Hotspot Cancer Mutation Impairs KAT8-mediated Nucleosomal Histone Acetylation.

KAT8 is an evolutionarily conserved lysine acetyltransferase that catalyzes histone acetylation at H4K16 or H4K5 and H4K8 through distinct protein complexes. It plays a pivotal role in male X chromosome dosage compensation in Drosophila and is implicated in the regulation of diverse cellular processes in mammals. Mutations and dysregulation of KAT8 have been reported in human neurodevelopmental disorders and various cancers. However, the precise mechanisms by which these mutations disrupt KAT8's normal function, leading to disease pathogenesis, remain largely unknown. In this study, we focus on a hotspot missense cancer mutation, the R98W point mutation within the Tudor-knot domain. Our study reveals that the R98W mutation leads to a reduction in global H4K16ac levels in cells and downregulates the expression of target genes. Mechanistically, we demonstrate that R98 is essential for KAT8-mediated acetylation of nucleosomal histones by modulating substrate accessibility.

TAZ2 truncation confers overactivation of p300 and cellular vulnerability to HDAC inhibition.

The histone acetyltransferase p300/CBP is composed of several conserved domains, among which, the TAZ2 domain is known as a protein-protein interaction domain that binds to E1A and various transcription factors. Here we show that TAZ2 has a HAT autoinhibitory function. Truncating p300/CBP at TAZ2 leads to hyperactive HAT and elevated histone H3K27 and H3K18 acetylation in cells. Mechanistically, TAZ2 cooperates with other HAT neighboring domains to maintain the HAT active site in a 'closed' state. Truncating TAZ2 or binding of transcription factors to TAZ2 induces a conformational change that 'opens' the active site for substrate acetylation. Importantly, genetic mutations that lead to p300/CBP TAZ2 truncations are found in human cancers, and cells with TAZ2 truncations are vulnerable to histone deacetylase inhibitors. Our study reveals a function of the TAZ2 domain in HAT autoinhibitory regulation and provides a potential therapeutic strategy for the treatment of cancers harboring p300/CBP TAZ2 truncations.

New indices system for quantifying the nexus between economic-social development, natural resources consumption, and environmental pollution in China during 1978-2018.

It is meaningful to study how China can maintain the sustainable utilization of natural resources and the continuous improvement of environmental conditions while ensuring the stable development of the economy and society. In this study, a new indices system was proposed for the analysis of nexus among social-economic-natural resource-environment complex systems following the DPSIR (Driving Force - Pressure - State - Impact - Respond) framework, CCD (Coupling Coordination Degree) analysis and VAR (Vector Auto-Regressive) model were applied for quantifying the synergy and trade-off of China in the nexus framework. Results showed that: (1) Although China's rapid development has caused big consumption of natural resources and increasing pollutants discharges during 1978-2018, China has not got into trouble of extreme resource depletion and ecosystem collapse. On the contrary, China guaranteed food supply, stopped forest degradation, and avoided pollution-induced healthy crises & life-shortening. (2) Adjustment of water pollution industries and the increase of wastewater treatment investment contributed 39% and 37% to the reduction of water pollutant discharge, respectively. The contribution of energy structure adjustment to acid rain control was 26%. The pollutants discharged in no less than 70% of the provinces are strictly controlled below the environmental capacity. The increase of fertilizer application and effective irrigated area contributed 32% to China's grain increase, and China's grain self-sufficiency rate has been maintained above 110%. The improvement of the water-saving irrigation rate contributed 28% to the reduction of water consumption. The reduction of comprehensive efficiency contributed 23.8% to the decrease in energy consumptions per GDP. The CCD assessment showed that China has entered a phase of pre-eminently coordinated development since 2013.

Effect and safety management of different thickness ginger mud moxibustion on ankylosing spondylitis with kidney yang deficiency / 中国实用护理杂志

Objective:To observe the curative effect and temperature safety management of ginger mud moxibustion of different thickness on ankylosing spondylitis patients with kidney yang deficiency type, and to explore the best curative effect combination and safety combination of ginger mud thickness in Du Meridian moxibustion.Methods:From March 2020 to March 2021, 90 patients with ankylosing spondylitis of kidney yang deficiency type who were treated with Du moxibustion in the Affiliated Hospital of Nanjing University of Chinese Medicine were selected. According to the thickness of ginger paste, they were randomly divided into 2 cm-thick ginger mud thickness group, 3 cm-thick ginger mud thickness group and 4 cm-thick ginger mud thickness group when the diameter and height of moxa wool were the same as 2 cm, 30 cases in each group. They were treated with Fu Yang Du moxibustion once a week for 60 minutes each time. Visual Analogue Scale (VAS), Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), Bath Ankylosing Spondylitis Functional Index (BASFI), traditional Chinese medicine syndrome score were used to evaluate the curative effect of spinal function before and after the intervention for 6 weeks. The time when moxibustion temperature reached 43 ℃ and moxibustion temperature maintained at 43-45 ℃ were analyzed for safety evaluation.Results:After moxibustion, VAS scores of 2 cm-thick ginger mud thickness group, 3 cm-thick ginger mud thickness group and 4 cm-thick ginger mud thickness group were 2.89 ± 0.96, 2.01 ± 0.69, 2.93 ± 1.23, BASDAI scores were 3.51 ± 0.94, 2.69 ± 0.68, 3.13 ± 0.96 and BASFI scores were 1.71 ± 0.99, 0.99 ± 0.36, 1.61 ± 0.50, the traditional Chinese medicine syndrome scores were 15.97 ± 4.61, 12.08 ± 3.21, 13.79 ± 3.58. The scores of the three groups were statistically significant ( F values were 6.51-19.22, all P<0.05) . After the intervention, there were significant differences in the scores between 2 cm-thick ginger mud thickness group and 3 cm-thick ginger mud thickness group, 2 cm-thick ginger mud thickness group and 4 cm-thick ginger mud thickness group, 3 cm-thick ginger mud thickness group and 4 cm-thick ginger mud thickness group ( t values were -6.61-4.56, all P<0.05). Conclusions:Du moxibustion is an effective method to treat ankylosing spondylitis of kidney yang deficiency type. The best curative effect and safety combination is moxa velvet diameter height 2 cm, ginger mud thickness 3 cm.

Hepatic CPT1A Facilitates Liver-Adipose Cross-Talk via Induction of FGF21 in Mice.

BACKGROUND & AIMS: Hepatosteatosis, defined as excessive intrahepatic lipid accumulation, represents the first step of NAFLD. When combined with additional cellular stress, this benign status progresses to local and systemic pathological conditions such as NASH and insulin resistance. However, the molecular events directly caused by hepatic lipid build-up, in terms of its impact on liver biology and peripheral organs, remain unclear. Carnitine palmitoyltransferase 1A (CPT1A) is the rate limiting enzyme for long chain fatty acid beta-oxidation in the liver. Here we utilise hepatocyte-specific Cpt1a knockout (LKO) mice to investigate the physiological consequences of abolishing hepatic long chain fatty acid metabolism. APPROACH & RESULTS: Compared to the wild-type (WT) littermates, high fat diet (HFD)-fed LKO mice displayed more severe hepatosteatosis but were otherwise protected against diet-induced weight gain, insulin resistance, hepatic ER stress, inflammation and damage. Interestingly, increased energy expenditure was observed in LKO mice, accompanied by enhanced adipose tissue browning. RNAseq analysis revealed that the peroxisome proliferator activator alpha (PPARα)- fibroblast growth factor 21 (FGF21) axis was activated in liver of LKO mice. Importantly, antibody-mediated neutralization of FGF21 abolished the healthier metabolic phenotype and adipose browning in LKO mice, indicating that the elevation of FGF21 contributes to the improved liver pathology and adipose browning in HFD-treated LKO mice. CONCLUSIONS: Liver with deficient CPT1A expression adopts a healthy steatotic status that protects against HFD-evoked liver damage and potentiates adipose browning in an FGF21-dependent manner. Inhibition of hepatic CPT1A may serve as a viable strategy for the treatment of obesity and NAFLD.

Hepatic CPT1A Facilitates Liver-Adipose Cross-Talk via Induction of FGF21 in Mice.

BACKGROUND & AIMS: Hepatosteatosis, defined as excessive intrahepatic lipid accumulation, represents the first step of NAFLD. When combined with additional cellular stress, this benign status progresses to local and systemic pathological conditions such as NASH and insulin resistance. However, the molecular events directly caused by hepatic lipid build-up, in terms of its impact on liver biology and peripheral organs, remain unclear. Carnitine palmitoyltransferase 1A (CPT1A) is the rate limiting enzyme for long chain fatty acid beta-oxidation in the liver. Here we utilise hepatocyte-specific Cpt1a knockout (LKO) mice to investigate the physiological consequences of abolishing hepatic long chain fatty acid metabolism. APPROACH & RESULTS: Compared to the wild-type (WT) littermates, high fat diet (HFD)-fed LKO mice displayed more severe hepatosteatosis but were otherwise protected against diet-induced weight gain, insulin resistance, hepatic ER stress, inflammation and damage. Interestingly, increased energy expenditure was observed in LKO mice, accompanied by enhanced adipose tissue browning. RNAseq analysis revealed that the peroxisome proliferator activator alpha (PPARα)- fibroblast growth factor 21 (FGF21) axis was activated in liver of LKO mice. Importantly, antibody-mediated neutralization of FGF21 abolished the healthier metabolic phenotype and adipose browning in LKO mice, indicating that the elevation of FGF21 contributes to the improved liver pathology and adipose browning in HFD-treated LKO mice. CONCLUSIONS: Liver with deficient CPT1A expression adopts a healthy steatotic status that protects against HFD-evoked liver damage and potentiates adipose browning in an FGF21-dependent manner. Inhibition of hepatic CPT1A may serve as a viable strategy for the treatment of obesity and NAFLD.

Discovery of Selective Small-Molecule Inhibitors for the ENL YEATS Domain.

Eleven-nineteen leukemia (ENL) protein is a histone acetylation reader essential for disease maintenance in acute leukemias, in particular, the mixed-lineage leukemia (MLL)-rearranged leukemia. In this study, we carried out high-throughput screening of a small-molecule library to identify inhibitors for the ENL YEATS domain. Structure-activity relationship studies of the hits and structure-based inhibitor design led to two compounds, 11 and 24, with IC50 values below 100 nM in inhibiting the ENL-acetyl-H3 interaction. Both compounds, and their precursor compound 7, displayed strong selectivity toward the ENL YEATS domain over all other human YEATS domains. Moreover, 7 exhibited on-target inhibition of ENL in cultured cells and a synergistic effect with the bromodomain and extraterminal domain inhibitor JQ1 in killing leukemia cells. Together, we have developed selective chemical probes for the ENL YEATS domain, providing the basis for further medicinal chemistry-based optimization to advance both basic and translational research of ENL.

Mechanistic insights into chromatin targeting by leukemic NUP98-PHF23 fusion.

Chromosomal NUP98-PHF23 translocation is associated with an aggressive form of acute myeloid leukemia (AML) and poor survival rate. Here, we report the molecular mechanisms by which NUP98-PHF23 recognizes the histone mark H3K4me3 and is inhibited by small molecule compounds, including disulfiram that directly targets the PHD finger of PHF23 (PHF23PHD). Our data support a critical role for the PHD fingers of NUP98-PHF23, and related NUP98-KDM5A and NUP98-BPTF fusions in driving leukemogenesis, and demonstrate that blocking this interaction in NUP98-PHF23 expressing AML cells leads to cell death through necrotic and late apoptosis pathways. An overlap of NUP98-KDM5A oncoprotein binding sites and H3K4me3-positive loci at the Hoxa/b gene clusters and Meis1 in ChIP-seq, together with NMR analysis of the H3K4me3-binding sites of the PHD fingers from PHF23, KDM5A and BPTF, suggests a common PHD finger-dependent mechanism that promotes leukemogenesis by this type of NUP98 fusions. Our findings highlight the direct correlation between the abilities of NUP98-PHD finger fusion chimeras to associate with H3K4me3-enriched chromatin and leukemic transformation.

Few-view CT image reconstruction using improved total variation regularization.

X-ray radiation is harmful to human health. Thus, obtaining a better reconstructed image with few projection view constraints is a major challenge in the computed tomography (CT) field to reduce radiation dose. In this study, we proposed and tested a new algorithm that combines penalized weighted least-squares using total generalized variation (PWLS-TGV) and dictionary learning (DL), named PWLS-TGV-DL to address this challenge. We first presented and tested this new algorithm and evaluated it through both data simulation and physical experiments. We then analyzed experimental data in terms of image qualitative and quantitative measures, such as the structural similarity index (SSIM) and the root mean square error (RMSE). The experiments and data analysis indicated that applying the new algorithm to CT data recovered images more efficiently and yielded better results than the traditional CT image reconstruction approaches.

Linifanib exerts dual anti-obesity effect by regulating adipocyte browning and formation.

Obesity is caused by energy imbalance and accompanied by adipocyte hypertrophy and hyperplasia. Therefore, both enhancement of adipocyte energy expenditure and inhibition of adipogenesis are viable ways to combat obesity. Using the Ucp1-2A-luciferase reporter animal model previously reported by us as a screening platform, a chemical compound Linifanib was identified as a potent inducer of UCP1 expression in primary inguinal adipocytes in vitro and in vivo. Signal pathway analyses showed that Linifanib promoted adipocyte browning by attenuating STAT3 phosphorylation. The effects of Linifanib on adipocyte browning were blocked by the compound, SD19, which activates the STAT3 signaling cascade. Linifanib also inhibited adipocyte differentiation, by blocking mitotic clonal expansion, which could be rescued by STAT3 activator. Taken together, our results indicate that Linifanib might serve as a potential drug for the treatment of obesity.

Production of a novel Poria cocos immunomodulatory protein in Pichia pastoris: cloning, expression, purification and activities assays.

In this study, the cDNA of immunomodulatory protein from Poria cocos (PCP) was amplified by reverse transcription polymerase chain reaction and used to transform P. Pastoris cells, resulting in rPCP expression as a secreted protein to a concentration of ~ 38 mg/L following methanol induction in shake flasks. Approximately 1.6 mg of high purity rPCP was obtained from a 100-mL culture by Ni+-affinity chromatography, and sodium dodecyl sulfate polyacrylamide gel electrophoresis results indicated rPCP as a homologous dimer glycoprotein formed by different molecular-weight monomers. Peptide-N-glycosidase F-mediated deglycosylation analysis showed the presence of an N-glycosylated rPCP monomer, and bioactivity assays showed that rPCP activity upregulated tumor necrosis factor (TNF)-α and interleukin-1ß transcription and increased TNF-α secretion from mouse macrophage RAW 264.7 cells. Shortly, we demonstrated successful purification of active rPCP from P. pastoris, which promoted further study of its biological activities and medical applications.

Discovery and optimization of 1-(1H-indol-1-yl)ethanone derivatives as CBP/EP300 bromodomain inhibitors for the treatment of castration-resistant prostate cancer.

The CREB (cAMP responsive element binding protein) binding protein (CBP) and its homolog EP300 have emerged as new therapeutic targets for the treatment of cancer and inflammatory diseases. Here we report the identification, optimization and evaluation of 1-(1H-indol-1-yl)ethanone derivatives as CBP/EP300 inhibitors starting from fragment-based virtual screening (FBVS). A cocrystal structure of the inhibitor (22e) in complex with CBP provides a solid structural basis for further optimization. The most potent compound 32h binds to the CBP bromodomain and has an IC50 value of 0.037 µM in the AlphaScreen assay which was 2 times more potent than the reported CBP bromodomain inhibitor SGC-CBP30 in our hands. 32h also exhibit high selectivity for CBP/EP300 over other bromodomain-containing proteins. Notably, the ester derivative (29h) of compound 32h markedly inhibits cell growth in several prostate cancer cell lines including LNCaP, 22Rv1 and LNCaP derived C4-2B. Compound 29h suppresses the mRNA expression of full length AR (AR-FL), AR target genes and other oncogene in LNCaP cells. 29h also reduces the expression of PSA, the biomarker of prostate cancer. CBP/EP300 inhibitor 29h represents a promising lead compound for the development of new therapeutics for the treatment of castration-resistant prostate cancer.

The natural compound, formononetin, extracted from Astragalus membranaceus increases adipocyte thermogenesis by modulating PPARγ activity.

BACKGROUND AND PURPOSE: Increasing energy expenditure through adipocyte thermogenesis is generally accepted as a promising strategy to mitigate obesity and its related diseases. However, few clinically effective and safe agents are known to promote adipocyte thermogenesis. In this study, 20 traditional Chinese herbal medicines were screened to examine whether they induced adipocyte thermogenesis. EXPERIMENTAL APPROACH: The effects of Chinese herbal medicines or components isolated from extracts of A. membranaceus, on adipocyte thermogenesis were analysed by assessing expression of uncoupling protein 1 (UCP1) by qPCR. Eight-week-old C57BL6/J male mice were fed a high-fat diet for 8 weeks and then randomized to two groups treated with vehicle or formononetin for another 8 weeks. Glucose tolerance tests and staining of adipose tissue with haematoxylin and eosin were carried out. Whole-body oxygen consumption was measured with an open-circuit indirect calorimetry system. KEY RESULTS: Extracts of A. membranaceus increased expression of Ucp1 in primary cultures of mouse adipocytes. Formononetin was the only known component of A. membranaceus extracts to increase adipocyte Ucp1 expression. Diet-induced obese mice treated with formononetin gained less weight and showed higher energy expenditure than untreated mice. In addition, formononetin binds directly with PPARγ. CONCLUSIONS AND IMPLICATION: Taken together, our study demonstrates that the Chinese herbal medicine from A. membranaceus and its constituent formononetin have the potential to reduce obesity and associated metabolic disorders. Our results suggest that formononetin regulates adipocyte thermogenesis as a non-classical PPARγ agonist.

Adipocyte SIRT1 controls systemic insulin sensitivity by modulating macrophages in adipose tissue.

Adipose tissue inflammation, characterized by augmented infiltration and altered polarization of macrophages, contributes to insulin resistance and its associated metabolic diseases. The NAD+-dependent deacetylase SIRT1 serves as a guardian against metabolic disorders in multiple tissues. To dissect the roles of SIRT1 in adipose tissues, metabolic phenotypes of mice with selective ablation of SIRT1 in adipocytes and myeloid cells were monitored. Compared to myeloid-specific SIRT1 depletion, mice with adipocyte-selective deletion of SIRT1 are more susceptible to diet-induced insulin resistance. The phenotypic changes in adipocyte-selective SIRT1 knockout mice are associated with an increased number of adipose-resident macrophages and their polarization toward the pro-inflammatory M1 subtype. Mechanistically, SIRT1 in adipocytes modulates expression and secretion of several adipokines, including adiponectin, MCP-1, and interleukin 4, which in turn alters recruitment and polarization of the macrophages in adipose tissues. In adipocytes, SIRT1 deacetylates the transcription factor NFATc1 and thereby enhances the binding of NFATc1 to the Il4 gene promoter. These findings suggest that adipocyte SIRT1 controls systemic glucose homeostasis and insulin sensitivity via the cross talk with adipose-resident macrophages.

Visualization and Quantification of Browning Using a Ucp1-2A-Luciferase Knock-in Mouse Model.

Both mammals and adult humans possess classic brown adipocytes and beige adipocytes, and the amount and activity of these adipocytes are considered key factors in combating obesity and its associated metabolic diseases. Uncoupling protein 1 (Ucp1) is the functional marker of both brown and beige adipocytes. To facilitate a reliable, easy, and sensitive measurement of Ucp1 expression both in vivo and in vitro, we generated a Ucp1-2A-luciferase knock-in mouse by deleting the stop codon for the mouse Ucp1 gene and replacing it with a 2A peptide. This peptide was followed by the luciferase coding sequence to recapitulate the expression of the Ucp1 gene at the transcriptional and translational levels. With this mouse, we discovered a cold-sensitive brown/beige adipose depot underneath the skin of the ears, which we named uBAT. Because of the sensitivity and high dynamic range of luciferase activity, the Ucp1-2A-luciferase mouse is useful for both in vitro quantitative determination and in vivo visualization of nonshivering thermogenesis. With the use of this model, we identified and characterized axitinib, an oral small-molecule tyrosine kinase inhibitor, as an effective browning agent.

Brown Adipogenic Reprogramming Induced by a Small Molecule.

Brown adipose tissue (BAT) has attracted considerable research interest because of its therapeutic potential to treat obesity and associated metabolic diseases. Augmentation of brown fat mass and/or its function may represent an attractive strategy to enhance energy expenditure. Using high-throughput phenotypic screening to induce brown adipocyte reprogramming in committed myoblasts, we identified a retinoid X receptor (RXR) agonist, bexarotene (Bex), that efficiently converted myoblasts into brown adipocyte-like cells. Bex-treated mice exhibited enlarged BAT mass, enhanced BAT function, and a modest browning effect in subcutaneous white adipose tissue (WAT). Expression analysis showed that Bex initiated several "browning" pathways at an early stage during brown adipocyte reprogramming. Our findings suggest RXRs as new master regulators that control brown and beige fat development and activation, unlike the common adipogenic regulator PPARγ. Moreover, we demonstrated that selective RXR activation may potentially offer a therapeutic approach to manipulate brown/beige fat function in vivo.

Harmine Induces Adipocyte Thermogenesis through RAC1-MEK-ERK-CHD4 Axis.

Harmine is a natural compound possessing insulin-sensitizing effect in db/db diabetic mice. However its effect on adipose tissue browning is unknown. Here we reveal that harmine antagonizes high fat diet-induced adiposity. Harmine-treated mice gained less weight on a high fat diet and displayed increased energy expenditure and adipose tissue thermogenesis. In vitro, harmine potently induced the expression of thermogenic genes in both brown and white adipocytes, which was largely abolished by inhibition of RAC1/MEK/ERK pathway. Post-transcriptional modification analysis revealed that chromodomain helicase DNA binding protein 4 (CHD4) is a potential downstream target of harmine-mediated ERK activation. CHD4 directly binds the proximal promoter region of Ucp1, which is displaced upon treatment of harmine, thereby serving as a negative modulator of Ucp1. Thus, here we reveal a new application of harmine in combating obesity via this off-target effect in adipocytes.

The discovery of novel and selective fatty acid binding protein 4 inhibitors by virtual screening and biological evaluation.

Adipocyte fatty acid binding protein (AFABP, FABP4) has been proven to be a potential therapeutic target for diabetes, atherosclerosis and inflammation-related diseases. In this study, a series of new scaffolds of small molecule inhibitors of FABP4 were identified by virtual screening and were validated by a bioassay. Fifty selected compounds were tested, which led to the discovery of seven hits. Structural similarity-based searches were then performed based on the hits and led to the identification of one high affinity compound 33b (Ki=0.29±0.07µM, ΔTm=8.5°C). This compound's effective blockade of inflammatory response was further validated by its ability to suppress pro-inflammatory cytokines induced by lipopolysaccharide (LPS) stimulation. Molecular dynamics simulation (MD) and mutagenesis studies validated key residues for its inhibitory potency and thus provide an important clue for the further development of drugs.

High level expression and purification of active recombinant human interleukin-15 in Pichia pastoris.

Interleukin-15 (IL-15) is a pleiotropic cytokine and a member of the four α-helix bundle family of cytokines which include IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. IL-15 exhibits a broad biological activity and induces the differentiation and proliferation of T, B and natural killer (NK) cells. In this study, a DNA fragment containing the mature human IL-15 sequence was cloned into pPICZaA vector, generating a fusion protein with the alpha factor signal sequence in the N-terminus and 6×His as well as c-Myc tags in the C-terminus. The resulting plasmid was integrated into the genome of Pichia pastoris strain X-33. Recombinant yeast transformants with high-level recombinant human IL-15 (rhIL-15) production were identified, which secrete as much as 75 mg/L rhIL-15 after 3 days of induction by methanol. The rhIL-15 was purified by Ni(+)-NTA affinity chromatography, followed by DEAE anion exchange, yielding over 95% highly purified rhIL-15. Mass spectrometry and MALDI-TOF-TOF analysis showed the purified rhIL-15 had larger molecular weights than expected, due to different degrees of N-linked glycosylation. The biological activity of the rhIL-15 proteins was measured by its ability to enhance cellular proliferation of CTLL-2 and NK cells. The results demonstrate that the experimental procedure we have reported here can produce a large amount of active recombinant human IL-15 from P. pastoris.