RapidQ: A reader-free microfluidic platform for the quantitation of antibodies against the SARS-CoV-2 spike protein.

We describe RapidQ, a fast, disposable, easy-to-use microfluidic assay for the quantitation of the anti-SARS-CoV-2 spike (S) protein IgG in plasma samples. The assay utilizes antigen-coated paramagnetic microbeads, which are induced to aggregate inside the RapidQ microfluidic device in the presence of the target antibody. Aggregation occurs via interaction between the biotinylated detection antibody and polymeric streptavidin. The mobility of the beads inside the two microchannels of the device depends on their aggregation state, with larger clusters moving at higher velocities under a given liquid flow rate. One of the microchannels incorporates a permanent magnet that captures arriving beads and forms a localized constriction that retards liquid flow. Since the constriction grows faster when the beads are more aggregated, the length of the liquid column accumulated downstream from the constriction relative to that of the unconstricted control channel is proportional to the sample antibody concentration. The assay demonstrates a detection limit of 4 µg/ml of monoclonal anti-S protein antibody diluted in plasma with CV ≤ 13%, as well as negative and positive percent agreements of 100% (95% CI: 92.75%-100%) and 100% (95% CI: 80.5%-100%), respectively, when compared to a nucleic acid amplification test used to identify COVID-19 positive individuals, whose samples were collected ≥17 d from a positive PCR test. Finally, the RapidQ assay was used to monitor the kinetics of antibody responses to COVID-19 vaccination in a small study cohort.

Development of a M cell-targeted microparticulate platform, BSK02™, for oral immunization against the ovarian cancer antigen, sperm protein 17.

Although it only accounts for approximately 5% of all female cancer cases, ovarian cancer (OC) ranks as the fifth leading cause of death due to cancer in women. We have evaluated the potential of an orally administered microparticulate vaccine incorporating an immunodominant epitope peptide derived from the cancer/testis antigen sperm protein 17 (SP17) aberrantly expressed in OC, to retard the progression of the disease. The peptide antigen and the immune-stimulatory toll-like receptor 9 ligand CpG oligonucleotide were incorporated into spray dried microparticles composed of enteric and sustained release polymers together with the Aleuria aurantia lectin targeting microfold cells present in the gut-associated lymphoid tissue. These particles were administered via oral route to mice challenged week prior with SP17-expressing ID8 OC cells. Analysis of splenocytes harvested from vaccinated mice revealed strong activation of IFN-γ+/CD8+ lymphocytes in response to re-stimulation with the SP17 antigen. Moreover, vaccinated animals showed significant retardation of ascites/tumor volume in comparison to placebo-treated animals four weeks after the tumor challenge (p = 0.005). Taken together, our results suggest that vaccination against SP17 using orally administered microparticles could potentially be used as an effective consolidation strategy for OC patients with residual tumor or high probability for relapse following first-line treatments. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 29-36, 2019.

Correction: Formation of lipid/peptide tubules by IAPP and temporin B on supported lipid membranes.

Correction for 'Formation of lipid/peptide tubules by IAPP and temporin B on supported lipid membranes' by Paavo K. J. Kinnunen et al., Soft Matter, 2015, DOI: 10.1039/b925228b.

Formation of lipid/peptide tubules by IAPP and temporin B on supported lipid membranes.

The conversion of various and to is accelerated by , which are also postulated to represent targets mediating the cytotoxicity of protofibrils. Yet, our understanding of the molecular details governing -catalyzed fibrillogenesis of precursors remains limited. To obtain insight into the intricate interplay of and biophysics we have recently introduced supported bilayers (SLBs) with fluorescent analogs as model biomembranes, observed by time-lapse . Here we demonstrate that human islet () induces within minutes of its application on bilayers the expulsion of numerous flexible tubules from the . Intriguingly, these flexible tubules gradually evolve into a network of straight tubes locally attached to the substrate. Two-color imaging of the and the fluorescently labeled revealed to be distributed along the . Similar linear tubules were observed with the antimicrobial temporin B and the non-amyloidogenic rat , revealing that the above mesoscopic perturbations are not related to formation by the human . Micromanipulation experiments revealed that the linearity of the tubules was caused by tension, stretching the tubules between their points of attachment to the substrate. After longer incubation times, for SLBs containing the oxidatively modified 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (, bearing a terminal carboxyl at the end of the chain) and human (but not the other ) some of the straight transformed into highly regular helices. This is likely to reflect tension originating from an efficient aggregation of the into parallelly aligned bundles, associated with tubes containing the oxidized , possibly together with a concomitant flow of along the tubules to the immobile aggregates attaching the tubules to the substrate, these two processes cause, upon shortening of the linear scaffold, the attached excess tubule to adopt a helical morphology, coiling around the core. The above studies are in line with the multiphasic kinetics of fibrillation in the presence of oxidized containing liposomes, assessed by fluorescence enhancement. In addition to demonstrating the feasibility of SLBs as biomimetic model system for studying -assisted fibrillation, our results accentuate the role of chemical composition in modulation of different stages of this process and the associated transformation of architecture. Accordingly, changes in the chemical nature of cellular arising from pathophysiological processes such as oxidative stress may participate in the triggering amyloidogenesis as well as amplification of its detrimental effects in vivo.

A hemi-fission intermediate links two mechanistically distinct stages of membrane fission.

Fusion and fission drive all vesicular transport. Although topologically opposite, these reactions pass through the same hemi-fusion/fission intermediate, characterized by a 'stalk' in which only the outer membrane monolayers of the two compartments have merged to form a localized non-bilayer connection. Formation of the hemi-fission intermediate requires energy input from proteins catalysing membrane remodelling; however, the relationship between protein conformational rearrangements and hemi-fusion/fission remains obscure. Here we analysed how the GTPase cycle of human dynamin 1, the prototypical membrane fission catalyst, is directly coupled to membrane remodelling. We used intramolecular chemical crosslinking to stabilize dynamin in its GDP·AlF4(-)-bound transition state. In the absence of GTP this conformer produced stable hemi-fission, but failed to progress to complete fission, even in the presence of GTP. Further analysis revealed that the pleckstrin homology domain (PHD) locked in its membrane-inserted state facilitated hemi-fission. A second mode of dynamin activity, fuelled by GTP hydrolysis, couples dynamin disassembly with cooperative diminishing of the PHD wedging, thus destabilizing the hemi-fission intermediate to complete fission. Molecular simulations corroborate the bimodal character of dynamin action and indicate radial and axial forces as dominant, although not independent, drivers of hemi-fission and fission transformations, respectively. Mirrored in the fusion reaction, the force bimodality might constitute a general paradigm for leakage-free membrane remodelling.

Intrapolypeptide interactions between the GTPase effector domain (GED) and the GTPase domain form the bundle signaling element in dynamin dimers.

Biochemical and structural studies of dynamin have shown that the C-terminus of the GTPase effector domain (GED) folds back and docks onto a platform created by the N- and C-terminal α-helices of the GTPase domain to form a three-helix bundle. While cross-linking studies suggested that insect cell-expressed dynamin existed as a domain-swapped dimer, X-ray structures of protein expressed in Escherichia coli failed to detect evidence of this domain swap. Here, by cross-linking several cysteine pair replacements and analyzing cross-linked species by matrix-assisted laser desorption ionization Mega time of flight, we conclude that dynamin is not domain-swapped and that GED-GTPase domain interactions occur in cis.

Dynamin-catalyzed membrane fission requires coordinated GTP hydrolysis.

Dynamin is the most-studied membrane fission machinery and has served as a paradigm for studies of other fission GTPases; however, several critical questions regarding its function remain unresolved. In particular, because most dynamin GTPase domain mutants studied to date equally impair both basal and assembly-stimulated GTPase activities, it has been difficult to distinguish their respective roles in clathrin-mediated endocytosis (CME) or in dynamin catalyzed membrane fission. Here we compared a new dynamin mutant, Q40E, which is selectively impaired in assembly-stimulated GTPase activity with S45N, a GTP-binding mutant equally defective in both basal and assembly-stimulated GTPase activities. Both mutants potently inhibit CME and effectively recruit other endocytic accessory proteins to stalled coated pits. However, the Q40E mutant blocks at a later step than S45N, providing additional evidence that GTP binding and/or basal GTPase activities of dynamin are required throughout clathrin coated pit maturation. Importantly, using in vitro assays for assembly-stimulated GTPase activity and membrane fission, we find that the latter is much more potently inhibited by both dominant-negative mutants than the former. These studies establish that efficient fission from supported bilayers with excess membrane reservoir (SUPER) templates requires coordinated GTP hydrolysis across two rungs of an assembled dynamin collar.

Interfacial behavior of cholesterol, ergosterol, and lanosterol in mixtures with DPPC and DMPC.

Binary mixtures of cholesterol, ergosterol, and lanosterol with phosphatidylcholines differing in the length of the saturated acyl chains, viz 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (DMPC), were analyzed using a Langmuir balance for recording force-area (pi-A) and surface potential-area (psi-A) isotherms. A progressive disappearance of the liquid expanded-liquid condensed transition was observed in mixed monolayers with DPPC after the increase in the content of all three sterols. For fluid DMPC matrix, no modulation of the monolayer phase behavior due to the sterols was evident with the exception of lanosterol, for which a pronounced discontinuity between mole fractions of X = 0.3 and X = 0.75 was discernible in the compression isotherms. Condensing and expanding effects in force-area (pi-A) isotherms due to varying X(sterols) and differences in the monolayer physical state were assessed from the values for the interfacial compression moduli. Surface potential measurements support the notion that cholesterol and ergosterol, but not lanosterol, reduce the penetration of water into the lipid monolayers. Examination of the excess free energy of mixing revealed an enhanced stability of binary monolayers containing cholesterol compared to those with ergosterol or lanosterol; the differences are emphasized in the range of surface pressure values found in natural membranes.

Oxidized phospholipids as potential molecular targets for antimicrobial peptides.

The effects of oxidatively modified phospholipids on the association with model biomembranes of four antimicrobial peptides (AMPs), temporin B and L, indolicidin, and LL-37(F27W) were studied by Langmuir balance and fluorescence spectroscopy. In keeping with previous reports the negatively charged phospholipid phosphatidylglycerol (PG) enhanced the intercalation of all four peptides into lipid monolayers and liposomal bilayers under low ionic strength conditions. Interestingly, similar effect was observed for 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), a zwitterionic oxidized phospholipid bearing an aldehyde function at the end of its truncated sn-2 acyl chain. Instead, the structurally similar 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC) containing a carboxylic moiety was less efficient in promoting the membrane association of these peptides. Physiological saline reduced the binding of the above peptides to membranes containing PG, whereas interactions with PoxnoPC were found to be insensitive to ionic strength. Notably, membrane intercalation of temporin L, the most surface active of the above peptides could be into PoxnoPC containing monolayers was strongly attenuated by methoxyamine, suggesting the importance of Schiff base formation between peptide amino groups and the lipid aldehyde function. PoxnoPC and similar aldehyde bearing oxidatively modified phospholipids could represent novel molecular targets for AMPs.

Interaction of cytochrome c with 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine: evidence for acyl chain reversal.

The conformational dynamics of the oxidatively modified phospholipid 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC) were assessed by observing by fluorescence energy transfer the association of the water-soluble cationic protein cytochrome c with micelles composed of this lipid. In keeping with reversal of the azelaoyl chain so as to expose its carboxyl function on the micelle surface, cytochrome c bound avidly to the micelles. In contrast, the aldehyde group bearing 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) interacted only weakly. While the physiological significance of the above interaction is uncertain, our results demonstrate that oxidative damage alters the physical properties of lipid bilayers, involving enrichment of the polar moieties of oxidatively modified lipid chains within the membrane surface.

Oxidized phospholipids as potential novel drug targets.

The interactions of three therapeutic agents, viz. the antipsychotics HPD and CPZ, and the antineoplastic anthracycline DOX, with oxidatively modified phospholipids were studied by monitoring the quenching of fluorescence of an incorporated pyrene-labeled lipid derivative. All three drugs bound avidly to the two oxidized PCs bearing either an aldehyde or carboxylic function at the end of the sn-2 nonanoyl chain, with the highest affinity measured between CPZ and the latter oxidized lipid. Subsequent dissociation of the above drugs from the oxidized lipids by DNA, acidic phospholipids, and NaCl revealed the binding of these drugs with the aldehyde lipid to be driven by hydrophobicity similarly to their binding to lysophosphatidylcholine, whereas a significant contribution of electrostatics was evident for the lipid with the carboxylic moiety. These results connect to previous experimental data, demonstrating the induction by these drugs of oxidative stress and binding to membrane phospholipids. These issues are elaborated with reference to their clinical use and side effects.

Characterization of two oxidatively modified phospholipids in mixed monolayers with DPPC.

The properties of two oxidatively modified phospholipids viz. 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) and 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), were investigated using a Langmuir balance, recording force-area (pi-A) isotherms and surface potential psi. In mixed monolayers with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) a progressive disappearance of the liquid expanded-liquid condensed transition and film expansion was observed with increasing content of the oxidized phospholipids. The above is in agreement with fluorescence microscopy of the monolayers, which revealed an increase in the liquid expanded region of DPPC monolayers. At a critical pressure pi(s) approximately 42 mN/m both Poxo- and PazePC induced a deflection in the pi-A isotherms, which could be rationalized in terms of reorientation of the oxidatively modified acyl chains into aqueous phase (adaptation of the so-called extended conformation), followed upon further film compression by solubilization of the oxidized phospholipids into the aqueous phase. Surface potential displayed a discontinuity at the same value of area/molecule, corresponding to the loss of the oxidized phospholipids from the monolayers. Our data support the view that lipid oxidation modifies both the small-scale structural dynamics of biological membranes as well as their more macroscopic lateral organization. Accordingly, oxidatively modified lipids can be expected to influence the organization and functions of membrane associated proteins.

Characterization of the main transition of dinervonoylphosphocholine liposomes by fluorescence spectroscopy.

The structural dynamics of the main phase transition of large unilamellar dinervonoylphosphocholine (DNPC) vesicles was investigated by steady state and time-resolved fluorescence spectroscopy of the membrane incorporated fluorescent lipid analog, 1-palmitoyl-2[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC). These data were supplemented by differential scanning calorimetry (DSC) and fluorescence anisotropy measured for 1-palmitoyl-2-(3-(diphenylhexatrienyl) propanoyl)-sn-glycero-3-phosphocholine (DPHPC). The collected data displayed several discontinuities in the course of the main transition and the pretransition. The discontinuities seen in the fluorescence properties may require modification of the existing models for phospholipid main transition as a first order process. From our previous study on dipalmitoylphosphocholine (DPPC), we concluded the transition to involve a first-order process resulting in the formation of an intermediate phase, which then converts into the liquid crystalline state by a second order process. Changes in the physical properties of the DNPC matrix influencing probe behavior were similar to those reported previously for PPDPC in DPPC. In gel state DNPC [(T-T(m))<-10] the high values for excimer/monomer emission ratio (I(e)/I(m)) suggest enrichment of the probe in clusters. In this temperature range, excimer fluorescence for PPDPC (mole fraction X(PPDPC)=0.02) is described by two formation times up to (T-T(m)) approximately -10, with a gradual disappearance of the fractional intensity (I(R1)) of the shorter formation time (tau(R1)) with increasing temperature up to (T-T(m)) approximately -10. This would be consistent with the initiation of the bilayer melting at the PPDPC clusters and the subsequent dispersion of the one population of PPDPC domains. A pronounced decrement in I(e) starts at (T-T(m))=-10, continuing until T(m) is reached. No decrease was observed in fluorescence quantum yield in contrast to our previous study on DPPC/PPDPC large unilamellar vesicles (LUVs) [J. Phys. Chem., B 107 (2003) 1251], suggesting that a lack of proper hydrophobic mismatch may prevent the formation of the previously reported PPDPC superlattice. With further increase in temperature and starting at (T-T(m)) approximately -1, I(e), tau(R2), and excimer decay times (tau(D)) reach plateaus while increment in trans-->gauche isomerization continues. This behavior is in keeping with an intermediate phase existing in the temperature range -1<(T-T(m))<4 and transforming into the liquid disordered phase as a second order process, the latter being completed when (T-T(m))-->4 and corresponding to approximately 50% of the total transition enthalpy.

Evidence for the lack of a specific interaction between cholesterol and sphingomyelin.

The putative specific interaction and complex formation by sphingomyelin and cholesterol was investigated. Accordingly, low contents (1 mol % each) of fluorescently labeled derivatives of these lipids, namely 1-palmitoyl-2[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PyrPC), n-[10-(1-pyrenyl)decanoyl]sphingomyelin (PyrSM), and increasing concentrations of cholesterol (up to 5 mol %), were included in large unilamellar vesicles composed of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or 1,2-dinervonoyl-sn-glycero-3-phosphocholine (DNPC), and the excimer/monomer fluorescence emission ratio (I(e)/I(m)) was measured. In DNPC below the main phase transition, the addition of up to 5 mol % cholesterol reduced I(e)/I(m) significantly. Except for this, cholesterol had only a negligible effect in both matrices and for both probes. We then compared the efficiency of resonance energy transfer from PyrPC and PyrSM to 22-(n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3beta-ol (NBDchol). An augmenting colocalization of the latter resonance energy transfer pair with temperature was observed in a DMPC matrix below the main phase transition. In contrast, compared to PyrSM the colocalization of PyrPC with NBDchol was more efficient in the longer DNPC matrix. These results could be confirmed using 5,6-dibromo-cholestan-3beta-ol as a collisional quencher for the pyrene-labeled lipids. The results indicate lack of a specific interaction between sphingomyelin and cholesterol, and further imply that hydrophobic mismatch between the lipid constituents could provide the driving force for the cosegregation of sphingomyelin and cholesterol in fluid phospholipid bilayers of thicknesses comparable to those found for biomembranes.