Cell Maps for Artificial Intelligence: AI-Ready Maps of Human Cell Architecture from Disease-Relevant Cell Lines.

This article describes the Cell Maps for Artificial Intelligence (CM4AI) project and its goals, methods, standards, current datasets, software tools , status, and future directions. CM4AI is the Functional Genomics Data Generation Project in the U.S. National Institute of Health's (NIH) Bridge2AI program. Its overarching mission is to produce ethical, AI-ready datasets of cell architecture, inferred from multimodal data collected for human cell lines, to enable transformative biomedical AI research.

Opposing roles of resident and infiltrating immune cells in the defence against Legionella longbeachae via IL-18R/IFN-γ/ROS axis in mice.

The immune response against Legionella longbeachae, a causative agent of the often-fatal Legionnaires' pneumonia, is poorly understood. Here we investigated the specific roles of tissue-resident alveolar macrophages (AM) and infiltrating phagocytes during infection with this pathogen. AM were the predominant cell type that internalized bacteria one day after infection. Three and five days after infection, AM numbers were greatly reduced while there was an influx of neutrophils and later monocyte-derived cells (MC) into lung tissue. AM carried greater numbers of viable L.longbeachae than neutrophils and MC, which correlated with a higher capacity of L.longbeachae to translocate bacterial effector proteins required for bacterial replication into the AM cytosol. Cell ablation experiments demonstrated that AM promoted infection whereas neutrophils and MC were required for efficient bacterial clearance. IL-18 was important for IFN-γ production by IL-18R+ NK cells and T cells which, in turn, stimulated ROS-mediated bactericidal activity in neutrophils resulting in restriction of L.longbeachae infection. Ciliated bronchiolar epithelial cells also expressed IL-18R but did not play a role in IL-18-mediated L.longbeachae clearance. Our results have identified opposing innate functions of tissue-resident and infiltrating immune cells during L.longbeachae infection that may be manipulated to improve protective responses.

Human fertilization in vivo and in vitro requires the CatSper channel to initiate sperm hyperactivation.

The infertility of many couples rests on an enigmatic dysfunction of the man's sperm. To gain insight into the underlying pathomechanisms, we assessed the function of the sperm-specific multisubunit CatSper-channel complex in the sperm of almost 2,300 men undergoing a fertility workup, using a simple motility-based test. We identified a group of men with normal semen parameters but defective CatSper function. These men or couples failed to conceive naturally and upon medically assisted reproduction via intrauterine insemination and in vitro fertilization. Intracytoplasmic sperm injection (ICSI) was, ultimately, required to conceive a child. We revealed that the defective CatSper function was caused by variations in CATSPER genes. Moreover, we unveiled that CatSper-deficient human sperm were unable to undergo hyperactive motility and, therefore, failed to penetrate the egg coat. Thus, our study provides the experimental evidence that sperm hyperactivation is required for human fertilization, explaining the infertility of CatSper-deficient men and the need of ICSI for medically assisted reproduction. Finally, our study also revealed that defective CatSper function and ensuing failure to hyperactivate represents the most common cause of unexplained male infertility known thus far and that this sperm channelopathy can readily be diagnosed, enabling future evidence-based treatment of affected couples.

Morphometric Analyses of Macrophages.

Cell morphology and motility drive the cellular capabilities to interact with the environment. For example, microglia, the longest known tissue-resident macrophages, show a highly branched process tree with which they continuously scan their environment. Computational image analysis allows to quantify morphology and/or motility from images of tissue-resident macrophages. Here, I describe a step-by-step protocol for analyzing the morphology (and motility) of macrophages with our recently described, freely available software MotiQ, which provides a broad band of parameters and thereby serves as a versatile tool for studies of morphology and motility.

Producing and Testing Prototype Tissue-Engineered 3D Tri-Leaflet Valved Stents on Biodegradable Poly-ε-Caprolactone Scaffolds.

Transcatheter pulmonary valve replacement is a minimally-invasive alternative treatment for right ventricular outflow tract dysfunction and has been rapidly evolving over the past years. Heart valve prostheses currently available still have major limitations. Therefore, one of the significant challenges for the future is the roll out of transcatheter tissue engineered pulmonary valve replacement to more patients. In the present study, biodegradable poly-ε-caprolactone (PCL) nanofiber scaffolds in the form of a 3D leaflet matrix were successfully seeded with human endothelial colony-forming cells (ECFCs), human induced pluripotent stem cell-derived MSCs (hMSCs), and porcine MSCs (pMSCs) for three weeks for the generation of 3D tissue-engineered tri-leaflet valved stent grafts. The cell adhesion, proliferation, and distribution of these 3D heart leaflets was analyzed using fluorescence microscopy and scanning electron microscopy (SEM). All cell lineages were able to increase the overgrown leaflet area within the three-week timeframe. While hMSCs showed a consistent growth rate over the course of three weeks, ECFSs showed almost no increase between days 7 and 14 until a growth spurt appeared between days 14 and 21. More than 90% of heart valve leaflets were covered with cells after the full three-week culturing cycle in nearly all leaflet areas, regardless of which cell type was used. This study shows that seeded biodegradable PCL nanofiber scaffolds incorporated in nitinol or biodegradable stents will offer a new therapeutic option in the future.

Cylicins are a structural component of the sperm calyx being indispensable for male fertility in mice and human.

Cylicins are testis-specific proteins, which are exclusively expressed during spermiogenesis. In mice and humans, two Cylicins, the gonosomal X-linked Cylicin 1 (Cylc1/CYLC1) and the autosomal Cylicin 2 (Cylc2/CYLC2) genes, have been identified. Cylicins are cytoskeletal proteins with an overall positive charge due to lysine-rich repeats. While Cylicins have been localized in the acrosomal region of round spermatids, they resemble a major component of the calyx within the perinuclear theca at the posterior part of mature sperm nuclei. However, the role of Cylicins during spermiogenesis has not yet been investigated. Here, we applied CRISPR/Cas9-mediated gene editing in zygotes to establish Cylc1- and Cylc2-deficient mouse lines as a model to study the function of these proteins. Cylc1 deficiency resulted in male subfertility, whereas Cylc2-/-, Cylc1-/yCylc2+/-, and Cylc1-/yCylc2-/- males were infertile. Phenotypical characterization revealed that loss of Cylicins prevents proper calyx assembly during spermiogenesis. This results in decreased epididymal sperm counts, impaired shedding of excess cytoplasm, and severe structural malformations, ultimately resulting in impaired sperm motility. Furthermore, exome sequencing identified an infertile man with a hemizygous variant in CYLC1 and a heterozygous variant in CYLC2, displaying morphological abnormalities of the sperm including the absence of the acrosome. Thus, our study highlights the relevance and importance of Cylicins for spermiogenic remodeling and male fertility in human and mouse, and provides the basis for further studies on unraveling the complex molecular interactions between perinuclear theca proteins required during spermiogenesis.

Male humans, mice and other animals produce sex cells known as sperm that seek out and fertilize egg cells from females. Sperm have a very distinctive shape with a head and a long tail that enables them to swim towards an egg. At the front of the sperm's head is a pointed structure known as the acrosome that helps the sperm to burrow into an egg cell. A structure known as the cytoskeleton is responsible for forming and maintaining the shape of acrosomes and other parts of cells. Two proteins, known as Cylicin 1 and Cylicin 2, are unique to the cytoskeleton of sperm, but their roles remain unclear. To investigate the role of the Cylicins during spermiogenesis, Schneider, Kovacevic et al. used an approach called CRISPR/Cas9-mediated gene-editing to generate mutant mice that were unable to produce either Cylicin 1 or Cylicin 2, or both proteins. The experiments found that healthy female mice were less likely to become pregnant when they mated with mutant males that lacked Cylicin 1 compared with males that had the protein. When they did become pregnant, the females had smaller litters of babies. Mutant male mice lacking Cylicin 2 or both Cylicin proteins (so-called "double" mutants), were infertile and mating with healthy female mice did not lead to any pregnancies. Further experiments found that the sperm of such mice had smaller heads than normal sperm, defective acrosomes, and curled tails that wrapped around the head. Schneider, Kovacevic et al. also examined the sperm of a human patient who had inherited genetic variants in the genes encoding both Cylicin proteins. Similar to the double mutant mice, the patient was infertile, and his sperm also had defective acrosomes and curled tails. These findings indicate that Cylicins are required to make the acrosome as sperm cells mature and help maintain the structure of the cytoskeleton of sperm. Further studies of Cylicins and other sperm proteins in mice may help us to understand some of the factors that contribute to male infertility in humans.

Pulmonary valve reconstruction for acquired pulmonary regurgitation in patients with treated congenital heart disease.

OBJECTIVES: Pulmonary valve regurgitation is a common problem after relief of right ventricular outflow tract (RVOT) obstruction with a transannular patch. Pulmonary valve replacement with a homograft or xenograft is the routine treatment. Longevity of biological valves and the availability of homografts are limited. Alternatives to restore RVOT competence are evaluated. The goal of this study was to present intermediate-term results for pulmonary valve reconstruction (PVr) in patients with severe regurgitation. METHODS: PVr was performed in 24 patients (August 2006‒July 2018). We analysed perioperative data, pre- and postoperative cardiac magnetic resonance (CMR) imaging studies, freedom from valve replacement and risk factors for pulmonary valve dysfunction. RESULTS: The underlying diagnoses were tetralogy of Fallot (n = 18, 75%), pulmonary stenosis (n = 5, 20.8%) and the double outlet right ventricle post banding procedure (n = 1, 4.2%). The median age was 21.5 (14.8-23.7) years. Main (n = 9, 37.5%) and branch pulmonary artery procedures (n = 6, 25%) and surgery of the RVOT (n = 16, 30.2%) were often part of the reconstruction. The median follow-up after the operation was 8.0 (4.7-9.7) years. Freedom from valve failure was 96% at 2 and 90% at 5 years. The mean longevity of the reconstructive surgery was 9.9 years (95% confidence interval: 8.8-11.1 years). CMR before and 6 months after surgery showed a reduction in the regurgitation fraction [41% (33-55) vs 20% (18-27) P = 0.00] and of the indexed right ventricular end-diastolic volume [156 ml/m2 (149-175) vs 116 ml/m2 (100-143), P = 0.004]. Peak velocity across the pulmonary valve (determined by CMR) half a year after surgery was 2.0, unchanged. CONCLUSIONS: PVr can be achieved with acceptable intermediate-term results and may delay pulmonary valve replacement.

Transcatheter versus surgical treatment for isolated superior sinus venosus atrial septal defect.

BACKGROUND: The superior sinus venosus atrial septal defect is a congenital communication between the left and right atria. Open surgical approach by patch closure has historically been the only treatment option. Recently, a transcatheter approach has been developed. This study aims to compare the efficacy and safety of surgical and transcatheter approach in treatment of sinus venosus atrial septal defect. METHODS: Between March 2010 and December 2020, 58 patients (median age: 45.4, range 14.8-73.8) underwent either surgical or transcatheter correction of superior sinus venosus atrial septal defect with partial anomalous pulmonary venous drainage. RESULTS: Twenty-four patients (median age: 35.4, range 14.8-66.8) underwent surgery while 34 patients (median age: 46.8, range 15.5-73.8) had a transcatheter treatment. During the catheterization era, 41 patients was considered suitable for a transcatheter closure. In 5 patients, surgery was the patient's or referring physician's choice. In 2 cases, the procedure was unsuccessful; the remaining 34 were successfully closed (94.4% of cases). Intensive care unit stay (median of 1 day, range 0.5-4, vs. 0, range 0-2, p < 0.0001) and hospital stay (median 7 days, range 2-15 vs. 2 days, range 1-12, p < 0.0001), were significantly longer in the surgery group. Total early complication rate, consisted on procedural and in-hospital complication, were higher in the surgical group (62.5% vs. 23.5%; p = 0.005). However, complications in both groups were clinically mild. At follow-up, a small residual shunt was present in 6 patients (surgery group: 2 pts; catheterization group: 4 pts; p: NS). Imaging studies showed significant improvement of right ventricular size and unobstructed pulmonary venous return in all patients. No late complications occurred at follow-up. CONCLUSIONS: Transcatheter correction of sinus venosus atrial septal defect is effective and safe in selected patients and may be considered as a valid alternative to surgery.

Twenty-four hour rhythmicity in mitochondrial network connectivity and mitochondrial respiration; a study in human skeletal muscle biopsies of young lean and older individuals with obesity.

OBJECTIVE: Mitochondrial network dynamics may play role in metabolic homeostasis. Whether mitochondrial network dynamics are involved in adaptations to day-night fluctuations in energy supply and demand is unclear. Here we visualized and quantified the mitochondrial network morphology in human skeletal muscle of young healthy lean and older individuals with obesity over the course of 24 h METHODS: Muscle biopsies taken at 5 timepoints over a 24-hour period obtained from young healthy lean and older metabolically impaired obese males were analyzed for mitochondrial network integrity with confocal laser scanning microscopy. Variation of level of fragmentation over the course of the day were aligned with variation of mitochondrial respiration over the day RESULTS: Young healthy lean individuals displayed a day-night rhythmicity in mitochondrial network morphology, which aligned with the day-night rhythmicity of mitochondrial respiratory capacity, with a more fused network coinciding with higher mitochondrial respiratory capacity. In the older individuals with obesity, the mitochondrial network was more fragmented overall compared to young healthy lean individuals and completely lacked 24 h rhythmicity, which was also true for the mitochondrial respiratory capacity CONCLUSIONS: Our data shows a paralleled rhythmicity between mitochondrial network morphology and mitochondrial oxidative capacity, which oscillates over the course of a mimicked real-life day in human skeletal muscle of young, healthy lean individuals. In older individuals with obesity, the lack of a 24-hour rhythmicity in mitochondrial network connectivity was also aligned with a lack in respiratory capacity. This suggests that 24-hour rhythmicity in mitochondrial network connectivity is a determinant of rhythmicity in mitochondrial respiratory capacity. Thus, restoring mitochondrial network integrity may promote mitochondrial respiratory capacity and hence contribute to blunting the metabolic aberrations in individuals with a disturbed 24-hour rhythmicity in metabolism, like older individuals with obesity.

Widespread amyloidogenicity potential of multiple myeloma patient-derived immunoglobulin light chains.

BACKGROUND: In a range of human disorders such as multiple myeloma (MM), immunoglobulin light chains (IgLCs) can be produced at very high concentrations. This can lead to pathological aggregation and deposition of IgLCs in different tissues, which in turn leads to severe and potentially fatal organ damage. However, IgLCs can also be highly soluble and non-toxic. It is generally thought that the cause for this differential solubility behaviour is solely found within the IgLC amino acid sequences, and a variety of individual sequence-related biophysical properties (e.g. thermal stability, dimerisation) have been proposed in different studies as major determinants of the aggregation in vivo. Here, we investigate biophysical properties underlying IgLC amyloidogenicity. RESULTS: We introduce a novel and systematic workflow, Thermodynamic and Aggregation Fingerprinting (ThAgg-Fip), for detailed biophysical characterisation, and apply it to nine different MM patient-derived IgLCs. Our set of pathogenic IgLCs spans the entire range of values in those parameters previously proposed to define in vivo amyloidogenicity; however, none actually forms amyloid in patients. Even more surprisingly, we were able to show that all our IgLCs are able to form amyloid fibrils readily in vitro under the influence of proteolytic cleavage by co-purified cathepsins. CONCLUSIONS: We show that (I) in vivo aggregation behaviour is unlikely to be mechanistically linked to any single biophysical or biochemical parameter and (II) amyloidogenic potential is widespread in IgLC sequences and is not confined to those sequences that form amyloid fibrils in patients. Our findings suggest that protein sequence, environmental conditions and presence and action of proteases all determine the ability of light chains to form amyloid fibrils in patients.

Phase separation and dynamical arrest of protein solutions dominated by short-range attractions.

The interplay of liquid-liquid phase separation (LLPS) and dynamical arrest can lead to the formation of gels and glasses, which is relevant for such diverse fields as condensed matter physics, materials science, food engineering, and the pharmaceutical industry. In this context, protein solutions exhibit remarkable equilibrium and non-equilibrium behaviors. In the regime where attractive and repulsive forces compete, it has been demonstrated, for example, that the location of the dynamical arrest line seems to be independent of ionic strength, so that the arrest lines at different ionic screening lengths overlap, in contrast to the LLPS coexistence curves, which strongly depend on the salt concentration. In this work, we show that the same phenomenology can also be observed when the electrostatic repulsions are largely screened, and the range and strength of the attractions are varied. In particular, using lysozyme in brine as a model system, the metastable gas-liquid binodal and the dynamical arrest line as well as the second virial coefficient have been determined for various solution conditions by cloud-point measurements, optical microscopy, centrifugation experiments, and light scattering. With the aim of understanding this new experimental phenomenology, we apply the non-equilibrium self-consistent generalized Langevin equation theory to a simple model system with only excluded volume plus short-range attractions, to study the dependence of the predicted arrest lines on the range of the attractive interaction. The theoretical predictions find a good qualitative agreement with experiments when the range of the attraction is not too small compared with the size of the protein.

Protein solutions close to liquid-liquid phase separation exhibit a universal osmotic equation of state and dynamical behavior.

Liquid-liquid phase separation (LLPS) of protein solutions is governed by highly complex protein-protein interactions. Nevertheless, it has been suggested that based on the extended law of corresponding states (ELCS), as proposed for colloids with short-range attractions, one can rationalize not only the thermodynamics, but also the structure and dynamics of such systems. This claim is systematically and comprehensively tested here by static and dynamic light scattering experiments. Spinodal lines, the isothermal osmotic compressibility κT and the relaxation rate of concentration fluctuations Γ are determined for protein solutions in the vicinity of LLPS. All these quantities are found to exhibit a corresponding-states behavior. This means that, for different solution conditions, these quantities are essentially the same if considered at similar reduced temperature or second virial coefficient. For moderately concentrated solutions, the volume fraction ϕ dependence of κT and Γ can be consistently described by Baxter's model of adhesive hard spheres. The off-critical, asymptotic T behavior of κT and Γ close to LLPS is consistent with the scaling laws predicted by mean-field theory. Thus, the present work aims at a comprehensive experimental test of the applicability of the ELCS to structural and dynamical properties of concentrated protein solutions.

Novel analytical tools reveal that local synchronization of cilia coincides with tissue-scale metachronal waves in zebrafish multiciliated epithelia.

Motile cilia are hair-like cell extensions that beat periodically to generate fluid flow along various epithelial tissues within the body. In dense multiciliated carpets, cilia were shown to exhibit a remarkable coordination of their beat in the form of traveling metachronal waves, a phenomenon which supposedly enhances fluid transport. Yet, how cilia coordinate their regular beat in multiciliated epithelia to move fluids remains insufficiently understood, particularly due to lack of rigorous quantification. We combine experiments, novel analysis tools, and theory to address this knowledge gap. To investigate collective dynamics of cilia, we studied zebrafish multiciliated epithelia in the nose and the brain. We focused mainly on the zebrafish nose, due to its conserved properties with other ciliated tissues and its superior accessibility for non-invasive imaging. We revealed that cilia are synchronized only locally and that the size of local synchronization domains increases with the viscosity of the surrounding medium. Even though synchronization is local only, we observed global patterns of traveling metachronal waves across the zebrafish multiciliated epithelium. Intriguingly, these global wave direction patterns are conserved across individual fish, but different for left and right noses, unveiling a chiral asymmetry of metachronal coordination. To understand the implications of synchronization for fluid pumping, we used a computational model of a regular array of cilia. We found that local metachronal synchronization prevents steric collisions, i.e., cilia colliding with each other, and improves fluid pumping in dense cilia carpets, but hardly affects the direction of fluid flow. In conclusion, we show that local synchronization together with tissue-scale cilia alignment coincide and generate metachronal wave patterns in multiciliated epithelia, which enhance their physiological function of fluid pumping.

Divergent remodeling of the skeletal muscle metabolome over 24 h between young, healthy men and older, metabolically compromised men.

24 h whole-body substrate metabolism and the circadian clock within skeletal muscle are both compromised upon metabolic disease in humans. Here, we assessed the 24 h muscle metabolome by serial muscle sampling performed under 24 h real-life conditions in young, healthy (YH) men versus older, metabolically compromised (OMC) men. We find that metabolites associated with the initial steps of glycolysis and hexosamine biosynthesis are higher in OMC men around the clock, whereas metabolites associated with glutamine-alpha-ketoglutarate, ketone, and redox metabolism are lower in OMC men. The night period shows the largest number of differently expressed metabolites. Both groups demonstrate 24 h rhythmicity in half of the metabolome, but rhythmic metabolites only partially overlap. Specific metabolites are only rhythmic in YH men (adenosine), phase shifted in OMC men (cis-aconitate, flavin adenine dinucleotide [FAD], and uridine diphosphate [UDP]), or have a reduced 24 h amplitude in OMC men (hydroxybutyrate and hippuric acid). Our data highlight the plasticity of the skeletal muscle metabolome over 24 h and large divergence across the metabolic health spectrum.

AdipoQ-a simple, open-source software to quantify adipocyte morphology and function in tissues and in vitro.

The different adipose tissues (ATs) can be distinguished according to their function. For example, white AT stores energy in form of lipids, whereas brown AT dissipates energy in the form of heat. These functional differences are represented in the respective adipocyte morphology; whereas white adipocytes contain large, unilocular lipid droplets, brown adipocytes contain smaller, multilocular lipid droplets. However, an automated, image analysis pipeline to comprehensively analyze adipocytes in vitro in cell culture as well as ex vivo in tissue sections is missing. We here present AdipoQ, an open-source software implemented as ImageJ plugins that allows us to analyze adipocytes in tissue sections and in vitro after histological and/or immunofluorescent labeling. AdipoQ is compatible with different imaging modalities and staining methods, allows batch processing of large datasets and simple post-hoc analysis, provides a broad band of parameters, and allows combining multiple fluorescent readouts. Therefore AdipoQ is of immediate use not only for basic research but also for clinical diagnosis.

Universal effective interactions of globular proteins close to liquid-liquid phase separation: Corresponding-states behavior reflected in the structure factor.

Intermolecular interactions in protein solutions, in general, contain many contributions. If short-range attractions dominate, the state diagram exhibits liquid-liquid phase separation (LLPS) that is metastable with respect to crystallization. In this case, the extended law of corresponding states (ELCS) suggests that thermodynamic properties are insensitive to details of the underlying interaction potential. Using lysozyme solutions, we investigate the applicability of the ELCS to the static structure factor and how far effective colloidal interaction models can help to rationalize the phase behavior and interactions of protein solutions in the vicinity of the LLPS binodal. The (effective) structure factor has been determined by small-angle x-ray scattering. It can be described by Baxter's adhesive hard-sphere model, which implies a single fit parameter from which the normalized second virial coefficient b2 is inferred and found to quantitatively agree with previous results from static light scattering. The b2 values are independent of protein concentration but systematically vary with temperature and solution composition, i.e., salt and additive content. If plotted as a function of temperature normalized by the critical temperature, the values of b2 follow a universal behavior. These findings validate the applicability of the ELCS to globular protein solutions and indicate that the ELCS can also be reflected in the structure factor.

Measurement of ciliary beating and fluid flow in the zebrafish adult telencephalon.

Motile cilia are hair-like structures that move and propel fluid, playing important roles in the physiology of organs. Here, we present a protocol to visualize and measure ciliary beating and cerebrospinal fluid (CSF) flow in the telencephalon of an adult zebrafish brain explant. We describe the preparation of brain explants, the recording of ciliary beating and CSF flow, and data analysis using ImageJ and MATLAB. These imaging and analysis techniques can be directly translated to other ciliated systems. For complete details on the use and execution of this protocol, please refer to D'Gama et al. (2021).

A cAMP signalosome in primary cilia drives gene expression and kidney cyst formation.

The primary cilium constitutes an organelle that orchestrates signal transduction independently from the cell body. Dysregulation of this intricate molecular architecture leads to severe human diseases, commonly referred to as ciliopathies. However, the molecular underpinnings how ciliary signaling orchestrates a specific cellular output remain elusive. By combining spatially resolved optogenetics with RNA sequencing and imaging, we reveal a novel cAMP signalosome that is functionally distinct from the cytoplasm. We identify the genes and pathways targeted by the ciliary cAMP signalosome and shed light on the underlying mechanisms and downstream signaling. We reveal that chronic stimulation of the ciliary cAMP signalosome transforms kidney epithelia from tubules into cysts. Counteracting this chronic cAMP elevation in the cilium by small molecules targeting activation of phosphodiesterase-4 long isoforms inhibits cyst growth. Thereby, we identify a novel concept of how the primary cilium controls cellular functions and maintains tissue integrity in a specific and spatially distinct manner and reveal novel molecular components that might be involved in the development of one of the most common genetic diseases, polycystic kidney disease.