functional assay

功能测定
  • 文章类型: Journal Article
    松柏是林业和农业中有效的授粉者,比其他蜜蜂具有更高的耐寒性。然而,其耐寒机制尚不清楚。水通道蛋白(AQP)作为细胞膜蛋白促进快速水流,帮助渗透调节。最近的研究强调了昆虫AQP在脱水和寒冷胁迫中的重要性。冷应激条件下的白棘芽孢杆菌的比较转录组分析揭示了四个AQP的上调,表明它们在耐寒性中的潜在作用。七个AQP-Eglp1,Eglp2,Eglp3,DRIP,PRIP,围兜,和AQP12L-已被鉴定为沙棘。这些在各种组织中广泛表达,特别是在消化道和马尔皮根小管中。对非洲爪狼卵母细胞表达系统中BterAQPs的功能分析显示出不同的水和甘油选择性,BterDrip具有最高的透水性。BterDrip的分子建模揭示了六个跨膜结构域,两个NPA图案,和AR/R收缩区域(Phe131,His256,Ser265和Arg271),可能有助于其水的选择性。沉默BterDRIP可加速冷应激条件下的白棘芽孢杆菌死亡率,强调BterDRIP在其耐寒性中的关键作用,并为其冷适应提供分子机制。
    Bombus terrestris are efficient pollinators in forestry and agriculture, with higher cold tolerance than other bees. Yet, their cold tolerance mechanism remains unclear. Aquaporins (AQPs) function as cell membrane proteins facilitating rapid water flow, aiding in osmoregulation. Recent studies highlight the importance of insect AQPs in dehydration and cold stress. Comparative transcriptome analysis of B. terrestris under cold stress revealed up-regulation of four AQPs, indicating their potential role in cold tolerance. Seven AQPs-Eglp1, Eglp2, Eglp3, DRIP, PRIP, Bib, and AQP12L-have been identified in B. terrestris. These are widely expressed in various tissues, particularly in the alimentary canal and Malpighian tubules. Functional analysis of BterAQPs in the Xenopus laevis oocytes expressing system showed distinct water and glycerol selectivity, with BterDrip exhibiting the highest water permeability. Molecular modeling of BterDrip revealed six transmembrane domains, two NPA motifs, and an ar/R constriction region (Phe131, His256, Ser265, and Arg271), likely contributing to its water selectivity. Silencing BterDRIP accelerated mortality in B. terrestris under cold stress, highlighting the crucial role of BterDRIP in their cold tolerance and providing a molecular mechanism for their cold adaptation.
    导出

    更多引用

    收藏

    翻译标题摘要

    我要上传

    求助全文

  • 文章类型: Journal Article
    UNASSIGNED: The ITGB6 gene encoding a protein that can regulate the integrin αvβ6 heterodimer protein expression in different status was shown to play an important role in multiple human cancers, such as brain cancer, colon cancer and oral cancer, and is related to clinical progression. This study aims to explore the function and the mechanism of the ITGB6 gene or protein in pancreatic cancer.
    UNASSIGNED: We examined the expression of ITGB6 in pancreatic cancer using immunohistochemistry and analyzed the relationship between the expression of ITGB6 and the clinicopathologic features in pancreatic cancer patients. In addition, a bioinformatic method was used to analyze the ITGB6 mRNA level in pancreatic tumor tissues compared with normal pancreatic tissues and to analyze the correlation between high KIF23 expression and prognosis in pancreatic cancer patients. Moreover, colony formation assay, MTT assay, cell scratch, cell invasion and western blot assays in vitro and a xenograft mouse model in vivo were performed to analyze the effect of KIF23 on proliferation and invasion of pancreatic cancer cells.
    UNASSIGNED: Increased expression of ITGB6 was significantly correlated with poor clinical outcome in both our clinical data and TCGA data of pancreatic cancer. Furthermore, functional assays revealed that ITGB6 knockdown in vivo and in vitro might inhibit cancer cell proliferation and the ability of invasion or migration.
    UNASSIGNED: Our data suggest that ITGB6 is associated with pancreatic cancer malignant progression. Hence, ITGB6 may serve as a potential target of pancreatic cancer for future research, and further study is needed.
    导出

    更多引用

    收藏

    翻译标题摘要

    我要上传

       PDF(Pubmed)

  • 文章类型: Journal Article
    乙型肝炎病毒(HBV)感染会导致严重的肝脏疾病,并且是全球范围内的健康问题。尽管疫苗是在婴儿出生后接种的,目前尚无有效的HBV感染药物。干扰素刺激基因(ISGs)是宿主中有助于抑制病毒的重要因素,C19orf66基因具有广泛的抗病毒谱。
    在这项研究中,对C19orf66基因中的三个SNP进行了测序和基因分型,并通过双荧光素酶报告基因测定对其潜在功能进行了预测和进一步验证。
    尽管HBV患者和对照组之间的基因型和等位基因频率没有显着差异,基因型和等位基因频率在HBsAg阳性的HBV患者和HBsAg阴性的HBV患者或对照之间显示出显着差异。rs77076061的基因型AA(P=0.009)和AT(P=0.019)在HBsAg阳性的HBV患者中显示出较高和较低的频率,分别。rs1979262基因型AG在HBsAg阳性的HBV患者(13.22%)比HBsAg阴性的患者(7.53%,P=0.036)或对照(8.48%,P=0.033)。rs1979262等位基因A的频率在HBsAg阳性患者(6.61%)高于HBsAg阴性患者(3.77%,P=0.042),而等位基因G则相反。此外,C19orf66基因中SNP的基因型与ALT之间的关联,AST,和DBIL水平也被鉴定。功能测定表明,SNP可能通过改变转录因子的连接来影响C19orf66的表达。
    总之,C19orf66基因多态性与HBV感染/生化指标的相关性在云南省首次被发现。
    Hepatitis B virus (HBV) infection causes serious liver diseases and is a healthy problem worldwide. Although vaccines are administered to infants after birth, there is no effective medicine for HBV infection. The interferon-stimulated genes (ISGs) are important factors in the host that can aid in restraining the virus, and the C19orf66 gene has a wide-antiviral spectrum.
    In this study, three SNPs in the C19orf66 gene were sequenced and genotyped, and their potential function were predicted and further verified by dual-luciferase reporter assay.
    Although no significant difference of genotype and allele frequency was observed between HBV patients and the controls, the genotype and allele frequency showed significant difference between HBV patients with HBsAg-positive and HBV patients with HBsAg-negative or controls. Genotype AA (P= 0.009) and AT (P= 0.019) of rs77076061 showed higher and lower frequency in HBV patients with HBsAg-positive than in patients with HBsAg-negative, respectively. Genotype AG of rs1979262 played a risk role in HBV patients with HBsAg-positive (13.22%) than in patients with HBsAg-negative (7.53%, P= 0.036) or controls (8.48%, P= 0.033). The frequency of allele A of rs1979262 was higher in patients with HBsAg-positive (6.61%) than in patients with HBsAg-negative (3.77%, P= 0.042), while it was the opposite for the allele G. Moreover, the associations between genotypes of SNPs in the C19orf66 gene and the ALT, AST, and DBIL level were also identified. The functional assay suggested that the SNPs might influence the C19orf66 expression by changing the connection of transcriptional factors.
    In summary, the association between genetic polymorphisms in the C19orf66 gene and HBV infection/biochemical indices of patients was firstly identified in Yunnan Province.
    导出

    更多引用

    收藏

    翻译标题摘要

    我要上传

       PDF(Pubmed)

  • 文章类型: Journal Article
    宫颈癌,女性癌症相关死亡的第二大原因,是由人乳头瘤病毒(HPV)引起的,性传播病毒.疫苗接种是预防病毒感染和随后发生宫颈癌的有效预防措施。酶联免疫吸附测定(ELISA)通常用于在临床前模型或临床志愿者中测量特异性结合抗体滴度并评估测试疫苗的免疫原性。得出滴度的两种方法,终点滴度(ET)和有效稀释,产生带有临界值的中位最大有效稀释倍数(ED50),被广泛使用。对于HPV,基于假病毒粒子的中和测定(PBNA)用于测量功能性抗体滴度。发现ELISA结合滴度和功能性PBNA滴度与疫苗中测试的所有9种HPV类型密切相关。与以往关于HPV16/18的研究一致。比较两种滴定法的PBNA结果,ED50方法显示出更高的精度和与PBNA结果更密切的相关性,针对单个类型和所有9种类型的汇总数据分析。当比较基于截止值的ET方法的滴定结果与使用整个稀释系列的所有数据点的ED50方法时,ED50方法显示出更好的精确度和与PBNA结果更强的相关性。
    Cervical cancer, the second leading cause of cancer-related deaths among women, is caused by human papillomavirus (HPV), a sexually transmitted virus. Vaccination is an effective preventive measure against viral infections and subsequent development of cervical cancer. Enzyme-linked immunosorbent assay (ELISA) is commonly used to measure specific binding antibody titers and assess the immunogenicity of test vaccines in preclinical models or clinical volunteers. Two methods of deriving titers, the endpoint titer (ET) and the effective dilution producing a median maximal effective fold of dilution (ED50) with a cut-off value, are widely used. For HPV, a pseudovirion-based neutralization assay (PBNA) is used to measure functional antibody titers. The ELISA binding titers and functional PBNA titers were found to be well-correlated for all nine HPV types tested in the vaccine, consistent with previous studies on HPV 16/18. Comparing the PBNA results with the two titration methods, the ED50 method showed higher precision and a closer correlation with PBNA results, both for individual types and pooled data analysis for all nine types. When comparing the titration results of the ET method based on a cut-off value with the ED50 method using all the data points across the dilution series, the ED50 method demonstrated better precision and a stronger correlation with PBNA results.
    导出

    更多引用

    收藏

    翻译标题摘要

    我要上传

    求助全文

  • 文章类型: Journal Article
    哺乳动物的光生态位变化与视觉能力的变化有关,主要由三种视觉色素介导,其中两个(SWS1和M/LWS)用于色觉,视紫红质(RH1)用于弱光视觉。为了进一步阐明哺乳动物视觉适应不同光环境的分子机制,已经进行了一项系统研究,其中包括跨不同群体的进化分析和体外测定。这里,我们从220个物种中收集了三种视蛋白的基因序列,覆盖了所有主要的哺乳动物进化枝。在筛选视锥细胞视蛋白基因缺失后,我们估计了这三个基因中每一个的选择压力,并比较了生活在明亮和昏暗环境中的物种所经历的选择水平。SWS1色素在生活在明亮光线环境中的物种中经历了加速进化,就像水生鲸目动物中的RH1一样,表明生态适应的潜在变化。为了进一步阐明这两种颜料的功能机理,然后,我们在代表性分类群中进行了定点突变。对于SWS1,鼠兔和小鼠的紫色和紫外线敏感性主要受到关键位点86和93处的取代的影响,这些取代具有强烈的上位相互作用。对于RH1,抹香鲸和牛序列之间的表型差异很大程度上是由195位点的替换造成的,这对于深潜物种的弱光感觉可能至关重要。已经在哺乳动物中发现了视觉色素的不同进化模式,对应于光生态位,尽管仍然需要额外的表型分析来充分解释功能机制。
    Photic niche shifts of mammals are associated with changing visual capabilities, primarily mediated by three visual pigments, two (SWS1 and M/LWS) of them for color vision and rhodopsin (RH1) for dim-light vision. To further elucidate molecular mechanisms of mammalian visual adaptations to different light environments, a systematic study incorporating evolutionary analyses across diverse groups and in vitro assays have been carried out. Here, we collected gene sequences for the three opsins from 220 species covering all major mammalian clades. After screening for cone opsin gene losses, we estimated selective pressures on each of the three genes and compared the levels of selection experienced by species living in bright- and dim-light environments. SWS1 pigment is shown to experience accelerated evolution in species living in bright-light environments as has RH1 in aquatic cetaceans, indicating potential shifts for ecological adaptations. To further elucidate the functional mechanisms for these two pigments, we then carried out site-directed mutagenesis in representative taxa. For SWS1, violet and ultraviolet sensitivities in the pika and mouse are mainly affected by substitutions at the critical sites 86 and 93, which have strong epistatic interaction. For RH1, the phenotypic difference between the sperm whale and bovine sequences is largely contributed by a substitution at site 195, which could be critical for dim-light sensation for deep-diving species. Different evolutionary patterns for the visual pigments have been identified in mammals, which correspond to photic niches, although additional phenotypic assays are still required to fully explain the functional mechanisms.
    导出

    更多引用

    收藏

    翻译标题摘要

    我要上传

    求助全文

  • 文章类型: Journal Article
    17β-羟基类固醇脱氢酶3型(17β-HSD3)缺乏症在中国46,XY性发育障碍(DSD)患者中很少报道。使用靶向下一代测序(NGS),从206名中国46名XYDSD患者中确定了7名17β-HSD3缺乏症患者。通过液相色谱-串联质谱法(LC-MS/MS)测量血清AD和T水平。进行计算机模拟和功能研究以评估HSD17B3变体的酶活性损害。在外显子剪接变体中进行小基因测定。我们的结果表明,在7例无关患者中发现了4种新的和5种报告的HSD17B3变体。患者在童年时期表现出隐秘的表现,青春期后表现出经典的男性化,T/AD比率<0.4。在具有p.N130S的单等位基因变体的患者中鉴定出从5'UTR到外显子1的杂合大缺失。虽然预计“可能致病”,只有p.S232P和p.S160F显著降低了17β-HSD3的酶活性。先前报告的\'错义\'变体c0.277G>A(p。E93K)显示对酶活性没有影响,但导致外显子3的异常剪接,并被重新分类为外显子剪接变体。在我们的研究中,一个废话,一个外显子拼接,一个删除,在17β-HSD3缺乏症患者中检测到一个大缺失和五个错义变异,扩大这种疾病的临床和分子特征。当遗传模式和功能研究不一致时,应谨慎解释计算机分析。
    17β-Hydroxysteroid dehydrogenase type 3 (17β-HSD3) deficiency is rarely reported in Chinese patients with 46, XY disorders of sexual development (DSD). Seven subjects with 17β-HSD3 deficiency were identified from 206 Chinese 46, XY DSD patients using targeted next-generation sequencing (NGS). Serum AD and T levels were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In silico and functional studies were performed to evaluate the enzymatic activity impairment of HSD17B3 variants. A minigene assay was performed in an exonic splicing variant. Our results showed that four novel and five reported HSD17B3 variants were identified in 7 unrelated patients. The patients showed cryptic presentation during childhood and classical virilization after puberty with T/AD ratio< 0.4. A heterozygous large deletion from the 5\'UTR to exon 1 was identified in a patient with a monoallelic variant of p.N130S. Although predicted to be \'likely pathogenic\', only p. S232P and p. S160F drastically reduced the enzymatic activity of 17β-HSD3. A previously reported \'missense\' variant c 0.277 G>A (p. E93K) was revealed to have no impact on enzyme activity but resulted in aberrant splicing of exon 3 and was reclassified as an exonic splicing variant. In our study, one nonsense, one exonic splicing, one deletion, one large deletion and five missense variants were detected in patients with 17β-HSD3 deficiency, expanding the clinical and molecular profile of this disorder. In silico analysis should be cautiously interpreted when the heredity pattern and functional study are inconsistent.
    导出

    更多引用

    收藏

    翻译标题摘要

    我要上传

    求助全文

  • 文章类型: Journal Article
    在哺乳动物嗅觉感觉神经元中表达的气味受体(ORs)对于嗅觉是必不可少的。然而,许多ORs的结构功能研究受到不成功的异源表达的阻碍。要理解并最终克服这一瓶颈,我们对80多个OR变体和嵌合体进行了异源表达和功能测定.结合文献资料和机器学习,我们发现跨膜结构域4(TM4)及其与相邻残基的相互作用对于OR功能表达很重要。数据突出了其中T4.62的关键作用。无法到达细胞膜的OR可以通过TM4中的修饰来挽救。因此,MOR256-3(Olfr124)中的此类修饰也改变了对气味剂的OR反应。T1614.62P导致MOR256-3在内质网(ER)中滞留,而T1614.62P/T1484.49A逆转了保留,并使受体转运到细胞膜。这项研究为哺乳动物ORs的广泛功能研究提供了新的线索。
    Odorant receptors (ORs) expressed in mammalian olfactory sensory neurons are essential for the sense of smell. However, structure-function studies of many ORs are hampered by unsuccessful heterologous expression. To understand and eventually overcome this bottleneck, we performed heterologous expression and functional assays of over 80 OR variants and chimeras. Combined with literature data and machine learning, we found that the transmembrane domain 4 (TM4) and its interactions with neighbor residues are important for OR functional expression. The data highlight critical roles of T4.62 therein. ORs that fail to reach the cell membrane can be rescued by modifications in TM4. Consequently, such modifications in MOR256-3 (Olfr124) also alter OR responses to odorants. T1614.62 P causes the retention of MOR256-3 in the endoplasmic reticulum (ER), while T1614.62 P/T1484.49 A reverses the retention and makes receptor trafficking to cell membrane. This study offers new clues toward wide-range functional studies of mammalian ORs.
    导出

    更多引用

    收藏

    翻译标题摘要

    我要上传

       PDF(Pubmed)

  • 文章类型: Journal Article
    UNASSIGNED: Family with sequence similarity 57 member A (FAM57A) is a membrane associated gene contributing to lung carcinogenesis. In hepatocellular carcinoma (HCC) and other cancers, whether FAM57A exerts similar roles has been rarely reported. Herein, the biological functions and clinical significance of FAM57A in HCC were explored.
    UNASSIGNED: Initially the differential expression of FAM57A between nontumor and HCC tissues was validated using a number of publicly accessible databases and immunohistochemistry (IHC). Then, the Kruskal-Wallis rank sum test or the Wilcoxon rank sum test as well as logistic regression were employed to analyze the association of FAM57A expression with clinical characteristics of HCC. The Cox regression and Kaplan-Meier analyses were conducted to assess the prognostic significance. Besides, with the coexpression analysis, Gene Ontology (GO), Gene Set Enrichment Analysis (GSEA), and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, the molecular pathomechanisms that were mediated by FAM57A in HCC were elucidated. Furthermore, the correlations between FAM57A expression and tumor-infiltrating immune cells (TIICs) or immune checkpoint genes were analyzed. Finally, in vitro cell functional assay was carried out to preliminarily verify the role of FAM57A in HCC.
    UNASSIGNED: FAM57A expression was demonstrated to be higher in HCC samples than in nontumor samples (all p-values <0.05), statistically correlated with clinicopathological characteristics (clinical stage, T stage, pathological grade), and inversely correlated to HCC patient survival. Univariate and multivariate Cox regression analyses showed that FAM57A expression could independently predict prognosis in HCC patients. Functional enrichment analyses further indicated that FAM57A was involved in multiple tumor-related pathways. FAM57A expression was positively correlated with TIICs, gene markers of TIICs, as well as immune checkpoint genes. Also, high expression of FAM57A predicted a poor prognosis for HCC based on immune cell subgroups. Functional assay of FAM57A knockdown significantly inhibited cell proliferation and induced cell apoptosis in HCC cells.
    UNASSIGNED: Our results indicated that FAM57A could be used as a biomarker to predict the prognosis and immunotherapy response for HCC patients and might function as an oncogene to promote HCC progression.
    导出

    更多引用

    收藏

    翻译标题摘要

    我要上传

       PDF(Pubmed)

  • 文章类型: Journal Article
    Sensory systems are attractive evolutionary models to address how organisms adapt to local environments that can cause ecological speciation. However, tests of these evolutionary models have focused on visual, auditory, and olfactory senses. Here, we show local adaptation of bitter taste receptor genes in two neighboring populations of a wild mammal-the blind mole rat Spalax galili-that show ecological speciation in divergent soil environments. We found that basalt-type bitter receptors showed higher response intensity and sensitivity compared with chalk-type ones using both genetic and cell-based functional analyses. Such functional changes could help animals adapted to basalt soil select plants with less bitterness from diverse local foods, whereas a weaker reception to bitter taste may allow consumption of a greater range of plants for animals inhabiting chalk soil with a scarcity of food supply. Our study shows divergent selection on food resources through local adaptation of bitter receptors, and suggests that taste plays an important yet underappreciated role in speciation.
    导出

    更多引用

    收藏

    翻译标题摘要

    我要上传

       PDF(Pubmed)

  • 文章类型: Journal Article
    水通道蛋白(AQP)在单细胞和多细胞生物体中跨细胞膜转运溶质。在这项研究中,水通道蛋白CsPrip在二化霉中被发现,一种重要的水稻害虫。CsPrip由两个变体组成,CsPrip_v1和CsPrip_v2;前者的变体<103bp比后者短,尽管两者都表现出相同的开放阅读框(ORF)。跨膜拓扑和蛋白质结构分析表明,CsPrip保留了水选择性昆虫AQPs的保守特征,包括六个跨膜结构域,两个保守的疏水天冬酰胺-脯氨酸-丙氨酸基序和芳香/精氨酸收缩区。在非洲爪的卵母细胞中的表达表明,CsPrip优先转运水和尿素,而不是海藻糖和甘油。CsPrip转录本在化脓杆菌幼虫的多个器官和组织中表达,并且在后肠和Malpighian小管中最丰富。CsPrip转录在成年男性中最高,并且在整个发育过程中相对稳定。CsPrip在幼虫中的表达被热胁迫显著改变,相对湿度水平会影响3龄和5龄幼虫的CsPrip转录。这项研究证实,水通道蛋白CsPrip在维持化脓杆菌中的水平衡方面具有多种关键功能。
    Aquaporin (AQP) transport solutes across cell membranes in both unicellular and multicellular organisms. In this study, the aquaporin CsPrip was identified in Chilo suppressalis, an important pest of rice. CsPrip was comprised of two variants, CsPrip_v1 and CsPrip_v2; the former variant was <103 bp was shorter than the latter, although both exhibited the same open reading frame (ORF). Transmembrane topology and protein structure analyses showed that CsPrip retained the conserved features of water-selective insect AQPs, including six transmembrane domains, two conserved hydrophobic asparagine-proline-alanine motifs and the aromatic/arginine constriction region. Expression in Xenopus oocytes revealed that CsPrip preferentially transported water and urea instead of trehalose and glycerol. The CsPrip transcript was expressed in multiple organs and tissues of C. suppressalis larvae and was most abundant in the hindgut and Malpighian tubules. CsPrip transcription was highest in male adults and was relatively stable throughout development. CsPrip expression in larvae was significantly altered by thermal stress, and relative humidity levels impacted CsPrip transcription in 3rd and 5th instar larvae. This study confirms that the aquaporin CsPrip performs multiple critical functions in maintaining water equilibrium in C. suppressalis.
    导出

    更多引用

    收藏

    翻译标题摘要

    我要上传

       PDF(Sci-hub)

公众号