Honokiol-enhanced cytotoxic T lymphocyte activity against cholangiocarcinoma cells mediated by dendritic cells pulsed with damage-associated molecular patterns

BACKGROUND Cholangiocarcinoma or biliary tract cancer has a high mortality rate resulting from late presentation and ineffective treatment strategy. Since immunotherapy by dendritic cells (DC) may be beneficial for cholangiocarcinoma treatment but their efficacy against cholangiocarcinoma was low. We suggest how such antitumor activity can be increased using cell lysates derived from an honokioltreated cholangiocarcinoma cell line (KKU-213L5). AIM To increase antitumour activity of DCs pulsed with cell lysates derived from honokiol-treated cholangiocarcinoma cell line (KKU-213L5). METHODS The effect of honokiol, a phenolic compound isolated from Magnolia officinalis, on choangiocarcinoma cells was investigated in terms of the cytotoxicity and the expression of damage-associated molecular patterns (DAMPs). DCs were loaded with tumour cell lysates derived from honokiol-treated cholangiocarcinoma cells their efficacy including induction of T lymphocyte proliferation, proinflammatory cytokine production and cytotoxicity effect on target cholangiocarcinoma cells were evaluated. RESULTS Honokiol can effectively activate cholangiocarcinoma apoptosis and increase the release of damage-associated molecular patterns. DCs loaded with cell lysates derived from honokiol-treated tumour cells enhanced priming and stimulated T lymphocyte proliferation and type I cytokine production. T lymphocytes stimulated with DCs pulsed with cell lysates of honokiol-treated tumour cells significantly increased specific killing of human cholangiocarcinoma cells compared to those associated with DCs pulsed with cell lysates of untreated cholangiocarcinoma cells. CONCLUSION The present findings suggested that honokiol was able to enhance the immunogenicity of cholangiocarcinoma cells associated with increased effectiveness of DC-based vaccine formulation. Treatment of tumour cells with honokiol offers a promising approach as an ex vivo DC-based anticancer vaccine.

Study on gene expression patterns and functional pathways of peripheral blood monocytes reveals potential molecular mechanism of surgical treatment for periodontitis

BACKGROUND Periodontitis is a chronic inflammation of periodontal supporting tissue caused by local factors. Periodontal surgery can change the gene expression of peripheral blood mononuclear cells. However, little is known about the potential mechanism of surgical treatment for periodontitis. AIM To explore the potential molecular mechanism of surgical treatment for periodontitis. METHODS First, based on the expression profiles of genes related to surgical treatment for periodontitis, a set of expression disorder modules related to surgical treatment for periodontitis were obtained by enrichment analysis. Subsequently, based on crosstalk analysis, we proved that there was a significant crosstalk relationship between module 3 and module 5. Finally, based on predictive analysis of multidimensional regulators, we identified a series of regulatory factors, such as endogenous genes, non-coding RNAs (ncRNAs), and transcription factors, which have potential regulatory effects on periodontitis. RESULTS A total of 337 genes related to surgical treatment for periodontitis were obtained, and 3896 genes related to periodontitis were amplified. Eight expression modules of periodontitis were obtained, involving the aggregation of 2672 gene modules. These modules are mainly involved in G-protein coupled receptor signaling pathway, coupled to cyclic nucleotide second messenger, and adenylate cyclasemodulating G-protein coupled receptor signaling pathway. In addition, eight endogenous genes (including EGF, RPS27A, and GNB3) were screened by network connectivity analysis. Finally, based on this set of potential dysfunction modules, 94 transcription factors (including NFKB1, SP1, and STAT3) and 1198 ncRNAs (including MALAT1, CRNDE, and ANCR) were revealed. These core regulators are thought to be involved in the potential molecular mechanism of periodontitis after surgical treatment. CONCLUSION Based on the results of this study, we can show biologists and pharmacists a new idea to reveal the potential molecular mechanism of surgical treatment for periodontitis, and provide valuable reference for follow-up treatment programs.

Damage-associated molecular patterns in inflammatory bowel disease:From biomarkers to therapeutic targets

The chronic inflammatory process underlying inflammatory bowel disease (IBD), comprising Crohn's disease and ulcerative colitis, derives from the interplay of several components in a genetically susceptible host. These components include environmental elements and gut microbiota a dysbiosis. For decades, immune abnormalities have been investigated as critically important in IBD pathogenesis, and attempts to develop effective therapies have predominantly targeted the immune system. Nevertheless, immune events represent only one of the constituents contributing to IBD pathogenesis within the context of the complex cellular and molecular network underlying chronic intestinal inflammation. These factors need to be appreciated within the milieu of nonimmune components. Damage-associated molecular patterns (DAMPs), which are essentially endogenous stress proteins expressed or released as a result of cell or tissue damage, have been shown to act as direct proinflammatory mediators. Excessive or persistent signalling mediated by such molecules can underlie several chronic inflammatory disorders, including IBD. The release of endogenous DAMPs amplifies the inflammatory response driven by immune and non-immune cells and promotes epigenetic reprogramming in IBD.The effects determine pathologic changes,which may sustain chronic intestinal inflammation and also underlie specific disease phenotypes.In addition to highlighting the potential use of DAMPs such as calprotectin as biomarkers,research on DAMPs may reveal novel mechanistic associations in IBD pathogenesis and is expected to uncover putative therapeutic targets.

Study on the Mechanism of Nanopatterning in Printed Electronics Based on Molecular Dynamics Simulation

In order to research the feasibility of using the selective adsorption principle to achieve automatic shaping of nano patterns,in this study,using the liquid gallium as the conductive ink and graphene as the printing plate surface,by changing the surface wettability of patterned areas on the nanoscale of graphene printed boards,the automatic formation of liquid gallium patterns on the graphene printed plate surface was simulated.The results indicated that liquid gallium can achieve automatic patterning on the surface of graphene patterned areas;the greater the interaction energy between gallium and carbon atoms,the clearer the pattern;gallium liquid is prone to remain in complex local positions of the pattern,making it difficult to shape the pattern;if the spacing between adjacent pattern lines is too large or too small,it will result in residual gallium liquid between the lines;increasing the thickness of the gallium film will cause the pattern to expand beyond the boundary,but increasing the thickness of the gallium film can also enhance the thickness and uniformity of the pattern lines.In summary,the principle of selective adsorption can be used to achieve the automatic formation of nano patterns,and the pattern formation effect is influenced by factors such as atomic interaction energy and pattern configuration.

Potential of damage associated molecular patterns in synergising radiation and the immune response in oesophageal cancer

BACKGROUND There is an intimate crosstalk between cancer formation,dissemination,treatment response and the host immune system,with inducing tumour cell death the ultimate therapeutic goal for most anti-cancer treatments.However,inducing a purposeful synergistic response between conventional therapies and the immune system remains evasive.The release of damage associated molecular patterns(DAMPs)is indicative of immunogenic cell death and propagation of established immune responses.However,there is a gap in the literature regarding the importance of DAMP expression in oesophageal adenocarcinoma(OAC)or by immune cells themselves.AIM To investigate the effects of conventional therapies on DAMP expression and to determine whether OAC is an immunogenic cancer.METHODS We investigated the levels of immunogenic cell death-associated DAMPs,calreticulin(CRT)and HMGB1 using an OAC isogenic model of radioresistance.DAMP expression was also assessed directly using ex vivo cancer patient T cells(n=10)and within tumour biopsies(n=9)both pre and post-treatment with clinically relevant chemo(radio)therapeutics.RESULTS Hypoxia in combination with nutrient deprivation significantly reduces DAMP expression by OAC cells in vitro.Significantly increased frequencies of T cell DAMP expression in OAC patients were observed following chemo-(radio)therapy,which was significantly higher in tumour tissue compared with peripheral blood.Patients with high expression of HMGB1 had a significantly better tumour regression grade(TRG 1-2)compared to low expressors.CONCLUSION In conclusion,OAC expresses an immunogenic phenotype with two distinct subgroups of high and low DAMP expressors,which correlated with tumour regression grade and lymphatic invasion.It also identifies DAMPs namely CRT and HMGB1 as potential promising biomarkers in predicting good pathological responses to conventional chemo(radio)therapies currently used in the multimodal management of locally advanced disease.

Heterotic Classes and Utilization Patterns in Chinese Foxtail Millet [<i>Setaria italica</i>(L.) P. Beauv]

Utilization of heterosis to develop hybrid cultivars can significantly increase yield of most crops including foxtail millet. However, previous foxtail millet hybrid cultivars have been largely developed from crosses between sterile lines and conventional varieties or between sterile lines and varieties that are geographically distent from the sterile lines. The research on classification of heterotic classes and determinetaion of heterotic patterns has not been reported, which results in uncertainty in selection of parents for crosses and delays progress in utilization of high yielding hybrids in large-scale commercial production. In this study, a core collection of 128 accessions from China was grouped into six classes using combined analyses of population structure, pedigree, and clustering. The classification was conducted based mainly on molecular clustering of genotypic data, also considered the population structure and mathematical clustering using phenotypic data, and was finally validated through pedigree analysis. According to the transgressive and superstandard heterosis for grain yield, plant height, panicle length, panicle diameter, single panicle weight, grain weight per panicle, and 1000-grain weight collected from an incomplete-diallel-cross experiment, we identified six superior heterosis patterns (C2/C1, C2/C4, C2/C5, C2/C6, C1/C5 and C4/C5) and four inferior heterosis patterns (C1/C3, C1/C4, C1/C6 and C4/C6), and explored their potential applications in millet hybrid breeding. This study laid a foundation for effective use of foxtail millet heterosis in improving millet hybrid yield.

Observation of Vortex Patterns in a Magnetized Dusty Plasma System

Vortex patterns of dust particles have been observed in a magnetized dusty plasma system. The formation mechanism of two-dimensional （2D） vortex patterns has been investigated by analysing the forces acting on dust particles and molecular dynamics （MD） simulations in a 2D confined magnetized dusty plasma. It has been found that with a weak confining electric field and a strong magnetic field, the particles＇ trajectories will form a vortex shape. The simulation results agree with our experimental observations. In our experiments, vortex patterns can be induced via circular rotation of particles by changing the rf （radio-frequency） power in a magnetized dusty plasma.

Effects of Different Cultivation Patterns of Wheat on Population Structure of Puccinia striiformis West.f. sp. tritici

The paper was to study the effects of different cultivation patterns( mix cultivation and monocultivation) of wheat on population structure of Puccinia striiformis West. f. sp. tritici in the fields. Five race-specific-markers( CY32,CY31,CY29,CY23 and Shuiyuan pathotype) were used to survey 113 infected samples collected from two cultivation patterns. The results indicated that frequency of race-specific-markers under monocultivation was higher than that under mix cultivation; the dominant race-specific-markers were CY32 and CY29 under monocultivation,and the frequency of detection were 81. 5% and 78. 5%,respectively. The dominant race-specific-markers were CY29 and Shuiyuan pathotype under mix cultivation,and the frequency of detection are 41. 7% and 18. 8%,respectively.Several race-specific-markers were detected in single infected leaf,and 41. 7% of infected single leaf were detected with more than two race-specific-markers,58. 3% of infected single leaf were detected with one race-specific-marker under mix cultivation pattern,while there were 75. 0% infected leaves with more than two race-specific-markers and 25. 0% infected single leaf detected with one race-specific-marker under monocultivation pattern. The results indicated that mix cultivation pattern of wheat can reduce races on single leaf,affect the distribution of races in infected leaves,and suppress the occurrence frequency of dominant races of P. striiformis in the fields significantly,subsequently reduced severity and prevalence of the disease.

Peri-Net-Pro: the neural processes with quantified uncertainty for crack patterns

This paper develops a deep learning tool based on neural processes(NPs)called the Peri-Net-Pro,to predict the crack patterns in a moving disk and classifies them according to the classification modes with quantified uncertainties.In particular,image classification and regression studies are conducted by means of convolutional neural networks(CNNs)and NPs.First,the amount and quality of the data are enhanced by using peridynamics to theoretically compensate for the problems of the finite element method(FEM)in generating crack pattern images.Second,case studies are conducted with the prototype microelastic brittle(PMB),linear peridynamic solid(LPS),and viscoelastic solid(VES)models obtained by using the peridynamic theory.The case studies are performed to classify the images by using CNNs and determine the suitability of the PMB,LBS,and VES models.Finally,a regression analysis is performed on the crack pattern images with NPs to predict the crack patterns.The regression analysis results confirm that the variance decreases when the number of epochs increases by using the NPs.The training results gradually improve,and the variance ranges decrease to less than 0.035.The main finding of this study is that the NPs enable accurate predictions,even with missing or insufficient training data.The results demonstrate that if the context points are set to the 10th,100th,300th,and 784th,the training information is deliberately omitted for the context points of the 10th,100th,and 300th,and the predictions are different when the context points are significantly lower.However,the comparison of the results of the 100th and 784th context points shows that the predicted results are similar because of the Gaussian processes in the NPs.Therefore,if the NPs are employed for training,the missing information of the training data can be supplemented to predict the results.

Nanoindentation Models of Monolayer Graphene and Graphyne under Point Load Pattern Studied by Molecular Dynamics

Molecular dynamics simulations are performed to study the nanoindentation models of monolayer suspended graphene and graphyne. Fullerenes are selected as indenters. Our results show that Young＇s modulus of monolayer-thick graphyne is almost half of that of graphene, which is estimated to be 0.50 TPa. The mechanical properties of graphene and graphyne are different in the presence of strain. A pre-tension has an important effect on the mechanical properties of a membrane. Both the pre-tension and Young＇s modulus plots demonstrate index behavior. The toughness of graphyne is stronger than that of graphene due to Young＇s modulus magnitude. Young＇s moduli of graphene and graphyne are almost independent of the size ratio of indenter to membrane.

Old and new damage‐associated molecular patterns (DAMPs) in autoimmune diseases

All organisms living in complex environments have evolved effective mechanisms of dynamic responses to extracellular stimuli.The immune system activates when damaged or injured cells release damage‐associated molecular patterns(DAMPs).In addition to well‐characterized DAMPs such as high‐mobility group box 1 and adenosine triphosphate,studies on new classes of DAMPs have emerged.Here,we review recent reports of a new class of isoprenoid‐derived DAMPs,including farnesyl pyrophosphate and geranylgeranyl pyrophosphate,both of which are pivotal metabolic inter-mediates of the mevalonate pathway.We also explore the roles of old and new DAMPs in autoimmune diseases that result from dysregulated inflammation.The findings highlight that understanding the functional mechanisms of DAMPs is important to enrich the DAMP family and decipher their immunoregulatory mechanisms to provide new therapeutics for the prevention and treatment of autoimmune diseases.

坏死性凋亡分子机制及与牙周炎的相关性

背景:近年来,随着细胞死亡研究的不断深入,坏死性凋亡逐渐成为学术界研究的热点和难点,其多种信号通路及蛋白在牙周炎的发生发展中起到不可忽视的作用,人们试图通过阻止细胞坏死性凋亡的发生,从而延缓牙周组织炎症的进程。目的:就坏死性凋亡的分子机制及其与牙周炎的相关性进行综述,期望为牙周炎的预防、诊断、治疗及预后提供新思路、新方向。方法:第一作者于2023年10月应用计算机检索PubMed、中国知网等数据库建库到2024年4月发表的相关文献,以“坏死性凋亡,细胞程序性死亡,牙周炎,牙周,免疫,炎症,受体相互作用蛋白激酶1,受体相互作用蛋白激酶3,混合系列蛋白激酶样结构域”为中文检索词;“necroptosis,programmed cell death,periodontitis,periodontal,immunity,inflammation,RIP1,RIP3,MLKL”为英文检索词,阅读每篇文献的文题、摘要、引言进行初步筛选,最终纳入56篇文献进行总结分析。结果与结论:①牙周炎的主要发病机制为牙周致病菌刺激机体,导致牙周微环境发生改变,打破原有免疫平衡并释放各种炎症因子,引发炎症反应造成牙周组织破坏,坏死性凋亡可以调控牙周组织免疫炎症反应,从而影响牙周炎的发生发展进程;②坏死性凋亡的发生与多种蛋白、信号通路及信号分子有关,已有动物实验证实了磷酸化混合系列蛋白激酶样结构域(pMLKL)的表达量与炎症程度有关,且在患有牙周炎小鼠的牙周组织中,RIP3和pMLKL高表达,通过阻断RIP3/MLKL通路,有利于减少牙周组织炎症、控制牙周炎的进展;③探究pMLKL是否可作为牙周炎的诊断指标,在牙周炎的预防、诊断及治疗上有很大价值。

Establishment,Characterization and Expression Pattern of a Spleen Cell Line,SMSP,Derived from Turbot(Scophthalmus maximus)

A turbot(Scophthalmus maximus)cell line named SMSP was obtained from the spleen.The origin of the cells was identified by morphology,chromosome number and COI gene.The optimal basic medium,serum concentration and growth temperature of the cells were detected.SMSP cell line is mainly composed of fibroblast-like cells.Most of the SMSP cells contained 44 chromosomes,and the sequence of COI gene confirmed that the cells were originated from turbot.The optimal culture conditions were 24℃,DMEM+10%FBS.The cell line had high transfection efficiency for siRNA and plasmid.After stimulation with lipopolysaccharide(LPS)or poly(I:C),the expressions of immune-related genes such as TNF-β,IL-12s,IL-10 and IL-1βwere up-regulated significantly in the early stage(P<0.05).This study will provide a model for exploring immune mechanism of turbot against pathogen in vitro.

High mobility group box 1 in the central nervous system:regeneration hidden beneath inflammation

High-mobility group box 1 was first discovered in the calf thymus as a DNA-binding nuclear protein and has been widely studied in diverse fields,including neurology and neuroscience.High-mobility group box 1 in the extracellular space functions as a pro-inflammatory damage-associated molecular pattern,which has been proven to play an important role in a wide variety of central nervous system disorders such as ischemic stroke,Alzheimer’s disease,frontotemporal dementia,Parkinson’s disease,multiple sclerosis,epilepsy,and traumatic brain injury.Several drugs that inhibit high-mobility group box 1 as a damage-associated molecular pattern,such as glycyrrhizin,ethyl pyruvate,and neutralizing anti-high-mobility group box 1 antibodies,are commonly used to target high-mobility group box 1 activity in central nervous system disorders.Although it is commonly known for its detrimental inflammatory effect,high-mobility group box 1 has also been shown to have beneficial pro-regenerative roles in central nervous system disorders.In this narrative review,we provide a brief summary of the history of high-mobility group box 1 research and its characterization as a damage-associated molecular pattern,its downstream receptors,and intracellular signaling pathways,how high-mobility group box 1 exerts the repair-favoring roles in general and in the central nervous system,and clues on how to differentiate the pro-regenerative from the pro-inflammatory role.Research targeting high-mobility group box 1 in the central nervous system may benefit from differentiating between the two functions rather than overall suppression of high-mobility group box 1.

Clinical distribution and molecular profiling on postoperative colorectal cancer patients with different traditional Chinese medicine syndromes

Background:Traditional Chinese medicine(TCM)syndrome,also named syndrome,are comprehensive and integral analyses of clinical information which helps to guide different individualized treatment prescriptions.Methods:Thirty healthy controls and 80 colorectal cancer(CRC)patients(including 33 Spleen Qi Deficiency syndrome,23 Dampness Heat syndrome,17 Blood Stasis syndrome and 7 other syndrome)were enrolled into this study.Human mRNAs were extracted from peripheral blood mononuclear cells.The gene expression for CRC patients with different TCM syndrome was determined by microarray and qRT-PCR.Results:Spleen Qi Deficiency,Dampness Heat and Blood Stasis were the most common syndromes in CRC patients.There is a significant difference was found in mRNA expression levels(especially for PIK3CA,STAT3,SOX9 and KDM5C)among Spleen Qi Deficiency,Dampness Heat and Blood Stasis syndrome groups.The higher mRNA levels of JNK1,TP53,MLH1,MSH6,PMS2,SOCS3,TCF7L2,FAM123B,PSAP,FBXW7,SALL4 and the lower expression of inflammatory cytokine IL-6 were found in Spleen Qi Deficiency group but not other syndrome types.The higher mRNA levels of KRAS,MUC16,EGFR,GRASP65,PIK3CA,MAPK7,CD24,STAT3,SLC11A1,Bcl-2,TXNDC17 and some inflammatory cytokines(IL-6,IL-23,TNF-a,CXCR4)were found in Dampness Heat group but not other syndrome types.Blood Stasis syndrome showed higher expression of SOX9,MLH1,MSH6,KDM5C,PCDH11X,PSAP and SALL4,and lower mRNA levels of PIK3CA,CD24,STAT3,CXCR4,TXNDC17 and TP53.The CRC patients with Dampness Heat syndrome might have a poor prognosis than other syndrome types.Conclusion:The identification of syndrome conditions had different impacts on CRC prognosis,and which might be related with different mRNA expression levels.Some oncogenes and pro-inflammatory cytokines were highly expressed in Dampness Heat group but not other syndrome types,suggesting that the CRC patients with Dampness Heat syndrome might have a poor prognosis.Our results prelimitarily uncovered the molecular basis of syndrome differences in CRC prognosis,a better understanding for TCM treatment of CRC.

Inertia-Centric Stability Analysis of a Planar Uniform Dust Molecular Cloud with Weak Neutral-Charged Dust Frictional Coupling

This paper adopts an inertia-centric evolutionary model to study the excitation mechanism of new gravito-electrostatic eigenmode structures in a one-dimensional（1-D） planar self-gravitating dust molecular cloud（DMC） on the Jeans scale.A quasi-neutral multi-fluid consisting of warm electrons,warm ions,neutral gas and identical inertial cold dust grains with partial ionization is considered.The grain-charge is assumed not to vary at the fluctuation evolution time scale.The neutral gas particles form the background,which is weakly coupled with the collapsing grainy plasma mass.The gravitational decoupling of the background neutral particles is justifiable for a higher inertial mass of the grains with higher neutral population density so that the Jeans mode frequency becomes reasonably large.Its physical basis is the Jeans assumption of a self-gravitating uniform medium adopted for fiducially analytical simplification by neglecting the zero-order field.So,the equilibrium is justifiably treated initially as ＂homogeneous＂.The efficacious inertial role of the thermal species amidst weak collisions of the neutral-charged grains is taken into account.A standard multiscale technique over the gravito-electrostatic equilibrium yields a unique pair of Korteweg-de Vries（KdV） equations.It is integrated numerically by the fourth-order Runge-Kutta method with multi-parameter variation for exact shape analyses.Interestingly,the model is conducive for the propagation of new conservative solitary spectral patterns.Their basic physics,parametric features and unique characteristics are discussed.The results go qualitatively in good correspondence with the earlier observations made by others.Tentative applications relevant to space and astrophysical environments are concisely highlighted.

四川盆地南部下寒武统筇竹寺组页岩孔隙结构特征与页岩气赋存模式

近年来,四川盆地南部地区(以下简称川南地区)下寒武统筇竹寺组页岩气勘探开发取得了实质性的突破,但是,关于筇竹寺组页岩气储层孔隙结构特征及其对页岩气赋存模式影响的研究还较为薄弱。为此,以川南地区W207井筇竹寺组页岩为例,以总有机碳含量(TOC)不小于1.0%为界,将筇竹寺组页岩划分出4个高有机碳页岩层段(H1—H4层),并基于大视域扫描电镜、流体注入法孔隙定量表征、三维分子结构建模与分子模拟等方法,研究了不同富有机质层段页岩孔隙特征,并着重分析了H3层页岩孔隙发育特征与页岩气赋存模式。研究结果表明:(1)筇竹寺组页岩纵向上孔隙发育差异较大,其中H3层页岩孔隙系统最为有利,发育粒缘缝—有机质纳米连通孔隙—有机质基质分子内孔隙3级孔隙网络,其矿物粒缘缝与有机质纳米孔隙提供了较大的游离气储集空间,页岩有机质分子结构中普遍发育微孔,提供了较大的吸附空间;(2)微孔对总吸附量的贡献随压力增高而降低,但在30.0 MPa时仍贡献了56%以上的总吸附气量;(3)高温高压下(30.0 MPa、70℃),页岩中游离气甲烷含量占总含气量的57%,当压力由30.0 MPa降低至16.5 MPa,游离气贡献总开采气量超过80%;(4)粒缘缝、有机质纳米连通孔隙和有机质基质分子内孔隙良好的配置关系为页岩气富集及开发提供了良好的连通体系,远离风化壳的超压层段有利于页岩孔隙发育。结论认为,川南地区筇竹寺组H3层页岩孔隙系统发育,具备页岩气高产地质条件,是筇竹寺组下一步重点关注层段,在此基础上寻找远离风化壳、孔隙发育的超压页岩储层是筇竹寺组选区选层的关键。

New substituted molecular classifications of advanced gastric adenocarcinoma:characteristics and probable treatment strategies

Background:Gastric adenocarcinoma(GA)is a heterogeneous tumor,and the accurate classification of GA is important.Previous classifications are based on molecular analysis and have not focused on GA with the primitive enterocyte phenotype(GAPEP),a unique subtype with a poor prognosis and frequent liver metastases.New substituted molecular(SM)classifications based on immunohistochemistry(IHC)are needed.Methods:According to the IHC staining results,we divided 582 cases into six types:mismatch repair deficient(dMMR),Epstein-Barr virus associated(EBVa),the primitive enterocyte phenotype(PEP),the epithelial mes-enchymal transition(EMT)phenotype,not otherwise specified/P53 mutated(NOS/P53m)and not otherwise specified/P53 wild-type(NOS/P53w).We analyzed the clinicopathological features,the immune microenviron-ment(PD-L1,CD8)and expression of HER2 and VEGFR2 of those types.Results:There were 31(5.3%)cases of the dMMR type,13(2.2%)cases of the EBVa type,44(7.6%)cases of the PEP type,122(21.0%)cases of the EMT type,127(21.8%)cases of the NOS/P53m type and 245(42.1%)cases of the NOS/P53w type.Patients with the dMMR type had the best survival(P<0.001).Patients with the EBVa type were younger(P<0.001)and had higher PD-L1 and CD8 expression(P<0.001)than other patients.Patients with the EMT type exhibited poor differentiation and a higher rate of abdominal metastasis.Patients with the NOS/P53m and PEP types had the worst survival rates and the highest PD-L1/HER2/VEGFR2 expression levels among all patients(P<0.001).Conclusion:Different SM classifications have different clinicopathological features and expression patterns,which indicate the probable clinical treatment strategies for these subtypes.

西方蜜蜂AKHR基因的生物信息学及表达模式分析

本研究对西方蜜蜂Apis mellifera脂动激素受体(Adipokinetic hormone receptor,AKHR)基因AmAKHR CDS区进行RT-PCR扩增和生物信息学分析,并测定了AmAKHR在工蜂不同组织和不同发育阶段的表达模式,旨在为进一步的功能研究提供参考和基础。通过PCR扩增AmAKHR的CDS,使用相关软件预测AmAKHR蛋白的理化性质、分子特征及蛋白互作网络。采用RT-qPCR技术检测AmAKHR在工蜂不同组织和不同发育阶段的相对表达量。结果表明,AmAKHR基因CDS长度为1050 bp,编码349个氨基酸;AmAKHR的相对分子量约为40.63 kDa,分子式为C1873H2939N471O484S26,含39个磷酸化位点,7个α-跨膜螺旋结构及8个保守基序,不含信号肽,主要分布于质膜;AmAKHR与小蜜蜂Apis florea的AKHR在进化树上聚为一支;AmAKHR的二级结构包含127个无规则卷曲,124个α-螺旋,87条延伸链和11个β-转角;AmAKHR与卵黄原蛋白和神经肽Corazonin等10个蛋白构成1个互作网络;AmAKHR在西方蜜蜂工蜂的毒腺、中肠、咽下腺、脂肪体、脑和触角等6个组织中差异表达,在脂肪体中的表达量最高,而在中肠中的表达量最低;AmAKHR在西方蜜蜂工蜂1日龄幼虫、3日龄幼虫、5日龄幼虫、2日龄预蛹、1日龄蛹中均有表达,且表达量随着日龄的增长而持续上调。研究结果揭示AmAKHR为疏水性蛋白和膜蛋白,并与卵黄原蛋白和神经肽等10个蛋白潜在互作,AmAKHR可能在西方蜜蜂工蜂的能量代谢、脂类分解和变态发育中发挥作用。

黄曲条跳甲短链气味结合蛋白基因PstrOBP-sc分子和功能特点分析

【目的】分析黄曲条跳甲(Phyllotreta striolata)短链气味结合蛋白(Odorant binding protein,OBP)基因PstrOBP-sc分子和功能特点,为揭示昆虫短链OBP基因的功能提供参考。【方法】利用逆转录聚合酶链反应(RT-PCR)克隆黄曲条跳甲短链Classic OBP基因,并对其进行生物信息学分析;通过逆转录聚合酶链反应技术分析短链OBP基因在黄曲条跳甲雌、雄成虫不同组织中的表达情况;构建重组表达载体异源表达短链OBP基因,以聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹法(Western blotting)验证目的蛋白;运用荧光结合试验检测短链OBP的配体结合能力。【结果】通过逆转录聚合酶链反应克隆验证,成功获得1条具有完整开放阅读框的黄曲条跳甲短链OBP基因PstrOBP-sc。PstrOBP-sc基因含有1个357 bp的开放阅读框,编码118个氨基酸残基,N末端具有1个由20个氨基酸残基组成的信号肽,成熟蛋白PSTROBP-SC仅由98个氨基酸残基组成,序列中含有6个保守的半胱氨酸,属于典型短链OBP基因。蛋白二级结构预测结果显示,PSTROBP-SC蛋白仅存在5个α-螺旋,且在第5个α-螺旋的下游仅有2个氨基酸残基。PstrOBP-sc基因组织表达模式及异源表达的PSTROBP-SC蛋白配体结合能力研究结果表明,PstrOBP-sc基因在黄曲条跳甲雌、雄成虫的所有供试组织中均有表达,而非特异性表达于嗅觉器官。同时,异源表达PSTROBP-SC不能结合荧光探针1-NPN。【结论】PstrOBP-sc基因的分子和功能特点不同于当前广泛研究的中长链OBP基因,其在生物体内很可能行使嗅觉以外的其他生理功能。