Signal Intensity Distribution Research Articles

Study on the damage mechanism and evolution model of preloaded sandstone subjected to freezing–thawing action based on the NMR technology

Abstract Investigating the damage degradation of rock during the freezing and thawing process is more consistent with the actual engineering environment, considering its internal initial damage. In this study, the effects of initial damage from preloading and subzero-temperature freezing–thawing on microscopic and macroscopic mechanical properties of sandstone were studied based on the nuclear magnetic resonance (NMR) technique. The results show that the P-wave velocity of the sample decreased, while the porosity increased as the initial damage level increased. The distribution of T2 signal intensity exposed to the low-temperature freezing–thawing–saturation treatment was rather larger than that under normal temperature conditions for samples with different levels of initial damage from preloading, indicating that the low-temperature freezing–thawing condition would promote the porosity and have an obvious increase in damage. A continuum damage model considering subzero-temperature freezing–thawing damage from different pore sizes was finally introduced to describe the damage evolution mechanism of sandstone. The research results can be used to quantitatively evaluate the damage evolution mechanism of sandstone treated by subzero-temperature freezing–thawing without mechanical tests. Furthermore, the analysis and research results show that the damage variables of sandstone obtained by the NMR theory were lower than those of classical rock mechanics theory.

Redefining the concept of residual renal function with kidney sodium MRI: a pilot study.

The concept of residual kidney function (RKF) is exclusively based upon urine volume and small solute clearance, making RKF challenging to assess in clinical practice. The aim of this study was to test the technical feasibility of obtaining useable 23Na-MRI kidney images in hemodialysis (HD) participants. We conducted an exploratory prospective study to quantify the cortico-medullary sodium gradient in healthy and HD participants. Participants fasted for eight hours prior to their study visit. Urine samples were collected to measure urinary osmolarity, before MRI. Proton and sodium pictures were merged; ROIs were delineated for the medulla and cortex when feasible. In cases where cortex could not be identified, we considered the cortico to medulla gradient (CMG) to be no longer present, resulting in a medulla-to-cortex ratio of 1. 17 healthy volunteers and 21 HD participants. Median (IQR) fasting medulla to cortex ratio was significantly higher 1.56 [1.5-1.61] in healthy volunteers compared to HD patients 1.22 [1.13-1.3], p<0.0001. Medulla to cortex ratio and median urinary osmolarity were correlated (r=0.87, p<0.0001) in the whole population. We found a significant association between HD vintage and medulla to cortex ratio whereas we did not find any association with urine volume. Sodium signal intensity distribution within healthy kidney describes two different peaks- relating to well defined cortex and medulla; whereas HD participants displays only a single peak indicative of the markedly lower sodium concentration. This study is only an exploratory study with a modest number of patients. the application of kidney sodium MRI to the study of RKF in patients receiving maintenance HD is practical and provides a previously unavailable ability to interrogate the function of remnant tubular function.

Radiomic Analysis in Pituitary Tumors: Current Knowledge and Future Perspectives.

Radiomic analysis has emerged as a valuable tool for extracting quantitative features from medical imaging data, providing in-depth insights into various contexts and diseases. By employing methods derived from advanced computational techniques, radiomics quantifies textural information through the evaluation of the spatial distribution of signal intensities and inter-voxel relationships. In recent years, these techniques have gained considerable attention also in the field of pituitary tumors, with promising results. Indeed, the extraction of radiomic features from pituitary magnetic resonance imaging (MRI) images has been shown to provide useful information on various relevant aspects of these diseases. Some of the key topics that have been explored in the existing literature include the association of radiomic parameters with histopathological and clinical data and their correlation with tumor invasiveness and aggressive behavior. Their prognostic value has also been evaluated, assessing their role in the prediction of post-surgical recurrence, response to medical treatments, and long-term outcomes. This review provides a comprehensive overview of the current knowledge and application of radiomics in pituitary tumors. It also examines the current limitations and future directions of radiomic analysis, highlighting the major challenges that need to be addressed before a consistent integration of these techniques into routine clinical practice.

Assessment of Brain Tumour Perfusion Using Early-Phase 18F-FET PET: Comparison with Perfusion-Weighted MRI

PurposeMorphological imaging using MRI is essential for brain tumour diagnostics. Dynamic susceptibility contrast (DSC) perfusion-weighted MRI (PWI), as well as amino acid PET, may provide additional information in ambiguous cases. Since PWI is often unavailable in patients referred for amino acid PET, we explored whether maps of relative cerebral blood volume (rCBV) in brain tumours can be extracted from the early phase of PET using O-(2-18F-fluoroethyl)-L-tyrosine (18F-FET).ProcedureUsing a hybrid brain PET/MRI scanner, PWI and dynamic 18F-FET PET were performed in 33 patients with cerebral glioma and four patients with highly vascularized meningioma. The time interval from 0 to 2 min p.i. was selected to best reflect the blood pool phase in 18F-FET PET. For each patient, maps of MR-rCBV, early 18F-FET PET (0–2 min p.i.) and late 18F-FET PET (20–40 min p.i.) were generated and coregistered. Volumes of interest were placed on the tumour (VOI-TU) and normal-appearing brain (VOI-REF). The correlation between tumour-to-brain ratios (TBR) of the different parameters was analysed. In addition, three independent observers evaluated MR-rCBV and early 18F-FET maps (18F-FET-rCBV) for concordance in signal intensity, tumour extent and intratumoural distribution.ResultsTBRs calculated from MR-rCBV and 18F-FET-rCBV showed a significant correlation (r = 0.89, p < 0.001), while there was no correlation between late 18F-FET PET and MR-rCBV (r = 0.24, p = 0.16) and 18F-FET-rCBV (r = 0.27, p = 0.11). Visual rating yielded widely agreeing findings or only minor differences between MR-rCBV maps and 18F-FET-rCBV maps in 93 % of the tumours (range of three independent raters 91–94%, kappa among raters 0.78–1.0).ConclusionEarly 18F-FET maps (0–2 min p.i.) in gliomas provide similar information to MR-rCBV maps and may be helpful when PWI is not possible or available. Further studies in gliomas are needed to evaluate whether 18F-FET-rCBV provides the same clinical information as MR-rCBV.

Late gadolinium enhancement entropy as a new measure of myocardial tissue heterogeneity for prediction of adverse cardiac events in patients with hypertrophic cardiomyopathy

ObjectivesEntropy is a new late gadolinium enhanced (LGE) cardiac magnetic resonance (CMR)–derived parameter that is independent of signal intensity thresholds. Entropy can be used to measure myocardial tissue heterogeneity by comparing full pixel points of tissue images. This study investigated the incremental prognostic value of left ventricular (LV) entropy in patients with hypertrophic cardiomyopathy (HCM).MethodsThis study enrolled 337 participants with HCM who underwent 3.0-T CMR. The LV entropy was obtained by calculating the probability distribution of the LV myocardial pixel signal intensities of the LGE sequence. Patients who underwent CMR imaging were followed up for endpoints. The primary endpoint was defined as readmission to the hospital owing to heart failure. The secondary endpoint was the composite of the primary endpoint, sudden cardiac death and non-cardiovascular death.ResultsDuring the median follow-up of 24 months ± 13 (standard deviation), 43 patients who reached the primary and secondary endpoints had a higher entropy (6.20 ± 0.45, p < 0.001). The patients with increased entropy (≥ 5.587) had a higher risk of the primary and secondary endpoints, compared with HCM patients with low entropy (p < 0.001 for both). In addition, Cox analysis showed that LV entropy provided significant prognostic value for predicting both primary and secondary endpoints (HR: 1.291 and 1.273, all p < 0.001). Addition of LV entropy to the multivariable model improved model performance and risk reclassification (p < 0.05).ConclusionLV entropy assessed by CMR was an independent predictor of primary and secondary endpoints. LV entropy assessment contributes to improved risk stratification in patients with HCM.Critical relevance statementMyocardial heterogeneity reflected by entropy the derived parameter of LGE has prognostic value for adverse events in HCM. The measurement of LV entropy helped to identify patients with HCM who were at risk for heart failure and sudden cardiac death.Key points• Left ventricular entropy can reflect myocardial heterogeneity in HCM patients.• Left ventricular entropy was significantly higher in HCM patients who reached endpoint events.• Left ventricular entropy helps to predict the occurrence of heart failure and death in HCM patients.Graphical

Entropy as a novel predictor of cardiovascular events in patients with left ventricular noncompaction

BackgroundThe risk stratification of left ventricular noncompaction (LVNC) remains ambiguous. LV entropy derived from late gadolinium enhancement (LGE) in cardiac magnetic resonance (CMR) as a novel measurement of myocardial heterogeneity may serve as the substrate of major adverse cardiovascular events (MACEs). This retrospective study aimed to investigate the value of LV entropy for predicting MACEs in LVNC patients. MethodsConsecutive patients who underwent CMR and met the diagnosis criteria of LVNC were included. All patients were follow-up for MACEs (cardiac death, ventricular arrhythmia requiring therapy or heart failure hospitalization), and their LV entropy derived from the distribution of pixel signal intensities in the LGE of the LV myocardium was analyzed. ResultsOne hundred and forty-three patients (mean age 40 years, 64.3% male) were followed for a median of 3.2 years, and forty-two (29.4%) experienced MACEs. Presenting of symptoms, left ventricular end-diastolic diameter (LVEDD), LV end-diastolic volume (LVEDV) index, LV end-systolic volume (LVESV) index, LV ejection fraction (LVEF), LGE extent, and LV entropy showed association with MACEs. LV entropy maintained independent association with MACEs (HR: 4.76, 95%CI 3.68–5.15, p < 0.001) in multivariable analysis. Entropy was also strong independent predictor of MACEs in patients with and without LGE (HR: 5.89, 95% CI4.18–7.73, p < 0.001; HR: 3.06, 95% CI:1.53–4.80, p = 0.013, respectively). ConclusionsLV entropy can predict MACEs in LVNC patients and provide incremental prognostic value on top of LVEF and LGE. Also, LV entropy may help risk stratification in LGE-negative LVNC patients.

Selection of optimal quantile protein biomarkers based on cell-level immunohistochemistry data

BackgroundProtein biomarkers of cancer progression and response to therapy are increasingly important for improving personalized medicine. Advanced quantitative pathology platforms enable measurement of protein expression in tissues at the single-cell level. However, this rich quantitative cell-by-cell biomarker information is most often not exploited. Instead, it is reduced to a single mean across the cells of interest or converted into a simple proportion of binary biomarker-positive or -negative cells.ResultsWe investigated the utility of retaining all quantitative information at the single-cell level by considering the values of the quantile function (inverse of the cumulative distribution function) estimated from a sample of cell signal intensity levels in a tumor tissue. An algorithm was developed for selecting optimal cutoffs for dichotomizing cell signal intensity distribution quantiles as predictors of continuous, categorical or survival outcomes. The proposed algorithm was used to select optimal quantile biomarkers of breast cancer progression based on cancer cells’ cell signal intensity levels of nuclear protein Ki-67, Proliferating cell nuclear antigen, Programmed cell death 1 ligand 2, and Progesterone receptor. The performance of the resulting optimal quantile biomarkers was validated and compared to the standard cancer compartment mean signal intensity markers using an independent external validation cohort. For Ki-67, the optimal quantile biomarker was also compared to established biomarkers based on percentages of Ki67-positive cells. For proteins significantly associated with PFS in the external validation cohort, the optimal quantile biomarkers yielded either larger or similar effect size (hazard ratio for progression-free survival) as compared to cancer compartment mean signal intensity biomarkers.ConclusionThe optimal quantile protein biomarkers yield generally improved prognostic value as compared to the standard protein expression markers. The proposed methodology has a broad application to single-cell data from genomics, transcriptomics, proteomics, or metabolomics studies at the single cell level.

Using Intratumor Heterogeneity of Immunohistochemistry Biomarkers to Classify Laryngeal and Hypopharyngeal Tumors Based on Histologic Features

Haralick texture features are used to quantify the spatial distribution of signal intensities within an image. In this study, the heterogeneity of proliferation (Ki-67 expression) and immune cells (CD45 expression) within tumors was quantified and used to classify histologic characteristics of larynx and hypopharynx carcinomas. Of 21 laryngectomy specimens, 74 whole-mount tumor slides were scored on histologic characteristics. Ki-67 and CD45 immunohistochemistry was performed, and all sections were digitized. The tumor area was annotated in QuPath. Haralick features independent of the diaminobenzidine intensity were extracted from the isolated diaminobenzidine signal to quantify intratumor heterogeneity. Haralick features from both Ki-67 and CD45 were used as input for a principal component analysis. A linear support vector machine was fitted to the first 4 principal components for classification and validated with a leave-one-patient-out cross-validation method. Significant differences in individual Haralick features were found between cohesive and noncohesive tumors for CD45 (angular second motion: P =.03, inverse difference moment: P =.009, and entropy: P =.02) and between the larynx and hypopharynx tumors for both CD45 (angular second motion: P =.03, inverse difference moment: P =.007, and entropy: P =.005) and Ki-67 (correlation: P =.003). Therefore, these features were used for classification. The linear classifier resulted in a classification accuracy of 85% for site of origin and 81% for growth pattern. A leave-one-patient-out cross-validation resulted in an error rate of 0.27 and 0.35 for both classifiers, respectively. In conclusion, we show a method to quantify intratumor heterogeneity of immunohistochemistry biomarkers using Haralick features. This study also shows the feasibility of using these features to classify tumors by histologic characteristics. The classifiers created in this study are a proof of concept because more data are needed to create robust classifiers, but the method shows potential for automated tumor classification.

Quantile Index Biomarkers Based on Single-Cell Expression Data

Current histocytometry methods enable single-cell quantification of biomolecules in tumor tissue sections by multiple detection technologies, including multiplex fluorescence-based immunohistochemistry or in situ hybridization. Quantitative pathology platforms can provide distributions of cellular signal intensity (CSI) levels of biomolecules across the entire cell populations of interest within the sampled tumor tissue. However, the heterogeneity of CSI levels is usually ignored, and the simple mean signal intensity value is considered a cancer biomarker. Here we consider the entire distribution of CSI expression levels of a given biomolecule in the cancer cell population as a predictor of clinical outcome. The proposed quantile index (QI) biomarker is defined as the weighted average of CSI distribution quantiles in individual tumors. The weight for each quantile is determined by fitting a functional regression model for a clinical outcome. That is, the weights are optimized so that the resulting QI has the highest power to predict a relevant clinical outcome. The proposed QI biomarkers were derived for proteins expressed in cancer cells of malignant breast tumors and demonstrated improved prognostic value compared with the standard mean signal intensity predictors. The R package Qindex implementing QI biomarkers has been developed. The proposed approach is not limited to immunohistochemistry data and can be based on any cell-level expressions of proteins or nucleic acids.

Ventilation and perfusion MRI at a 0.35 T MR-Linac: feasibility and reproducibility study

BackgroundHybrid devices that combine radiation therapy and MR-imaging have been introduced in the clinical routine for the treatment of lung cancer. This opened up not only possibilities in terms of accurate tumor tracking, dose delivery and adapted treatment planning, but also functional lung imaging. The aim of this study was to show the feasibility of Non-uniform Fourier Decomposition (NuFD) MRI at a 0.35 T MR-Linac as a potential treatment response assessment tool, and propose two signal normalization strategies for enhancing the reproducibility of the results.MethodsTen healthy volunteers (median age 28 ± 8 years, five female, five male) were repeatedly scanned at a 0.35 T MR-Linac using an optimized 2D+t balanced steady-state free precession (bSSFP) sequence for two coronal slice positions. Image series were acquired in normal free breathing with breaks inside and outside the scanner as well as deep and shallow breathing. Ventilation- and perfusion-weighted maps were generated for each image series using NuFD. For intra-volunteer ventilation map reproducibility, a normalization factor was defined based on the linear correlation of the ventilation signal and diaphragm position of each scan as well as the diaphragm motion amplitude of a reference scan. This allowed for the correction of signal dependency on the diaphragm motion amplitude, which varies with breathing patterns. The second strategy, which can be used for ventilation and perfusion, eliminates the dependency on the signal amplitude by normalizing the ventilation/perfusion maps with the average ventilation/perfusion signal within a selected region-of-interest (ROI). The position and size dependency of this ROI was analyzed. To evaluate the performance of both approaches, the normalized ventilation/perfusion-weighted maps were compared and the deviation of the mean ventilation/perfusion signal from the reference was calculated for each scan. Wilcoxon signed-rank tests were performed to test whether the normalization methods can significantly improve the reproducibility of the ventilation/perfusion maps.ResultsThe ventilation- and perfusion-weighted maps generated with the NuFD algorithm demonstrated a mostly homogenous distribution of signal intensity as expected for healthy volunteers regardless of the breathing maneuver and slice position. Evaluation of the ROI’s size and position dependency showed small differences in the performance. Applying both normalization strategies improved the reproducibility of the ventilation by reducing the median deviation of all scans to 9.1%, 5.7% and 8.6% for the diaphragm-based, the best and worst performing ROI-based normalization, respectively, compared to 29.5% for the non-normalized scans. The significance of this improvement was confirmed by the Wilcoxon signed rank test with p, <, 0.01 at alpha , =, 0.05. A comparison of the techniques against each other revealed a significant difference in the performance between best ROI-based normalization and worst ROI (p, =, 0.01) and between best ROI-based normalization and scaling factor (p, =, 0.02), but not between scaling factor and worst ROI (p, =, 0.71). Using the ROI-based approach for the perfusion-maps, the uncorrected deviation of 10.2% was reduced to 5.3%, which was shown to be significant (p, <, 0.01).ConclusionsUsing NuFD for non-contrast enhanced functional lung MRI at a 0.35 T MR-Linac is feasible and produces plausible ventilation- and perfusion-weighted maps for volunteers without history of chronic pulmonary diseases utilizing different breathing patterns. The reproducibility of the results in repeated scans significantly benefits from the introduction of the two normalization strategies, making NuFD a potential candidate for fast and robust early treatment response assessment of lung cancer patients during MR-guided radiotherapy.

Time-resolved ICP-MS analysis of mineral element contents and distribution patterns in spermatogenic cells of different types

Mineral elements play an important role in the spermatogenesis, maturation, and fertilization of sperm. It is of great scientific significance to study the role of mineral elements in spermatogenesis by accurately measuring the content of elements in different spermatogenic cells and analyzing the distribution pattern of elements in spermatogenesis. Here, time-resolved inductively coupled plasma mass spectrometry (ICP-MS) was used to analyze the content and distribution patterns of mineral elements in spermatogenic cells of different types at the single cell level. Firstly, spermatogonia, spermatocytes, round spermatids and elongating spermatids were successfully isolated from testis of mice of different weeks of age by differential adherent method and discontinuous bovine serum albumin (BSA) density gradient method. Then, signal profiles and elemental distributions of 24Mg, 31P, 52Cr, 55Mn, 56Fe and 66Zn in spermatogenic cells were obtained with dwell time at 0.1 ms. Based on the results of acid digestion, we derived a formula to calculate element content in single cell from peak area for each element, and the feasibility and universality of the formula in the quantitative detection of single cell elements were verified by sperm samples to a certain extent. The detection results of element content in single cell showed that the content of 31P in elongating spermatids was significantly higher than that in spermatogonia, spermatocytes and round spermatids (P < 0.01), and the distribution range was wider. However, the 52Cr and 56Fe content of elongating spermatids was lower than that of spermatogonia, spermatocytes and round spermatids (P < 0.05). When spermatogonia developed into round spermatids, the contents of 55Mn and 66Zn in single cell increased significantly (P < 0.05), then decreased to the lowest in elongating spermatids. In addition, the significant decrease of 52Cr, 55Mn, 56Fe and 66Zn content in elongating spermatids also be visually observed from the center of the fitting curve of the element signal intensity distribution moving to the left. This study provides an elemental view of the changes in elemental content at various stages of spermatogenesis at the single-cell level. Time-resolved ICP-MS is used to detect mineral elements content and distribution patterns in spermatogenic cells of testis, which is helpful to better explore the stages and modes of action of various elements in spermatogenesis, and provide direct evidence for revealing the effects of element content changes on spermatogenesis and semen quality regulation.

The deconvolved distribution estimator: enhancing reionization-era CO line-intensity mapping analyses with a cross-correlation analogue for one-point statistics

ABSTRACT We present the deconvolved distribution estimator (DDE), an extension of the voxel intensity distribution (VID), in the context of future observations proposed as part of the CO Mapping Array Project (COMAP). The DDE exploits the fact that the observed VID is a convolution of correlated signal intensity distributions and uncorrelated noise or interloper intensity distributions. By deconvolving the individual VID of two observables away from their joint VID in a Fourier-space operation, the DDE suppresses sensitivity to interloper emission while maintaining sensitivity to correlated components. The DDE thus improves upon the VID by reducing the relative influence of uncorrelated noise and interloper biases, which is useful in the context of COMAP observations that observe different rotational transitions of CO from the same comoving volume in different observing frequency bands. Fisher forecasts suggest that the theoretical sensitivity in the DDE allows significant improvements in constraining power compared to either the cross power spectrum or the individual VID data, and matches the constraining power of the combination of all other one- and two-point summary statistics. Future work should further investigate the covariance and model-dependent behaviour of this novel one-point cross-correlation statistic.

Image analysis workflows to reveal the spatial organization of cell nuclei and chromosomes

ABSTRACT Nucleus, chromatin, and chromosome organization studies heavily rely on fluorescence microscopy imaging to elucidate the distribution and abundance of structural and regulatory components. Three-dimensional (3D) image stacks are a source of quantitative data on signal intensity level and distribution and on the type and shape of distribution patterns in space. Their analysis can lead to novel insights that are otherwise missed in qualitative-only analyses. Quantitative image analysis requires specific software and workflows for image rendering, processing, segmentation, setting measurement points and reference frames and exporting target data before further numerical processing and plotting. These tasks often call for the development of customized computational scripts and require an expertise that is not broadly available to the community of experimental biologists. Yet, the increasing accessibility of high- and super-resolution imaging methods fuels the demand for user-friendly image analysis workflows. Here, we provide a compendium of strategies developed by participants of a training school from the COST action INDEPTH to analyze the spatial distribution of nuclear and chromosomal signals from 3D image stacks, acquired by diffraction-limited confocal microscopy and super-resolution microscopy methods (SIM and STED). While the examples make use of one specific commercial software package, the workflows can easily be adapted to concurrent commercial and open-source software. The aim is to encourage biologists lacking custom-script-based expertise to venture into quantitative image analysis and to better exploit the discovery potential of their images. Abbreviations: 3D FISH: three-dimensional fluorescence in situ hybridization; 3D: three-dimensional; ASY1: ASYNAPTIC 1; CC: chromocenters; CO: Crossover; DAPI: 4',6-diamidino-2-phenylindole; DMC1: DNA MEIOTIC RECOMBINASE 1; DSB: Double-Strand Break; FISH: fluorescence in situ hybridization; GFP: GREEN FLUORESCENT PROTEIN; HEI10: HUMAN ENHANCER OF INVASION 10; NCO: Non-Crossover; NE: Nuclear Envelope; Oligo-FISH: oligonucleotide fluorescence in situ hybridization; RNPII: RNA Polymerase II; SC: Synaptonemal Complex; SIM: structured illumination microscopy; ZMM (ZIP: MSH4: MSH5 and MER3 proteins); ZYP1: ZIPPER-LIKE PROTEIN 1.

Radial distribution of pump and signal intensities in step index EDFA for LP11 mode in Kerr nonlinear condition

Abstract In an all-optical communication system, an erbium-doped fiber amplifier performs a very significant role. The effectiveness of the operation of this kind of amplifier depends on different parameters of the amplifier. Variation of the intensities of pump and signal with distance along the radius of the fiber from the core axis is one such significant parameter. In our present case, we have studied the distribution of the intensities of both the pump and signal along the radius of the fiber in an erbium-doped dual-mode fiber amplifier for the LP11 mode. In the present case, some step-index fibers of different normalized frequencies have opted. Our study is an application of the Chebyshev technique expressing the LP11 modal field in the form of a power series. A little computation is required for the prediction of the concerned results by this technique. Results obtained from this study show an excellent match with those found by the rigorous finite element method establishing its accuracy. This study using such a user-friendly and accurate technique will be helpful to the optical engineers involved in this domain.

Characterization of Asymmetrically Fed Er3+/Yb3+ Co-Doped Double Core Integrated Waveguides

The output signal intensity distribution of several fs-laser written double-core active waveguides was registered. The ratio between cores output powers exhibited a strong dependence on the input pump power when both input pump and signal powers were directly coupled to one of the cores. A model based on the coupled-mode theory which incorporates non-identical cores and longitudinally variable signal gain coefficients was implemented. Simulations based on this model are compared to experiments. A good agreement is achievable if small differences between cores’ parameters (within the manufacturing precision limits) are allowed.

Poster 155: Muscle-Derived Beige Adipose Precursors Secrete Promyogenic Exosomes that Treat Rotator Cuff Muscle Degeneration in Mice and are Identified in Humans by Single-Cell RNA Sequencing

Objectives:Muscle atrophy, fibrosis, and fatty infiltration are common to a variety of sports-related and degenerative conditions and are thought to be irreversible. Fibroadipogenic progenitors (FAPs) are multipotent resident muscle stem cells with the capacity to differentiate into fibrogenic as well as white- and beige adipose tissue (WAT; BAT). FAPs that have assumed a BAT differentiation state (FAP-BAT) have proven efficacious in treating muscle degeneration in a variety of injury models. The purpose of this study was to characterize the subpopulation of murine FAPs with FAP-BAT activity, determine whether their pro-myogenic effect is mediated via exosomes, and analyze human FAPs for an analogous pro-myogenic, exosome-rich subpopulation. Given their non-myogenic differentiation capacity, it was hypothesized that FAP-BAT exerts a pro-myogenic effect on the injured muscle environment through the secretion of exosomes.Methods:FAPs from UCP1-reporter mice were isolated via FACS and sorted according to the differential intensity of the UCP1 signal observed: negative for UCP1 (UCP1-), intermediate intensity (UCP1+), and high intensity (UCP1++). Bulk RNAseq was performed on UCP1-, UCP1+, and UCP1++ FAPs to evaluate distinct characteristics of each population. Exosomes were harvested from UCP1++ (Exo-FB) and UCP1- (Exo-nFB) cells using cushioned-density gradient ultracentrifugation and used to treat C2C12 cells and MEFs in vitro, and the myotube fusion index was assessed. Exo-FB and Exo-nFB were then used to treat WT C57B/L6J mice that had undergone massive rotator cuff tear. At 6 weeks mice were sacrificed and supraspinatus muscles were harvested and analyzed for muscle atrophy, fibrosis, fatty infiltration, and UCP1 expression. Single-cell RNA sequencing (scRNAseq) was then performed on FAPs isolated from human rotator cuff muscle that were treated with mirabegron or standard media to assess for the presence of a parallel promyogenic subpopulation of FAP-BAT cells in humans.Results:Flow cytometry analysis of sorted UCP1-reporter mouse FAPs revealed a trimodal distribution of UCP1 signal intensity, which correlated with three distinct transcriptomic profiles characterized with bulk RNAseq. UCP1++ cells were marked by high mitochondrial gene expression, BAT markers, and exosome surface makers (Table 1). UCP1- cells were marked by fibrogenic markers and UCP1+ cells were characterized differential enrichment of WAT markers (Table 1). Exo-FB treatment of C2C12 cells resulted in robust myotube fusion, with a myotube fusion index of 33.3 ± 6.3% compared to 7.1 ± 3.2% with Exo-nFB (p <0.001) and 8.8 ± 3.3% with exosome-free media (p = 0.0014), while treatment of MEFs resulted in transdifferentiation into myotubes (Fig. 1). Mice that were treated with Exo-FB at the time of rotator cuff injury demonstrated markedly reduced muscle atrophy and FI compared to treatment with Exo-nFB or PBS (Fig. 2). ScRNAseq of human FAPs from the rotator cuff revealed 6 distinct subpopulations of human FAPs, with differential expression of mitochondrial genes, certain BAT markers, and exosome surface markers noted within one of the subpopulations (Fig. 3).Conclusions:FAP-BAT cells comprise a subpopulation of FAPs with upregulated mitochondrial gene expression and exosome production that mediates promyogenic effects in vitro and in vivo, and are present as a transcriptomically similar subpopulation of FAPs in humans.Table 1.Highly Differentially Expressed Genes of UCP1++ FAPsFigure 1.C2C12 cells and MEFs cultured for 14 days with Exo-FB(2 x 109), Exo-nFB(2 x 109), or exosome-free media, and stained for myosin heavy chain (MF20)j scale bar = 50µm (A), Myotube fusion index was calculated as the percentage of nuclei per field contained within an MF20+ myotube. n-3 replicates per condition; ** indicates p<0.005, *** indicates p<0.001.Figure 3.Light microscoay and immunofluorescence of human FAPs grown in standard media (top row) or mirabegron treated media (bottom row); scale bar =50µm; red arrows indicates predominant perilipin expression, green arrow indicates predominant UCP1 expression, yellow arrow indicates expression of both UCP1 and perilipin (A) UMAP plot of human scRNAseq data demonstrating 5 distinct clusters of human FAPs identified with Seurat (B) UMAP plot demonstrating distribution of human FAPs by in vitro treatment conditions: standard media or mirabegron (c). Dot plot of top 20 differentially expressed genes by treatment condition based upon adjusted p-value; “Average Expression” denotes z-score from mean overaal gene expression (D). Dot plot demonstrating unifrom increase in mitochondrial gene expression with mirabegron (E). Dot plot showing 13 genes that are significantly differentially expressed in both murine UCP1++ FAPs and human FAP subpopulations (F). Dot plot demonstrating additional characterstic genes of interest stratified by cluster. Red arrows indicates populations favorted to contain FAP-BAT cells at varying differentiations stages; green arrow highlights Adrb2 receptor expression; blue arrow highlights exosome surface markers CD63 and CD81(G).

Near-Infrared Fluorescence Axillary Reverse Mapping (ARM) Procedure in Invasive Breast Cancer: Relationship between Fluorescence Signal in ARM Lymph Nodes and Clinical Outcomes

Simple SummaryNear-infrared fluorescence axillary reverse mapping (ARM) is a promising procedure for identification and preservation of arm lymphatic drainage during axillary lymph node dissection (ALND). We included 109 patients to analyze the indocyanine green fluorescence signal in ARM lymph nodes after resection. The fluorescence signal from ARM lymph nodes were compared with clinical findings to determine the importance of this criterion on the potential management of patients with ALND. ARM lymph nodes were identified in 94.5% of cases. The mean normalized fluorescence signal intensity value was 0.47 with no significant signal difference between metastatic and non-metastatic ARM lymph nodes. Only the preoperative diagnosis of metastasis in the axillary nodes of patients was significantly associated with a higher ARM node fluorescence signal intensity. Although preliminary results did not show that fluorescence signal intensity is a reliable diagnostic tool, the NIR fluorescence ARM procedure may be useful for ARM lymph node identification.The near-infrared (NIR) fluorescence axillary reverse mapping (ARM) procedure is a promising tool to identify and preserve arm lymphatic drainage during axillary lymph node dissection (ALND). The ARMONIC clinical trial was conducted to validate the technique on a large cohort of patients and to analyze the predictive clinical factors for ARM lymph node metastasis. For the first time, the fluorescence signal intensity from the ARM lymph nodes was measured and correlated with clinical findings. A total of 109 patients with invasive breast cancer and indications of mastectomy and ALND underwent the NIR fluorescence ARM procedure. Indocyanine green was administered by intradermal injection followed by intraoperative identification and resection of the ARM lymph nodes with NIR fluorescence camera guidance. The fluorescence signal intensity and signal distribution were then measured ex vivo and compared with clinical outcomes. ARM lymph nodes were successfully identified by fluorescence in 94.5% of cases. The mean normalized fluorescence signal intensity value was 0.47 with no significant signal difference between metastatic and non-metastatic ARM lymph nodes (p = 0.3728). At the microscopic level, the fluorescence signal distribution was focally intense in lymphoid tissue areas. Only the preoperative diagnosis of metastasis in the axillary nodes of patients was significantly associated with a higher ARM node fluorescence signal intensity (p = 0.0253), though it was not significantly associated with the pathological nodal (pN) status (p = 0.8081). Based on an optimal cut-off fluorescence value, the final sensitivity and specificity of the NIR fluorescence ARM procedure for ARM lymph node metastatic involvement were 64.7% and 47.3%, respectively. Although our preliminary results did not show that fluorescence signal intensity is a reliable diagnostic tool, the NIR fluorescence ARM procedure may be useful for ARM lymph node identification. Clinical trial registration: NCT02994225.

Muscle-Derived Beige Adipose Precursors Secrete Promyogenic Exosomes That Treat Rotator Cuff Muscle Degeneration in Mice and Are Identified in Humans by Single-Cell RNA Sequencing

Background: Muscle atrophy, fibrosis, and fatty infiltration are common to a variety of sports-related and degenerative conditions and are thought to be irreversible. Fibroadipogenic progenitors (FAPs) are multipotent resident muscle stem cells with the capacity to differentiate into fibrogenic as well as white and beige adipose tissue (BAT). FAPs that have assumed a BAT differentiation state (FAP-BAT) have proven efficacious in treating muscle degeneration in numerous injury models. Purpose: To characterize the subpopulation of murine FAPs with FAP-BAT activity, determine whether their promyogenic effect is mediated via exosomes, and analyze human FAPs for an analogous promyogenic exosome-rich subpopulation. Study Design: Controlled laboratory study. Methods: FAPs from UCP1 reporter mice were isolated via fluorescence-activated cell sorting and sorted according to the differential intensity of the UCP1 signal observed: negative for UCP1 (UCP1–), intermediate intensity (UCP1+), and high intensity (UCP1++). Bulk RNA sequencing was performed on UCP1–, UCP1+, and UCP1++ FAPs to evaluate distinct characteristics of each population. Exosomes were harvested from UCP1++ FAP-BAT exosomes (Exo-FB) as well as UCP1– non–FAP-BAT exosomes (Exo-nFB) cells using cushioned-density gradient ultracentrifugation and used to treat C2C12 cells and mouse embryonic fibroblasts in vitro, and the myotube fusion index was assessed. Exo-FB and Exo-nFB were then used to treat wild type C57B/L6J mice that had undergone a massive rotator cuff tear. At 6 weeks mice were sacrificed, and supraspinatus muscles were harvested and analyzed for muscle atrophy, fibrosis, fatty infiltration, and UCP1 expression. Single-cell RNA sequencing was then performed on FAPs isolated from human muscle that were treated with the beta-agonist formoterol or standard media to assess for the presence of a parallel promyogenic subpopulation of FAP-BAT cells in humans. Results: Flow cytometry analysis of sorted UCP1 reporter mouse FAPs revealed a trimodal distribution of UCP1 signal intensity, which correlated with 3 distinct transcriptomic profiles characterized with bulk RNA sequencing. UCP1++ cells were marked by high mitochondrial gene expression, BAT markers, and exosome surface makers; UCP1– cells were marked by fibrogenic markers; and UCP1+ cells were characterized differential enrichment of white adipose tissue markers. Exo-FB treatment of C2C12 cells resulted in robust myotube fusion, while treatment of mouse embryonic fibroblasts resulted in differentiation into myotubes. Treatment of cells with Exo-nFB resulted in poor myotube formation. Mice that were treated with Exo-FB at the time of rotator cuff injury demonstrated markedly reduced muscle atrophy and fatty infiltration as compared with treatment with Exo-nFB or phosphate-buffered saline. Single-cell RNA sequencing of human FAPs from the rotator cuff revealed 6 distinct subpopulations of human FAPs, with one subpopulation demonstrating the presence of UCP1+ beige adipocytes with a distinct profile of BAT, mitochondrial, and extracellular vesicle–associated markers. Conclusion: FAP-BAT cells form a subpopulation of FAPs with upregulated beige gene expression and exosome production that mediate promyogenic effects in vitro and in vivo, and they are present as a transcriptomically similar subpopulation of FAPs in humans. Clinical Relevance: FAP-BAT cells and their exosomes represent a potential therapeutic avenue for treating rotator cuff muscle degeneration.

Iron-sensitive MR imaging of the primary motor cortex to differentiate hereditary spastic paraplegia from other motor neuron diseases.

Hereditary spastic paraplegia (HSP) is a group of genetic neurodegenerative diseases characterised by upper motor neuron (UMN) impairment of the lower limbs. The differential diagnosis with primary lateral sclerosis (PLS) and amyotrophic lateral sclerosis (ALS) can be challenging. As microglial iron accumulation was reported in the primary motor cortex (PMC) of ALS cases, here we assessed the radiological appearance of the PMC in a cohort of HSP patients using iron-sensitive MR imaging and compared the PMC findings among HSP, PLS, and ALS patients. We included 3-T MRI scans of 23 HSP patients, 7 PLS patients with lower limb onset, 8 ALS patients with lower limb and prevalent UMN onset (UMN-ALS), and 84 ALS patients with any other clinical picture. The PMC was visually rated on 3D T2*-weighted images as having normal signal intensity, mild hypointensity, or marked hypointensity, and differences in the frequency distribution of signal intensity among the diseases were investigated. The marked hypointensity in the PMC was visible in 3/22 HSP patients (14%), 7/7 PLS patients (100%), 6/8 UMN-ALS patients (75%), and 35/84 ALS patients (42%). The frequency distribution of normal signal intensity, mild hypointensity, and marked hypointensity in HSP patients was different than that in PLS, UMN-ALS, and ALS patients (p < 0.01 in all cases). Iron-sensitive imaging of the PMC could provide useful information in the diagnostic work - up of adult patients with a lower limb onset UMN syndrome, as the cortical hypointensity often seen in PLS and ALS cases is apparently rare in HSP patients. • The T2* signal intensity of the primary motor cortex was investigated in patients with HSP, PLS with lower limb onset, and ALS with lower limb and prevalent UMN onset (UMN-ALS) using a clinical 3-T MRI sequence. • Most HSP patients had normal signal intensity in the primary motor cortex (86%); on the contrary, all the PLS and the majority of UMN-ALS patients (75%) had marked cortical hypointensity. • The T2*-weighted imaging of the primary motor cortex could provide useful information in the differential diagnosis of sporadic adult-onset UMN syndromes.

Mitigating the effects of choroidal hyper- and hypo-transmission defects on choroidal vascularity index assessments using optical coherence tomography.

Changes in choroidal vascularity index (CVI) are associated with multiple choroid-related ocular diseases. CVI is calculated as the area/volume ratio of vessels in the choroid, which could be affected by alterations in regional signal intensities due to hypo-transmission defects (hypoTDs) caused by drusen and retinal pigment epithelium (RPE) detachments, and hyper-transmission defects (hyperTDs) caused by the absence of RPE. To develop a simulation model to verify the CVI assessments in eyes with hyper/hypoTDs and demonstrate that accurate CVIs can be achieved after attenuation correction on swept-source optical coherence tomography (SS-OCT). A simulation model was developed on 6×6 mm macular scans from normal subjects. Signal intensity in a cylindrical region below RPE was altered to mimic hyper/hypoTDs. CVIs were compared inside and outside the simulated regions before and after attenuation correction. CVI assessments of OCT scans from patients with hyperTDs due to geographic atrophy (GA) and from patients with hypoTDs due to drusen that subsequently resolved with the disappearance of the hypoTDs were compared with and without attenuation correction. Ten normal eyes were recruited to generate the hyper/hypoTD simulation model. In eyes with hypoTDs, CVIs were overestimated, and in eyes with hyperTDs, the CVIs were underestimated (P<0.001). After attenuation correction, the uneven distribution of signal intensity was eliminated and the resulting CVI showed no significant difference compared with the 'ground truth', which is measured from the original scans. Attenuation correction successfully eliminated the influence of hyperTDs caused by GA on CVI measurements (n=38). Quantitatively, no significant difference was found in the CVIs of eyes before and after drusen collapse with attenuation correction (n=8). The simulation model could reveal the impact of hypo/hyperTDs on CVI quantification in eyes with choroid-involved ocular diseases. The importance of attenuation correction to ensure accuracy in choroidal vessel segmentation was demonstrated by analyzing eyes with GA or drusen.