Cancer SLC6A6-mediated taurine uptake transactivates immune checkpoint genes and induces exhaustion in CD8+ T cells.

Taurine is used to bolster immunity, but its effects on antitumor immunity are unclear. Here, we report that cancer-related taurine consumption causes T cell exhaustion and tumor progression. The taurine transporter SLC6A6 is correlated with aggressiveness and poor outcomes in multiple cancers. SLC6A6-mediated taurine uptake promotes the malignant behaviors of tumor cells but also increases the survival and effector function of CD8+ T cells. Tumor cells outcompete CD8+ T cells for taurine by overexpressing SLC6A6, which induces T cell death and malfunction, thereby fueling tumor progression. Mechanistically, taurine deficiency in CD8+ T cells increases ER stress, promoting ATF4 transcription in a PERK-JAK1-STAT3 signaling-dependent manner. Increased ATF4 transactivates multiple immune checkpoint genes and induces T cell exhaustion. In gastric cancer, we identify a chemotherapy-induced SP1-SLC6A6 regulatory axis. Our findings suggest that tumoral-SLC6A6-mediated taurine deficiency promotes immune evasion and that taurine supplementation reinvigorates exhausted CD8+ T cells and increases the efficacy of cancer therapies.

PKM2 functions as a histidine kinase to phosphorylate PGAM1 and increase glycolysis shunts in cancer.

Phosphoglycerate mutase 1 (PGAM1) is a key node enzyme that diverts the metabolic reactions from glycolysis into its shunts to support macromolecule biosynthesis for rapid and sustainable cell proliferation. It is prevalent that PGAM1 activity is upregulated in various tumors; however, the underlying mechanism remains unclear. Here, we unveil that pyruvate kinase M2 (PKM2) moonlights as a histidine kinase in a phosphoenolpyruvate (PEP)-dependent manner to catalyze PGAM1 H11 phosphorylation, that is essential for PGAM1 activity. Moreover, monomeric and dimeric but not tetrameric PKM2 are efficient to phosphorylate and activate PGAM1. In response to epidermal growth factor signaling, Src-catalyzed PGAM1 Y119 phosphorylation is a prerequisite for PKM2 binding and the subsequent PGAM1 H11 phosphorylation, which constitutes a discrepancy between tumor and normal cells. A PGAM1-derived pY119-containing cell-permeable peptide or Y119 mutation disrupts the interaction of PGAM1 with PKM2 and PGAM1 H11 phosphorylation, dampening the glycolysis shunts and tumor growth. Together, these results identify a function of PKM2 as a histidine kinase, and illustrate the importance of enzyme crosstalk as a regulatory mode during metabolic reprogramming and tumorigenesis.

O-GlcNAcylation of ATP-citrate lyase couples glucose supply to lipogenesis for rapid tumor cell proliferation.

Elevated lipid synthesis is one of the best-characterized metabolic alterations in cancer and crucial for membrane expansion. As a key rate-limiting enzyme in de novo fatty acid synthesis, ATP-citrate lyase (ACLY) is frequently up-regulated in tumors and regulated by posttranslational modifications (PTMs). Despite emerging evidence showing O-GlcNAcylation on ACLY, its biological function still remains unknown. Here, we observed a significant upregulation of ACLY O-GlcNAcylation in various types of human tumor cells and tissues and identified S979 as a major O-GlcNAcylation site. Importantly, S979 O-GlcNAcylation is required for substrate CoA binding and crucial for ACLY enzymatic activity. Moreover, it is sensitive to glucose fluctuation and decisive for fatty acid synthesis as well as tumor cell proliferation. In response to EGF stimulation, both S979 O-GlcNAcylation and previously characterized S455 phosphorylation played indispensable role in the regulation of ACLY activity and cell proliferation; however, they functioned independently from each other. In vivo, streptozocin treatment- and EGFR overexpression-induced growth of xenograft tumors was mitigated once S979 was mutated. Collectively, this work helps comprehend how cells interrogate the nutrient enrichment for proliferation and suggests that although mammalian cell proliferation is controlled by mitogen signaling, the ancient nutrition-sensing mechanism is conserved and still efficacious in the cells of multicellular organisms.

Exercise-induced ß-hydroxybutyrate promotes Treg cell differentiation to ameliorate colitis in mice.

Increasing attention is being paid to the mechanistic investigation of exercise-associated chronic inflammatory disease improvement. Ulcerative colitis (UC) is one type of chronic inflammatory bowel disease with increasing incidence and prevalence worldwide. It is known that regular moderate aerobic exercise (RMAE) reduces the incidence or risk of UC, and attenuates disease progression in UC patients. However, the mechanisms of this RMAE's benefit are still under investigation. Here, we revealed that ß-hydroxybutyrate (ß-HB), a metabolite upon prolonged aerobic exercise, could contribute to RMAE preconditioning in retarding dextran sulfate sodium (DSS)-induced mouse colitis. When blocking ß-HB production, RMAE preconditioning-induced colitis amelioration was compromised, whereas supplementation of ß-HB significantly rescued impaired ß-HB production-associated defects. Meanwhile, we found that RMAE preconditioning significantly caused decreased colonic Th17/Treg ratio, which is considered to be important for colitis mitigation; and the downregulated Th17/Treg ratio was associated with ß-HB. We further demonstrated that ß-HB can directly promote the differentiation of Treg cell rather than inhibit Th17 cell generation. Furthermore, ß-HB increased forkhead box protein P3 (Foxp3) expression, the core transcriptional factor for Treg cell, by enhancing histone H3 acetylation in the promoter and conserved noncoding sequences of the Foxp3 locus. In addition, fatty acid oxidation, the key metabolic pathway required for Treg cell differentiation, was enhanced by ß-HB treatment. Lastly, administration of ß-HB without exercise significantly boosted colonic Treg cell and alleviated colitis in mice. Together, we unveiled a previously unappreciated role for exercise metabolite ß-HB in the promotion of Treg cell generation and RMAE preconditioning-associated colitis attenuation.

8-Oxoguanine DNA glycosylase 1 selectively modulates ROS-responsive NF-κB targets through recruitment of MSK1 and phosphorylation of RelA/p65 at Ser276.

Nuclear factor kappa B (NF-κB) activity is regulated by various posttranslational modifications, of which Ser276 phosphorylation of RelA/p65 is particularly impacted by reactive oxygen species (ROS). This modification is responsible for selective upregulation of a subset of NF-κB targets; however, the precise mechanism remains elusive. ROS have the ability to modify cellular molecules including DNA. One of the most common oxidation products is 8-oxo-7,8-dihydroguanine (8-oxoGua), which is repaired by the 8-oxoguanine DNA glycosylase1 (OGG1)-initiated base excision repair pathway. Recently, a new function of OGG1 has been uncovered. OGG1 binds to 8-oxoGua, facilitating the occupancy of NF-κB at promoters and enhancing transcription of pro-inflammatory cytokines and chemokines. In the present study, we demonstrated that an interaction between DNA-bound OGG1 and mitogen-and stress-activated kinase 1 is crucial for RelA/p65 Ser276 phosphorylation. ROS scavenging or OGG1 depletion/inhibition hindered the interaction between mitogen-and stress-activated kinase 1 and RelA/p65, thereby decreasing the level of phospho-Ser276 and leading to significantly lowered expression of ROS-responsive cytokine/chemokine genes, but not that of Nfkbis. Blockade of OGG1 binding to DNA also prevented promoter recruitment of RelA/p65, Pol II, and p-RNAP II in a gene-specific manner. Collectively, the data presented offer new insights into how ROS signaling dictates NF-κB phosphorylation codes and how the promoter-situated substrate-bound OGG1 is exploited by aerobic mammalian cells for timely transcriptional activation of ROS-responsive genes.

Methyl jasmonate-induced bHLH42 mediates tissue-specific accumulation of anthocyanins via regulating flavonoid metabolism-related pathways in Caitai.

Anthocyanin is a type of plant secondary metabolite beneficial to human health. The anthocyanin content of vegetable and fruit crops signifies their nutritional quality. However, the molecular mechanism of anthocyanin accumulation, especially tissue-specific accumulation, in Caitai, as well as in other Brassica rapa varieties, remains elusive. In the present study, taking advantage of three kinds of Caitai cultivars with diverse colour traits between leaves and stems, we conducted a comparative transcriptome analysis and identified the molecular pathway of anthocyanin biosynthesis in Caitai leaves and stems, respectively. Our further investigations demonstrate that bHLH42, which is robustly induced by MeJA, closely correlates with tissue-specific accumulation of anthocyanins in Caitai; bHLH42 upregulates the expression of flavonoid/anthocyanin biosynthetic pathway genes to activate anthocyanin biosynthesis pathway, importantly, overexpression of bHLH42 significantly improves the anthocyanin content of Caitai. Our analysis convincingly suggests that bHLH42 induced by jasmonic acid signalling plays a crucial role in tissue-specific accumulation of anthocyanins in Caitai.

Butyrate enhances CPT1A activity to promote fatty acid oxidation and iTreg differentiation.

Inducible regulatory T (iTreg) cells play a crucial role in immune suppression and are important for the maintenance of immune homeostasis. Mounting evidence has demonstrated connections between iTreg differentiation and metabolic reprogramming, especially rewiring in fatty acid oxidation (FAO). Previous work showed that butyrate, a specific type of short-chain fatty acid (SCFA) readily produced from fiber-rich diets through microbial fermentation, was critical for the maintenance of intestinal homeostasis and capable of promoting iTreg generation by up-regulating histone acetylation for gene expression as an HDAC inhibitor. Here, we revealed that butyrate could also accelerate FAO to facilitate iTreg differentiation. Moreover, butyrate was converted, by acyl-CoA synthetase short-chain family member 2 (ACSS2), into butyryl-CoA (BCoA), which up-regulated CPT1A activity through antagonizing the association of malonyl-CoA (MCoA), the best known metabolic intermediate inhibiting CPT1A, to promote FAO and thereby iTreg differentiation. Mutation of CPT1A at Arg243, a reported amino acid required for MCoA association, impaired both MCoA and BCoA binding, indicating that Arg243 is probably the responsible site for MCoA and BCoA association. Furthermore, blocking BCoA formation by ACSS2 inhibitor compromised butyrate-mediated iTreg generation and mitigation of mouse colitis. Together, we unveil a previously unappreciated role for butyrate in iTreg differentiation and illustrate butyrate-BCoA-CPT1A axis for the regulation of immune homeostasis.

Ultrasensitive detection of H2O2 via electrochemical sensor by graphene synergized with MOF-on-MOF nanozymes.

An electrochemical sensor was developed for the detection of hydrogen peroxide (H2O2), utilizing the synergistic effects of graphene (Gr) and MOF-on-MOF nanozymes (FeCu-NZs). Initially, Fe-MOF with peroxide-like activity is synthesized using a solvothermal method. Subsequently, the organic ligand on its surface binds Cu2+, enhancing the enzyme-like activity further. The resulting FeCu-NZs exhibit a distinctive electrochemical signal in response to H2O2. Moreover, integrating FeCu-NZs with Gr significantly amplifies the electrochemical signal and effectively reduces the sensor's detection limit. The developed sensor exhibited linear ranges of 0.1-3800 µM, with a limit of detection (LOD) of 0.06 µM. Additionally, FeCu-NZs catalyze H2O2 to generate abundant •OH radicals, and colorimetric detection of H2O2 is facilitated using the color rendering principle of 3,3',5,5'-tetramethylbenzidine (TMB). Notably, this detection method was applied to determine  H2O2 concentrations in real samples, achieving a recovery exceeding 95.7%. In summary, this research provides a practical platform for the construction of traditional nanozymes and the integration of electrochemical systems, which have broad applications in food analysis, environmental monitoring, and medical diagnosis.

HDAC inhibitors as a potential therapy for chemotherapy-induced neuropathic pain.

Cancer, a chronic disease characterized by uncontrolled cell development, kills millions of people globally. The WHO reported over 10 million cancer deaths in 2020. Anticancer medications destroy healthy and malignant cells. Cancer treatment induces neuropathy. Anticancer drugs cause harm to spinal cord, brain, and peripheral nerve somatosensory neurons, causing chemotherapy-induced neuropathic pain. The chemotherapy-induced mechanisms underlying neuropathic pain are not fully understood. However, neuroinflammation has been identified as one of the various pathways associated with the onset of chemotherapy-induced neuropathic pain. The neuroinflammatory processes may exhibit varying characteristics based on the specific type of anticancer treatment delivered. Neuroinflammatory characteristics have been observed in the spinal cord, where microglia and astrocytes have a significant impact on the development of chemotherapy-induced peripheral neuropathy. The patient's quality of life might be affected by sensory deprivation, loss of consciousness, paralysis, and severe disability. High cancer rates and ineffective treatments are associated with this disease. Recently, histone deacetylases have become a novel treatment target for chemotherapy-induced neuropathic pain. Chemotherapy-induced neuropathic pain may be treated with histone deacetylase inhibitors. Histone deacetylase inhibitors may be a promising therapeutic treatment for chemotherapy-induced neuropathic pain. Common chemotherapeutic drugs, mechanisms, therapeutic treatments for neuropathic pain, and histone deacetylase and its inhibitors in chemotherapy-induced neuropathic pain are covered in this paper. We propose that histone deacetylase inhibitors may treat several aspects of chemotherapy-induced neuropathic pain, and identifying these inhibitors as potentially unique treatments is crucial to the development of various chemotherapeutic combination treatments.

Immobilized horseradish peroxidase on boric acid modified polyoxometalate molecularly imprinted polymer for biocatalytic degradation of phenol in wastewater: Optimized immobilization, degradation and toxicity assessment.

The degradation of phenol from wastewater is crucial for environmental protection. Biological enzymes, such as horseradish peroxidase (HRP), have shown great potential in the degradation of phenol. In this research, we prepared a hollow CuO/Cu2O octahedron adsorbent with a carambola matrix shape through the hydrothermal method. The surface of the adsorbent was modified by silane emulsion self-assembly, where 3-aminophenyl boric acid (APBA) and polyoxometalate (PW9) were combined with silanization reagents and grafted onto the surface. The adsorbent was then molecularly imprinted with dopamine to obtain boric acid modified polyoxometalate molecularly imprinted polymer (Cu@B@PW9@MIPs). This adsorbent was used to immobilize HRP, which served as a biological enzyme catalyst from horseradish. The adsorbent was characterized, and its synthetic conditions, experimental conditions, selectivity, reproducibility, and reusability were evaluated. The maximum adsorption amount of HRP under optimized conditions was 159.1 mg g-1, as determined using high-performance liquid chromatography (HPLC). At pH 7.0, the immobilized enzyme showed a high efficiency of up to 90.0% in removing phenol, after 20 min of reaction with 25 mmol L-1 H2O2 and 0.20 mg mL-1 Cu@B@PW9@HRP. Growth tests of aquatic plants confirmed that the adsorbent reduced harm. Gas chromatography-mass spectrometry (GC-MS) tests revealed that the degraded phenol solution contained about fifteen phenol derivatives intermediates. This adsorbent has the potential to become a promising biological enzyme catalyst for dephenolization.

Construction of the molecularly imprinted adsorbent based on shaddock peel biochar sphere for highly sensitive detection of ribavirin in food and water resources.

Ribavirin (RBV) that is not metabolically released into the environment can contaminate the environment and even make organisms resistant to it. Therefore, it is of great significance to establish a simple and effective method for adsorbing RBV in the environment. In this study, a novel biochar-based boronate affinity molecularly imprinted polymers (C@H@B-MIPs) were synthesized. This is the first time that shaddock peel biochar sphere was used as a carrier for specific recognition of RBV. The polymerization conditions were optimized and the binding properties of RBV were studied. Benefiting from the synergistic effect of boronate affinity and surface imprinting, the C@H@B-MIPs showed rapid equilibrium kinetics of 15 min, high adsorption capacity of 18.30 mg g-1, and excellent reusability for RBV. The linear range was 0.05-100 mg L-1, and the detection limit was 0.023 mg L-1. This method was triumphant applied to the selective adsorption of RBV in food and water resources with recovery rates of 81.4-97.7%. This study provides a practical platform for the manufacture of efficient biomass-based adsorbents.

LMP2-mRNA lipid nanoparticle sensitizes EBV-related tumors to anti-PD-1 therapy by reversing T cell exhaustion.

BACKGROUND: Targeting EBV-proteins with mRNA vaccines is a promising way to treat EBV-related tumors like nasopharyngeal carcinoma (NPC). We assume that it may sensitize tumors to immune checkpoint inhibitors. RESULTS: We developed an LMP2-mRNA lipid nanoparticle (C2@mLMP2) that can be delivered to tumor-draining lymph nodes. C2@mLMP2 exhibited high transfection efficiency and lysosomal escape ability and induced an increased proportion of CD8 + central memory T cells and CD8 + effective memory T cells in the spleen of the mice model. A strong synergistic anti-tumor effect of C2@mLMP2 in combination with αPD-1 was observed in tumor-bearing mice. The mechanism was identified to be associated with a reverse of CD8 + T cell exhaustion in the tumor microenvironment. The pathological analysis further proved the safety of the vaccine and the combined therapy. CONCLUSIONS: This is the first study proving the synergistic effect of the EBV-mRNA vaccine and PD-1 inhibitors for EBV-related tumors. This study provides theoretical evidence for further clinical trials that may expand the application scenario and efficacy of immunotherapy in NPC.

Open Tubular Capillary Electrochromatography-Mass Spectrometry for Analysis of Underivatized Amino Acid Enantiomers with a Porous Layer-Gold Nanoparticle-Modified Chiral Column.

By developing a novel chiral column, we integrate open tubular capillary electrochromatography into sheathless mass spectrometry (MS) for efficient analysis of underivatized amino acid enantiomers. The chiral column is easily fabricated by modifying the inner surface of a capillary with a three-dimensional porous layer (PL, thickness ∼ 90 nm, pore size ∼ 30 nm) and gold nanoparticles and by introducing a chiral selector, thiol ß-cyclodextrin (SH-ß-CD), onto the modified surface via Au-S bonds. This approach greatly enhances the specific surface area and thus the ratio of the stationary phase to mobile phase and interaction between the stationary phase and analytes. The proposed PLOT@Au@CD column is coupled to the sheathless CE-ESI-MS system for chiral analysis of amino acid enantiomers. No derivatization of amino acids is required for chiral analysis, and baseline separation of a total of 15 pairs of amino acid enantiomers is achieved within 17 min with high column efficiencies of 5.60 × 104 to 1.82 × 106 N/m, high resolutions of 1.51-10.0, and low limits of detection between 0.02 and 0.09 µg/mL. The separation efficiency and MS intensity are only slightly decreased over 60 runs or after usage for 15 days, showing excellent repeatability and stability of the PLOT@Au@CD column. The proposed method is successfully applied to the determination of amino acid enantiomers in vinegar samples with satisfactory accuracy. Our study provides a new approach for developing a chiral stationary phase in the chromatographic separation technique, which can be easily coupled to sensitive MS detection, thus it would be of value for various applications in the fields of chiral analysis.

Measuring healthy female nulliparous pubovisceral muscle from diffusion kurtosis imaging.

This study explores the feasibility of using diffusion kurtosis imaging (DKI) in the pelvic floor region and assesses the water diffusivity of the pubovisceral muscle. Twenty-seven healthy young nulliparous females underwent DKI at 3.0 T that included 15 gradient directions and three b values (0, 750, and 1500 s/mm2 ). The diffusion tensor and diffusion kurtosis metrics values of the pubovisceral muscle were measured after image processing. Two independent sample t-tests, a paired-samples t-test, and a nonparametric hypothesis test were performed as appropriate to compare the differences among different metrics. Twenty-six subjects (mean ± standard deviation age, 25 ± 2 years) were successfully analyzed by measuring the diffusion tensor and diffusion kurtosis metrics of the bilateral pubovisceral muscles. The metrics included mean kurtosis, axial kurtosis, radial kurtosis, fractional anisotropy, mean diffusivity, axial diffusivity, and radial diffusivity. We found no statistically significant differences for these measurement values between the left and right pubovisceral muscles (p = 0.271-0.931). However, radial kurtosis was greater than axial kurtosis in both pubovisceral muscles (p < 0.001) and axial diffusivity was lower than radial diffusivity in both pubovisceral muscles (p < 0.001). We deem the application of DKI technology to the pelvic floor region to be feasible.

Dual roles of ANGPTL4 in multiple inflammatory responses in stomatitis mice.

BACKGROUND: Stomatitis is inflammation of the oral mucosa. Angiopoietin-like protein 4 (ANGPTL4) has pleiotropic functions both anti-inflammatory and pro-inflammatory properties. In the present study, we tested whether there is a correlation between increased ANGPTL4 expression and inflammation in stomatitis mice and the mechanisms involved. METHODS AND RESULTS: In this study, the oral mucosa of mice was burned with 90% phenol and intraperitoneal injection of 5-fluorouracil to establish the model of stomatitis mice. The pathological changes of stomatitis mice were observed by H&E staining of paraffin section. The expressions of cytokines and ANGPTL4 were detected by fluorescence quantitative PCR, and the protein levels of ANGPTL4 were detected by western blot. Compared with control group, the oral mucosal structure of model mice was damaged. The expression of ANGPTL4 were significantly increased concomitantly with elevated production of anti-inflammatory cytokine (peroxisome proliferator-activated receptor alpha) and pro-inflammatory cytokines [nuclear transcription factor-kappa B, interleukin-6 (IL-6), IL-1ß, and tumor necrosis factor-α] in mice with stomatitis. CONCLUSIONS: This study suggests that ANGPTL4 may be a double-edged sword in multiple inflammatory responses in stomatitis mice.

Analgesic effect of nobiletin against neuropathic pain induced by the chronic constriction injury of the sciatic nerve in mice.

Neuropathic pain is chronic pain resulting from central or peripheral nerve damage that remains difficult to treat. Current evidence suggests that nobiletin, isolated from Citrus reticulata Blanco, possesses analgesic and neuroprotective effects. However, its effect on neuropathic pain has not been reported. This study evaluated the analgesic effect of nobiletin on neuropathic pain induced by chronic constriction injury (CCI) in mice. In vivo, mice were intragastrically administered with nobiletin (30, 60, 120 mg/kg) for eight consecutive days, respectively. Our study indicated that nobiletin ameliorated mechanical allodynia, cold allodynia and thermal hyperalgesia on CCI mice at doses that do not induce significant sedation. Moreover, nobiletin could ameliorate axonal and myelin injury of the sciatic nerve and further restore abnormal sciatic nerve electrical activity on CCI mice. In vitro studies indicated that nobiletin could suppress the proteins and mRNA expression of the IRF5/P2X4R/BDNF signalling pathway in fibronectin-induced BV2 cells. Overall, our results indicated that nobiletin might exert an analgesic effect on CCI-induced neuropathic pain in mice by inhibiting the IRF5/P2X4R/BDNF signalling pathway in spinal microglia. This study provided a novel potential therapeutic drug for neuropathic pain and new insights into the pharmacological action of nobiletin.

Metal oxide-based macroporous ordered double affinity molecularly imprinted polymer for specific separation and enrichment of glycoprotein from food samples: a co-modification of DMSA and boronate affinity.

Metal oxide-based macroporous ordered double affinity molecularly imprinted polymers (D-MIPs) were developed as solid phase extraction (SPE) adsorbents for the specific identification of ovalbumin (OVA) under physiological pH conditions prior to ultraviolet visible (UV-vis) spectrophotometric detection. Herein, macroporous alumina (MA) was used as a matrix; dimercaptosuccinic acid (DMSA) and 3-aminophenylboric acid (APBA) were employed as dual-functional monomers; APBA is a self-polymerizing monomer. The effects of synthesis conditions, SPE conditions as well as selectivity, reproducibility, and reusability were studied. The co-modification of DMSA and boronate affinity renders the adsorbent exhibiting a high adsorption capacity (114.4 mg g-1) and short equilibrium time (30 min). The surface imprinting technology causes the adsorbent to have high selectivity towards OVA. The OVA recovery range is 91.1-99.6%. This study provides a promising method for the enrichment of OVA and other cis-diol-containing analytes in complex biological samples. A novel metal oxide-based macroporous ordered nanoparticle with a combination of DMSA and boronate affinity was successfully prepared for specific separation and enrichment of glycoprotein from complex biological samples.

HBeAg mediates inflammatory functions of macrophages by TLR2 contributing to hepatic fibrosis.

BACKGROUND: We and others have confirmed activation of macrophages plays a critical role in liver injury and fibrogenesis during HBV infection. And we have also proved HBeAg can obviously induce the production of macrophage inflammatory cytokines compared with HBsAg and HBcAg. However, the receptor and functional domain of HBeAg in macrophage activation and its effects and mechanisms on hepatic fibrosis remain elusive. METHODS: The potentially direct binding receptors of HBeAg were screened and verified by Co-IP assay. Meanwhile, the function domain and accessible peptides of HBeAg for macrophage activation were analyzed by prediction of surface accessible peptide, construction, and synthesis of truncated fragments. Furthermore, effects and mechanisms of the activation of hepatic stellate cells induced by HBeAg-treated macrophages were investigated by Transwell, CCK-8, Gel contraction assay, Phospho Explorer antibody microarray, and Luminex assay. Finally, the effect of HBeAg in hepatic inflammation and fibrosis was evaluated in both human and murine tissues by immunohistochemistry, immunofluorescence, ELISA, and detection of liver enzymes. RESULTS: Herein, we verified TLR-2 was the direct binding receptor of HBeAg. Meanwhile, C-terminal peptide (122-143 aa.) of core domain in HBeAg was critical for macrophage activation. But arginine-rich domain of HBcAg hided this function, although HBcAg and HBeAg shared the same core domain. Furthermore, HBeAg promoted the proliferation, motility, and contraction of hepatic stellate cells (HSCs) in a macrophage-dependent manner, but not alone. PI3K-AKT-mTOR and p38 MAPK signaling pathway were responsible for motility phenotype of HSCs, while the Smad-dependent TGF-ß signaling pathway for proliferation and contraction of them. Additionally, multiple chemokines and cytokines, such as CCL2, CCL5, CXCL10, and TNF-α, might be key mediators of HSC activation. Consistently, HBeAg induced transient inflammation response and promoted early fibrogenesis via TLR-2 in mice. Finally, clinical investigations suggested that the level of HBeAg is associated with inflammation and fibrosis degrees in patients infected with HBV. CONCLUSIONS: HBeAg activated macrophages via the TLR-2/NF-κB signal pathway and further exacerbated hepatic fibrosis by facilitating motility, proliferation, and contraction of HSCs with the help of macrophages.

Mutations in ASH1L confer susceptibility to Tourette syndrome.

Tourette syndrome (TS) is a childhood-onset neuropsychiatric disorder characterized by repetitive motor movements and vocal tics. The clinical manifestations of TS are complex and often overlap with other neuropsychiatric disorders. TS is highly heritable; however, the underlying genetic basis and molecular and neuronal mechanisms of TS remain largely unknown. We performed whole-exome sequencing of a hundred trios (probands and their parents) with detailed records of their clinical presentations and identified a risk gene, ASH1L, that was both de novo mutated and associated with TS based on a transmission disequilibrium test. As a replication, we performed follow-up targeted sequencing of ASH1L in additional 524 unrelated TS samples and replicated the association (P value = 0.001). The point mutations in ASH1L cause defects in its enzymatic activity. Therefore, we established a transgenic mouse line and performed an array of anatomical, behavioral, and functional assays to investigate ASH1L function. The Ash1l+/- mice manifested tic-like behaviors and compulsive behaviors that could be rescued by the tic-relieving drug haloperidol. We also found that Ash1l disruption leads to hyper-activation and elevated dopamine-releasing events in the dorsal striatum, all of which could explain the neural mechanisms for the behavioral abnormalities in mice. Taken together, our results provide compelling evidence that ASH1L is a TS risk gene.

Screening and heterologous expression of flavone synthase and flavonol synthase to catalyze hesperetin to diosmetin.

OBJECTIVES: In this study, 44 flavone synthases (FNS) and flavonol synthases (FLS) from different origins were collected. The instability index and conserved domain of the enzymes were analyzed through bioinformatics analysis, the results of which allowed us to screen suitable enzymes for constructing recombinant Escherichia coli. Defective enzymes were selected as controls. RESULTS: Native- and sodium dodecyl sulfate-polyacrylamide gel electrophoresis were conducted to isolate the heterologously expressed proteins. Liquid chromatography-mass spectrometry, 1H nuclear magnetic resonance, and ultra-performance liquid chromatography were performed to qualitatively and quantitatively analyze the products. The cellular transformation results showed that recombinant E. coli catalyzed the synthesis of diosmetin from hesperetin, and in vitro catalysis showed that heterologously expressed FNS/FLS played a catalytic role in this reaction. AnFNS (from Angelica archangelica) showed the highest substrate conversion (38.80% for cellular transformation, 12.93% for in vitro catalysis). CONCLUSIONS: The catalytic capacity of FNS/FLS from different origins exhibited the expected results, indicating that bioinformatics analysis is useful for screening enzymes. In addition, the catalytic properties of AnFNS and CaFLS (from Camellia sinensis) differed significantly, although these enzymes are structurally similar. Based on this difference, C-2 was predicted as the key site for FNS/FLS catalytic synthesis of diosmetin rather than C-3.