Cross-Species Single-Cell Analysis Reveals Divergence of the Primate Microglia Program.

Microglia, the brain-resident immune cells, are critically involved in many physiological and pathological brain processes, including neurodegeneration. Here we characterize microglia morphology and transcriptional programs across ten species spanning more than 450 million years of evolution. We find that microglia express a conserved core gene program of orthologous genes from rodents to humans, including ligands and receptors associated with interactions between glia and neurons. In most species, microglia show a single dominant transcriptional state, whereas human microglia display significant heterogeneity. In addition, we observed notable differences in several gene modules of rodents compared with primate microglia, including complement, phagocytic, and susceptibility genes to neurodegeneration, such as Alzheimer's and Parkinson's disease. Our study provides an essential resource of conserved and divergent microglia pathways across evolution, with important implications for future development of microglia-based therapies in humans.

Rapid unleashing of macrophage efferocytic capacity via transcriptional pause release.

During development, inflammation or tissue injury, macrophages may successively engulf and process multiple apoptotic corpses via efferocytosis to achieve tissue homeostasis1. How macrophages may rapidly adapt their transcription to achieve continuous corpse uptake is incompletely understood. Transcriptional pause/release is an evolutionarily conserved mechanism, in which RNA polymerase (Pol) II initiates transcription for 20-60 nucleotides, is paused for minutes to hours and is then released to make full-length mRNA2. Here we show that macrophages, within minutes of corpse encounter, use transcriptional pause/release to unleash a rapid transcriptional response. For human and mouse macrophages, the Pol II pause/release was required for continuous efferocytosis in vitro and in vivo. Interestingly, blocking Pol II pause/release did not impede Fc receptor-mediated phagocytosis, yeast uptake or bacterial phagocytosis. Integration of data from three genomic approaches-precision nuclear run-on sequencing, RNA sequencing, and assay for transposase-accessible chromatin using sequencing (ATAC-seq)-on efferocytic macrophages at different time points revealed that Pol II pause/release controls expression of select transcription factors and downstream target genes. Mechanistic studies on transcription factor EGR3, prominently regulated by pause/release, uncovered EGR3-related reprogramming of other macrophage genes involved in cytoskeleton and corpse processing. Using lysosomal probes and a new genetic fluorescent reporter, we identify a role for pause/release in phagosome acidification during efferocytosis. Furthermore, microglia from egr3-deficient zebrafish embryos displayed reduced phagocytosis of apoptotic neurons and fewer maturing phagosomes, supporting defective corpse processing. Collectively, these data indicate that macrophages use Pol II pause/release as a mechanism to rapidly alter their transcriptional programs for efficient processing of the ingested apoptotic corpses and for successive efferocytosis.

Live imaging of neuronal degradation by microglia reveals a role for v0-ATPase a1 in phagosomal fusion in vivo.

A significant proportion of neurons in the brain undergo programmed cell death. In order to prevent the diffusion of damaging degradation products, dying neurons are quickly digested by microglia. Despite the importance of microglia in several neuronal pathologies, the mechanism underlying their degradation of neurons remains elusive. Here, we exploit a microglial population in the zebrafish to study this process in intact living brains. In vivo imaging reveals that digestion of neurons occurs in compartments arising from the progressive fusion of vesicles. We demonstrate that this fusion is mediated by the v0-ATPase a1 subunit. By applying live pH indicators, we show that the a1 subunit mediates fusion between phagosomes and lysosomes during phagocytosis, a function that is independent of its proton pump activity. As a real-time description of microglial phagocytosis in vivo, this work advances our understanding of microglial-mediated neuronal degeneration, a hallmark of many neuronal diseases.

Drowning in Children and Predictive Parameters: A 15-Year Multicenter Retrospective Analysis.

BACKGROUND: Drowning is a serious and underestimated public health problem, with the highest morbidity and mortality reported among children. Data regarding pediatric outcomes of drowning are often inadequate, and data collection is poorly standardized among centers. This study aims to provide an overview of a drowning pediatric population in pediatric emergency department, focusing on its main characteristics and management and evaluating prognostic factors. METHODS: This is a retrospective multicenter study involving eight Italian Pediatric Emergency Departments. Data about patients between 0 to 16 years of age who drowned between 2006 and 2021 were collected and analyzed according to the Utstein-style guidelines for drowning. RESULTS: One hundred thirty-five patients (60.9% males, median age at the event 5; interquartile range, 3-10) were recruited and only those with known outcome were retained for the analysis (133). Nearly 10% had a preexisting medical conditions with epilepsy being the most common comorbidity. One third were hospitalized in the intensive care unit (ICU) and younger males had a higher rate of ICU admission than female peers. Thirty-five patients (26.3%) were hospitalized in a medical ward while 19 (14.3%) were discharged from the emergency department and 11 (8.3%) were discharged after a brief medical observation less than 24 hours. Six patients died (4.5%). Medium stay in the ED was approximately 40 hours. No difference in terms of ICU admission was found between cardiopulmonary resuscitation performed by bystanders or trained medical personnel ( P = 0.388 vs 0.390). CONCLUSIONS: This study offers several perspectives on ED victims who drowned. One of the major finding is that no difference in outcomes was seen in patients who received cardiopulmonary resuscitation performed by bystanders or medical services, highlighting the importance of a prompt intervention.

Rapid clearance of cellular debris by microglia limits secondary neuronal cell death after brain injury in vivo.

Moderate or severe traumatic brain injury (TBI) causes widespread neuronal cell death. Microglia, the resident macrophages of the brain, react to injury by migrating to the lesion site, where they phagocytose cellular debris. Microglial phagocytosis can have both beneficial (e.g. debris clearance) and detrimental (e.g. respiratory burst, phagoptosis) consequences. Hence, whether the overall effect of microglial phagocytosis after brain injury in vivo is neuroprotective or neurotoxic is not known. Here, we establish a system with which to carry out dynamic real-time analyses of the mechanisms regulating cell death after brain injury in vivo We show that mechanical injury to the larval zebrafish brain induces distinct phases of primary and secondary cell death. Excitotoxicity contributes to secondary cell death in zebrafish, reflecting findings from mammals. Microglia arrive at the lesion site within minutes of injury, where they rapidly engulf dead cells. Importantly, the rate of secondary cell death is increased when the rapid removal of cellular debris by microglia is reduced pharmacologically or genetically. In summary, our results provide evidence that microglial debris clearance is neuroprotective after brain injury in vivo.

A well-appearing infant with a sudden anisocoria.

Case presentationA 10-month-old boy was admitted to the emergency department due to a sudden onset of left unilateral mydriasis (figure 1). His medical history was unremarkable. A minor head trauma 2 days before was reported, without alarming signs or symptoms. His mother was putting him to sleep, after coming back from work, when she noticed a different pupil size and promptly went to the ED with her husband. The parents denied any use of medications, including nebulised therapy or direct contact with plants. The child was well appearing and his vital signs were within the standard age limits. His extraocular motility was normal as well as the rest of his neurological and physical examination. Parents' behaviour was somehow remarkable. Even though the child was not suffering, the mother seemed very worried while the father was nervous and aggressive, repeatedly asking for a discharge.

CaSiAn: a Calcium Signaling Analyzer tool.

Summary: Ca2+ is a central second messenger in eukaryotic cells that regulates many cellular processes. Recently, we have indicated that typical Ca2+ signals are not purely oscillatory as widely assumed, but exhibit stochastic spiking with cell type and pathway specific characteristics. Here, we present the Calcium Signaling Analyzer (CaSiAn), an open source software tool that allows for quantifying these signal characteristics including individual spike properties and time course statistics in a semi-automated manner. CaSiAn provides an intuitive graphical user interface allowing experimentalists to easily process a large amount of Ca2+ signals, interactively tune peak detection, revise statistical measures and access the quantified signal properties as excel or text files. Availability and implementation: CaSiAn is implemented in Java and available on Github (https://github.com/mmahsa/CaSiAn) as well as on the project page (http://r3lab.uni.lu/web/casa). Supplementary information: Supplementary data are available at Bioinformatics online.

Somatic symptom disorder should be suspected in children with alleged chronic Lyme disease.

We report a case series of seven children admitted to a tertiary level pediatric ward for long-lasting physical symptoms with a previous diagnosis of chronic Lyme disease. In these children, medical history and clinical features were strongly suggestive of a psychopathological disorder, mainly a somatic symptom disorder. What is Known: • There is an increasing number of diagnoses of chronic Lyme disease both in North America and in Europe. Adults receive this diagnosis to explain chronic physical complaints often with negative history and serology. What is New: • Somatic symptom disorder should be suspected in children and adolescents with non-specific symptoms diagnosed with chronic Lyme disease.

Needle-related pain and distress management during needle-related procedures in children with and without intellectual disability.

Children with intellectual disability frequently undergo needle-related procedures for diagnosis or treatment. Nevertheless, only a few studies deal with pain and distress management during the procedure in this population of children. This study aimed to investigate the number of anxiety and pain management techniques performed during needle procedure in children with intellectual disability (cases) compared to a population of children without intellectual disability (controls). This multicenter cohort study was performed from July 2016 to January 2018 in the pediatric ward of four urban hospitals in Italy. Eligible subjects were children with and without intellectual disability, from 4 to 17 years old, who needed venipuncture or intravenous cannulation for diagnosis or treatment. Use of topical anesthesia, distraction techniques, and physical or verbal comfort during procedures were recorded. Pain and anxiety scores were also recorded. Forty-seven cases and 94 controls were recruited. Three pain- and anxiety-relieving techniques were performed during the procedure in 12 (25%) cases and in 10 controls (11%); two techniques were performed in 23 (50%) cases and in 26 (28%) controls; 12 (25%) cases and 52 (55%) controls received only one.Conclusion: In this series, children with intellectual disability received significantly more relieving techniques, but experienced more pain and anxiety when compared to children without intellectual disability. What is Known: • Children with intellectual disability experience more episodes of pain than cognitively healthy ones, and almost 10% of these episodes are due to medical procedures. What is New: • Children with intellectual disability despite receiving more relieving techniques during a needle-related procedure experienced more pain and anxiety when compared to healthy children.

Autophosphorylation and Pin1 binding coordinate DNA damage-induced HIPK2 activation and cell death.

Excessive genome damage activates the apoptosis response. Protein kinase HIPK2 is a key regulator of DNA damage-induced apoptosis. Here, we deciphered the molecular mechanism of HIPK2 activation and show its relevance for DNA damage-induced apoptosis in cellulo and in vivo. HIPK2 autointeracts and site-specifically autophosphorylates upon DNA damage at Thr880/Ser882. Autophosphorylation regulates HIPK2 activity and mutation of the phosphorylation-acceptor sites deregulates p53 Ser46 phosphorylation and apoptosis in cellulo. Moreover, HIPK2 autophosphorylation is conserved between human and zebrafish and is important for DNA damage-induced apoptosis in vivo. Mechanistically, autophosphorylation creates a binding signal for the phospho-specific isomerase Pin1. Pin1 links HIPK2 activation to its stabilization by inhibiting HIPK2 polyubiquitination and modulating Siah-1-HIPK2 interaction. Concordantly, Pin1 is required for DNA damage-induced HIPK2 stabilization and p53 Ser46 phosphorylation and is essential for induction of apotosis both in cellulo and in zebrafish. Our results identify an evolutionary conserved mechanism regulating DNA damage-induced apoptosis.

Glial Cell Development and Function in the Zebrafish Central Nervous System.

Over the past decades the zebrafish has emerged as an excellent model organism with which to study the biology of all glial cell types in nervous system development, plasticity, and regeneration. In this review, which builds on the earlier work by Lyons and Talbot in 2015, we will summarize how the relative ease to manipulate the zebrafish genome and its suitability for intravital imaging have helped understand principles of glial cell biology with a focus on oligodendrocytes, microglia, and astrocytes. We will highlight recent findings on the diverse properties and functions of these glial cell types in the central nervous system and discuss open questions and future directions of the field.

Timeline of diagnosed pain causes in children with severe neurological impairment.

Objective: Pain's causes in children with severe cognitive impairment may be challenging to diagnose. This study aimed to investigate if there is a relationship between pain causes and the age of children. Methods: We conducted a multicenter retrospective study in three Italian Pediatric Units. Eligible subjects were patients from 1 to 18 years with severe neurological impairment. We collected data regarding diagnoses, pain causes and medical or surgical procedures. The timing of pain episodes was categorized into age-related periods: infants and toddlers (0-24 months), preschool children (3-5 years), schoolchildren (6-12 years), and adolescents (13-17 years). Results: Eighty children with severe neurological impairment were enrolled. The mean age was 11 years (±5.8). Gastroenterological pain was most common in the first years of life (p = 0.004), while orthopaedic and tooth pain was the most typical in schoolchildren and adolescents (p = 0.001 and p = 0.02). Concerning surgical procedures, PEG placement and gastric fundoplication were significantly more common in the first 5 years of age (p = 0.03), and heart surgery was typical of infants (p = 0.04). Orthopaedic surgery was more commonly reported in older children and adolescents (p < 0.001). Conclusions: Some causes of pain are more frequent in children with severe neurological impairment in defined age-related periods. Specific age-related pain frequencies may help physicians in the diagnostic approach.

Animal models for studying microglia: the first, the popular, and the new.

Microglia, the resident phagocytes of brain, have been intensively studied since their discovery in the 1920s. There is no doubt that the possibility of culturing microglia in vitro has advanced enormously our understanding of these cells. However, as we know today, that microglia react to even small changes in the brain, it is crucial to also study these cells by preserving as much as possible their natural environment. Nowadays, advances in imaging technologies and transgenic cell labeling methods allow the direct observation of cells at work. These in vivo approaches have already changed our view on microglia by showing that these cells are active even in the healthy adult brain. As today, there is upcoming evidence that microglia can directly influence neuronal activity, understanding their roles and, in particular, their interactions with neurons is of great importance. The aim of this review is to illustrate three animal models that are currently used for microglial research and to discuss their characteristics and advantages by presenting recent achievements in microglial research. In our view the availability of different systems for studying microglia will lead to a more comprehensive understanding of their functions.

T2-Low Asthma: A Discussed but Still Orphan Disease.

Asthma affects 10% of the worldwide population; about 5% of cases are severe with the need for target therapies such as biologics. All the biologics approved for asthma hit the T2 pathway of inflammation. T2-high asthma is classified as allergic and non-allergic, whereas T2-low asthma can be further defined as paucigranulocytic asthma, Type 1 and Type-17 inflammation and the neutrophilic form that accounts for 20-30% of all patients with asthma. Neutrophilic asthma's prevalence is even higher in patients with severe or refractory asthma. We searched Medline and PubMed archives from the past ten years for articles with the subsequent titles: "neutrophilic asthma", "non-type 2 asthma" and "paucigranulocytic asthma". We identified 177 articles; 49 were considered relevant by the title and 33 by the reading of the abstract. Most of these articles are reviews (n = 19); only 6 are clinical trials. No study identified an effective treatment. We used the literature reported by these articles to search for further biologic treatments that target pathways different from T2. We identified 177 articles, 93 of which were considered relevant for the review and included in the present article. In conclusion, T2-low asthma remains poorly investigated in terms of biomarkers, especially as a therapeutic orphan disease.

Transient Receptor Potential Ankyrin 1 (TRPA1) Methylation and Chronic Pain: A Systematic Review.

BACKGROUND AND OBJECTIVE: Chronic pain represents a major global health issue in terms of psycho-physiological, therapeutic, and economic burden, not limited to adults but also to the pediatric age. Despite its great impact, its molecular mechanisms have still not been completely unraveled. Focusing on the impact of epigenetics in the pain complex trait, we assessed the association between chronic pain and the methylation pattern of TRPA1, a key gene related to pain sensitivity. METHODS: We conducted a systematic review retrieving articles from three different databases. After deduplication, 431 items were subjected to manual screening, and then 61 articles were selected and screened again. Of these, only six were maintained for meta-analysis and analyzed using specific R packages. RESULTS: Six articles were divided into two groups (group 1: comparison of mean methylation levels between healthy subjects and patients with chronic pain; group 2: correlation between mean methylation levels and pain sensation). A non-significant mean difference was obtained from the analysis of group 1 with a value of 3.97 (95% C.I. -7.79; 15.73). Analysis of group 2 showed a high level of variability between studies (correlation = 0.35, 95% C.I. -0.12; 0.82) due to their heterogeneity (I2 = 97%, p < 0.01). CONCLUSIONS: Despite the high variability observed in the different studies analyzed, our results suggest that hypermethylation and increased pain sensitivity could be connected, possibly due to the variation of TRPA1 expression.