Associations between whole grains intake and new-onset hypertension: a prospective cohort study.

IMPORTANCE: Epidemiological evidences regarding the association between whole grain intake and the risk of new-onset hypertension are still controversial. OBJECTIVE: We aimed to investigate the relationship between whole grain intake and new-onset hypertension and examine possible effect modifiers in the general population. METHODS: A total of 10,973 participants without hypertension from the China Health and Nutrition Survey were enrolled, with follow-up beginning in 1997 and ending in 2015. Whole grain intake was assessed by 3 consecutive 24-h dietary recalls combined with a household food inventory. Multivariable hazard ratios (HRs) and 95% confidence intervals (CIs) were estimated using Cox proportional hazards regression model after adjusting for potential risk factors. RESULTS: During a median follow-up of 7.0 years, 3,733 participants developed new-onset hypertension. The adjusted HRs (95% CIs) were as follows: for quartile 2 (HR: 0.52; 95% CI: 0.47-0.57), quartile 3 (HR: 0.46; 95% CI: 0.42-0.51), and quartile 4 (HR: 0.35; 95% CI: 0.31-0.38), compared with quartile 1. Different types of whole grain types, including wheat (adjusted HR, 0.35; 95% CI, 0.32-0.39), maize (adjusted HR, 0.50; 95% CI, 0.42-0.59), and millet (adjusted HR, 0.38; 95% CI, 0.30-0.48), showed significant associations with a reduced risk of hypertension. The association between whole grain intake and new-onset hypertension was stronger in individuals with older age (P for interaction < 0.001) and higher BMI (P for interaction < 0.001). CONCLUSION: Higher consumption of whole grains was significantly associated with a lower risk of new-onset hypertension. This study provides further evidence supporting the importance of increasing whole grain intake for hypertension prevention among Chinese adults.

Bevacizumab induces ferroptosis and enhances CD8+ T cell immune activity in liver cancer via modulating HAT1 and increasing IL-9.

Bevacizumab is a recombinant humanized monoclonal immunoglobulin (Ig) G1 antibody of VEGF, and inhibits angiogenesis and tumor growth in hepatocellular carcinoma (HCC). Ferroptosis, a new form of regulated cell death function independently of the apoptotic machinery, has been accepted as an attractive target for pharmacological intervention; the ferroptosis pathway can enhance cell immune activity of anti-PD1 immunotherapy in HCC. In this study we investigated whether and how bevacizumab regulated ferroptosis and immune activity in liver cancer. Firstly, we performed RNA-sequencing in bevacizumab-treated human liver cancer cell line HepG2 cells, and found that bevacizumab significantly altered the expression of a number of genes including VEGF, PI3K, HAT1, SLC7A11 and IL-9 in liver cancer, bevacizumab upregulated 37 ferroptosis-related drivers, and downregulated 17 ferroptosis-related suppressors in particular. We demonstrated that bevacizumab triggered ferroptosis in liver cancer cells by driving VEGF/PI3K/HAT1/SLC7A11 axis. Clinical data confirmed that the expression levels of VEGF were positively associated with those of PI3K, HAT1 and SLC7A11 in HCC tissues. Meanwhile, we found that bevacizumab enhanced immune cell activity in tumor immune-microenvironment. We identified that HAT1 up-regulated miR-143 targeting IL-9 mRNA 3'UTR in liver cancer cells; bevacizumab treatment resulted in the increase of IL-9 levels and its secretion via VEGF/PI3K/HAT1/miR-143/IL-9 axis, which led to the inhibition of tumor growth in vivo through increasing the release of IL-2 and Granzyme B from activated CD8+ T cells. We conclude that in addition to inhibiting angiogenesis, bevacizumab induces ferroptosis and enhances CD8+ T cell immune activity in liver cancer. This study provides new insight into the mechanisms by which bevacizumab synergistically modulates ferroptosis and CD8+ T cell immune activity in liver cancer.

Inhibition of USP7 enhances CD8+ T cell activity in liver cancer by suppressing PRDM1-mediated FGL1 upregulation.

Lymphocyte activation gene 3 (LAG3), an immune checkpoint molecule expressed on activated T cells, functions as a negative regulator of immune responses. Persistent antigen exposure in the tumor microenvironment results in sustained LAG3 expression on T cells, contributing to T cell dysfunction. Fibrinogen-like protein 1 (FGL1) has been identified as a major ligand of LAG3, and FGL1/LAG3 interaction forms a novel immune checkpoint pathway that results in tumor immune evasion. In addition, ubiquitin-specific peptidase 7 (USP7) plays a crucial role in cancer development. In this study we investigated the role of USP7 in modulation of FGL1-mediated liver cancer immune evasion. We showed that knockdown of USP7 or treatment with USP7 inhibitor P5091 suppressed liver cancer growth by promoting CD8+ T cell activity in Hepa1-6 xenograft mice and in HepG2 or Huh7 cells co-cultured with T cells, whereas USP7 overexpression produced the opposite effect. We found that USP7 upregulated FGL1 in HepG2 and Huh7 cells by deubiquitination of transcriptional factor PR domain zinc finger protein 1 (PRDM1), which transcriptionally activated FGL1, and attenuated the CD8+ T cell activity, leading to the liver cancer growth. Interestingly, USP7 could be transcriptionally stimulated by PRDM1 as well in a positive feedback loop. P5091, an inhibitor of USP7, was able to downregulate FGL1 expression, thus enhancing CD8+ T cell activity. In an immunocompetent liver cancer mouse model, the dual blockade of USP7 and LAG3 resulted in a superior antitumor activity compared with anti-LAG3 therapy alone. We conclude that USP7 diminishes CD8+ T cell activity by a USP7/PRDM1 positive feedback loop on FGL1 production in liver cancer; USP7 might be a promising target for liver cancer immunotherapy.

The catalytic mechanism of the mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2).

Methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) is a new drug target that is expressed in cancer cells but not in normal adult cells, which provides an Achilles heel to selectively kill cancer cells. Despite the availability of crystal structures of MTHFD2 in the inhibitor- and cofactor-bound forms, key information is missing due to technical limitations, including (a) the location of absolutely required Mg2+ ion, and (b) the substrate-bound form of MTHFD2. Using computational modeling and simulations, we propose that two magnesium ions are present at the active site whereby (i) Arg233, Asp225, and two water molecules coordinate [Formula: see text], while [Formula: see text] together with Arg233 stabilize the inorganic phosphate (Pi); (ii) Asp168 and three water molecules coordinate [Formula: see text], and [Formula: see text] further stabilizes Pi by forming a hydrogen bond with two oxygens of Pi; (iii) Arg201 directly coordinates the Pi; and (iv) through three water-mediated interactions, Asp168 contributes to the positioning and stabilization of [Formula: see text], [Formula: see text] and Pi. Our computational study at the empirical valence bond level allowed us also to elucidate the detailed reaction mechanisms. We found that the dehydrogenase activity features a proton-coupled electron transfer with charge redistribution connected to the reorganization of the surrounding water molecules which further facilitates the subsequent cyclohydrolase activity. The cyclohydrolase activity then drives the hydration of the imidazoline ring and the ring opening in a concerted way. Furthermore, we have uncovered that two key residues, Ser197/Arg233, are important factors in determining the cofactor (NADP+/NAD+) preference of the dehydrogenase activity. Our work sheds new light on the structural and kinetic framework of MTHFD2, which will be helpful to design small molecule inhibitors that can be used for cancer treatment.

Aspirin modulates succinylation of PGAM1K99 to restrict the glycolysis through NF-κB/HAT1/PGAM1 signaling in liver cancer.

Aspirin as a chemopreventive agent is able to restrict the tumor growth. Phosphoglycerate mutase 1 (PGAM1) is a key enzyme of glycolysis, playing an important role in the development of cancer. However, the underlying mechanism by which aspirin inhibits the proliferation of cancer cells is poorly understood. This study aims to identify the effects of aspirin on modulating PGAM1 enzymatic activities in liver cancer. Here, we found that aspirin attenuated the PGAM1 succinylation to suppress the PGAM1 enzymatic activities and glycolysis in hepatoma cells. Mechanically, aspirin remarkably reduced the global succinylation levels of hepatoma cells, including the PGAM1 succinylation, which led to the block of conversion from 3-phosphoglycerate (3-PG) to 2-phosphoglycerate (2-PG) in cells. Interestingly, RNA-seq analysis identified that aspirin could significantly decrease the levels of histone acetyltransferase 1 (HAT1), a writer of PGAM1 succinylation, in liver cancer. As a target of aspirin, NF-κB p65 could effectively up-regulate the expression of HAT1 in the system, resulting in the increase of PGAM1 enzymatic activities. Moreover, we observed that the PGAM1-K99R mutant failed to rescue the aspirin-induced inhibition of PGAM1 activities, glycolysis, and proliferation of hepatoma cells relative to PGAM1-WT. Functionally, aspirin down-regulated HAT1 and decreased the PGAM1 succinylation levels in the tumor tissues from mice treated with aspirin in vivo. Thus, we conclude that aspirin modulates PGAM1K99 succinylation to restrict the PGAM1 activities and glycolysis through NF-κB p65/HAT1/PGAM1 signaling in liver cancer. Our finding provides new insights into the mechanism by which aspirin inhibits glycolysis in hepatocellular carcinoma.

Aspirin triggers ferroptosis in hepatocellular carcinoma cells through restricting NF-κB p65-activated SLC7A11 transcription.

A number of studies have shown that aspirin, as commonly prescribed drug, prevents the development of hepatocellular carcinoma (HCC). Ferroptosis as a dynamic tumor suppressor plays a vital role in hepatocarcinogenesis. In this study we investigated whether aspirin affected ferroptosis in liver cancer cells. RNA-seq analysis revealed that aspirin up-regulated 4 ferroptosis-related drivers and down-regulated 5 ferroptosis-related suppressors in aspirin-treated HepG2 cells. Treatment with aspirin (4 mM) induced remarkable ferroptosis in HepG2 and Huh7 cells, which was enhanced by the ferroptosis inducer erastin (10 µM). We demonstrated that NF-κB p65 restricted ferroptosis in HepG2 and Huh7 cells through directly binding to the core region of SLC7A11 promoter and activating the transcription of ferroptosis inhibitor SLC7A11, whereas aspirin induced ferroptosis through inhibiting NF-κB p65-activated SLC7A11 transcription. Overexpression of p65 rescued HepG2 and Huh7 cells from aspirin-induced ferroptosis. HCC patients with high expression levels of SLC7A11 and p65 presented lower survival rate. Functionally, NF-κB p65 blocked the aspirin-induced ferroptosis in vitro and in vivo, which was attenuated by erastin. We conclude that aspirin triggers ferroptosis by restricting NF-κB-activated SLC7A11 transcription to suppress the growth of HCC. These results provide a new insight into the mechanism by which aspirin regulates ferroptosis in hepatocarcinogenesis. A combination of aspirin and ferroptosis inducer may provide a potential strategy for the treatment of HCC in clinic.

Heat shock protein family A member 8 serving as a co-activator of transcriptional factor ETV4 up-regulates PHLDA2 to promote the growth of liver cancer.

Heat shock protein family A member 8 (HSPA8) participates in the folding or degradation of misfolded proteins under stress and plays critical roles in cancer. In this study, we investigated the function of HSPA8 in the development of liver cancer. By analyzing the TCGA transcriptome dataset, we found that HSPA8 was upregulated in 134 clinical liver cancer tissue samples, and positively correlated with poor prognosis. IHC staining showed the nuclear and cytoplasmic localization of HSPA8 in liver cancer cells. Knockdown of HSPA8 resulted in a decrease in the proliferation of HepG2 and Huh-7 cells. ChIP-seq and RNA-seq analysis revealed that HSPA8 bound to the promoter of pleckstrin homology-like domain family A member 2 (PHLDA2) and regulated its expression. The transcription factor ETV4 in HepG2 cells activated PHLDA2 transcription. HSPA8 and ETV4 could interact with each other in the cells and colocalize in the nucleus. From a functional perspective, we demonstrated that HSPA8 upregulated PHDLA2 through the coactivating transcription factor ETV4 to enhance the growth of liver cancer in vitro and in vivo. From a therapeutic perspective, we identified both HSPA8 and PHDLA2 as novel targets in the treatment of HCC. In conclusion, this study demonstrates that HSPA8 serves as a coactivator of ETV4 and upregulates PHLDA2, leading to the growth of HCC, and is a potential therapeutic target in HCC treatment.

Liver-derived metabolites as signaling molecules in fatty liver disease.

Excessive fat accumulation in the liver has become a major health threat worldwide. Unresolved fat deposition in the liver can go undetected until it develops into fatty liver disease, followed by steatohepatitis, fibrosis, cirrhosis, and eventually hepatocellular carcinoma. Lipid deposition in the liver is governed by complex communication, primarily between metabolic organs. This can be mediated by hormones, organokines, and also, as has been more recently discovered, metabolites. Although how metabolites from peripheral organs affect the liver is well documented, the effect of metabolic players released from the liver during the development of fatty liver disease or associated comorbidities needs further attention. Here we focus on interorgan crosstalk based on metabolites released from the liver and how these molecules act as signaling molecules in peripheral tissues. Due to the liver's specific role, we are covering lipid and bile mechanism-derived metabolites. We also discuss the high sucrose intake associated with uric acid release from the liver. Excessive fat deposition in the liver during fatty liver disease development reflects disrupted metabolic processes. As a response, the liver secretes a variety of signaling molecules as well as metabolites which act as a footprint of the metabolic disruption. In the coming years, the reciprocal exchange of metabolites between the liver and other metabolic organs will gain further importance and will help to better understand the development of fatty liver disease and associated diseases.

Proof-of-Concept Method to Study Uncharacterized Methyltransferases Using PRDM15.

The PRDM family of methyltransferases has been implicated in cellular proliferation and differentiation and is deregulated in human diseases, most notably in cancer. PRDMs are related to the SET domain family of methyltransferases; however, from the 19 PRDMs only a few PRDMs with defined enzymatic activities are known. PRDM15 is an uncharacterized transcriptional regulator, with significant structural disorder and lack of defined small-molecule binding pockets. Many aspects of PRDM15 are yet unknown, including its structure, substrates, reaction mechanism, and its methylation profile. Here, we employ a series of computational approaches for an exploratory investigation of its potential substrates and reaction mechanism. Using the knowledge of PRDM9 and current knowledge of PRDM15 as basis, we tried to identify genuine substrates of PRDM15. We start from histone-based peptides and learn that the native substrates of PRDM15 may be non-histone proteins. In the future, a combination of sequence-based approaches and signature motif analysis may provide new leads. In summary, our results provide new information about the uncharacterized methyltransferase, PRDM15.

Histidine protonation states are key in the LigI catalytic reaction mechanism.

Lignin is one of the world's most abundant organic polymers, and 2-pyrone-4,6-dicarboxylate lactonase (LigI) catalyzes the hydrolysis of 2-pyrone-4,6-dicarboxylate (PDC) in the degradation of lignin. The pH has profound effects on enzyme catalysis and therefore we studied this in the context of LigI. We found that changes of the pH mostly affects surface residues, while the residues at the active site are more subject to changes of the surrounding microenvironment. In accordance with this, a high pH facilitates the deprotonation of the substrate. Detailed free energy calculations by the empirical valence bond (EVB) approach revealed that the overall hydrolysis reaction is more likely when the three active site histidines (His31, His33 and His180) are protonated at the ɛ site, however, protonation at the δ site may be favored during specific steps of the reaction. Our studies have uncovered the determinant role of the protonation state of the active site residues His31, His33 and His180 in the hydrolysis of PDC.

Na+ Sensitivity of the KAT2-Like Channel Is a Common Feature of Cucurbits and Depends on the S5-P-S6 Segment.

Inhibition of Shaker K+ channel activity by external Na+ was previously reported in the melon (Cucumis melo L.) inwardly rectifying K+ channel MIRK and was hypothesized to contribute to salt tolerance. In this study, two inward Shaker K+ channels, CsKAT2 from cucumber (Cucumis sativus) and ClKAT2 from watermelon (Citrullus lanatus), were identified and characterized in Xenopus oocytes. Both channels were inwardly rectifying K+ channels with higher permeability to potassium than other monovalent cations and more active when external pH was acidic. Similarly to MIRK, their activity displayed an inhibition by external Na+, thus suggesting a common feature in Cucurbitaceae (Cucumis spp., Citrullus spp.). CsKAT2 and ClKAT2 are highly expressed in guard cells. After 24 h of plant treatment with 100 mM NaCl, the three KAT2-like genes were significantly downregulated in leaves and guard cells. Reciprocal chimeras were obtained between MIRK and Na+-insensitive AtKAT2 cDNAs. The chimera where the MIRK S5-P-S6 segment was replaced by that from AtKAT2 no longer showed Na+ sensitivity, while the inverse chimera gained Na+ sensitivity. These results provide evidence that the molecular basis of the channel blockage by Na+ is located in the S5-P-S6 region. Comparison of the electrostatic property in the S5-P-S6 region in AtKAT2 and MIRK revealed four key amino acid residues potentially governing Na+ sensitivity.

PRMT5 confers lipid metabolism reprogramming, tumour growth and metastasis depending on the SIRT7-mediated desuccinylation of PRMT5 K387 in tumours.

The protein arginine methyltransferase 5 (PRMT5), which is highly expressed in tumour tissues, plays a crucial role in cancer development. However, the mechanism by which PRMT5 promotes cancer growth is poorly understood. Here, we report that PRMT5 contributes to lipid metabolism reprogramming, tumour growth and metastasis depending on the SIRT7-mediated desuccinylation of PRMT5 K387 in tumours. Mass spectrometric analysis identified PRMT5 lysine 387 as its succinylation site. Moreover, the desuccinylation of PRMT5 K387 enhances the methyltransferase activity of PRMT5. SIRT7 catalyses the desuccinylation of PRMT5 in cells. The SIRT7-mediated dessuccinylation of PRMT5 lysine 387 fails to bind to STUB1, decreasing PRMT5 ubiquitination and increasing the interaction between PRMT5 and Mep50, which promotes the formation of the PRMT5-Mep50 octamer. The PRMT5-Mep50 octamer increases PRMT5 methyltransferase activity, leading to arginine methylation of SREBP1a. The symmetric dimethylation of SREBP1a increases the levels of cholesterol, fatty acid, and triglyceride biogenesis in the cells, escaping degradation through the ubiquitin-proteasome pathway. Functionally, the desuccinylation of PRMT5 K387 promotes lipid metabolism reprogramming, tumour growth and metastasis in vitro and in vivo in tumours.

IFN-α inhibits HBV transcription and replication by promoting HDAC3-mediated de-2-hydroxyisobutyrylation of histone H4K8 on HBV cccDNA minichromosome in liver.

The epigenetic modification of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a crucial role in cccDNA transcription and viral persistence. Interferon-α (IFN-α) is a pivotal agent against HBV cccDNA. However, the mechanism by which IFN-α modulates the epigenetic regulation of cccDNA remains poorly understood. In this study, we report that IFN-α2b enhances the histone deacetylase 3 (HDAC3)-mediated de-2-hydroxyisobutyrylation of histone H4 lysine 8 (H4K8) on HBV cccDNA minichromosome to restrict the cccDNA transcription in liver. By screening acetyltransferases and deacetylases, we identified that HDAC3 was an effective restrictor of HBV transcription and replication. Moreover, we found that HDAC3 was able to mediate the de-2-hydroxyisobutyrylation of H4K8 in HBV-expressing hepatoma cells. Then, the 2-hydroxyisobutyrylation of histone H4K8 (H4K8hib) was identified on the HBV cccDNA minichromosome, promoting the HBV transcription and replication. The H4K8hib was regulated by HDAC3 depending on its deacetylase domain in the system. The low level of HDAC3 and high level of H4K8hib were observed in the liver tissues from HBV-infected human liver-chimeric mice. The levels of H4K8hib on HBV cccDNA minichromosome were significantly elevated in the liver biopsy specimens from clinical hepatitis B patients, which was consistent with the high transcriptional activity of cccDNA. Strikingly, IFN-α2b effectively facilitated the histone H4K8 de-2-hydroxyisobutyrylation mediated by HDAC3 on the HBV cccDNA minichromosome in primary human hepatocytes and hepatoma cells, leading to the inhibition of HBV transcription and replication. Our finding provides new insights into the mechanism by which IFN-α modulates the epigenetic regulation of HBV cccDNA minichromosome.

Does a MediDiet With Additional Extra Virgin Olive Oil and Pistachios Reduce the Incidence of Gestational Diabetes?

OBJECTIVE: The present study aimed to evaluate gestational diabetes mellitus (GDM) incidence in pregnant women following the Mediterranean diet (MedDiet) with the addition of extra virgin olive oil (EVOO) and pistachios. METHODS: A total of 560 pregnant patients were enrolled in the present study. The MedDiet was introduced in both the interventional group (IG) and the control group. The women in the IG received 40 mL of EVOO every day along with 25 to 30 g of roasted pistachios. The incidence of GDM was recorded along with specific maternal and neonatal outcomes. RESULTS: The nutritional scores and MedDiet adherence screener scores were not statistically different between the groups at baseline, but the difference was statistically significant and higher in the IG at 24 to 28 weeks (P = .001) and at 36 to 38 weeks (P = .001). GDM was diagnosed in 51 (20.4%) women in the control group and 34 (13.6%) women in the IG. The MedDiet significantly reduced GDM incidence (P = .02) after adjusting for confounding factors. CONCLUSION: The present study shows that dietary intervention in pregnant women, including a MedDiet and increased consumption of EVOO and pistachios, decreases the incidence of GDM.

Efficient Protein-Protein Couplings Mediated by Small Molecules under Mild Conditions.

Protein-protein coupling reactions under physiological conditions that do not impact the three-dimensional structures of the proteins are in high demand. Owing to the combination of phenylsulfonyl and aldehyde groups in 5-fluoro-4-(phenylsulfonyl)picolinaldehyde (FPPA), the fluorine substituent shows high reactivity toward free thiols. In FPPA, the fluorine is more reactive than phenylsulfonyl for free thiols. Thus the first quantitative nucleophilic substitution can be followed by selective substitution of phenylsulfonyl by an additional thiol or cyclization of aldehyde with a 1,2-aminothiol molecule. The FPPA mediated protein-protein coupling proceeds efficiently under mild conditions, resulting in stable protein conjugates. This coupling method has negligible 3D structural perturbations on the target proteins, and it produces overall intact, nearly traceless, and native structural folds of proteins. It is highly suitable for reconstruction of proteins that are difficult to make and segmental isotopic labeling of multidomain proteins.

Inhibition of SOCS6 confers radioresistance in esophageal squamous cell carcinoma.

Esophageal cancer is one of the most common cancer of the digestive system and radiotherapy is widely applied in advanced esophageal cancer treatment, however radioresistance (RR) is one of the major reasons for radiotherapy failure. There is limited knowledge on the mechanisms that cause RR, here we identify suppressors of cytokine signaling 6 (SOCS6) is a negative regulator of radioresistance in ESCC cells. SOCS6 deficiency in ESCC cells conferred radioresistance in vitro and in vivo by increasing radiation-induced G2/M arrest, DNA damage repair and inhibiting radiation-induced apoptosis. Moreover, the transcriptome sequencing analysis demonstrates that the transcription of SOCS6 was partially p53-dependent. Importantly we found that highly correlated SOCS6 and P53 express lower in RR esophageal cancer tissues compare with radiosensitive ones. Collectedly our study uncovers that SOCS6, as a downstream effector of p53, is a key regulator involved in the radioresistance of ESCC.

Aspirin modulates 2-hydroxyisobutyrylation of ENO1K281 to attenuate the glycolysis and proliferation of hepatoma cells.

Aspirin can efficiently inhibit the glycolysis and proliferation of cancer cells, however, the underlying mechanism is poorly understood. Here, we report that aspirin attenuates the glycolysis and proliferation of hepatoma cells through modulating the levels of lysine 2-hydroxyisobutyrylation (Khib) of enolase 1 (ENO1). We found that aspirin decreased the levels of glucose consumption and lactate production in hepatoma cells. Moreover, 4 mM aspirin reduced the activities of ENO1, a key enzyme of glycolysis, and decreased the levels of ENO1 Khib in the cells. Interestingly, we identified that 4 mM aspirin could decrease the levels of Khib on many proteins by using pan Khib antibody in the cells. Interestingly, the activities of ENO1 could be rescued by the transient overexpression of ENO1, but not by ENO1 mutant (K281R). Moreover, we identified that the C646, an inhibitor of p300 which is a writer of Khib, could reduce the levels of ENO1 Khib, resulting in the decrease of ENO1 activities. The treatment with PDTC, an inhibitor of NF-κB which is a target of aspirin, could work well as C646 in the cells. Both of aspirin and C646 (or PDTC) displayed a stronger effect than the single treatment in the system. Functionally, ENO1, but not ENO1 mutant (K281R), could rescue the aspirin-induced inhibition of proliferation of liver cancer cells in vitro, suggesting that ENO1K281 is involved in the aspirin-mediated inhibition of liver cancer. Our finding provides new insights into the mechanism by which aspirin attenuates the glycolysis and proliferation of hepatoma cells.

Virtual screening of potentially endocrine-disrupting chemicals against nuclear receptors and its application to identify PPARγ-bound fatty acids.

Nuclear receptors (NRs) are key regulators of energy homeostasis, body development, and sexual reproduction. Xenobiotics binding to NRs may disrupt natural hormonal systems and induce undesired adverse effects in the body. However, many chemicals of concerns have limited or no experimental data on their potential or lack-of-potential endocrine-disrupting effects. Here, we propose a virtual screening method based on molecular docking for predicting potential endocrine-disrupting chemicals (EDCs) that bind to NRs. For 12 NRs, we systematically analyzed how multiple crystal structures can be used to distinguish actives and inactives found in previous high-throughput experiments. Our method is based on (i) consensus docking scores from multiple structures at a single functional state (agonist-bound or antagonist-bound), (ii) multiple functional states (agonist-bound and antagonist-bound), and (iii) multiple pockets (orthosteric site and alternative sites) of these NRs. We found that the consensus enrichment from multiple structures is better than or comparable to the best enrichment from a single structure. The discriminating power of this consensus strategy was further enhanced by a chemical similarity-weighted scoring scheme, yielding better or comparable enrichment for all studied NRs. Applying this optimized method, we screened 252 fatty acids against peroxisome proliferator-activated receptor gamma (PPARγ) and successfully identified 3 previously unknown fatty acids with Kd = 100-250 µM including two furan fatty acids: furannonanoic acid (FNA) and furanundecanoic acid (FUA), and one cyclopropane fatty acid: phytomonic acid (PTA). These results suggested that the proposed method can be used to rapidly screen and prioritize potential EDCs for further experimental evaluations.

Exploring alternative catalytic mechanisms of the Cas9 HNH domain.

Understanding the reaction mechanism of CRISPR-associated protein 9 (Cas9) is crucial for the application of programmable gene editing. Despite the availability of the structures of Cas9 in apo- and substrate-bound forms, the catalytically active structure is still unclear. Our first attempt to explore the catalytic mechanism of Cas9 HNH domain has been based on the reasonable assumption that we are dealing with the same mechanism as endonuclease VII, including the assumption that the catalytic water is in the first shell of the Mg2+ . Trying this mechanism with the cryo-EM structure forced us to induce significant structural change driven by the movement of K848 (or other positively charged residue) close to the active site to facilitate the proton transfer step. In the present study, we explore a second reaction mechanism where the catalytic water is in the second shell of the Mg2+ and assume that the cryo-EM structure by itself is a suitable representation of a catalytic-ready structure. The alternative mechanism indicates that if the active water is from the second shell, then the calculated reaction barrier is lower compared with the corresponding barrier when the water comes from the first shell.

Identification of Serratia marcescens isolated from Antheraea pernyi eggs and determination of bacterial pathogenicity and transmission pathway.

The Chinese oak silkworm, Antheraea pernyi, is an important alternate host for mass production of Trichogramma parasitoids, which play a vital role in the biological control of lepidopterous pests in China. The quality of A. pernyi eggs is particularly important for mass production of these parasitoids. In this study, a pathogenic bacterium, isolated and purified from A. pernyi eggs that had turned gray in color, was identified as Serratia marcescens. We used morphology, biochemistry and 16S rDNA analysis to characterize the strain, which was named "APE strain". Serratia marcescens APE strain was determined to be the causal bacterium associated with the disease in the eggs, verified by a test based on Koch's Postulates. We tested the pathogenicity of S. marcescens APE strain on A. pernyi eggs; the percentage of diseased (gray) eggs reached 57.78% when uninfected eggs were exposed to a concentration of 1 × 109 cfu/mL bacterial suspension for 7 h. S. marcescens was transmitted mechanically by Trichogramma parasitoids. The transmission rate was 25.56%. In a horizontal transmission test, the highest percentage of uninfected eggs that developed infections was 51.43% after being treated with contents of diseased eggs for 12 h. In a vertical transmission test, the number of infected eggs per treated adult female was 63.8-92.3 after treatment with different S. marcescens concentrations, significantly lower than the 304.3 eggs per female in the control group. Furthermore, the percentage of infected (gray) eggs produced by adult moths exposed to bacteria in the larval stage was 80.5-85.3%.