Structure, function, and recombinant production of EGFL7.

The secreted factor Epidermal growth factor-like protein 7 (EGFL7) is involved in angiogenesis, vasculogenesis, as well as neurogenesis. Importantly, EGFL7 is also implicated in various pathological conditions, including tumor angiogenesis in human cancers. Thus, understanding the mechanisms through which EGFL7 regulates and promotes blood vessel formation is of clear practical importance. One principle means by which EGFL7's function is investigated is via the expression and purification of the recombinant protein. This mini-review describes three methods used to produce recombinant EGFL7 protein. First, a brief overview of EGFL7's genetics, structure, and function is provided. This is followed by an examination of the advantages and disadvantages of three common expression systems used in the production of recombinant EGFL7; (i) Escherichia coli (E. coli), (ii) human embryonic kidney (HEK) 293 cells or other mammalian cells, and (iii) a baculovirus-based Sf9 insect cell expression system. Based on the available evidence, we conclude that the baculovirus-based Sf9 insect cell expression currently has the advantages of producing active recombinant EGFL7 in the native conformation with the presence of acceptable posttranslational modifications, while providing sufficient yield and stability for experimental purposes.

Microglia in retinal angiogenesis and diabetic retinopathy.

Diabetic retinopathy has a high probability of causing visual impairment or blindness throughout the disease progression and is characterized by the growth of new blood vessels in the retina at an advanced, proliferative stage. Microglia are a resident immune population in the central nervous system, known to play a crucial role in regulating retinal angiogenesis in both physiological and pathological conditions, including diabetic retinopathy. Physiologically, they are located close to blood vessels and are essential for forming new blood vessels (neovascularization). In diabetic retinopathy, microglia become widely activated, showing a distinct polarization phenotype that leads to their accumulation around neovascular tufts. These activated microglia induce pathogenic angiogenesis through the secretion of various angiogenic factors and by regulating the status of endothelial cells. Interestingly, some subtypes of microglia simultaneously promote the regression of neovascularization tufts and normal angiogenesis in neovascularization lesions. Modulating the state of microglial activation to ameliorate neovascularization thus appears as a promising potential therapeutic approach for managing diabetic retinopathy.

N-Cyanopiperazines as Specific Covalent Inhibitors of the Deubiquitinating Enzyme UCHL1.

Cyanamides have emerged as privileged scaffolds in covalent inhibitors of deubiquitinating enzymes (DUBs). However, many compounds with a cyanopyrrolidine warhead show cross-reactivity toward small subsets of DUBs or toward the protein deglycase PARK7/DJ-1, hampering their use for the selective perturbation of a single DUB in living cells. Here, we disclose N'-alkyl,N-cyanopiperazines as structures for covalent enzyme inhibition with exceptional specificity for the DUB UCHL1 among 55 human deubiquitinases and with effective target engagement in cells. Notably, transitioning from 5-membered pyrrolidines to 6-membered heterocycles eliminated PARK7 binding and introduced context-dependent reversibility of the isothiourea linkage to the catalytic cysteine of UCHL1. Compound potency and specificity were analysed by a range of biochemical assays and with a crystal structure of a cyanopiperazine in covalent complex with UCHL1. The structure revealed a compound-induced conformational restriction of the cross-over loop, which underlies the observed inhibitory potencies. Through the rationalization of specificities of different cyanamides, we introduce a framework for the investigation of protein reactivity of bioactive nitriles of this compound class. Our results represent an encouraging case study for the refining of electrophilic compounds into chemical probes, emphasizing the potential to engineer specificity through subtle chemical modifications around the warhead.

c-Cbl Regulates Murine Subventricular Zone-Derived Neural Progenitor Cells in Dependence of the Epidermal Growth Factor Receptor.

The localization, expression, and physiological role of regulatory proteins in the neurogenic niches of the brain is fundamental to our understanding of adult neurogenesis. This study explores the expression and role of the E3-ubiquitin ligase, c-Cbl, in neurogenesis within the subventricular zone (SVZ) of mice. In vitro neurosphere assays and in vivo analyses were performed in specific c-Cbl knock-out lines to unravel c-Cbl's role in receptor tyrosine kinase signaling, including the epidermal growth factor receptor (EGFR) pathway. Our findings suggest that c-Cbl is significantly expressed within EGFR-expressing cells, playing a pivotal role in neural stem cell proliferation and differentiation. However, c-Cbl's function extends beyond EGFR signaling, as its loss upon knock-out stimulated progenitor cell proliferation in neurosphere cultures. Yet, this effect was not detected in hippocampal progenitor cells, reflecting the lack of the EGFR in the hippocampus. In vivo, c-Cbl exerted only a minor proneurogenic influence with no measurable impact on the formation of adult-born neurons. In conclusion, c-Cbl regulates neural stem cells in the subventricular zone via the EGFR pathway but, likely, its loss is compensated by other signaling modules in vivo.

A20 regulates lymphocyte adhesion in murine neuroinflammation by restricting endothelial ICOSL expression in the CNS.

A20 is a ubiquitin-modifying protein that negatively regulates NF-κB signaling. Mutations in A20/TNFAIP3 are associated with a variety of autoimmune diseases, including multiple sclerosis (MS). We found that deletion of A20 in central nervous system (CNS) endothelial cells (ECs) enhances experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. A20ΔCNS-EC mice showed increased numbers of CNS-infiltrating immune cells during neuroinflammation and in the steady state. While the integrity of the blood-brain barrier (BBB) was not impaired, we observed a strong activation of CNS-ECs in these mice, with dramatically increased levels of the adhesion molecules ICAM-1 and VCAM-1. We discovered ICOSL to be expressed by A20-deficient CNS-ECs, which we found to function as adhesion molecules. Silencing of ICOSL in CNS microvascular ECs partly reversed the phenotype of A20ΔCNS-EC mice without reaching statistical significance and delayed the onset of EAE symptoms in WT mice. In addition, blocking of ICOSL on primary mouse brain microvascular ECs impaired the adhesion of T cells in vitro. Taken together, we propose that CNS EC-ICOSL contributes to the firm adhesion of T cells to the BBB, promoting their entry into the CNS and eventually driving neuroinflammation.

The origin of brain malignancies at the blood-brain barrier.

Despite improvements in extracranial therapy, survival rate for patients suffering from brain metastases remains very poor. This is coupled with the incidence of brain metastases continuing to rise. In this review, we focus on core contributions of the blood-brain barrier to the origin of brain metastases. We first provide an overview of the structure and function of the blood-brain barrier under physiological conditions. Next, we discuss the emerging idea of a pre-metastatic niche, namely that secreted factors and extracellular vesicles from a primary tumor site are able to travel through the circulation and prime the neurovasculature for metastatic invasion. We then consider the neurotropic mechanisms that circulating tumor cells possess or develop that facilitate disruption of the blood-brain barrier and survival in the brain's parenchyma. Finally, we compare and contrast brain metastases at the blood-brain barrier to the primary brain tumor, glioma, examining the process of vessel co-option that favors the survival and outgrowth of brain malignancies.

How divergent are children's divergent movements? The role of cognition and expertise in a class-randomized cross-over trial.

This study aimed at investigating (1) the cognitive and motor predictors of divergent movement ability (DMA) in childhood and (2) the role of sport and enriched physical education (PE) experience. Participants were 165 fifth graders, aged 10-11 years, with different histories (onset and duration) of participation in enriched PE. They were assessed in cognitive/attentional and motor/sport skills and active play/sport habits at baseline, and six months later in DMA. Results of regression analyses showed an overall prediction of DMA by sport practice and a nuanced prediction of individual DMA indices (fluency, flexibility, originality) by decision making and spatial attention ability. Linear mixed models revealed better DMA in children exposed to enriched PE, with differential effects on DMA indices depending on its duration and earlier/later onset. The results identify novel cognitive determinants of children's DMA and suggest that sport practice and experience of designed enrichment in PE may benefit DMA.

Acute exercise and children's cognitive functioning: What is the optimal dose of cognitive challenge?

Acute bouts of exercise have the potential to benefit children's cognition. Inconsistent evidence on the role of qualitative exercise task characteristics calls for further investigation of the cognitive challenge level in exercise. Thus, the study aim was to investigate which "dose" of cognitive challenge in acute exercise benefits children's cognition, also exploring the moderating role of individual characteristics. In a within-subject experimental design, 103 children (Mage = 11.1, SD = 0.9, 48% female) participated weekly in one of three 15-min exergames followed by an Attention Network task. Exergame sessions were designed to keep physical intensity constant (65% HRmax) and to have different cognitive challenge levels (low, mid, high; adapted to the ongoing individual performance). ANOVAs performed on variables that reflect the individual functioning of attention networks revealed a significant effect of cognitive challenge on executive control efficiency (reaction time performances; p = .014, ƞ2p = .08), with better performances after the high-challenge condition compared to lower ones (ps < .015), whereas alerting and orienting were unaffected by cognitive challenge (ps > .05). ANOVAs performed on variables that reflect the interactive functioning of attention networks revealed that biological sex moderated cognitive challenge effects. For males only, the cognitive challenge level influenced the interactive functioning of executive control and orienting networks (p = .004; ƞ2p = .07). Results suggest that an individualized and adaptive cognitively high-challenging bout of exercise is more beneficial to children's executive control than less challenging ones. For males, the cognitive challenge in an acute bout seems beneficial to maintain executive control efficiency also when spatial attention resources cannot be validly allocated in advance. Results are interpreted referring to the cognitive stimulation hypothesis and arousal theory.

Editorial for "Impact of Regorafenib on Endothelial Transdifferentiation of Glioblastoma Stem-like Cells".

Glioblastoma multiforme (GBM) is the most frequently occurring form of malignant primary brain tumor in adults [...].

Distinct Lineage of Slow-Cycling Cells Amidst the Prevailing Heterogeneity in Glioblastoma.

Glioblastoma (GBM) is the most aggressive form of primary brain tumor in adults [...].

Migration-Associated Transportome and Therapeutic Potential in Glioblastoma Multiforme (GBM).

GBM is a highly aggressive and very common malignant form of primary brain tumors in adults [...].

Leave the screen: The influence of everyday behaviors on self-reported interoception.

The influence of physical activity on interoception is apparent, however little is known about within-person variability following physical activity and sedentary behavior in daily life. To test this, 70 healthy adults (Mage 21.67 ± 2.50) wore thigh-mounted accelerometers for 7-days, with self-reported interoception recorded on movement-triggered smartphones. Participants additionally reported the predominant activity type performed across the last 15 min. Investigating this timeframe, multi-level analyses revealed that each one-unit increase in physical activity was associated with an increase in self-reported interoception (B = 0.0025, p = .013), whereas contrastingly, each one-minute increase in sedentary behavior was associated with a decrease (B = -0.06. p = .009). Investigating the influence of different activity types in comparison to screen time behavior, both partaking in exercise (B = 4.48, p < .001) and daily-life physical activity (B = 1.21, p < .001) were associated with an increase in self-reported interoception. Regarding other behavior categories, non-screen time behavior both with (B = 1.13, p < .001) and without (B = 0.67, p = .004) social interaction were also associated with an increase in self-reported interoception compared to screen-time behavior. Extending from previous laboratory-based studies, these findings indicate that physical activity influences interoceptive processes in real-life, further supplemented by the novel and contrasting findings regarding sedentary behavior. Furthermore, associations with activity type reveal important mechanistic information, highlighting the importance of reducing screen-time behavior to preserve and support interoceptive perceptions. Findings can be used to inform health recommendations for reducing screen-time behavior and guiding evidence-based physical activity interventions to promote interoceptive processes.

The Role of Posterior Neural Plate-Derived Presomitic Mesoderm (PSM) in Trunk and Tail Muscle Formation and Axis Elongation.

Elongation of the posterior body axis is distinct from that of the anterior trunk and head. Early drivers of posterior elongation are the neural plate/tube and notochord, later followed by the presomitic mesoderm (PSM), together with the neural tube and notochord. In axolotl, posterior neural plate-derived PSM is pushed posteriorly by convergence and extension of the neural plate. The PSM does not go through the blastopore but turns anteriorly to join the gastrulated paraxial mesoderm. To gain a deeper understanding of the process of axial elongation, a detailed characterization of PSM morphogenesis, which precedes somite formation, and of other tissues (such as the epidermis, lateral plate mesoderm and endoderm) is needed. We investigated these issues with specific tissue labelling techniques (DiI injections and GFP+ tissue grafting) in combination with optical tissue clearing and 3D reconstructions. We defined a spatiotemporal order of PSM morphogenesis that is characterized by changes in collective cell behaviour. The PSM forms a cohesive tissue strand and largely retains this cohesiveness even after epidermis removal. We show that during embryogenesis, the PSM, as well as the lateral plate and endoderm move anteriorly, while the net movement of the axis is posterior.

The Glioma Immune Landscape: A Double-Edged Sword for Treatment Regimens.

Immune cells constitute a major part of the tumor microenvironment, thereby playing an important role in regulating tumor development. They interact with tumor cells, resulting in the suppression or promotion of glioma development. Therefore, in recent years, scientists have focused on immunotherapy that involves enhancing the immune response to fight the battle against cancer more effectively. While it has shown success against different cancer types, immunotherapy faces major roadblocks in glioma treatment. These involve the blood brain barrier, tumor heterogeneity and an immunosuppressive glioma microenvironment, among other factors. Additionally, the interaction of the peripheral immune system with the central nervous system provides another challenge for immunotherapeutic regimens. For modulating different immune cell populations to counter glioma cells, it is important to expand our knowledge about their role within the glioma microenvironment; therefore, herein, we review the different immune cell populations found in the glioma microenvironment and navigate through the various shortcomings of current immunotherapies for glioma. We conclude by providing an insight into ongoing pre-clinical and clinical trials for glioma therapies.

Dose-response relation between the duration of a cognitively challenging bout of physical exercise and children's cognition.

Acute bouts of physical exercise have the potential to benefit children's cognition. Inconsistent evidence calls for systematic investigations of dose-response relations between quantitative (intensity and duration) and qualitative (modality) exercise characteristics. Thus, in this study the optimal duration of an acute cognitively challenging physical exercise to benefit children's cognition was investigated, also exploring the moderating role of individual characteristics. In a within-subject experimental design, 104 children (Mage = 11.5, SD = 0.8, 51% female) participated weekly in one of four exergaming conditions of different durations (5, 10, 15, 20 min) followed by an Attention Network task (ANT-R). Exergame sessions were designed to keep physical intensity constant (65% HRmax ) and to have a high cognitive challenge level (adapted to the individual ongoing performance). Repeated measures ANOVAs revealed a significant effect of exercise duration on reaction times (RTs; p = 0.009, ƞ2 p = 0.11), but not on response accuracy. Post hoc analyses showed faster information processing speed after 15 min of exercise compared to 10 min (p = 0.019, ƞ2 p = 0.09). Executive control, alerting and orienting performances and interactions were unaffected by exercise duration (ps > 0.05). Among individual characteristics, habitual physical activity moderated duration effects on RTs. For more active children, exercise duration influenced the interaction between executive control and orienting (p = 0.034; ƞ2 p = 0.17) with best performances after the 15 min duration. Results suggest that an acute 15 min cognitively high-challenging bout of physical exercise enhances allocable resources, which in turn facilitate information processing, and-for more active children only-also executive processes. Results are interpreted according to the arousal theory and cognitive stimulation hypothesis.

EGFL7 loss correlates with increased VEGF-D expression, upregulating hippocampal adult neurogenesis and improving spatial learning and memory.

Neural stem cells reside in the subgranular zone, a specialized neurogenic niche of the hippocampus. Throughout adulthood, these cells give rise to neurons in the dentate gyrus, playing an important role in learning and memory. Given that these core cognitive processes are disrupted in numerous disease states, understanding the underlying mechanisms of neural stem cell proliferation in the subgranular zone is of direct practical interest. Here, we report that mature neurons, neural stem cells and neural precursor cells each secrete the neurovascular protein epidermal growth factor-like protein 7 (EGFL7) to shape this hippocampal niche. We further demonstrate that EGFL7 knock-out in a Nestin-CreERT2-based mouse model produces a pronounced upregulation of neurogenesis within the subgranular zone. RNA sequencing identified that the increased expression of the cytokine VEGF-D correlates significantly with the ablation of EGFL7. We substantiate this finding with intraventricular infusion of VEGF-D upregulating neurogenesis in vivo and further show that VEGF-D knock-out produces a downregulation of neurogenesis. Finally, behavioral studies in EGFL7 knock-out mice demonstrate greater maintenance of spatial memory and improved memory consolidation in the hippocampus by modulation of pattern separation. Taken together, our findings demonstrate that both EGFL7 and VEGF-D affect neurogenesis in the adult hippocampus, with the ablation of EGFL7 upregulating neurogenesis, increasing spatial learning and memory, and correlating with increased VEGF-D expression.

Structural basis for specific inhibition of the deubiquitinase UCHL1.

Ubiquitination regulates protein homeostasis and is tightly controlled by deubiquitinases (DUBs). Loss of the DUB UCHL1 leads to neurodegeneration, and its dysregulation promotes cancer metastasis and invasiveness. Small molecule probes for UCHL1 and DUBs in general could help investigate their function, yet specific inhibitors and structural information are rare. Here we report the potent and non-toxic chemogenomic pair of activity-based probes GK13S and GK16S for UCHL1. Biochemical characterization of GK13S demonstrates its stereoselective inhibition of cellular UCHL1. The crystal structure of UCHL1 in complex with GK13S shows the enzyme locked in a hybrid conformation of apo and Ubiquitin-bound states, which underlies its UCHL1-specificity within the UCH DUB family. Phenocopying a reported inactivating mutation of UCHL1 in mice, GK13S, but not GK16S, leads to reduced levels of monoubiquitin in a human glioblastoma cell line. Collectively, we introduce a set of structurally characterized, chemogenomic probes suitable for the cellular investigation of UCHL1.

Gamma Irradiation Triggers Immune Escape in Glioma-Propagating Cells.

Glioblastoma multiforme is the most common and devastating form of brain tumor for which only palliative radio- and chemotherapy exists. Although some clinical studies on vaccination approaches have shown promising efficacy due to their potential to generate long-term immune surveillance against cancer cells, the evasion mechanisms preventing therapy response are largely uncharacterized. Here, we studied the response of glioblastoma-propagating cells (GPCs) to clinically relevant doses of γ radiation. GPCs were treated with 2.5 Gy of γ radiation in seven consecutive cellular passages to select for GPCs with increased colony-forming properties and intrinsic or radiation-induced resistance (rsGPCs). Quantitative proteomic analysis of the cellular signaling platforms of the detergent-resistant membranes (lipid rafts) in GPCs vs. rsGPCs revealed a downregulation of the MHC class I antigen-processing and -presentation machinery. Importantly, the radio-selected GPCs showed reduced susceptibility towards cytotoxic CD8+ T-cell-mediated killing. While previous studies suggested that high-dose irradiation results in enhanced antigen presentation, we demonstrated that clinically relevant sub-lethal fractionated irradiation results in reduced expression of components of the MHC class I antigen-processing and -presentation pathway leading to immune escape.

Giving Ideas Some Legs or Legs Some Ideas? Children's Motor Creativity Is Enhanced by Physical Activity Enrichment: Direct and Mediated Paths.

Approaches to foster motor creativity differ according to whether creative movements are assumed to be enacted creative ideas, or solutions to emerging motor problems that arise from task and environmental constraints. The twofold aim of the current study was to investigate whether (1) an enriched physical education (PE) intervention delivered with a joint constraints-led and cognitive stimulation approach fosters motor creativity, and the responsiveness to the intervention is moderated by baseline motor and cognitive skills and sex; (2) the intervention may benefit motor creativity through gains in motor coordination, executive function, and creative thinking. Ninety-five children, aged 6-9 years, participated in a 6-month group randomized trial with specialist-led enriched PE vs. generalist-led conventional PE. Before and after the intervention, Bertsch's Test of Motor Creativity, Movement Assessment Battery for Children, Random Number Generation task and Torrance Test of Creative Thinking were administered. Linear mixed models were run accounting for the random effects of data clusters. Multiple mediation analysis was performed to assess whether motor coordination, executive function and creative thinking mediated any improvement of motor creativity. Results showed that (1) specialist-led enriched PE, compared to generalist-led conventional practice, elicited a more pronounced improvement in all motor creativity dimensions (fluency, flexibility, and originality) independently of baseline levels of motor and cognitive skills and sex; and (2) improved motor creativity was partially mediated by improved motor coordination and, as regards motor flexibility, also by improved inhibitory ability. In conclusion, enriching PE with tailored manipulations of constraints and variability may enhance the ability to create multiple and original task-pertinent movements both directly and through indirect paths. The results are discussed extending to motor creativity a theoretical framework that distinguishes different creativity modes. The intervention may have fostered the generation of creative movements directly through the exposure to variation in constraints, activating the sensorimotor 'flow' mode of creativity that bypasses higher-order cognition, but also indirectly through a systematic and conscious convergence on solutions, activating the 'deliberate' mode of creativity that relies on inhibition to reject common or task-inappropriate movement categories.