Spatial multimodal analysis of transcriptomes and metabolomes in tissues.

We present a spatial omics approach that combines histology, mass spectrometry imaging and spatial transcriptomics to facilitate precise measurements of mRNA transcripts and low-molecular-weight metabolites across tissue regions. The workflow is compatible with commercially available Visium glass slides. We demonstrate the potential of our method using mouse and human brain samples in the context of dopamine and Parkinson's disease.

Characterization of Effects of Compressed Sensing on High Spectral and Spatial Resolution (HiSS) MRI with Comparison to SENSE.

High Spectral and Spatial resolution (HiSS) MRI shows high diagnostic performance in the breast. Acceleration methods based on k-space undersampling could allow stronger T2*-based image contrast and/or higher spectral resolution, potentially increasing diagnostic performance. An agar/oil phantom was prepared with water-fat boundaries perpendicular to the readout and phase encoding directions in a breast coil. HiSS MRI was acquired at 3T, at sensitivity encoding (SENSE) acceleration factors R of up to 10, and the R = 1 dataset was used to simulate corresponding compressed sensing (CS) accelerations. Image quality was evaluated by quantifying noise and artifact levels. Effective spatial resolution was determined via modulation transfer function analysis. Dispersion vs. absorption (DISPA) analysis and full width at half maximum (FWHM) quantified spectral lineshape changes. Noise levels remained constant with R for CS but amplified with SENSE. SENSE preserved the spatial resolution of HiSS MRI, while CS reduced it in the phase encoding direction. SENSE showed no effect on FWHM or DISPA markers, while CS increased FWHM. Thus, CS might perform better in noise-limited or geometrically constrained applications, but in geometric configurations specific to breast MRI, spectral analysis might be compromised, decreasing the diagnostic performance of HiSS MRI.

A topographic atlas defines developmental origins of cell heterogeneity in the human embryonic lung.

The lung contains numerous specialized cell types with distinct roles in tissue function and integrity. To clarify the origins and mechanisms generating cell heterogeneity, we created a comprehensive topographic atlas of early human lung development. Here we report 83 cell states and several spatially resolved developmental trajectories and predict cell interactions within defined tissue niches. We integrated single-cell RNA sequencing and spatially resolved transcriptomics into a web-based, open platform for interactive exploration. We show distinct gene expression programmes, accompanying sequential events of cell differentiation and maturation of the secretory and neuroendocrine cell types in proximal epithelium. We define the origin of airway fibroblasts associated with airway smooth muscle in bronchovascular bundles and describe a trajectory of Schwann cell progenitors to intrinsic parasympathetic neurons controlling bronchoconstriction. Our atlas provides a rich resource for further research and a reference for defining deviations from homeostatic and repair mechanisms leading to pulmonary diseases.

Impact of Histology and Tumor Grade on Clinical Outcomes Beyond 5 Years of Follow-Up in a Large Cohort of Renal Cell Carcinomas.

INTRODUCTION: The optimal length for clinical follow-up of renal cell carcinoma (RCC) patients is unclear. We evaluated the impact of ISUP/WHO tumor grade and histological subtype on short- and long-term survival and risk of recurrence/metastasis in a large cohort of RCC patients. PATIENTS AND METHODS: We studied 1679 RCC patients from a single referral center in Italy. Adjusted hazard ratios for overall survival were estimated using Cox regression models. Adjusted absolute risk of developing recurrence or metastasis was computed considering competing risks of mortality. RESULTS: During up to 13 years of follow-up, 175 (10.4%) RCC patients died, of whom 92% beyond 5 years. Hazard ratio of grade IV clear cell carcinomas (ccRCC) was 3.82 compared to grade II. Notably, 33% of recurrences and 56% of distant metastases occurred beyond 5 years of follow-up. The estimated probabilities of recurrence/metastasis were 15% and 45% within and beyond 5 years of follow-up, respectively. After 5 years, the absolute risk of recurrences increased also for papillary renal cell carcinoma type I (35.2%) and grade I ccRCC (17%). CONCLUSION: After 5 years of follow-up, both risk of mortality and recurrences or metastases were high and were modified by histological types and tumor grade. These data strongly support histology- and grade-tailored surveillance strategies and long-term follow-up for RCC patients.

Tumor cells in light-chain amyloidosis and myeloma show distinct transcriptional rewiring of normal plasma cell development.

Although light-chain amyloidosis (AL) and multiple myeloma (MM) are characterized by tumor plasma cell (PC) expansion in bone marrow (BM), their clinical presentation differs. Previous attempts to identify unique pathogenic mechanisms behind such differences were unsuccessful, and no studies have investigated the differentiation stage of tumor PCs in patients with AL and MM. We sought to define a transcriptional atlas of normal PC development in secondary lymphoid organs (SLOs), peripheral blood (PB), and BM for comparison with the transcriptional programs (TPs) of tumor PCs in AL, MM, and monoclonal gammopathy of undetermined significance (MGUS). Based on bulk and single-cell RNA sequencing, we observed 13 TPs during transition of normal PCs throughout SLOs, PB, and BM. We further noted the following: CD39 outperforms CD19 to discriminate newborn from long-lived BM-PCs; tumor PCs expressed the most advantageous TPs of normal PC differentiation; AL shares greater similarity to SLO-PCs whereas MM is transcriptionally closer to PB-PCs and newborn BM-PCs; patients with AL and MM enriched in immature TPs had inferior survival; and protein N-linked glycosylation-related TPs are upregulated in AL. Collectively, we provide a novel resource to understand normal PC development and the transcriptional reorganization of AL and other monoclonal gammopathies.

Morphometric analysis of atypical glandular cells correctly classifies normal, reactive, and atypical cells in cervical smears.

The 2014 Bethesda System diagnostic criteria for atypical glandular cells (AGC) aid in the classification of atypical cells in cervical cytology. Anyway, AGC diagnosis remains challenging, due to low frequencies of this finding (approximately 0.5%-1% of Pap test results), abundance of AGC mimics, and significant interobserver variability. We developed an algorithm based on nuclear areas parameter that can help to differentiate AGC from Normal and Reactive glandular cells. Nuclear areas and perimeters were measured on 16 Pap smears with AGC and 18 with Reactive glandular cells of women aged between 30 and 77. Glandular cells from nonpathological Pap smears were used as controls. For each case, the means, medians, standard deviations, and the minimum and maximum values of both nuclear areas and perimeters of the cells of interest were calculated. The nuclear area analysis showed a 100% specificity in discriminating Normal from Altered cells (either Reactive or AGC), whereas the nuclear perimeter analysis showed a lower specificity (87.5%). Both nuclear area and perimeter variability analysis resulted in high specificity values in distinguishing Reactive cells from AGC. Therefore, a stepwise two-step algorithm using nuclear areas to discriminate Normal from Altered cells, and nuclear area variability to distinguish Reactive from AGC, allowed us to reliably classify the cells into these three categories. The morphometric analysis of nuclear area is a valuable and reliable aid in AGC diagnosis and standardization, easily integrable into common automatic algorithms.

Transcriptional profiling of circulating tumor cells in multiple myeloma: a new model to understand disease dissemination.

The reason why a few myeloma cells egress from the bone marrow (BM) into peripheral blood (PB) remains unknown. Here, we investigated molecular hallmarks of circulating tumor cells (CTCs) to identify the events leading to myeloma trafficking into the bloodstream. After using next-generation flow to isolate matched CTCs and BM tumor cells from 32 patients, we found high correlation in gene expression at single-cell and bulk levels (r ≥ 0.94, P = 10-16), with only 55 genes differentially expressed between CTCs and BM tumor cells. CTCs overexpressed genes involved in inflammation, hypoxia, or epithelial-mesenchymal transition, whereas genes related with proliferation were downregulated in CTCs. The cancer stem cell marker CD44 was overexpressed in CTCs, and its knockdown significantly reduced migration of MM cells towards SDF1-α and their adhesion to fibronectin. Approximately half (29/55) of genes differentially expressed in CTCs were prognostic in patients with newly-diagnosed myeloma (n = 553; CoMMpass). In a multivariate analysis including the R-ISS, overexpression of CENPF and LGALS1 was significantly associated with inferior survival. Altogether, these results help understanding the presence of CTCs in PB and suggest that hypoxic BM niches together with a pro-inflammatory microenvironment induce an arrest in proliferation, forcing tumor cells to circulate in PB and seek other BM niches to continue growing.

Preoperative Assessment of Neural Elements in Lumbar Spinal Stenosis by Upright Magnetic Resonance Imaging: An Implication for Routine Practice?

Introduction Lumbar spinal stenosis (LSS) is a kinetic-dependent disease typically aggravating during spinal loading. To date, assessment of LSS is usually performed with magnetic resonance imaging (MRI). However, conventional supine MRI is associated with significant drawbacks as it does not truly reflect physiological loads, experienced by discoligamentous structures during erect posture. Consequently, supine MRI often fails to reveal the source of pain and/or disability caused by LSS. The present study sought to assess neural dimensions via MRI in supine, upright, and upright-hyperlordotic position in order to evaluate the impact of patient positioning on neural narrowing. Therefore, radiological measures such as neuroforaminal dimensions, central canal volume, sagittal listhesis, and lumbar lordosis at spinal level L4/5 were extracted and stratified according to patient posture. Materials and methods Overall, 10 subjects were enclosed in this experimental study. MRI was performed in three different positions: (1) 0° supine (SP), (2) 80° upright (UP), and (3) 80° upright + hyperlordotic (HY) posture. Upright MRI was conducted utilizing a 0.25T open-configuration scanner equipped with a rotatable examination bed allowing for true standing MRI. Radiographic outcome of upright MRI imaging was extracted and evaluated according to patient positioning. Results Upright MRI-based assessment of neural dimensions was successfully accomplished in all subjects. Overall, radiographic parameters revealed a significant decrease of neural dimensions from supine to upright position: Specifically, mean foraminal area decreased from SP to UP by 13.3% (P ≤ 0.05) as well as from SP to HY position by 21% (P ≤ 0.05). Supplementation of hyperlordosis did not result in additional narrowing of neural elements (P ≥ 0.05). Furthermore, central canal volume revealed a decrease of 7% at HY and 8% at UP compared to SP position (P ≥ 0.05). Assessment of lumbar lordosis yielded in a significant increase when assessed at HY (+22.1%) or UP (+8.7%) compared to SP (P ≤ 0.05). Conclusions Our data suggest that neuroforaminal dimensions assessed by conventional supine MRI are potentially overestimated in patients with LSS. Especially, in patients having occult disease not visualized on conventional imaging modalities, upright MRI allows for a precise, clinically relevant, and at the same time non-invasive evaluation of neural elements in LSS when neural decompression is considered.

The Lumbar Spine as a Dynamic Structure Depicted in Upright MRI.

Spinal canal stenosis is a dynamic phenomenon that becomes apparent during spinal loading. Current diagnostic procedures have considerable short comings in diagnosing the disease to full extend, as they are performed in supine situation. Upright MRI imaging might overcome this diagnostic gap.This study investigated the lumbar neuroforamenal diameter, spinal canal diameter, vertebral body translation, and vertebral body angles in 3 different body positions using upright MRI imaging.Fifteen subjects were enrolled in this study. A dynamic MRI in 3 different body positions (at 0° supine, 80° upright, and 80° upright + hyperlordosis posture) was taken using a 0.25 T open-configuration scanner equipped with a rotatable examination bed allowing a true standing MRI.The mean diameter of the neuroforamen at L5/S1 in 0° position was 8.4 mm on the right and 8.8 mm on the left, in 80° position 7.3 mm on the right and 7.2 mm on the left, and in 80° position with hyperlordosis 6.6 mm (P < 0.05) on the right and 6.1 mm on the left (P < 0.001).The mean area of the neuroforamen at L5/S1 in 0° position was 103.5 mm on the right and 105.0 mm on the left, in 80° position 92.5 mm on the right and 94.8 mm on the left, and in 80° position with hyperlordosis 81.9 mm on the right and 90.2 mm on the left.The mean volume of the spinal canal at the L5/S1 level in 0° position was 9770 mm, in 80° position 10600 mm, and in 80° position with hyperlordosis 9414 mm.The mean intervertebral translation at level L5/S1 was 8.3 mm in 0° position, 9.9 mm in 80° position, and 10.1 mm in the 80° position with hyperlordosis.The lordosis angle at level L5/S1 was 49.4° in 0° position, 55.8° in 80° position, and 64.7 mm in the 80° position with hyperlordosis.Spinal canal stenosis is subject to a dynamic process, that can be displayed in upright MRI imaging. The range of anomalies is clinically relevant and dynamic positioning of the patient during MRI can provide essential diagnostic information which are not attainable with other methods.

Extremity-dedicated low-field MRI shows good diagnostic accuracy and interobserver agreement for the diagnosis of the acutely injured knee.

OBJECTIVES: To estimate diagnostic accuracy and interobserver agreement of extremity-dedicated low-field magnetic resonance imaging (lfMRI) for meniscal tears, anterior cruciate ligament (ACL) tears and knee fractures. METHODS: We enrolled 62 patients with acute knee trauma and 19 patients with suspected knee fracture. Arthroscopy/Computed tomography was regarded the gold standard for cruciate ligament and meniscal tears/fractures. RESULTS: Arthroscopy showed 39 meniscal tears. Sensitivity/Specificity of lfMRI was 95.8%/97.4% (reader 1)/100%/100% (reader 2) for medial and 93.3%/100% (reader 1)/93.3%/93.6% (reader 2) for lateral meniscal tears. Sensitivity/Specificity was 100% for ACL tears and fractures. Interobserver agreement was very good. CONCLUSION: lfMRI showed reproducible high sensitivity and specificity for the diagnosis of the acutely injured knee.

Weight-bearing MR imaging as an option in the study of gravitational effects on the vocal tract of untrained subjects in singing phonation.

Magnetic Resonance Imaging (MRI) of subjects in a supine position can be used to evaluate the configuration of the vocal tract during phonation. However, studies of speech phonation have shown that gravity can affect vocal tract shape and bias measurements. This is one of the reasons that MRI studies of singing phonation have used professionally trained singers as subjects, because they are generally considered to be less affected by the supine body position and environmental distractions. A study of untrained singers might not only contribute to the understanding of intuitive singing function and aid the evaluation of potential hazards for vocal health, but also provide insights into the effect of the supine position on singers in general. In the present study, an open configuration 0.25 T MRI system with a rotatable examination bed was used to study the effect of body position in 20 vocally untrained subjects. The subjects were asked to sing sustained tones in both supine and upright body positions on different pitches and in different register conditions. Morphometric measurements were taken from the acquired images of a sagittal slice depicting the vocal tract. The analysis concerning the vocal tract configuration in the two body positions revealed differences in 5 out of 10 measured articulatory parameters. In the upright position the jaw was less protruded, the uvula was elongated, the larynx more tilted and the tongue was positioned more to the front of the mouth than in the supine position. The findings presented are in agreement with several studies on gravitational effects in speech phonation, but contrast with the results of a previous study on professional singers of our group where only minor differences between upright and supine body posture were observed. The present study demonstrates that imaging of the vocal tract using weight-bearing MR imaging is a feasible tool for the study of sustained phonation in singing for vocally untrained subjects.

Influence of knee flexion angle and weight bearing on the Tibial Tuberosity-Trochlear Groove (TTTG) distance for evaluation of patellofemoral alignment.

PURPOSE: The aim of the present study was to investigate the influence of knee flexion and weight bearing on the Tibial Tuberosity-Trochlear Groove (TTTG) distance. MATERIALS AND METHODS: Magnetic resonance imaging of the knee was carried out in 8 healthy volunteers. An open 0.25 T scanner equipped with a C-shaped permanent tilting magnet allowing examinations in weight-bearing conditions was used for the present investigation. A 3D gradient-echo sequence with axial slice orientation was obtained in a lying and an upright position with the knee straight and at 30° of knee flexion. The medial, central and lateral trochlear heights as well as the TTTG were determined. RESULTS: The mean medial trochlear height was 76.2 ± 4%, the central trochlear height was 72.2 ± 3%, and lateral trochlear height was 82.9 ± 3 %. The mean TTTG distance was 11.6 ± 4.4 mm in lying position at 0° knee flexion and 7.3 ± 2.9 mm (n.s.) at 30° knee flexion. Under weight bearing, the mean TTTG was significantly smaller at both 0° knee flexion 6.3 ± 3.2 mm (p = 0.040) and 30° knee flexion 4.9 ± 3.9 mm (p = 0.006) compared to the lying position with 0° knee flexion. CONCLUSION: Tibial Tuberosity-Trochlear Groove distance depends on both knee flexion angle and weight bearing. The latter only seems to be of relevance in full extension.

Accurate semiautomatic assessment of ligament length variations from MRI data.

PURPOSE: A semiautomatic method for the assessment of ligament length variations during different joint positions based on MRI data is proposed. METHODS: Ligament lengths are represented as distances between points marking characteristic locations in the ligament insertion regions on the bones. These points are defined manually for one single reference joint position and for all other joint positions they are automatically mapped with high accuracy to the correct locations using image registration methods. The methodology is validated using data from 16 volunteers depicting the coracoclavicular ligaments in the left shoulder during different arm abductions. RESULTS: The method yielded a superior reproducibility of the point locations over different joint positions compared to manual point marking. Significant ligament length variations were found for different abductions which was not possible with manual measurements. Acquisition related geometric distortions and inaccuracies during the registration and segmentation process were small. CONCLUSIONS: The proposed method provides superior accuracy for the in vivo analysis of ligament dynamics compared to manual measurements. This permits a better understanding of the ligament behavior during joint motion and offers new possibilities for presurgical planning which to date has not been possible with manual data analysis.

The effect of supine and upright position on vocal tract configurations during singing--a comparative study in professional tenors.

INTRODUCTION: Previous studies using dynamic real-time magnetic resonance imaging (MRI) to analyze vocal tract configurations in singers are limited by the fact that the image acquisitions were performed in the supine position. The aim of this study was to examine differences of the vocal tract shape in professional tenors between supine and upright positions. MATERIAL AND METHODS: The vocal tract profiles of nine professional Western classically trained tenors were analyzed using a rotating MRI scanner (0.25 T). The singers performed sustained tones in an ascending scale from C4 (262 Hz) to A4 (440 Hz) on the vowel /a/ in supine and upright positions, starting in modal register and continuing to their stage voice above passaggio or changing to falsetto register, respectively. RESULTS: Many articulators such as lip opening, jaw opening, tongue position, and uvula position were not affected by the subjects' positions. However, the larynx was found to be higher (P<0.001) and the jaw more protruded (P<0.001) for the supine position. The general changes associated with pitch and register were not affected by these systematic differences. CONCLUSIONS: The effect of supine versus upright position on the vocal tract shape is considered to be rather small in professional tenors. The modifications in the vocal tract associated with register and pitch are not affected to a great extent by the position.

Tendon graft fixation sites at the coracoid process for reconstruction of the coracoclavicular ligaments: a kinematic evaluation of three different surgical techniques.

PURPOSE: The virtual graft length kinematics of 3 operative techniques were investigated and compared with kinematics of the native coracoclavicular ligaments. METHODS: Thirteen healthy volunteers underwent magnetic resonance imaging (MRI) of the shoulder in 30° increments of abduction (0° to 120°). A 3-dimensional model of the coracoid process (CP) and the clavicle (CL) was created. Footprints of the conoid and the trapezoid ligament were identified. At the CP the potential fixation sites of 3 techniques for reconstruction of the coracoclavicular ligaments (CCLs) were marked. The techniques investigated were (1) horizontal transcoracoid drilling (TH), (2) transclavicular-transcoracoid drilling (TT), and (3) tendon graft passage underneath (PU) the coracoid process. Distances between the clavicular and coracoidal footprints of the coracoclavicular ligaments and to the virtual footprints on the coracoid process were determined for each abduction increment. RESULTS: All methods investigated resulted in a significantly longer virtual trapezoidal graft (P = .001). In PU, in addition, the virtual conoidal graft was significantly longer. TT resulted in a virtual conoidal graft and conoid ligament of equal length. TH showed identical length and distance regulation of the virtual conoidal graft and the conoid ligament, but significant shortening of the virtual trapezoidal graft during abduction. PU showed isometry of the virtual trapezoidal and conoidal grafts. CONCLUSIONS: None of the described procedures for graft fixation restores the kinematics of the native coracoclavicular ligaments. Graft fixation techniques should be chosen with respect to the preoperative type of instability. Persisting isolated vertical instability might benefit from fixation of the conoidal grafts at the native clavicular footprint. For horizontal clavicular instabilities, techniques more preserving of trapezoid ligament kinematics might be favorable. CLINICAL RELEVANCE: The data suggest that the technique of fixation in conoid and trapezoid ligament reconstruction should depend on the underlying type of instability.

A stress MRI of the shoulder for evaluation of ligamentous stabilizers in acute and chronic acromioclavicular joint instabilities.

PURPOSE: To show the feasibility of a stress magnetic resonance imaging (MRI) as a new method for simultaneous evaluation of the morphology and the functional integrity of the acromioclavicular joint (ACJ) ligamentous stabilizers. MATERIALS AND METHODS: MRI of four volunteers, 10 patients with acute, and six with chronic ACJ injuries was performed using a 0.25 T open MRI scanner. A 2D-proton-density and a 3D-gradient-echo sequence at rest and under 6.5 kg shoulder traction were performed. Comparative measurements of the coracoclavicular and the acromioclavicular distance were performed. Additionally, the conoid and trapezoid ligament lengths were measured with multiplanar reconstructions. RESULTS: MRI at rest correctly identified tears of the coracoclavicular and the acromioclavicular ligaments in eight patients suffering acute ACJ injuries. Stress application helped to distinguish between partial and complete coracoclavicular ligament tears in two cases. Insufficiency of the ACJ ligaments was present in all acute and chronic ACJ injuries. Stress application in chronic ACJ ligaments revealed isolated insufficiency of the conoid ligament in three cases and of the trapezoid ligament in one case. Combined insufficiency was present in two cases. CONCLUSION: Stress MRI facilitates simultaneous acquisition of morphologic and functional information of the ACJ stabilizers. In acute ACJ injuries it helps to distinguish between partial and complete ligament tears. In chronic ACJ injuries it provides functional information of the ligament regrinds.

In vivo analysis of coracoclavicular ligament kinematics during shoulder abduction.

BACKGROUND: Anatomic reconstruction of the coracoclavicular ligaments for the treatment of acromioclavicular joint separations provides superior biomechanical stability compared with other procedures. Clavicular and coracoidal footprints of the conoid ligament (CL) and the trapezoid ligament (TL) are well described. So far, little is known about their kinematics and the changes of the coracoclavicular distance during shoulder abduction. HYPOTHESIS: The coracoclavicular distance along the coracoclavicular ligaments changes significantly with shoulder abduction and weightbearing. STUDY DESIGN: Descriptive laboratory study. METHODS: With use of an open magnetic resonance imaging scanner, the shoulders of 13 healthy volunteers were examined in supine and sitting positions. Three-dimensional magnetic resonance images of the shoulders were obtained in 30° increments of abduction (0°-120°). A manual segmentation of the scapula, the clavicle, and the coracoclavicular ligaments was performed. The insertion points of the coracoclavicular ligaments were identified, and automated measures along the ligamentous course were carried out. RESULTS: During transfer from the lying to sitting position, the coracoclavicular distance showed significant lengthening of 3 mm along the center of the CL, which significantly increased another 3 mm during shoulder abduction to a total lengthening of 6 mm. In the supine position, the coracoclavicular distance along the TL did not elongate significantly. In the sitting position, the distance along the medial portion of the TL shortened significantly, whereas the distance along the center portion did not elongate significantly during shoulder abduction. CONCLUSION: The distances between the coracoclavicular insertion points depend on both patient and shoulder positioning. To prevent overconstraining of the graft, the CL should be fixated during 90° to 120° of shoulder abduction in a sitting position. Isometric reconstruction of the TL can be achieved if precise fixation of the graft at the centers of the conoidal and clavicular footprints is performed.

Spatial and temporal coherence of filtered thermal light.

When a filter is placed in front of a double slit illuminated by a primary source of finite extent, the theory of partial coherence predicts that in general the interference fringes do not acquire unit visibility even as the passband of the filter is made arbitrarily narrow. The effect of reducing the filter bandwidth is that the visibility of the fringes tends to the modulus of the spectral degree of coherence and that more interference fringes become visible. A systematic experimental verification of these theoretical predictions is lacking so far and is provided here from the use of a highly sensitive CCD camera.