Discovery of a small-molecule inhibitor that traps Polθ on DNA and synergizes with PARP inhibitors.

The DNA damage response (DDR) protein DNA Polymerase θ (Polθ) is synthetic lethal with homologous recombination (HR) factors and is therefore a promising drug target in BRCA1/2 mutant cancers. We discover an allosteric Polθ inhibitor (Polθi) class with 4-6 nM IC50 that selectively kills HR-deficient cells and acts synergistically with PARP inhibitors (PARPi) in multiple genetic backgrounds. X-ray crystallography and biochemistry reveal that Polθi selectively inhibits Polθ polymerase (Polθ-pol) in the closed conformation on B-form DNA/DNA via an induced fit mechanism. In contrast, Polθi fails to inhibit Polθ-pol catalytic activity on A-form DNA/RNA in which the enzyme binds in the open configuration. Remarkably, Polθi binding to the Polθ-pol:DNA/DNA closed complex traps the polymerase on DNA for more than forty minutes which elucidates the inhibitory mechanism of action. These data reveal a unique small-molecule DNA polymerase:DNA trapping mechanism that induces synthetic lethality in HR-deficient cells and potentiates the activity of PARPi.

Alkyne as a Latent Warhead to Covalently Target SARS-CoV-2 Main Protease.

There is an urgent need for improved therapy to better control the ongoing COVID-19 pandemic. The main protease Mpro plays a pivotal role in SARS-CoV-2 replications, thereby representing an attractive target for antiviral development. We seek to identify novel electrophilic warheads for efficient, covalent inhibition of Mpro. By comparing the efficacy of a panel of warheads installed on a common scaffold against Mpro, we discovered that the terminal alkyne could covalently modify Mpro as a latent warhead. Our biochemical and X-ray structural analyses revealed the irreversible formation of the vinyl-sulfide linkage between the alkyne and the catalytic cysteine of Mpro. Clickable probes based on the alkyne inhibitors were developed to measure target engagement, drug residence time, and off-target effects. The best alkyne-containing inhibitors potently inhibited SARS-CoV-2 infection in cell infection models. Our findings highlight great potentials of alkyne as a latent warhead to target cystine proteases in viruses and beyond.

Monitoring silver diamine fluoride application with optical coherence tomography and thermal imaging: An in vitro proof of concept study.

OBJECTIVES: The purpose of this study was to show that optical coherence tomography (OCT) and thermal imaging can be used to monitor changes in the structure and activity of caries lesions over time after treatment with silver diamine fluoride (SDF). METHODS: Artificial caries lesions were formed on enamel and dentin bovine blocks. Each block was partitioned into five windows with the central three windows exposed to a demineralization solution to create lesions: one sound window served as a sound control (SC), one sound window was exposed to SDF to serve as a test control (SCT), one lesion window served as a lesion control (LC), one lesion window received one application of SDF (L1), while the other lesion window received two applications of SDF (L2). Each window was scanned using OCT before SDF application, and every week subsequently, for 12 weeks after initial SDF treatment. Changes in the mean intensity and the width of the peak of increased reflectivity due to the lesion and SDF along with the intensity at a depth of 180 µm from the surface representing optical penetration through the lesion were monitored. Changes in the heat lost, ΔQ (temperature integrated over time) of each window during drying with air were also monitored using a thermal imaging camera. Transverse microradiography (TMR), and high-resolution microscopy were also used for the analysis of selected samples. RESULTS: The reflectivity and optical penetration of sound and lesion areas of enamel and dentin manifested significant changes in OCT images after SDF application. Thermal imaging showed significant differences in ΔQ indicative of permeability changes in the sound and lesion areas of enamel and dentin after SDF application.

Use of unbiased metagenomic and transcriptomic analyses to investigate the association between feline calicivirus and feline chronic gingivostomatitis in domestic cats.

OBJECTIVE: To identify associations between microbes and host genes in cats with feline chronic gingivostomatitis (FCGS), a debilitating inflammatory oral mucosal disease with no known cause, compared with healthy cats and cats with periodontitis (control cats). ANIMALS: 19 control cats and 23 cats with FCGS. PROCEDURES: At least 1 caudal oral mucosal swab specimen was obtained from each cat. Each specimen underwent unbiased metatranscriptomic next-generation RNA sequencing (mNGS). Filtered mNGS reads were aligned to all known genetic sequences from all organisms and to the cat transcriptome. The relative abundances of microbial and host gene read alignments were compared between FCGS-affected cats and control cats and between FCGS-affected cats that did and did not clinically respond to primary treatment. Assembled feline calicivirus (FCV) genomes were compared with reverse transcription PCR (RT-PCR) primers commonly used to identify FCV. RESULTS: The only microbe strongly associated with FCGS was FCV, which was detected in 21 of 23 FCGS-affected cats but no control cats. Problematic base pair mismatches were identified between the assembled FCV genomes and RT-PCR primers. Puma feline foamy virus was detected in 9 of 13 FCGS-affected cats that were refractory to treatment and 5 healthy cats but was not detected in FCGS-affected cats that responded to tooth extractions. The most differentially expressed genes in FCGS-affected cats were those associated with antiviral activity. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that FCGS pathogenesis has a viral component. Many FCV strains may yield false-negative results on RT-PCR-based assays. Coinfection of FCGS-affected cats with FCV and puma feline foamy virus may adversely affect response to treatment.

High Contrast Reflectance Imaging of Enamel Demineralization and Remineralization at 1950-nm for the Assessment of Lesion Activity.

BACKGROUND AND OBJECTIVES: Previous studies have shown that large changes in the diffuse reflectivity of caries lesions during drying with air can be used to assess lesion activity. The largest changes occur at short wavelength infrared (SWIR) wavelengths coincident with high water absorption. The strongest water absorption in the SWIR occurs at 1950 nm. In this study changes in the reflectivity of simulated lesions with varying degrees of remineralization was measured at 1500-2340 nm and at 1950 nm as the samples were dried with air. STUDY DESIGN/MATERIALS AND METHODS: Twenty bovine enamel surfaces each with five treatment windows were exposed to two demineralization/remineralization regimens to produce simulated lesions of varying depth, severity, and mineral gradients. An extended range tungsten-halogen lamp with a long pass filter (1500-2340 nm) and a broadband amplified spontaneous emission source centered near the peak of the water-absorption band at 1950-nm were used as light sources and an extended range InGaAs camera (1000-2340 nm) was used to acquire reflected light images as the samples were dried with air. Lesions were also assessed using digital microscopy, polarized light microscopy, optical coherence tomography, and transverse microradiography. RESULTS: Both wavelength ranges showed extremely high lesion contrast (>0.9) for all six lesion treatment windows in both models. The change in contrast (ΔI) was significantly higher for the 1950 nm broadband source for all the intact lesion windows compared with the 1500-2340 nm wavelength range. CONCLUSION: SWIR light at 1950 nm yields extremely high contrast of demineralization and appears to be the optimum wavelength for the assessment of lesion activity on tooth coronal surfaces. Lasers Surg. Med. 00:00-00, 2020. © 2020 Wiley Periodicals LLC.

A SWIR imaging handpiece for the clinical assessment of lesion activity via dehydration. Preclinical Assessment.

New imaging methods are needed to assess the activity of caries lesions on tooth surfaces. Recent studies have shown that changes in the contrast of lesions during dehydration with air at SWIR wavelengths can be used to determine if lesions are active or arrested. In this study changes in the reflectance of caries lesions during dehydration with air was monitored at 1500-1750-nm on extracted teeth using an imaging system with an InGaAs camera, a light source and a 3D printed handpiece with an integrated air nozzle suitable for clinical use. Lesion structure was also assessed with optical coherence tomography and microCT for comparison. This small preclinical study demonstrated that a 3D printed appliance with integrated air for dehydration can be used to acquire SWIR dehydration curves similar to those acquired previously for benchtop imaging systems.

A dual handheld SWIR transillumination/reflectance probe for imaging lesions on tooth occlusal and proximal surfaces.

We developed a clinical probe capable of acquiring simultaneous short wavelength infrared (SWIR) reflectance and occlusal transillumination images of lesions on tooth proximal and occlusal surfaces to reduce the potential of false positives. The dual probe is 3D-printed and the imaging system uses a Ge-enhanced camera and fiber-optic light sources that use SWIR light at 1300-nm for occlusal transillumination and SWIR 1450-nm light for reflectance measurements. The purpose of this study was to test the performance of the probe on extracted teeth prior to commencing clinical studies. The dual probe was used to image extracted teeth with proximal and occlusal lesions. SWIR images of each tooth were compared with micro-CT images to assess performance.

SWIR, Thermal and CP-OCT imaging probes for the in vivo assessment of the activity of root caries lesions.

New imaging technologies are needed for the clinical assessment of lesions on root surfaces. It is not sufficient to simply detect caries lesions; methods are needed to assess lesion depth, structural composition and activity to determine if chemical intervention has the potential to be effective and if remineralization has occurred. Lesions were monitored using CP-OCT during lesion dehydration to assess the lesion structure and any shrinkage. Thermal imaging at 6-10 µm wavelengths and short wavelength-IR imaging at 1450-1750-nm were used to monitor thermal emission during lesion dehydration to assess lesion activity. Imaging probes were custom fabricated for clinical use. We present the first clinical results of a small feasibility study employing CP-OCT, thermal and SWIR imaging to assess lesion activity in vivo on thirty test subjects with suspected root caries lesions.

Image-Guided Ablation of Dental Calculus From Root Surfaces Using a DPSS Er:YAG Laser.

BACKGROUND AND OBJECTIVES: Recent studies have demonstrated that near-infrared (IR) imaging can be used to acquire high-contrast images of root caries and calculus on the root surfaces of extracted teeth at wavelengths longer than 1450 nm. The purpose of this study was to determine if image-guided laser ablation can be used to selectively remove calculus from tooth surfaces with minimal damage to the underlying sound cementum and dentin. MATERIALS AND METHODS: In this study, sequential near-IR images at 1500-1700 nm were used to guide a diode-pumped (DPSS) Er:YAG laser for the removal of calculus from the root surfaces of 10 extracted teeth. The selectivity of removal was assessed using digital microscopy, optical coherence tomography, and surface profilometry. RESULTS: Calculus was removed rapidly with minimal damage to the underlying sound cementum and dentin. Image-guided ablation achieved high-selectivity, the mean volume of calculus removal was more than 27 times higher than the mean loss of cementum. CONCLUSIONS: We have demonstrated that near-IR image-guided laser ablation can be used for the selective removal of calculus from root surfaces ex vivo. Additionally, we have demonstrated that a diode-pumped solid-state Er:YAG laser is well suited for selective removal. Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Selective removal of dental calculus with a diode-pumped Er:YAG laser.

Selective removal of dental calculus with high precision is best accomplished using lasers operating at high pulse repetition rates focused to a small spot size to limit damage to sound tissues. Conventional flash-lamp pumped Er:YAG lasers are poorly suited for this purpose, but new diode-pumped solid state (DPSS) Er:YAG lasers have become available operating at high pulse repetition rates. The purpose of this study was to determine if image-guided laser ablation can be used to selectively remove calculus from tooth surfaces with minimal damage to the underlying sound cementum and dentin. A DPSS Er:YAG laser system was used to selectively remove calculus from ten extracted teeth using sequential SWIR images at 1500-1750-nm. The selectivity of removal was assessed using digital microscopy and optical coherence tomography. Calculus was removed with minimal damage to the underlying sound cementum and dentin.

Use of a DPSS Er:YAG laser for the selective removal of composite from tooth surfaces.

New diode-pumped solid state (DPSS) Er:YAG lasers have become available operating at high pulse repetition rates. These lasers are ideally suited for integration with laser scanning systems for the selective removal of dental decay and composite restorative materials from tooth surfaces. The purpose of this study was to determine if a DPSS Er:YAG laser system is suitable for the selective removal of composite from tooth surfaces. Relative ablation rates of composite and enamel were determined and composite was removed from tooth surfaces using a DPSS Er:YAG laser. Composite was removed very rapidly with ablation rates approaching 50-µm per pulse. A fluence of ~50 J/cm2 appeared optimal for the removal of composite and damage to the enamel was limited to less than 100-µm after the removal of composite as thick as 700-800-µm; however, dentin is removed at similar rates to composite. The DPSS Er:YAG laser appears to be better suited for the removal of composite than conventional flash-lamp pumped Er:YAG lasers since composite is ablated at higher rates than dental enamel and the high pulse repetition rates enable greater selectivity while maintaining high removal rates.

Influence of Multi-Wavelength Laser Irradiation of Enamel and Dentin Surfaces on Surface Morphology and Permeability.

UV and IR lasers can be used to specifically target protein, water, and the mineral phase of dental hard tissues to produce varying changes in surface morphology. In this study, we irradiated enamel and dentin surfaces with various combinations of lasers operating at 0.355, 2.94, and 9.4 µm, exposed those surfaces to topical fluoride, and subsequently evaluated the influence of these changes on surface morphology and permeability. Digital microscopy and surface dehydration rate measurements were used to monitor changes in the samples overtime. The surface morphology and permeability (dehydration rate) varied markedly with the different laser treatments on enamel. On dentin, fluoride was most effective in reducing the permeability.

Influence of multi-wavelength laser irradiation of enamel and dentin surfaces at 0.355, 2.94, and 9.4 µm on surface morphology, permeability, and acid resistance.

OBJECTIVE: Ultraviolet (UV) and infrared (IR) lasers can be used to specifically target protein, water, and mineral, respectively, in dental hard tissues to produce varying changes in surface morphology, permeability, reflectivity, and acid resistance. The purpose of this study was to explore the influence of laser irradiation and topical fluoride application on the surface morphology, permeability, reflectivity, and acid resistance of enamel and dentin to shed light on the mechanism of interaction and develop more effective treatments. METHODS: Twelve bovine enamel surfaces and twelve bovine dentin surfaces were irradiated with various combinations of lasers operating at 0.355 (Freq.-tripled Nd:YAG (UV) laser), 2.94 (Er:YAG laser), and 9.4 µm (CO2 laser), and surfaces were exposed to an acidulated phosphate fluoride gel and an acid challenge. Changes in the surface morphology, acid resistance, and permeability were measured using digital microscopy, polarized light microscopy, near-IR reflectance, fluorescence, polarization sensitive-optical coherence tomography (PS-OCT), and surface dehydration rate measurements. RESULTS: Different laser treatments dramatically influenced the surface morphology and permeability of both enamel and dentin. CO2 laser irradiation melted tooth surfaces. Er:YAG and UV lasers, while not melting tooth surfaces, showed markedly different surface roughness. Er:YAG irradiation led to significantly rougher enamel and dentin surfaces and led to higher permeability. There were significant differences in acid resistance among the various treatment groups. CONCLUSION: Surface dehydration measurements showed significant changes in permeability after laser treatments, application of fluoride and after exposure to demineralization. CO2 laser irradiation was most effective in inhibiting demineralization on enamel while topical fluoride was most effective for dentin surfaces. Lasers Surg. Med. 49:913-927, 2017. © 2017 Wiley Periodicals, Inc.

High-Contrast Reflectance Imaging of Composite Restorations Color-Matched to Tooth Structure at 1000-2300-nm.

A major advantage of composite restoration materials is that they can be color matched to the tooth. However, this presents a challenge when composites fail and they need to be replaced. Dentists typically spend more time repairing and replacing composites than placing new restorations. We have shown in previous studies that high-contrast images of composite can be acquired in occlusal transmission mode at near-IR wavelengths coincident with higher water absorption. The purpose of this study was to determine if similar high-contrast images can be acquired in reflectance mode at longer wavelengths where water absorption is even higher. Extracted human teeth with existing composite restoration (n=14) were imaged at wavelengths from 900-2300 using an extended range InGaAs camera. Our results indicate that NIR wavelengths longer than 1400-nm coincident with higher water absorption yield the highest contrast between dental composites and tooth structure in reflectance.

Selective removal of dental composite with a diode-pumped Er:YAG laser.

Selective removal of dental composite with high precision is best accomplished using lasers operating at high pulse repetition rates focused to a small spot size. Conventional flash-lamp pumped Er:YAG lasers are poorly suited for this purpose, but new diode-pumped Er:YAG lasers have become available operating at high pulse repetition rates. The purpose of this study was to compare the ablation rates and selectivity of enamel and composite for a 30 W diode-pumped Er:YAG laser operating with a pulse duration of 30-50-µs and evaluate it's suitability for the selective removal of composite from tooth surfaces. The depth of ablation and changes in surface morphology were assessed using digital microscopy. The fluence range of 30-50 J/cm2 appeared optimal for the removal of composite, and damage to sound enamel was limited to less than 100-µm after the removal of composite as thick as 700-800-µm. Future studies will focus on the use of methods of feedback to further increase selectivity.

Use of 2D images of depth and integrated reflectivity to represent the severity of demineralization in cross-polarization optical coherence tomography.

Several studies have demonstrated the potential of cross-polarization optical coherence tomography (CP-OCT) to quantify the severity of early caries lesions (tooth decay) on tooth surfaces. The purpose of this study is to show that 2D images of the lesion depth and the integrated reflectivity can be used to accurately represent the severity of early lesions. Simulated early lesions of varying severity were produced on tooth samples using simulated lesion models. Methods were developed to convert the 3D CP-OCT images of the samples to 2D images of the lesion depth and lesion integrated reflectivity. Calculated lesion depths from OCT were compared with lesion depths measured from histological sections examined using polarized light microscopy. The 2D images of the lesion depth and integrated reflectivity are well suited for visualization of early demineralization.

Multispectral Near-Infrared Imaging of Composite Restorations in Extracted Teeth.

One major advantage of composite restoration materials is that they can be color matched to the tooth. However, this presents a challenge when composites fail and they need to be replaced. Dentists typically spend more time repairing and replacing composites than placing new restorations. Previous studies have shown that near-infrared imaging can be used to distinguish between sound enamel and decay due to the differences in light scattering. The purpose of this study was to use a similar approach and exploit differences in light scattering to attain high contrast between composite and tooth structure. Extracted human teeth with composites (n=16) were imaged in occlusal transmission mode at wavelengths of 1300-nm, 1460-nm and 1550-nm using an InGaAs image sensor with a tungsten halogen light source with spectral filters. All samples were also imaged in the visible range using a high definition 3D digital microscope. Our results indicate that NIR wavelengths at 1460-nm and 1550-nm, coincident with higher water absorption yield the highest contrast between dental composites and tooth structure.

Near-IR imaging of cracks in teeth.

Dental enamel is highly transparent at near-IR wavelengths and several studies have shown that these wavelengths are well suited for optical transillumination for the detection and imaging of tooth decay. We hypothesize that these wavelengths are also well suited for imaging cracks in teeth. Extracted teeth with suspected cracks were imaged at several wavelengths in the near-IR from 1300-1700-nm. Extracted teeth were also examined with optical coherence tomography to confirm the existence of suspected cracks. Several teeth of volunteers were also imaged in vivo at 1300-nm to demonstrate clinical potential. In addition we induced cracks in teeth using a carbon dioxide laser and imaged crack formation and propagation in real time using near-IR transillumination. Cracks were clearly visible using near-IR imaging at 1300-nm in both in vitro and in vivo images. Cracks and fractures also interfered with light propagation in the tooth aiding in crack identification and assessment of depth and severity.

Imaging Early Demineralization on Tooth Occlusal Surfaces with a High Definition InGaAs Camera.

In vivo and in vitro studies have shown that high contrast images of tooth demineralization can be acquired in the near-IR due to the high transparency of dental enamel. The purpose of this study is to compare the lesion contrast in reflectance at near-IR wavelengths coincident with high water absorption with those in the visible, the near-IR at 1300-nm and with fluorescence measurements for early lesions in occlusal surfaces. Twenty-four human molars were used in this in vitro study. Teeth were painted with an acid-resistant varnish, leaving a 4×4 mm window in the occlusal surface of each tooth exposed for demineralization. Artificial lesions were produced in the exposed windows after 1 & 2-day exposure to a demineralizing solution at pH 4.5. Lesions were imaged using NIR reflectance at 3 wavelengths, 1310, 1460 and 1600-nm using a high definition InGaAs camera. Visible light reflectance, and fluorescence with 405-nm excitation and detection at wavelengths greater than 500-nm were also used to acquire images for comparison. Crossed polarizers were used for reflectance measurements to reduce interference from specular reflectance. The contrast of both the 24 hr and 48 hr lesions were significantly higher (P<0.05) for NIR reflectance imaging at 1460-nm and 1600-nm than it was for NIR reflectance imaging at 1300-nm, visible reflectance imaging, and fluorescence. The results of this study suggest that NIR reflectance measurements at longer near-IR wavelengths coincident with higher water absorption are better suited for imaging early caries lesions.

High contrast reflectance imaging of simulated lesions on tooth occlusal surfaces at near-IR wavelengths.

INTRODUCTION: In vivo and in vitro studies have shown that high contrast images of tooth demineralization can be acquired in the near-infrared (near-IR) without the interference of stain. The purpose of this study is to compare the lesion contrast in reflectance at near-IR wavelengths coincident with high water absorption with those in the visible, the near-IR at 1,300 nm and with fluorescence measurements for early lesions in occlusal surfaces. METHODS: Twenty-four human molars were used in this in vitro study. Teeth were painted with an acid-resistant varnish, leaving a 4 × 4 mm window in the occlusal surface of each tooth exposed for demineralization. Artificial lesions were produced in the exposed windows after 1- and 2-day exposure to a demineralizing solution at pH 4.5. Lesions were imaged using near-IR reflectance at three wavelengths, 1,300, 1,460, and 1,600 nm using a high definition InGaAs camera. Visible light reflectance, and fluorescence with 405 nm excitation and detection at wavelengths greater than 500 nm were also used to acquire images for comparison. Crossed polarizers were used for reflectance measurements to reduce interference from specular reflectance. RESULTS: The contrast of both the 1- and 2-day lesions were significantly higher (P < 0.05) for near-IR reflectance imaging at 1,460 and 1,600 nm than it was for near-IR reflectance imaging at 1,300 nm, visible reflectance imaging, and fluorescence. CONCLUSION: The markedly higher contrast at 1,460 and 1,600 nm wavelengths, coincident with higher water absorption, suggest that these wavelengths are better suited than 1,300 nm for imaging early/shallow demineralization on tooth surfaces.