化妆品眼刺激性评价中角膜损伤生物标志物研究

针对离体兔眼(IRE)试验只适用于筛选严重眼刺激性(UN GHS第1类)的缺陷,建立IRE试验结合基于细胞活性生物标记物的荧光染色方法用于评价化妆品眼刺激性。通过IRE试验条件的探索,包括测试浓度和后暴露时间,对市售化妆品进行眼刺激性检测,同时对角膜进行冰冻切片并染色,鬼笔环肽对丝状肌动蛋白进行染色,DAPI染色胞核后,测量损伤深度(depth of injury,DOI),并比较体外DOI、体内DOI及Draize试验结果,建立预测模型。结果显示通过DOI检测,可对化妆品无刺激性至中刺激性进行检测,表明在离体兔眼中,基于细胞活力的DOI测量可作为角膜损伤的生物标志物,适用于化妆品眼刺激性的评价。

Numerical simulation study on the effect of temperature on the restricted diffusion in porous media

It is of great significance to study how temperature affects the restricted diffusion in pores for an accurate evaluation of reservoir physical properties by using nuclear magnetic resonance(NMR)diffusion-transverse relaxation(D-T2)spectrum under reservoir temperature conditions.In this paper,we simulate the restricted diffusion and twodimensional(2D)NMR D-T2 spectra of water molecules at different temperatures using random-walk method.The one-dimensional(1D)restricted diffusion simulation results show that the diffusion coefficient in the pore at room temperature decays with the diffusion time and eventually reaches a plateau.Under the condition of long-time diffusion,the ratio of restricted diffusion coefficient to bulk diffusion coefficient at different temperatures tends to be the same constant.With the increase in temperature,the simulated D-T2 spectra also gradually move upward.The simulated D-T2 spectra at all temperatures are consistent with the Pade interpolation equation.In addition,the results calculated by Pade interpolation equation demonstrate that the degree of temperature influence on the D-T2 spectrum of rock is quantitatively related to the pore radius,porosity and cementation index.

A Novel Surgery Classification for Endoscopic Approaches to Middle Ear Cholesteatoma

This study aimed to develop a novel surgery classification for an endoscopic approach to middle ear cholesteatoma.We retrospectively analyzed the surgical approaches and outcomes of patients with middle ear cholesteatoma.Middle ear cholesteatoma surgeries were divided into four types and two special types as follows:type I,attic retraction pocket,which only requires tympanostomy tube placement or retraction pocket resection and cartilage reconstruction;typeⅡ,cholesteatoma which is limited to the attic or in which endoscopy can confirm complete removal of mastoid cholesteatoma lesions,including type Ⅱa,requiring only use of a curette,and type Ⅱ b,requiring use of an electric drill or chisel;type Ⅲ,cholesteatoma not limited to the attic,in which endoscopy cannot confirm complete removal of mastoid cholesteatoma lesions,requiring the combined use of endoscope and microscope to perform endoscopic tympanoplasty and"Canal Wall Up"mastoidectomy;type Ⅳ,extensive involvement of mastoid cavity cholesteatoma lesions and/or cases with a potential risk of complications,removal of which can only be performed under a microscope for"Canal Wall Down"mastoidectomy.In addition,there were two special types:"difficult external auditory canal"and congenital cholesteatoma in children.In our system,type I and type U middle ear cholesteatoma surgery was completely performed under an endoscope alone.However,estimating the extent of the lesions,determining the choice of mastoid opening and reestablishing ventilation are the key points for an endoscopic approach to middle ear cholesteatoma.The classification of endoscopic middle ear cholesteatoma surgery may benefit the selection of surgical indications.

Systematic screening reveals synergistic interactions that overcome MAPK inhibitor resistance in cancer cells

Objective:Effective adjuvant therapeutic strategies are urgently needed to overcome MAPK inhibitor(MAPKi)resistance,which is one of the most common forms of resistance that has emerged in many types of cancers.Here,we aimed to systematically identify the genetic interactions underlying MAPKi resistance,and to further investigate the mechanisms that produce the genetic interactions that generate synergistic MAPKi resistance.Methods:We conducted a comprehensive pair-wise sgRNA-based high-throughput screening assay to identify synergistic interactions that sensitized cancer cells to MAPKi,and validated 3 genetic combinations through competitive growth,cell viability,and spheroid formation assays.We next conducted Kaplan-Meier survival analysis based on The Cancer Genome Atlas database and conducted immunohistochemistry to determine the clinical relevance of these synergistic combinations.We also investigated the MAPKi resistance mechanisms of these validated synergistic combinations by using co-immunoprecipitation,Western blot,qRTPCR,and immunofluorescence assays.Results:We constructed a systematic interaction network of MAPKi resistance and identified 3 novel synergistic combinations that effectively targeted MAPKi resistance(ITGB3+IGF1R,ITGB3+JNK,and HDGF+LGR5).We next analyzed their clinical relevance and the mechanisms by which they sensitized cancer cells to MAPKi exposure.Specifically,we discovered a novel protein complex,HDGF-LGR5,that adaptively responded to MAPKi to enhance cancer cell stemness,which was up-or downregulated by the inhibitors of ITGB3+JNK or ITGB3+IGF1R.Conclusions:Pair-wise sgRNA library screening provided systematic insights into elucidating MAPKi resistance in cancer cells.ITGB3-+IGF1R-targeting drugs(cilengitide+linsitinib)could be used as an effective therapy for suppressing the adaptive formation of the HDGF-LGR5 protein complex,which enhanced cancer stemness during MAPKi stress.

Comparative Study on Effects of Rheum officinale and Wine Prepared Rheum palmatum Dachengqi Decoction on Laxative Effect of Mice

[Objectives] To study the differences in the effects of Rheum officinale and wine prepared Rheum palmatum Dachengqi Decoction(DCQ) on laxative effect of mice.[Methods]First,72 mice were randomly divided into positive control group,blank control group,6 and10 g/kg dose R.officinale DCQ groups,6 and 10 g/kg dose wine prepared R.palmatum DCQ groups.After administration at the dose of20 m L/kg,metabolic cage method and carbon powder propulsion were used to conduct Na+-K+-ATPasc activity experiment,and the comparison was made for the differences in the normal laxative effect of R.officinale and wine prepared R.palmatum on DCQ,the carbon powder propulsion rate,and Na+-K+-ATPasc activit.[Results]There are differences in the laxative effect of R.officinale and wine prepared R.palmatum on DCQ,but their differences in changes are,to some extent,connected with R.officinale and wine prepared R.palmatum.From comparison of R.officinale DCQ group,wine prepared R.palmatum DCQ group,blank control group,and positive control group,it is found that there are differences in the normal laxative effect of normal mice,carbon powder propulsion,and inhibition of Na+-K+-ATPase activity.In addition,the comparison of drug administration groups indicates that the differences in the effects of R.officinale DCQ group are most significant,and there are significant differences in the 6 and 10 g/kg groups.[Conclusions]There are certain differences in the influence of R.officinale and wine prepared R.palmatum Dachengqi Decoction on normal laxative effect of mice,which may be connected with the fact that changes in main chemical components of king drugs R.officinale and wine prepared R.palmatum lead to changes in the dissolution amount of anthraquinones in the compound Dachengqi Decoction.

Water Extraction Process of Chinese Herbal Compound Man Gan Ning

[Objectives]To optimize the water extraction process of Chinese Herbal Compound Man Gan Ning and establish a method for its extraction and content determination.[Methods]The L9(34)orthogonal test was carried out,the comprehensive scores of ginsenoside Rb1 content,notoginsenoside R1 content,tanshinone I content and saikosaponin A content in the compound extract were used as evaluation indicators to explore the effects of water addition,the water addition,decoction time and decoction times on the water extraction process,and verification tests were carried out.[Results]The optimized water extraction process was as follows:adding 9 times of water in the first extraction,and extracting for 90 min;adding 7 times of water in the second extraction,and extracting for 60 min;adding 7 times of water in the third extraction,and extracting for 60 min.[Conclusions]The optimized water extraction process is stable and feasible,and can be used for the extraction of various herbs in Compound Man Gan Ning.It can also provide experimental basis for the formulation development of Compound Man Gan Ning sustained release tablet.

Effects of Different Processed Products of Ilex hainanensis Merr on Rats with Hyperlipidemia

[Objectives] This study aimed to investigate the hypolipidemic effects of different processed products of Ilex hainanensis Merr to provide experimental basis for the research on the hypolipidemic mechanism of I. hainanensis Merr. [Methods] SD rats were fed high-fat diet to establish hyperlipidemia rat models. The hyperlipidemia rats were administered with different processed products of I. hainanensis Merr by gavage to investigate the preventive and therapeutic effects of the medicinal material. [Results] The levels of total cholesterol(TC), triglyceride(TG), low density lipoprotein cholesterol(LDL-C) in the rats fed high-fat diet were significantly higher than those in of the control group(P<0.01), indicating successful modeling. Among different processing methods, the hypolipidemic effects were in the order as baking=traditional processing=frying=microwaving>drying in shade. Among different baking times, the hypolipidemic effects ranked as 180 ℃>200 ℃=220 ℃. Among different baking times, the hypolipidemic effects ranked as 20 min=40 min>30 min. The hypolipidemic effects of processed product of I. hainanensis Merr with different amounts of active ingredients were different. [Conclusions] The processed products of I. hainanensis Merr all had hypolipidemic effects, but the effects were different. This study will provide a scientific basis for expanding the resources of I. hainanensis Merr.

Effects of Dachengqi Decoctions Made from Raw Rhubarb and Vinegar-processed Rhubarb on Levels of Endotoxin,NO and TNF-αin Serum

[Objectives]The effects of Dachengqi decoctions made from raw rhubarb and vinegar-processed rhubarb on serum endotoxin,NO and TNF-αlevels were investigated.[Methods]Total 105 mice were randomly divided into Dachengqi decoction made from raw rhubarb groups(6,10 g/kg),Dachengqi decoction made from vinegar-processed rhubarb groups(6,10 g/kg),positive control group,blank control group and model group.After administration at a dose of 20 mL/kg,the levels of endotoxin,NO and TNF-αin serum were determined.The effects of Dachengqi decoctions made from raw rhubarb and vinegar-processed rhubarb on serum endotoxin,NO and TNF-αlevels in mice were compared.[Results]Dachengqi decoctions made from raw rhubarb and vinegar-processed rhubarb both increased the NO level and reduced the endotoxin and TNF-αlevels in serum of ABP mice.[Conclusions]Dachengqi decoctions made from raw rhubarb and vinegar-processed rhubarb have different effects on endotoxin,NO and TNF-αcontents.These changes correspond to the effects of Dachengqi Decoctions made from raw rhubarb and vinegar-processed rhubarb.The change in the content of rhubarb anthraquinones has a certain effect on the efficacy of Dachengqi decoction.

Building digital life systems for future biology and medicine

The rapid development of biological technology (BT) and information technology (IT) especially of genomics and artificial intelligence (AI) is bringing great potential for revolutionizing future medicine. We propose the concept and framework of Digital Life Systems or dLife as a new paradigm to unleash this potential. It includes the multi-scale and multi-granule measure and representation of life in the digital space, the mathematical and/or computational modeling of the biology behind physiological and pathological processes, and ultimately cyber twins of healthy or diseased human body in the virtual space that can be used to simulate complex biological processes and deduce effects of medical treatments. We advocate that dLife is the route toward future AI precision medicine and should be the new paradigm for future biological and medical research.

Explore the dominant factor in prime editing via a view of DNA processing

Prime editing is a revolutionary gene-editing method that is capable of introducing insertions,deletions and base substitutions into the genome.However,the editing efficiency of Prime Editor(PE)is limited by the DNA repair process.Here,we show that overexpression of the flap structure-specific endonuclease 1(FEN1)and the DNA ligase 1(LIG1)increases the efficiency of prime editing,which is similar to the dominant negative mutL homolog 1(MLH1dn).In addition,MLH1 is still the dominant factor over FEN1 and LIG1 in prime editing.Our results help to further understand the relationship of proteins involved in prime editing and envisage future directions for the development of PE.

基于无网格法的Timoshenko曲梁动力分析

曲梁具有外形美观、受力性能良好的优点,故在工程中得到广泛应用.论文基于移动最小二乘近似和一阶剪切变形理论,提出一种对Timoshenko曲梁自由振动和受迫振动进行分析的无网格方法.通过一系列离散点离散曲梁,建立曲梁无网格模型,然后推导曲梁势能和动能方程,通过哈密顿原理给出曲梁自由振动和受迫振动的控制方程,因为论文方法不能直接施加边界条件,所以使用完全转换法处理本质边界条件,最后求解方程得到频率和振动模态.文末通过算例验证了论文方法的有效性,且通过收敛性分析表明论文方法具有较好的收敛性,并进一步分析了不同边界条件、跨高比和变截面变曲率对曲梁自由振动和受迫振动的影响,将计算结果与文献解或ABAQUS解进行对比分析,表明论文方法具有较高的精度,且适用于实际工程情况.

An inducible CRISPR-ON system for controllable gene activation in human pluripotent stem cells

Human pluripotent stem cells （hPSCs） are an important system to study early human development, model human diseases, and develop cell replacement therapies. However, genetic manipulation of hPSCs is challenging and a method to simultaneously activate multiple genomic sites in a controllable manner is sorely needed. Here, we constructed a CRISPR.ON system to efficiently upregulate endogenous genes in hPSCs. A doxycycline （Dox） inducible dCasg-VP64-p65-Rta （dCas9-VPR） transcription activator and a reverse Tet transactivator （rtTA） expression cassette were knocked into the two alleles of the AAVS1 locus to generate an iVPR hESC line. We showed that the dCas9-VPR level could be precisely and reversibly controlled by the addition and withdrawal of Dox. Upon transfection of multiplexed gRNA plasmid targeting the NANOG pro- moter and Dox induction, we were able to control NANOG gene expression from its endogenous locus. Interestingly, an elevated NANOG level promoted naive pluripotsnt gene expression, enhanced cell survival and clonogenicity, and enabled hESCs to integrate with the inner cell mass （ICM） of mouse blastocysts in vitro. Thus, iVPR cells provide a convenient platform for gene function studies as well as high-throughput screens in hPSCs.

小胶质细胞抑制剂在神经性疼痛和吗啡耐受中的调节机制和治疗潜力

小胶质细胞是参与调节神经性疼痛(NPP)和吗啡耐受性的重要细胞。在确定它们的生理结构后,已经阐明了有关其可塑性和极性的信息,但关于这种类型的细胞在NPP和吗啡耐受性中的作用,仍有许多知识要学习。小胶质细胞通过控制中枢神经系统的损伤和对疾病的内源性免疫反应,介导健康和疾病的多种功能。小胶质细胞活化可导致阿片样物质系统活性改变,而NPP的特征在于对吗啡的抗性。在这里,我们研究小胶质细胞的调节机制,并综述了小胶质细胞抑制剂抑制NPP和吗啡耐受性的潜力。靶向的神经胶质活化是治疗NPP和预防吗啡耐受的临床有前途的方法。最后,我们为小胶质细胞抑制剂的未来研究提出了建议。

Regulation by competition:a hidden layer of gene regulatory network

Background:Molecular competition brings about trade-offs of shared limited resources among the cellular components,and thus introduces a hidden layer of regulatory mechanism by connecting components even without direct physical interactions.Several molecular competition scenarios have been observed recently,but there is still a lack of systematic quantitative understanding to reveal the essence of molecular competition.Methods:Here,by abstracting the analogous competition mechanism behind diverse molecular systems,we built a unified coarse-grained competition motif model to systematically integrate experimental evidences in these processes and analyzed general properties shared behind them from steady-state behavior to dynamic responses.Results:We could predict in what molecular environments competition would reveal threshold behavior or display a negative linear dependence.We quantified how competition can shape regulator-target dose-response curve,modulate dynamic response speed,control target expression noise,and introduce correlated fluctuations between targets.Conclusions:This work uncovered the complexity and generality of molecular competition effect as a hidden layer of gene regulatory network,and therefore provided a unified insight and a theoretical framework to understand and employ competition in both natural and synthetic systems.

Improved Fractional-order Damping Method for Voltage-controlled DFIG System Under Weak Grid

When a doubly-fed induction generator(DFIG)is connected to a weak grid,the coupling between the grid and the DFIG itself will increase,which will cause stability problems.It is difficult to maintain the tracking accuracy and robustness of the phase-locked loop(PLL)in the weak grid,and the risk of instability of the current-controlled DFIG(CC-DFIG)system will increase.In this paper,a new type of voltage-controlled DFIG(VC-DFIG)mode is adopted,which is a grid-forming structure that can independently support the voltage and frequency with a certain adaptability in the weak grid.A small-signal impedance model of the VC-DFIG system is also established.The impedance of DFIG inevitably generates coupling with the grid impedance in the weak grid,especially in parallel compensation grids,and results in resonance.On the basis of the VC-DFIG,impedance stability analysis is performed to study the influences of the control structure and short-circuit ratio.Then,a feedforward damping method is proposed to modify the impedance of the VC-DFIG system at resonance frequencies.The proposed fractional order damping is utilized,which can enhance the robustness and rapidity of resonance suppression under parameter fluctuations.Finally,the experimental results are presented to validate the effectiveness of the proposed control strategy.

Construction, characterization and application of a genomewide promoter library in Saccharomyces cerevisiae

Promoters are critical elements to control gene expression but could behave differently under various growth conditions. Here we report the construction of a genome-wide promoter library, in which each native promoter in Saccharomyces cerevisiae was cloned upstream of a yellow fluorescent protein （YFP） reporter gene. Nine libraries were arbitrarily defined and assembled in bacteria. The resulting pools of promoters could be prepared and transformed into a yeast strain either as centromeric plasmids or integrated into a genomic locus upon enzymatic treatment. Using fluorescence activated cell sorting, we classified the yeast strains based on YFP fluorescence intensity and arbitrarily divided the entire library into 12 bins, representing weak to strong promoters. Several strong promoters were identified from the most active bins and their activities were assayed under different growth conditions. Finally, these promoters were applied to drive the expression of genes in xylose utilization to improve fermentation efficiency. Together, this library could provide a quick solution to identify and utilize desired promoters under user-defined growth conditions.

A quantitative understanding of microRNA- mediated competing endogenous RNA regulation

MicroRNA （miRNA） plays key roles in post-transcriptional regulations. Recently, a competing endogenous RNA （ceRNA） hypothesis has been proposed that miRNA targets could communicate and regulate each other through titrating shared miRNAs, which provides a new layer of gene regulation. Though a number of ceRNAs playing biological functions have been identified, the ceRNA hypothesis remains controversial. Recent experimental and theoretical studies argued that the modulation of a single RNA species could hardly change the expression level of competing miRNA targets through ceRNA effect under normal physiological conditions. Here, we reviewed a common framework to model miRNA regulations, and summarized the current theoretical and experimental studies for quantitative understanding ceRNA effect. By revisiting a coarse-grained ceRNA model, we proposed that network topology could significantly influence the competing effect and ceRNA regulation at protein level could be much stronger than that at RNA level. We also provided a conditional independent binding equation to describe miRNA relative repression on different target, which could be applied to quantify siRNA off-target effect.

Construction of a CRISPR-based paired-sgRNA library for chromosomal deletion of long non-coding RNAs

Background:Derived from an adaptive bacterial immune system,the clustered regularly interspaced palindromic repeats(CRISPR)/CRISPR-associated 9(Cas9)system has shown great potential in high-throughput functional genomic screening,especially for protein-coding genes.However,it is still challenging to apply the similar strategy to study non-coding genomic elements such as long non-coding RNAs(lncRNAs)or clusters of microRNAs,because short insertions or deletions may not be sufficient to generate loss-of-function phenotypes.Methods:Here,we presented a systematic strategy for designing a CRISPR-based paired-sgRNA library for highthroughput screening in non-coding regions.Due to the abundance of lncRNAs and their diverse regulatory roles in vivo,we repurposed microarray datasets to select 600 highly expressed lncRNAs in non-small-cell lung cancer and designed two schemes for lncRNA deletion with^20 paired-sgRNAs for each lncRNA.Through Golden-Gate assembly,we generated a pooled CRISPR-based library with a total of 12,878 sgRNA pairs.Results:Over 80%of paired-sgRNAs were recovered from final pooled library with a relarively even distribution.Cleavage efficiency of sgRNA pairs was validated through experiments of transient transfection and viral infection.Moreover,randomly selected paired-sgRNAs showed that efficient deletion of genomic DNA could be achieved with a deletion size within the range of 500 to 3000 bp.Conclusions:In summary,we have demonstrated a strategy to design and construct a pooled paired-sgRNA library to generate genomic deletion in the lncRNA regions,validated their deletion efficiency and explored the relationship of deletion efficiency with respect to deletion size.This method would be also suitable for investigation of other uncharacterized non-coding genomic regions in mammalian cells in an efficient and cost-efTective manner.

Donor-acceptor 2D covalent organic frameworks for efficient heterogeneous photocatalyticα-oxyamination

Covalent organic frameworks(COFs)have received widespread interest due to their high porosity,excellent crystallinity,tailorable structures,and broad application prospects.It has been demonstrated that proper combination and arrangement of electron donor and acceptor units in 2D conjugated COF lattice could promote efficient charge separation and electron transfer,and thus is beneficial for photocatalysis.In this article,three donor-acceptor(D-A)2D COFs were prepared by Schiff base reaction of electron acceptor 4,4′,4′′,4′′′-(benzo[1,2-d:4,5-d′]bis(oxazole)-2,4,6,8-tetrayl)tetraaniline(BBO)with different electron donors:thieno[3,2-b]thiophene-2,5-dicarbaldehyde(TT),benzo[1,2-b:4,5-b’]dithiophene-2,6-dicarboxaldehyde(BDT)and terephthalaldehyde(Ph),respectively.These D-A 2D COFs exhibited prominent photocatalytic activity towardsα-oxyamination of 1,3-dicarbonyl with 2,2,6,6-tetramethyl-1-piperdinyloxy(TEMPO)upon visible light irradiation.Among these D-ABBO-COFs,DTT-ABBO-COF exhibited the highest photocatalytic rates,which can be ascribed to the more negative highest occupied molecular orbital(HOMO)and narrower bandgap.The excellent stability,high activity and superior recyclability render DTT-ABBO-COF as a potential and environmentally friendly heterogeneous catalyst forα-oxyamination.

Direct pore engineering of 2D imine covalent organic frameworks via sub-stoichiometric synthesis

Two-dimensional covalent organic frameworks(2D COFs)bearing various topologies can be precisely designed based on the symmetry of monomers.Their pore environment can be further regulated via either pre-functionalization of the monomers or post-modification of the skeleton.Among them,sub-stoichiometric synthesis is an efficient method which can mediate uncondensed functional moieties periodically arranged in the COF skeletons.Herein,a series of imine-linked 2D COFs with specifically decorated formyl or amino groups were selectively synthesized via sub-stoichiometric formyl transamination.The molar ratios of the monomers directly determine the structures of the resulting COFs with periodic vertex vacancies.The COF with polar amino sites could efficiently capture CO_(2) and H_(2)O.This work demonstrates tuning the stoichiometry of the monomers could facilely modulate the functions of the pore channels.It also provides insights into constructing novel COFs with complex structures for targeted applications.