Plasma collagen neoepitopes are associated with multiorgan disease in the ACCESS and GRADS sarcoidosis cohorts.

INTRODUCTION: The pathogenesis of sarcoidosis involves tissue remodelling mediated by the accumulation of abnormal extracellular matrix, which is partly the result of an imbalance in collagen synthesis, cross-linking and degradation. During this process, collagen fragments or neoepitopes, are released into the circulation. The significance of these circulating collagen neoepitopes in sarcoidosis remains unknown. METHODS: We employed plasma samples from patients with sarcoidosis enrolled in A Case Control Etiologic Study of Sarcoidosis (ACCESS) and Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS), and healthy control patients recruited from the Yale community. Plasma concentrations of type III and VI collagen degradation (C3M and C6M) and formation (PRO-C3 and PRO-C6) were quantified via neoepitope-specific competitive ELISA, and statistical associations were sought with clinical phenotypes. RESULTS: Relative to healthy controls, the plasma of both sarcoidosis cohorts was enriched for C3M and C6M, irrespective of corticosteroid use and disease duration. While circulating collagen neoepitopes were independent of Scadding stage, there was a significant association between multiorgan disease and PRO-C3, PRO-C6 and C3M in the ACCESS cohort; PRO-C3 and C6M displayed this property in GRADS. These findings were unrelated to plasma levels of interleukin-4 (IL-4), IL-5, IL-6, IL-9, IL-10 and IL-13. Moreover, PRO-C3 was associated with dermatological disease in both cohorts. DISCUSSION: In two well-characterised sarcoidosis cohorts, we discovered that the plasma is enriched for neoepitopes of collagen degradation (C3M and C6M). In multiorgan disease, there was an association with circulating neoepitopes of type III formation (PRO-C3), perhaps mediated by dermatological sarcoidosis. Further investigation in this arena has the potential to foster new insights into the pathogenic mechanisms of this complex disease.

cGAS Expression is Enhanced in Systemic Sclerosis Associated Interstitial Lung Disease and Stimulates Inflammatory Myofibroblast Activation.

Objective: The lungs of patients with Systemic Sclerosis Associated Interstitial Lung Disease (SSc-ILD) contain inflammatory myofibroblasts arising in association with fibrotic stimuli and perturbed innate immunity. The innate immune DNA binding receptor Cyclic GMP-AMP synthase (cGAS) is implicated in inflammation and fibrosis, but its involvement in SSc-ILD remains unknown. We examined cGAS expression, activity, and therapeutic potential in SSc-ILD using cultured fibroblasts, precision cut lung slices (PCLS), and a well-accepted animal model. Methods: Expression and localization of cGAS, cytokines, and type 1 interferons were evaluated in SSc-ILD lung tissues, bronchoalveolar lavage (BAL), and isolated lung fibroblasts. CGAS activation was assessed in a publicly available SSc-ILD single cell RNA sequencing dataset. Production of cytokines, type 1 interferons, and αSMA elicited by TGFß1 or local substrate stiffness were measured in normal human lung fibroblasts (NHLFs) via qRT-PCR, ELISA, and immunofluorescence. Small molecule cGAS inhibition was tested in cultured fibroblasts, human PCLS, and the bleomycin pulmonary fibrosis model. Results: SSc-ILD lung tissue and BAL are enriched for cGAS, cytokines, and type 1 interferons. The cGAS pathway shows constitutive activation in SSc-ILD fibroblasts and is inducible in NHLFs by TGFß1 or mechanical stimuli. In these settings, and in human PCLS, cGAS expression is paralleled by the production of cytokines, type 1 interferons, and αSMA that are mitigated by a small molecule cGAS inhibitor. These findings are recapitulated in the bleomycin mouse model. Conclusion: cGAS signaling contributes to pathogenic inflammatory myofibroblast phenotypes in SSc-ILD. Inhibiting cGAS or its downstream effectors represents a novel therapeutic approach.

Toll-like Receptor 9 Inhibition Mitigates Fibroproliferative Responses in Translational Models of Pulmonary Fibrosis.

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease for which current treatment options only slow clinical progression. Previously, we identified a subset of patients with IPF with an accelerated disease course associated with fibroblast expression of Toll-Like Receptor 9 (TLR9) mediated by interactions with its ligand mitochondrial DNA (mtDNA). OBJECTIVES: We aimed to show that TLR9 activation induces fibroproliferative responses that are abrogated by its antagonism by using two commercially-available indirect inhibitors and a proprietary, selective direct small molecule inhibitor. METHODS: We employed two independent cohorts of patients with IPF, multiple in vitro fibroblast cell culture platforms, an in vivo mouse model, and an ex vivo human precision cut lung slices system to investigate the clinical and biologic significance of TLR9 in this disease. MEASUREMENTS AND MAIN RESULTS: In two independent IPF cohorts, plasma mtDNA activates TLR9 in a manner associated with the expression of MCP-1, IL-6, TNFα, and IP-10 and worsened transplant-free survival. Our cell culture platform showed that TLR9 mediates fibroblast activation via TGFß1 and stiff substrates, and that its antagonism, particularly direct inhibition, ameliorates this process, including production of these TLR9 associated pharmacodynamic endpoints. We further demonstrated that direct TLR9 inhibition mitigates these fibroproliferative responses in our in vivo and ex vivo models of pulmonary fibrosis. CONCLUSIONS: In this novel study, we found that direct TLR9 inhibition mitigates fibroproliferative responses in preclinical models of pulmonary fibrosis. Our work demonstrates the therapeutic potential of direct TLR9 antagonism in IPF and related fibrotic lung diseases.

Intranasal neomycin evokes broad-spectrum antiviral immunity in the upper respiratory tract.

Respiratory virus infections in humans cause a broad-spectrum of diseases that result in substantial morbidity and mortality annually worldwide. To reduce the global burden of respiratory viral diseases, preventative and therapeutic interventions that are accessible and effective are urgently needed, especially in countries that are disproportionately affected. Repurposing generic medicine has the potential to bring new treatments for infectious diseases to patients efficiently and equitably. In this study, we found that intranasal delivery of neomycin, a generic aminoglycoside antibiotic, induces the expression of interferon-stimulated genes (ISGs) in the nasal mucosa that is independent of the commensal microbiota. Prophylactic or therapeutic administration of neomycin provided significant protection against upper respiratory infection and lethal disease in a mouse model of COVID-19. Furthermore, neomycin treatment protected Mx1 congenic mice from upper and lower respiratory infections with a highly virulent strain of influenza A virus. In Syrian hamsters, neomycin treatment potently mitigated contact transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In healthy humans, intranasal application of neomycin-containing Neosporin ointment was well tolerated and effective at inducing ISG expression in the nose in a subset of participants. These findings suggest that neomycin has the potential to be harnessed as a host-directed antiviral strategy for the prevention and treatment of respiratory viral infections.

Damage sensing through TLR9 Promotes Viral Clearance and Recovery During Influenza Infection.

Host response aimed at eliminating the infecting pathogen, as well as the pathogen itself, can cause tissue injury. Tissue injury leads to the release of a myriad of cellular components including mitochondrial DNA, which the host senses through pattern recognition receptors. How the sensing of tissue injury by the host shapes the anti-pathogen response remains poorly understood. In this study, we utilized mice that are deficient in toll-like receptor-9 (TLR9), which binds to unmethylated CpG DNA sequences such as those present in bacterial and mitochondrial DNA. To avoid direct pathogen sensing by TLR9, we utilized the influenza virus, which lacks ligands for TLR9, to determine how damage sensing by TLR9 contributes to anti-influenza immunity. Our data show that TLR9-mediated sensing of tissue damage promotes an inflammatory response during early infection, driven by the myeloid cells and associated cytokine responses. Along with the diminished inflammatory response, the absence of damage sensing through TLR9 led to impaired viral clearance manifested as a higher and prolonged influenza burden in the lung. The absence of TLR9 led to extensive infection of myeloid cells including monocytes and macrophages rendering them highly inflammatory, despite having a low initial inflammatory response. The persistent inflammation driven by infected myeloid cells led to persistent lung injury and impaired recovery in influenza-infected TLR9-/- mice. Further, we show elevated circulating TLR9 ligands in the plasma samples of patients with influenza, demonstrating its clinical relevance. Overall, over data show an essential role of damage sensing through TLR9 in promoting anti-influenza immunity.

Targeting the NLRP3 inflammasome and associated cytokines in scleroderma associated interstitial lung disease.

SSc-ILD (scleroderma associated interstitial lung disease) is a complex rheumatic disease characterized in part by immune dysregulation leading to the progressive fibrotic replacement of normal lung architecture. Because improved treatment options are sorely needed, additional study of the fibroproliferative mechanisms mediating this disease has the potential to accelerate development of novel therapies. The contribution of innate immunity is an emerging area of investigation in SSc-ILD as recent work has demonstrated the mechanistic and clinical significance of the NLRP3 inflammasome and its associated cytokines of TNFα (tumor necrosis factor alpha), IL-1ß (interleukin-1 beta), and IL-18 in this disease. In this review, we will highlight novel pathophysiologic insights afforded by these studies and the potential of leveraging this complex biology for clinical benefit.

Use of a dental surveyor to ensure optimal seating of implant overdenture attachments.

Many implant overdenture attachments accommodate divergent abutments. However, there can be instances where the denture base resin surrounding the abutment may impede seating by binding on the axial surface(s) of the abutment. This article describes the use of a dental surveyor to aid clinicians in determining where the resin denture base might be preventing the seating of overdenture attachments. The surveyor can be used for judicious adjustment to allow optimal seating of the attachments.

Digital scanning to aid guiding plane and rest seat preparations for removable partial dentures.

This article describes the use of a digital intraoral scanner to aid clinicians making rest seat and guiding plane preparations, simplifying the procedure and reducing the time needed to analyze the abutment modifications. No additional equipment is required other than a diagnostic cast and a digital scanner with preparation comparison software.

Coexistence of ice clusters and liquid-like water clusters on the Ru(0001) surface.

The RAIRS spectra of water adsorbed on Ru(0001) at 85 K are recorded from 600 cm-1 to 4000 cm-1. Measured at water coverages from 0.13 ML to 2.0 ML, the RAIRS spectra suggest that chemisorption of water on Ru(0001) depends on coverage. Water adsorbs on a clean Ru surface as chemisorbed ice-like clusters (likely through an O-Ru bond) up to 0.33 ML. Above this coverage, the chemisorbed layer saturates. Upon more exposure, water adsorbs as a liquid-like H-bonded layer without bonding to the Ru substrate. The chemisorbed water absorbs 7 times less IR per molecule than the liquid-like structure, which indicates that the orientation of the chemisorbed water is more parallel to the surface. Additionally, the influence of water-Ru bonding on H-bonding is reflected in the OH symmetric stretching mode. Under perturbation from water-Ru bonding, a large red shift (40 cm-1) in the free OH stretching frequency is observed in the chemisorbed clusters. By deconvoluting the main H-bonded OH stretching peak into five Gaussian sub-bands at 2945 ± 5 cm-1, 3210 ± 5 cm-1, 3300 ± 15 cm-1, 3430 ± 5 cm-1 and 3570 ± 10 cm-1, changes in the H-bonding network are rationalized in terms of H-bonding motifs. The donor-acceptor-acceptor motif is significant only in the chemisorbed clusters. On the other hand, the donor-acceptor motif dominates in the liquid-like structure, which increases the disorder present in the adlayer. Although chemisorption is suppressed above 0.33 ML, no structural changes in the ice-like clusters are observed up to multilayer coverage. Therefore, ice-like and liquid-like water coexist in a meta-stable state at 85 K.

Two-octave spanning supercontinuum generation in stoichiometric silicon nitride waveguides pumped at telecom wavelengths.

We demonstrate supercontinuum generation in stoichiometric silicon nitride (Si3N4 in SiO2) integrated optical waveguides, pumped at telecommunication wavelengths. The pump laser is a mode-locked erbium fiber laser at a wavelength of 1.56 µm with a pulse duration of 120 fs. With a waveguide-internal pulse energy of 1.4 nJ and a waveguide with 1.0 µm × 0.9 µm cross section, designed for anomalous dispersion across the 1500 nm telecommunication range, the output spectrum extends from the visible, at around 526 nm, up to the mid-infrared, at least to 2.6 µm, the instrumental limit of our detection. This output spans more than 2.2 octaves (454 THz at the -30 dB level). The measured output spectra agree well with theoretical modeling based on the generalized nonlinear Schrödinger equation. The infrared part of the supercontinuum spectra shifts progressively towards the mid-infrared, well beyond 2.6 µm, by increasing the width of the waveguides.

On-chip visible-to-infrared supercontinuum generation with more than 495 THz spectral bandwidth.

We report ultra-broadband supercontinuum generation in high-confinement Si3N4 integrated optical waveguides. The spectrum extends through the visible (from 470 nm) to the infrared spectral range (2130 nm) comprising a spectral bandwidth wider than 495 THz, which is the widest supercontinuum spectrum generated on a chip.

High confinement, high yield Si(3)N(4) waveguides for nonlinear optical applications.

In this paper we present a novel fabrication technique for silicon nitride (Si(3)N(4)) waveguides with a thickness of up to 900 nm, which are suitable for nonlinear optical applications. The fabrication method is based on etching trenches in thermally oxidized silicon and filling the trenches with Si(3)N(4). Using this technique no stress-induced cracks in the Si(3)N(4) layer were observed resulting in a high yield of devices on the wafer. The propagation losses of the obtained waveguides were measured to be as low as 0.4 dB/cm at a wavelength of around 1550 nm.

The effectiveness of using a patient simulator to teach light-curing skills.

BACKGROUND: The authors evaluated the effectiveness of using a patient simulator (MARC Patient Simulator [MARC PS], BlueLight analytics, Halifax, Nova Scotia, Canada), to instruct dental students (DS) on how to deliver energy optimally to a restoration from a curing light. Five months later, the authors evaluated the retention of the instruction provided to the DS. METHODS: Toward the end of the DS' first year of dental education, the authors evaluated the light-curing techniques of one-half of the class of first-year DS (Group 1) before and after receiving instruction by means of the patient simulator. Five months later, they retested DS in Group 1 and tested the remaining first-year DS who were then second-year DS and who had received no instruction by means of the patient simulator (Group 2). They gave DS in Group 1 and Group 2 MARC PS instruction and retested them. The authors also the tested fourth-year DS (Group 3) and dentists (Group 4) by using the MARC PS before giving any instruction by means of the MARC PS. RESULTS: The results of one-way analysis of variance (ANOVA) showed that there were no significant differences in the ability of dentists and DS to light cure a simulated restoration before they received instruction by means of the patient simulator (P = .26). The results of two-way ANOVA and Fisher protected least significant difference tests showed that after receiving instruction by means of the patient simulator, DS delivered significantly more energy to a simulated restoration, and this skill was retained. There were no significant differences between DS in Group 1 and Group 2 after they had received instruction by means of the patient simulator. CONCLUSIONS: The abilities of dentists and DS to light cure a simulated restoration were not significantly different. Hands-on teaching using a patient simulator enhanced the ability of DS to use a curing light. This skill was retained for at least five months. PRACTICAL IMPLICATIONS: The education provided to dentists and DS is insufficient to teach them how to deliver the optimum amount of energy from a curing light. Better teaching and understanding of the importance of light curing is required.

Integrated CARS source based on seeded four-wave mixing in silicon nitride.

We present a theoretical investigation of an integrated nonlinear light source for coherent anti-Stokes Raman scattering (CARS) based on silicon nitride waveguides. Wavelength tunable and temporally synchronized signal and idler pulses are obtained by using seeded four-wave mixing. We find that the calculated input pump power needed for nonlinear wavelength generation is more than one order of magnitude lower than in previously reported approaches based on optical fibers. The tuning range of the wavelength conversion was calculated to be 1418 nm to 1518 nm (idler) and 788 nm to 857 nm (signal), which corresponds to a coverage of vibrational transitions from 2350 cm-1 to 2810 cm-1. A maximum conversion efficiency of 19.1% at a peak pump power of 300 W is predicted.

The noise-limited-resolution for stimulated emission depletion microscopy of diffusing particles.

With recent developments in microscopy, such as stimulated emission depletion (STED) microscopy, far-field imaging at resolutions better than the diffraction limit is now a commercially available technique. Here, we show that, in the special case of a diffusive regime, the noise-limited resolution of STED imaging is independent of the saturation intensity of the fluorescent label. Thermal motion limits the signal integration time, which, for a given excited-state lifetime, limits the total number of photons available for detection.

Ellipsometry with randomly varying polarization states.

We show that, under the right conditions, one can make highly accurate polarization-based measurements without knowing the absolute polarization state of the probing light field. It is shown that light, passed through a randomly varying birefringent material has a well-defined orbit on the Poincar sphere, which we term a generalized polarization state, that is preserved. Changes to the generalized polarization state can then be used in place of the absolute polarization states that make up the generalized state, to measure the change in polarization due to a sample under investigation. We illustrate the usefulness of this analysis approach by demonstrating fiber-based ellipsometry, where the polarization state of the probe light is unknown, and, yet, the ellipsometric angles of the investigated sample (Ψ and Δ) are obtained with an accuracy comparable to that of conventional ellipsometry instruments by measuring changes to the generalized polarization state.

Effect of instruction on dental students’ ability to light-cure a simulated restoration.

OBJECTIVE: To measure the light energy that dental students delivered to a simulated Class I restoration before training, immediately after training and 4 months after training. METHODS: Thirty-eight (38) dental students used a single light-emitting diode curing light (SmartLite iQ2, Dentsply) to cure, for 10 seconds, a simulated Class I restoration positioned in the Managing Accurate Resin Curing - Patient Simulator (BlueLight analytics inc.). The students then attended an instructional lecture and received individualized instruction on optimizing their light-curing technique. The students were retested immediately after instruction and again 4 months later (without further instruction). The irradiance and energy delivered during light-curing were calculated for each student at all 3 time points. Mean values were calculated and compared. RESULTS: Before instruction, the students delivered between 0.1 and 7.2 J/cm2 of energy (mean ± standard deviation [SD] 4.1 ± 1.7 J/cm2). After instruction, the same students delivered between 5.8 and 7.5 J/cm2 of energy (mean ± SD 6.7 ± 0.4 J/cm2). Analysis of variance and Fisher's Protected Least Significant Difference tests showed that instruction with the patient simulator led to a significant improvement in the amount of energy delivered and that the students retained this information. When retested 4 months later, the students delivered between 4.2 and 7.9 J/cm2 of energy (mean ± SD 6.1 ±1.1 J/cm2). Although this was less energy than immediately after instruction, the decline was not significant (p = 0.44). CONCLUSIONS: Provision of immediate feedback on light-curing technique and instruction on how to avoid mistakes led to a significant and lasting improvement in the amount of energy delivered by the students.

Incoherently pumped continuous wave optical parametric oscillator broadened by non-collinear phasematching.

In this paper, we report on a singly resonant optical parametric oscillator (OPO) pumped by an amplified spontaneous emission (ASE) source. The pump focusing conditions allow non-collinear phasematching, which resulted in a 230 nm (190 cm(-1)) spectral bandwidth. Calculations indicate that such phasematching schemes may be used to further broaden OPO spectral bandwidths.

One-Watt level mid-IR output, singly resonant, continuous-wave optical parametric oscillator pumped by a monolithic diode laser.

We report more than 1.1 Watt of idler power at 3373 nm in a singly resonant optical parametric oscillator (SRO), directly pumped by a single-frequency monolithic tapered diode laser. The SRO is based on a periodically poled MgO:LiNbO3 crystal in a four mirror cavity and is excited by 8.05 W of 1062 nm radiation. The SRO pump power at threshold is 4 W. The internal slope-efficiency and conversion efficiency reach 89% and 44% respectively. The signal and idler waves are temperature tuned in the range of 1541 to 1600 nm and 3154 to 3415 nm respectively. To the best of our knowledge, this is the highest output obtained for a diode pumped optical parametric oscillator (OPO), and the first time a SRO is directly pumped by a monolithic tapered diode laser.

Detection and characterization of carbon contamination on EUV multilayer mirrors.

In this paper, we detect and characterize the carbon contamination layers that are formed during the illumination of extreme ultraviolet (EUV) multilayer mirrors. The EUV induced carbon layers were characterized ex situ using spectroscopic ellipsometry (SE) and laser generated surface acoustic waves (LG-SAW). We show that both LG-SAW and SE are very sensitive for measuring carbon layers, even in the presence of the highly heterogeneous structure of the multilayer. SE has better overall sensitivity, with a detection limit of 0.2 nm, while LG-SAW has an estimated detection limit of 2 nm. In addition, SE reveals that the optical properties of the EUV induced carbon contamination layer are consistent with the presence of a hydrogenated, polymeric like carbon. On the other hand, LG-SAW reveals that the EUV induced carbon contamination layer has a low Young's modulus (<100 GPa), which means that the layer is mechanically soft. We compare the limits of detection and quantification of the two techniques and discuss their prospective for monitoring carbon contamination build up on EUV optics.