How much (ATP) does it cost to build a trypanosome? A theoretical study on the quantity of ATP needed to maintain and duplicate a bloodstream-form Trypanosoma brucei cell.

ATP hydrolysis is required for the synthesis, transport and polymerization of monomers for macromolecules as well as for the assembly of the latter into cellular structures. Other cellular processes not directly related to synthesis of biomass, such as maintenance of membrane potential and cellular shape, also require ATP. The unicellular flagellated parasite Trypanosoma brucei has a complex digenetic life cycle. The primary energy source for this parasite in its bloodstream form (BSF) is glucose, which is abundant in the host's bloodstream. Here, we made a detailed estimation of the energy budget during the BSF cell cycle. As glycolysis is the source of most produced ATP, we calculated that a single parasite produces 6.0 x 1011 molecules of ATP/cell cycle. Total biomass production (which involves biomass maintenance and duplication) accounts for ~63% of the total energy budget, while the total biomass duplication accounts for the remaining ~37% of the ATP consumption, with in both cases translation being the most expensive process. These values allowed us to estimate a theoretical YATP of 10.1 (g biomass)/mole ATP and a theoretical [Formula: see text] of 28.6 (g biomass)/mole ATP. Flagellar motility, variant surface glycoprotein recycling, transport and maintenance of transmembrane potential account for less than 30% of the consumed ATP. Finally, there is still ~5.5% available in the budget that is being used for other cellular processes of as yet unknown cost. These data put a new perspective on the assumptions about the relative energetic weight of the processes a BSF trypanosome undergoes during its cell cycle.

Correction: How much (ATP) does it cost to build a trypanosome? A theoretical study on the quantity of ATP needed to maintain and duplicate a bloodstream-form Trypanosoma brucei cell.

[This corrects the article DOI: 10.1371/journal.ppat.1011522.].

Gradient method for accurate affinity determinations.

The value of the affinity constants (kd, ka, and KD) that are determined by label free interaction analysis methods are strongly affected by the ligand density at the sensor surface [1]. This paper outlines a new SPR-imaging method that applies a ligand density gradient enabling the analyte response to be extrapolated to Rmax = 0 µRIU. The mass transport limited region is used to determine the analyte concentration. Cumbersome optimization procedures for tuning the ligand density is prevented and surface dependent effects as rebinding, strong biphasic behavior etcetera are minimized. The method can be fully automated for e.g. accurate determination of the quality of antibodies from commercial sources.

Kinetic characterization of SPR-based biomarker assays enables quality control, calibration free measurements and robust optimization for clinical application.

Biomarker measurements are essential for the early diagnosis of complex diseases. However, many current biomarker assays lack sensitivity and multiplexing capacity, work in a narrow detection range and importantly lack real time quality control opportunities, which hampers clinical translation. In this paper, we demonstrate a toolbox to kinetically characterize a biomarker measurement assay using Surface Plasmon Resonance imaging (SPRi) with ample opportunities for real time quality control by exploiting quantitative descriptions of the various biomolecular interactions. We show an accurate prediction of SPRi measurements at both low and high concentrations of various analytes with deviations <5% between actual measurements and predicted measurement. The biphasic binding sites model was accurate for fitting the experimental curves and enables optimal detection of heterophilic antibodies, cross-reactivity, spotting irregularities and/or other confounders. The toolbox can also be used to create a (simulated) calibration curve, enabling calibration-free measurements with good recovery, it allows for easy assay optimizations, and could help bridge the gap to bring new biomarker assays to the clinic.

Nanoparticle Enhancement Cascade for Sensitive Multiplex Measurements of Biomarkers in Complex Fluids with Surface Plasmon Resonance Imaging.

There is a large unmet need for reliable biomarker measurement systems for clinical application. Such systems should meet challenging requirements for large scale use, including a large dynamic detection range, multiplexing capacity, and both high specificity and sensitivity. More importantly, these requirements need to apply to complex biological samples, which require extensive quality control. In this paper, we present the development of an enhancement detection cascade for surface plasmon resonance imaging (SPRi). The cascade applies an antibody sandwich assay, followed by neutravidin and a gold nanoparticle enhancement for quantitative biomarker measurements in small volumes of complex fluids. We present a feasibility study both in simple buffers and in spiked equine synovial fluid with four cytokines, IL-1ß, IL-6, IFN-γ, and TNF-α. Our enhancement cascade leads to an antibody dependent improvement in sensitivity up to 40 000 times, resulting in a limit of detection as low as 50 fg/mL and a dynamic detection range of more than 7 logs. Additionally, measurements at these low concentrations are highly reliable with intra- and interassay CVs between 2% and 20%. We subsequently showed this assay is suitable for multiplex measurements with good specificity and limited cross-reactivity. Moreover, we demonstrated robust detection of IL-6 and IL-1ß in spiked undiluted equine synovial fluid with small variation compared to buffer controls. In addition, the availability of real time measurements provides extensive quality control opportunities, essential for clinical applications. Therefore, we consider this method is suitable for broad application in SPRi for multiplex biomarker detection in both research and clinical settings.

Surface Plasmon Resonance is an Analytically Sensitive Method for Antigen Profiling of Extracellular Vesicles.

BACKGROUND: Identification, enumeration, and characterization of extracellular vesicles (EVs) are hampered by the small size of EVs, a low refractive index, and low numbers of antigens on their surface. METHODS: We investigated the potential of a 48-multiplex surface plasmon resonance imaging (SPRi) system to perform EV phenotyping. Antigen surface density of 11 antigens was measured on the human breast cancer cell lines HS578T, MCF7, and SKBR3 and their EVs by use of both SPRi and the widely used flow cytometry (FCM). RESULTS: For cells, the SPRi and FCM signals for antigen exposure correlated (RHS578T cells2 = 0.66, RMCF7 cells2 = 0.78, RSKBR3 cells2 = 0.60). With regard to EVs, SPRi detected 31 out of 33 tested antibody-EV pairs, whereas our flow cytometer detected 5 antibody-EV pairs because of high blank and isotype control signals. For HS578T-derived EVs, the SPRi and FCM signals correlated (R2HS578T EVs = 0.98). However, on MCF7- and SKBR3-derived EVs, insufficient antigens were detected by our flow cytometer. To confirm that the SPRi responses correlated with mean antigen density on EVs, the SPRi responses of EVs were correlated with antigen density on parental cells as measured by FCM (RHS578T2 = 0.77, RMCF72 = 0.49, RSKBR32 = 0.52). CONCLUSIONS: SPRi responses correlate with mean antigen density. Moreover, SPRi detects lower antigen-exposure levels than FCM because SPRi measures an ensemble of EVs binding to the sensor surface, whereas FCM detects antigens of single EV.

C-reactive protein enhances IgG-mediated phagocyte responses and thrombocytopenia.

Immune-mediated platelet destruction is most frequently caused by allo- or autoantibodies via Fcγ receptor-dependent phagocytosis. Disease severity can be predicted neither by antibody isotype nor by titer, indicating that other factors play a role. Here we show that the acute phase protein C-reactive protein (CRP), a ligand for Fc receptors on phagocytes, enhances antibody-mediated platelet destruction by human phagocytes in vitro and in vivo in mice. Without antiplatelet antibodies, CRP was found to be inert toward platelets, but it bound to phosphorylcholine exposed after oxidation triggered by antiplatelet antibodies, thereby enhancing platelet phagocytosis. CRP levels were significantly elevated in patients with allo- and autoantibody-mediated thrombocytopenias compared with healthy controls. Within a week, intravenous immunoglobulin treatment in children with newly diagnosed immune thrombocytopenia led to significant decrease of CRP levels, increased platelet numbers, and clinically decreased bleeding severity. Furthermore, the higher the level of CRP at diagnosis, the longer it took before stable platelet counts were reached. These data suggest that CRP amplifies antibody-mediated platelet destruction and may in part explain the aggravation of thrombocytopenia on infections. Hence, targeting CRP could offer new therapeutic opportunities for these patients.

Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling.

The adult stem cell marker Lgr5 and its relative Lgr4 are often co-expressed in Wnt-driven proliferative compartments. We find that conditional deletion of both genes in the mouse gut impairs Wnt target gene expression and results in the rapid demise of intestinal crypts, thus phenocopying Wnt pathway inhibition. Mass spectrometry demonstrates that Lgr4 and Lgr5 associate with the Frizzled/Lrp Wnt receptor complex. Each of the four R-spondins, secreted Wnt pathway agonists, can bind to Lgr4, -5 and -6. In HEK293 cells, RSPO1 enhances canonical WNT signals initiated by WNT3A. Removal of LGR4 does not affect WNT3A signalling, but abrogates the RSPO1-mediated signal enhancement, a phenomenon rescued by re-expression of LGR4, -5 or -6. Genetic deletion of Lgr4/5 in mouse intestinal crypt cultures phenocopies withdrawal of Rspo1 and can be rescued by Wnt pathway activation. Lgr5 homologues are facultative Wnt receptor components that mediate Wnt signal enhancement by soluble R-spondin proteins. These results will guide future studies towards the application of R-spondins for regenerative purposes of tissues expressing Lgr5 homologues.

Modeling single cell antibody excretion on a biosensor.

We simulated, using Comsol Multiphysics, the excretion of antibodies by single hybridoma cells and their subsequent binding on a surface plasmon resonance imaging (SPRi) sensor. The purpose was to confirm that SPRi is suitable to accurately quantify antibody (anti-EpCAM) excretion. The model showed that antibody loss by diffusion away from the sensor was less than 1%. Unexpectedly, more than 99% of the excreted antibodies were captured on the sensor. These data prove the remarkable phenomenon that the SPRi output of cellular antibody excretion and its subsequent binding, performed under the conditions described here, is directly usable for quantification of single cell antibody production rates.

Interpolation method for accurate affinity ranking of arrayed ligand-analyte interactions.

The values of the affinity constants (kd, ka, and KD) that are determined by label-free interaction analysis methods are affected by the ligand density. This article outlines a surface plasmon resonance (SPR) imaging method that yields high-throughput globally fitted affinity ranking values using a 96-plex array. A kinetic titration experiment without a regeneration step has been applied for various coupled antibodies binding to a single antigen. Globally fitted rate (kd and ka) and dissociation equilibrium (KD) constants for various ligand densities and analyte concentrations are exponentially interpolated to the KD at Rmax = 100 RU response level (KD(R100)).

Quantification of antibody production of individual hybridoma cells by surface plasmon resonance imaging.

Surface plasmon resonance imaging (SPRi) is most frequently used for the label-free measurement of biomolecular interactions. Here we explore the potential of SPRi to measure antibody production of individual hybridoma cells. As a model system, cells from a hybridoma, producing monoclonal antibodies recognizing epithelial cell adhesion molecule (EpCAM), were used. Recombinant human EpCAM protein was immobilized on an SPR sensor and hybridoma cells were introduced into an IBIS MX96 SPR imager and the SPRi response was followed for 10h. SPRi responses were detected on the spots of the sensor only where ligands of the produced antibody were present. By measuring the SPRi signals on individual cells the antibody production of the individual cells was measured and production rates were calculated. For 53 single EpCAM hybridoma cells the production ranged from 0.16 to 11.95 pg (mean 2.96p g per cell, SD 2.51) over a period of 10 h. Antibody excretion per cell per hour ranged from 0.02 to 1.19 pg (mean 0.30, SD 0.25). Here we demonstrate for the first time that antibody production of individual cells can be measured and quantified by SPRi, opening a new avenue for measuring excretion products of individual cells.

Label-free cell profiling.

A surface plasmon resonance (SPR) array imaging method is outlined for label-free cell profiling. Red blood cells (RBCs) were injected into a flow chamber on top of a spotted sensor surface. Spots contained antibodies to various RBC membrane antigens. A typical sensorgram showed an initial response corresponding to cell sedimentation (S) followed by a specific upward response (T) corresponding to specific binding of cells during a critical wash step. The full analysis cycle for RBC profiling was less than 6 min. The sensor surface could be regenerated at least 100 times, allowing the determination of a cell surface antigen profile of RBCs.

Use of tissue expansion to facilitate liver and small bowel transplant in young children with contracted abdominal cavities.

Liver and small bowel transplant is an established treatment for infants with IFALD. Despite organ reduction techniques, mortality on the waiting list remains high due to shortage of size-matched pediatric donors. Small abdominal cavity volume due to previous intestinal resection poses a significant challenge to achieve abdominal closure post-transplant. Seven children underwent tissue expansion of abdominal skin prior to multiorgan transplant. In total, 17 tissue expanders were placed subcutaneously in seven children. All seven subjects underwent re-exploration to deal with complications: hematoma, extrusion, infection, or port related. Three expanders had to be removed. Four children went on to have successful combined liver and small bowel transplant. Two children died on the waiting list of causes not related to the expander and one child died from sepsis attributed to an infected expander. Tissue expansion can generate skin to facilitate closure of abdomen post-transplant, thus allowing infants with small abdominal volumes to be considered for transplant surgery. Tissue expansion in children with end-stage liver disease and portal hypertension is associated with a very high complication rate and needs to be closely monitored during the expansion process.

Method for estimating the single molecular affinity.

Affinity constants (k(d), k(a), and K(D)) can be determined by methods that apply immobilized ligands such as immunoassays and label-free biosensor technologies. This article outlines a new surface plasmon resonance (SPR) array imaging method that yields affinity constants that can be considered as the best estimate of the affinity constant for single biomolecular interactions. Calculated rate (k(d) and k(a)) and dissociation equilibrium (K(D)) constants for various ligand densities and analyte concentrations are extrapolated to the K(D) at the zero response level (K(D)(R0)). By applying this method to an LGR5-exo-Fc-RSPO1-FH interaction couple, the K(D)(R0) was determined as 3.1 nM.

Porous surfaces: stability and recovery of coronaviruses.

The role of indirect contact in the transmission of SARS-CoV-2 is not clear. SARS-CoV-2 persists on dry surfaces for hours to days; published studies have largely focused on hard surfaces with less research being conducted on different porous surfaces, such as textiles. Understanding the potential risks of indirect transmission of COVID-19 is useful for settings where there is close contact with textiles, including healthcare, manufacturing and retail environments. This article aims to review current research on porous surfaces in relation to their potential as fomites of coronaviruses compared to non-porous surfaces. Current methodologies for assessing the stability and recovery of coronaviruses from surfaces are also explored. Coronaviruses are often less stable on porous surfaces than non-porous surfaces, for example, SARS-CoV-2 persists for 0.5 h-5 days on paper and 3-21 days on plastic; however, stability is dependent on the type of surface. In particular, the surface properties of textiles differ widely depending on their construction, leading to variation in the stability of coronaviruses, with longer persistence on more hydrophobic materials such as polyester (1-3 days) compared to highly absorbent cotton (2 h-4 days). These findings should be considered where there is close contact with potentially contaminated textiles.

Presence and strength of binding of IgM, IgG and IgA antibodies against SARS-CoV-2 during CoViD-19 infection.

Surface Plasmon Resonance imaging (SPRi) was used to determine the presence and strength of binding of IgG, IgM and IgA against the Receptor Binding Domain (RBD) of SARS-CoV-2 in sera of 119 CoViD-19 patients. The SPRi assay measures the antibody isotype levels and the strength of binding to the RBD of ultimate 384 patient samples in one run. It turns out that during the course of the disease, the IgG levels and strength of binding increased while generally the IgM and IgA levels go down. Recovered patients all show high strength of binding of the IgG type to the RBD protein. The anti-RBD immunoglobulins SPRi assay provides additional insights in the immune status of patients recovering from CoViD-19 and this new method can furthermore be applied for the assessment of the quality of the immune reaction of healthy individuals to SARS-CoV-2 in vaccination programs.

High throughput surface plasmon resonance imaging method for clinical detection of presence and strength of binding of IgM, IgG and IgA antibodies against SARS-CoV-2 during CoViD-19 infection.

Surface Plasmon Resonance imaging (SPRi) was used to determine the presence and strength of binding of IgG, IgM and IgA against the Receptor Binding Domain (RBD) of SARS-CoV-2 in sera of 102 CoViD-19 and non-CoViD-19 patients. The SPRi assay simultaneously measures the antibody isotype levels and the strength of binding to the RBD of ultimate 384 patient samples in one run. It turns out that during the course of the disease, the IgG levels and strength of binding increased while generally the IgM and IgA levels go down. Recovered patients all show high strength of binding of the IgG type to the RBD protein. The anti-RBD immunoglobulins SPRi assay provides additional insights in the immune status of patients recovering from CoViD-19. This new high throughput method can be applied for the assessment of the quality of the immune reaction of healthy individuals to SARS-CoV-2 and its mutants in vaccination programs.•Surface Plasmon Resonance imaging is an unprecedented technology for high throughput screening of antibody profiling of CoViD19 patients.•Fingerprinting of isotypes IgM, IgG and IgA can be performed for 384 patients in one run.•An affinity maturation effect was shown for patients recovering from CoViD19.

Electrokinetic label-free screening chip: a marriage of multiplexing and high throughput analysis using surface plasmon resonance imaging.

We present an electrokinetic label-free biomolecular screening chip (Glass/PDMS) to screen up to 10 samples simultaneously using surface plasmon resonance imaging (iSPR). This approach reduces the duration of an experiment when compared to conventional experimental methods. This new device offers a high degree of parallelization not only for analyte samples, but also for multiplex analyte interactions where up to 90 ligands are immobilized on the sensing surface. The proof of concept has been demonstrated with well-known biomolecular interactant pairs. The new chip can be used for high throughput screening applications and kinetics parameter extraction, simultaneously, of interactant-protein complex formation.

Electrokinetic lab-on-a-biochip for multi-ligand/multi-analyte biosensing.

We present a simple electrokinetic lab-on-a-biochip (EKLB) with four microchannels integrated with a surface plasmon resonance imaging (iSPR) label-free biosensor that is operated using a single electrical voltage for the simultaneous transport of reagents in all microchannels without conventional fluidic plumbing. We demonstrate the utility of the simple approach with various biosensing experiments, including single injection kinetics (multiple varied ligand densities and single analyte concentration), one shot kinetics (single ligand densities and multiple varied analyte concentrations), and multi-ligand/multianalyte detection. In all cases, the binding kinetics and affinity were extracted using a conventional 1:1 interaction model. Since the reagent transport is done with a single electrical voltage source, scaling up to hundreds to thousands of simultaneous experiments is straightforward.

IgG Subclass Determines Suppression Versus Enhancement of Humoral Alloimmunity to Kell RBC Antigens in Mice.

It has long been appreciated that immunoglobulins are not just the effector endpoint of humoral immunity, but rather have a complex role in regulating antibody responses themselves. Donor derived anti-RhD IgG has been used for over 50 years as an immunoprophylactic to prevent maternal alloimmunization to RhD. Although anti-RhD has dramatically decreased rates of hemolytic disease of the fetus and newborn (for the RhD alloantigen), anti-RhD also fails in some cases, and can even paradoxically enhance immune responses in some circumstances. Attempts to generate a monoclonal anti-RhD have largely failed, with some monoclonals suppressing less than donor derived anti-RhD and others enhancing immunity. These difficulties likely result, in part, because the mechanism of anti-RhD remains unclear. However, substantial evidence exists to reject the common explanations of simple clearance of RhD + RBCs or masking of antigen. Donor derived anti-RhD is a mixture of 4 different IgG subtypes. To the best of our knowledge an analysis of the role different IgG subtypes play in immunoregulation has not been carried out; and, only IgG1 and IgG3 have been tested as monoclonals. Multiple attempts to elicit alloimmune responses to human RhD epitopes in mice have failed. To circumvent this limitation, we utilize a tractable animal model of RBC alloimmunization using the human Kell glycoprotein as an antigen to test the effect of IgG subtype on immunoregulation by antibodies to RBC alloantigens. We report that the ability of an anti-RBC IgG to enhance, suppress (at the level of IgM responses), or have no effect is a function of the IgG subclass in this model system.