing structures for assay processing, a porous membrane and all dried reagents required for the analysis. Additionally, a blister containing a washing buffer is connected to a new aliquoting structure enabling the serial aliquoting of washing buffer for repetitive bound-free separation steps. The proof-of-concept for two immunoassays is shown in the cartridge with each requiring only 30 µL of whole blood or plasma as the sample material. The detection of the cardiac marker Troponin T with a functional sensitivity of 7.55 ng L-1 (cv = 10%) within 11 minutes is shown based on samples from ten donors which were measured with six breadboard instruments to prove the platform capability for highly sensitive measurements at diagnostic relevant concentrations. Furthermore an assay for the cardiac marker NT-proBNP (five donors, six instruments) with a time-to-result of 12 minutes demonstrates that high-titer analytes (43 to 16.566 ng L-1) can be measured as well. A method comparison of our platform with a state-of-the-art laboratory analyzer proves an excellent correlation of the measured analyte concentrations. All results are obtained from injection moulded cartridges and all components of the platform are compatible for mass production.

A new immunochemistry platform for a guideline-compliant cardiac troponin T assay at the point of care: proof of principle.

BACKGROUND: A multitude of troponin assays for the point-of-care (POC) have been developed showing a lack of analytical sensitivity and precision. We present a new platform solution for the high-sensitivity detection of cardiac troponin T (cTnT) in a 30 µL whole blood sample with a turnaround time of 11 min. METHODS: The immunoassay was completely run in a ready-to-use plastic disposable, a centrifugal microfluidic disc with fully integrated reagents. After the sample application, the assay was automatically processed by separating the cellular blood components via centrifugation, followed by incubation of a defined volume from the generated plasma with the immunoreagents. The fluorescence in the signal zone of a membrane was measured after its washing for the cTnT quantitation. RESULTS: A calibration curve, measured in whole blood samples spiked with native human cTnT, was generated covering a range up to a concentration of approximately 8300 ng/L. The lower detection limit was determined to be 3.0 ng/L. At a concentration of 14 ng/L, the 99th percentile value from the high-sensitivity cardiac troponin T (hs-cTnT) assay in the Elecsys® system, the imprecision (CV) was 3.8%. A CV profile indicated that the functional sensitivity for a CV <10% was 6.8 ng/L. The assay did not show any significant cross-reaction with human skeletal troponin T. We observed an excellent correlation with the hs-TnT Elecsys® assay for 49 clinical plasma samples (r=0.9744). CONCLUSIONS: The described technology shows that an analytical performance for a highly sensitive determination of cTnT can be achieved in a POC setting.

Reduction of disulphide bonds unmasks potent antimicrobial activity of human ß-defensin 1.

Human epithelia are permanently challenged by bacteria and fungi, including commensal and pathogenic microbiota. In the gut, the fraction of strict anaerobes increases from proximal to distal, reaching 99% of bacterial species in the colon. At colonic mucosa, oxygen partial pressure is below 25% of airborne oxygen content, moreover microbial metabolism causes reduction to a low redox potential of -200 mV to -300 mV in the colon. Defensins, characterized by three intramolecular disulphide-bridges, are key effector molecules of innate immunity that protect the host from infectious microbes and shape the composition of microbiota at mucosal surfaces. Human ß-defensin 1 (hBD-1) is one of the most prominent peptides of its class but despite ubiquitous expression by all human epithelia, comparison with other defensins suggested only minor antibiotic killing activity. Whereas much is known about the activity of antimicrobial peptides in aerobic environments, data about reducing environments are limited. Herein we show that after reduction of disulphide-bridges hBD-1 becomes a potent antimicrobial peptide against the opportunistic pathogenic fungus Candida albicans and against anaerobic, Gram-positive commensals of Bifidobacterium and Lactobacillus species. Reduced hBD-1 differs structurally from oxidized hBD-1 and free cysteines in the carboxy terminus seem important for the bactericidal effect. In vitro, the thioredoxin (TRX) system is able to reduce hBD-1 and TRX co-localizes with reduced hBD-1 in human epithelia. Hence our study indicates that reduced hBD-1 shields the healthy epithelium against colonisation by commensal bacteria and opportunistic fungi. Accordingly, an intimate interplay between redox-regulation and innate immune defence seems crucial for an effective barrier protecting human epithelia.

An unfolded CH1 domain controls the assembly and secretion of IgG antibodies.

A prerequisite for antibody secretion and function is their assembly into a defined quaternary structure, composed of two heavy and two light chains for IgG. Unassembled heavy chains are actively retained in the endoplasmic reticulum (ER). Here, we show that the C(H)1 domain of the heavy chain is intrinsically disordered in vitro, which sets it apart from other antibody domains. It folds only upon interaction with the light-chain C(L) domain. Structure formation proceeds via a trapped intermediate and can be accelerated by the ER-specific peptidyl-prolyl isomerase cyclophilin B. The molecular chaperone BiP recognizes incompletely folded states of the C(H)1 domain and competes for binding to the C(L) domain. In vivo experiments demonstrate that requirements identified for folding the C(H)1 domain in vitro, including association with a folded C(L) domain and isomerization of a conserved proline residue, are essential for antibody assembly and secretion in the cell.

The structural analysis of shark IgNAR antibodies reveals evolutionary principles of immunoglobulins.

Sharks and other cartilaginous fish are the phylogenetically oldest living organisms that rely on antibodies as part of their adaptive immune system. They produce the immunoglobulin new antigen receptor (IgNAR), a homodimeric heavy chain-only antibody, as a major part of their humoral adaptive immune response. Here, we report the atomic resolution structure of the IgNAR constant domains and a structural model of this heavy chain-only antibody. We find that despite low sequence conservation, the basic Ig fold of modern antibodies is already present in the evolutionary ancient shark IgNAR domains, highlighting key structural determinants of the ubiquitous Ig fold. In contrast, structural differences between human and shark antibody domains explain the high stability of several IgNAR domains and allowed us to engineer human antibodies for increased stability and secretion efficiency. We identified two constant domains, C1 and C3, that act as dimerization modules within IgNAR. Together with the individual domain structures and small-angle X-ray scattering, this allowed us to develop a structural model of the complete IgNAR molecule. Its constant region exhibits an elongated shape with flexibility and a characteristic kink in the middle. Despite the lack of a canonical hinge region, the variable domains are spaced appropriately wide for binding to multiple antigens. Thus, the shark IgNAR domains already display the well-known Ig fold, but apart from that, this heavy chain-only antibody employs unique ways for dimerization and positioning of functional modules.

The structure of a folding intermediate provides insight into differences in immunoglobulin amyloidogenicity.

Folding intermediates play a key role in defining protein folding and assembly pathways as well as those of misfolding and aggregation. Yet, due to their transient nature, they are poorly accessible to high-resolution techniques. Here, we made use of the intrinsically slow folding reaction of an antibody domain to characterize its major folding intermediate in detail. Furthermore, by a single point mutation we were able to trap the intermediate in equilibrium and characterize it at atomic resolution. The intermediate exhibits the basic beta-barrel topology, yet some strands are distorted. Surprisingly, two short strand-connecting helices conserved in constant antibody domains assume their completely native structure already in the intermediate, thus providing a scaffold for adjacent strands. By transplanting these helical elements into beta(2)-microglobulin, a highly homologous member of the same superfamily, we drastically reduced its amyloidogenicity. Thus, minor structural differences in an intermediate can shape the folding landscape decisively to favor either folding or misfolding.

Processing of proteins by the molecular chaperone Hsp104.

The molecular chaperone Hsp104 is an AAA+ ATPase (ATPase associated with a variety of cellular activities) from yeast that catalyzes protein disaggregation. Using mutagenesis, we impaired nucleotide binding or hydrolysis in the two nucleotide-binding domains (NBD) of Hsp104 and analyzed the consequences for chaperone function by monitoring ATP hydrolysis, polypeptide binding, polypeptide processing, and disaggregation. Our results reveal that ATP binding to NBD1 serves as a central regulatory switch for the chaperone; it triggers binding of polypeptides, and stimulates ATP hydrolysis in the C-terminal NBD2 by more than two orders of magnitude, implying that ATP hydrolysis in this domain is important for disaggregation. Moreover, we show that Hsp104 actively unfolds its polypeptide substrates during processing, demonstrating that AAA+ proteins involved in disaggregation share a common threading mechanism with AAA+ proteins mediating protein unfolding/degradation.

Effect of the mGluR5-NAM Basimglurant on Behavior in Adolescents and Adults with Fragile X Syndrome in a Randomized, Double-Blind, Placebo-Controlled Trial: FragXis Phase 2 Results.

Preclinical data suggest that inhibition of the metabotropic glutamate receptor 5 (mGluR5) receptor might hold therapeutic benefits in Fragile X syndrome (FXS). Treatment of Fmr1 knockout mice with mGluR5-negative allosteric modulators (NAMs) has been reported to correct a broad range of phenotypes related to FXS. The early short-term clinical trials with mGluR5 NAMs, including basimglurant, assessing the effects in individuals with FXS, were supportive of further exploration in larger, well-controlled trials. We evaluated basimglurant, a potent and selective mGluR5 NAM, in a 12-week, double-blind, parallel-group study of 183 adults and adolescents (aged 14-50, mean 23.4 years) with FXS. Individuals with an FMR1 full mutation were randomized to placebo or one of two doses of basimglurant. The primary efficacy endpoint was the change from baseline in behavioral symptoms using the Anxiety Depression and Mood Scale (ADAMS) total score. All treatment arms showed marked behavioral improvements from baseline to week 12 with less improvement in the basimglurant 1.5 mg arm than placebo; however, basimglurant 0.5 mg was inferior to placebo in the ADAMs total score. Treatment with basimglurant was overall well-tolerated. A higher incidence of adverse events classified as psychiatric disorders were reported in patients treated with basimglurant, including three patients with hallucinations or psychosis. In this phase 2 clinical trial, basimglurant did not demonstrate improvement over placebo. Evaluation of the overall risk-benefit in younger patient populations is an important consideration for the design of potential further investigations of efficacy with this class of medications.

Conformational selection in substrate recognition by Hsp70 chaperones.

Hsp70s are molecular chaperones involved in the folding and assembly of proteins. They recognize hydrophobic amino acid stretches in their substrate binding groove. However, a detailed understanding of substrate specificity is still missing. Here, we use the endoplasmic reticulum-resident Hsp70 BiP to identify binding sites in a natural client protein. Two sites are mutually recognized and form stable Hsp70-substrate complexes. In silico and in vitro analyses revealed an extended substrate conformation as a crucial factor for interaction and show an unexpected plasticity of the substrate binding groove. The basic binding mechanism is conserved among different Hsp70s.

Substrate discrimination of the chaperone BiP by autonomous and cochaperone-regulated conformational transitions.

The endoplasmic reticulum is the site of folding, assembly and quality control for proteins of the secretory pathway. The ATP-regulated Hsp70 chaperone BiP (heavy chain-binding protein), together with cochaperones, has important roles in all of these processes. The functional cycle of Hsp70s is determined by conformational transitions that are required for substrate binding and release. Here, we used the intrinsically disordered C(H)1 domain of antibodies as an authentic substrate protein and analyzed the conformational cycle of BiP by single-molecule and ensemble Förster resonance energy transfer (FRET) measurements. Nucleotide binding resulted in concerted domain movements of BiP. Conformational transitions of the lid domain allowed BiP to discriminate between peptide and protein substrates. A major BiP cochaperone in antibody folding, ERdj3, modulated the conformational space of BiP in a nucleotide-dependent manner, placing the lid subdomain in an open, protein-accepting state.