Genome-wide analysis identifies NR4A1 as a key mediator of T cell dysfunction.

T cells become dysfunctional when they encounter self antigens or are exposed to chronic infection or to the tumour microenvironment1. The function of T cells is tightly regulated by a combinational co-stimulatory signal, and dominance of negative co-stimulation results in T cell dysfunction2. However, the molecular mechanisms that underlie this dysfunction remain unclear. Here, using an in vitro T cell tolerance induction system in mice, we characterize genome-wide epigenetic and gene expression features in tolerant T cells, and show that they are distinct from effector and regulatory T cells. Notably, the transcription factor NR4A1 is stably expressed at high levels in tolerant T cells. Overexpression of NR4A1 inhibits effector T cell differentiation, whereas deletion of NR4A1 overcomes T cell tolerance and exaggerates effector function, as well as enhancing immunity against tumour and chronic virus. Mechanistically, NR4A1 is preferentially recruited to binding sites of the transcription factor AP-1, where it represses effector-gene expression by inhibiting AP-1 function. NR4A1 binding also promotes acetylation of histone 3 at lysine 27 (H3K27ac), leading to activation of tolerance-related genes. This study thus identifies NR4A1 as a key general regulator in the induction of T cell dysfunction, and a potential target for tumour immunotherapy.

Negative regulation of IL-17-mediated signaling and inflammation by the ubiquitin-specific protease USP25.

Interleukin 17 (IL-17) is important in infection and autoimmunity; how it signals remains poorly understood. In this study, we identified the ubiquitin-specific protease USP25 as a negative regulator of IL-17-mediated signaling and inflammation. Overexpression of USP25 inhibited IL-17-triggered signaling, whereas USP25 deficiency resulted in more phosphorylation of the inhibitor IκBα and kinase Jnk and higher expression of chemokines and cytokines, as well as a prolonged half-life for chemokine CXCL1-encoding mRNA after treatment with IL-17. Consistent with that, Usp25(-/-) mice showed greater sensitivity to IL-17-dependent inflammation and autoimmunity in vivo. Mechanistically, stimulation with IL-17 induced the association of USP25 with the adaptors TRAF5 and TRAF6, and USP25 induced removal of Lys63-linked ubiquitination in TRAF5 and TRAF6 mediated by the adaptor Act1. Thus, our results demonstrate that USP25 is a deubiquitinating enzyme (DUB) that negatively regulates IL-17-triggered signaling.

Trade-offs between diaspore dispersal and dormancy within a spike of the invasive annual grass Aegilops tauschii.

MAIN CONCLUSION: Differences in dispersal and dormancy of heteromorphic diaspores of Aegilos tauschii may increase its flexibility to invade/occupy weedy unpredictable habitats by spreading risk in space and time. In plant species that produce dimorphic seeds, there often is a negative relationship between dispersal and dormancy, with high dispersal-low dormancy in one morph and low dispersal-high dormancy in the other, which may function as a bet-hedging strategy that spreads the risk of survival and ensures reproductive success. However, the relationship between dispersal and dormancy and its ecological consequences in invasive annual grasses that produce heteromorphic diaspores is not well studied. We compared dispersal and dormancy responses of diaspores from the basal (proximal) to the distal position on compound spikes of Aegilops tauschii, an invasive grass with heteromorphic diaspores. Dispersal ability increased and degree of dormancy decreased as diaspore position on a spike increased from basal to distal. There was a significant positive correlation between length of awns and dispersal ability, and awn removal significantly promoted seed germination. Germination was positively correlated with GA concentration and negatively correlated with ABA concentration, and the ABA: GA ratio was high in seeds with low germination/high dormancy. Thus, there was a continuous inverse-linear relationship between diaspore dispersal ability and degree of dormancy. This negative relationship between diaspore dispersal and degree of dormancy at different positions on a spike of Aegilops tauschii may facilitate seedling survival in space and time.

Ductile machining of optical micro-structures on single crystal germanium by elliptical vibration assisted sculpturing.

Microstructure surfaces are widely and deeply applied in optical fields to decrease the volume of an optical system and realize sophisticated optical requirements. This paper studies the machining of optical micro-structures with micro-scale height on single crystal germanium by elliptical vibration assisted sculpturing (EVAS) method in which two vibration amplitudes keep constant. Due to the superimposition of low-frequency sculpture path and high-frequency elliptical locus, the investigations are focused on cutting plane to describe motion trajectory of the tool tip of cutting tool for desired complex motion path and to calculate the sculpture path of elliptical vibration cutting device to make corresponding path of tool tip be the envelope of target profile. A comparative experiment is firstly conducted to verify that elliptical locus would deteriorate machining accuracy without accurate compensation for the ellipse. Following that, 2D and 3D sinusoidal surfaces and a picture, whose structural heights are all not less than 2.0µm, are experimentally machined on single crystal germanium. Satisfying experimental results demonstrate the feasibility of EVAS method for fabricating optical micro-structures on germanium.

Transcription factor achaete-scute homologue 2 initiates follicular T-helper-cell development.

In immune responses, activated T cells migrate to B-cell follicles and develop into follicular T-helper (TFH) cells, a recently identified subset of CD4(+) T cells specialized in providing help to B lymphocytes in the induction of germinal centres. Although Bcl6 has been shown to be essential in TFH-cell function, it may not regulate the initial migration of T cells or the induction of the TFH program, as exemplified by C-X-C chemokine receptor type 5 (CXCR5) upregulation. Here we show that expression of achaete-scute homologue 2 (Ascl2)--a basic helix-loop-helix (bHLH) transcription factor--is selectively upregulated in TFH cells. Ectopic expression of Ascl2 upregulates CXCR5 but not Bcl6, and downregulates C-C chemokine receptor 7 (CCR7) expression in T cells in vitro, as well as accelerating T-cell migration to the follicles and TFH-cell development in vivo in mice. Genome-wide analysis indicates that Ascl2 directly regulates TFH-related genes whereas it inhibits expression of T-helper cell 1 (TH1) and TH17 signature genes. Acute deletion of Ascl2, as well as blockade of its function with the Id3 protein in CD4(+) T cells, results in impaired TFH-cell development and germinal centre response. Conversely, mutation of Id3, known to cause antibody-mediated autoimmunity, greatly enhances TFH-cell generation. Thus, Ascl2 directly initiates TFH-cell development.

Aquaporin4 Knockout Aggravates Early Brain Injury Following Subarachnoid Hemorrhage Through Impairment of the Glymphatic System in Rat Brain.

BACKGROUND: It is reported that the expression of aquaporin4 (AQP4) in the brain is increased and leads to the brain edema after subarachnoid hemorrhage (SAH). In this study, by using AQP4 knockout rat model, the opposite role of AQP4 in early brain injury following SAH through modulation of interstitial fluid (ISF) transportation in the brain glymphatic system had been explored. METHODS: The SAH model was established using endovascular perforation method, the AQP4 knockout rat model was generated using TALENs (transcription activator-like (TAL) effector nucleases) technique. The animals were randomly divided into four groups: sham (n = 16), AQP4-/-sham (n = 16), SAH (n = 24), and AQP4-/-SAH groups (n = 27). The roles of AQP4 in the brain water content and neurological function were detected. In addition, immunohistochemistry and Nissl staining were applied to observe the effects of AQP4 on the blood-brain barrier (BBB) integrity and the loss of neurons in the hippocampus. To explore the potential mechanism of these effects, the distribution of Gd-DTPA (interstitial fluid indicator) injected from cisterna magna was evaluated with MRI. RESULTS: Following SAH, AQP4 knockout could significantly increase the water content in the whole brain and aggravate the neurological deficits. Furthermore, the loss of neuron and BBB disruption in hippocampus were also exacerbated. The MRI results indicated that the ISF transportation in the glymphatic system of AQP4 deficit rat was significantly injured. CONCLUSION: AQP4 facilitates the ISF transportation in the brain to eliminate the toxic factors; AQP4 knockout will aggravate the early brain injury following SAH through impairment of the glymphatic system.

The olfactory stimulation slows down the substance clearance in the extracellular space of the hippocampus in rat brain.

Accelerating the clearance of toxin in the brain extracellular space (ECS) has grown a promising strategy for treating some central nervous system diseases. As oldest sensory system, we know little about the influence of olfaction on the brain, but preclinical studies such as treatment of neurological diseases through it are in the ascendant. This makes it important to clarify the effects of olfaction on brain ECS and interstitial fluid (ISF) drainage. In this study, the effect of olfactory stimulation (eugenol, EUG) on ISF flow in hippocampus and its association with aquaporin 4 (Aqp4) had been investigated. The results show that eugenol can significantly increase the activity of hippocampal neurons, but reduce the clearance and diffusion rates of Gd-DTPA and A-594 in hippocampus. Similarly, eugenol inhalation slows down the rate of Gd-DTPA in CSF entering the hippocampus and its clearance. And knockout of Aqp4 gene aggravated these processes. In vitro results showed that after Aqp4 gene silencing, astrocytes grew slowly, with significantly decreased cells number, less nuclei, atrophied bodies and shorter processes. These results concluded that olfactory stimulation can change the ECS structure of the hippocampus, slow down the ISF drainage, and improve the function of neurons, while Aqp4 plays important roles.

Ubc9 Is Required for Positive Selection and Late-Stage Maturation of Thymocytes.

SUMOylation is an important posttranslational modification that regulates protein function in diverse biological processes. However, its role in early T cell development has not been genetically studied. UBC9 is the only E2 enzyme for all SUMOylation. In this study, by selectively deleting Ubc9 gene in T cells, we have investigated the functional roles of SUMOylation in T cell development. Loss of Ubc9 results in a significant reduction of CD4 and CD8 single-positive lymphocytes in both thymus and periphery. Ubc9-deficient cells exhibit defective late-stage maturation post the initial positive selection with increased apoptosis and impaired proliferation, among which attenuated IL-7 signaling was correlated with the decreased survival of Ubc9-deficent CD8 single-positive cells. Furthermore, NFAT nuclear retention induced by TCR signals was regulated by SUMOylation during thymocytes development. Our study thus reveals a novel posttranslational mechanism underlying T cell development.

Cutting edge: Smad2 and Smad4 regulate TGF-ß-mediated Il9 gene expression via EZH2 displacement.

IL-9 is a proallergic cytokine produced by a newly proposed Th cell subset, Th9. Th9 cells can be generated by treatment of naive T cells with TGF-ß and IL-4 in vitro. However, it is still not clear how TGF-ß signaling regulates Th9 differentiation. In this study, we demonstrate that Smad2 and Smad4, two transcriptional factors activated by TGF-ß signaling, are required for Th9 differentiation in vitro. Deficiency of Smad2 or Smad4 in T cells resulted in impaired IL-9 expression, which was coincident with enrichment of repressive chromatin modification histone H3 K27 trimethylation and enhanced EZH2 binding to the Il9 locus. Pharmacologic inhibition of EZH2 partially rescued IL-9 production in Smad-deficient Th9 cells. Smad proteins may displace EZH2 directly from the Il9 locus, because Smad2 and Smad4 can bind EZH2. Our data shed light on the molecular mechanisms underlying Th9 cell differentiation, revealing that the TGF-ß-Smad2/4-signaling pathway regulates IL-9 production through an epigenetic mechanism.

Real-time monitoring of atmospheric ammonia based on modifier-enhanced vacuum ultraviolet photoionization ion mobility spectrometry.

Ammonia (NH3) plays an important role in the atmospheric environment such as the formation of PM2.5, the concentration monitoring of which could hence help in the air quality assessment. In this study, a method for quantitative monitoring of atmospheric NH3 was developed based on modifier-enhanced selectivity detection using a homemade vacuum ultraviolet photoionization ion mobility spectrometry (VUV-PI-IMS). To enhance the resolution and sensitivity of measuring NH3, 2-butanone as the gas modifier was introduced into the drift tube with the drift gas. Atmospheric NH3 can be selectively detected, where the peak-to-peak resolution (RP-P) of 7.69 was obtained. The product ions were identified to be [C4H8O]2NH4+ by using a homemade time-of-flight mass spectrometer. The calculated limit of detection (LOD) was 0.39 ppbv improving about 10 times. For the most common concentration variation of atmospheric NH3 in the range of 10-100 ppbv, the linear curve was obtained with R2 of 0.997. Lastly, the VUV-PI-IMS was used to monitor the evolution of atmospheric NH3 near our laboratory and mounted on a car for monitoring the regional distribution of atmospheric NH3 in Dalian, China. The results also showed that VUV-PI-IMS has a promising application prospect in monitoring the concentration of atmospheric NH3 and supporting the air quality assessment.

Seed position in spikelet as a contributing factor to the success of the winter annual invasive grass Aegilops tauschii.

Seed position - dependent effects on seed dormancy/germination are well documented at the inflorescence/infructescence level, but less is known about seeds at different positions within a dispersal unit. For the invasive winter annual grass Aegilops tauschii, we quantified morphology, mass and dormancy/germination of seeds from basal (1), middle (2), and distal (3) positions in two spikelet types (Left and Right). We also investigated seedling emergence, survival, plant size and seed production of plants from seeds in different spikelet positions of two spikelet types under different soil nutrient and water conditions. We found that these seed, seedling and plant traits performed as mirror images between the Left and Right spikelet types. The middle seed was significantly the longest and had the maximum mass, while the basal seed was the shortest and had medium mass. Middle seeds had the highest increase in mass during imbibition and the highest germination percentages and rates, while basal seeds had the lowest. Seedling emergence and survival, plant size and seed production for each position of seeds were highest in the added fertilizer combined with regular watering treatment and lowest in the no fertilizer combined with natural moisture, while height of plants derived from the middle and the distal seeds was significantly higher than that of plants derived from the basal seeds under all soil nutrient and water conditions. Seedling survival, number of tillers per plant and seed production per plant from the middle and distal seeds were significantly lower than those from basal seeds under all soil nutrient and water treatments. The considerable variation in seedling emergence and survival, plant size and seed production between seeds in different positions in the spikelet results in much flexibility in all stages of the life cycle, thereby likely contributing to the invasiveness of A. tauschii.

Evaluation of the Glymphatic System With Diffusion Tensor Imaging-Along the Perivascular Space in Cancer Pain.

Cancer pain (CP) is one of the most common symptoms affecting life quality, and there is considerable variation in pain experience among patients with malignant tumors. Previously, it has been found that the fluid drainage function in the brain can be regulated by peripheral pain stimulation. However, the relationship between cancer pain and functional changes of the glymphatic system (an important pathway for fluid drainage in the brain) remains unclear. In this study, 97 participants were enrolled, which included 40 participants in the cancer pain (CP) group, 27 participants in the painless cancer (PLC) group and 30 participants in the control (NC) group. Differences in glymphatic system function among the three groups and between before and after pain pharmacological intervention were analyzed by measuring diffusivity and the index along the perivascular space (ALPS index) using diffusion tensor imaging. We found that diffusivity and the ALPS index were significantly lower in the CP group than in the PLC and NC group and increased following intervention with pain relief. Moreover, the ALPS index was negatively correlated with the degree of pain in the CP group. The present study verified that alterations in glymphatic function are closely related to cancer pain, and the quantification of functional changes reflects pain severity. Our findings support the use of neuroimaging biomarkers for cancer pain assessment and indicate that pain can be alleviated by regulating brain function status.

Phosphorylation of Nur77 by the MEK-ERK-RSK cascade induces mitochondrial translocation and apoptosis in T cells.

Nur77, an orphan nuclear receptor, plays a key role in apoptosis in T cells. In cancer cell lines, Nur77 can induce apoptosis through the intrinsic apoptotic pathway, but the mechanism by which Nur77 kills T cells remains controversial. In this study, we provide biochemical, pharmacological, and genetic evidence demonstrating that Nur77 induces apoptosis through the activation of the intrinsic pathway in T cells. We also show that Nur77 is a physiological substrate of the MEK-ERK-RSK cascade. Specifically, we demonstrate that RSK phosphorylates Nur77 at serine 354 and this modulates Nur77 nuclear export and intracellular translocation during T cell death. Our data reveal that Nur77 phosphorylation and mitochondrial targeting, regulated by RSK, defines a role for the MEK1/2-ERK1/2 cascade in T cell apoptosis.

Stimulation Modeling on Three-Dimensional Anisotropic Diffusion of MRI Tracer in the Brain Interstitial Space.

Purpose: To build a mathematical model based magnetic resonance (MR) method to simulate drug anisotropic distribution in vivo in the interstitial space (ISS) of the brain. Materials and Methods: An injection of signal intensity-related gadolinium-diethylenetriaminepentaacetic acid (Gd-DTPA), which is an exogenous drug, was administered, and its diffusion was traced in the ISS of the brain using MRI. Dynamic MRI scans were performed to monitor and record the changes in signal intensity in each pixel of the region of interest. The transport parameters were calculated using the modified equation to simulate three-dimensional anisotropic diffusion, which was resolved using a Laplace transform and a linear regressive model. Results: After Gd-DTPA was introduced into the caudate nucleus, its distribution was demonstrated in real time. As the Gd-DTPA gradually cleared, the associated hyperintensity attenuated over time. The average diffusion coefficient (D) and the clearance rate constant (k) were (1.305 ± 0.364) × 10-4 mm2/s and (1.40 ± 0.206) × 10-5 s-1, respectively. Discussion: The combination of trace-based MRI and modified diffusion mathematical models can visualize and measure the three-dimensional anisotropic distribution of drugs in the ISS of the brain.

The effects of dihydroxyphenyl lactic acid on alleviating blood-brain barrier injury following subarachnoid hemorrhage in rats.

BACKGROUND: In this study, the protective effects of 3,4-dihydroxyphenyl lactic acid (DLA) on blood-brain barrier (BBB) injury following subarachnoid hemorrhage (SAH) has been explored. METHODS: Male Sprague-Dawley rats (weight 300-350 g) were used to establish the SAH model using the endovascular perforation method. The animals were randomly divided into four groups: sham (n = 40), SAH (n = 46), SAH + vehicle (n = 44), and SAH + DLA (n = 40) treatment groups. At 1 h after SAH, either DLA (10 mg/kg) or normal saline (vehicle) was administered by femoral vein injection. The effects of DLA on mortality, neurological function, brain water content, and BBB were observed. Additionally, immunohistochemistry and western blot techniques were applied to investigate the mechanism of action of DLA. RESULTS: We found that the administration of DLA (10 mg/kg) following SAH could improve neurological functions, reduce brain water content, and maintain BBB integrity. The expression of pro-inflammatory and pro-apoptotic factors such as toll-like receptor 4 (TLR4), NF-κB (p-p65), tumor necrosis factor-α, p-p38 MAPK, p-p53, and caspase-3 were significantly increased after SAH. These same factors were markedly attenuated following treatment with DLA. CONCLUSIONS: These findings showed that DLA can alleviate BBB injury following SAH through its anti-inflammatory and anti-apoptotic effects via suppression of TLR4 and its downstream NF-κB and p38 MAPK pathways.

Erratum: Publisher Correction: Endogenous formaldehyde is a memory-related molecule in mice and humans.

[This corrects the article DOI: 10.1038/s42003-019-0694-x.].

Endogenous formaldehyde is a memory-related molecule in mice and humans.

Gaseous formaldehyde is an organic small molecule formed in the early stages of earth's evolution. Although toxic in high concentrations, formaldehyde plays an important role in cellular metabolism and, unexpectedly, is found even in the healthy brain. However, its pathophysiological functions in the brain are unknown. Here, we report that under physiological conditions, spatial learning activity elicits rapid formaldehyde generation from mitochondrial sarcosine dehydrogenase (SARDH). We find that elevated formaldehyde levels facilitate spatial memory formation by enhancing N-methyl-D-aspartate (NMDA) currents, but that high formaldehyde concentrations gradually inactivate the NMDA receptor by cross-linking NR1 subunits to NR2B via the C232 residue. We also report that in mice with aldehyde dehydrogenase-2 (ALDH2) knockout, formaldehyde accumulation due to hypofunctional ALDH2 impairs memory, consistent with observations of Alzheimer's disease patients. We also find that formaldehyde deficiency caused by mutation of the mitochondrial SARDH gene in children with sarcosinemia or in mice with Sardh deletion leads to cognitive deficits. Hence, we conclude that endogenous formaldehyde regulates learning and memory via the NMDA receptor.

The Drainage of Interstitial Fluid in the Deep Brain is Controlled by the Integrity of Myelination.

In searching for the drainage route of the interstitial fluid (ISF) in the deep brain, we discovered a regionalized ISF drainage system as well as a new function of myelin in regulating the drainage. The traced ISF from the caudate nucleus drained to the ipsilateral cortex along myelin fiber tracts, while in the opposite direction, its movement to the adjacent thalamus was completely impeded by a barrier structure, which was identified as the converged, compact myelin fascicle. The regulating and the barrier effects of myelin were unchanged in AQP4-knockout rats but were impaired as the integrity of boundary structure of drainage system was destroyed in a demyelinated rat model. We thus proposed that the brain homeostasis was maintained within each ISF drainage division locally, rather than across the brain as a whole. A new brain division system and a new pathogenic mechanism of demyelination are therefore proposed.

New insight into Alzheimer's disease: Light reverses Aß-obstructed interstitial fluid flow and ameliorates memory decline in APP/PS1 mice.

INTRODUCTION: Pharmacological therapies to treat Alzheimer's disease (AD) targeting "Aß" have failed for over 100 years. Low levels of laser light can disassemble Aß. In this study, we investigated the mechanisms that Aß-blocked extracellular space (ECS) induces memory disorders in APP/PS1 transgenic mice and addressed whether red light (RL) at 630 nm rescues cognitive decline by reducing Aß-disturbed flow of interstitial fluid (ISF). METHODS: We compared the heating effects on the brains of rats illuminated with laser light at 630, 680, and 810 nm for 40 minutes, respectively. Then, a light-emitting diode with red light at 630 nm (LED-RL) was selected to illuminate AD mice. The changes in the structure of ECS in the cortex were examined by fluorescent double labeling. The volumes of ECS and flow speed of ISF were quantified by magnetic resonance imaging. Spatial memory behaviors in mice were evaluated by the Morris water maze. Then, the brains were sampled for biochemical analysis. RESULTS: RL at 630 nm had the least heating effects than other wavelengths associated with ~49% penetration ratio into the brains. For the molecular mechanisms, Aß could induce formaldehyde (FA) accumulation by inactivating FA dehydrogenase. Unexpectedly, in turn, FA accelerated Aß deposition in the ECS. However, LED-RL treatment not only directly destroyed Aß assembly in vitro and in vivo but also activated FA dehydrogenase to degrade FA and attenuated FA-facilitated Aß aggregation. Subsequently, LED-RL markedly smashed Aß deposition in the ECS, recovered the flow of ISF, and rescued cognitive functions in AD mice. DISCUSSION: Aß-obstructed ISF flow is the direct reason for the failure of the developed medicine delivery from superficial into the deep brain in the treatment of AD. The phototherapy of LED-RL improves memory by reducing Aß-blocked ECS and suggests that it is a promising noninvasive approach to treat AD.