Lysosomes, caspase-mediated apoptosis, and cytoplasmic activation of P21, but not cell senescence, participate in a redundant fashion in embryonic morphogenetic cell death.

Micromass cultures of embryonic limb skeletal progenitors replicate the tissue remodelling processes observed during digit morphogenesis. Here, we have employed micromass cultures in an in vitro assay to study the nature of cell degeneration events associated with skeletogenesis. In the assay, "naive" progenitors obtained from the autopod aggregate to form chondrogenic nodules and those occupying the internodular spaces exhibit intense apoptosis and progressive accumulation of larger cells, showing intense SA-ß-Gal histochemical labelling that strictly overlaps with the distribution of neutral red vital staining. qPCR analysis detected intense upregulation of the p21 gene, but P21 immunolabelling showed cytoplasmic rather than the nuclear distribution expected in senescent cells. Semithin sections and transmission electron microscopy confirmed the presence of canonical apoptotic cells, degenerated cell fragments in the process of phagocytic internalization by the neighbouring cells, and large vacuolated cells containing phagosomes. The immunohistochemical distribution of active caspase 3, cathepsin D, and ß-galactosidase together with the reduction in cell death by chemical inhibition of caspases (Q-VAD) and lysosomal cathepsin D (Pepstatin A) supported a redundant implication of both pathways in the dying process. Chemical inhibition of P21 (UC2288) revealed a complementary role of this factor in the dying process. In contrast, treatment with the senolytic drug Navitoclax increased cell death without changing the number of cells positive for SA-ß-Gal. We propose that this model of tissue remodelling involves the cooperative activation of multiple degradation routes and, most importantly, that positivity for SA-ß-Gal reflects the occurrence of phagocytosis, supporting the rejection of cell senescence as a defining component of developmental tissue remodelling.

Anionic Phospholipids Shift the Conformational Equilibrium of the Selectivity Filter in the KcsA Channel to the Conductive Conformation: Predicted Consequences on Inactivation.

Here, we report an allosteric effect of an anionic phospholipid on a model K+ channel, KcsA. The anionic lipid in mixed detergent-lipid micelles specifically induces a change in the conformational equilibrium of the channel selectivity filter (SF) only when the channel inner gate is in the open state. Such change consists of increasing the affinity of the channel for K+, stabilizing a conductive-like form by maintaining a high ion occupancy in the SF. The process is highly specific in several aspects: First, lipid modifies the binding of K+, but not that of Na+, which remains unperturbed, ruling out a merely electrostatic phenomenon of cation attraction. Second, no lipid effects are observed when a zwitterionic lipid, instead of an anionic one, is present in the micelles. Lastly, the effects of the anionic lipid are only observed at pH 4.0, when the inner gate of KcsA is open. Moreover, the effect of the anionic lipid on K+ binding to the open channel closely emulates the K+ binding behaviour of the non-inactivating E71A and R64A mutant proteins. This suggests that the observed increase in K+ affinity caused by the bound anionic lipid should result in protecting the channel against inactivation.

Rapid artificial intelligence solutions in a pandemic-The COVID-19-20 Lung CT Lesion Segmentation Challenge.

Artificial intelligence (AI) methods for the automatic detection and quantification of COVID-19 lesions in chest computed tomography (CT) might play an important role in the monitoring and management of the disease. We organized an international challenge and competition for the development and comparison of AI algorithms for this task, which we supported with public data and state-of-the-art benchmark methods. Board Certified Radiologists annotated 295 public images from two sources (A and B) for algorithms training (n=199, source A), validation (n=50, source A) and testing (n=23, source A; n=23, source B). There were 1,096 registered teams of which 225 and 98 completed the validation and testing phases, respectively. The challenge showed that AI models could be rapidly designed by diverse teams with the potential to measure disease or facilitate timely and patient-specific interventions. This paper provides an overview and the major outcomes of the COVID-19 Lung CT Lesion Segmentation Challenge - 2020.

A plant-like battery: a biodegradable power source ecodesigned for precision agriculture.

The natural environment has always been a source of inspiration for the research community. Nature has evolved over thousands of years to create the most complex living systems, with the ability to leverage inner and outside energetic interactions in the most efficient way. This work presents a flow battery profoundly inspired by nature, which mimics the fluid transport in plants to generate electric power. The battery was ecodesigned to meet a life cycle for precision agriculture (PA) applications; from raw material selection to disposability considerations, the battery is conceived to minimize its environmental impact while meeting PA power requirements. The paper-based fluidic system relies on evaporation as the main pumping force to pull the reactants through a pair of porous carbon electrodes where the electrochemical reaction takes place. This naturally occurring transpiration effect enables to significantly expand the operational lifespan of the battery, overcoming the time-limitation of current capillary-based power sources. Most relevant parameters affecting the battery performance, such as evaporation flow and redox species degradation, are thoroughly studied to carry out device optimization. Flow rates and power outputs comparable to those of capillary-based power sources are achieved. The prototype practicality has been demonstrated by powering a wireless plant-caring device. Standardized biodegradability and phytotoxicity assessments show that the battery is harmless to the environment at the end of its operational lifetime. Placing sustainability as the main driver leads to the generation of a disruptive battery concept that aims to address societal needs within the planetary environmental boundaries.

Regulation of Developmental Cell Death in the Animal Kingdom: A Critical Analysis of Epigenetic versus Genetic Factors.

The present paper proposes a new level of regulation of programmed cell death (PCD) in developing systems based on epigenetics. We argue against the traditional view of PCD as an altruistic "cell suicide" activated by specific gene-encoded signals with the function of favoring the development of their neighboring progenitors to properly form embryonic organs. In contrast, we propose that signals and local tissue interactions responsible for growth and differentiation of the embryonic tissues generate domains where cells retain an epigenetic profile sensitive to DNA damage that results in its subsequent elimination in a fashion reminiscent of what happens with scaffolding at the end of the construction of a building. Canonical death genes, including Bcl-2 family members, caspases, and lysosomal proteases, would reflect the downstream molecular machinery that executes the dying process rather than being master cell death regulatory signals.

Transforming growth factor beta signaling: The master sculptor of fingers.

Transforming growth factor beta (TGFß) constitutes a large and evolutionarily conserved superfamily of secreted factors that play essential roles in embryonic development, cancer, tissue regeneration, and human degenerative pathology. Studies of this signaling cascade in the regulation of cellular and tissue changes in the three-dimensional context of a developing embryo have notably advanced in the understanding of the action mechanism of these growth factors. In this review, we address the role of TGFß signaling in the developing limb, focusing on its essential function in the morphogenesis of the autopod. As we discuss in this work, modern mouse genetic experiments together with more classical embryological approaches in chick embryos, provided very valuable information concerning the role of TGFß and Activin family members in the morphogenesis of the digits of tetrapods, including the formation of phalanxes, digital tendons, and interphalangeal joints. We emphasize the importance of the Activin and TGFß proteins as digit inducing factors and their critical interaction with the BMP signaling to sculpt the hand and foot morphology.

Modeling the Differentiation of Embryonic Limb Chondroprogenitors by Cell Death and Cell Senescence in High Density Micromass Cultures and Their Regulation by FGF Signaling.

Considering the importance of programmed cell death in the formation of the skeleton during embryonic development, the aim of the present study was to analyze whether regulated cell degeneration also accompanies the differentiation of embryonic limb skeletal progenitors in high-density tridimensional cultures (micromass cultures). Our results show that the formation of primary cartilage nodules in the micromass culture assay involves a patterned process of cell death and cell senescence, complementary to the pattern of chondrogenesis. As occurs in vivo, the degenerative events were preceded by DNA damage detectable by γH2AX immunolabeling and proceeded via apoptosis and cell senescence. Combined treatments of the cultures with growth factors active during limb skeletogenesis, including FGF, BMP, and WNT revealed that FGF signaling modulates the response of progenitors to signaling pathways implicated in cell death. Transcriptional changes induced by FGF treatments suggested that this function is mediated by the positive regulation of the genetic machinery responsible for apoptosis and cell senescence together with hypomethylation of the Sox9 gene promoter. We propose that FGF signaling exerts a primordial function in the embryonic limb conferring chondroprogenitors with their biological properties.

Effects of Berberine on the Chondrogenic Differentiation of Embryonic Limb Skeletal Progenitors.

INTRODUCTION: Berberine (BBR) is an isoquinoline plant alkaloid with demonstrated anti-inflammatory, anti-tumor and immunosuppressive pharmacological properties that functions via multiple signaling pathways and epigenetic modulators. Numerous studies have proposed BBR as a promising therapeutic agent for joint cartilage degeneration, and other connective tissue diseases. PURPOSE AND METHODS: This work aimed to evaluate the effects of BBR on the growth and differentiation of embryonic skeletal progenitors using the limb mesoderm micromass culture assay. RESULTS: Our findings show that at difference of its apoptotic influence on a variety of tumor tissues, cell death was not induced in skeletal progenitors by the addition of 12 or 25 µM BBR concentration to the culture medium. Morphological and transcriptional analysis revealed dual and opposite effects of BBR treatments on chondrogenesis depending on the stage of differentiation of the cultured progenitors. At early stage of culture, BBR was a potent chondrogenic inhibitor, while chondrogenesis was intensified in treatments at advanced stages of culture. The chondrogenic promoting effect was accompanied by a moderate upregulation of gene markers of prehypertrophic cartilage, including ColXa1, alkaline phosphatase Alpl, Runx2, and Indian Hedgehog Ihh. We further observed a positive transcriptional influence of BBR in the expression of DNA methyltransferase genes, Dnmt1, Dnmt3a and Dnmt3b, suggesting a potential involvement of epigenetic factors in its effects. CONCLUSION: Our study uncovers a new pharmacological influence of BBR in cartilage differentiation that must be taken into account in designing clinical protocols for its employment in the treatment of cartilage degenerative diseases.

Chest X-ray lung imaging features in pediatric COVID-19 and comparison with viral lower respiratory infections in young children.

RATIONALE: Chest radiography (CXR) is a noninvasive imaging approach commonly used to evaluate lower respiratory tract infections (LRTIs) in children. However, the specific imaging patterns of pediatric coronavirus disease 2019 (COVID-19) on CXR, their relationship to clinical outcomes, and the possible differences from LRTIs caused by other viruses in children remain to be defined. METHODS: This is a cross-sectional study of patients seen at a pediatric hospital with polymerase chain reaction (PCR)-confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (n = 95). Patients were subdivided in infants (0-2 years, n = 27), children (3-10 years, n = 27), and adolescents (11-19 years, n = 41). A sample of young children (0-2 years, n = 68) with other viral lower respiratory infections (LRTI) was included to compare their CXR features with the subset of infants (0-2 years) with COVID-19. RESULTS: Forty-five percent of pediatric patients with COVID-19 were hospitalized and 20% required admission to intensive care unit (ICU). The most common abnormalities identified were ground-glass opacifications (GGO)/consolidations (35%) and increased peribronchial markings/cuffing (33%). GGO/consolidations were more common in older individuals and perihilar markings were more common in younger subjects. Subjects requiring hospitalization or ICU admission had significantly more GGO/consolidations in CXR (p < .05). Typical CXR features of pediatric viral LRTI (e.g., hyperinflation) were more common in non-COVID-19 viral LRTI cases than in COVID-19 cases (p < .05). CONCLUSIONS: CXR may be a complemental exam in the evaluation of moderate or severe pediatric COVID-19 cases. The severity of GGO/consolidations seen in CXR is predictive of clinically relevant outcomes. Hyperinflation could potentially aid clinical assessment in distinguishing COVID-19 from other types of viral LRTI in young children.

Development and evaluation of a machine learning-based point-of-care screening tool for genetic syndromes in children: a multinational retrospective study.

BACKGROUND: Delays in the diagnosis of genetic syndromes are common, particularly in low and middle-income countries with limited access to genetic screening services. We, therefore, aimed to develop and evaluate a machine learning-based screening technology using facial photographs to evaluate a child's risk of presenting with a genetic syndrome for use at the point of care. METHODS: In this retrospective study, we developed a facial deep phenotyping technology based on deep neural networks and facial statistical shape models to screen children for genetic syndromes. We trained the machine learning models on facial photographs from children (aged <21 years) with a clinical or molecular diagnosis of a genetic syndrome and controls without a genetic syndrome matched for age, sex, and race or ethnicity. Images were obtained from three publicly available databases (the Atlas of Human Malformations in Diverse Populations of the National Human Genome Research Institute, Face2Gene, and the dataset available from Ferry and colleagues) and the archives of the Children's National Hospital (Washington, DC, USA), in addition to photographs taken on a standard smartphone at the Children's National Hospital. We designed a deep learning architecture structured into three neural networks, which performed image standardisation (Network A), facial morphology detection (Network B), and genetic syndrome risk estimation, accounting for phenotypic variations due to age, sex, and race or ethnicity (Network C). Data were divided randomly into 40 groups for cross validation, and the performance of the model was evaluated in terms of accuracy, sensitivity, and specificity in both the total population and stratified by race or ethnicity, age, and sex. FINDINGS: Our dataset included 2800 facial photographs of children (1318 [47%] female and 1482 [53%] male; 1576 [56%] White, 432 [15%] African, 430 [15%] Hispanic, and 362 [13%] Asian). 1400 children with 128 genetic conditions were included (the most prevalent being Williams-Beuren syndrome [19%], Cornelia de Lange syndrome [17%], Down syndrome [16%], 22q11.2 deletion [13%], and Noonan syndrome [12%] syndrome) in addition to 1400 photographs of matched controls. In the total population, our deep learning-based model had an accuracy of 88% (95% CI 87-89) for the detection of a genetic syndrome, with 90% sensitivity (95% CI 88-92) and 86% specificity (95% CI 84-88). Accuracy was greater in White (90%, 89-91) and Hispanic populations (91%, 88-94) than in African (84%, 81-87) and Asian populations (82%, 78-86). Accuracy was also similar in male (89%, 87-91) and female children (87%, 85-89), and similar in children younger than 2 years (86%, 84-88) and children aged 2 years or older (eg, 89% [87-91] for those aged 2 years to <5 years). INTERPRETATION: This genetic screening technology could support early risk stratification at the point of care in global populations, which has the potential accelerate diagnosis and reduce mortality and morbidity through preventive care. FUNDING: Children's National Hospital and Government of Abu Dhabi.

SCA-1/Ly6A Mesodermal Skeletal Progenitor Subpopulations Reveal Differential Commitment of Early Limb Bud Cells.

At early developmental stages, limb bud mesodermal undifferentiated cells are morphologically indistinguishable. Although the identification of several mesodermal skeletal progenitor cell populations has been recognized, in advanced stages of limb development here we identified and characterized the differentiation hierarchy of two new early limb bud subpopulations of skeletal progenitors defined by the differential expression of the SCA-1 marker. Based on tissue localization of the mesenchymal stromal cell-associated markers (MSC-am) CD29, Sca-1, CD44, CD105, CD90, and CD73, we identified, by multiparametric analysis, the presence of cell subpopulations in the limb bud capable of responding to inductive signals differentially, namely, sSca+ and sSca- cells. In concordance with its gene expression profile, cell cultures of the sSca+ subpopulation showed higher osteogenic but lower chondrogenic capacity than those of sSca-. Interestingly, under high-density conditions, fibroblast-like cells in the sSca+ subpopulation were abundant. Gain-of-function employing micromass cultures and the recombinant limb assay showed that SCA-1 expression promoted tenogenic differentiation, whereas chondrogenesis is delayed. This model represents a system to determine cell differentiation and morphogenesis of different cell subpopulations in similar conditions like in vivo. Our results suggest that the limb bud is composed of a heterogeneous population of progenitors that respond differently to local differentiation inductive signals in the early stages of development, where SCA-1 expression may play a permissive role during cell fate.

Facial analysis technology for the detection of Down syndrome in the Democratic Republic of the Congo.

Down syndrome is one of the most common chromosomal anomalies affecting the world's population, with an estimated frequency of 1 in 700 live births. Despite its relatively high prevalence, diagnostic rates based on clinical features have remained under 70% for most of the developed world and even lower in countries with limited resources. While genetic and cytogenetic confirmation greatly increases the diagnostic rate, such resources are often non-existent in many low- and middle-income countries, particularly in Sub-Saharan Africa. To address the needs of countries with limited resources, the implementation of mobile, user-friendly and affordable technologies that aid in diagnosis would greatly increase the odds of success for a child born with a genetic condition. Given that the Democratic Republic of the Congo is estimated to have one of the highest rates of birth defects in the world, our team sought to determine if smartphone-based facial analysis technology could accurately detect Down syndrome in individuals of Congolese descent. Prior to technology training, we confirmed the presence of trisomy 21 using low-cost genomic applications that do not need advanced expertise to utilize and are available in many low-resourced countries. Our software technology trained on 132 Congolese subjects had a significantly improved performance (91.67% accuracy, 95.45% sensitivity, 87.88% specificity) when compared to previous technology trained on individuals who are not of Congolese origin (p < 5%). In addition, we provide the list of most discriminative facial features of Down syndrome and their ranges in the Congolese population. Collectively, our technology provides low-cost and accurate diagnosis of Down syndrome in the local population.

Rapid Artificial Intelligence Solutions in a Pandemic - The COVID-19-20 Lung CT Lesion Segmentation Challenge.

Artificial intelligence (AI) methods for the automatic detection and quantification of COVID-19 lesions in chest computed tomography (CT) might play an important role in the monitoring and management of the disease. We organized an international challenge and competition for the development and comparison of AI algorithms for this task, which we supported with public data and state-of-the-art benchmark methods. Board Certified Radiologists annotated 295 public images from two sources (A and B) for algorithms training (n=199, source A), validation (n=50, source A) and testing (n=23, source A; n=23, source B). There were 1,096 registered teams of which 225 and 98 completed the validation and testing phases, respectively. The challenge showed that AI models could be rapidly designed by diverse teams with the potential to measure disease or facilitate timely and patient-specific interventions. This paper provides an overview and the major outcomes of the COVID-19 Lung CT Lesion Segmentation Challenge - 2020.

Histone Epigenetic Signatures in Embryonic Limb Interdigital Cells Fated to Die.

During limb formation in vertebrates with free digits, the interdigital mesoderm is eliminated by a massive degeneration process that involves apoptosis and cell senescence. The degradation process is preceded by intense DNA damage in zones located close to methylated DNA, accompanied by the activation of the DNA repair response. In this study, we show that trimethylated histone 3 (H3K4me3, H3K9me3, and H3K27me3) overlaps with zones positive for 5mC in the nuclei of interdigital cells. This pattern contrasts with the widespread distribution of acetylated histones (H3K9ac and H4ac) and the histone variant H3.3 throughout the nucleoplasm. Consistent with the intense labeling of acetylated histones, the histone deacetylase genes Hdac1, Hdac2, Hdac3, and Hdac8, and at a more reduced level, Hdac10, are expressed in the interdigits. Furthermore, local treatments with the histone deacetylase inhibitor trichostatin A, which promotes an open chromatin state, induces massive cell death and transcriptional changes reminiscent of, but preceding, the physiological process of interdigit remodeling. Together, these findings suggest that the epigenetic profile of the interdigital mesoderm contributes to the sensitivity to DNA damage that precedes apoptosis during tissue regression.

Cell death in the developing vertebrate limb: A locally regulated mechanism contributing to musculoskeletal tissue morphogenesis and differentiation.

Our aim is to critically review current knowledge of the function and regulation of cell death in the developing limb. We provide a detailed, but short, overview of the areas of cell death observed in the developing limb, establishing their function in morphogenesis and structural development of limb tissues. We will examine the functions of this process in the formation and growth of the limb primordia, formation of cartilaginous skeleton, formation of synovial joints, and establishment of muscle bellies, tendons, and entheses. We will analyze the plasticity of the cell death program by focusing on the developmental potential of progenitors prior to death. Considering the prolonged plasticity of progenitors to escape from the death process, we will discuss a new biological perspective that explains cell death: this process, rather than secondary to a specific genetic program, is a consequence of the tissue building strategy employed by the embryo based on the formation of scaffolds that disintegrate once their associated neighboring structures differentiate.

Confluence of Cellular Degradation Pathways During Interdigital Tissue Remodeling in Embryonic Tetrapods.

Digits develop in the distal part of the embryonic limb primordium as radial prechondrogenic condensations separated by undifferentiated mesoderm. In a short time interval the interdigital mesoderm undergoes massive degeneration to determine the formation of free digits. This fascinating process has often been considered as an altruistic cell suicide that is evolutionarily-regulated in species with different degrees of digit webbing. Initial descriptions of interdigit remodeling considered lysosomes as the primary cause of the degenerative process. However, the functional significance of lysosomes lost interest among researcher and was displaced to a secondary role because the introduction of the term apoptosis. Accumulating evidence in recent decades has revealed that, far from being a unique method of embryonic cell death, apoptosis is only one among several redundant dying mechanisms accounting for the elimination of tissues during embryonic development. Developmental cell senescence has emerged in the last decade as a primary factor implicated in interdigit remodeling. Our review proposes that cell senescence is the biological process identified by vital staining in embryonic models and implicates lysosomes in programmed cell death. We review major structural changes associated with interdigit remodeling that may be driven by cell senescence. Furthermore, the identification of cell senescence lacking tissue degeneration, associated with the maturation of the digit tendons at the same stages of interdigital remodeling, allowed us to distinguish between two functionally distinct types of embryonic cell senescence, "constructive" and "destructive."

The methylation status of the embryonic limb skeletal progenitors determines their cell fate in chicken.

Digits shape is sculpted by interdigital programmed cell death during limb development. Here, we show that DNA breakage in the periphery of 5-methylcytosine nuclei foci of interdigital precursors precedes cell death. These cells showed higher genome instability than the digit-forming precursors when exposed to X-ray irradiation or local bone morphogenetic protein (BMP) treatments. Regional but not global DNA methylation differences were found between both progenitors. DNA-Methyl-Transferases (DNMTs) including DNMT1, DNMT3B and, to a lesser extent, DNMT3A, exhibited well-defined expression patterns in regions destined to degenerate, as the interdigital tissue and the prospective joint regions. Dnmt3b functional experiments revealed an inverse regulation of cell death and cartilage differentiation, by transcriptional regulation of key genes including Sox9, Scleraxis, p21 and Bak1, via differential methylation of CpG islands across their promoters. Our findings point to a regulation of cell death versus chondrogenesis of limb skeletal precursors based on epigenetic mechanisms.

SegSRGAN: Super-resolution and segmentation using generative adversarial networks - Application to neonatal brain MRI.

BACKGROUND AND OBJECTIVE: One of the main issues in the analysis of clinical neonatal brain MRI is the low anisotropic resolution of the data. In most MRI analysis pipelines, data are first re-sampled using interpolation or single image super-resolution techniques and then segmented using (semi-)automated approaches. In other words, image reconstruction and segmentation are then performed separately. In this article, we propose a methodology and a software solution for carrying out simultaneously high-resolution reconstruction and segmentation of brain MRI data. METHODS: Our strategy mainly relies on generative adversarial networks. The network architecture is described in detail. We provide information about its implementation, focusing on the most crucial technical points (whereas complementary details are given in a dedicated GitHub repository). We illustrate the behavior of the proposed method for cortex analysis from neonatal MR images. RESULTS: The results of the method, evaluated quantitatively (Dice, peak signal-to-noise ratio, structural similarity, number of connected components) and qualitatively on a research dataset (dHCP) and a clinical one (Epirmex), emphasize the relevance of the approach, and its ability to take advantage of data-augmentation strategies. CONCLUSIONS: Results emphasize the potential of our proposed method/software with respect to practical medical applications. The method is provided as a freely available software tool, which allows one to carry out his/her own experiments, and involve the method for the super-resolution reconstruction and segmentation of arbitrary cerebral structures from any MR image dataset.