Coupling Differential Centrifugation with Exonuclease Treatment and Size Exclusion Chromatography (DIFSEC) for Purification of mtDNA from Mammalian Cells.

Direct analysis of mtDNA using PCR-free methods is limited by the presence of persistent, contaminating nucleic acids originating from the nuclear genome, even following stringent mitochondrial isolations. Here we describe a method developed in our laboratory that couples existing, commercially available mtDNA isolation protocols with exonuclease treatment and size exclusion chromatography (DIFSEC). This protocol produces highly enriched mtDNA extracts from small-scale cell culture, with near-undetectable nuclear DNA contamination.

Real-world evaluation of a novel technology for quantitative simultaneous antibody detection against multiple SARS-CoV-2 antigens in a cohort of patients presenting with COVID-19 syndrome.

An evaluation of a rapid portable gold-nanotechnology measuring SARS-CoV-2 IgM, IgA and IgG antibody concentrations against spike 1 (S1), spike 2 (S) and nucleocapsid (N) was conducted using serum samples from 74 patients tested for SARS-CoV-2 RNA on admission to hospital, and 47 historical control patients from March 2019. 59 patients were RNA(+) and 15 were RNA(-). A serum (±) classification was derived for all three antigens and a quantitative serological profile was obtained. Serum(+) was identified in 30% (95% CI 11-48) of initially RNA(-) patients, in 36% (95% CI 17-54) of RNA(+) patients before 10 days, 77% (95% CI 67-87) between 10 and 20 days and 95% (95% CI 86-100) after 21 days. The patient-level diagnostic accuracy relative to RNA(±) after 10 days displayed 88% sensitivity (95% CI 75-95) and 75% specificity (95% CI 22-99), although specificity compared with historical controls was 100% (95%CI 91-100). This study provides robust support for further evaluation and validation of this novel technology in a clinical setting and highlights challenges inherent in assessment of serological tests for an emerging disease such as COVID-19.

Accuracy and precision analysis for a biophotonic assay of C-reactive protein.

A multiplexed biophotonic assay platform has been developed using the localised particle plasmon in gold nanoparticles assembled in an array and functionalised for two assays: total IgG and C-reactive protein (CRP). A protein A/G (PAG) assay, calibrated with a NIST reference material, shows a maximum surface coverage of θmax = 7.13 ± 0.19 mRIU, equivalent to 1.5 ng mm-2 of F(ab)-presenting antibody. The CRP capture antibody has an equivalent surface binding density of θmax = 2.95 ± 0.41 mRIU indicating a 41% capture antibody availability. Free PAG binding to the functionalised anti-CRP surface shows that only 47 ± 3% of CRP capture antibodies are correctly presenting Fab regions for antigen capture. The accuracy and precision of the CRP sensor assay was assessed with 54 blood samples containing spiked CRP in the range 2-160 mg L-1. The mean accuracy was 0.42 mg L-1 with Confidence Interval (CI) at 95% from -14.7 to 13.8 mg L-1 and the precision had a Coefficient of Variation (CV) of 10.6% with 95% CI 0.9%-20.2%. These biophotonic platform performance metrics indicate a CRP assay with 2-160 mg L-1 dynamic range, performed in 8 minutes from 5 µL of whole blood without sample preparation.

A rapid and quantitative technique for assessing IgG monomeric purity, calibrated with the NISTmAb reference material.

The fraction of intact monomer in a sample (moles/moles), the monomeric purity, is measured as a quality control in therapeutic monoclonal antibodies but is often unknown in research samples and remains a major source of variation in quantitative antibody-based techniques such as immunoassay development. Here, we describe a novel multiplex technique for estimating the monomeric purity and antigen affinity of research grade antibody samples. Light scattering was used to simultaneously observe the mass of antibody binding to biosensor surfaces functionalised with antigen (revealing Fab binding kinetics) or protein A/G (PAG). Initial estimates of monomeric purity in 7 antibody samples including a therapeutic infliximab biosimilar were estimated by observing a mass deficit on the PAG surface compared to the NISTmAb standard of high monomeric purity. Monomeric purity estimates were improved in a second step by observing the mass of antigen binding to the mass of antibody on the PAG surface. The NISTmAb and infliximab biosimilar displayed tightly controlled stoichiometries for antigen binding of 1.31 ± 0.57 and 1.71 ± 0.16 (95% confidence interval)-within the theoretical limit of 1-2 antigens per antibody depending on avidity. The other antibodies in the panel displayed antigen binding stoichiometries in the range 0.06-1.15, attributed to lower monomeric purity. The monomeric purity estimates were verified by electrospray ionization mass spectrometry (ESI), the gold standard technique for structural characterization of antibodies. ESI data indicated that the NISTmAb and infliximab biosimilar samples had monomeric purity values of 93.5% and 94.7%, respectively, whilst the research grade samples were significantly lower (54-89%). Our results demonstrate rapid quality control testing for monomeric purity of antibody samples (< 15 min) which could improve the reproducibility of antibody-based experiments.

Towards salivary C-reactive protein as a viable biomarker of systemic inflammation.

C-reactive protein (CRP) is a commonly used marker of systemic inflammation, routinely measured in serum blood samples. However salivary samples offer a non-invasive and easily accessible alternative which would improve point of care (POC) testing for inflammation. Two major challenges restrict the use of saliva: the influence of the oral environment on CRP and its local production; and collecting a standardised sample given patient-dependent salivary flow rates. Here we review the reported studies of salivary CRP in humans as a potential marker of systemic inflammation and how the challenges can be overcome. Salivary CRP currently poorly reflects systemic inflammation as it does not consistently and strongly correlate with serum CRP. The mean and one standard deviation reported R2 values are 0.53 ±â€¯0.23 from 14 studies. An improved understanding of the key challenges and implemented solutions are needed to optimise salivary CRP use. Firstly, control for the effects of local oral inflammation. Screening for oral trauma is one option, however this could drastically limit the number of patients suitable for salivary CRP testing and the number of professionals able to use the POC test. Secondly, the role of a dilution biomarker is considered controlling for salivary flow rate which dilutes serum CRP by ~104; a variable and likely-patient specific factor. The ideal dilution biomarker should have many of the pharmacokinetic, sensitivity and specificity characteristics of CRP. The potential for positive acute phase protein serum amyloid A (SAA) and negative acute phase protein albumin is considered and the characteristics of any correction function discussed. Currently, however, there are no available strategies to make salivary CRP a reliable quantitative measure of serum CRP and hence POC systemic inflammation testing.

Structure of PINK1 and mechanisms of Parkinson's disease-associated mutations.

Mutations in the human kinase PINK1 (hPINK1) are associated with autosomal recessive early-onset Parkinson's disease (PD). hPINK1 activates Parkin E3 ligase activity, involving phosphorylation of ubiquitin and the Parkin ubiquitin-like (Ubl) domain via as yet poorly understood mechanisms. hPINK1 is unusual amongst kinases due to the presence of three loop insertions of unknown function. We report the structure of Tribolium castaneum PINK1 (TcPINK1), revealing several unique extensions to the canonical protein kinase fold. The third insertion, together with autophosphorylation at residue Ser205, contributes to formation of a bowl-shaped binding site for ubiquitin. We also define a novel structural element within the second insertion that is held together by a distal loop that is critical for TcPINK1 activity. The structure of TcPINK1 explains how PD-linked mutations that lie within the kinase domain result in hPINK1 loss-of-function and provides a platform for the exploration of small molecule modulators of hPINK1.

Kinetic Analysis of the Multivalent Ligand Binding Interaction between Protein A/G and IgG: A Standard System Setting.

Recombinant protein A/G (PAG) has a sequence coding for eight IgG binding sites and has enhanced interspecies affinity. High-frequency sampling of a PAG titration with IgG produces concentration profiles that are sensitive to the kinetic availability of the binding sites. The full kinetic model developed here for IgG binding sequentially to PAG shows only two distinct kinetic processes, describing an initial rapid association of two antibodies to PAG with a rate constant k-fast = (1.86 ± 0.08) × 106 M-1 s-1 and a slower antibody binding process to all remaining sites, k-slow = (1.24 ± 0.05) × 104 M-1 s-1. At equilibrium (after 1 h), the maximum IgG occupancy of PAG is 2.8 ± 0.5, conflicting with the genetic evidence of eight binding sites and suggesting significant steric hindrance of the neighboring IgG binding sites. The phosphate-buffered saline (PBS) solution defines a standard system setting, and this may be compared with other settings. The mean association rate of PAG-IgGn in the standard setting is 282 ± 20% higher than when PAG is tethered to a surface. A systems biology approach requires that a model parameter set that defines a system in a standard setting should be transferable to another system. The transfer of parameters between settings may be performed using activity coefficients characterizing an effective concentration of species in a system, ai = γici. The activity correction, γ, for the eight-site occupancy is γ = 0.35 ± 0.06, and mapping from the standard setting to the solution setting suggests γPAG-IgG = 0.4 ± 0.03. The role of activity coefficients and transferability of kinetic parameters between system settings is discussed.

Resistance exercise initiates mechanistic target of rapamycin (mTOR) translocation and protein complex co-localisation in human skeletal muscle.

The mechanistic target of rapamycin (mTOR) is a central mediator of protein synthesis in skeletal muscle. We utilized immunofluorescence approaches to study mTOR cellular distribution and protein-protein co-localisation in human skeletal muscle in the basal state as well as immediately, 1 and 3 h after an acute bout of resistance exercise in a fed (FED; 20 g Protein/40 g carbohydrate/1 g fat) or energy-free control (CON) state. mTOR and the lysosomal protein LAMP2 were highly co-localised in basal samples. Resistance exercise resulted in rapid translocation of mTOR/LAMP2 towards the cell membrane. Concurrently, resistance exercise led to the dissociation of TSC2 from Rheb and increased in the co-localisation of mTOR and Rheb post exercise in both FED and CON. In addition, mTOR co-localised with Eukaryotic translation initiation factor 3 subunit F (eIF3F) at the cell membrane post-exercise in both groups, with the response significantly greater at 1 h of recovery in the FED compared to CON. Collectively our data demonstrate that cellular trafficking of mTOR occurs in human muscle in response to an anabolic stimulus, events that appear to be primarily influenced by muscle contraction. The translocation and association of mTOR with positive regulators (i.e. Rheb and eIF3F) is consistent with an enhanced mRNA translational capacity after resistance exercise.

Time-course analysis of C3a and C5a quantifies the coupling between the upper and terminal Complement pathways in vitro.

An in vitro zymosan-activation of the Complement system, through the lectin and alternative pathways, was performed in pooled human serum over a 24h time-course. Activation was quantitatively monitored by measuring the concentration of the upper Complement pathway fragment, C3a and the terminal pathway fragment, C5a. Upper Complement showed a maximum activation of 39% and the time-to-maximum activation reduced 8-fold, as a highly non-linear function of the zymosan dose. The C3a:C5a molar ratio rose to a maximum of 1100:1, before terminal pathway activation was initiated; indicating a flux threshold. This threshold appears to be exceeded once more than 31% of C3 molecules are activated. Above this threshold, significant activation of terminal pathway was observed; reducing the molar ratio to 17:1. The C5a/C3a molar ratio was used to determine the terminal pathway activation relative to total Complement activation and ranged from 0.1-0.8%. This depicts upper Complement activation to be 49-fold larger than terminal activation, a figure consistent with the observed density of the membrane attack complex in the membrane of cells. Our results thus indicate that the relative activity of opsonisation is ~50-fold greater than membrane attack complex formation, in vitro, in the pooled serum phenotype. The results suggest a potential clinical application, where an in vitro analysis of a patient on admission, or prior to a surgical procedure, would indicate their upper Complement activation capacity, with activation of C3 measured thereafter, or post-operatively. A patient with an exhausted upper Complement capacity may be vulnerable to infections and complications, such as sepsis.

Label-free Fab and Fc affinity/avidity profiling of the antibody complex half-life for polyclonal and monoclonal efficacy screening.

A unified approach to affinity screening for Fab and Fc interactions of an antibody for its antigen and FcγR receptor has been developed. An antigen array is used for the Fab affinity and cross-reactivity screening and protein A/G proxy is the FcγR receptor. The affinities are derived using a simple 1:1 binding model with a consistent error analysis. The association and dissociation kinetics are measured over optimised times for accurate determination. The Fab/Fc affinities are derived for ten antibodies: mAb-actin (mouse), pAb-BSA (sheep), pAb-collagen V (rabbit), pAb-CRP (goat), mAb-F1 (mouse), mAbs (mouse) 7.3, 12.3, 29.3, 36.3 and 46.3 raised against LcrV in Yersinia pestis. The rate of the dissociation of antigen-antibody complexes relates directly to their immunological function as does the Fc-FcγR complex and a new half-life plot has been defined with a Fab/Fc half-life range of 17-470 min. The upper half-life value points to surface avidity. Two antibodies that are protective as an immunotherapy define a Fab half-life >250 min and an Fc half-life >50 min as characteristics of ideal interactions which can form the basis of an antibody screen for immunotherapy.

Kinetic epitope mapping of monoclonal antibodies raised against the Yersinia pestis virulence factor LcrV.

Five monoclonal antibodies, mAb7.3, mAb29.3, mAb46.3, mAb12.3 and mAb36.3, raised to the LcrV virulence factor from Yersinia pestis were characterised for their Fab affinity against the purified protein and their Fc affinity to Protein A/G as a proxy for the FcγR receptor. Kinetic measurements were performed label-free in a localised particle plasmon array reader. The Fc-ProteinA/G complex first-order half-life was determined for each antibody and fell in the range of 0.8-3.8h. The Fab first-order half-lives had ranged from 3.4 to 9.2h although two antibodies, mAb12.3 and mAb36.3, showed low affinity interactions. Competitive binding studies of mixtures of the Fab-active antibodies were performed to measure the relative binding efficiency of one antibody in the presence of the other. A geometric relative positioning of the epitopes of mAb7.3, mAb29.3 and mAb46.3 was determined based on the footprint locus of the antibody and the percentage of competitive binding. The two known protective antibodies mAb7.3 and mAb29.3 showed greater interference, indicating epitopes close to one another compared to the non-protective mAb46.3 antibody. The Fab-Fc complex half-life screen and epitope mapping are potentially useful tools in the screening of therapeutic antibodies or vaccine candidates.

A gold nanoparticle-linked glycoconjugate vaccine against Burkholderia mallei.

Burkholderia mallei are Gram-negative bacteria, responsible for the disease glanders. B. mallei has recently been classified as a Tier 1 agent owing to the fact that this bacterial species can be weaponised for aerosol release, has a high mortality rate and demonstrates multi-drug resistance. Furthermore, there is no licensed vaccine available against this pathogen. Lipopolysaccharide (LPS) has previously been identified as playing an important role in generating host protection against Burkholderia infection. In this study, we present gold nanoparticles (AuNPs) functionalised with a glycoconjugate vaccine against glanders. AuNPs were covalently coupled with one of three different protein carriers (TetHc, Hcp1 and FliC) followed by conjugation to LPS purified from a non-virulent clonal relative, B. thailandensis. Glycoconjugated LPS generated significantly higher antibody titres compared with LPS alone. Further, they improved protection against a lethal inhalation challenge of B. mallei in the murine model of infection. FROM THE CLINICAL EDITOR: Burkholderia mallei is associated with multi-drug resistance, high mortality and potentials for weaponization through aerosol inhalation. The authors of this study present gold nanoparticles (AuNPs) functionalized with a glycoconjugate vaccine against this Gram negative bacterium demonstrating promising results in a murine model even with the aerosolized form of B. Mallei.

Differential gene regulation in the Ag nanoparticle and Ag(+)-induced silver stress response in Escherichia coli: a full transcriptomic profile.

We report the whole-transcriptome response of Escherichia coli bacteria to acute treatment with silver nanoparticles (AgNPs) or silver ions [Ag(I)] as silver nitrate using gene expression microarrays. In total, 188 genes were regulated by both silver treatments, 161 were up-regulated and 27 were down-regulated. Significant regulation was observed for heat shock response genes in line with protein denaturation associated with protein structure vulnerability indicating Ag(I)-labile -SH bonds. Disruption to iron-sulphur clusters led to the positive regulation of iron-sulphur assembly systems and the expression of genes for iron and sulphate homeostasis. Further, Ag ions induced a redox stress response associated with large (>600-fold) up-regulation of the E. coli soxS transcriptional regulator gene. Ag(I) is isoelectronic with Cu(I), and genes associated with copper homeostasis were positively regulated indicating Ag(I)-activation of copper signalling. Differential gene expression was observed for the silver nitrate and AgNP silver delivery. Nanoparticle delivery of Ag(I) induced the differential regulation of 379 genes; 309 genes were uniquely regulated by silver nanoparticles and 70 genes were uniquely regulated by silver nitrate. The differential silver nanoparticle-silver nitrate response indicates that the toxic effect of labile Ag(I) in the system depends upon the mechanism of delivery to the target cell.

Glycosylation characterization of human and porcine fibrinogen proteins by lectin-binding biophotonic microarray imaging.

Lectin binding has been studied using the particle plasmon light-scattering properties of gold nanoparticles printed into an array format. Performance of the kinetic assay is evaluated from a detailed analysis of the binding of concanavalin A (ConA) and wheat germ agglutinin (WGA) to their target monosaccharides indicating affinity constants in the order of KD ∼10 nM for the lectin-monosaccharide interaction. The detection limits for the lectins following a 200 s injection time were determined as 10 ng/mL or 0.23 nM and 100 ng/mL or 0.93 nM, respectively. Subsequently, a nine-lectin screen was performed on the porcine and human fibrinogen glycoproteins. The observed spectra of lectin-protein specific binding rates result in characteristic patterns that evidently correlate with the structure of the glycans and allow one to distinguish between glycosylation of the porcine and human fibrinogens. The array technology has the potential to perform a multilectin screen of large numbers of proteins providing information on protein glycosylation and their microheterogeneity.

Growth phenotype screening of Schizosaccharomyces pombe using a Lensless microscope.

The Lensless microscope has a large field of view and allows the capture of the diffraction pattern from a large number of cells simultaneously. A simple algorithm to measure intensity changes in the Airy Disc First Fringe (ADFF) has been derived to follow the growth characteristics of the unicellular yeast Schizosaccharomyces pombe. The performance of the algorithm is calibrated using comparison between optical image and ADFF analysis of polystyrene microspheres with known dimensions and has an accuracy of 5% over all lengths above the diffraction-limited measurements. We have observed the growth characteristics of S. pombe for N=100 cells to determine the growth phenotype distributions of Length (L(t=0)) and width (W(t=0)) on arrival at the surface, lag phase adjustment to the new growth conditions (B), the length at birth, LB, and cell cycle length, tcell. The observed cell width distribution has a median width of 3.9 (±0.1) µm, as expected, but a non-normal distribution. Similarly, all growth parameters studied, L(t=0), LB and cell cycle time are phenotypes with non-normal distributions but with medians consistent with the literature values.

Whole-cell Escherichia coli-based bio-sensor assay for dual zinc oxide nanoparticle toxicity mechanisms.

A whole-cell biosensor assay for dual ZnO nanoparticle toxicity mechanisms has been developed based on the transcriptional response of Escherichia coli to: (1) Zn(2+) from ZnO nanoparticle dissolution with genes zntA (Zn(2+) efflux) and znuABC (Zn(2+) uptake); and (2) redox stress from ZnO nanoparticle photo-electron production under ultraviolet light with genes soxS and katG. Both processes occur in a dispersion of ZnO nanoparticles leading to toxicity. ZnO nanoparticle dissolution was measured independently by ICP-MS and photo-radical generation was confirmed by the stochiometric reduction of the redox dye, 2, 6-dichloroindolphenol (DCPIP). The whole-cell biosensor can detect both toxicity mechanisms and is a species-specific assay capable of discriminating between ZnO nanoparticles and the Zn(2+) dissolution product.

Measurement of the localised plasmon penetration depth for gold nanoparticles using a non-invasive bio-stacking method.

We have used the formation of a bio-probe stack with up to 24 steps on gold nanoparticle and continuous gold surfaces to characterize the penetration depth of the plasmon field in a non-invasive manner by only involving biomolecules from standard bio-assays. An alternating anti-goat rabbit IgG and anti-rabbit IgG bio-probe stack is polymerized on protein A/G functionalized gold surfaces. The change in plasmon excitation angle or light scattering decreases exponentially with each stacking step although the bio-integrity of the antibody epitope is maintained. The exponential decay in the derived kinetic parameters is attributed to the change in the penetration depth and the step size is calibrated using a commercial continuous gold surface plasmon resonance surface to be 17.5 ± 0.8 nm, consistent with the expected dimension of the antibody. The penetration depth of the gold spherical nanoparticles of diameter 90 ± 13 nm is determined to be 93 ± 10 nm.

Crystal structure of a soluble form of human CD73 with ecto-5'-nucleotidase activity.

CD73 is a dimeric ecto-5'-nucleotidase that is expressed on the exterior side of the plasma membrane. CD73 has important regulatory functions in the extracellular metabolism of certain nucleoside monophosphates, in particular adenosine monophosphate, and has been linked to a number of pathological conditions such as cancer and myocardial ischaemia. Here, we present the crystal structure of a soluble form of human soluble CD73 (sCD73) at 2.2 Å resolution, a truncated form of CD73 that retains ecto-5'-nucleotidase activity. With this structure we obtained insight into the dimerisation of CD73, active site architecture, and a sense of secondary modifications of the protein. The crystal structure reveals a conserved loop that is directly involved in the dimer-dimer interaction showing that the two subunits of the dimer are not linked by disulfide bridges. Using biophotonic microarray imaging we were able to confirm glycosylation of the enzyme and show that the enzyme is decorated with a variety of oligosaccharide structures. The crystal structure of sCD73 will aid the design of inhibitors or activator molecules for the treatment of several diseases and prove useful in explaining the possible roles of single nucleotide polymorphisms in physiology and disease.

Differential immuno-kinetic assays of allergen-specific binding for peanut allergy serum analysis.

A label-free nanoparticle array platform has been used to detect total peanut allergen-specific binding from whole serum of patients suffering from peanut allergy. The serum from 10 patients was screened against a four-allergen panel of cat and dog dander, dust mite and peanut allergen protein Ara h1. The IgE and IgG contributions to the total specific-binding protein load to Ara h1 were identified using two secondary IgG- and IgE-specific antibodies and were found to contribute less than 50 % of the total specific protein load. The total mass of IgE, IgE and the unresolved specific-binding protein ΔsBP for Ara h1 provides a new serum profile for high-RAST-grade patients 5 and 6 with the IgG/IgE ratio of 4 ± 2 and ΔsBP/IgE ratio of 17 ± 11, neither of which is protective for the small patient cohort.

Whole blood screening of antibodies using label-free nanoparticle biophotonic array platform.

A gold nanoparticle, localised plasmon array biosensor using light scattering has been employed in the detection of allergen-specific antibodies in whole blood and sera. The array sensor was functionalized with four different allergens, cat dander (Fel d1), dust mite (Der p1), peanut allergen (Ara h1) and dog dander (Can f1) and immuno-kinetic assay was performed to detect their respective anti-allergen IgG antibodies. Specific positive responses to antibodies at a concentration of 25 nM were observed for Fel d1, Der p1, and Ara h1 allergens, while the Can f1 channel served as a reference control. The sensitivity was further enhanced using a secondary anti-IgG detection antibodies to give a limit of detection of 2 nM. The results indicate the potential for nanoparticle scattering multiplexed arrays to screen unprepared blood samples at point-of-care for assays of complex samples such as the whole blood.