Potent Henipavirus Neutralization by Antibodies Recognizing Diverse Sites on Hendra and Nipah Virus Receptor Binding Protein.

Hendra (HeV) and Nipah (NiV) viruses are emerging zoonotic pathogens in the Henipavirus genus causing outbreaks of disease with very high case fatality rates. Here, we report the first naturally occurring human monoclonal antibodies (mAbs) against HeV receptor binding protein (RBP). All isolated mAbs neutralized HeV, and some also neutralized NiV. Epitope binning experiments identified five major antigenic sites on HeV-RBP. Animal studies demonstrated that the most potent cross-reactive neutralizing mAbs, HENV-26 and HENV-32, protected ferrets in lethal models of infection with NiV Bangladesh 3 days after exposure. We solved the crystal structures of mAb HENV-26 in complex with both HeV-RBP and NiV-RBP and of mAb HENV-32 in complex with HeV-RBP. The studies reveal diverse sites of vulnerability on RBP recognized by potent human mAbs that inhibit virus by multiple mechanisms. These studies identify promising prophylactic antibodies and define protective epitopes that can be used in rational vaccine design.

Single-Molecule Real-Time 3D Imaging of the Transcription Cycle by Modulation Interferometry.

Many essential cellular processes, such as gene control, employ elaborate mechanisms involving the coordination of large, multi-component molecular assemblies. Few structural biology tools presently have the combined spatial-temporal resolution and molecular specificity required to capture the movement, conformational changes, and subunit association-dissociation kinetics, three fundamental elements of how such intricate molecular machines work. Here, we report a 3D single-molecule super-resolution imaging study using modulation interferometry and phase-sensitive detection that achieves <2 nm axial localization precision, well below the few-nanometer-sized individual protein components. To illustrate the capability of this technique in probing the dynamics of complex macromolecular machines, we visualize the movement of individual multi-subunit E. coli RNA polymerases through the complete transcription cycle, dissect the kinetics of the initiation-elongation transition, and determine the fate of σ70 initiation factors during promoter escape. Modulation interferometry sets the stage for single-molecule studies of several hitherto difficult-to-investigate multi-molecular transactions that underlie genome regulation.

Memory B Cells that Cross-React with Group 1 and Group 2 Influenza A Viruses Are Abundant in Adult Human Repertoires.

Human B cell antigen-receptor (BCR) repertoires reflect repeated exposures to evolving influenza viruses; new exposures update the previously generated B cell memory (Bmem) population. Despite structural similarity of hemagglutinins (HAs) from the two groups of influenza A viruses, cross-reacting antibodies (Abs) are uncommon. We analyzed Bmem compartments in three unrelated, adult donors and found frequent cross-group BCRs, both HA-head directed and non-head directed. Members of a clonal lineage from one donor had a BCR structure similar to that of a previously described Ab, encoded by different gene segments. Comparison showed that both Abs contacted the HA receptor-binding site through long heavy-chain third complementarity determining regions. Affinities of the clonal-lineage BCRs for historical influenza-virus HAs from both group 1 and group 2 viruses suggested that serial responses to seasonal influenza exposures had elicited the lineage and driven affinity maturation. We propose that appropriate immunization regimens might elicit a comparably broad response.

Structural Activation of Pro-inflammatory Human Cytokine IL-23 by Cognate IL-23 Receptor Enables Recruitment of the Shared Receptor IL-12Rß1.

Interleukin-23 (IL-23), an IL-12 family cytokine, plays pivotal roles in pro-inflammatory T helper 17 cell responses linked to autoimmune and inflammatory diseases. Despite intense therapeutic targeting, structural and mechanistic insights into receptor complexes mediated by IL-23, and by IL-12 family members in general, have remained elusive. We determined a crystal structure of human IL-23 in complex with its cognate receptor, IL-23R, and revealed that IL-23R bound to IL-23 exclusively via its N-terminal immunoglobulin domain. The structural and functional hotspot of this interaction partially restructured the helical IL-23p19 subunit of IL-23 and restrained its IL-12p40 subunit to cooperatively bind the shared receptor IL-12Rß1 with high affinity. Together with structural insights from the interaction of IL-23 with the inhibitory antibody briakinumab and by leveraging additional IL-23:antibody complexes, we propose a mechanistic paradigm for IL-23 and IL-12 whereby cognate receptor binding to the helical cytokine subunits primes recruitment of the shared receptors via the IL-12p40 subunit.

Vertical growth dynamics of biofilms.

During the biofilm life cycle, bacteria attach to a surface and then reproduce, forming crowded, growing communities. Many theoretical models of biofilm growth dynamics have been proposed; however, difficulties in accurately measuring biofilm height across relevant time and length scales have prevented testing these models, or their biophysical underpinnings, empirically. Using white light interferometry, we measure the heights of microbial colonies with nanometer precision from inoculation to their final equilibrium height, producing a detailed empirical characterization of vertical growth dynamics. We propose a heuristic model for vertical growth dynamics based on basic biophysical processes inside a biofilm: diffusion and consumption of nutrients and growth and decay of the colony. This model captures the vertical growth dynamics from short to long time scales (10 min to 14 d) of diverse microorganisms, including bacteria and fungi.

Dark-field computed tomography reaches the human scale.

X-ray computed tomography (CT) is one of the most commonly used three-dimensional medical imaging modalities today. It has been refined over several decades, with the most recent innovations including dual-energy and spectral photon-counting technologies. Nevertheless, it has been discovered that wave-optical contrast mechanisms-beyond the presently used X-ray attenuation-offer the potential of complementary information, particularly on otherwise unresolved tissue microstructure. One such approach is dark-field imaging, which has recently been introduced and already demonstrated significantly improved radiological benefit in small-animal models, especially for lung diseases. Until now, however, dark-field CT could not yet be translated to the human scale and has been restricted to benchtop and small-animal systems, with scan durations of several minutes or more. This is mainly because the adaption and upscaling to the mechanical complexity, speed, and size of a human CT scanner so far remained an unsolved challenge. Here, we now report the successful integration of a Talbot-Lau interferometer into a clinical CT gantry and present dark-field CT results of a human-sized anthropomorphic body phantom, reconstructed from a single rotation scan performed in 1 s. Moreover, we present our key hardware and software solutions to the previously unsolved roadblocks, which so far have kept dark-field CT from being translated from the optical bench into a rapidly rotating CT gantry, with all its associated challenges like vibrations, continuous rotation, and large field of view. This development enables clinical dark-field CT studies with human patients in the near future.

Interferometric Biosensor for High Sensitive Label-Free Recording of HiPS Cardiomyocytes Contraction in Vitro.

Heart disease remains a leading cause of global mortality, underscoring the need for advanced technologies to study cardiovascular diseases and develop effective treatments. We introduce an innovative interferometric biosensor for high-sensitivity and label-free recording of human induced pluripotent stem cell (hiPSC) cardiomyocyte contraction in vitro. Using an optical cavity, our device captures interference patterns caused by the contraction-induced displacement of a thin flexible membrane. First, we demonstrate the capability to quantify spontaneous contractions and discriminate between contraction and relaxation phases. We calculate a contraction-induced vertical membrane displacement close to 40 nm, which implies a traction stress of 34 ± 4 mN/mm2. Finally, we investigate the effects of a drug compound on contractility amplitude, revealing a significant reduction in contractile forces. The label-free and high-throughput nature of our biosensor may enhance drug screening processes and drug development for cardiac treatments. Our interferometric biosensor offers a novel approach for noninvasive and real-time assessment of cardiomyocyte contraction.

Red-on-Yellow Queen: Bio-Layer Interferometry Reveals Functional Diversity Within Micrurus Venoms and Toxin Resistance in Prey Species.

Snakes in the family Elapidae largely produce venoms rich in three-finger toxins (3FTx) that bind to the α 1 subunit of nicotinic acetylcholine receptors (nAChRs), impeding ion channel activity. These neurotoxins immobilize the prey by disrupting muscle contraction. Coral snakes of the genus Micrurus are specialist predators who produce many 3FTx, making them an interesting system for examining the coevolution of these toxins and their targets in prey animals. We used a bio-layer interferometry technique to measure the binding interaction between 15 Micrurus venoms and 12 taxon-specific mimotopes designed to resemble the orthosteric binding region of the muscular nAChR subunit. We found that Micrurus venoms vary greatly in their potency on this assay and that this variation follows phylogenetic patterns rather than previously reported patterns of venom composition. The long-tailed Micrurus tend to have greater binding to nAChR orthosteric sites than their short-tailed relatives and we conclude this is the likely ancestral state. The repeated loss of this activity may be due to the evolution of 3FTx that bind to other regions of the nAChR. We also observed variations in the potency of the venoms depending on the taxon of the target mimotope. Rather than a pattern of prey-specificity, we found that mimotopes modeled after snake nAChRs are less susceptible to Micrurus venoms and that this resistance is partly due to a characteristic tryptophan → serine mutation within the orthosteric site in all snake mimotopes. This resistance may be part of a Red Queen arms race between coral snakes and their prey.

Real-Time Study of the Specific Interactions of Lactoferrin with Mimicked Heparan Sulfate Meshes Using Ordered Porous Layer Interferometry.

Heparan sulfate (HS) meshes within the glycocalyx on cell surfaces have protein recognition ability and have been crucial for gaining insights into vital bioprocesses, such as viral infection, cancer development, and inflammation. The protein recognition ability is determined by the mesh property and compositions of HS, although little attention has been paid to the effect of the mesh property on the recognition. An in-depth specificity study of protein-HS-mesh recognition is essential to illustrate related biological functions. Here, ordered porous layer interferometry is applied to study the interaction behavior between mimicked HS meshes and lactoferrin (LF). Our work aimed at mimicking HS meshes with heparin, a widely used substitute of HS, and analyzing the specific LF-heparin-mesh interaction mechanism by inhibiting the nonspecific interaction in a blended sample. We found that the counterion release-based electrostatic interaction is dominant in the specific LF-heparin-mesh recognition. Furthermore, we detail the contributions of nonspecific and specific interactions to the recognition. We illustrate that the concentrated charge distribution of the proteins appears to be primarily related to this robust, specific recognition.

Development of a Methodology Based on Optical Interferometry for Measuring Fibrinolytic Activity.

Fibrinolytic activity assay is particularly important for the detection, diagnosis, and treatment of cardiovascular disease and the development of fibrinolytic drugs. A novel efficacious strategy for real-time and label-free dynamic detection of fibrinolytic activity based on ordered porous layer interferometry (OPLI) was developed. Fibrin or a mixture of fibrin and plasminogen (Plg) was loaded into the highly ordered silica colloidal crystal (SCC) film scaffold to construct a fibrinolytic response interference layer to measure fibrinolytic activity with different mechanisms of action. Fibrinolytic enzyme-triggered fibrinolysis led to the migration of interference fringes in the interferogram, which could be represented by optical thickness changes (ΔOT) tracked in real time by the OPLI system. The morphology and optical property of the fibrinolytic response interference layer were characterized, and the Plg content in the fibrinolytic response interference layer and experimental parameters of the system were optimized. The method showed adequate sensitivity for the fibrinolytic activity of lumbrokinase and streptokinase, with wide linear ranges of 12-6000 and 10-2000 U/mL, respectively. Compared with the traditional fibrin plate method, it has a lower detection limit and higher linearity. The whole kinetic process of fibrinolysis by these two fibrinolytic drug models was recorded in real time, and the Michaelis constant and apparent kinetic parameters were calculated. Importantly, some other blood proteins were less interfering with this system, and it showed reliability in fibrin activity detection in real whole blood samples. This study established a better and more targeted research method of in vitro fibrinolysis and provided dynamic monitoring data for the analysis of fibrinolytic activity of whole blood.

Detection of bladder cancer cells using quantitative interferometric label-free imaging flow cytometry.

Bladder cancer is one of the most common cancers with a high recurrence rate. Patients undergo mandatory yearly scrutinies, including cystoscopies, which makes bladder cancer highly distressing and costly. Here, we aim to develop a non-invasive, label-free method for the detection of bladder cancer cells in urine samples, which is based on interferometric imaging flow cytometry. Eight urothelial carcinoma and one normal urothelial cell lines, along with red and white blood cells, imaged quantitatively without staining by an interferometric phase microscopy module while flowing in a microfluidic chip, and classified by two machine-learning algorithms, based on deep-learning semantic segmentation convolutional neural network and extreme gradient boosting. Furthermore, urine samples obtained from bladder-cancer patients and healthy volunteers were imaged, and classified by the system. We achieved accuracy and area under the curve (AUC) of 99% and 97% for the cell lines on both machine-learning algorithms. For the real urine samples, the accuracy and AUC were 96% and 96% for the deep-learning algorithm and 95% and 93% for the gradient-boosting algorithm, respectively. By combining label-free interferometric imaging flow cytometry with high-end classification algorithms, we achieved high-performance differentiation between healthy and malignant cells. The proposed technique has the potential to supplant cystoscopy in the bladder cancer surveillance and diagnosis space.

Advanced control strategies for continuous capture of monoclonal antibodies based upon biolayer interferometry.

The semi and fully continuous production of monoclonal antibodies (mAbs) has been gaining traction as a lower cost, and efficient production of mAbs to broaden patient access. To be truly flexible and adaptive to process demands, the industry has lacked sufficient advanced control strategies. The variation of the upstream product concentration typically cannot be handled by the downstream capture step, which is configured for a constant feed concentration and fixed binding capacity. This inflexibility leads to losses of efficiency and product yield. This study shows that these challenges can be overcome by a novel advanced control strategy concept that includes dynamic control throughout a perfusion bioreactor, with cell retention by alternating tangential flow, integrated with simulated moving bed (SMB) multi-column chromatography. The automation workflow and advanced control strategy were implemented through the use of a visual programming development environment. This enabled dynamic flow control across the upstream and downstream process integrated with a dynamic column loading of the SMB. A sensor prototype, based on continuous biolayer interferometry measurements was applied to detect mAb breakthrough within the last column flow-through to manage column switching. This novel approach provided higher specificity and lower background signal compared to commonly used spectroscopy methods, resulting in an optimized resin utilization while simultaneously avoiding product loss. The dynamic loading was found to provide a twofold increase of the mAb concentration in the eluate compared to a conservative approach with a predefined recipe with similar impurity removal. This concept shows that advanced control strategies can lead to significant process efficiency and yield improvement.

Nanoporous Polystyrene Inverse Opal Materials with Optical Interference Properties for Label-Free Biosensing.

Colloidal crystal nanomaterials have been proven to be valuable substrates for optical-based biosensing due to their ordered macroporous nanostructure and brilliant optical properties. In this work, silica colloidal crystal (SCC) thin films, as well as polystyrene-SCC composite films and inverse opal (IO) polystyrene films fabricated using SCC as templates, are investigated for their application as substrate materials in optical interferometric biosensors. The SCC films formed by the self-assembly of silica colloidal crystals have the most densely packed nano-3D structure, also known as the opal structure. IO films are fabricated by filling the opal pores of SCC with polystyrene and then removing the template, resulting in an interconnected nano-3D ordered macroporous structure, as indicated by the name inverse opal. The performance of the three materials was compared and discussed based on an ordered porous layer interferometry optical platform, focusing on refractive index response, protein adsorption response, and biomolecular interaction response. These results could potentially offer innovative material support for the advancement of label-free optical biosensors, which can be used for more biological/biochemical/biomolecular reaction monitoring studies.

Measurement of enzyme activity of insoluble substrates based on ordered porous layer interferometry and the application in evaluation of thrombolytic drugs.

The development of innovative methods for real-time surveillance of enzymatic activity determination processes is essential, particularly for insoluble substrate enzymatic assessments. In this work, a novel method for enzymatic activity determination was devised by assembling a 190 nm silica colloidal crystal (SCC) film onto a glass slide, coupled with Ordered Porous Layer Interferometry (OPLI) technology. By fixing the substrate of the enzyme on the surface of the silica sphere, a solid-liquid interface can be formed for monitoring enzymatic activity. The enzymatic activity is gauged by the change in the SCC film's thickness caused by the digestion of the loaded substrate. The procedure of chymotrypsin-mediated casein digestion was documented in real time, facilitating the examination of chymotrypsin's activity and kinetics. The newly-developed enzymatic activity determination method demonstrated exceptional sensitivity towards chymotrypsin activity, with a linear range spanning 0.0505-2.02 units per mg. Additionally, the method was extended to the assessment of fibrinolysis enzyme activity and kinetic analysis, yielding promising results. Therefore, this technique can serve as a real-time, user-friendly, cost-effective novel approach for enzymatic activity determination, providing fresh perspectives for enzymatic activity determination studies.

Comparative analysis of aptamers binding to SARS-CoV-2 N protein using capillary electrophoresis and bio-layer interferometry.

Aptamers are increasingly employed in SARS-CoV-2 theragnostics in recent years. Characterization of aptamers, testing affinity and kinetic parameters (e.g., equilibrium dissociation constant (KD), kon, and koff), can be done by several methods and influenced by many factors. This study aims to characterize the binding of aptamers to SARS-CoV-2 nucleocapsid (N) protein using capillary electrophoresis (CE) and bio-layer interferometry (BLI). These two analytical methods differ by how the aptamer binds to its target protein once the aptamer, as a capture ligand, is partitioned in solution (CE) or immobilized on the biosensor (BLI). With CE, the KD values of the N-binding aptamers (tNSP1, tNSP2, and tNSP3) were determined to be 18 ± 4 nM, 45 ± 11 nM, and 32 ± 7 nM, respectively, while the KD measurements by BLI yielded 4.8 ± 0.6, 4.5 ± 0.5, and 2.9 ± 0.3 nM, respectively. CE results showed a higher KD across all aptamers tested. The differences in the steric hindrance and confirmational structures of the aptamers immobilized on the BLI biosensors versus those suspended in the CE sample solution affect the molecular interactions between aptamers and the target proteins. Moreover, the buffer composition including pH and ionic strength can influence the stability of aptamer structures, or aptamer-protein complexes. All these variables affect the binding and calculated KD. In this sense, a KD value alone is not sufficient to make comparisons between aptamers; instead, the entire experimental setup should also be considered. This is particularly important when implementing aptamers in different bioanalytical systems.

Impact of the preservation media on ex vivo bone samples for full field mechanical testing.

The preservation method to store bone tissue for posterior analysis is a widespread practice. However, the method's potential influence on the material's mechanical properties is often overlooked during single-point experimentation. Saline and formaldehyde solutions are the most common among the employed preservation media. A full field analysis of the mice femoral bone deformation using non-destructive optical techniques is conducted to assess the influence of the storage media on the viscoelastic properties of the tissue. Three different groups are subjected to a standard three-point bending test. The first group is the control, with fresh post-mortem samples. The second and third groups used saline and formaldehyde solutions, respectively. During the mechanical test, the bone's surface and internal deformation are monitored simultaneously using digital holographic interferometry and Fourier-domain optical coherence tomography. A mechanical comparison among the three groups is presented. The results show that after 48 h of immersion in saline solution, the mice bones keep their viscoelastic behavior similar to fresh bones. Meanwhile, 48 h in formaldehyde modifies the response and affects the marrow structure. The high sensitivity of the optical phase also makes it possible to observe changes in the anisotropy of the samples. As a comparison, Raman spectroscopy analyzes the three bone groups to prove that the preservation media does not affect a single-point inspection.

Comparison of measurements and calculated lens power using three biometers: a Scheimpflug tomographer with partial coherence interferometry and two swept source optical coherence tomographers.

PURPOSE: To compare the biometric measurements obtained from the Pentacam AXL Wave, IOLMaster 700, and ANTERION and calculate the recommended intraocular lens power using the Barrett Formulae. METHODS: This was a retrospective cross-sectional study of patients who underwent biometry using the Pentacam AXL Wave, IOLMaster 700, and ANTERION. Flat keratometry (K1), steep keratometry (K2), anterior chamber depth (ACD), and axial length (AL) from each device were measured and compared. These parameters were used to calculate the recommended IOL powers using the Barrett formula. RESULTS: The study included 252 eyes of 153 patients. The IOLMaster had the highest acquisition rate among the two biometers. The Pentacam obtained the shortest mean AL, the IOLMaster measured the highest mean keratometry values, and the ANTERION measured the highest mean ACD. In terms of pairwise comparisons, keratometry and axial length were not significantly different between the Pentacam-IOLMaster and ANTERION-IOLMaster groups, while the rest of the pairwise comparisons were statistically significant. In nontoric and toric eyes, 35-45% of patients recommended the same sphere of IOL power. In another 30-40%, the Pentacam and ANTERION recommended an IOL power one step greater than that of the IOLMaster-derived data. 50% of the study population recommended the same toric-cylinder IOL power. CONCLUSIONS: The Pentacam AXL Wave, IOLMaster 700, and ANTERION can reliably provide data for IOL power calculations; however, these data are not interchangeable. In nontoric and toric eyes, 35-45% of cases recommended the same sphere IOL power, and in another 30-40%, the Pentacam and ANTERION recommended one-step higher IOL power than the IOLMaster-derived data. In targeting emmetropia, selecting the first plus IOL power is advisable when using the Pentacam and ANTERION to approximate the IOL power calculations recommended by the IOLMaster 700.

Effects of short-term exposure to red or near-infrared light on axial length in young human subjects.

PURPOSE: To determine whether visible light is needed to elicit axial eye shortening by exposure to long wavelength light. METHODS: Incoherent narrow-band red (620 ± 10 nm) or near-infrared (NIR, 875 ± 30 nm) light was generated by an array of light-emitting diodes (LEDs) and projected monocularly in 17 myopic and 13 non-myopic subjects for 10 min. The fellow eye was occluded. Light sources were positioned 50 cm from the eye in a dark room. Axial length (AL) was measured before and after the exposure using low-coherence interferometry. RESULTS: Non-myopic subjects responded to red light with significant eye shortening, while NIR light induced minor axial elongation (-13.3 ± 17.3 µm vs. +6.5 ± 11.6 µm, respectively, p = 0.005). Only 41% of the myopic subjects responded to red light exposure with a decrease in AL and changes were therefore, on average, not significantly different from those observed with NIR light (+0.2 ± 12.1 µm vs. +1.1 ± 11.2 µm, respectively, p = 0.83). Interestingly, there was a significant correlation between refractive error and induced changes in AL after exposure to NIR light in myopic eyes (r(15) = -0.52, p = 0.03) and induced changes in AL after exposure to red light in non-myopic eyes (r(11) = 0.62, p = 0.02), with more induced axial elongation with increasing refractive error. CONCLUSIONS: Incoherent narrow-band red light at 620 nm induced axial shortening in 77% of non-myopic and 41% of myopic eyes. NIR light did not induce any significant changes in AL in either refractive group, suggesting that the beneficial effect of red laser light therapy on myopia progression requires visible stimulation and not simply thermal energy.

Protein-Protein Binding Kinetics by Biolayer Interferometry.

The specific kinetics and thermodynamics of protein-protein interactions underlie the molecular mechanisms of cellular functions; hence the characterization of these interaction parameters is central to the quantitative understanding of physiological and pathological processes. Many methods have been developed to study protein-protein interactions, which differ in various features including the interaction detection principle, the sensitivity, whether the method operates in vivo, in vitro, or in silico, the temperature control, the use of labels, immobilization, the amount of sample required, the number of measurements that can be accomplished simultaneously, or the cost. Bio-Layer Interferometry (BLI) is a label-free biophysical method to measure the kinetics of protein-protein interactions. Label-free interaction assays are a broad family of methods that do not require protein modifications (other than immobilization) or labels such as fusions with fluorescent proteins or transactivating domains or chemical modifications like biotinylation or reaction with radionuclides. Besides BLI, other label-free techniques that are widely used for determining protein-protein interactions include surface plasmon resonance (SPR), thermophoresis, and isothermal titration calorimetry (ITC), among others.

High-resolution multicontrast tomography with an X-ray microarray anode-structured target source.

Multicontrast X-ray imaging with high resolution and sensitivity using Talbot-Lau interferometry (TLI) offers unique imaging capabilities that are important to a wide range of applications, including the study of morphological features with different physical properties in biological specimens. The conventional X-ray TLI approach relies on an absorption grating to create an array of micrometer-sized X-ray sources, posing numerous limitations, including technical challenges associated with grating fabrication for high-energy operations. We overcome these limitations by developing a TLI system with a microarray anode-structured target (MAAST) source. The MAAST features an array of precisely controlled microstructured metal inserts embedded in a diamond substrate. Using this TLI system, tomography of a Drum fish tooth with high resolution and tri-contrast (absorption, phase, and scattering) reveals useful complementary structural information that is inaccessible otherwise. The results highlight the exceptional capability of high-resolution multicontrast X-ray tomography empowered by the MAAST-based TLI method in biomedical applications.