Nonlinear Elastic Bottlebrush Polymer Hydrogels Modulate Actomyosin Mediated Protrusion Formation in Mesenchymal Stromal Cells.

The nonlinear elasticity of many tissue-specific extracellular matrices is difficult to recapitulate without the use of fibrous architectures, which couple strain-stiffening with stress relaxation. Herein, bottlebrush polymers are synthesized and crosslinked to form poly(ethylene glycol)-based hydrogels and used to study how strain-stiffening behavior affects human mesenchymal stromal cells (hMSCs). By tailoring the bottlebrush polymer length, the critical stress associated with the onset of network stiffening is systematically varied, and a unique protrusion-rich hMSC morphology emerges only at critical stresses within a biologically accessible stress regime. Local cell-matrix interactions are quantified using 3D traction force microscopy and small molecule inhibitors are used to identify cellular machinery that plays a critical role in hMSC mechanosensing of the engineered, strain-stiffening microenvironment. Collectively, this study demonstrates how covalently crosslinked bottlebrush polymer hydrogels can recapitulate strain-stiffening biomechanical cues at biologically relevant stresses and be used to probe how nonlinear elastic matrix properties regulate cellular processes.

Integrative multi-omic profiling of adult mouse brain endothelial cells and potential implications in Alzheimer's disease.

The blood-brain barrier (BBB) is primarily manifested by a variety of physiological properties of brain endothelial cells (ECs), but the molecular foundation for these properties remains incompletely clear. Here, we generate a comprehensive molecular atlas of adult brain ECs using acutely purified mouse ECs and integrated multi-omics. Using RNA sequencing (RNA-seq) and proteomics, we identify the transcripts and proteins selectively enriched in brain ECs and demonstrate that they are partially correlated. Using single-cell RNA-seq, we dissect the molecular basis of functional heterogeneity of brain ECs. Using integrative epigenomics and transcriptomics, we determine that TCF/LEF, SOX, and ETS families are top-ranked transcription factors regulating the BBB. We then validate the identified brain-EC-enriched proteins and transcription factors in normal mouse and human brain tissue and assess their expression changes in mice with Alzheimer's disease. Overall, we present a valuable resource with broad implications for regulation of the BBB and treatment of neurological disorders.

Photo-expansion microscopy enables super-resolution imaging of cells embedded in 3D hydrogels.

Hydrogels are extensively used as tunable, biomimetic three-dimensional cell culture matrices, but optically deep, high-resolution images are often difficult to obtain, limiting nanoscale quantification of cell-matrix interactions and outside-in signalling. Here we present photopolymerized hydrogels for expansion microscopy that enable optical clearance and tunable ×4.6-6.7 homogeneous expansion of not only monolayer cell cultures and tissue sections, but cells embedded within hydrogels. The photopolymerized hydrogels for expansion microscopy formulation relies on a rapid photoinitiated thiol/acrylate mixed-mode polymerization that is not inhibited by oxygen and decouples monomer diffusion from polymerization, which is particularly beneficial when expanding cells embedded within hydrogels. Using this technology, we visualize human mesenchymal stem cells and their interactions with nascently deposited proteins at <120 nm resolution when cultured in proteolytically degradable synthetic polyethylene glycol hydrogels. Results support the notion that focal adhesion maturation requires cellular fibronectin deposition; nuclear deformation precedes cellular spreading; and human mesenchymal stem cells display cell-surface metalloproteinases for matrix remodelling.

4D Printing of Extrudable and Degradable Poly(Ethylene Glycol) Microgel Scaffolds for Multidimensional Cell Culture.

Granular synthetic hydrogels are useful bioinks for their compatibility with a variety of chemistries, affording printable, stimuli-responsive scaffolds with programmable structure and function. Additive manufacturing of microscale hydrogels, or microgels, allows for the fabrication of large cellularized constructs with percolating interstitial space, providing a platform for tissue engineering at length scales that are inaccessible by bulk encapsulation where transport of media and other biological factors are limited by scaffold density. Herein, synthetic microgels with varying degrees of degradability are prepared with diameters on the order of hundreds of microns by submerged electrospray and UV photopolymerization. Porous microgel scaffolds are assembled by particle jamming and extrusion printing, and semi-orthogonal chemical cues are utilized to tune the void fraction in printed scaffolds in a logic-gated manner. Scaffolds with different void fractions are easily cellularized post printing and microgels can be directly annealed into cell-laden structures. Finally, high-throughput direct encapsulation of cells within printable microgels is demonstrated, enabling large-scale 3D culture in a macroporous biomaterial. This approach provides unprecedented spatiotemporal control over the properties of printed microporous annealed particle scaffolds for 2.5D and 3D tissue culture.

Utilisation of skin blood flow as a precursor for pressure injury development in persons with acute spinal cord injury: A proof of concept.

People with spinal cord injury (SCI) are at high risk of developing a pressure injury. It is unclear why some people with SCI develop pressure injury while others with similar predisposing risk factors do not during acute hospitalisation. This may hinder healthcare utilisation to prevent pressure injuries. The purpose of the study was to examine the proof-of-concept objective bedside skin blood flow measurements before a pressure injury develops in spinal cord injured patients during acute hospitalisation. This was an observational study. All participants had acute traumatic SCI and were pressure injury-free upon enrollment. Skin blood flow patterns were collected at both heels under two circumstances: localised pressure for reactive hyperemia, and localised heating for heat hyperemia. Our results showed that reactive and heat hyperemia were successfully induced in all eleven participants. Two participants developed pressure injury and nine did not have pressure injury at discharge. Heat hyperemia was smaller in participants with pressure injury. No difference was observed in reactive hyperemia between the groups. In conclusion, skin blood flow measurements could be obtained at bedside during acute hospitalisation of SCI for the purpose of research. Further examination of a larger group is warranted to determine clinical use of heat hyperemia pattern as predictor for pressure injury development.

Stress Relaxation and Composition of Hydrazone-Crosslinked Hybrid Biopolymer-Synthetic Hydrogels Determine Spreading and Secretory Properties of MSCs.

The extracellular matrix plays a critical role in mechanosensing and thereby influences the secretory properties of bone-marrow-derived mesenchymal stem/stromal cells (MSCs). As a result, interest has grown in the development of biomaterials with tunable properties for the expansion and delivery of MSCs that are used in cell-based therapies. Herein, stress-relaxing hydrogels are synthesized as hybrid networks containing both biopolymer and synthetic macromer components. Hyaluronic acid is functionalized with either aldehyde or hydrazide groups to form covalent adaptable hydrazone networks, which are stabilized by poly(ethylene glycol) functionalized with bicyclononyne and heterobifunctional small molecule crosslinkers containing azide and benzaldehyde moieties. Tuning the composition of these gels allows for controlled variation in the characteristic timescale for stress relaxation and the amount of stress relaxed. Over this compositional space, MSCs are observed to spread in formulations with higher degrees of adaptability, with aspect ratios of 1.60 ± 0.18, and YAP nuclear:cytoplasm ratios of 6.5 ± 1.3. Finally, a maximum MSC pericellular protein thickness of 1.45 ± 0.38 µm occurred in highly stress-relaxing gels, compared to 1.05 ± 0.25 µm in non-adaptable controls. Collectively, this study contributes a new understanding of the role of compositionally defined stress relaxation on MSCs mechanosensing and secretion.

Granular PEG hydrogels mediate osteoporotic MSC clustering via N-cadherin influencing the pro-resorptive bias of their secretory profile.

Postmenopausal osteoporosis results from a pro-resorptive bone environment, which decreases bone mineral density causing increased fracture risk. Bone marrow derived mesenchymal stem/stromal cells (MSCs) secrete factors involved in bone homeostasis, but osteoporosis mediated changes to their secretions remain understudied. Herein, we examined the secretome of MSCs isolated from ovariectomized rats (OVX rMSCs), a model of post-menopausal osteoporosis, as a function of cell-cell interactions. Specifically, we controlled clustering of OVX and SHAM rMSCs by assembling them in granular hydrogels synthesized from poly(ethylene glycol) microgels with average diameters of ∼10, 100, and 200 µm. We directed both the sizes of rMSC clusters (single cells to ∼30 cells/cluster) and the percentages of cells within clusters (∼20-90%) by controlling the scaffold pore dimensions. Large clusters of OVX rMSCs had a pro-resorptive secretory profile, with increased concentrations of Activin A, CXCL1, CX3CL1, MCP-1, TIMP-1, and TNF-ɑ, compared to SHAM rMSCs. As this pro-resorptive bias was only observed in large cell clusters, we characterized the expression of several cadherins, mediators of cell-cell contacts. N-cadherin expression was elevated (∼4-fold) in OVX relative to SHAM rMSCs, in both cell clusters and single cells. Finally, TIMP-1 and MCP-1 secretion was only decreased in large cell clusters of OVX rMSCs when N-cadherin interactions were blocked, highlighting the dependence of OVX rMSC secretion of pro-resorptive cytokines on N-cadherin mediated cell-cell contacts. Further elucidation of the N-cadherin mediated osteoporotic MSC secretome may have implications for developing therapies for postmenopausal osteoporosis. STATEMENT OF SIGNIFICANCE: Postmenopausal osteoporosis is a prevalent bone disorder that affects tens of millions of women worldwide. This disease is characterized by severe bone loss resulting from a pro-resorptive bone marrow environment, where the rates of bone resorption outpace the rates of bone deposition. The paracrine factors secreted by bone marrow MSCs can influence cell types responsible for bone homeostasis, but the osteoporosis-mediated changes to MSC secretory properties remains understudied. In this study, we used PEG-based porous granular scaffolds to study the influence of cell clustering on the secretory properties of osteoporotic MSCs. We observed increased secretion of several pro-resorptive factors by osteoporotic MSCs in large clusters. Further, we explored the dependence of this altered secretion profile on N-cadherin mediated cell-cell contacts.

Hot-processed virgin coconut oil abrogates cisplatin-induced nephrotoxicity by restoring redox balance in rats compared to fermentation-processed virgin coconut oil.

Virgin coconut oil (VCO) is a functional food oil prepared from fresh coconut kernel either by hot-processed (HPVCO) or fermentation-processed (FPVCO). The FPVCO has been widely explored for its pharmacological efficacy; while HPVCO, which has traditional uses, is less explored. The present study compared the phenolic content and nephroprotective effect of both these oils in male Wistar rats. In vitro antioxidant activity was estimated in terms of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferric reducing antioxidant power and ex vivo lipid peroxidation inhibition. In in vivo models, the rats were pretreated orally with of FPVCO or HPVCO (doses 2 and 4 mL/kg) for seven days and nephrotoxicity was induced by the single intraperitoneal injection of cisplatin (10 mg/kg). The results indicated significantly higher polyphenol content in HPVCO (400.3 ± 5.8 µg/mL) than that of FPVCO (255.5 ± 5.8 µg/mL). Corroborating with the increased levels of polyphenols, the in vitro antioxidant potential was significantly higher in the HPVCO. Further, pretreatment with these VCO preparations protected the rats against the cisplatin-induced nephrotoxicity, with higher extent by HPVCO. The renal function markers like urea, creatinine and total bilirubin were significantly reduced (p < 0.05) with HPVCO pretreatment. Apart from the nephroprotective effects, HPVCO also abrogated the cisplatin-induced myelosuppression and hepatotoxicity. The restoration of hepato-renal function by the pretreatment of HPVCO was well corroborated with the improvement in functional antioxidants and subsequent reduction in renal lipid peroxidation. Supporting these observations, renal histology revealed reduced glomerular/tubular congestion and necrosis. Thus, the study concludes that HPVCO may be better functional food than FPVCO.

Mesenchymal stem cell-inspired microgel scaffolds to control macrophage polarization.

There is a desire in regenerative medicine to create biofunctional materials that can control and direct cell function in a precise manner. One particular stem cell of interest, human mesenchymal stem cells (hMSCs), can function as regulators of the immunogenic response and aid in tissue regeneration and wound repair. Here, a porous hydrogel scaffold assembled from microgel subunits was used to recapitulate part of this immunomodulatory behavior. The scaffolds were used to culture a macrophage cell line, while cytokines were delivered exogenously to polarize the macrophages to either a pro-inflammatory (M1) or alternatively activated (M2a) phenotypes. Using a cytokine array, interleukin 10 (IL-10) was identified as one key anti-inflammatory factor secreted by hMSCs in pro-inflammatory conditions; it was elevated (125 ± 25 pg/ml) in pro-inflammatory conditions compared to standard medium (6 ± 10 pg/ml). The ability of hMSC laden scaffolds to reverse the M1 phenotype was then examined, even in the presence of exogenous pro-inflammatory cytokines. Co-culture of M1 and M2 macrophages with hMSCs reduced the secretion of TNFα, a pro-inflammatory cytokine even in the presence of pro-inflammatory stimulatory factors. Next, IL-10 was supplemented in the medium or tethered directly to the microgel subunits; both methods limited the secretion of pro-inflammatory cytokines of encapsulated macrophages even in pro-inflammatory conditions. Cumulatively, these results reveal the potential of biofunctional microgel-based scaffolds as acellular therapies to present anti-inflammatory cytokines and control the immunogenic cascade.

Engineering the MSC Secretome: A Hydrogel Focused Approach.

The therapeutic benefits of exogenously delivered mesenchymal stromal/stem cells (MSCs) have been largely attributed to their secretory properties. However, clinical translation of MSC-based therapies is hindered due to loss of MSC regenerative properties during large-scale expansion and low survival/retention post-delivery. These limitations might be overcome by designing hydrogel culture platforms to modulate the MSC microenvironment. Hydrogel systems could be engineered to i) promote MSC proliferation and maintain regenerative properties (i.e., stemness and secretion) during ex vivo expansion, ii) improve MSC survival, retention, and engraftment in vivo, and/or iii) direct the MSC secretory profile using tailored biochemical and biophysical cues. Herein, it is reviewed how hydrogel material properties (i.e., matrix modulus, viscoelasticity, dimensionality, cell adhesion, and porosity) influence MSC secretion, mediated through cell-matrix and cell-cell interactions. In addition, it is highlighted how biochemical cues (i.e., small molecules, peptides, and proteins) can improve and direct the MSC secretory profile. Last, the authors' perspective is provided on future work toward the understanding of how microenvironmental cues influence the MSC secretome, and designing the next generation of biomaterials, with optimized biophysical and biochemical cues, to direct the MSC secretory profile for improved clinical translation outcomes.

Cell-free DNA (cfDNA) and Exosome Profiling from a Year-Long Human Spaceflight Reveals Circulating Biomarkers.

Liquid biopsies based on cell-free DNA (cfDNA) or exosomes provide a noninvasive approach to monitor human health and disease but have not been utilized for astronauts. Here, we profile cfDNA characteristics, including fragment size, cellular deconvolution, and nucleosome positioning, in an astronaut during a year-long mission on the International Space Station, compared to his identical twin on Earth and healthy donors. We observed a significant increase in the proportion of cell-free mitochondrial DNA (cf-mtDNA) inflight, and analysis of post-flight exosomes in plasma revealed a 30-fold increase in circulating exosomes and patient-specific protein cargo (including brain-derived peptides) after the year-long mission. This longitudinal analysis of astronaut cfDNA during spaceflight and the exosome profiles highlights their utility for astronaut health monitoring, as well as cf-mtDNA levels as a potential biomarker for physiological stress or immune system responses related to microgravity, radiation exposure, and the other unique environmental conditions of spaceflight.

A novel NGS library preparation method to characterize native termini of fragmented DNA.

Biological and chemical DNA fragmentation generates DNA molecules with a variety of termini, including blunt ends and single-stranded overhangs. We have developed a Next Generation Sequencing (NGS) assay, XACTLY, to interrogate the termini of fragmented DNA, information traditionally lost in standard NGS library preparation methods. Here we describe the XACTLY method, showcase its sensitivity and specificity, and demonstrate its utility in in vitro experiments. The XACTLY assay is able to report relative abundances of all lengths and types (5' and 3') of single-stranded overhangs, if present, on each DNA fragment with an overall accuracy between 80-90%. In addition, XACTLY retains the sequence of each native DNA molecule after fragmentation and can capture the genomic landscape of cleavage events at single nucleotide resolution. The XACTLY assay can be applied as a novel research and discovery tool for fragmentation analyses and in cell-free DNA.

Phototunable Viscoelasticity in Hydrogels Through Thioester Exchange.

Mechanical cues are delivered to resident cells by the extracellular matrix and play an important role in directing cell processes, ranging from embryonic development and cancer metastasis to stem cell differentiation. Recently, cellular responses to viscoelastic and elastic mechanical cues have been studied; however, questions remain as to how cells identify and transduce these cues differently. We present a synthetic cell culture substrate with viscoelastic properties based on thioester exchange chemistry that can be modulated in situ with the photoinitiated thiol-ene 'click' reaction. With this method, stress relaxation in thioester hydrogels with an average relaxation time of 740,000 s can be switched off in the presence of cells without change to the elastic modulus. NIH 3T3 fibroblasts, cultured for 48 h on viscoelastic compared to elastic thioester substrates, displayed increased cell area (660-560 µm2) and increased nuclear to cytoplasmic YAP/TAZ ratios (2.4 to 2.2) when cultured on elastic compared to viscoelastic hydrogels, respectively. Next, when the viscoelasticity was switched off after 24 h, the fibroblasts responded to this change and exhibited an average cell area of 540 µm2, and nuclear to cytoplasmic YAP/TAZ ratio of 2.1, approaching that of the control elastic gels. Phototunable viscoelastic thioester hydrogels provide a tunable materials system to investigate time-dependent cellular responses to viscoelasticity and should prove useful for understanding the dynamics of mechanoresponsive cellular pathways.

Porous bio-click microgel scaffolds control hMSC interactions and promote their secretory properties.

Human mesenchymal stem/stromal cells (hMSCs) are known to secrete numerous cytokines that signal to endogenous cells and aid in tissue regeneration. However, the role that biomaterial scaffolds can play in controlling hMSC secretory properties has been less explored. Here, microgels were co-assembled with hMSCs using three different microgel populations, with large (190 ± 100 µm), medium (110 ± 60 µm), and small (13 ± 6 µm) diameters, to create distinct porous environments that influenced hMSC clustering. Cells embedded in large diameter microgel networks resided in large clusters (~40 cells), compared to small clusters (~6 cells) observed in networks using medium diameter microgels and primarily single cells in small diameter microgel networks. Using a cytokine microarray, an overall increase in secretion was observed in scaffolds that promoted hMSC clustering, with over 60% of the measured cytokines most elevated in the large diameter microgel networks. N-cadherin interactions were identified as partially mediating these differences, so the microgel formulations were modified with an N-cadherin epitope, HAVDI, to mimic cell-cell interactions. Results revealed increased secretory properties for hMSCs in HAVDI functionalized scaffolds, even the non-clustered cells in small diameter microgel networks. Together, these results demonstrate opportunities for microgel-based scaffold systems for hMSC delivery and tailoring of porous materials properties to promote their secretory potential.

A ligation-based single-stranded library preparation method to analyze cell-free DNA and synthetic oligos.

BACKGROUND: Cell-free DNA (cfDNA), present in circulating blood plasma, contains information about prenatal health, organ transplant reception, and cancer presence and progression. Originally developed for the genomic analysis of highly degraded ancient DNA, single-stranded DNA (ssDNA) library preparation methods are gaining popularity in the field of cfDNA analysis due to their efficiency and ability to convert short, fragmented DNA into sequencing libraries without altering DNA ends. However, current ssDNA methods are costly and time-consuming. RESULTS: Here we present an efficient ligation-based single-stranded library preparation method that is engineered to produce complex libraries in under 2.5 h from as little as 1 nanogram of input DNA without alteration to the native ends of template molecules. Our method, called Single Reaction Single-stranded LibrarY or SRSLY, ligates uniquely designed Next-Generation Sequencing (NGS) adapters in a one-step combined phosphorylation/ligation reaction that foregoes end-polishing. Using synthetic DNA oligos and cfDNA, we demonstrate the efficiency and utility of this approach and compare with existing double-stranded and single-stranded approaches for library generation. Finally, we demonstrate that cfDNA NGS data generated from SRSLY can be used to analyze DNA fragmentation patterns to deduce nucleosome positioning and transcription factor binding. CONCLUSIONS: SRSLY is a versatile tool for converting short and fragmented DNA molecules, like cfDNA fragments, into sequencing libraries while retaining native lengths and ends.

Longitudinal multi-omics of host-microbe dynamics in prediabetes.

Type 2 diabetes mellitus (T2D) is a growing health problem, but little is known about its early disease stages, its effects on biological processes or the transition to clinical T2D. To understand the earliest stages of T2D better, we obtained samples from 106 healthy individuals and individuals with prediabetes over approximately four years and performed deep profiling of transcriptomes, metabolomes, cytokines, and proteomes, as well as changes in the microbiome. This rich longitudinal data set revealed many insights: first, healthy profiles are distinct among individuals while displaying diverse patterns of intra- and/or inter-personal variability. Second, extensive host and microbial changes occur during respiratory viral infections and immunization, and immunization triggers potentially protective responses that are distinct from responses to respiratory viral infections. Moreover, during respiratory viral infections, insulin-resistant participants respond differently than insulin-sensitive participants. Third, global co-association analyses among the thousands of profiled molecules reveal specific host-microbe interactions that differ between insulin-resistant and insulin-sensitive individuals. Last, we identified early personal molecular signatures in one individual that preceded the onset of T2D, including the inflammation markers interleukin-1 receptor agonist (IL-1RA) and high-sensitivity C-reactive protein (CRP) paired with xenobiotic-induced immune signalling. Our study reveals insights into pathways and responses that differ between glucose-dysregulated and healthy individuals during health and disease and provides an open-access data resource to enable further research into healthy, prediabetic and T2D states.

Rescuing mesenchymal stem cell regenerative properties on hydrogel substrates post serial expansion.

The use of human mesenchymal stem/stromal cells (hMSCs) in most clinical trials requires millions of cells/kg, necessitating ex vivo expansion typically on stiff substrates (tissue culture polystyrene [TCPS]), which induces osteogenesis and replicative senescence. Here, we quantified how serial expansion on TCPS influences proliferation, expression of hMSC-specific surface markers, mechanosensing, and secretome. Results show decreased proliferation and surface marker expression after five passages (P5) and decreased mechanosensing ability and cytokine production at later passages (P11-P12). Next, we investigated the capacity of poly(ethylene glycol) hydrogel matrices (E ~ 1 kPa) to rescue hMSC regenerative properties. Hydrogels reversed the reduction in cell surface marker expression observed at P5 on TCPS and increased secretion of cytokines for P11 hMSCs. Collectively, these results show that TCPS expansion significantly changes functional properties of hMSCs. However, some changes can be rescued by using hydrogels, suggesting that tailoring material properties could improve in vitro expansion methods.

Distinct transcriptomic and exomic abnormalities within myelodysplastic syndrome marrow cells.

To provide biologic insights into mechanisms underlying myelodysplastic syndromes (MDS) we evaluated the CD34+ marrow cells transcriptome using high-throughput RNA sequencing (RNA-Seq). We demonstrated significant differential gene expression profiles (GEPs) between MDS and normal and identified 41 disease classifier genes. Additionally, two main clusters of GEPs distinguished patients based on their major clinical features, particularly between those whose disease remained stable versus patients who transformed into acute myeloid leukemia within 12 months. The genes whose expression was associated with disease outcome were involved in functional pathways and biologic processes highly relevant for MDS. Combined with exomic analysis we identified differential isoform usage of genes in MDS mutational subgroups, with consequent dysregulation of distinct biologic functions. This combination of clinical, transcriptomic and exomic findings provides valuable understanding of mechanisms underlying MDS and its progression to a more aggressive stage and also facilitates prognostic characterization of MDS patients.

Gpr124 is essential for blood-brain barrier integrity in central nervous system disease.

Although blood-brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt-ß-catenin signaling. Constitutive activation of Wnt-ß-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption.