Aminopeptidase N/CD13 Crosslinking Promotes the Activation and Membrane Expression of Integrin CD11b/CD18.

The ß2 integrin CD11b/CD18, also known as complement receptor 3 (CR3), and the moonlighting protein aminopeptidase N (CD13), are two myeloid immune receptors with overlapping activities: adhesion, migration, phagocytosis of opsonized particles, and respiratory burst induction. Given their common functions, shared physical location, and the fact that some receptors can activate a selection of integrins, we hypothesized that CD13 could induce CR3 activation through an inside-out signaling mechanism and possibly have an influence on its membrane expression. We revealed that crosslinking CD13 on the surface of human macrophages not only activates CR3 but also influences its membrane expression. Both phenomena are affected by inhibitors of Src, PLCγ, Syk, and actin polymerization. Additionally, after only 10 min at 37 °C, cells with crosslinked CD13 start secreting pro-inflammatory cytokines like interferons type 1 and 2, IL-12p70, and IL-17a. We integrated our data with a bioinformatic analysis to confirm the connection between these receptors and to suggest the signaling cascade linking them. Our findings expand the list of features of CD13 by adding the activation of a different receptor via inside-out signaling. This opens the possibility of studying the joint contribution of CD13 and CR3 in contexts where either receptor has a recognized role, such as the progression of some leukemias.

Thermodynamic analysis of an entropically driven, high-affinity nanobody-HIV p24 interaction.

Protein-protein interactions are fundamental to life processes. Complementary computational, structural, and biophysical studies of these interactions enable the forces behind their specificity and strength to be understood. Antibody fragments such as single-chain antibodies have the specificity and affinity of full antibodies but a fraction of their size, expediting whole molecule studies and distal effects without exceeding the computational capacity of modeling systems. We previously reported the crystal structure of a high-affinity nanobody 59H10 bound to HIV-1 capsid protein p24 and deduced key interactions using all-atom molecular dynamics simulations. We studied the properties of closely related medium (37E7) and low (48G11) affinity nanobodies, to understand how changes of three (37E7) or one (48G11) amino acids impacted these interactions; however, the contributions of enthalpy and entropy were not quantified. Here, we report the use of qualitative and quantitative experimental and in silico approaches to separate the contributions of enthalpy and entropy. We used complementary circular dichroism spectroscopy and molecular dynamics simulations to qualitatively delineate changes between nanobodies in isolation and complexed with p24. Using quantitative techniques such as isothermal titration calorimetry alongside WaterMap and Free Energy Perturbation protocols, we found the difference between high (59H10) and medium (37E7) affinity nanobodies on binding to HIV-1 p24 is entropically driven, accounted for by the release of unstable waters from the hydrophobic surface of 59H10. Our results provide an exemplar of the utility of parallel in vitro and in silico studies and highlight that differences in entropic interactions between amino acids and water molecules are sufficient to drive orders of magnitude differences in affinity.

Ultra-sensitive molecular detection of gene fusions from RNA using ASPYRE.

BACKGROUND: RNA is a critical analyte for unambiguous detection of actionable mutations used to guide treatment decisions in oncology. Currently available methods for gene fusion detection include molecular or antibody-based assays, which suffer from either being limited to single-gene targeting, lack of sensitivity, or long turnaround time. The sensitivity and predictive value of next generation sequencing DNA-based assays to detect fusions by sequencing intronic regions is variable, due to the extensive size of introns. The required depth of sequencing and input nucleic acid required can be prohibitive; in addition it is not certain that predicted gene fusions are actually expressed. RESULTS: Herein we describe a method based on pyrophosphorolysis to include detection of gene fusions from RNA, with identical assay steps and conditions to detect somatic mutations in DNA [1], permitting concurrent assessment of DNA and RNA in a single instrument run. CONCLUSION: The limit of detection was under 6 molecules/ 6 µL target volume. The workflow and instrumentation required are akin to PCR assays, and the entire assay from extracted nucleic acid to sample analysis can be completed within a single day.

Single-copy detection of somatic variants from solid and liquid biopsy.

Accurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants.

Spin-enhanced nanodiamond biosensing for ultrasensitive diagnostics.

The quantum spin properties of nitrogen-vacancy defects in diamond enable diverse applications in quantum computing and communications1. However, fluorescent nanodiamonds also have attractive properties for in vitro biosensing, including brightness2, low cost3 and selective manipulation of their emission4. Nanoparticle-based biosensors are essential for the early detection of disease, but they often lack the required sensitivity. Here we investigate fluorescent nanodiamonds as an ultrasensitive label for in vitro diagnostics, using a microwave field to modulate emission intensity5 and frequency-domain analysis6 to separate the signal from background autofluorescence7, which typically limits sensitivity. Focusing on the widely used, low-cost lateral flow format as an exemplar, we achieve a detection limit of 8.2 × 10-19 molar for a biotin-avidin model, 105 times more sensitive than that obtained using gold nanoparticles. Single-copy detection of HIV-1 RNA can be achieved with the addition of a 10-minute isothermal amplification step, and is further demonstrated using a clinical plasma sample with an extraction step. This ultrasensitive quantum diagnostics platform is applicable to numerous diagnostic test formats and diseases, and has the potential to transform early diagnosis of disease for the benefit of patients and populations.

Minor groove binder modification of widely used TaqMan hydrolysis probe for detection of dengue virus reduces risk of false-negative real-time PCR results for serotype 4.

Dengue is a vector-transmitted viral infection that is a significant cause of morbidity and mortality in humans worldwide, with over 50 million apparent cases and a fatality rate of 2.5 % of 0.5 million severe cases per annum in recent years. Four serotypes are currently co-circulating. Diagnosis of infection may be by polymerase chain reaction, serology or rapid antigen test for NS1. Both pan-serotype and serotype-specific genome detection assays have been described, however, achieving adequate sensitivity with pan-serotype assays has been challenging. Indeed, as we show here, inspection of components and cycling parameters of a pan-serotype RT-qPCR assay in use in laboratories worldwide revealed insufficient probe stability to accommodate potential nucleotide mismatches, resulting in false-negatives. A minor-groove binder (MGB)-modified version of the probe was designed and its performance compared with that of the original probe in 32 samples. Eight of the samples were undetected by the original probe but detected by the MGB modified probe and six out of seven of these that could be serotyped belonged to serotype 4. Sequencing of the region targeted by the probe in these samples revealed two mismatches which were also universally present in all other serotype 4 sequences in a public database. We therefore recommend adoption of this MGB modification in order to reduce the risk of false-negative results, especially with dengue serotype 4 infections.

p24 revisited: a landscape review of antigen detection for early HIV diagnosis.

: Despite major advances in HIV testing, early detection of infection at the point of care (PoC) remains a key challenge. Although rapid antibody PoC and laboratory-based nucleic acid amplification tests dominate the diagnostics market, the viral capsid protein p24 is recognized as an alternative early virological biomarker of infection. However, the detection of ultra-low levels of p24 at the PoC has proven challenging. Here we review the landscape of p24 diagnostics to identify knowledge gaps and barriers and help shape future research agendas. Five hundred and seventy-four research articles to May 2018 that propose or evaluate diagnostic assays for p24 were identified and reviewed. We give a brief history of diagnostic development, and the utility of p24 as a biomarker in different populations such as infants, the newly infected, those on preexposure prophylaxis and self-testers. We review the performance of commercial p24 assays and consider elements such as immune complex disruption, resource-poor settings, prevalence, and assay antibodies. Emerging and ultrasensitive assays are reviewed and show a number of promising approaches but further translation has been limited. We summarize studies on the health economic benefits of using antigen testing. Finally, we speculate on the future uses of high-performance p24 assays, particularly, if available in self-test format.

Quantifying Biomolecular Binding Constants using Video Paper Analytical Devices.

A novel ultra-low-cost biochemical analysis platform to quantify protein dissociation binding constants and kinetics using paper microfluidics is reported. This approach marries video imaging with one of humankind's oldest materials: paper, requiring no large, expensive laboratory equipment, complex microfluidics or external power. Temporal measurements of nanoparticle-antibody conjugates binding on paper is found to follow the Langmuir Adsorption Model. This is exploited to measure a series of antibody-antigen dissociation constants on paper, showing excellent agreement with a gold-standard benchtop interferometer. The concept is demonstrated with a camera and low-end smartphone, 500-fold cheaper than the reference method, and can be multiplexed to measure ten reactions in parallel. These findings will help to widen access to quantitative analytical biochemistry, for diverse applications spanning disease diagnostics, drug discovery, and environmental analysis in resource-limited settings.

Self-Swabbing for Virological Confirmation of Influenza-Like Illness Among an Internet-Based Cohort in the UK During the 2014-2015 Flu Season: Pilot Study.

BACKGROUND: Routine influenza surveillance, based on laboratory confirmation of viral infection, often fails to estimate the true burden of influenza-like illness (ILI) in the community because those with ILI often manage their own symptoms without visiting a health professional. Internet-based surveillance can complement this traditional surveillance by measuring symptoms and health behavior of a population with minimal time delay. Flusurvey, the UK's largest crowd-sourced platform for surveillance of influenza, collects routine data on more than 6000 voluntary participants and offers real-time estimates of ILI circulation. However, one criticism of this method of surveillance is that it is only able to assess ILI, rather than virologically confirmed influenza. OBJECTIVE: We designed a pilot study to see if it was feasible to ask individuals from the Flusurvey platform to perform a self-swabbing task and to assess whether they were able to collect samples with a suitable viral content to detect an influenza virus in the laboratory. METHODS: Virological swabbing kits were sent to pilot study participants, who then monitored their ILI symptoms over the influenza season (2014-2015) through the Flusurvey platform. If they reported ILI, they were asked to undertake self-swabbing and return the swabs to a Public Health England laboratory for multiplex respiratory virus polymerase chain reaction testing. RESULTS: A total of 700 swab kits were distributed at the start of the study; from these, 66 participants met the definition for ILI and were asked to return samples. In all, 51 samples were received in the laboratory, 18 of which tested positive for a viral cause of ILI (35%). CONCLUSIONS: This demonstrated proof of concept that it is possible to apply self-swabbing for virological laboratory testing to an online cohort study. This pilot does not have significant numbers to validate whether Flusurvey surveillance accurately reflects influenza infection in the community, but highlights that the methodology is feasible. Self-swabbing could be expanded to larger online surveillance activities, such as during the initial stages of a pandemic, to understand community transmission or to better assess interseasonal activity.

Ultra-rapid, sensitive and specific digital diagnosis of HIV with a dual-channel SAW biosensor in a pilot clinical study.

Despite widened access to HIV testing, around half of those infected worldwide are unaware of their HIV-positive status and linkage to care remains a major challenge. Current rapid HIV tests are typically analogue risking incorrect interpretation, no facile electronic data capture, poor linkage to care and data loss for public health. Smartphone-connected diagnostic devices have potential to dramatically improve access to testing and patient retention with electronic data capture and wireless connectivity. We report a pilot clinical study of surface acoustic wave biosensors based on low-cost components found in smartphones to diagnose HIV in 133 patient samples. We engineered a small, portable, laboratory prototype and dual-channel biochips, with in-situ reference control coating and miniaturised configuration, requiring only 6 µL plasma. The dual-channel biochips were functionalized by ink-jet printing with capture coatings to detect either anti-p24 or anti-gp41 antibodies, and a reference control. Biochips were tested with 31 plasma samples from patients with HIV, and 102 healthy volunteers. SH-SAW biosensors showed excellent sensitivity, specificity, low sample volumes and rapid time to result, and were benchmarked to commercial rapid HIV tests. Testing for individual biomarkers found sensitivities of 100% (anti-gp41) and 64.5% (anti-p24) (combined sensitivity of 100%) and 100% specificity, within 5 min. All positive results were recorded within 60 s of sample addition with an electronic readout. Next steps will focus on a smartphone-connected device prototype and user-friendly app interface for larger scale evaluation and field studies, towards our ultimate goal of a new generation of affordable, connected point-of-care HIV tests.

Platinum Nanocatalyst Amplification: Redefining the Gold Standard for Lateral Flow Immunoassays with Ultrabroad Dynamic Range.

Paper-based lateral flow immunoassays (LFIAs) are one of the most widely used point-of-care (PoC) devices; however, their application in early disease diagnostics is often limited due to insufficient sensitivity for the requisite sample sizes and the short time frames of PoC testing. To address this, we developed a serum-stable, nanoparticle catalyst-labeled LFIA with a sensitivity surpassing that of both current commercial and published sensitivities for paper-based detection of p24, one of the earliest and most conserved biomarkers of HIV. We report the synthesis and characterization of porous platinum core-shell nanocatalysts (PtNCs), which show high catalytic activity when exposed to complex human blood serum samples. We explored the application of antibody-functionalized PtNCs with strategically and orthogonally modified nanobodies with high affinity and specificity toward p24 and established the key larger nanoparticle size regimes needed for efficient amplification and performance in LFIA. Harnessing the catalytic amplification of PtNCs enabled naked-eye detection of p24 spiked into sera in the low femtomolar range (ca. 0.8 pg·mL-1) and the detection of acute-phase HIV in clinical human plasma samples in under 20 min. This provides a versatile absorbance-based and rapid LFIA with sensitivity capable of significantly reducing the HIV acute phase detection window. This diagnostic may be readily adapted for detection of other biomolecules as an ultrasensitive screening tool for infectious and noncommunicable diseases and can be capitalized upon in PoC settings for early disease detection.

Towards an ultra-rapid smartphone- connected test for infectious diseases.

The development is reported of an ultra-rapid, point-of-care diagnostic device which harnesses surface acoustic wave (SAW) biochips, to detect HIV in a finger prick of blood within 10 seconds (sample-in-result-out). The disposable quartz biochip, based on microelectronic components found in every consumer smartphone, is extremely fast because no complex labelling, amplification or wash steps are needed. A pocket-sized control box reads out the SAW signal and displays results electronically. High analytical sensitivity and specificity are found with model and real patient blood samples. The findings presented here open up the potential of consumer electronics to cut lengthy test waiting times, giving patients on the spot access to potentially life-saving treatment and supporting more timely public health interventions to prevent disease transmission.

Unravelling the Molecular Basis of High Affinity Nanobodies against HIV p24: In Vitro Functional, Structural, and in Silico Insights.

Preventing the spread of infectious diseases remains an urgent priority worldwide, and this is driving the development of advanced nanotechnology to diagnose infections at the point of care. Herein, we report the creation of a library of novel nanobody capture ligands to detect p24, one of the earliest markers of HIV infection. We demonstrate that these nanobodies, one tenth the size of conventional antibodies, exhibit high sensitivity and broad specificity to global HIV-1 subtypes. Biophysical characterization indicates strong 690 pM binding constants and fast kinetic on-rates, 1 to 2 orders of magnitude better than monoclonal antibody comparators. A crystal structure of the lead nanobody and p24 was obtained and used alongside molecular dynamics simulations to elucidate the molecular basis of these enhanced performance characteristics. They indicate that binding occurs at C-terminal helices 10 and 11 of p24, a negatively charged region of p24 complemented by the positive surface of the nanobody binding interface involving CDR1, CDR2, and CDR3 loops. Our findings have broad implications on the design of novel antibodies and a wide range of advanced biomedical applications.

Tuneable plasmonic gold dendrimer nanochains for sensitive disease detection.

We report the development of a tuneable plasmonic nanochain immunoassay with increased sensitivity over traditional monodisperse nanoparticle lateral flow tests. Our approach takes advantage of the unique self-assembling properties of polyamidoamine dendrimers with gold nanoparticles in aqueous media to create one-dimensional nanochains, with a distinct red to blue colour change, attributable to a longitudinal plasmon resonance, which can be readily detected by eye and a digital camera. We optimise and characterise nanochain formation and stability using UV-visible spectroscopy, transmission electron microscopy and dynamic light scattering. As a proof-of-principle we focus on the application of nanochains for point-of-care diagnostics for p24, an important biomarker of early HIV infections and successfully detect p24 with a limit of detection of 5 ng ml-1 in pseudo-serum, 4 fold more sensitive than comparable studies with gold nanoparticles. These findings and underlying concepts highlight the potential of advanced functional organic-inorganic composite nanomaterials to diagnose infections, with broad applicability to non-communicable diseases.

Dynamic Perturbations of the T-Cell Receptor Repertoire in Chronic HIV Infection and following Antiretroviral Therapy.

HIV infection profoundly affects many parameters of the immune system and ultimately leads to AIDS, yet which factors are most important for determining resistance, pathology, and response to antiretroviral treatment - and how best to monitor them - remain unclear. We develop a quantitative high-throughput sequencing pipeline to characterize the TCR repertoires of HIV-infected individuals before and after antiretroviral therapy, working from small, unfractionated samples of peripheral blood. This reveals the TCR repertoires of HIV(+) individuals to be highly perturbed, with considerably reduced diversity as a small proportion of sequences are highly overrepresented. HIV also causes specific qualitative changes to the repertoire including an altered distribution of V gene usage, depletion of public TCR sequences, and disruption of TCR networks. Short-term antiretroviral therapy has little impact on most of the global damage to repertoire structure, but is accompanied by rapid changes in the abundance of many individual TCR sequences, decreases in abundance of the most common sequences, and decreases in the majority of HIV-associated CDR3 sequences. Thus, high-throughput repertoire sequencing of small blood samples that are easy to take, store, and process can shed light on various aspects of the T-cell immune compartment and stands to offer insights into patient stratification and immune reconstitution.

Evolution of cocirculating varicella-zoster virus genotypes during a chickenpox outbreak in Guinea-Bissau.

UNLABELLED: Varicella-zoster virus (VZV), a double-stranded DNA alphaherpesvirus, is associated with seasonal outbreaks of varicella in nonimmunized populations. Little is known about whether these outbreaks are associated with a single or multiple viral genotypes and whether new mutations rapidly accumulate during transmission. Here, we take advantage of a well-characterized population cohort in Guinea-Bissau and produce a unique set of 23 full-length genome sequences, collected over 7 months from eight households. Comparative sequence analysis reveals that four distinct genotypes cocirculated among the population, three of which were present during the first week of the outbreak, although no patients were coinfected, which indicates that exposure to infectious virus from multiple sources is common during VZV outbreaks. Transmission of VZV was associated with length polymorphisms in the R1 repeat region and the origin of DNA replication. In two cases, these were associated with the formation of distinct lineages and point to the possible coevolution of these loci, despite the lack of any known functional link in VZV or related herpesviruses. We show that these and all other sequenced clade 5 viruses possess a distinct R1 repeat motif that increases the acidity of an ORF11p protein domain and postulate that this has either arisen or been lost following divergence of the major clades. Thus, sequencing of whole VZV genomes collected during an outbreak has provided novel insights into VZV biology, transmission patterns, and (recent) natural history. IMPORTANCE: VZV is a highly infectious virus and the causative agent of chickenpox and shingles, the latter being particularly associated with the risk of painful complications. Seasonal outbreaks of chickenpox are very common among young children, yet little is known about the dynamics of the virus during person-to-person to transmission or whether multiple distinct viruses seed and/or cocirculate during an outbreak. In this study, we have sequenced chickenpox viruses from an outbreak in Guinea-Bissau that are supported by detailed epidemiological data. Our data show that multiple different virus strains seeded and were maintained throughout the 6-month outbreak period and that viruses transmitted between individuals accumulated new mutations in specific genomic regions. Of particular interest is the potential coevolution of two distinct parts of the genomes and our calculations of the rate of viral mutation, both of which increase our understanding of how VZV evolves over short periods of time in human populations.

What factors underlie children's susceptibility to semantic and phonological false memories? investigating the roles of language skills and auditory short-term memory.

Two experiments investigated the cognitive skills that underlie children's susceptibility to semantic and phonological false memories in the Deese/Roediger-McDermott procedure (Deese, 1959; Roediger & McDermott, 1995). In Experiment 1, performance on the Verbal Similarities subtest of the British Ability Scales (BAS) II (Elliott, Smith, & McCulloch, 1997) predicted correct and false recall of semantic lures. In Experiment 2, performance on the Yopp-Singer Test of Phonemic Segmentation (Yopp, 1988) did not predict correct recall, but inversely predicted the false recall of phonological lures. Auditory short-term memory was a negative predictor of false recall in Experiment 1, but not in Experiment 2. The findings are discussed in terms of the formation of gist and verbatim traces as proposed by fuzzy trace theory (Reyna & Brainerd, 1998) and the increasing automaticity of associations as proposed by associative activation theory (Howe, Wimmer, Gagnon, & Plumpton, 2009).

Deep sequencing of viral genomes provides insight into the evolution and pathogenesis of varicella zoster virus and its vaccine in humans.

Immunization with the vOka vaccine prevents varicella (chickenpox) in children and susceptible adults. The vOka vaccine strain comprises a mixture of genotypes and, despite attenuation, causes rashes in small numbers of recipients. Like wild-type virus, the vaccine establishes latency in neuronal tissue and can later reactivate to cause Herpes zoster (shingles). Using hybridization-based methodologies, we have purified and sequenced vOka directly from skin lesions. We show that alleles present in the vaccine can be recovered from the lesions and demonstrate the presence of a severe bottleneck between inoculation and lesion formation. Genotypes in any one lesion appear to be descended from one to three vaccine-genotypes with a low frequency of novel mutations. No single vOka haplotype and no novel mutations are consistently present in rashes, indicating that neither new mutations nor recombination with wild type are critical to the evolution of vOka rashes. Instead, alleles arising from attenuation (i.e., not derived from free-living virus) are present at lower frequencies in rash genotypes. We identify 11 loci at which the ancestral allele is selected for in vOka rash formation and show genotypes in rashes that have reactivated from latency cannot be distinguished from rashes occurring immediately after inoculation. We conclude that the vOka vaccine, although heterogeneous, has not evolved to form rashes through positive selection in the mode of a quasispecies, but rather alleles that were essentially neutral during the vaccine production have been selected against in the human subjects, allowing us to identify key loci for rash formation.

Specific capture and whole-genome sequencing of viruses from clinical samples.

Whole genome sequencing of viruses directly from clinical samples is integral for understanding the genetics of host-virus interactions. Here, we report the use of sample sparing target enrichment (by hybridisation) for viral nucleic acid separation and deep-sequencing of herpesvirus genomes directly from a range of clinical samples including saliva, blood, virus vesicles, cerebrospinal fluid, and tumour cell lines. We demonstrate the effectiveness of the method by deep-sequencing 13 highly cell-associated human herpesvirus genomes and generating full length genome alignments at high read depth. Moreover, we show the specificity of the method enables the study of viral population structures and their diversity within a range of clinical samples types.

Binding of more than one Tva800 molecule is required for ASLV-A entry.

BACKGROUND: Understanding the mechanism by which viruses enter their target cell is an essential part of understanding their infectious cycle. Previous studies have focussed on the multiplicity of viral envelope proteins that need to bind to their cognate receptor to initiate entry. Avian sarcoma and leukosis virus Envelope protein (ASLV Env) mediates entry via a receptor, Tva, which can be attached to the cell surface either by a phospholipid anchor (Tva800) or a transmembrane domain (Tva950). In these studies, we have now investigated the number of target receptors necessary for entry of ASLV Env-pseudotyped virions. RESULTS: Using titration and modelling experiments we provide evidence that binding of more than one receptor, probably two, is needed for entry of virions via Tva800. However, binding of just one Tva950 receptor is sufficient for successful entry. CONCLUSIONS: The different modes of attachment of Tva800 and Tva950 to the cell membrane have important implications for the utilisation of these proteins as receptors for viral binding and/or uptake.