Guidelines for application of high-content screening in traditional Chinese medicine:concept,equipment,and troubleshooting

High-content screening(HCS)technology combines automated high-speed imaging hardware and single-cell quantitative analysis.It can greatly accelerate data acquisition in cellular fluorescence imaging and is a powerful research technique in traditional Chinese medicine(TCM).An increasing number of laboratories and platforms,including TCM laboratories,have begun utilizing HCS systems.However,this technology is still in its infancy in TCM research and there is a lack of sufficient experience with the associated concepts,instrument configurations,and analysis methods.To improve the understanding of HCS among researchers in the field of TCM,this paper summarizes the concept of HCS,software and hardware configuration,the overall research process,as well as common problems and related solutions of HCS in TCM research based on our team’s previous research experience,providing several research examples and an outlook on future perspectives,aiming to provide a technical guide for HCS in TCM research.

Temperature-programmed pyrolysis of magnesium ammonium phosphate and removal of ammonia-nitrogen by its pyrolysate

In order to achieve the dual goals of complete deamination of magnesium ammonium phosphate（MAP） and ensure the pyrolysate＇s good removal properties towards ammonia-nitrogen, a temperature-programmed method for the pyrolysis of MAP was studied, as well as the thermodynamic and kinetic processes involved in the removal reaction system between MAP pyrolysate and aqueous ammonium. It was found that the pyrolysis method and pyrolysis final temperature had significant effects on the MAP pyrolysate＇s removal properties towards aqueous ammonium, and the following conditions were deemed to be more appropriate： pyrolysis final temperature and heating rate at 180 ℃ and 5 ℃/min, respectively, and a pH level of 9.5 for the removal reaction system. The resultant ammonium removal capacity by the MAP pyrolysate was 95.62 mg/g. After 120 min, the removal rate with an initial concentration of 1000 mg/L was 82%. The kinetic and thermodynamic results indicated that the removal of aqueous ammonium by MAP pyrolysate was the exchange process between H^＋ and NH4^＋ via MAP precipitation. The kinetics complied with the Lagergren quasi second-order model with an equilibrium time of 120 min, while the isothermal curves complied with the Freundlich model.

Bromocriptine protects perilesional spinal cord neurons from lipotoxicity after spinal cord injury

Recent studies have revealed that lipid droplets accumulate in neurons after brain injury and evoke lipotoxicity,damaging the neurons.However,how lipids are metabolized by spinal cord neurons after spinal cord injury remains unclear.Herein,we investigated lipid metabolism by spinal cord neurons after spinal cord injury and identified lipid-lowering compounds to treat spinal cord injury.We found that lipid droplets accumulated in perilesional spinal cord neurons after spinal cord injury in mice.Lipid droplet accumulation could be induced by myelin debris in HT22 cells.Myelin debris degradation by phospholipase led to massive free fatty acid production,which increased lipid droplet synthesis,β-oxidation,and oxidative phosphorylation.Excessive oxidative phosphorylation increased reactive oxygen species generation,which led to increased lipid peroxidation and HT22 cell apoptosis.Bromocriptine was identified as a lipid-lowering compound that inhibited phosphorylation of cytosolic phospholipase A2 by reducing the phosphorylation of extracellular signal-regulated kinases 1/2 in the mitogen-activated protein kinase pathway,thereby inhibiting myelin debris degradation by cytosolic phospholipase A2 and alleviating lipid droplet accumulation in myelin debris-treated HT22 cells.Motor function,lipid droplet accumulation in spinal cord neurons and neuronal survival were all improved in bromocriptine-treated mice after spinal cord injury.The results suggest that bromocriptine can protect neurons from lipotoxic damage after spinal cord injury via the extracellular signal-regulated kinases 1/2-cytosolic phospholipase A2 pathway.

Identification of fangchinoline as a broad-spectrum enterovirus inhibitor through reporter virus based high-content screening

Hand,foot,and mouth disease(HFMD)is a common pediatric illness mainly caused by enteroviruses,which are important human pathogens.Currently,there are no available antiviral agents for the therapy of enterovirus infection.In this study,an excellent high-content antiviral screening system utilizing the EV-A71-eGFP reporter virus was developed.Using this screening system,we screened a drug library containing 1042 natural compounds to identify potential EV-A71 inhibitors.Fangchinoline(FAN),a bis-benzylisoquinoline alkaloid,exhibits potential inhibitory effects against various enteroviruses that cause HFMD,such as EV-A71,CV-A10,CV-B3 and CV-A16.Further investigations revealed that FAN targets the early stage of the enterovirus life cycle.Through the selection of FAN-resistant EV-A71 viruses,we demonstrated that the VP1 protein could be a potential target of FAN,as two mutations in VP1(E145G and V258I)resulted in viral resistance to FAN.Our research suggests that FAN is an efficient inhibitor of EV-A71 and has the potential to be a broad-spectrum antiviral drug against human enteroviruses.

高盐化工废水回用处理工艺研究

针对高盐化工废水和中水回用原水水质特点,分析二者中需要去除的重难点污染物,为两套水处理系统选择适合的工艺技术路线,其中污水处理站工艺采用“脱硫破氰+气浮+水解酸化+两级A/O+内置MBR+臭氧氧化”,系统出水满足《农田灌溉水质标准》(GB 5084—2021);中水回用系统工艺采用“化学软化沉淀+V型滤池+DTLRO反渗透”,出水水质达到《城市污水再生利用工业用水水质》(GB/T 19923—2005)表1敞开式循环冷却水水质标准;项目可为同类型高盐化工废水处理回用提供工艺路线设计参考。

Preparation of hollow B–SiO2@TiO2 composites and their photocatalytic performances for degradation of ammonia-nitrogen and green algae in aqueous solution

Hollow B–SiO2@TiO2 composites were prepared by the wet chemical deposition method starting from TiCl4 and hollow B–SiO2 microspheres.TiO2 layers composed of anatase TiO2 nanoparticles were coated on the surfaces of the hollow B–SiO2 microspheres probably through the formation of Ti—O—Si and Ti—O—B bonds.A great number of—OH groups were also present at the TiO2 coating layers.The presence of Ti—O—Si bonds and Ti—O—B bonds resulted in the formation of defects in the TiO2 coating layers,which decreased the band gap of the TiO2 coating layers to ca.3.0 eV and endowed the TiO2 coating layers with visible light absorption performance.The buoyancy hollow B–SiO2@TiO2 composites exhibited high photocatalytic activities for the degradation of ammonia-nitrogen and green algae.The conversion of ammonia-nitrogen reached 65%when the degradation of ammonia-nitrogen(43 mg·L-1 at pH value of 8)was catalyzed by the B–SiO2@TiO2(100:10)composite under the simulated solar light irradiation at 35°C for 660 min.The green algae(5 mg·L-1)were almost completely degraded over the B–SiO@TiO2(100:20)photocatalyst under the visible light irradiation at 35°C for 510 min.

Indoor experiment and numerical simulation study of ammonia-nitrogen migration rules in soil column

The riverbank soil is a natural purifying agent for the polluted river water(Riverbank filtration, RBF). This is of great importance to groundwater safety along the riverbank. This paper examines the migration and transformation rules of ammonia-nitrogen in three typical types of sand soil using the indoor leaching experiment of soil column, and then makes comparison with the indoor experiment results in combination with the numerical simulation method. The experiment process shows that the change in ammonia-nitrogen concentration goes through three stages including "removal-water saturation-saturation". As the contents of clay particles in soil sample increase, the removal of ammonia-nitrogen from soil sample will take more time and gain higher ratio. During the removal period, the removal ratio of Column 1, Column 2 and Column 3 averages 68.8%(1-12 d), 74.6%(1-22 d) and 91.1%(1-26 d). The ammonia-nitrogen removal ratio shows no noticeable change as the depth of soil columns varies. But it is found that the ammonia-nitrogen removal ratio is the least of the whole experiment when the soil columns are at the depth of 15 cm. It can be preliminary inferred that the natural purifying performance of soil along the river for ammonia-nitrogen in river water mainly depends on the proportion of fine particles in soil. HYDRUS-1D model is used to simulate this experiment process, analyze the change of the bottom observation holes by time and depth in three columns(the tenth day), and make comparison with the experiment result. The coefficients of determination for fitting curves of Column 1, Column 2 and Column 3 are 0.953, 0.909, 0.882 and 0.955, 0.740, 0.980 separately. Besides, this paper examines the contribution of absorption, mineralization and nitrification in the simulation process. In the early removal stage, mineralization plays a dominant role and the maximum contribution rate of mineralization is 99%. As time goes by, absorption starts to function and gradually assumes a dominant position. In the middle and late removal stage, nitrification in Column 1 and Column 2 makes more contribution than mineralization. So the experiment result of the ammonia-nitrogen concentration is 0.6% and 2.4% lower than that in effluent and the maximum contribution ratio of nitrification is -4.53% and -5.10% respectively when only the function of absorption is considered. The mineralization in Column 1 and Column 2 in the middle and late removal stage still plays a more important role than nitrification. So the experiment result is 1.4% higher than that in effluent and the maximum contribution ratio of nitrification is -2.51% when only the function of absorption is considered. Therefore, absorption, mineralization and nitrification make different contributions during different part of the stage. This means that the natural purifying performance of soil along the river for ammonia-nitrogen in river water not only depends on the proportion of fine particles in soil, but depends on the mineralization and nitrification environment. This can offer some insights into the protection and recovery of groundwater along the riverbank.

Brain organoids are new tool for drug screening of neurological diseases

At the level of in vitro drug screening,the development of a phenotypic analysis system with highcontent screening at the core provides a strong platform to support high-throughput drug screening.There are few systematic reports on brain organoids,as a new three-dimensional in vitro model,in terms of model stability,key phenotypic fingerprint,and drug screening schemes,and particula rly rega rding the development of screening strategies for massive numbers of traditional Chinese medicine monomers.This paper reviews the development of brain organoids and the advantages of brain organoids over induced neurons or cells in simulated diseases.The paper also highlights the prospects from model stability,induction criteria of brain organoids,and the screening schemes of brain organoids based on the characteristics of brain organoids and the application and development of a high-content screening system.

鸟粪石沉淀法处理高氨氮稀土废水

以贺州市某稀土冶炼厂萃取分离稀土元素时产生的高氨氮废水为研究对象,进行鸟粪石沉淀法脱氮试验研究,设计4因素3水平正交试验,确定影响因素大小,得到适宜反应条件组合,进行pH、n(Mg):n(N)、n(P):n(N)、反应时间等4因素优化试验,考察各因素对于氨氮去除率及剩余总磷质量浓度的影响。结果显示,对于氨氮去除率与剩余TP质量浓度,影响因素依次为pH、反应时间、n(Mg):n(N)、n(P):n(N)。在pH=9.3,n(Mg):n(N):n(P)=2.2:1:2,反应时间20 min,氨氮质量浓度由4 420 mg/L降低至1 440 mg/L,氨氮去除率可达到67.40%,剩余TP质量浓度为0.9mg/L;较高的Ca2+质量浓度会影响MAP法对氨氮去除率,但有利于降低出水TP质量浓度;过长的反应时间会破坏已形成的MAP沉淀体系。

碳氮比对高含固率污泥厌氧消化的影响

在35℃、含固率10%、接种量20%的条件下,对碳氮比(C/N)分别为8、12、15的3组污泥进行97 d的厌氧消化试验,分析消化过程中p H、NH_4^+-N、挥发性固体(VS)去除率及产气情况,并采用聚合酶链式反应-变性梯度凝胶电泳(PCR-DGGE)技术分析微生物群落结构的变化。试验结果表明,8~13 d完成系统的启动,且消化过程中没有产生酸抑制现象。3组试验的单位VS产气率分别达到572、492和621 m L/g;VS降解效率分别达到57.1%、51.9%和51.0%;C/N比为8时,累积产气量最多;3组试验污泥DGGE图谱显示,碳氮比越低,细菌群落多样性越丰富,优势菌群群落密度也越大。因此,C/N为8时高含固率污泥厌氧消化效果更好。

高氨氮含量废水的处理方法及研究现状

针对高氨氮含量废水成分复杂、水质变化大的特点,叙述了物化脱氮法、传统和新型的生物脱氮法及物化-生物联合脱氮法在处理中高氨氮含量废水应用情况及其优缺点。为了使废水中的氨氮达到去除最大化、减小二次污染、降低投资和运行成本,采用多种技术联用的手段来处理高氨氮含量废水,可实现优势互补。对于出水要求较高的废水,一般需先进行预处理,再采用物化法、生物法或物化与生物联用法进行处理。

3种臭氧双层滤料滤池对水中有机物及氨氮去除效果研究

针对平原水库夏季有机物及氨氮含量高的问题进行了研究。采用滤前曝臭氧的方式,改变上层滤料的类型,对比3种滤池对水中有机物及氨氮的去除效果。结果表明,在臭氧处理前水的pH为6.84~7.32,COD_(Mn)为6.1~7.3mg/L、UV254为0.162~0.194 cm^(-1)、NH_3-N的质量浓度为1.5~2.0 mg/L的条件下,臭氧-煤砂滤池对水中COD_(Mn)、UV_(254)及NH_3-N的去除率分别为60.5%、87.3%和73.2%,臭氧-活性无烟煤-砂滤池对水中COD_(Mn)、UV_(254)及NH_3-N的去除率分别为81.3%、93.4%和88.3%,臭氧-炭砂滤池对水中COD_(Mn)、UV_(254)及NH_3-N的去除率分别为84.5%、95.2%和92.2%。3种滤池对浊度的去除率达到93%以上。相比传统煤砂滤池,采用臭氧与活性滤料联用能够提高滤池的生化性能,对季节性高有机物、高氨氮含量原水有较好的处理效果。

几种改性/型沸石的制备及其应用

天然沸石经过改性/型处理,可充分发挥其吸附性与离子交换性。Na型斜发沸石能有效处理高铁锰饮用水;多孔改性沸石球对污水中氨氮的去除率达到80%以上;粉煤灰合成的P型沸石有较好的吸附能力和较好催化能力。应用改性/型沸石成本低,效果好。文章介绍了主要的实验室改性/型沸石的制备及应用,以期得到实践推广。

高氨氮含量废水组合吹脱处理工艺实验研究

针对氨氮吹脱法结合吹脱装置及工艺对氨氮去除效果的研究工作较少的情况,基于氨氮吹脱过程的气液传质机理,进行了单一填料塔、旋流塔及填料与旋流塔组合工艺进行废水氨氮吹脱处理工艺对氨氮处理效果的实验研究。结果表明,组合氨氮吹脱处理工艺的氨氮去除率达到89.87%,比单一氨氮吹脱处理工艺的氨氮去除率提高25%以上。其中填料与旋流塔组合氨氮处理的优化工艺条件为:废水pH 11,空气体积流量60 m^(3)/h,进水体积流量20~25 L/h,温度60℃。

Ammonia nitrogen removal from aqueous solution using zeolite modified by microwave-sodium acetate

The characteristics of the zeolite modified by microwave and sodium acetate and its sorption of ammonia-nitrogen from simulated water sample were investigated.The results show that the modified zeolite by microwave-sodium acetate(SMMZ)has a high sorption efficiency and removal performance.The ammonia-nitrogen removal rate of SMMZ reaches 92.90%.The surface of SMMZ becomes loose and some pores appear,the specific surface area,total pore volume and average pore diameter increase after modification.Compared to the natural zeolite,SMMZ has a more concentrated pore size distribution in the range of 0-10 nm.The cation exchange capacity(CEC)of SMMZ is higher than that of the natural zeolite.And the ammonia nitrogen removal rate is consistent with the change of CEC.The SMMZ possesses rapid sorption and slow balance characteristics and ammonia-nitrogen sorption is consistent with both Langmuir adsorption isotherm model and Freundlich adsorption isotherm model.The adsorption kinetics of ammonia-nitrogen follows the pseudo-second order kinetic model.

Development of a high-content screening method to evaluate the cellular population-based biological activity of new siRNA delivering reagent

Normally, cellular responses to modified siRNAs or new siRNA delivery systems have been studied in group cell behavior by PCR, western blotting and fluorescence microscopy. In this study, we present a novel high-content screening （HCS） strategy to evaluate a novel delivery system （named CLD） of siRNA therapeutics, with which both the content of intracellular siRNAs and changes in protein expressing levels have been quantified in group cells and cellular population. We also observed that with the better cell uptake, CLD provided siRNA therapeutics （siBraf） better antitumor capability. This novel strategy was proved to be with efficiency, accuracy and high competency to adherent cell lines, thus making siRNA research more simplified.

低碳氮比规模化养猪场废水处理改造工程实例

针对某养猪场养猪废水高NH_4^+-N含量和低碳氮比等特点,采用上流式厌氧污泥床(UASB)+分段进水2级A/O+芬顿氧化+折点氯化除氮组合工艺对原有处理装置进行改造。原处理工艺无针对性,只进行厌氧+好氧生化处理,很难将废水中难降解物质去除,且脱氮除磷效果差,导致出水无法满足GB 8978-1996排放要求。改造后的实际运行结果表明,装置运行稳定,出水COD≤100 mg/L,NH4+-N、TP的质量浓度分别≤15、≤0.5 mg/L,达到GB8978-1996规定的一级排放标准。组合工艺充分利用现有设施,处理效率高、运行能耗低、管理简单方便,可有效保障出水各项指标稳定达标。

Screening of the ubiquitin-proteasome system activators for anti-Alzheimer’s disease by the high-content fluorescence imaging system

Ubiquitin-proteasome system(UPS)plays an important role in neurodegenerative diseases,such as Alzheimer’s disease(AD),Parkinson’s disease(PD),and Huntington’s disease(HD).The discovery of UPS activators for anti-neurodegenerative diseases is becoming increasingly important.In this study,we aimed to identify potential UPS activators using the high-throughput screening method with the high-content fluorescence imaging system and validate the neuroprotective effect in the cell models of AD.At first,stable YFP-CL1 HT22 cells were successfully constructed by transfecting the YFP-CL1 plasmid into HT22 cells,together with G418 screening.The degradation activity of the test compounds via UPS was monitored by detecting the YFP fluorescence intensity reflected by the ubiquitin-proteasome degradation signal CL1.By employing the high-content fluorescence imaging system,together with stable YFP-CL1 HT22 cells,the UPS activators were successfully screened from our established TCM library.The representative images were captured and analyzed,and quantification of the YFP fluorescence intensity was performed by flow cytometry.Then,the neuroprotective effect of the UPS activators was investigated in pEGFP-N1-APP(APP),pRK5-EGFP-Tau P301L(Tau P301L),or pRK5-EGFP-Tau(Tau)transiently transfected HT22 cells using fluorescence imaging,flow cytometry,and Western blot.In conclusion,our study established a high-content fluorescence imaging system coupled with stable YFP-CL1 HT22 cells for the highthroughput screening of the UPS activators.Three compounds,namely salvianolic acid A(SAA),salvianolic acid B(SAB),and ellagic acid(EA),were identified to significantly decrease YFP fluorescence intensity,which suggested that these three compounds are UPS activators.The identified UPS activators were demonstrated to clear AD-related proteins,including APP,Tau,and Tau P301L.Therefore,these findings provide a novel insight into the discovery and development of anti-AD drugs.

原料性能对加氢裂化装置长周期运行的影响

结合长庆石化120×104t/a加氢裂化装置运行数据,从原料油预处理过程,原料油的硫、氮含量、重杂质含量、金属含量及原料油干点几方面进行分析,总结出维持加氢裂化装置长周期运行的改进措施:鉴于原料油罐倒罐频繁,建议增加一具原料油罐,以保证进料前的充分沉降,除去原料油中的固体颗粒和水,减少原料油过滤器反冲洗频次;加强与上游装置的联系,严格常减压装置减压塔切割效率,密切监控原料油性质变化,增加色度分析法,监控VGO中重杂质含量,以指导加氢裂化生产,改善装置操作;由于减压蜡油硫低氮高,需要向系统补充一定量的硫,同时保持注水,减少循环氢中的氨浓度,但注水增加后,又伴随着循环氢中硫化氢的损失,因此从经济上考虑,需要保持三者的平衡,才能保证催化剂活性,维持装置的长周期运行;如果改善原料性质,维持循环氢中的硫化氢含量,降低氨含量,或者选用适合低硫原料油的催化剂,可提高装置运行周期。

煤气化变换工艺冷凝液氨氮脱除技术的应用

降低煤气化变换工艺冷凝液汽提过程的蒸汽消耗以及冷凝液中氨氮含量,是煤化工生产必须解决的问题。简要介绍了煤气化变换工艺冷凝液汽提原理和汽提工艺流程,采用高闪气热量对变换高温冷凝液和低温冷凝液中的氨氮进行汽提处理,以降低气化凝液中的氨氮,并回收有效气。经过汽提处理后,高温冷凝液和低温冷凝液中氨氮明显降低,均在200 mg/L以下;蒸汽消耗量明显降低,处理1 t高、低温变换冷凝液,蒸汽消耗量为21.1 kg。