Fungal melanin suppresses airway epithelial chemokine secretion through blockade of calcium fluxing.

Respiratory infections caused by the human fungal pathogen Aspergillus fumigatus are a major cause of mortality for immunocompromised patients. Exposure to these pathogens occurs through inhalation, although the role of the respiratory epithelium in disease pathogenesis has not been fully defined. Employing a primary human airway epithelial model, we demonstrate that fungal melanins potently block the post-translational secretion of the chemokines CXCL1 and CXCL8 independent of transcription or the requirement of melanin to be phagocytosed, leading to a significant reduction in neutrophil recruitment to the apical airway both in vitro and in vivo. Aspergillus-derived melanin, a major constituent of the fungal cell wall, dampened airway epithelial chemokine secretion in response to fungi, bacteria, and exogenous cytokines. Furthermore, melanin muted pathogen-mediated calcium fluxing and hindered actin filamentation. Taken together, our results reveal a critical role for melanin interaction with airway epithelium in shaping the host response to fungal and bacterial pathogens.

BTK inhibitor-induced defects in human neutrophil effector activity against Aspergillus fumigatus are restored by TNF-α.

Inhibition of Bruton's tyrosine kinase (BTK) through covalent modifications of its active site (e.g., ibrutinib [IBT]) is a preferred treatment for multiple B cell malignancies. However, IBT-treated patients are more susceptible to invasive fungal infections, although the mechanism is poorly understood. Neutrophils are the primary line of defense against these infections; therefore, we examined the effect of IBT on primary human neutrophil effector activity against Aspergillus fumigatus. IBT significantly impaired the ability of neutrophils to kill A. fumigatus and potently inhibited reactive oxygen species (ROS) production, chemotaxis, and phagocytosis. Importantly, exogenous TNF-α fully compensated for defects imposed by IBT and newer-generation BTK inhibitors and restored the ability of neutrophils to contain A. fumigatus hyphal growth. Blocking TNF-α did not affect ROS production in healthy neutrophils but prevented exogenous TNF-α from rescuing the phenotype of IBT-treated neutrophils. The restorative capacity of TNF-α was independent of transcription. Moreover, the addition of TNF-α immediately rescued ROS production in IBT-treated neutrophils, indicating that TNF-α worked through a BTK-independent signaling pathway. Finally, TNF-α restored effector activity of primary neutrophils from patients on IBT therapy. Altogether, our data indicate that TNF-α rescued the antifungal immunity block imposed by inhibition of BTK in primary human neutrophils.

Noise Tolerant Photonic Bowtie Grating Environmental Sensor.

Resonant photonic refractive index sensors have made major advances based on their high sensitivity and contact-less readout capability, which is advantageous in many areas of science and technology. A major issue for the technological implementation of such sensors is their response to external influences, such as vibrations and temperature variations; the more sensitive a sensor, the more susceptible it also becomes to external influences. Here, we introduce a novel bowtie-shaped sensor that is highly responsive to refractive index variations while compensating for temperature changes and mechanical (linear and angular) vibrations. We exemplify its capability by demonstrating the detection of salinity to a precision of 0.1%, corresponding to 2.3 × 10-4 refractive index units in the presence of temperature fluctuations and mechanical vibrations. As a second exemplar, we detected bacteria growth in a pilot industrial environment. Our results demonstrate that it is possible to translate high sensitivity resonant photonic refractive index sensors into real-world environments.

Candida albicans extracellular vesicles trigger type I IFN signalling via cGAS and STING.

The host type I interferon (IFN) pathway is a major signature of inflammation induced by the human fungal pathogen, Candida albicans. However, the molecular mechanism for activating this pathway in the host defence against C. albicans remains unknown. Here we reveal that mice lacking cyclic GMP-AMP synthase (cGAS)-stimulator of IFN genes (STING) pathway components had improved survival following an intravenous challenge by C. albicans. Biofilm-associated C. albicans DNA packaged in extracellular vesicles triggers the cGAS-STING pathway as determined by induction of interferon-stimulated genes, IFNß production, and phosphorylation of IFN regulatory factor 3 and TANK-binding kinase 1. Extracellular vesicle-induced activation of type I IFNs was independent of the Dectin-1/Card9 pathway and did not require toll-like receptor 9. Single nucleotide polymorphisms in cGAS and STING potently altered inflammatory cytokine production in human monocytes challenged by C. albicans. These studies provide insights into the early innate immune response induced by a clinically significant fungal pathogen.

Photonic Characterisation of Indium Tin Oxide as a Function of Deposition Conditions.

Indium tin oxide (ITO) has recently gained prominence as a photonic nanomaterial, for example, in modulators, tuneable metasurfaces and for epsilon-near-zero (ENZ) photonics. The optical properties of ITO are typically described by the Drude model and are strongly dependent on the deposition conditions. In the current literature, studies often make several assumptions to connect the optically measured material parameters to the electrical properties of ITO, which are not always clear, nor do they necessarily apply. Here, we present a comprehensive study of the structural, electrical, and optical properties of ITO and showed how they relate to the deposition conditions. We use guided mode resonances to determine the dispersion curves of the deposited material and relate these to structural and electrical measurements to extract all relevant material parameters. We demonstrate how the carrier density, mobility, plasma frequency, electron effective mass, and collision frequency vary as a function of deposition conditions, and that the high-frequency permittivity (쵰) can vary significantly from the value of 쵰 = 3.9 that many papers simply assume to be a constant. The depth of analysis we demonstrate allows the findings to be easily extrapolated to the photonic characterisation of other transparent conducting oxides (TCOs), whilst providing a much-needed reference for the research area.

Cross-kingdom anti-inflammatory effects of fungal melanin on airway epithelium by post-translational blockade of chemokine secretion.

Respiratory infections caused by the human fungal pathogens, Aspergillus fumigatus and Cryptococcus neoformans, are a major cause of mortality for immunocompromised patients. Exposure to these pathogens occurs through inhalation, although the role of the respiratory epithelium in disease pathogenesis has not been defined. Employing a primary human airway epithelial model, we demonstrate that fungal melanins potently block the post-translational secretion of CXCL1 and CXCL8 independent of transcription or the requirement of melanin to be phagocytosed, leading to a significant reduction of neutrophils to the apical airway both in vitro and in vivo. Aspergillus-derived melanin, a major constituent of the fungal cell wall, has far-reaching effects, dampening airway epithelial chemokine production in response to fungi, bacteria, and exogenous cytokines. Taken together, our results reveal a critical role for melanin interaction with airway epithelium in shaping the host response to fungal and bacterial pathogens.

The C-Type Lectin Receptor Dectin-2 Is a Receptor for Aspergillus fumigatus Galactomannan.

Aspergillus fumigatus is a ubiquitous environmental mold that causes significant mortality particularly among immunocompromised patients. The detection of the Aspergillus-derived carbohydrate galactomannan in patient serum and bronchoalveolar lavage fluid is the major biomarker used to detect A. fumigatus infection in clinical medicine. Despite the clinical relevance of this carbohydrate, we lack a fundamental understanding of how galactomannan is recognized by the immune system and its consequences. Galactomannan is composed of a linear mannan backbone with galactofuranose sidechains and is found both attached to the cell surface of Aspergillus and as a soluble carbohydrate in the extracellular milieu. In this study, we utilized fungal-like particles composed of highly purified Aspergillus galactomannan to identify a C-type lectin host receptor for this fungal carbohydrate. We identified a novel and specific interaction between Aspergillus galactomannan and the C-type lectin receptor Dectin-2. We demonstrate that galactomannan bound to Dectin-2 and induced Dectin-2-dependent signaling, including activation of spleen tyrosine kinase, gene transcription, and tumor necrosis factor alpha (TNF-α) production. Deficiency of Dectin-2 increased immune cell recruitment to the lungs but was dispensable for survival in a mouse model of pulmonary aspergillosis. Our results identify a novel interaction between galactomannan and Dectin-2 and demonstrate that Dectin-2 is a receptor for galactomannan, which leads to a proinflammatory immune response in the lung. IMPORTANCE Aspergillus fumigatus is a fungal pathogen that causes serious and often fatal disease in humans. The surface of Aspergillus is composed of complex sugar molecules. Recognition of these carbohydrates by immune cells by carbohydrate lectin receptors can lead to clearance of the infection or, in some cases, benefit the fungus by dampening the host response. Galactomannan is a carbohydrate that is part of the cell surface of Aspergillus but is also released during infection and is found in patient lungs as well as their bloodstreams. The significance of our research is that we have identified Dectin-2 as a mammalian immune cell receptor that recognizes, binds, and signals in response to galactomannan. These results enhance our understanding of how this carbohydrate interacts with the immune system at the site of infection and will lead to broader understanding of how release of galactomannan by Aspergillus effects the immune response in infected patients.

Mutation in protein disulfide isomerase A3 causes neurodevelopmental defects by disturbing endoplasmic reticulum proteostasis.

Recessive gene mutations underlie many developmental disorders and often lead to disabling neurological problems. Here, we report identification of a homozygous c.170G>A (p.Cys57Tyr or C57Y) mutation in the gene coding for protein disulfide isomerase A3 (PDIA3, also known as ERp57), an enzyme that catalyzes formation of disulfide bonds in the endoplasmic reticulum, to be associated with syndromic intellectual disability. Experiments in zebrafish embryos show that PDIA3C57Y expression is pathogenic and causes developmental defects such as axonal disorganization as well as skeletal abnormalities. Expression of PDIA3C57Y in the mouse hippocampus results in impaired synaptic plasticity and memory consolidation. Proteomic and functional analyses reveal that PDIA3C57Y expression leads to dysregulation of cell adhesion and actin cytoskeleton dynamics, associated with altered integrin biogenesis and reduced neuritogenesis. Biochemical studies show that PDIA3C57Y has decreased catalytic activity and forms disulfide-crosslinked aggregates that abnormally interact with chaperones in the endoplasmic reticulum. Thus, rare disease gene variant can provide insight into how perturbations of neuronal proteostasis can affect the function of the nervous system.

Dielectric nanohole array metasurface for high-resolution near-field sensing and imaging.

Dielectric metasurfaces support resonances that are widely explored both for far-field wavefront shaping and for near-field sensing and imaging. Their design explores the interplay between localised and extended resonances, with a typical trade-off between Q-factor and light localisation; high Q-factors are desirable for refractive index sensing while localisation is desirable for imaging resolution. Here, we show that a dielectric metasurface consisting of a nanohole array in amorphous silicon provides a favourable trade-off between these requirements. We have designed and realised the metasurface to support two optical modes both with sharp Fano resonances that exhibit relatively high Q-factors and strong spatial confinement, thereby concurrently optimizing the device for both imaging and biochemical sensing. For the sensing application, we demonstrate a limit of detection (LOD) as low as 1 pg/ml for Immunoglobulin G (IgG); for resonant imaging, we demonstrate a spatial resolution below 1 µm and clearly resolve individual E. coli bacteria. The combined low LOD and high spatial resolution opens new opportunities for extending cellular studies into the realm of microbiology, e.g. for studying antimicrobial susceptibility.

Attachment and antibiotic response of early-stage biofilms studied using resonant hyperspectral imaging.

Many bacterial species readily develop biofilms that act as a protective matrix against external challenge, e.g., from antimicrobial treatment. Therefore, biofilms are often responsible for persistent and recurring infections. Established methods for studying biofilms are either destructive or focus on the biofilm's surface. A non-destructive method that is sensitive to the underside of the biofilm is highly desirable, as it allows studying the penetration of antibiotics through the film. Here, we demonstrate that the high surface sensitivity of resonant hyperspectral imaging provides this capability. The method allows us to monitor the early stages of Escherichia coli biofilm formation, cell attachment and microcolony formation, in-situ and in real-time. We study the response of the biofilm to a number of different antibiotics and verify our observations using confocal microscopy. Based on this ability to closely monitor the surface-bound cells, resonant hyperspectral imaging gives new insights into the antimicrobial resistance of biofilms.

Performance limitations of resonant refractive index sensors with low-cost components.

Resonant biosensors are attractive for diagnostics because they can detect clinically relevant biomarkers with high sensitivity and in a label-free fashion. Most of the current solutions determine their detection limits in a highly stabilised laboratory environment, which does, however, not apply to real point-of-care applications. Here, we consider the more realistic scenario of low-cost components and an unstabilised environment and consider the related design implications. We find that sensors with lower quality-factor resonances are more fault tolerant, that a filtered LED lightsource is advantageous compared to a diode laser, and that a CMOS camera is preferable to a CCD camera for detection. We exemplify these findings with a guided mode resonance sensor and experimentally determine a limit of detection of 5.8 ± 1.7×10-5 refractive index units (RIU), which is backed up by a model identifying the various noise sources. Our findings will inform the design of high performance, low cost biosensors capable of operating in a real-world environment.

Extended Kalman Filtering Projection Method to Reduce the 3σ Noise Value of Optical Biosensors.

Optical biosensors have experienced a rapid growth over the past decade because of their high sensitivity and the fact that they are label-free. Many optical biosensors rely on tracking the change in a resonance signal or an interference pattern caused by the change in refractive index that occurs upon binding to a target biomarker. The most commonly used method for tracking such a signal is based on fitting the data with an appropriate mathematical function, such as a harmonic function or a Fano, Gaussian, or Lorentz function. However, these functions have limited fitting efficiency because of the deformation of data from noise. Here, we introduce an extended Kalman filter projection (EKFP) method to address the problem of resonance tracking and demonstrate that it improves the tolerance to noise, reduces the 3σ noise value, and lowers the limit of detection (LOD). We utilize the method to process the data of experiments for detecting the binding of C-reactive protein in a urine matrix with a chirped guided mode resonance sensor and are able to improve the LOD from 10 to 1 pg/mL. Our method reduces the 3σ noise value of this measurement compared to a simple Fano fit from 1.303 to 0.015 pixels. These results demonstrate the significant advantage of the EKFP method to resolving noisy data of optical biosensors.

Common-path interferometric label-free protein sensing with resonant dielectric nanostructures.

Research toward photonic biosensors for point-of-care applications and personalized medicine is driven by the need for high-sensitivity, low-cost, and reliable technology. Among the most sensitive modalities, interferometry offers particularly high performance, but typically lacks the required operational simplicity and robustness. Here, we introduce a common-path interferometric sensor based on guided-mode resonances to combine high performance with inherent stability. The sensor exploits the simultaneous excitation of two orthogonally polarized modes, and detects the relative phase change caused by biomolecular binding on the sensor surface. The wide dynamic range of the sensor, which is essential for fabrication and angle tolerance, as well as versatility, is controlled by integrating multiple, tuned structures in the field of view. This approach circumvents the trade-off between sensitivity and dynamic range, typical of other phase-sensitive modalities, without increasing complexity. Our sensor enables the challenging label-free detection of procalcitonin, a small protein (13 kDa) and biomarker for infection, at the clinically relevant concentration of 1 pg mL-1, with a signal-to-noise ratio of 35. This result indicates the utility for an exemplary application in antibiotic guidance, and opens possibilities for detecting further clinically or environmentally relevant small molecules with an intrinsically simple and robust sensing modality.

Ly6a Differential Expression in Blood-Brain Barrier Is Responsible for Strain Specific Central Nervous System Transduction Profile of AAV-PHP.B.

Adeno-associated virus (AAV) gene therapy for neurological diseases was revolutionized by the discovery that AAV9 crosses the blood-brain barrier (BBB) after systemic administration. Transformative results have been documented in various inherited diseases, but overall neuronal transduction efficiency is relatively low. The recent development of AAV-PHP.B with ∼60-fold higher efficiency than AAV9 in transducing the adult mouse brain was the major first step toward acquiring the ability to deliver genes to the majority of cells in the central nervous system (CNS). However, little is known about the mechanism utilized by AAV to cross the BBB, and how it may diverge across species. In this study, we show that AAV-PHP.B is ineffective for systemic CNS gene transfer in the inbred strains BALB/cJ, BALB/cByJ, A/J, NOD/ShiLtJ, NZO/HILtJ, C3H/HeJ, and CBA/J mice, but it is highly potent in C57BL/6J, FVB/NJ, DBA/2J, 129S1/SvImJ, and AKR/J mice and also the outbred strain CD-1. We used the power of classical genetics to uncover the molecular mechanisms AAV-PHP.B engages to transduce CNS at high efficiency, and by quantitative trait locus mapping we identify a 6 Mb region in chromosome 15 with an logarithm of the odds (LOD) score ∼20, including single nucleotide polymorphisms in the coding region of 9 different genes. Comparison of the publicly available data on the genome sequence of 16 different mouse strains, combined with RNA-seq data analysis of brain microcapillary endothelia, led us to conclude that the expression level of Ly6a is likely the determining factor for differential efficacy of AAV-PHP.B in transducing the CNS across different mouse strains.

Impact of Treatment Time on the Long-Term Outcome of Stroke Patients Treated With Mechanical Thrombectomy.

OBJECTIVE: To assess the long-term functional outcome of stroke in patients treated with mechanical thrombectomy (MT) performed during work hours (on-hours) versus after-hours, weekends, and official holidays (off-hours). METHODS: Data on all patients receiving MT at a comprehensive stroke center was collected between December 2014-December 2016. Our primary outcomes were the discharge and 90-day modified Rankin Scale (mRS). We developed propensity scores for off-hours treatment and used inverse probability of treatment weights to address confounding. We estimated logistic regression to assess the relationship between off-hours treatment and favorable patient outcomes. Independent variables include receiving thrombectomy during the off-hours, admission National Institute of Health Stroke Scale (NIHSS), door to groin time in minutes, age, and race. RESULTS: During the study period, 80 (41%) patients underwent thrombectomy during on-hours and 116 (59%) during off-hours. Mean age was 69.1 years for the on-hours group and 64.1 years for the off-hours group (P = .02). There were no statistically significant differences in median admission NIHSS, rate of alteplase administration, mean time from last known well to thrombectomy, rate of revascularization, and rate of hemorrhagic transformation between the 2 groups. Logistic regression analysis showed the probability of a favorable outcome at discharge (mRS ≤ 2) is 12.6 % lower for off-hours patients (P = .038, [95%CI -.25 to -.01]). For patients with a 90-day mRS (n = 117), the probability of a favorable outcome was 18.7% lower for those treated during the off-hours (P = .029, [95%CI -.36 to -.02]). CONCLUSIONS: There is a higher probability of a good functional outcome in acute ischemic stroke patients who receive MT when performed during regular work hours.

Long-Term Functional Outcome of Telestroke Patients Treated Under Drip-and-Stay Paradigm Compared with Patients Treated in a Comprehensive Stroke Center: A Single Center Experience.

Objective:The purpose of this study is to compare long-term functional outcome for patients who receive intravenous alteplase (tPA) at a primary stroke center (spoke) through telestroke consultations and remain at the spoke (drip-and-stay) with that for patients who receive tPA at the comprehensive stroke center (hub).Methods:Data on baseline characteristics, stroke severity on presentation, door to needle (DTN) time, the rate of symptomatic intracerebral hemorrhage (sICH) and long-term outcomes for all patients evaluated at the Medical University of South Carolina (MUSC) hub and MUSC telestroke network spoke sites between January 2016 and March 2017 were collected. Eligible patients received tPA at either the spoke or hub location during the study period. Patients who received mechanical thrombectomy were excluded from the study. Functional outcome was assessed with 90-day modified Rankin Scale (mRS). Descriptive statistics were used to compare patient demographics and clinical outcomes across the two groups.Results:Total of 426 were identified (60 hub patients and 366 drip-and-stay patients). There were no significant differences in patient age, sex, admission National Institute of Health Stroke Scale (NIHSS), sICH, or DTN times between the two groups. mRS of 0-2 at 90 days was achieved in 37 (61.7%) of the hub and in 255 (69.7%) in the drip-and-stay patients (p = 0.216). On regression analysis, there was no difference in the adjusted relative risk of having a lower mRS between drip-and-stay and hub patients (incidence rate ratio 1.14, p = 0.278, 95% confidence interval [0.9-1.43]).Conclusion:Our study shows no difference in the long-term functional outcome for patients who received tPA through telestroke consultation and remained at spoke hospitals (drip-and-stay) compared with patients who received tPA at the hub.

Shaping of Shared Autonomous Solutions With Minimal Interaction.

A fundamental problem in creating successful shared autonomy systems is enabling efficient specification of the problem for which an autonomous system can generate a solution. We present a general paradigm, Interactive Shared Solution Shaping (IS3), broadly applied to shared autonomous systems where a human can use their domain knowledge to interactively provide feedback during the autonomous planning process. We hypothesize that this interaction process can be optimized so that with minimal interaction, near-optimal solutions can be achieved. We examine this hypothesis in the space of resource-constrained mobile search and surveillance and show that without directly instructing a robot or complete communication of a believed target distribution, the human teammate is able to successfully shape the generation of an autonomous search route. This ability is demonstrated in three experiments that show (1) the IS3 approach can improve performance in that routes generated from interactions in general reduce the variance of the target detection performance, and increase overall target detection; (2) the entire IS3 autonomous route generation system's performance, including cost of interaction along with movement cost, experiences a tradeoff between performance vs. numbers of interactions that can be optimized; (3) the IS3 autonomous route generation system is able to perform within constraints by generating tours that stay under budget when executed by a real robot in a realistic field environment.

Chirped guided-mode resonance biosensor.

Advanced biomedical diagnostic technologies fulfill an important role in improving health and well-being in society. A large number of excellent technologies have already been introduced and have given rise to the "lab-on-a-chip" paradigm. Most of these technologies, however, require additional instrumentation for interfacing and readout, so they are often confined to the laboratory and are not suitable for use in the field or in wider clinical practice. Other technologies require a light coupling element, such as a grating coupler or a fiber coupler, which complicates packaging. Here, we introduce a novel biosensor based on a chirped guided-mode resonant grating. The chirped grating combines the sensing function with the readout function by translating spectral information into spatial information that is easily read out with a simple CMOS camera. We demonstrate a refractive index sensitivity of 137 nm/RIU and an extrapolated limit of detection of 267 pM for the specific binding of an immunoglobulin G antibody. The chirped guided-mode resonance approach introduces a new degree of freedom for sensing biomedical information that combines high sensitivity with autonomous operation. We estimate that the cost of components is U.S. $10 or less when mass manufactured, so the technology has the potential to truly transform point-of-care applications.

High speed e-beam writing for large area photonic nanostructures - a choice of parameters.

Photonic nanostructures are used for many optical systems and applications. However, some high-end applications require the use of electron-beam lithography (EBL) to generate such nanostructures. An important technological bottleneck is the exposure time of the EBL systems, which can exceed 24 hours per 1 cm(2). Here, we have developed a method based on a target function to systematically increase the writing speed of EBL. As an example, we use as the target function the fidelity of the Fourier Transform spectra of nanostructures that are designed for thin film light trapping applications, and optimize the full parameter space of the lithography process. Finally, we are able to reduce the exposure time by a factor of 5.5 without loss of photonic performance. We show that the performances of the fastest written structures are identical to the original ones within experimental error. As the target function can be varied according to different purposes, the method is also applicable to guided mode resonant grating and many other areas. These findings contribute to the advancement of EBL and point towards making the technology more attractive for commercial applications.

Dual gratings for enhanced light trapping in thin-film solar cells by a layer-transfer technique.

Thin film solar cells benefit significantly from the enhanced light trapping offered by photonic nanostructures. The thin film is typically patterned on one side only due to technological constraints. The ability to independently pattern both sides of the thin film increases the degrees of freedom available to the designer, as different functions can be combined, such as the reduction of surface reflection and the excitation of quasiguided modes for enhanced light absorption. Here, we demonstrate a technique based on simple layer transfer that allows us to independently pattern both sides of the thin film leading to enhanced light trapping. We used a 400 nm thin film of amorphous hydrogenated silicon and two simple 2D gratings for this proof-of-principle demonstration. Since the technique imposes no restrictions on the design parameters, any type of structure can be made.