Site-Specific m6A Erasing via Conditionally Stabilized CRISPR-Cas13b Editor

N6-methyladenosine (m⁶A) on RNAs plays an important role in regulating various biological processes and CRIPSR technology has been employed for programmable m⁶A editing. However, the bulky size of CRISPR protein and constitutively expressed CRISPR/RNA editing enzymes can interfere with the native function of target RNAs and cells. Herein, we reported a conditional m⁶A editing platform (FKBP*-dCas13b-ALK) based on a ligand stabilized dCas13 editor. The inducible expression of this m⁶A editing system was achieved by adding or removing the Shield-1 molecule. We further demonstrated that the targeted recruitment of dCas13b-m⁶A eraser fusion protein and site-specific m⁶A erasing were achieved under the control of Shield-1. Moreover, the release and degradation of dCas13b fusion protein occurred faster than the restoration of m⁶A on the target RNAs after Shield-1 removal, which provides an ideal opportunity to study the m⁶A function with minimal steric interference from bulky dCas13b fusion protein.     

Chemical Validation of Mycobacterium tuberculosis Phosphopantetheine Adenylyltransferase Using Fragment Linking and CRISPR Interference**

The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a K_D <20 μM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.     

Stereoselective Synthesis of the O-antigen of A. baumannii ATCC 17961 Using Long-Range Levulinoyl Group Participation

Herein, we described the first synthesis of the pentasaccharide and decasaccharide of the A. baumannii ATCC 17961 O-antigen for developing a synthetic carbohydrate-based vaccine against A. baumannii infection. The efficient synthesis of the rare sugar 2,3-diacetamido-glucuronate was achieved using our recently introduced organocatalytic glycosylation method. We found, for the first time, that long-range levulinoyl group participation via a hydrogen bond can result in a significantly improved β-selectivity in glycosylations. This solves the stereoselectivity problem of highly branched galactose acceptors. The proposed mechanism was supported by control experiments and DFT computations. Benefiting from the long-range levulinoyl group participation strategy, the pentasaccharide donor and acceptor were obtained via an efficient [2+1+2] one-pot glycosylation method and were used for the target decasaccharide synthesis.     

Rapid separation and binding configuration prediction of the components in Danshen decoction to endothelin A receptor using affinity chromatography and molecular dynamics simulation

As a famous traditional Chinese formula, Danshen Decoction has the potential to relieve the pain of pulmonary arterial hypertension patients, however, the functional components remain unknown. Herein, we reported a method to screen the functional components in Danshen Decoction targeting endothelin receptor A, an accepted target for the treatment of the disease. The receptor was functionalized on the macroporous silica gel through an epidermal growth factor receptor fusion tag and its covalent inhibitor. Using the affinity gel as the stationary phase, the bioactive compound was identified as salvianolic acid B by mass spectrometry. The binding kinetic parameter (dissociation rate constants k_d) of salvianolic acid B with the receptor was determined via peak profiling. Using the specific ligands of the receptor as probes, the binding configuration prediction of salvianolic acid B with the receptor was performed by molecular dynamics simulation. Our results indicated that salvianolic acid B is a potential bioactive compound in Danshen Decoction targeting the receptor. This work showed that receptor chromatography in combination with molecular dynamics simulation is applicable to predicting the binding kinetics and configuration of a ligand to a receptor, providing crucial insight for the rational design of drugs that recognize functional proteins.     

机械剥离石墨烯修饰电极检测食品接触材料中双酚A的迁移量

采用机械剥离石墨烯修饰电极快速检测食品接触材料中双酚A的迁移量。X射线电子衍射表征显示机械剥离石墨烯表面不存在含氧官能团,与化学还原石墨烯相比,机械剥离石墨烯对双酚A具有更好的电催化性能,降低了双酚A的氧化过电位,提高了电流响应。在优化的试验条件下,双酚A的浓度在1.0×10-7~1.5×10-5mol·L-1范围内与氧化峰电流呈线性关系,检出限(3S/N)为3.0×10-8mol·L-1。采用该电极对食品模拟物中的双酚A进行检测,加标回收率在85.1%~104%之间,测定值的相对标准偏差(n=6)在2.7%~5.9%之间。     

Synthesis of PEGylated hyaluronic acid for loading dichloro(1,2-diaminocyclohexane)platinum(Ⅱ)(DACHPt) in nanoparticles for cancer treatment

Dichloro(1,2-diaminocyclohexane)platinum(II)(DACHPt), a cisplatin(CDDP) analog, has shown lower toxicity than CDDP and no cross-resistance with CDDP in many CDDP-resistant cancers. PEGylated hyaluronan(m PEG-HA) is an m PEG conjugated with hyaluronan biodegradable polymer which is a naturally occurring biopolymer in the interstitium, is primarily cleared by the lymphatic system. m PEGhyaluronan–DACHPt(PEG-HA–Pt) conjugate could circulate long-term in the bloodstream and increase DACHPt concentration in the tumor site and decrease systemic toxicity. m PEG-HA conjugates with the range of 1%–5% substitution were synthesized, and the structures were confirmed by1 H NMR and IR. The particle size of DACHPt incorporated with m PEG-HA was about 86 nm and the loading content and efficiency were about 19%(w/w) and 86%, respectively. The synthesized m PEG-HA with different PEG substitution degrees presented non toxicity, and the cell viability of DACHPt loaded in m PEG-HA nanoparticles increased with increasing doses of DACHPt. DACHPt release from nanoparticles slightly decreased with increasing PEG substitution degree from 1% to 5% at 37 8C, pH 7.4 PBS solution. The DACHPt loaded in m PEG-HA nanoparticles significantly inhibited the growth of A549 xenografts in nude mice when compared to the DACHPt loaded in HA nanoparticles and the control group after 4 weeks treatment(p 0.01 compared with control). The body weight change curve shows that the mice weight loss was less than 5% by treating with both DACHPt loaded in m PEG-HA and HA nanoparticles. In conclusion, a novel DACHPt loaded m PEG-HA delivery system was developed with sustained release and increased platinum concentration in the tumor.     

新型7-O-(芳基氨基甲酸酯)-布雷菲德菌素A的合成及其抗肿瘤活性

芳胺与三光气反应制得芳基异氰酸酯(2a~2n);2a~2n分别对布雷菲德菌素A(3)的7-羟基进行结构修饰,合成了14个新型布雷菲德菌素A氨基甲酸酯类衍生物——7-O-(芳基氨基甲酸酯)-布雷菲德菌素A(4a~4n),其结构经1H NMR,13C NMR和ESI-HR-MS表征。初步的抗肿瘤活性测定结果表明,4i~4k均丧失抑制食管癌细胞株TE-1活性。分子对接研究揭示,引入3后,4的分子空间位阻增大,导致不能与靶位结合,致使抗肿瘤活性丧失。     

Magnetic CuO nanoparticles supported on graphene oxide as an efficient catalyst for A3-coupling synthesis of propargylamines

Magnetically separable CuO nanoparticles supported on graphene oxide (Fe₃O₄ NPs/GO-CuO NPs) is synthesized and characterized for the preparation of propargylamines in EtOH, at 90℃. Fe₃O₄ NPs/GO-CuO NPs is found to be an efficient catalyst for the A³-coupling of aldehydes, amines, and alkynes through C-H activation. Both aromatic and aliphatic aldehydes and alkynes are combined with secondary amines to provide a wide range of propargylamines in moderate to excellent yields.     

Medium effect on dielectric relaxation behaviors of 4A zeolite bulk dispersion system

Dielectric spectra of 4A zeolite particles bulked in deionized water (4A/W) or cyclohexane (4A/C) were measured respectively under different temperatures, and two dielectric relaxations were found in both of 4A/W and 4A/C. Because of existence of water molecules in pores, both low and high frequency relaxation times (τ_LFR and τ_HFR) of 4A/Wdecreasedmore sharply than 4A/C with increasing temperature. According to the temperature dependence of τ_LFR and τ_HFR, the activation energies of Na⁺ interacting with framework under hydrated and dehydrated environments were calculated respectively, and the original properties of Na⁺ in dehydrated zeolite were obtained by analyzing the dielectric properties of 4A/C. Furthermore, a transient state of Na⁺ motion with temperature was found in 4A/C system.     

HPLC-DAD分析金线莲中天麻素和天麻苷元

研究超声-微波协同法提取金线莲中的天麻素和天麻苷元,并对提取条件进行了优化.应用高效液相-二极管阵列检测法分析提取物中的天麻素和天麻苷元.研究结果表明:金线莲中天麻素、天麻苷元和其他杂质成分能够很好的分离,且两者的含量分别为1.430及0.562 mg/g,相对标准偏差为0.94%和0.25%.方法准确、快速、灵敏度高、重现性好.