Automatic Segmentation and Quantification of Nigrosome-1 Neuromelanin and Iron in MRI: A Candidate Biomarker for Parkinson's Disease.

BACKGROUND: There is a lack of automated tools for the segmentation and quantification of neuromelanin (NM) and iron in the nigrosome-1 (N1). Existing tools evaluate the N1 sign, i.e., the presence or absence of the "swallow-tail" in iron-sensitive MRI, or globally analyze the MRI signal in an area containing the N1, without providing a volumetric delineation. PURPOSE: Present an automated method to segment the N1 and quantify differences in N1's NM and iron content between Parkinson's disease (PD) patients and healthy controls (HCs). Study whether N1 degeneration is clinically related to PD and could be used as a biomarker of the disease. STUDY TYPE: Prospective. SUBJECTS: Seventy-one PD (65.3 ± 10.3 years old, 34 female/37 male); 30 HC (62.7 ± 7.8 years old, 17 female/13 male). FIELD STRENGTH/SEQUENCE: 3 T Anatomical T1-weighted MPRAGE, NM-MRI T1-weighted gradient with magnetization transfer, susceptibility-weighted imaging (SWI). ASSESSMENT: N1 was automatically segmented in SWI images using a multi-image atlas, populated with healthy N1 structures manually annotated by a neurologist. Relative NM and iron content were quantified and their diagnostic performance assessed and compared with the substantia nigra pars compacta (SNc). The association between image parameters and clinically relevant variables was studied. STATISTICAL TESTS: Nonparametric tests were used (Mann-Whitney's U, chi-square, and Friedman tests) at P = 0.05. RESULTS: N1's relative NM content decreased and relative iron content increased in PD patients compared with HCs (NM-CRHC = 22.55 ± 1.49; NM-CRPD = 19.79 ± 1.92; NM-nVolHC = 2.69 × 10-5 ± 1.02 × 10-5 ; NM-nVolPD = 1.18 × 10-5 ± 0.96 × 10-5 ; Iron-CRHC = 10.51 ± 2.64; Iron-CRPD = 19.35 ± 7.88; Iron-nVolHC = 0.72 × 10-5 ± 0.81 × 10-5 ; Iron-nVolPD = 2.82 × 10-5 ± 2.04 × 10-5 ). Binary logistic regression analyses combining N1 and SNc image parameters yielded a top AUC = 0.955. Significant correlation was found between most N1 parameters and both disease duration (ρNM-CR = -0.31; ρiron-CR = 0.43; ρiron-nVol = 0.46) and the motor status (ρNM-nVol = -0.27; ρiron-CR = 0.33; ρiron-nVol = 0.28), suggesting NM reduction along with iron accumulation in N1 as the disease progresses. DATA CONCLUSION: This method provides a fully automatic N1 segmentation, and the analyses performed reveal that N1 relative NM and iron quantification improves diagnostic performance and suggest a relative NM reduction along with a relative iron accumulation in N1 as the disease progresses. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 1.

In vitro modeling of polyclonal infection dynamics within the human airways by Haemophilus influenzae differential fluorescent labeling.

IMPORTANCE: Genomic diversity of nontypeable H. influenzae strains confers phenotypic heterogeneity. Multiple strains of H. influenzae can be simultaneously isolated from clinical specimens, but we lack detailed information about polyclonal infection dynamics by this pathogen. A long-term barrier to our understanding of this host-pathogen interplay is the lack of genetic tools for strain engineering and differential labeling. Here, we present a novel plasmid toolkit named pTBH (toolbox for Haemophilus), with standardized modules for fluorescent or bioluminescent labeling, adapted to H. influenzae requirements but designed to be versatile so it can be utilized in other bacterial species. We present detailed experimental and quantitative image analysis methods, together with proof-of-principle examples, and show the ample possibilities of 3D microscopy, combined with quantitative image analysis, to model H. influenzae polyclonal infection lifestyles and unravel the co-habitation and co-infection dynamics of this respiratory pathogen.

Synplex: In Silico Modeling of the Tumor Microenvironment From Multiplex Images.

Multiplex immunofluorescence is a novel, high-content imaging technique that allows simultaneous in situ labeling of multiple tissue antigens. This technique is of growing relevance in the study of the tumor microenvironment, and the discovery of biomarkers of disease progression or response to immune-based therapies. Given the number of markers and the potential complexity of the spatial interactions involved, the analysis of these images requires the use of machine learning tools that rely for their training on the availability of large image datasets, extremely laborious to annotate. We present Synplex, a computer simulator of multiplexed immunofluorescence images from user-defined parameters: i. cell phenotypes, defined by the level of expression of markers and morphological parameters; ii. cellular neighborhoods based on the spatial association of cell phenotypes; and iii. interactions between cellular neighborhoods. We validate Synplex by generating synthetic tissues that accurately simulate real cancer cohorts with underlying differences in the composition of their tumor microenvironment and show proof-of-principle examples of how Synplex could be used for data augmentation when training machine learning models, and for the in silico selection of clinically relevant biomarkers. Synplex is publicly available at https://github.com/djimenezsanchez/Synplex.

Brainstem neuromelanin and iron MRI reveals a precise signature for idiopathic and LRRK2 Parkinson's disease.

Neuromelanin (NM) loss in substantia nigra pars compacta (SNc) and locus coeruleus (LC) reflects neuronal death in Parkinson's disease (PD). Since genetically-determined PD shows varied clinical expressivity, we wanted to accurately quantify and locate brainstem NM and iron, to discover whether specific MRI patterns are linked to Leucine-rich repeat kinase 2 G2019S PD (LRRK2-PD) or idiopathic Parkinson's disease (iPD). A 3D automated MRI atlas-based segmentation pipeline (3D-ABSP) for NM/iron-sensitive MRI images topographically characterized the SNc, LC, and red nucleus (RN) neuronal loss and calculated NM/iron contrast ratio (CR) and normalized volume (nVol). Left-side NM nVol was larger in all groups. PD had lower NM CR and nVol in ventral-caudal SNc, whereas iron increased in lateral, medial-rostral, and caudal SNc. The SNc NM CR reduction was associated with psychiatric symptoms. LC CR and nVol discriminated better among subgroups: LRRK2-PD had similar LC NM CR and nVol as that of controls, and larger LC NM nVol and RN iron CR than iPD. PD showed higher iron SNc nVol than controls, especially among LRRK2-PD. ROC analyses showed an AUC > 0.92 for most pairwise subgroup comparisons, with SNc NM being the best discriminator between HC and PD. NM measures maintained their discriminator power considering the subgroup of PD patients with less than 5 years of disease duration. The SNc iron CR and nVol increase was associated with longer disease duration in PD patients. The 3D-ABSP sensitively identified NM and iron MRI patterns strongly correlated with phenotypic PD features.

Weakly supervised deep learning to predict recurrence in low-grade endometrial cancer from multiplexed immunofluorescence images.

Predicting recurrence in low-grade, early-stage endometrial cancer (EC) is both challenging and clinically relevant. We present a weakly-supervised deep learning framework, NaroNet, that can learn, without manual expert annotation, the complex tumor-immune interrelations at three levels: local phenotypes, cellular neighborhoods, and tissue areas. It uses multiplexed immunofluorescence for the simultaneous visualization and quantification of CD68 + macrophages, CD8 + T cells, FOXP3 + regulatory T cells, PD-L1/PD-1 protein expression, and tumor cells. We used 489 tumor cores from 250 patients to train a multilevel deep-learning model to predict tumor recurrence. Using a tenfold cross-validation strategy, our model achieved an area under the curve of 0.90 with a 95% confidence interval of 0.83-0.95. Our model predictions resulted in concordance for 96,8% of cases (κ = 0.88). This method could accurately assess the risk of recurrence in EC, outperforming current prognostic factors, including molecular subtyping.

NaroNet: Discovery of tumor microenvironment elements from highly multiplexed images.

Understanding the spatial interactions between the elements of the tumor microenvironment -i.e. tumor cells. fibroblasts, immune cells- and how these interactions relate to the diagnosis or prognosis of a tumor is one of the goals of computational pathology. We present NaroNet, a deep learning framework that models the multi-scale tumor microenvironment from multiplex-stained cancer tissue images and provides patient-level interpretable predictions using a seamless end-to-end learning pipeline. Trained only with multiplex-stained tissue images and their corresponding patient-level clinical labels, NaroNet unsupervisedly learns which cell phenotypes, cell neighborhoods, and neighborhood interactions have the highest influence to predict the correct label. To this end, NaroNet incorporates several novel and state-of-the-art deep learning techniques, such as patch-level contrastive learning, multi-level graph embeddings, a novel max-sum pooling operation, or a metric that quantifies the relevance that each microenvironment element has in the individual predictions. We validate NaroNet using synthetic data simulating multiplex-immunostained images where a patient label is artificially associated to the -adjustable- probabilistic incidence of different microenvironment elements. We then apply our model to two sets of images of human cancer tissues: 336 seven-color multiplex-immunostained images from 12 high-grade endometrial cancer patients; and 382 35-plex mass cytometry images from 215 breast cancer patients. In both synthetic and real datasets, NaroNet provides outstanding predictions of relevant clinical information while associating those predictions to the presence of specific microenvironment elements.

Complement C5a induces the formation of neutrophil extracellular traps by myeloid-derived suppressor cells to promote metastasis.

Myeloid-derived suppressor cells (MDSCs) play a major role in cancer progression. In this study, we investigated the mechanisms by which complement C5a increases the capacity of polymorphonuclear MDSCs (PMN-MDSCs) to promote tumor growth and metastatic spread. Stimulation of PMN-MDSCs with C5a favored the invasion of cancer cells via a process dependent on the formation of neutrophil extracellular traps (NETs). NETosis was dependent on the production of high mobility group box 1 (HMGB1) by cancer cells. Moreover, C5a induced the surface expression of the HMGB1 receptors TLR4 and RAGE in PMN-MDSCs. In a mouse lung metastasis model, inhibition of C5a, C5a receptor-1 (C5aR1) or NETosis reduced the number of circulating-tumor cells (CTCs) and the metastatic burden. In support of the translational relevance of these findings, C5a was able to stimulate migration and NETosis in PMN-MDSCs obtained from lung cancer patients. Furthermore, myeloperoxidase (MPO)-DNA complexes, as markers of NETosis, were elevated in lung cancer patients and significantly correlated with C5a levels. In conclusion, C5a induces the formation of NETs from PMN-MDSCs in the presence of cancer cells, which may facilitate cancer cell dissemination and metastasis.

Development and multimodal characterization of an elastase-induced emphysema mouse disease model for the COPD frequent bacterial exacerbator phenotype.

Chronic obstructive pulmonary disease (COPD) patients undergo infectious exacerbations whose frequency identifies a clinically meaningful phenotype. Mouse models have been mostly used to separately study both COPD and the infectious processes, but a reliable model of the COPD frequent exacerbator phenotype is still lacking. Accordingly, we first established a model of single bacterial exacerbation by nontypeable Haemophilus influenzae (NTHi) infection on mice with emphysema-like lesions. We characterized this single exacerbation model combining both noninvasive in vivo imaging and ex vivo techniques, obtaining longitudinal information about bacterial load and the extent of the developing lesions and host responses. Bacterial load disappeared 48 hours post-infection (hpi). However, lung recovery, measured using tests of pulmonary function and the disappearance of lung inflammation as revealed by micro-computed X-ray tomography, was delayed until 3 weeks post-infection (wpi). Then, to emulate the frequent exacerbator phenotype, we performed two recurrent episodes of NTHi infection on the emphysematous murine lung. Consistent with the amplified infectious insult, bacterial load reduction was now observed 96 hpi, and lung function recovery and disappearance of lesions on anatomical lung images did not happen until 12 wpi. Finally, as a proof of principle of the use of the model, we showed that azithromycin successfully cleared the recurrent infection, confirming this macrolide utility to ameliorate infectious exacerbation. In conclusion, we present a mouse model of recurrent bacterial infection of the emphysematous lung, aimed to facilitate investigating the COPD frequent exacerbator phenotype by providing complementary, dynamic information of both infectious and inflammatory processes.

Heterogenous presence of neutrophil extracellular traps in human solid tumours is partially dependent on IL-8.

Neutrophil extracellular traps (NETs) are webs of extracellular nuclear DNA extruded by dying neutrophils infiltrating tissue. NETs constitute a defence mechanism to entrap and kill fungi and bacteria. Tumours induce the formation of NETs to the advantage of the malignancy via a variety of mechanisms shown in mouse models. Here, we investigated the presence of NETs in a variety of human solid tumours and their association with IL-8 (CXCL8) protein expression and CD8+ T-cell density in the tumour microenvironment. Multiplex immunofluorescence panels were developed to identify NETs in human cancer tissues by co-staining with the granulocyte marker CD15, the neutrophil marker myeloperoxidase and citrullinated histone H3 (H3Cit), as well as IL-8 protein and CD8+ T cells. Three ELISA methods to detect and quantify circulating NETs in serum were optimised and utilised. Whole tumour sections and tissue microarrays from patients with non-small cell lung cancer (NSCLC; n = 14), bladder cancer (n = 14), melanoma (n = 11), breast cancer (n = 31), colorectal cancer (n = 20) and mesothelioma (n = 61) were studied. Also, serum samples collected retrospectively from patients with metastatic melanoma (n = 12) and NSCLC (n = 34) were ELISA assayed to quantify circulating NETs and IL-8. NETs were detected in six different human cancer types with wide individual variation in terms of tissue density and distribution. At least in NSCLC, bladder cancer and metastatic melanoma, NET density positively correlated with IL-8 protein expression and inversely correlated with CD8+ T-cell densities. In a series of serum samples from melanoma and NSCLC patients, a positive correlation between circulating NETs and IL-8 was found. In conclusion, NETs are detectable in formalin-fixed human biopsy samples from solid tumours and in the circulation of cancer patients with a considerable degree of individual variation. NETs show a positive association with IL-8 and a trend towards a negative association with CD8+ tumour-infiltrating lymphocytes. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Muscular and Tendon Degeneration after Achilles Rupture: New Insights into Future Repair Strategies.

Achilles tendon rupture is a frequent injury with an increasing incidence. After clinical surgical repair, aimed at suturing the tendon stumps back into their original position, the repaired Achilles tendon is often plastically deformed and mechanically less strong than the pre-injured tissue, with muscle fatty degeneration contributing to function loss. Despite clinical outcomes, pre-clinical research has mainly focused on tendon structural repair, with a lack of knowledge regarding injury progression from tendon to muscle and its consequences on muscle degenerative/regenerative processes and function. Here, we characterize the morphological changes in the tendon, the myotendinous junction and muscle belly in a mouse model of Achilles tendon complete rupture, finding cellular and fatty infiltration, fibrotic tissue accumulation, muscle stem cell decline and collagen fiber disorganization. We use novel imaging technologies to accurately relate structural alterations in tendon fibers to pathological changes, which further explain the loss of muscle mechanical function after tendon rupture. The treatment of tendon injuries remains a challenge for orthopedics. Thus, the main goal of this study is to bridge the gap between clinicians' knowledge and research to address the underlying pathophysiology of ruptured Achilles tendon and its consequences in the gastrocnemius. Such studies are necessary if current practices in regenerative medicine for Achilles tendon ruptures are to be improved.

The use of mixed collagen-Matrigel matrices of increasing complexity recapitulates the biphasic role of cell adhesion in cancer cell migration: ECM sensing, remodeling and forces at the leading edge of cancer invasion.

The migration of cancer cells is highly regulated by the biomechanical properties of their local microenvironment. Using 3D scaffolds of simple composition, several aspects of cancer cell mechanosensing (signal transduction, EMC remodeling, traction forces) have been separately analyzed in the context of cell migration. However, a combined study of these factors in 3D scaffolds that more closely resemble the complex microenvironment of the cancer ECM is still missing. Here, we present a comprehensive, quantitative analysis of the role of cell-ECM interactions in cancer cell migration within a highly physiological environment consisting of mixed Matrigel-collagen hydrogel scaffolds of increasing complexity that mimic the tumor microenvironment at the leading edge of cancer invasion. We quantitatively show that the presence of Matrigel increases hydrogel stiffness, which promotes ß1 integrin expression and metalloproteinase activity in H1299 lung cancer cells. Then, we show that ECM remodeling activity causes matrix alignment and compaction that favors higher tractions exerted by the cells. However, these traction forces do not linearly translate into increased motility due to a biphasic role of cell adhesions in cell migration: at low concentration Matrigel promotes migration-effective tractions exerted through a high number of small sized focal adhesions. However, at high Matrigel concentration, traction forces are exerted through fewer, but larger focal adhesions that favor attachment yielding lower cell motility.

NMF-RI: blind spectral unmixing of highly mixed multispectral flow and image cytometry data.

MOTIVATION: Recent advances in multiplex immunostaining and multispectral cytometry have opened the door to simultaneously visualizing an unprecedented number of biomarkers both in liquid and solid samples. Properly unmixing fluorescent emissions is a challenging task, which normally requires the characterization of the individual fluorochromes from control samples. As the number of fluorochromes increases, the cost in time and use of reagents becomes prohibitively high. Here, we present a fully unsupervised blind spectral unmixing method for the separation of fluorescent emissions in highly mixed spectral data, without the need for control samples. To this end, we extend an existing method based on non-negative Matrix Factorization, and introduce several critical improvements: initialization based on the theoretical spectra, automated selection of 'sparse' data and use of a re-initialized multilayer optimizer. RESULTS: Our algorithm is exhaustively tested using synthetic data to study its robustness against different levels of colocalization, signal to noise ratio, spectral resolution and the effect of errors in the initialization of the algorithm. Then, we compare the performance of our method to that of traditional spectral unmixing algorithms using novel multispectral flow and image cytometry systems. In all cases, we show that our blind unmixing algorithm performs robust unmixing of highly spatially and spectrally mixed data with an unprecedently low computational cost. In summary, we present the first use of a blind unmixing method in multispectral flow and image cytometry, opening the door to the widespread use of our method to efficiently pre-process multiplex immunostaining samples without the need of experimental controls. AVAILABILITY AND IMPLEMENTATION: https://github.com/djimenezsanchez/Blind_Unmixing_NMF_RI/ contains the source code and all datasets used in this manuscript. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Low cost gaze estimation: knowledge-based solutions.

Eye tracking technology in low resolution scenarios is not a completely solved issue to date. The possibility of using eye tracking in a mobile gadget is a challenging objective that would permit to spread this technology to non-explored fields. In this paper, a knowledge based approach is presented to solve gaze estimation in low resolution settings. The understanding of the high resolution paradigm permits to propose alternative models to solve gaze estimation. In this manner, three models are presented: a geometrical model, an interpolation model and a compound model, as solutions for gaze estimation for remote low resolution systems. Since this work considers head position essential to improve gaze accuracy, a method for head pose estimation is also proposed. The methods are validated in an optimal framework, I2Head database, which combines head and gaze data. The experimental validation of the models demonstrates their sensitivity to image processing inaccuracies, critical in the case of the geometrical model. Static and extreme movement scenarios are analyzed showing the higher robustness of compound and geometrical models in the presence of user's displacement. Accuracy values of about 3° have been obtained, increasing to values close to 5° in extreme displacement settings, results fully comparable with the state-of-the-art.

Dynamic Atlas-Based Segmentation and Quantification of Neuromelanin-Rich Brainstem Structures in Parkinson Disease.

We present a dynamic atlas composed of neuromelanin-enhanced magnetic resonance brain images of 40 healthy subjects. The performance of this atlas is evaluated on the fully automated segmentation of two paired neuromelanin-rich brainstem healthy structures: the substantia nigra pars compacta and the locus coeruleus. We show that our dynamic atlas requires in average 60% less images and, therefore, 60% less computation time than a static multi-image atlas while achieving a similar segmentation performance. Then, we show that by applying our dynamic atlas, composed of healthy subjects, to the segmentation and neuromelanin quantification of a set of brain images of 39 Parkinson disease patients, we are able to find significant quantitative differences in the level of neuromelanin between healthy subjects and Parkinson disease patients, thus opening the door to the use of these structures as image biomarkers in future computer aided diagnosis systems for the diagnosis of Parkinson disease.

Optimizing interoperability between video-oculographic and electromyographic systems.

A new system is presented that enhances the interoperability between a video-oculographic (VOG) system for mouse movement control and an electromyographic (EMG) system for mouse click detection. The proposed VOG-EMG system combines gaze and muscle information to minimize the number of undesired clicks due to involuntary activations and environmental noise. We tested the system with 24 subjects, comparing three different configurations: one in which the VOG and EMG systems worked independently and two in which we used VOG gaze information to improve the EMG click detection. Results show that the number of false-positive click detections can be reduced when VOG and EMG information is combined. In addition, the third configuration, including extra processing, can reduce the activation delay produced because of the combined use of the VOG and EMG systems. The new VOG-EMG system is meant to be used in noisy environments in which the number of false clicks may impeach a reliable human-computer interaction.