Feature Papers in Optical Sensors 2022.

Today, optical sensors are the subject of a very significant number of studies and applications [...].

Characterizing Emerging Canine H3 Influenza Viruses.

The continual emergence of novel influenza A strains from non-human hosts requires constant vigilance and the need for ongoing research to identify strains that may pose a human public health risk. Since 1999, canine H3 influenza A viruses (CIVs) have caused many thousands or millions of respiratory infections in dogs in the United States. While no human infections with CIVs have been reported to date, these viruses could pose a zoonotic risk. In these studies, the National Institutes of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) network collaboratively demonstrated that CIVs replicated in some primary human cells and transmitted effectively in mammalian models. While people born after 1970 had little or no pre-existing humoral immunity against CIVs, the viruses were sensitive to existing antivirals and we identified a panel of H3 cross-reactive human monoclonal antibodies (hmAbs) that could have prophylactic and/or therapeutic value. Our data predict these CIVs posed a low risk to humans. Importantly, we showed that the CEIRS network could work together to provide basic research information important for characterizing emerging influenza viruses, although there were valuable lessons learned.

Targeting Ribosomal Frameshifting as an Antiviral Strategy: From HIV-1 to SARS-CoV-2.

Treatment of HIV-1 has largely involved targeting viral enzymes using a cocktail of inhibitors. However, resistance to these inhibitors and toxicity in the long term have pushed the field to identify new therapeutic targets. To that end, -1 programmed ribosomal frameshifting (-1 PRF) has gained attention as a potential node for therapeutic intervention. In this process, a ribosome moves one nucleotide backward in the course of translating a mRNA, revealing a new reading frame for protein synthesis. In HIV-1, -1 PRF allows the virus to regulate the ratios of enzymatic and structural proteins as needed for correct viral particle assembly. Two RNA structural elements are central to -1 PRF in HIV: a slippery sequence and a highly conserved stable hairpin called the HIV-1 frameshifting stimulatory signal (FSS). Dysregulation of -1 PRF is deleterious for the virus. Thus, -1 PRF is an attractive target for new antiviral development. It is important to note that HIV-1 is not the only virus exploiting -1 PRF for regulating production of its proteins. Coronaviruses, including the COVID-19 pandemic virus SARS-CoV-2, also rely on -1 PRF. In SARS-CoV-2 and other coronaviruses, -1 PRF is required for synthesis of RNA-dependent RNA polymerase and several other nonstructural proteins. Coronaviruses employ a more complex RNA structural element for regulating -1 PRF called a pseudoknot. The purpose of this Account is primarily to review the development of molecules targeting HIV-1 -1 PRF. These approaches are case studies illustrating how the entire pipeline from screening to the generation of high-affinity leads might be implemented. We consider both target-based and function-based screening, with a particular focus on our group's approach beginning with a resin-bound dynamic combinatorial library (RBDCL) screen. We then used rational design approaches to optimize binding affinity, selectivity, and cellular bioavailability. Our tactic is, to the best of our knowledge, the only study resulting in compounds that bind specifically to the HIV-1 FSS RNA and reduce infectivity of laboratory and drug-resistant strains of HIV-1 in human cells. Lessons learned from strategies targeting -1 PRF HIV-1 might provide solutions in the development of antivirals in areas of unmet medical need. This includes the development of new frameshift-altering therapies for SARS-CoV-2, approaches to which are very recently beginning to appear.

Expanding the known structure space for RNA binding: a test of 2,5-diketopiperazine.

As the importance of RNA as a therapeutic target has become increasingly recognized, the need for new chemotypes able to bind RNA has grown in significance. We hypothesized that diketopiperazines (DKPs), common substructures in natural products and protein-targeting therapeutic agents, could serve as effective scaffolds for targeting RNA. To confirm this hypothesis, we designed and synthesized two analogs, one incorporating a DKP and one not, of compounds previously demonstrated to bind an RNA critical to the life cycle of HIV-1 with high affinity and specificity. Prior to compound synthesis, calculations employing density functional methods and molecular mechanics conformational searches were used to confirm that the DKP could present functionality in a similar (albeit not identical) orientation to the non DKP-containing compound. We found that both the DKP-containing and parent compound had similar affinities to the target RNA as measured by surface plasmon resonance (SPR). Both compounds were found to have modest but equal anti-HIV activity. These results establish the feasibility of using DKPs to target RNA.

Waveguide-Enhanced Raman Spectroscopy (WERS): An Emerging Chip-Based Tool for Chemical and Biological Sensing.

Photonic chip-based methods for spectroscopy are of considerable interest due to their applicability to compact, low-power devices for the detection of small molecules. Waveguide-enhanced Raman spectroscopy (WERS) has emerged over the past decade as a particularly interesting approach. WERS utilizes the evanescent field of a waveguide to generate Raman scattering from nearby analyte molecules, and then collects the scattered photons back into the waveguide. The large interacting area and strong electromagnetic field provided by the waveguide allow for significant enhancements in Raman signal over conventional approaches. The waveguide can also be coated with a molecular class-selective sorbent material to concentrate the analyte, thus further increasing the Raman signal. This review provides an overview of the historical development of WERS and highlights recent theoretical and experimental achievements with the technique.

Development of Methods for Specific Capture of Biological Targets on Aluminum Substrates: Application to Bacillus subtilis Spore Detection as a Model for Anthrax.

Many (if not most) biosensors rely on functional silane coatings as a first step toward covalent immobilization of specific capture molecules. While methods for silanization of silica (SiO2) surfaces are very well developed, less has been done to develop and characterize silanization methods for alternative substrates, such as alumina (Al2O3). In particular, the behavior of Al2O3 coatings grown on aluminum under ambient conditions has not been studied. To address this issue, we have tested solution-phase deposition of two silanes on Al2O3 (3-aminopropyl triethoxysilane and 3-triethoxysilyl)propylsuccinic anhydride) and their applicability to analyte-specific biosensing. Contact angle measurements and imaging via Scanning Electron Microsopy (SEM) were employed to characterize surfaces. We find that 3-aminopropyl triethoxysilane produces well-behaved films and demonstrate that this surface can undergo further reaction with glutaraldehyde followed by an anti-Bacillus subtilis antibody to yield functionalized Al2O3 surfaces capable of specific capture of B. subtilis spores (a model of B. anthracis, the causative organism of Anthrax). In contrast, 3-triethoxysilyl)propylsuccinic anhydride did not behave well with Al/Al2O3 under the reaction conditions tested. In addition to providing specific protocols for Al/Al2O3 functionalization, this work highlights the importance of surface chemistry assessment in the development of new sensors.

StaphAIR: A Label-Free Antigen Microarray Approach to Detecting Anti-Staphylococcus aureus Antibody Responses in Orthopedic Infections.

Arrayed imaging reflectometry (AIR) is an optical biosensor platform for simple, multiplex measurement of antigen-specific antibody responses in patient blood samples. Here, we report the development of StaphAIR, an 8-plex Staphylococcus aureus antigen array on the AIR platform for profiling antigen-specific anti-S. aureus humoral immune responses. Initial validation experiments with mouse and humanized monoclonal antibodies against the S. aureus autolysin glucosaminidase (Gmd) domain, and subsequent testing with dilution series of pooled positive human serum confirmed analytically robust behavior of the array, with all antigens displaying Langmuir-type dose-response curves. Testing a cohort of 82 patients with S. aureus musculoskeletal infections (MSKI) and 30 healthy individuals enabled discrimination of individual patient responses to different S. aureus antigens, with statistical significance between osteomyelitis patients and controls obtained overall for four individual antigens (IsdA, IsdB, Gmd, and SCIN). Multivariate analyses of the antibody titers obtained from StaphAIR revealed its utility as a potential diagnostic tool for detecting S. aureus MSKI (area under the receiver operating characteristic curve (AUC) > 0.85). We conclude that StaphAIR has utility as a high-throughput immunoassay for studying and diagnosing osteomyelitis in patients.

Associations of Serum Calciprotein Particle Size and Transformation Time With Arterial Calcification, Arterial Stiffness, and Mortality in Incident Hemodialysis Patients.

RATIONALE & OBJECTIVE: Characteristics of the transformation of primary to secondary calciprotein particles (CPPs) in serum, including the size of secondary CPP (CPP2) aggregates and the time of transformation (T50), may be markers for arterial calcification in patients undergoing hemodialysis (HD). We examined the associations of CPP2 aggregate size and T50 with arterial calcification in incident HD patients. STUDY DESIGN: Prospective cohort study. SETTING & PARTICIPANTS: Incident HD patients (n=402with available CPP2 measures and n=388with available T50 measures) from the Predictors of Arrhythmic and Cardiovascular Risk in End-Stage Renal Disease (PACE) Study PREDICTORS: Serum CPP2 size and T50 at baseline. OUTCOMES: Primary outcomes were baseline coronary artery and thoracic aorta calcifications. Exploratory outcomes included baseline arterial stiffness, measured by pulse wave velocity (PWV) and ankle brachial index, and longitudinally, repeat measures of PWV and all-cause mortality. ANALYTICAL APPROACH: Tobit regression, multiple linear regression, Poisson regression, linear mixed-effects regression, and Cox proportional hazards regression. RESULTS: Mean age was 55±13 years, 41% were women, 71% were Black, and 57% had diabetes mellitus. Baseline CPP2 size and T50 were correlated with baseline fetuin A level (r=-0.59 for CPP2 and 0.44 for T50; P<0.001 for both), but neither was associated with baseline measures of arterial calcification or arterial stiffness. Baseline CPP2 size and T50 were not associated with repeat measures of PWV. During a median follow-up of 3.5 (IQR, 1.7-6.2) years, larger CPP2 was associated with higher risk for mortality (HR, 1.17 [95% CI, 1.05-1.31] per 100nm larger CPP2 size) after adjusting for demographics and comorbid conditions, but there was no association between baseline T50 and risk for mortality. LIMITATIONS: Possible imprecision in assays, small sample size, limited generalizability to incident HD populations with different racial composition, and residual confounding. CONCLUSIONS: In incident HD patients, neither CPP2 size nor T50 was associated with prevalent arterial calcification and stiffness. Larger CPP2 was associated with risk for mortality, but this finding needs to be confirmed in future studies.

Label-Free, Multiplex Glycan Microarray Biosensor for Influenza Virus Detection.

Newly emerging influenza viruses adapted from animal species pose significant pandemic threats to public health. An understanding of hemagglutinin (HA) receptor-binding specificity to host receptors is key to studying the adaptation of influenza viruses in humans. This information may be particularly useful for predicting the emergence of a pandemic outbreak. Therefore, high-throughput sensing technologies able to profile HA receptor binding can facilitate studies of influenza virus evolution and adaptation in humans. As a step toward this goal, we have prepared glycan-based receptor analogue microarrays on the Arrayed Imaging Reflectometry (AIR) platform. These arrays demonstrate label-free, multiplex detection and discrimination between human and avian influenza viruses. Microarrays consisting of glycan probes with 2,6 and 2,3 linkages were prepared. After first confirming their ability to capture lectins (carbohydrate-binding proteins) with known specificities, we observed that the arrays were able to discriminate between and quantify human pandemic influenza A/California/07/2009 (H1N1pdm) and avian A/Netherlands/1/2000 (H13N8) influenza viruses, respectively. As the method may be expanded to large numbers of glycans (>100) and virus subtypes (H1-H18), we anticipate it can be applied to systematically evaluate influenza virus adaptation in humans. In turn, this will facilitate global influenza surveillance and serve as a new tool enabling health organizations, governments, research institutes, and laboratories to react quickly in the face of a pandemic outbreak.

Monitoring Serum Spike Protein with Disposable Photonic Biosensors Following SARS-CoV-2 Vaccination.

While mRNA vaccines have been well-studied in vitro and in animals prior to their use in the human population during the Covid-19 pandemic, their exact mechanisms of inducing immunity are still being elucidated. The large-scale collection of data necessary to fully understand these mechanisms, and their variability across heterogeneous populations, requires rapid diagnostic tests that accurately measure the various biomarkers involved in the immune response following vaccination. Recently, our lab developed a novel "Disposable Photonics" platform for rapid, label-free, scalable diagnostics that utilizes photonic ring resonator sensor chips combined with plastic micropillar cards able to provide passive microfluidic flow. Here, we demonstrate the utility of this system in confirming the presence of SARS-CoV-2 spike protein in the serum of recently vaccinated subjects, as well as tracking a post-vaccination rise in anti-SARS-CoV-2 antibodies. A maximum concentration in SARS-CoV-2 spike protein was detected one day after vaccination and was reduced below detectable levels within 10 days. This highlights the applicability of our rapid photonic sensor platform for acquiring the data necessary to understand vaccine mechanisms on a large scale, as well as individual patient responses to SARS-CoV-2 mRNA vaccines.

A Stable Biotin-Streptavidin Surface Enables Multiplex, Label-Free Protein Detection by Aptamer and Aptamer-Protein Arrays Using Arrayed Imaging Reflectometry.

While label-free multiplex sensor technology enables "mixing and matching" of different capture molecules in principle, in practice this has been rarely (if ever) demonstrated. To fill this gap, we developed protocols for the preparation of mixed aptamer-protein arrays on the arrayed imaging reflectometry (AIR) sensing platform using streptavidin as a common attachment point for both biotinylated proteins and aptamers. Doing so required overcoming the noted instability of dried streptavidin monolayers on surfaces. After characterizing this degradation, stable surfaces were obtained using a commercial microarray product. Microarraying through the layer of stabilizer then provided mixed aptamer-antibody arrays. We demonstrate that sensor arrays prepared in this manner are suitable for several probes (thrombin and TGF-ß1 aptamers; avi-tagged protein) and targets.

Antibacterial Copper-Hydroxyapatite Composite Coatings via Electrochemical Synthesis.

Antibacterial copper-hydroxyapatite (Cu-HA) composite coatings on titanium were synthesized using a novel process consisting of two consecutive electrochemical reactions. In the first stage, HA nanocrystals were grown on titanium using the cathodic electrolytic synthesis. The HA-coated titanium was then used as the cathode in a second reaction stage to electrochemically reduce Cu2+ ions in solution to metallic Cu nanoparticles. Reaction conditions were found that result in nanoscale Cu particles growing on the surface of the HA crystals. The two-stage synthesis allows facile control of copper content in the HA coatings. Antibacterial activity was measured by culturing Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive) in the presence of coatings having varying copper contents. The coatings displayed copper concentration-dependent antibacterial activity against both types of bacteria, likely due to the slow release of copper ions from the coatings. The observation of antibacterial activity from a relatively low loading of copper on the bioactive HA support suggests that multifunctional implant coatings can be developed to supplement or supplant prophylactic antibiotics used in implant surgery that are responsible for creating resistant bacteria strains.

Patients with advanced chronic kidney disease and vascular calcification have a large hydrodynamic radius of secondary calciprotein particles.

BACKGROUND: The size of secondary calciprotein particles (CPP2) and the speed of transformation (T50) from primary calciprotein particles (CPP1) to CPP2 in serum may be associated with vascular calcification (VC) in patients with chronic kidney disease (CKD). METHODS: We developed a high throughput, microplate-based assay using dynamic light scattering (DLS) to measure the transformation of CPP1 to CPP2, hydrodynamic radius (Rh) of CPP1 and CPP2, T50 and aggregation of CPP2. We used this DLS assay to test the hypothesis that a large Rh of CPP2 and/or a fast T50 are associated with VC in 45 participants with CKD Stages 4-5 (22 without VC and 23 with VC) and 17 healthy volunteers (HV). VC was defined as a Kauppila score >6 or an Adragao score ≥3. RESULTS: CKD participants with VC had larger cumulants Rh of CPP2 {370 nm [interquartile range (IQR) 272-566]} compared with CKD participants without VC [212 nm (IQR 169-315)] and compared with HV [168 nm (IQR 145-352), P < 0.01 for each]. More CPP2 were in aggregates in CKD participants with VC than those without VC (70% versus 36%). The odds of having VC increased by 9% with every 10 nm increase in the Rh of CPP2, after adjusting for age, diabetes, serum calcium and phosphate [odds ratio 1.09, 95% confidence interval (CI) 1.03, 1.16, P = 0.005]. The area under the receiver operating characteristic curve for VC of CPP2 size was 0.75 (95% CI 0.60, 0.90). T50 was similar in CKD participants with and without VC, although both groups had a lower T50 than HV. CONCLUSIONS: Rh of CPP2, but not T50, is independently associated with VC in patients with CKD Stages 4-5.

Enhancing the ligand efficiency of anti-HIV compounds targeting frameshift-stimulating RNA.

Ribosomal frameshifting, a process whereby a translating ribosome is diverted from one reading frame to another on a contiguous mRNA, is an important regulatory mechanism in biology and an opportunity for therapeutic intervention in several human diseases. In HIV, ribosomal frameshifting controls the ratio of Gag and Gag-Pol, two polyproteins critical to the HIV life cycle. We have previously reported compounds able to selectively bind an RNA stemloop within the Gag-Pol mRNA; these compounds alter the production of Gag-Pol in a manner consistent with increased frameshifting. Importantly, they also display antiretroviral activity in human T-cells. Here, we describe new compounds with significantly reduced molecular weight, but with substantially maintained affinity and anti-HIV activity. These results suggest that development of more "ligand efficient" enhancers of ribosomal frameshifting is an achievable goal.

Crowd on a Chip: Label-Free Human Monoclonal Antibody Arrays for Serotyping Influenza.

Rapid changes in influenza A virus (IAV) antigenicity create challenges in surveillance, disease diagnosis, and vaccine development. Further, serological methods for studying antigenic properties of influenza viruses often rely on animal models and therefore may not fully reflect the dynamics of human immunity. We hypothesized that arrays of human monoclonal antibodies (hmAbs) to influenza could be employed in a pattern-recognition approach to expedite IAV serology and to study the antigenic evolution of newly emerging viruses. Using the multiplex, label-free Arrayed Imaging Reflectometry (AIR) platform, we have demonstrated that such arrays readily discriminated among various subtypes of IAVs, including H1, H3 seasonal strains, and avian-sourced human H7 viruses. Array responses also allowed the first determination of antigenic relationships among IAV strains directly from hmAb responses. Finally, correlation analysis of antibody binding to all tested IAV subtypes allowed efficient identification of broadly reactive clones. In addition to specific applications in the context of understanding influenza biology with potential utility in "universal" flu vaccine development, these studies validate AIR as a platform technology for studying antigenic properties of viruses and also antibody properties in a high-throughput manner. We further anticipate that this approach will facilitate advances in the study of other viral pathogens.

HMMER web server: 2015 update.

The HMMER website, available at http://www.ebi.ac.uk/Tools/hmmer/, provides access to the protein homology search algorithms found in the HMMER software suite. Since the first release of the website in 2011, the search repertoire has been expanded to include the iterative search algorithm, jackhmmer. The continued growth of the target sequence databases means that traditional tabular representations of significant sequence hits can be overwhelming to the user. Consequently, additional ways of presenting homology search results have been developed, allowing them to be summarised according to taxonomic distribution or domain architecture. The taxonomy and domain architecture representations can be used in combination to filter the results according to the needs of a user. Searches can also be restricted prior to submission using a new taxonomic filter, which not only ensures that the results are specific to the requested taxonomic group, but also improves search performance. The repertoire of profile hidden Markov model libraries, which are used for annotation of query sequences with protein families and domains, has been expanded to include the libraries from CATH-Gene3D, PIRSF, Superfamily and TIGRFAMs. Finally, we discuss the relocation of the HMMER webserver to the European Bioinformatics Institute and the potential impact that this will have.

Methane Ebullition in Temperate Hydropower Reservoirs and Implications for US Policy on Greenhouse Gas Emissions.

The United States is home to 2198 dams actively used for hydropower production. With the December 2015 consensus adoption of the United Nations Framework Convention on Climate Change Paris Agreement, it is important to accurately quantify anthropogenic greenhouse gas emissions. Methane ebullition, or methane bubbles originating from river or lake sediments, has been shown to account for nearly all methane emissions from tropical hydropower reservoirs to the atmosphere. However, distinct ebullitive methane fluxes have been studied in comparatively few temperate hydropower reservoirs globally. This study measures ebullitive and diffusive methane fluxes from two eastern Washington reservoirs, and synthesizes existing studies of methane ebullition in temperate, boreal, and tropical hydropower reservoirs. Ebullition comprises nearly all methane emissions (>97%) from this study's two eastern Washington hydropower reservoirs to the atmosphere. Summer methane ebullition from these reservoirs was higher than ebullition in six southeastern U.S. hydropower reservoirs, however it was similar to temperate reservoirs in other parts of the world. Our literature synthesis suggests that methane ebullition from temperate hydropower reservoirs can be seasonally elevated compared to tropical climates, however annual emissions are likely to be higher within tropical climates, emphasizing the possible range of methane ebullition fluxes and the need for the further study of temperate reservoirs. Possible future changes to the Intergovernmental Panel on Climate Change and UNFCCC guidelines for national greenhouse gas inventories highlights the need for accurate assessment of reservoir emissions.

Probing the geometric constraints of RNA binding via dynamic covalent chemistry.

Dynamic Combinatorial Chemistry (DCC) has proven to be a reliable method for identifying hit compounds for target nucleic acid (DNA and RNA) sequences. Typically, these hit compounds are subjected to a lengthy process of optimization via traditional medicinal chemistry. Here, we examine the potential of DCC to also generate and test variations on a hit compound as a method for probing the binding site of an RNA-targeted compound. Specifically, we demonstrate that addition of linker dithiols to a disulfide library containing a known binder to the HIV-1 frameshift-stimulatory RNA (a critical regulator of the HIV life cycle) can yield a mixture of new bridged structures incorporating the dithiol, depending on dithiol structure. Equilibration of this library with the HIV FSS RNA resulted in selection of the original disulfide in preference to bridged structures, suggesting incorporation of the bridge is not compatible with this particular binding site. Application of this strategy to other RNA targets should allow for rapidly profiling the affinity of modified compounds.

Identification of activators of ERK5 transcriptional activity by high-throughput screening and the role of endothelial ERK5 in vasoprotective effects induced by statins and antimalarial agents.

Because ERK5 inhibits endothelial inflammation and dysfunction, activating ERK5 might be a novel approach to protecting vascular endothelial cells (ECs) against various pathological conditions of the blood vessel. We have identified small molecules that protect ECs via ERK5 activation and determined their contribution to preventing cardiac allograft rejection. Using high-throughput screening, we identified certain statins and antimalarial agents including chloroquine, hydroxychloroquine, and quinacrine as strong ERK5 activators. Pitavastatin enhanced ERK5 transcriptional activity and Kruppel-like factor-2 expression in cultured human and bovine ECs, but these effects were abolished by the depletion of ERK5. Chloroquine and hydroxychloroquine upregulated ERK5 kinase activity and inhibited VCAM-1 expression in an ERK5-dependent but MAPK/ERK kinase 5- and Kruppel-like factor 2/4-independent manner. Leukocyte rolling and vascular reactivity were used to evaluate endothelial function in vivo, and we found that EC-specific ERK5 knockout (ERK5-EKO) mice exhibited increased leukocyte rolling and impaired vascular reactivity, which could not be corrected by pitavastatin. The role of endothelial ERK5 in acute cardiac allograft rejection was also examined by heterotopic grafting of the heart obtained from either wild-type or ERK5-EKO mice into allomismatched recipient mice. A robust increase in both inflammatory gene expression and CD45-positive cell infiltration into the graft was observed. These tissue rejection responses were inhibited by pitavastatin in wild-type but not ERK5-EKO hearts. Our study has identified statins and antimalarial drugs as strong ERK5 activators and shown that ERK5 activation is preventive of endothelial inflammation and dysfunction and acute allograft rejection.

iPfam: a database of protein family and domain interactions found in the Protein Data Bank.

The database iPfam, available at http://ipfam.org, catalogues Pfam domain interactions based on known 3D structures that are found in the Protein Data Bank, providing interaction data at the molecular level. Previously, the iPfam domain-domain interaction data was integrated within the Pfam database and website, but it has now been migrated to a separate database. This allows for independent development, improving data access and giving clearer separation between the protein family and interactions datasets. In addition to domain-domain interactions, iPfam has been expanded to include interaction data for domain bound small molecule ligands. Functional annotations are provided from source databases, supplemented by the incorporation of Wikipedia articles where available. iPfam (version 1.0) contains >9500 domain-domain and 15 500 domain-ligand interactions. The new website provides access to this data in a variety of ways, including interactive visualizations of the interaction data.