Establishment of a Multiplex Detection Method for Common Bacteria in Blood Based on Human Mannan-Binding Lectin Protein-Conjugated Magnetic Bead Enrichment Combined with Recombinase-Aided PCR Technology

Objective Recombinase-aided polymerase chain reaction(RAP)is a sensitive,single-tube,two-stage nucleic acid amplification method.This study aimed to develop an assay that can be used for the early diagnosis of three types of bacteremia caused by Staphylococcus aureus(SA),Pseudomonas aeruginosa(PA),and Acinetobacter baumannii(AB)in the bloodstream based on recombinant human mannanbinding lectin protein(M1 protein)-conjugated magnetic bead(M1 bead)enrichment of pathogens combined with RAP.Methods Recombinant plasmids were used to evaluate the assay sensitivity.Common blood influenza bacteria were used for the specific detection.Simulated and clinical plasma samples were enriched with M1 beads and then subjected to multiple recombinase-aided PCR(M-RAP)and quantitative PCR(qPCR)assays.Kappa analysis was used to evaluate the consistency between the two assays.Results The M-RAP method had sensitivity rates of 1,10,and 1 copies/μL for the detection of SA,PA,and AB plasmids,respectively,without cross-reaction to other bacterial species.The M-RAP assay obtained results for<10 CFU/mL pathogens in the blood within 4 h,with higher sensitivity than qPCR.M-RAP and qPCR for SA,PA,and AB yielded Kappa values of 0.839,0.815,and 0.856,respectively(P<0.05).Conclusion An M-RAP assay for SA,PA,and AB in blood samples utilizing M1 bead enrichment has been developed and can be potentially used for the early detection of bacteremia.

胸腔积液沉渣包埋组织TTF-1、Galectin-3的表达水平与肺腺癌恶性生物学行为间的关系

目的探讨胸腔积液沉渣包埋组织甲状腺转录因子1(TTF-1)、半乳糖凝集素-3(Galectin-3)的表达水平与肺腺癌恶性生物学行为间的关系。方法选取2020年10月至2023年3月在该院就诊的肺腺癌患者163例,根据分化程度分为高分化组(26例)、中分化组(78例)、低分化组(59例)。对比3组胸腔积液沉渣包埋组织TTF-1、Galectin-3及恶性生物学行为相关基因(p16基因mRNA)表达水平,Spearman秩相关系数分析胸腔积液沉渣包埋组织TTF-1、Galectin-3与p16基因mRNA表达水平的相关性,多元线性回归分析胸腔积液沉渣包埋组织TTF-1、Galectin-3与p16基因mRNA表达水平间的依存关系,限制性立方样条图分析胸腔积液沉渣包埋组织TTF-1、Galectin-3与p16基因mRNA表达水平间的剂量-效应关系。结果低分化组胸腔积液沉渣包埋组织TTF-1、Galectin-3表达水平高于中分化组、高分化组,p16基因mRNA表达水平低于中分化组、高分化组,中分化组胸腔积液沉渣包埋组织TTF-1、Galectin-3表达水平高于高分化组,p16基因mRNA表达水平低于高分化组,差异有统计学意义(P<0.05);胸腔积液沉渣包埋组织TTF-1(r=-0.752,P<0.001)、Galectin-3(r=-0.811,P<0.001)表达水平与p16基因mRNA表达水平呈负相关;胸腔积液沉渣包埋组织Galectin-3表达水平与p16基因mRNA表达水平间无线性依存关系(P>0.05),胸腔积液沉渣包埋组织TTF-1表达水平与p16基因mRNA表达水平间有线性依存关系(P=0.049);当胸腔积液沉渣包埋组织TTF-1表达水平<4分、胸腔积液沉渣包埋组织Galectin-3表达水平<3分时,对p16基因mRNA表达作用最显著。结论胸腔积液沉渣包埋组织TTF-1、Galectin-3表达水平与肺腺癌恶性生物学行为关系密切,早期检测对临床实现精准干预避免病情恶化具有重要意义。

The lectin gene TRpL1 of tetraploid Robinia pseudoacacia L.response to salt stress

Lectins are natural proteins in animals,plants,and microorganisms and can be divided into 12 families.These lectins play important roles in various environmental stresses.Some polyploid plants show tolerance to environmental stresses and to insect pests.However,the mechanism of stress tolerance is unclear.Tetraploid Robinia pseudoacacia(4×)under salt stress showed higher tolerance than diploid R.pseudoacacia(2×).As lectin can improve stress tolerance,it was questioned whether the stress resistance of polyploid plants was related to the lectin protein.In this study,salt resistance of lectin gene TRpL1 was verified by its over-expression in plants.In addition,salt resistance of lectin protein by E.coli strains was detected.The data revealed that the over-expression transgenic plants of TRpL1 showed better salt tolerance than control plants under salt stress,and the TRpL1-expressing strain also grew better in the medium with added NaCl.Therefore,tetraploid plants can resist salt stress through TRpL1 protein regulation.

Characterization of a CD209-Like Lectin in Black Rockfish(Sebastes schlegelii):Structure Features and Binding Activities as a Pathogen Recognition Receptor

CD209,a transmembrane lectin belonging to the C-type lectin family,can recognize carbohydrates on the surface of host cells and invading pathogens,and play an important role in cell adhesion and migration,pathogen recognition and immune activation.Although well characterized in mammals,CD209 is still under-researched in fish.Here,we report a CD209-like gene,which was named SsCD209like,in black rockfish Sebastes schlegelii,and analyzed its structure features,expression patterns and ligand-binding activities.SsCD209like displays structural similarities to mammalian CD209s,with a cytosolic tail at N-terminus,a transmembrane region and an extracellular part containing a neck region and a CRD at C-terminus.The extracellular region and the neck region of SsCD-209like can both form dimers,which is different with the tetramer in human homologue.This result demonstrates the multimerization of CD209 homologue in fish for the first time.The EPN motif,a functional motif participating in sugar binding and affinity determination,is conserved in the CRD of SsCD209like,which is consistent with the higher binding strength of this lectin to L-fucose,D-GlcNAc and D-mannose.The binding of SsCD209like to different bacteria strains and bacteria-derived pathogen associated molecular patterns(PAMPs)are also observed in a dose-dependent manner.Results in this study show the sequence and structure features of SsCD209like and demonstrate its binding properties as a pathogen recognition receptor,which promotes our understanding of CD209 homologues in fish and provides basis for more in-depth studies of this molecule in the future.

A novel lectin from mushroom Phellodon melaleucus displays hemagglutination activity,and antitumor activity in a B16 melanoma mouse model

A novel lectin(termed PML)was purified from fruiting bodies of the edible mushroom Phellodon melaleucus(division Basidiomycota)by ion exchange,hydrophobic interaction,and gel filtration chromatographies,with overall titer recovery~60%and 20-fold purification.PML displayed hemagglutination activity 13319 units/mg toward rabbit erythrocytes.SDS-PAGE and gel filtration analyses revealed that PML is a homodimeric lectin with a molecular weight of 28.8 kDa.PML hemagglutination activity was not inhibited by various simple sugars or their derivatives,but was enhanced by cations Ca^(2+),Mg^(2+),Zn^(2+),and Cu^(2+).The activity was stable in pH range 6–9 and in the temperature range 20–60°C.Circular dichroism(CD)spectroscopic analysis showed that PML was composed primarily ofβ-sheets with lowα-helix content.In a B16 melanoma mouse model,PML treatment significantly inhibited tumor growth,and increased cytokine IL-10 content.Our findings suggest that PML is a potential anticancer therapeutic agent.

Effect of a Thermal Spring Water on Carbohydrate-Protein Interactions in In-Vitro Models Implicating Normal Human Keratinocytes and Recombinant Lectins

Background: Sugar moiety of macromolecules is today very well known for its implications in many biological recognition mechanisms including cell-cell, extracellular matrix-cell and/or bacteria-cell interactions. In this context lectins, which are carbohydrate-binding proteins displaying a high affinity for sugar groups of other molecules, are of a great importance, notably in immune response involving bacteria, viruses and fungi. As protein-carbohydrate interactions are often mediated by ions such as calcium, zinc or magnesium, we were prompted to study the effect of a thermal spring water (which contains this type of component) on interactions existing between: 1) osidic receptors of human normal keratinocytes and 2) two lectins greatly implicated in the immune response mechanisms (i.e. the dectin-1 and the langerin), and their ligands. Materials and Methods: In a first series of experiments, we studied the effect of increasing concentrations of a thermal spring water on interactions existing between glycosylated molecules and the osidic receptors expressed at the normal human keratinocytes surface. In a second step, and in order to better understand the putative effect of our thermal spring water on the immune response, we analyzed its effect on the interactions existing between the dectin-1 (implicated in the recognition of bacteria, viruses and fungi) and the langerin (expressed by Langerhans cells, the immune cells of the cutaneous tissue), and their ligands in a model using recombinant human lectins and appropriate binding molecules. Results: We showed here that our thermal spring water was able to reinforce interactions between keratinocytes osidic receptors and some of their ligands, in a dose-related manner: From 8% to 55% of increase with 10% to 30% (v/v) of thermal spring water. In the second part of our studies, we also showed that our thermal spring water was able to modulate interactions between dectin-1 and langerin and their ligands through a biphasic effect: Interactions were enhanced by more than 40% and 20% respectively with 10% of thermal spring water, and return to their basal level or lower for higher concentrations. Conclusion: The tested thermal spring water, probably due to its ionic composition, could significantly affect interactions of osidic receptors with their ligands. This property could be of a great interest to help immune system to maintain an appropriate “vigilance state” by using the thermal water at up to a concentration of 10%, and by avoiding any runaway reaction in case of aggression, by using concentrations higher than 10%. .

磁共振成像灌注参数与COL5A2、Galectin-3的相关性及对食管癌的诊断价值

目的探讨磁共振成像(MRI)灌注参数与V型胶原蛋白α2链(COL5A2)、半乳糖苷凝集素-3(Galectin-3)的相关性及对食管癌的诊断价值。方法选取2020年12月—2021年12月我院收治的疑似食管癌患者100例,以内镜和活检病理检查为金标准,最终确诊为食管癌患者的55例为观察组,其余45例食管炎病变患者为对照组。所有患者行磁共振成像检查及COL5A2、Galectin-3水平测定,对比两组MRI灌注参数,分析食管癌患者MRI灌注参数与肿瘤标志物水平的相关性,分析MRI灌注参数对食管癌的诊断价值。结果与对照组相比,观察组K^(trans)、K_(ep)、V_(e)水平较高(P<0.05);与Ⅰ~Ⅱ期食管癌患者相比,Ⅲ~Ⅳ期食管癌患者K trans、K ep、V e水平较高(P<0.05);与Ⅰ~Ⅱ期食管癌患者相比,Ⅲ~Ⅳ期食管癌COL5A2、Galectin-3水平升高(P<0.05);食管癌患者K^(trans)、K_(ep)、V_(e)、COL5A2、Galectin-3均与病情分期呈正相关(P<0.05);K^(trans)、K_(ep)、V_(e)均与COL5A2、Galectin-3水平呈正相关,差异具有统计学意义(P<0.05);与磁共振成像、COL5A2、Galectin-3单一诊断相比,联合诊断对食管癌的预测价值较高(P<0.05)。结论MRI灌注参数与血清Galectin-3、COL5A2三者联合检测准确度及灵敏度较高于单一检测,弥补了单一检测的不足,三者联合对不同分期食管癌患者具有一定的诊断价值。

LPS诱导小鼠脑小胶质细胞的激活及Tomato lectin的表达变化

探讨小胶质细胞对侧脑室注射内毒素脂多糖(lipopolysaccharide,LPS)刺激的反应及时程变化。采用组织化学方法,观察小鼠单次注射LPS入侧脑室后,Tomatolectin阳性的小胶质细胞于不同时间点在脑内的分布及表达的时程变化。LPS注射24h后,Tomatolectin组织化学染色显示血管内皮细胞、静息的和不同激活状态的小胶质细胞均呈阳性反应,其中Tomatolectin阳性的小胶质细胞明显被激活,2d时达表达高峰,巨噬细胞样的细胞和小胶质细胞呈现出肥大的、阿米巴样或杆状细胞等不同激活状态下的形态特征。这些激活的小胶质细胞主要分布于侧脑室周围的脑实质结构,如海马、隔区、胼胝体、纹状体,第三脑室周围的下丘脑及其它间脑结构。本结果提示:LPS能诱导小鼠脑室周围的脑实质内的小胶质细胞被激活,并上调小胶质细胞内Toma-tolectin的表达,这些细胞可能参与脑内刺激的免疫调节。

Cloning and Sequencing of a Lectin Protein Gene from the Roots of Sophora flavescens

A lectin protein(SFL) with molecular weight about 32 kD which markedly agglutinated rabbit and human red blood cells was purified from the roots of Sophora flavescens Ait. This protein, and apparently inhibited the growth of Fusarium vasinfectum Atk., Gibberella saubinetii (Mont.) Sacc., and Piricularia oryzae Cav. A set of degenerate PCR primer was synthesized according to the N-terminal sequence of the purified protein. The full-length cDNA coding the lectin was cloned by RT-PCR and 5'-RACE and sequenced (GenBank AF285121). The deduced amino acid sequence indicates that a preprotein with 284 amino acid residues is firstly translated and then processed to a mature protein with 254 amino acids. A N-Glycosylation site is the Asn 182 residue.

Identification of a Lectin Gene Induced in Rice in Response to Magnaporthe grisea Infection

By mRNA differential display, eight induced cDNAs were obtained from rice leaves infected with an incompatible race 131 of Magnaporthe grisea, and one of these cDNAs was highly similar to salt-induced mannose-binding lectin gene. Using this fragment as a probe, a full length cDNA was isolated from a nice cDNA library, which was constructed using mRNA from the incompatible race-infected leaves. Sequence analysis indicates that the cDNA encodes a protein of 15 kD with 145 amino, acids and shares 96% identity at nucleotide level with MRL and salT, but is identical to MRL at amino acid level. Genomic Southern blotting shows that there are two mannose-binding lectin genes in rice genome. Northern blotting analysis indicates that the gene was strongly and specifically induced in rice leaves infected with the incompatible race, suggesting that the lectin induction be involved in the defense of rice to M. grisea.

Transformation of Pea Lectin Gene and Parasponia Haemoglobin Gene into Rice and Their Expressions

Lectin and leghemoglobin in legumes play the important roles, respectively, in recognition of host plants to their rhizobial bacteria, and lowering the oxygen partial pressure around bacteroids and protecting nitrogenase from oxygen in symbiotic nitrogen-fixing nodules. In order to extend the host range of the rhizobial bacteria and to make them fix nitrogen in non-legumes, pea lectin gene (pl) and Parasponia hemoglobin gene ( phl,) have been constructed into a plant expression vector (pCBHUL) and the vector pCBHUL was introduced into rice calli from immature young embryos by particle bombardment. After the calli were regenerated into plantlets on the resistant-selecting media containing hygromycin, they were identified by PCR and Southern blot hybridization. It was indicated that the pi and phb genes were integrated into nucleic genome of the transformed rice plants. GUS activity and the product of the pi gene were determined by GUS staining, Western blot and in situ hybridization at translational level. Eighteen out of 40 plants resistant to hygromycin were positively identified by PCR analysis with the rate of 45%. The pi gene was expressed in 3 out of 18 plants with 17% and 7.5% in 40 plants. The results may provide a clue for exploring whether Rhizobium leguminosarum by. viceae could extend its host range and make the transgenic rice plants have the possibility of being symbiotic, or associative to nitrogen fixation.

Research on the Lectin in Muscle Tissue of Varicorhinus macrolepis

[Objective] The paper aimed at researching lectins in muscles of Varicorhinus macrolepis and providing scientific basis for researching the adaptation mechanism and immune response of V.macrolepis to environment,which were advantageous for the protection and reproduction of V.macrolepis.[Method] V.macrolepis was used as test materials for the hemagglutination test by dialdehyde fixation to prove the existence of lectins in muscle crude homogenate of Varicorhinus macrolepis and study the physical and chemical characters.[Result] Lectins in muscle crude homogenate of V.macrolepis had shown hemagglutination effects on erythrocytes of six types of animals and had the maximum hemagglutination activity against rabbit erythrocytes,which belonged to the S-type lectins with optimal pH ranged from 4 to 8 and optimal temperature at 60 ℃.Results from the saccharide inhibition test had indicated that the sucrose was the only kind of saccharide which had inhibited the hemagglutination,suggesting that sucrose had played an important role in the process of recognition and aggregation of lectins.[Conclusion] It had been speculated that the optimal pH ranges for thermal sensitivity and hemagglutination activity of lectins in different types of aquatic organisms were similar.

蒙药嘎日迪-13对脑缺血大鼠不同时相海马中IL-1β IL-8、ICAM-1、P-selectin表达的影响

目的观察蒙药嘎日迪-13对脑缺血大鼠不同时相海马中IL-1β、IL-8、ICAM-1、P-selectin表达的影响。方法 360只雄性SD大鼠随机分为脑缺血模型组、假手术组、嘎日迪-13大、中、小剂量组,每组又随机分为6个时相组,灌胃给药,一次/d,连续27 d后,线栓法制备大鼠大脑中动脉栓塞模型,神经功能评分后免疫组化法测定海马IL-1β、IL-8、ICAM-1、P-selectin的表达。结果嘎日迪-13明显降低脑缺血大鼠神经功能评分,并明显降低脑缺血大鼠海马IL-1β、IL-8、ICAM-1、P-selectin的表达水平。结论蒙药嘎日迪-13对大鼠脑缺血有较好的保护作用,其机制可能与降低IL-1β、IL-8、ICAM-1、P-selectin等因子的表达水平有关。

Lectin法分析uNK细胞在人妊娠母胎界面的分布变化

通过Lectin法分析uNK细胞在人妊娠早、晚期母胎界面的分布变化。结果发现:(1)在妊娠早期的蜕膜中,uNK细胞主要有两种形式,一种是成熟uNK细胞,细胞体积较大,胞浆和胞膜呈现强烈的棕黄色染色,分布于蜕膜的血管中。另一种是未成熟uNK细胞,细胞体积较小,胞核呈现黄色染色,分布于蜕膜细胞之间。(2)在妊娠早期的绒毛组织中,uNK主要是未成熟uNK,分布于绒毛间质之间。(3)与妊娠早期蜕膜的uNK细胞数相比,至妊娠晚期,蜕膜的uNK细胞数量明显减少(P<0.01)。(4)在妊娠晚期,绒毛组织不表达uNK细胞。结论:uNK细胞主要分布于子宫蜕膜组织,且随着妊娠的进展,其表达数量明显下降。这种时空性的表达变化可能与其在调控滋养层细胞的侵袭、血管重建及免疫耐受方面密切相关。

斑节对虾1种lectin基因的电子克隆及分析

以中国明对虾的c-lectin(DQ167572)基因为探针,搜索EST数据库,获得了日本对虾的1个lectin序列,通过电子克隆扩增到了该基因,并对基因进行了生物信息学分析。

呼吸系统功能状态与sICAM-1、sVCAM-1、E-selectin的相关性

目的探讨呼吸系统功能状态与E-选择素(E-selectin)、可溶性细胞间黏附分子1(soluble inte FAellular adhesion molecule-1,sICAM-1)、可溶性血管细胞黏附分子1(souble vascular cell adhesion molecule 1,sVCAM-1)的相关性。方法选取体检中心健康体检者216名,根据HRA系统检测的呼吸系统功能状态的结果,将研究对象分为呼吸功能正常组,呼吸功能轻度改变组,支气管、呼吸道炎性反应状态组三组,分别编号为A、B、C组。清晨空腹采血,留取血清,测定sICAM-1、sVCAM-1、Eselectin的含量。结果与A组相比,吸烟使B、C组呼吸系统功能状态改变的危险性增大(OR=1.216,95%CI:0.6~2.466;OR=3.2,95%CI:1.502~6.819)。随着呼吸系统功能状态炎性程度的增加,E-selectin、sICAM-1、sVCAM-1的浓度明显增高,且差异有统计学意义(P均<0.01)。A组中E-selectin、sICAM-1、sVCAM-1的浓度在吸烟组和非吸烟组的差异均无统计学意义;B组中,与非吸烟组相比,吸烟组sICAM-1、sVCAM-1明显增高(P均<0.01);在C组中,吸烟组中E-selectin、sICAM-1、sVCAM-1明显高于非吸烟组,且差异均有统计学意义(P均<0.01)。结论随着呼吸系统功能状态严重程度的增高,E-selectin、sICAM-1、sVCAM-1的浓度明显增高。吸烟者血清E-selectin、sICAM-1、sVCAM-1的水平增加。

MBL及其补体激活的LECTIN途径

MBL是一种血清C型凝集素 ,是机体先天免疫的重要分子之一 ,本文在简要总述MBL的结构和功能的基础上 ,对MBL参与的凝集素途径进行阐述。

Use of lectin microarray to differentiate gastric cancer from gastric ulcer

AIM: To investigate the feasibility of lectin microarray for differentiating gastric cancer from gastric ulcer. METHODS: Twenty cases of human gastric cancer tissue and 20 cases of human gastric ulcer tissue were collected and processed. Protein was extracted from the frozen tissues and stored. The lectins were dissolved in buffer, and the sugar-binding specificities of lectins and the layout of the lectin microarray were summarized. The median of the effective data points for each lectin was globally normalized to the sum of medians of all effective data points for each lectin in one block. Formalin-fixed paraffin-embedded gastric cancer tissues and their corresponding gastric ulcer tissues were subjected to Ag retrieval. Biotinylated lectin was used as the primary antibody and HRP-streptavidin as the secondary antibody. The glycopatterns of glycoprotein in gastric cancer and gastric ulcer specimens were determined by lectin microarray, and then validated by lectin histochemistry. Data are presented as mean +/- SD for the indicated number of independent experiments. RESULTS: The glycosylation level of gastric cancer was significantly higher than that in ulcer. In gastric cancer, most of the lectin binders showed positive signals and the intensity of the signals was stronger, whereas the opposite was the case for ulcers. Significant differences in the pathological score of the two lectins were apparent between ulcer and gastric cancer tissues using the same lectin. For MPL and VVA, all types of gastric cancer detected showed stronger staining and a higher positive rate in comparison with ulcer, especially in the case of signet ring cell carcinoma and intra-mucosal carcinoma. GalNAc bound to MPL showed a significant increase. A statistically significant association between MPL and gastric cancer was observed. As with MPL, there were significant differences in VVA staining between gastric cancer and ulcer. CONCLUSION: Lectin microarray can differentiate the different glycopatterns in gastric cancer and gastric ulcer, and the lectins MPL and VVA can be used as biomarkers. (C) 2014 Baishideng Publishing Group Co., Limited. All rights reserved.

Mannose-binding lectin and maladies of the bowel and liver

Mannose-binding lectin (MBL) is a pattern-recognition molecule that binds to characteristic carbohydrate mo-tifs present on the surface of many different pathogens. MBL binding stimulates the immune system via the lectin pathway of complement activation. In certain clinical situations, often characterized by pre-existing immune compromise, MBL deficiency increases the risk of infec-tious and other disease-specific complications. Many of the key pathogenic processes inherent to common gastroenterological diseases, such as infection, immuno-logical damage, and carcinogenesis, have been linked to MBL. This editorial reviews the biology of MBL, outlines key disease associations to document the breadth of influence of MBL, and finally, highlights the relevance of MBL to both gastroenterological health and disease.

人P-选择素胞外区Lectin片段真核表达载体的构建与鉴定

目的 构建人P 选择素 (P selectin)胞外区凝集素 (Lectin)片段的真核表达载体。方法 从人血小板中提取总RNA ,经RT PCR得到P 选择素cDNA基因 ,用PCR方法扩增其中的Lectin片段 ,并将其克隆到真核表达载体 pcDNA4/HisMaxA中 ,构建了PCMV 启动控制基因表达质粒 pcDNA4/HisMaxA P selectin’sLectin。结果 通过酶切和基因序列分析确定重组入载体 pcDNA4/HisMaxA的片段为目的基因的核苷酸序列。 结论 重组质粒 pcDNA4/HisMaxA P selectin’sLectin的成功构建 ,为在真核细胞中高效表达该表位片段对应蛋白并制备其抗体作进一步研究提供基础。