Plant phase extraction: A method for enhanced discovery of the RNA-binding proteome and its dynamics in plants.

RNA-binding proteins (RBPs) play critical roles in posttranscriptional gene regulation. Current methods of systematically profiling RBPs in plants have been predominantly limited to proteins interacting with polyadenylated (poly(A)) RNAs. We developed a method called plant phase extraction (PPE), which yielded a highly comprehensive RNA-binding proteome (RBPome), uncovering 2,517 RBPs from Arabidopsis (Arabidopsis thaliana) leaf and root samples with a highly diverse array of RNA-binding domains. We identified traditional RBPs that participate in various aspects of RNA metabolism and a plethora of non-classical proteins moonlighting as RBPs. We uncovered constitutive and tissue-specific RBPs essential for normal development and, more importantly, revealed RBPs crucial for salinity stress responses from a RBP-RNA dynamics perspective. Remarkably, 40% of the RBPs are non-polyA RBPs that were not previously annotated as RBPs, signifying the advantage of PPE in unbiasedly retrieving RBPs. We propose that intrinsically disordered regions contribute to their non-classical binding and provide evidence that enzymatic domains from metabolic enzymes have additional roles in RNA binding. Taken together, our findings demonstrate that PPE is an impactful approach for identifying RBPs from complex plant tissues and pave the way for investigating RBP functions under different physiological and stress conditions at the posttranscriptional level.

Tryptophan Metabolism in Central Nervous System Diseases: Pathophysiology and Potential Therapeutic Strategies.

The metabolism of L-tryptophan (TRP) regulates homeostasis, immunity, and neuronal function. Altered TRP metabolism has been implicated in the pathophysiology of various diseases of the central nervous system. TRP is metabolized through two main pathways, the kynurenine pathway and the methoxyindole pathway. First, TRP is metabolized to kynurenine, then kynurenic acid, quinolinic acid, anthranilic acid, 3-hydroxykynurenine, and finally 3-hydroxyanthranilic acid along the kynurenine pathway. Second, TRP is metabolized to serotonin and melatonin along the methoxyindole pathway. In this review, we summarize the biological properties of key metabolites and their pathogenic functions in 12 disorders of the central nervous system: schizophrenia, bipolar disorder, major depressive disorder, spinal cord injury, traumatic brain injury, ischemic stroke, intracerebral hemorrhage, multiple sclerosis, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease. Furthermore, we summarize preclinical and clinical studies, mainly since 2015, that investigated the metabolic pathway of TRP, focusing on changes in biomarkers of these neurologic disorders, their pathogenic implications, and potential therapeutic strategies targeting this metabolic pathway. This critical, comprehensive, and up-to-date review helps identify promising directions for future preclinical, clinical, and translational research on neuropsychiatric disorders.

Maternal exposure to dibutyl phthalate regulates MSH6 crotonylation to impair homologous recombination in fetal oocytes.

Homologous recombination (HR) during early oogenesis repairs programmed double-strand breaks (DSBs) to ensure female fertility and offspring health. The exposure of fetal ovaries to endocrine disrupting chemicals (EDCs) can cause reproductive disorders in the adulthood. The EDC dibutyl phthalate (DBP) is widely distributed in flexible plastic products, leading to ubiquitous human exposure. Here, we report that maternal exposure to DBP caused gross aberrations in meiotic prophase I of fetal oocytes, including delayed progression, impaired DNA damage response, uncoupled localization of DMC1 and RAD51, and decreased HR. However, programmed DSBs were efficiently repaired. DBP exposure negatively regulated lysine crotonylation (Kcr) of MSH6. Similar meiotic defects were observed in fetal ovaries with targeted disruption of Msh6, and mutation of K544cr of MSH6 impaired its association with Ku70, thereby promoting non-homologous end joining (NHEJ) and inhibiting HR. Unlike mature F1 females, F2 female mice exhibited premature follicular activation, precocious puberty, and anxiety-like behaviors. Therefore, DBP can influence early meiotic events, and Kcr of MSH6 may regulate preferential induction of HR or NHEJ for DNA repair during meiosis.

Study on the mechanism of various exogenous proteins with different inhibitions on wheat starch digestion: From the distribution behaviors of protein in the starch matrix.

This study aimed to compare the effect of various exogenous proteins on wheat starch (WS) digestion and assess the relevant mechanisms based on the distribution behaviors of exogenous proteins in the starch matrix. Rice protein (RP), soy protein isolate (SPI), and whey protein isolate (WPI) all effectively suppressed the rapid digestion of WS but with different modes. RP increased the slowly digestible starch content, while SPI and WPI increased the resistant starch content. Fluorescence images showed that RP aggregated and competed for effective space with starch granules, while SPI and WPI formed continuous network structures among the starch matrix. These distribution behaviors endowed different reductions in starch digestion by influencing the gelatinization and ordered structure of starch. Pasting and water mobility results suggested all exogenous proteins inhibited the water migration and swelling of starch. Simultaneously, X-ray diffraction and Fourier transform infrared spectroscopy analysis showed that exogenous proteins improved the ordered structures of starch. RP had a more significant effect on the long-term ordered structure, while SPI and WPI had a more effective effect on the short-term ordered structure. These findings will enrich the theory of exogenous protein inhibiting starch digestion and inspire the applications in low-glycemic index food.

Decidual derived exosomal miR-99a-5p targets Ppp2r5a to inhibit trophoblast invasion in response to CeO2NPs exposure.

BACKGROUND: The biological effects of cerium dioxide nanoparticles (CeO2NPs), a novel material in the biomedical field, have attracted widespread attention. Our previous study confirmed that exposure to CeO2NPs during pregnancy led to abnormal trophoblast invasion during early placental development, thereby impairing placental development. The potential mechanisms may be related to low-quality decidualization triggered by CeO2NPs exposure, such as an imbalance in trophoblast invasion regulators secreted by decidual cells. However, the intermediate link mediating the "dialogue" between decidual cells and trophoblasts during this process remains unclear. As an important connection between cells, exosomes participate in the "dialogue" between endometrial cells and trophoblasts. Exosomes transfer bioactive microRNA into target cells, which can target and regulate the level of mRNA in target cells. RESULTS: Here, we constructed a mice primary uterine stromal cell-induced decidualization model in vitro, and detected the effect of CeO2NPs exposure on the expression of decidual-derived exosomal miRNAs by high-throughput sequencing. Bioinformatics analysis and dual-luciferase reporter assays were performed to identify target genes of the screened key miRNAs in regulating trophoblast invasion. Finally, the role of the screened miRNAs and their target genes in regulating trophoblast (HTR-8/SVneo cells) invasion was confirmed. The results showed that CeO2NPs exposure inhibited trophoblast invasion by promoting miR-99a-5p expression in decidual-derived exosomes, and Ppp2r5a is a potential target gene for miR-99a-5p to inhibit trophoblast invasion. CONCLUSIONS: This study revealed the molecular mechanism by which CeO2NPs exposure inhibits trophoblast invasion from the perspective of decidual derived exosomal miRNAs. These results will provide an experimental basis for screening potential therapeutic targets for the negative biological effects of CeO2NPs exposure and new ideas for studying the mechanism of damage to trophoblast cells at the decidual-foetal interface by harmful environmental or occupational factors.

Arabidopsis HOT3/eIF5B1 constrains rRNA RNAi by facilitating 18S rRNA maturation.

Ribosome biogenesis is essential for protein synthesis in gene expression. Yeast eIF5B has been shown biochemically to facilitate 18S ribosomal RNA (rRNA) 3' end maturation during late-stage 40S ribosomal subunit assembly and gate the transition from translation initiation to elongation. But the genome-wide effects of eIF5B have not been studied at the single-nucleotide resolution in any organism, and 18S rRNA 3' end maturation is poorly understood in plants. Arabidopsis HOT3/eIF5B1 was found to promote development and heat stress acclimation by translational regulation, but its molecular function remained unknown. Here, we show that HOT3 is a late-stage ribosome biogenesis factor that facilitates 18S rRNA 3' end processing and is a translation initiation factor that globally impacts the transition from initiation to elongation. By developing and implementing 18S-ENDseq, we revealed previously unknown events in 18S rRNA 3' end maturation or metabolism. We quantitatively defined processing hotspots and identified adenylation as the prevalent nontemplated RNA addition at the 3' ends of pre-18S rRNAs. Aberrant 18S rRNA maturation in hot3 further activated RNA interference to generate RDR1- and DCL2/4-dependent risiRNAs mainly from a 3' portion of 18S rRNA. We further showed that risiRNAs in hot3 were predominantly localized in ribosome-free fractions and were not responsible for the 18S rRNA maturation or translation initiation defects in hot3. Our study uncovered the molecular function of HOT3/eIF5B1 in 18S rRNA maturation at the late 40S assembly stage and revealed the regulatory crosstalk among ribosome biogenesis, messenger RNA (mRNA) translation initiation, and siRNA biogenesis in plants.

The N-terminal extension of Arabidopsis ARGONAUTE 1 is essential for microRNA activities.

microRNAs (miRNAs) regulate target gene expression through their ARGONAUTE (AGO) effector protein, mainly AGO1 in Arabidopsis thaliana. In addition to the highly conserved N, PAZ, MID and PIWI domains with known roles in RNA silencing, AGO1 contains a long, unstructured N-terminal extension (NTE) of little-known function. Here, we show that the NTE is indispensable for the functions of Arabidopsis AGO1, as a lack of the NTE leads to seedling lethality. Within the NTE, the region containing amino acids (a.a.) 91 to 189 is essential for rescuing an ago1 null mutant. Through global analyses of small RNAs, AGO1-associated small RNAs, and miRNA target gene expression, we show that the region containing a.a. 91-189 is required for the loading of miRNAs into AGO1. Moreover, we show that reduced nuclear partitioning of AGO1 did not affect its profiles of miRNA and ta-siRNA association. Furthermore, we show that the 1-to-90a.a. and 91-to-189a.a. regions of the NTE redundantly promote the activities of AGO1 in the biogenesis of trans-acting siRNAs. Together, we report novel roles of the NTE of Arabidopsis AGO1.

MicroRNA156 conditions auxin sensitivity to enable growth plasticity in response to environmental changes in Arabidopsis.

MicroRNAs (miRNAs) play diverse roles in plant development, but whether and how miRNAs participate in thermomorphogenesis remain ambiguous. Here we show that HYPONASTIC LEAVES 1 (HYL1)-a key component of miRNA biogenesis-acts downstream of the thermal regulator PHYTOCHROME INTERACTING FACTOR 4 in the temperature-dependent plasticity of hypocotyl growth in Arabidopsis. A hyl1-2 suppressor screen identified a dominant dicer-like1 allele that rescues hyl1-2's defects in miRNA biogenesis and thermoresponsive hypocotyl elongation. Genome-wide miRNA and transcriptome analysis revealed microRNA156 (miR156) and its target SQUAMOSA PROMOTER-BINDING-PROTEIN-LIKE 9 (SPL9) to be critical regulators of thermomorphogenesis. Surprisingly, perturbation of the miR156/SPL9 module disengages seedling responsiveness to warm temperatures by impeding auxin sensitivity. Moreover, miR156-dependent auxin sensitivity also operates in the shade avoidance response at lower temperatures. Thus, these results unveil the miR156/SPL9 module as a previously uncharacterized genetic circuit that enables plant growth plasticity in response to environmental temperature and light changes.

Alternations in the multilevel structures of chickpea protein during fermentation and their relationship with digestibility.

This study investigated the effects of fermentation on in vitro protein digestibility of chickpeas and their relationship with the variations of multilevel structures of chickpea protein. The results showed that lactobacillus fermentation not only increased the solubility of chickpea protein but also enhanced the hydrolysis of protein during gastric and intestinal digestion by altering the multilevel structures of chickpea protein. The degree of hydrolysis, free amino acid content, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that macromolecule chickpea protein was hydrolyzed during fermentation. Raman and UV spectroscopy scans indicated that the α-helix content increased while the content of ß-sheet in chickpea protein dropped significantly after fermentation. As for fermented chickpea protein, the aromatic acid residues were gradually more exposed than the unfermented chickpea protein, and the intramolecular disulfide bond was generally converted to the intermolecular form. Our findings showed that fermentation changed the multilevel structures of chickpea protein, degrading spherical structures into looser states that were more responsible for their effective hydrolysis during digestion. Furthermore, better digestibility of chickpea protein would stimulate the use of chickpea fermentation in food products.

Single-cell transcriptome analysis and in vitro differentiation of testicular cells reveal novel insights into male sterility of the interspecific hybrid cattle-yak.

BACKGROUND: Interspecific hybridization plays vital roles in enriching animal diversity, while male hybrid sterility (MHS) of the offspring commonly suffered from spermatogenic arrest constitutes the postzygotic reproductive isolation. Cattle-yak, the hybrid offspring of cattle (Bos taurus) and yak (Bos grunniens) can serve as an ideal MHS animal model. Although meiotic arrest was found to contribute to MHS of cattle-yak, yet the cellular characteristics and developmental potentials of male germline cell in pubertal cattle-yak remain to be systematically investigated. RESULTS: Single-cell RNA-seq analysis of germline and niche cell types in pubertal testis of cattle-yak and yak indicated that dynamic gene expression of developmental germ cells was terminated at late primary spermatocyte (meiotic arrest) and abnormal components of niche cell in pubertal cattle-yak. Further in vitro proliferation and differentially expressed gene (DEG) analysis of specific type of cells revealed that undifferentiated spermatogonia of cattle-yak exhibited defects in viability and proliferation/differentiation potentials. CONCLUSION: Comparative scRNA-seq and in vitro proliferation analysis of testicular cells indicated that not only meiotic arrest contributed to MHS of cattle-yak. Spermatogenic arrest of cattle-yak may originate from the differentiation stage of undifferentiated spermatogonia and niche cells of cattle-yak may provide an adverse microenvironment for spermatogenesis.

Effects of low-dose dexamethasone on inflammatory factors in drainage fluid and wound healing after thyroid surgery during perioperative period.

This study explored the effect of perioperative use of low-dose dexamethasone on inflammatory factors in drainage fluid and wound healing after thyroid surgery. In the prospective, double-blinded, randomised controlled study, adults who underwent elective thyroidectomy received 0.1 mg/kg of intravenous dexamethasone or a matching volume of placebo (saline) after induction of general anaesthesia. The primary outcome was IL6 and IL10 concentration in drainage at 24 hours postoperative. The secondary endpoint was the SBSES (modified Stony Brook Scar Evaluation Scale) total score at 1 week postoperative. From 8 July to 17 December 2020, 64 patients (mean [SD] age, 40.42 [9.52]; 13 males [20.31%]) were recruited, received operation, and completed the 1-month follow-up. Inflammatory factors in drainage did not differ between the two groups but only had significant differences at different timepoint. The dexamethasone group patients had a higher SBSES total score at 1 week after the treatment but, without statistical significance (dexamethasone vs placebo: 3.13 ± 1.24 vs 2.97 ± 0.93, P = .571). The dexamethasone group patients had a higher SBSES total score (dexamethasone vs placebo: 3.103 ± 1.148 vs 2.868 ± 0.827, P = .011) and colour score (dexamethasone vs placebo: 0.603 ± 0.493 vs 0.412 ± 0.496, P = .026) at 1-week follow-up than the placebo group patients. Preoperative single small-dose intravenous dexamethasone did not show to improve wound healing quality nor reduce incision inflammation but may release pain, and reduce tissue angiogenesis, and thus the scar redness.

INDETERMINATE1 autonomously regulates phosphate homeostasis upstream of the miR399-ZmPHO2 signaling module in maize.

The macronutrient phosphorus is essential for plant growth and development. Plants have evolved multiple strategies to increase the efficiency of phosphate (Pi) acquisition to protect themselves from Pi starvation. However, the crosstalk between Pi homeostasis and plant development remains to be explored. Here, we report that overexpressing microRNA399 (miR399) in maize (Zea mays) is associated with premature senescence after pollination. Knockout of ZmPHO2 (Phosphate 2), a miR399 target, resulted in a similar premature senescence phenotype. Strikingly, we discovered that INDETERMINATE1 (ID1), a floral transition regulator, inhibits the transcription of ZmMIR399 genes by directly binding to their promoters, alleviating the repression of ZmPHO2 by miR399 and ultimately contributing to the maintenance of Pi homeostasis in maize. Unlike ZmMIR399 genes, whose expression is induced by Pi deficiency, ID1 expression was independent of the external inorganic orthophosphate status, indicating that ID1 is an autonomous regulator of Pi homeostasis. Furthermore, we show that ZmPHO2 was under selection during maize domestication and cultivation, resulting in a more sensitive response to Pi starvation in temperate maize than in tropical maize. Our study reveals a direct functional link between Pi-deprivation sensing by the miR399-ZmPHO2 regulatory module and plant developmental regulation by ID1.

Efficacy and safety assessment of traditional Chinese medicine for erectile dysfunction: A meta-analysis and trial sequential analysis.

BACKGROUND: Several patients with erectile dysfunction do not accept or benefit from conventional therapy with phosphodiesterase type 5 inhibitors; thus, alternative and complementary therapies are in need. Traditional Chinese medicine has been treating erectile dysfunction in China, but its clinical value is inconclusive. OBJECTIVE: To systematically evaluate the efficacy and safety of traditional Chinese medicine in treating erectile dysfunction. METHODS: Randomized controlled trials were retrieved from a comprehensive search in the literature published in the past decade from the Web of Science, PubMed, Embase, Cochrane Library, SinoMed, China National Knowledge Internet, WanFang, and VIP. We performed a meta-analysis of the International Index of Erectile Function 5 questionnaire scores, clinical recovery rates, and testosterone levels using Review Manager 5.4 software. The trial sequential analysis was conducted to check the results. RESULTS: A total of 45 trials with 5016 patients were included. Meta-analysis results showed that traditional Chinese medicine effectively improved the International Index of Erectile Function 5 questionnaire scores (weighted mean difference = 3.78, 95% confidence interval: 3.12, 4.44; p < 0.001), clinical recovery rates (risk ratio = 1.57, 95% confidence interval: 1.38, 1.79; p < 0.001), testosterone levels (weighted mean difference = 2.42, 95% confidence interval: 1.59, 3.25; p < 0.001) compared with the controls. The single and add-on applications of traditional Chinese medicine could improve the International Index of Erectile Function 5 questionnaire score (p < 0.001). The trial sequential analysis confirmed the robustness of the analysis of the International Index of Erectile Function 5 questionnaire scores. A significant difference in the incidence of adverse effects between the treatment and control groups was not observed (risk ratio = 0.82, 95% confidence interval: 0.65, 1.05; p = 0.12). CONCLUSION: Traditional Chinese medicine can gain better responses in improving the International Index of Erectile Function 5 questionnaire scores, clinical recovery rates, and testosterone levels as an alternative and complementary treatment, with no increase in side effects. However, more standardized, long-term, traditional Chinese medicine and integrative therapy clinical trials are needed to support the clinical application of traditional Chinese medicine.

Role of phenolic acids with different functional groups in the regulation of starch digestion in simulated dietary intake patterns.

This study investigated the effects of phenolic acids with different functional groups (cinnamic acid: CIA, caffeic acid: CA, ferulic acid: FA) on corn starch (CS) digestibility by simulating dietary intake patterns (co-heating and non-co-heating) and their mechanism. Both treatments could reduce the digestibility of CS. Compared to the non-co-heating treatment, the resistant starch content of 10 % CA co-heating samples increased by 8.36 %. The co-heating case led to a decrease in the trough viscosity, peak viscosity, and final viscosity of CS. Phenolic acids reduced the short-range order of CS, which was due to the interaction through hydrogen bonding by co-heating. The contribution was most pronounced for CA which contained more hydroxyl groups on the benzene ring. Quartz Crystal microbalance tests further confirmed that different absorption of phenolic acids to CS was caused by their hydroxyl groups on the benzene ring. These results demonstrated that the functional groups of phenolic acids were a controllable factor in inhibiting starch digestion, and co-heating could be considered a promising method to control starch digestion and an advocating way to ingest phenolic supplements.

Dysregulated glycolysis underpins high-fat-associated endometrial decidualization impairment during early pregnancy in mice.

Pregnancy complications are more likely to occur in obese women because of defective decidualization. However, the specific mechanism of glycolysis in decidual modulation associated with obesity remains unknown. Therefore, we explored the role of glycolysis in the endometrium of obese pregnant mice during decidualization. C57BL/6J mice were fed a high-fat diet (HFD) to induce obesity. All obesity related parameters were significantly higher in the HFD mice than control. Furthermore, the HFD mice had fewer implantation sites, a smaller decidual area growth, and decreased decidualization marker protein expression than control. The HFD mice also had significantly decreased lactate production and glycolytic enzyme expression. To confirm the functional role of glycolysis during the decidual period in obese pregnant mice, we extracted endometrial stromal cells (ESCs) and treated them with oleic acid (OA) and palmitic acid (PA) to mimic a high-fat environment. Decidualization and glycolysis were significantly restricted in the OA-and PA-treated groups. Moreover, we administered a glycolytic inhibitor, 2-DG, and an agonist, pioglitazone. 2-DG treatment considerably decreased the cells' glycolysis and decidualization. However, pioglitazone treatment improved glycolysis and alleviated defective decidualization. In conclusion, obesity-induced endometrial glycolysis modifications and key glycolytic enzyme downregulation during early pregnancy might cause abnormal decidualization, leading to an unsustainable pregnancy.

Proliferative exhausted CD8+ T cells exacerbate long-lasting anti-tumor effects in human papillomavirus-positive head and neck squamous cell carcinoma.

The survival prognosis of human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC) is largely different, and little is known about the anti-tumor mechanism of tumor-infiltrated exhausted CD8+ T cells (Tex) in HNSCC. We performed cell-level multi-omics sequencing on human HNSCC samples to decipher the multi-dimensional characteristics of Tex cells. A proliferative exhausted CD8+ T cell cluster (P-Tex) which was beneficial to survival outcomes of patients with HPV-positive HNSCC was identified. Interestingly, P-Tex cells expressed CDK4 genes as high as cancer cells, which could be simultaneously inhibited by CDK4 inhibitors and might be a potential reason for the ineffectiveness of CDK4 inhibitors in treating HPV-positive HNSCC. P-Tex cells could aggregate in the antigen-presenting cell niches and activate certain signaling pathways. Together, our findings suggest a promising role for P-Tex cells in the prognosis of patients with HPV-positive HNSCC by providing modest but persistent anti-tumor effects.

The impact of enhancing publicity and commemoration of body donors at Zhengzhou University, China.

As a fundamental subject of medical education, human anatomy plays a critical role in the development of medical science. However, because of multiple factors including cultural conservativism and limited social understanding, China is facing a particularly severe shortage of bodies donated for anatomy education. Zhengzhou University (ZZU) has continued to uphold whole-body dissection as the preferred method for medical students to learn anatomy. For this study, records of registered individuals (who have signed a body donation agreement) and donors (whose bodies have been received) from 2001 to 2020 were collected and analyzed. The aim of this study was to explore the factors influencing the body donation program (BDP) at ZZU, and then reinforce the social understanding for the BDP. The results showed a significant increase in the numbers of both registered individuals and donors since 2015, which is the year the publicity and commemoration in honor of donors were increased. There were no significant differences between the biological male and female sexes in the registered individuals, but the number of male donors (12.85 ± 10.86, per year) was significantly higher than that of female donors (4.75 ± 4.53, per year). The current donor profile at ZZU is male in his 60/70s, while the profile of registered individuals is male or female in their 60s. Strengthening the publicity and commemoration in honor of donors may contribute to the implementation of BDPs.

An enriched environment improves long-term functional outcomes in mice after intracerebral hemorrhage by mechanisms that involve the Nrf2/BDNF/glutaminase pathway.

Post-stroke depression exacerbates neurologic deficits and quality of life. Depression after ischemic stroke is known to some extent. However, depression after intracerebral hemorrhage (ICH) is relatively unknown. Increasing evidence shows that exposure to an enriched environment (EE) after cerebral ischemia/reperfusion injury has neuroprotective effects in animal models, but its impact after ICH is unknown. In this study, we investigated the effect of EE on long-term functional outcomes in mice subjected to collagenase-induced striatal ICH. Mice were subjected to ICH with the standard environment (SE) or ICH with EE for 6 h/day (8:00 am-2:00 pm). Depressive, anxiety-like behaviors and cognitive tests were evaluated on day 28 with the sucrose preference test, tail suspension test, forced swim test, light-dark transition experiment, morris water maze, and novel object recognition test. Exposure to EE improved neurologic function, attenuated depressive and anxiety-like behaviors, and promoted spatial learning and memory. These changes were associated with increased expression of transcription factor Nrf2 and brain-derived neurotrophic factor (BDNF) and inhibited glutaminase activity in the perihematomal tissue. However, EE did not change the above behavioral outcomes in Nrf2-/- mice on day 28. Furthermore, exposure to EE did not increase BDNF expression compared to exposure to SE in Nrf2-/- mice on day 28 after ICH. These findings indicate that EE improves long-term outcomes in sensorimotor, emotional, and cognitive behavior after ICH and that the underlying mechanism involves the Nrf2/BDNF/glutaminase pathway.