Single Reflectance Research Articles

Biofabricated osteoblast-derived nanovesicles as extracellular vesicle mimics for bone repair

Osteoblast-derived extracellular vesicles (EVs) have demonstrated therapeutic utility for bone repair as transporters of key biomolecules capable of accelerating biomineralisation and tissue repair. The clinical translation of these biologically derived nanoparticles, however remains limited due to scalability, heterogeneity and standardisation issues. Herein we investigate the generation of nanovesicles (NVs) from mineralising osteoblasts by extrusion directly compared against natural EV counterparts from the same parental cells.Mineralising osteoblast-derived EVs (MO-EVs) were isolated via ultracentrifugation from cell culture media. The parental osteoblasts were then processed via serial extrusion to <200 nm. EVs and NVs were characterised by comparing their size, concentration and morphology. The presence of tetraspanin markers was detected by Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS). Osteoblasts viability and metabolic activity was assessed after both EV and NV-treatment before comparing their mineralising potency via alizarin red staining.EVs and NVs exhibited similar diameters of approximately 100 nm with a vesicular morphology. EVs were found to be richer in proteins and exhibited a significantly more negative ζ-potential compared to NVs. SP-IRIS analysis confirmed the presence of CD9, CD63 and CD81 in EVs. At both 1 and 10 μg/mL, EVs and NVs reduced the metabolic activity of osteoblasts, however, this was not associated with any cytotoxic effects. The biomineralisation study performed using osteoblasts showed that only EVs significantly increased mineral deposition (p < 0.05) compared to untreated control.In this study, we have established for the first time the biofabrication of cell-derived nanovesicles as a promising alternative to extracellular vesicles derived from mineralising osteoblasts.

Fluorescence detection of pituitary neuroendocrine tumour during endoscopic transsphenoidal surgery using bevacizumab-800CW: a non-randomised, non-blinded, single centre feasibility and dose finding trial [DEPARTURE trial

Achieving endocrine remission by gross total resection is challenging in pituitary neuroendocrine tumours (PitNETs) with cavernous sinus invasion. This study aims to assess the safety, feasibility, and optimal dose for intraoperative fluorescence imaging as an added instrument to discriminate PitNET from surrounding tissue using bevacizumab-800CW, targeting vascular endothelial growth factor A (VEGF-A). In part I, dose-escalation (0-4∙5-10-25mg) was performed in 4 groups of 3 patients with PitNETs Knosp grade 3-4. In part II, after interim analysis, the 10mg and 25mg groups were expanded to a total of 6 patients. Quantitative fluoroscence molecular endoscopy consisted of wide field fluorescence molecular endoscopy and multi-diameter single fiber reflectance / single fiber fluorescence spectroscopy. Mean fluorescence intensity (MFI) of the fresh surgical specimen was calculated and VEGF-staining was performed. Eighteen patients were included. All doses were well tolerated. Three serious adverse events were registered, but none were tracer-related. Part I showed an adequate in-vivo tumour-to-background ratio for both 10mg (TBR 2∙00 [1∙86, 2∙19]) and 25mg (TBR 2∙10, [1∙86, 2∙58]). Part II revealed a substantially higher MFI in the 25mg group. With both 10mg and 25mg a statistically significant difference between tumour and surrounding tissue was detected (p < 0∙0001). All surgical specimens had VEGF-A expression. This study demonstrates the safety and feasibility of quantitative fluorescence molecular endoscopy during PitNET surgery. Both 10mg and 25mg bevacizumab-800CW result in clear differentiation in-vivo, with improved contrast ex-vivo (MFI) in the 25mg group. NCT04212793 / Study Details| Detection of PitNET Tissue During TSS Using Bevacizumab800CW| ClinicalTrials.gov.

Confirming size-exclusion chromatography as a clinically relevant extracellular vesicles separation method from 1mL plasma through a comprehensive comparison of methods

BackgroundExtracellular vesicles (EVs) are amongst the most promising candidates for developing blood-based biomarkers. However, patient sample availability is a key barrier to translational research whilst most biobanks store samples of 1.5mL volume or less. To date, there is no consensus on the most suitable method of EV separation and current techniques frequently require large volumes of biofluids, complicated technology, technical expertise, or significant operating costs, which prevents their widespread adoption by less EV-focussed laboratories. Therefore, there is a need for an easy and reproducible method that separates representative EVs from clinically relevant 1mL volumes of plasma prior to subsequent biomarker identification.MethodsIn this study, EVs were separated from a clinically relevant 1mL volume of human plasma using four different separation techniques: size exclusion chromatography (SEC), differential ultracentrifugation, precipitation, and immunoaffinity magnetic bead capture. The EVs were characterised using several orthogonal techniques (protein quantification, nanoparticle tracking analysis, transmission electron microscopy, Western blot, single particle interferometric reflectance imaging sensing, and mass spectrometry-based proteomics) to comprehensively compare the separated samples.ResultsWe provide examples of anticipated results highlighting that SEC-processed samples have greater protein quantification yield, greater particle yield of the expected size for EVs, and sufficient EV purity, which facilitates effective EV cargo assessment by proteomics. Moreover, we confirm significant overlap with known EV-related proteins within the Vesiclepedia database. Additionally, using single particle interferometric reflectance imaging sensing (Leprechaun®), we identify that SEC has the most representative surface tetraspanin distribution of the separated EV population compared to unprocessed plasma.DiscussionGiven that SEC requires minimal expertise, no complicated technology and can separate EVs within 90 min, this comparison reinforces SEC as a clinically relevant EV separation method from 1mL of plasma making it suitable for widespread implementation.

Abstract 3633: Multi-parametric characterization of extracellular vesicles in the aqueous humor of uveal melanoma eyes undergoing brachytherapy

Abstract Introduction: Aqueous humor (AH), fluid in the anterior chamber of the eye, is a liquid biopsy source of tumor information in uveal melanoma (UM). Extracellular vesicles (EVs) have recently been characterized in the AH of retinoblastoma eyes, however, have not been investigated in UM. EVs are small membrane-enclosed particles secreted by virtually every cell type with a role in intercellular communications. We hypothesize that tumor-derived EVs are detectable in the AH of UM eyes and that understanding the type of vesicles present may help us better understand their function, particularly for cancer. Small extracellular vesicles (sEVs) have been shown to promote tumorigenesis, treatment resistance, and metastasis, leading to the discovery of clinically-relevant biomarkers for multiple cancer types. As the diversity and role of sEVs in the AH of UM patients has never previously been reported, this project is a first step towards investigating new biomarkers of clinical utility. Our aim is to use single particle analysis to characterize sEV subpopulations in the AH of UM patients by quantifying their size, concentration, and phenotypes based on cell surface markers, specifically tetraspanin co-expression patterns. Methods: sEVs were analyzed from paired pre- and post-brachytherapy AH samples collected from 19 UM patients. AH samples from 5 glaucoma patients served as the control cohort. Unprocessed AH underwent Nanoparticle Tracking Analysis for size quantification and Single Particle-Interferometric Reflectance Imaging Sensor analysis for fluorescent particle count and tetraspanin immunophenotyping; counts were subsequently converted to percentages for analysis. Statistics were performed using Mann-Whitney U, ANOVA, and Tukey’s tests. Results: AH derived from UM eyes (pre-brachytherapy) displayed a broader sEV size distribution than glaucoma eyes, with more diverse sEV subpopulations. Both tumor and nontumor AH samples demonstrated a dominant CD63+ sEV subpopulation. A significantly higher percentage of CD63/81+ sEVs (P= 0.0217) and CD9/63/81+ sEVs (P= 0.0146) were found in post-brachytherapy samples compared to pre-brachytherapy. Conclusions: sEV subpopulations in UM eyes demonstrate increased heterogeneity in comparison to nontumor eyes. Our results are consistent with prior studies reporting that the CD63+ sEV subtype is an AH-specific biomarker enriched across multiple ocular diseases. The increase in CD63/81+ sEVs and CD9/63/81+ sEVs after brachytherapy is presumably a result of radiation-induced release of these vesicles, suggesting a tumor-derived origin of these subpopulations. CD63/81+ sEV subpopulation was previously reported to also be a tumor-derived subpopulation in AH of retinoblastoma eyes. Further study of the cargo carried by these sEV subpopulations may uncover molecular mechanisms pivotal to UM and other ocular cancers. Citation Format: Shreya Sirivolu, Chen-Ching Peng, Paolo Neviani, Jesse L. Berry, Liya Xu. Multi-parametric characterization of extracellular vesicles in the aqueous humor of uveal melanoma eyes undergoing brachytherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3633.

Increased circulating platelet-derived extracellular vesicles in severe COVID-19 disease.

Coagulation disturbances are major contributors to COVID-19 pathogenicity, but limited data exist on the involvement of extracellular vesicles (EVs) and residual cells (RCs). Fifty hospitalized COVID-19 patients stratified by their D-dimer levels into high (>1.5 mg/L, n = 15) or low (≤1.5 mg/l, n = 35) and 10 healthy controls were assessed for medium-sized EVs (mEVs; 200-1000 nm) and large EVs/RCs (1000-4000 nm) by high sensitivity flow cytometry. EVs were analyzed for CD61, CD235a, CD45, and CD31, commonly used to detect platelets, red blood cells, leukocytes or endothelial cells, respectively, whilst phosphatidyl serine EVs/RCs were detected by lactadherin-binding implicating procoagulant catalytic surface. Small EV detection (sEVs; 50-200 nm) and CD41a (platelet integrin) colocalization with general EV markers CD9, CD63, and CD81 were performed by single particle interferometric reflectance imaging sensor. Patients with increased D-dimer exhibited the highest number of RCs and sEVs irrespective of cell origin (p < .05). Platelet activation, reflected by increased CD61+ and lactadherin+ mEV and RC levels, associated with coagulation disturbances. Patients with low D-dimer could be discriminated from controls by tetraspanin signatures of the CD41a+ sEVs, suggesting the changes in the circulating platelet sEV subpopulations may offer added prognostic value during COVID progression.

Using only the red-edge bands is sufficient to detect tree stress: A case study on the early detection of PWD using hyperspectral drone images

Pine wilt disease (PWD) is a destructive disease of pine trees caused by the pine wood nematode and early identification is crucial for disease control. Hyperspectral drone imagery has the potential to achieve early detection, although specific methods have not been sufficiently explored, including the spectral characteristics of early infections and the most efficient identification methods. This study aimed to examine the spectral responses to early infection, quantify the separability of healthy and early infections, and compare the accuracy and efficiency of different methods including single bands, vegetation indices (VIs) and 1st derivative reflectance and indices. We collected hyperspectral drone data in southeast China and used linear discriminant analysis (LDA) to determine the separability of healthy trees and trees at an early stage of infection. We also used bands with the same wavelengths as Sentienl-2 images (denoted S2 bands) to propose a standard for band selection.We found that it was possible to separate healthy trees and those at an early stage of infection around 0.71–0.78 using different methods. Among individual bands, the red-edge bands had the highest separability of 0.74. Using standard vegetation indices resulted in separability between 0.67 and 0.71; in addition, we proposed three new indices that achieved separability between 0.73 and 0.75. The 1st derivative reflectance at 714 nm had the highest separability of 0.78 in this study, while using the 1st derivative reflectance indices was slightly less accurate. The classification accuracy was also slightly lower when using Random Forest (RF) with all bands, sensitive bands, and S2 bands.We conclude that, of the methods tested, the red-edge bands are most sensitive to early infection, and using the 1st derivative reflectance at 714 nm or using the 1st derivative reflectance at the red-edge and blue-edge inflection point (REIP and BEIP) was sufficient for early identification. Acquiring and using additional wavelengths hardly improved the classification. Using wavelengths similar to those in the Sentienl-2 images achieved similar results, and thus can be used as a standard for dimensionality reduction of hyperspectral data pertaining to forest disease. The newly proposed VIs and 1st derivative reflectances at the yellow-edge inflection point (YEIP) and BEIP delivered better performance than the other tested indices, and could be alternatives for early identification. This study proposes simple and practical methods for early identification of wilt and provides insights for efficient data acquisition and data reduction.

Tetraspanin profiles of serum extracellular vesicles reflect functional limitations and pain perception in knee osteoarthritis.

Emerging evidence suggests that extracellular vesicles (EVs) can play roles in inflammatory processes and joint degradation in primary osteoarthritis (OA), a common age-associated joint disease. EV subpopulations express tetraspanins and platelet markers that may reflect OA pathogenesis. The present study investigated the associations between these EV surface markers and articular cartilage degradation, subjectively and objectively assessed pain, and functional limitations in primary knee OA (KOA). Serum EVs were determined by high-sensitivity flow cytometry (large CD61+ EVs) and single particle interferometric reflectance imaging sensor (small CD41+, CD63+, CD81+, and CD9+ EVs) from end-stage KOA patients and controls (n = 8 per group). Knee pain and physical functions were assessed with several health- and pain-related questionnaires, established measurements of physical medicine, and neuromuscular examination. The obtained data were analyzed using supervised and unsupervised univariate and multivariate models. With the combined dataset of cartilage thickness, knee function, pain, sensation, and EV molecular signatures, we identified highly correlated groups of variables and found several EV markers that were statistically significant predictors of pain, physical limitations, and other aspects of well-being for KOA patients, for instance CD41+/CD63+/CD9+ small EVs associated with the range of motion of the knee, physical performance, and pain sensitivity. Particular serum EV subpopulations showed clear associations with KOA pain and functional limitations, suggesting that their implications in OA pathophysiology warrant further study.

Near-mid infrared spectroscopy of carbonaceous chondrites: Insights into spectral variation due to aqueous alteration and thermal metamorphism in asteroids

Carbonaceous chondrites (CCs) are windows into the early Solar System and the histories of their parent bodies. Their infrared spectral signatures are powerful proxies for deciphering their composition and evolution history, but still present formidable challenges in relation to determining the degree of secondary processing such as aqueous alteration and thermal metamorphism via comprehensive data and mid-infrared feature. In our study, we delved into the infrared spectra spanning 1–25 μm of 17 CCs, with distinct petrological characteristics and varying degrees of alteration. Through this investigation, we uncovered distinct spectral patterns that shed light on the processes of alteration and metamorphism. As aqueous alteration intensifies, two key spectral features, the 3 μm-region absorption feature associated with OH-bearing minerals and water, and the 6 μm band indicative of water molecules, both grow in intensity. Simultaneously, their band centers shift towards shorter wavelengths. Moreover, as alteration progresses, a distinctive absorption feature emerges near 2.72 μm, resembling the OH absorption feature found in serpentine and saponite minerals. Comparison of aqueous alteration to laboratory-heated CCs suggests that the 3 μm region OH/H2O absorption feature differs between CC heated to less than or more than ∼300 °C. Further insights are gained by examining the vibrational features of silicate minerals, notably influencing the 10 μm and 20 μm regions. The 12.4 μm /11.4 μm reflectance ratio diminishes, and the reflectance peak in the 9–14 μm range shifts towards shorter wavelengths. These changes are attributed to the transformation of anhydrous silicates into phyllosilicates. In the 15–25 μm region, the influence of thermal metamorphism becomes evident and results in the appearance of more spectral features, the single reflectance peak at 22.1 μm undergoes a transformation into two distinct peaks at 19 μm and 25 μm, which is primarily attributed to the increased presence of anhydrous silicates and olivine recrystallization. These findings offer novel insights into the volatile-rich compositions of C-complex asteroids and the thermal evolution histories of their parent bodies.

Measuring the Optical Properties of Highly Diffuse Materials.

Measuring the optical properties of highly diffuse materials is a challenge as it could be related to the white colour or an oversaturation of pixels in the acquisition system. We used a spatially resolved method and adapted a nonlinear trust-region algorithm to the fit Farrell diffusion theory model. We established an inversion method to estimate two optical properties of a material through a single reflectance measurement: the absorption and the reduced scattering coefficient. We demonstrate the validity of our method by comparing results obtained on milk samples, with a good fitting and a retrieval of linear correlations with the fat content, given by R2 scores over 0.94 with low p-values. The values of absorption coefficients retrieved vary between 1 × 10-3 and 8 × 10-3 mm-1, whilst the values of the scattering coefficients obtained from our method are between 3 and 8 mm-1 depending on the percentage of fat in the milk sample, and under the assumption of the anisotropy factor g>0.8. We also measured and analyzed the results on white paint and paper, although the paper results were difficult to relate to indicators. Thus, the method designed works for highly diffuse isotropic materials.

Syntheses and characterization of quaternary selenites and tellurite of antimony, NaSbSe2O7, AgSbSe2O7 and Na2Sb4Te2O14

NaSbSe2O7, AgSbSe2O7 and Na2Sb4Te2O14 compounds have been synthesized by solid state reactions and characterized by single crystal X-ray diffraction, thermal analysis, reflectance spectroscopy, and second harmonic generation (SHG) response studies. The isostructural NaSbSe2O7 and AgSbSe2O7 selenites crystallize in centrosymmetric Cmcm space group with respective orthorhombic unit cell parameter values of a = 10.0704(4) and 10.0449(5) Å, b = 8.3074(4) and 8.3825(5) Å and c = 7.1449(3) and 7.1438(5) Å. Their one-dimensional structure consists of Na+ or Ag+ cations and anionic [SbSe2O7]- chains made up of SbO6 octahedra and SeO3 trigonal pyramids. Na2Sb4Te2O14 is a mixed-valent compound, which crystallizes in noncentrosymmetric P3121 space group with hexagonal unit cell parameter values of a = 7.32670(10) Å and c = 18.5150(5) Å. It has a three-dimensional hexagonal-tungsten-oxide (HTO) related anionic [Sb4Te2O14]2- structural framework and shows powder SHG response equivalent to 0.4 × KDP.

CD63/81 Small Extracellular Vesicles in the Aqueous Humor are Retinoblastoma Associated.

Although biopsy is contraindicated in retinoblastoma (RB), the aqueous humor (AH) is a robust liquid biopsy source of molecular tumor information, facilitating biomarker discovery. Small extracellular vesicles (sEVs), promising biomarker candidates across multiple cancers, were recently identified in RB AH, but relationships between sEVs and RB clinical features are unknown. We analyzed sEVs in 37 AH samples from 18 RB eyes of varying International Intraocular Retinoblastoma Classification (IIRC) groups and explored clinical correlations. Ten samples were collected at diagnosis (DX) and 27 during treatment (Tx). Unprocessed AH underwent Single Particle-Interferometric Reflectance Imaging Sensor (SP-IRIS) analysis for fluorescent particle count and tetraspanin immunophenotyping; counts were subsequentially converted to percentages for analysis. Comparing DX and Tx samples, a higher percentage of CD63/81+ sEVs was found in DX AH (16.3 ± 11.6% vs. 5.49 ± 3.67% P = 0.0009), with a more homogenous mono-CD63+ sEV population seen in Tx AH (43.5 ± 14.7% vs. 28.8 ± 9.38%, P = 0.0073). Among DX samples, CD63/81+ sEVs were most abundant in group E eyes (n = 2) compared to group D (n = 6) by count (2.75 × 105 ± 3.40 × 105 vs. 5.95 × 103 ± 8.16 × 103, P = 0.0006), and to group A+B (n = 2) by count (2.75 × 105 ± 3.40 × 105 vs. 2.73 × 102 ± 2.59 × 102, P = 0.0096) and percentage (32.1 ± 7.98% vs. 7.79 ± 0.02%, P = 0.0187). CD63/81+ sEVs enrich AH from RB eyes before treatment and those with more significant tumor burden, suggesting they are tumor-derived. Future research into their cargo may reveal mechanisms of cellular communication via sEVs in RB and novel biomarkers.

La orina más allá de los electrolitos: diagnóstico a través de las vesículas extracelulares

Background and objectiveExtracellular vesicles (EVs) reflect the pathophysiological state of their cells of origin and are a reservoir of renal information accessible in urine. When biopsy is not an option, EVs present themselves as sentinels of function and damage, providing a non-invasive approach. However, the analysis of EVs in urine requires prior isolation, which slows down and hinders translation to clinical practice. The aim of this study is to show the applicability of the “single particle interferometric reflectance imaging sensor” (SP-IRIS) technology through the ExoView® platform for the direct analysis of urine EVs and proteins involved in renal function. Materials and methodsThe ExoView® technology enables the quantification and phenotyping of EVs present in urine and the quantification of their membrane and internal proteins. We have applied this technology to the quantification of urinary EVs and their proteins with renal tubular expression, amnionless (AMN) and secreted frizzled-related protein 1 (SFRP1), using only 5μl of urine. Tubular expression was confirmed by immunohistochemistry. ResultsThe mean size of the EVs analysed was 59±16nm for those captured by tetraspanin CD63, 61±16nm for those captured by tetraspanin CD81, and 59±10 nm for tetraspanin CD9, with CD63 being the majority EVs subpopulation in urine (48.92%). The distribution of AMN and SFRP1 in the three capture tetraspanins turned out to be similar for both proteins, being expressed mainly in CD63 (48.23% for AMN and 52.1% for SFRP1). ConclusionsThis work demonstrates the applicability and advantages of the ExoView® technique for the direct analysis of urine EVs and their protein content in relation to the renal tubule. The use of minimum volumes, 5μl, and the total analysis time not exceeding three hours facilitate the translation of EVs to daily clinical practice as source of diagnostic information.

Abstract 6717: Extracellular vesicles in diagnostic retinoblastoma aqueous humor correlate with disease stage and clinical outcome: a pilot study

Abstract Introduction: Retinoblastoma (RB), a pediatric retinal malignancy, is unique in that it cannot be biopsied, impeding biomarker discovery. Aqueous humor (AH) is an ocular fluid that is an established source of tumor information, serving as a surrogate liquid biopsy. Extracellular vesicles (EVs) are promising biomarker candidates, and were recently established in RB AH. EVs are membrane-enclosed secretory vesicles carrying bioactive molecular information, which can be phenotyped using validated tetraspanin protein markers. Currently, relationships between EVs and RB clinical features are unknown, but establishing such relationships is necessary to determine the utility of EVs as RB biomarkers. Methods: We quantified and phenotyped EVs in 37 AH samples from 18 RB eyes with varying International Intraocular Retinoblastoma Classification (IIRC) stages and explored clinical correlations. Samples were collected from 10 eyes at diagnosis (DX) and 27 eyes during treatment (Tx). By outcome, 6 DX eyes were enucleated and 4 were salvaged. DX samples were collected from one A, one B, six D and two E eyes by IIRC group. 10μL of unprocessed AH underwent Single Particle-Interferometric Reflectance Imaging Sensor (SP-IRIS) (Exoview R100) analysis for fluorescent-based particle count and immunophenotyping of tetraspanin expression (CD63/81/9). Counts were converted to percentages. Statistics were done with Mann-Whitney U, ANOVA, and Tukey’s tests. Results: Samples from DX eyes (n=10) contained a higher number of EVs than samples from Tx eyes (n=27) (1.68 x 107 ± 4.28 x 107 per mL vs. 2.10x 106 ± 1.33 x 106 per mL) by SP-IRIS analysis. By percentage, DX samples were enriched with CD63/81+ EVs (P&amp;lt;0.001) while Tx samples had more homogenous mono-CD63+ dominant EV populations (P=0.007). Enrichment of CD63/81+ EVs was significantly higher in enucleated AH (n=6) than salvaged AH (n=4) when compared by count (P=0.038) and marginally higher when compared by percentage (P=0.067). The CD9/81+ EV population percentage was significant in salvaged eyes (P=0.01). By IIRC, CD63/81+ EVs were most abundant in Group E eyes (n= 2) when compared to Group D eyes (n=6) by count (P&amp;lt;0.001), and when compared to Group A+B eyes (n=2) by count (P=0.01) and percentage (P=0.019). Group A+B eyes had a larger percentage of CD9/81+ EVs than did Group E eyes (P=0.043). Conclusions: EV quantity in RB AH decreases with treatment, and phenotypic expression profiles vary by treatment status and clinical outcome. TheCD63/81+ EV subpopulation is higher in eyes with advanced Group E disease and those requiring enucleation, while the CD9/81+ subpopulation is higher in eyes with less advanced Group A+B disease and those salvaged with treatment. Our data suggests that EV phenotypes correlate with RB clinical features, and further research into these subpopulations offers the prospect of establishing critical RB biomarkers. Citation Format: Sarah Pike, Chen-Ching Peng, Paolo Neviani, Jesse L. Berry, Liya Xu. Extracellular vesicles in diagnostic retinoblastoma aqueous humor correlate with disease stage and clinical outcome: a pilot study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6717.

Receptor activator of nuclear factor-kappa B is enriched in CD9-positive extracellular vesicles released by osteoclasts.

Receptor activator of nuclear factor-kappa B (RANK)-containing extracellular vesicles (EVs) bind RANK-Ligand (RANKL) on osteoblasts, and thereby simultaneously inhibit bone resorption and promote bone formation. Because of this, they are attractive candidates for therapeutic bone anabolic agents. Previously, RANK was detected in 1 in every 36 EVs from osteoclasts by immunogold electron microscopy. Here, we have sought to characterize the subpopulation of EVs from osteoclasts that contains RANK in more detail. The tetraspanins CD9 and CD81 were localized in osteoclasts by immunofluorescence. EVs were visualized by transmission electron microscopy. A Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) and immunoaffinity isolations examined whether RANK is enriched in specific types of EVs. Immunofluorescence showed CD9 was mostly on or near the plasma membrane of osteoclasts. In contrast, CD81 was localized deeper in the osteoclast's cytosolic vesicular network. By interferometry, both CD9 and CD81 positive EVs from osteoclasts were small (56-83 nm in diameter), consistent with electron microscopy. The CD9 and CD81 EV populations were mostly distinct, and only 22% of the EVs contained both markers. RANK was detected by SP-IRIS in 2%-4% of the CD9-containing EVs, but not in CD81-positive EVs, from mature osteoclasts. Immunomagnetic isolation of CD9-containing EVs from conditioned media of osteoclasts removed most of the RANK. A trace amount of RANK was isolated with CD81. RANK was enriched in a subset of the CD9-positive EVs. The current study provides the first report of selective localization of RANK in subsets of EVs.

Near-Infrared Absorption Coefficients in Kidney Stone Minerals and Their Relation to Crystal Structure

To quantitatively understand laser ablation of kidney stones by near-infrared (NIR) lasers, accurate measurements of physical properties related to heating and mechanical failure are needed. While light scattering varies among individual stones, absorption coefficients for the materials that form the bulk of each kidney stone are measurable when scattering is controlled. We developed methods to grow single crystals with lengths and widths >20 μm for the minerals that form kidney stones: calcium oxalate monohydrate, calcium oxalate dihydrate, calcium hydrogen phosphate dihydrate, hydroxyapatite, anhydrous uric acid, and magnesium ammonium phosphate hexahydrate. These single crystals were used with an infrared microscope to measure their NIR absorption coefficients from 1.67 to 2.5 μm. The strong correlation between single crystal NIR spectra and diffuse reflectance NIR spectra indicates little anisotropy in absorption. Most minerals absorbed more strongly at 1.94 μm than 2.12 μm, the wavelengths of thulium fiber and holmium:YAG surgical lasers, respectively. For water bearing minerals, we attribute absorption mainly to water combination modes and assign NIR absorption peaks to specific water molecules and the OH bonds thereof where possible. Reported absorption coefficients can be used to quantitatively compare both laboratory investigations of ablation and surgical experience to physical properties for the first time.

Optical fiber sensors for monitoring cement paste carbonation

The use of concrete has been widespread in our society in housing and infrastructure, despite the environmental cost associated with its production. Its decay poses a social, economic, and environmental problem. Currently, the carbonation of cement paste is monitored through the measurement of its pH, with several optical fiber sensors (OFS) have been produced for this purpose. In the current work the focus is, also, on the carbonation monitoring of cement paste through an OFS, but not through pH measurements. Single fiber reflectance spectroscopy, previously employed to measure cement paste durability, is used to monitor the discoloration of cement paste caused by carbonation. As the carbonation front reaches the fiber tip embedded in the cement paste, the signal reflected onto the fiber increases. The accelerated carbonation of two limestone cement paste samples in an atmosphere of 100% CO2 was successfully monitored. The applicability of the sensor for operational use with ambient CO2 was confirmed through the measurement of carbonation at 3% CO2. The cross interference from water ingress and egress was also evaluated, and it didn’t hinder the measurements of carbonation. Therefore, a novel OFS capable of measuring cement paste carbonation and durability, was achieved.

UAV Remote Sensing Prediction Method of Winter Wheat Yield Based on the Fused Features of Crop and Soil

The early and accurate acquisition of crop yields is of great significance for maintaining food market stability and ensuring global food security. Unmanned aerial vehicle (UAV) remote sensing offers the possibility of predicting crop yields with its advantages of flexibility and high resolution. However, most of the existing remote sensing yield estimation studies focused solely on crops but did not fully consider the influence of soil on yield formation. As an integrated system, the status of crop and soil together determines the final yield. Compared to crop-only yield prediction, the approach that additionally considers soil background information will effectively improve the accuracy and reduce bias in the results. In this study, a novel method for segmenting crop and soil spectral images based on different vegetation coverage is first proposed, in which pixels of crop and soil can be accurately identified by determining the discriminant value Q. On the basis of extracting crop and soil waveband’s information by individual pixel, an innovative approach, projected non-negative matrix factorization based on good point set and matrix cross fusion (PNMF-MCF), was developed to effectively extract and fuse the yield-related features of crop and soil. The experimental results on winter wheat show that the proposed segmentation method can accurately distinguish crop and soil pixels under complex soil background of four different growth periods. Compared with the single reflectance of crop or soil and the simple combination of crop and soil reflectance, the fused yield features spectral matrix FP obtained with PNMF−MCF achieved the best performance in yield prediction at the flowering, flag leaf and pustulation stages, with R2 higher than 0.7 in these three stages. Especially at the flowering stage, the yield prediction model based on PNMF-MCF had the highest R2 with 0.8516 and the lowest RMSE with 0.0744 kg/m2. Correlation analysis with key biochemical parameters (nitrogen and carbon, pigments and biomass) of yield formation showed that the flowering stage was the most vigorous season for photosynthesis and the most critical stage for yield prediction. This study provides a new perspective and complete framework for high-precision crop yield forecasting using UAV remote sensing technology.

Cu(II)-Catalysed Hydrocarboxylation of Imines Utilizing CO2 to Synthesize α-Unsaturated Aminocarboxylic Acids

Here, we report the Cu(II)-photocatalysed hydrocarboxylation of imines (C=N) from a series of synthesized Schiff Base derivatives, namely (E)-1-(4-((4-methylbenzylidene)amino)phenyl)ethanone, (E)-1-(3-((5-bromo-2-hydroxybenzylidene)amino)phenyl)ethanone, (E)-4-((5-bromo-2-hydroxybenzylidene)amino)-1,5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one, and (E)-1,5-dimethyl-4-((4-methylbenzylidene)amino)-2-phenyl-1H-pyrazol-3(2H)-one, with carbon dioxide (CO2) to generate disubstituted amino acids. Under mild conditions (atmospheric pressure of CO2, room temperature, and 30 W Blue LED light), good to excellent yields confirming the formation of substituted amino acid unsaturated acid derivatives were obtained. Single crystal X-ray diffraction (SC-XRD) and UV-Vis diffuse reflectance spectroscopy (UV-Vis-DRS) confirmed the square pyramidal geometry of the Cu(II) photocatalyst. Docking and DFT calculations of the substituted amino acid unsaturated acid derivatives showed their potential as antimicrobial molecules.

Opportunities and pitfalls in (sub)diffuse reflectance spectroscopy

For a long time, steady-state reflectance spectroscopy measurements have been performed so that diffusion theory could be used to extract tissue optical properties from the reflectance. The development of subdiffuse techniques, such as Single Fiber Reflectance Spectroscopy and subdiffuse SFDI, provides new opportunities for clinical applications since they have the key advantage that they are much more sensitive to the details of the tissue scattering phase function in comparison to diffuse techniques. Since the scattering phase function is related to the subcellular structure of tissue, subdiffuse measurements have the potential to provide a powerful contrast between healthy and diseased tissue. In the subdiffuse regime, the interrogated tissue volumes are much smaller than in the diffuse regime. Whether a measurement falls within the diffuse or subdiffuse regime depends on tissue optical properties and the distance between the source and detector fiber for fiber-optic techniques or the projected spatial frequency for hyperspectral imaging and SFDI. Thus, the distance between source and detector fibers or the projected spatial frequency has important implications for clinical applications of reflectance spectroscopy and should be carefully selected, since it influences which tissue optical properties the technique is sensitive to and the size of the tissue volume that is interrogated. In this paper, we will review the opportunities and pitfalls in steady-state reflectance spectroscopy in the subdiffuse and the diffuse regime. The discussed opportunities can guide the choice of either the diffuse or subdiffuse regime for a clinical application, and the discussed pitfalls can ensure these are avoided to enable the development of robust diagnostic algorithms. We will first discuss the relevant basics of light-tissue interaction. Next, we will review all the tissue scattering phase functions that have been measured and investigate which scattering phase function models are representative of tissue. Subsequently, we will discuss the sensitivity of diffuse and subdiffuse techniques to tissue optical properties and we will explore the difference in the interrogation depth probed by diffuse and subdiffuse techniques.

Single vesicle analysis of CD47 association with integrins and tetraspanins on extracellular vesicles released by T lymphoblast and prostate carcinoma cells.

CD47 regulates the trafficking of specific coding and noncoding RNAs into extracellular vesicles (EVs), and the RNA contents of CD47+ EVs differ from that of CD63+ EVs released by the same cells. Single particle interferometric reflectance imaging sensing combined with immunofluorescent imaging was used to analyse the colocalization of tetraspanins, integrins, and CD47 on EVs produced by wild type and CD47‐deficient Jurkat T lymphoblast and PC3 prostate carcinoma cell lines. On Jurkat cell‐derived EVs, β1 and α4 integrin subunits colocalized predominantly with CD47 and CD81 but not with CD63 and CD9, conserving the known lateral interactions between these proteins in the plasma membrane. Although PC3 cell‐derived EVs lacked detectable α4 integrin, specific association of CD81 with β1 and CD47 was preserved. Loss of CD47 expression in Jurkat cells significantly reduced β1 and α4 levels on EVs produced by these cells while elevating CD9+, CD63+, and CD81+ EVs. In contrast, loss of CD47 in PC3 cells decreased the abundance of CD63+ and CD81+ EVs. These data establish that CD47+ EVs are mostly distinct from EVs bearing the tetraspanins CD63 and CD9, but CD47 also indirectly regulates the abundance of EVs bearing these non‐interacting tetraspanins via mechanisms that remain to be determined.