Development of Immune Response to Experimental Bovine Trichophyton vervucosum Infection

Abstract— Cell mediated and humoral immune responses to experimental Trichophyton verrucosum infection were assessed by sequential cutaneous biopsies, antibody assessments and microscopic monitoring of fungal presence. Histopathologic examination showed the accrual of lymphocytes and other inflammatory cells in the dermis of infected sites. Immunoperoxidase staining of frozen sections with monoclonal antibody preparations revealed an influx of macrophages, BoCD4+ and B0CD8+ lymphocytes and γδ T cells from the 5th day to the 33rd day of infection. A moderate influx of B cells was observed. Protein G-colloidal gold staining revealed the presence of immunoglobulins in the dermis and superficial epidermal layers. Trichophyton specific serum antibodies appeared between days 33 and 55. Microscopic assessment of infected tissues revealed an increase in T, verrucosum elements (mycelium and ectothrix spores) from days 19 to 55. Fungal elements in infected areas did not decrease until after both humoral and cell mediated elements of the immune response were established. These responses imply a combination of cell mediated and humoral events were associated with T. verrucosum immunity and clearance in the calf. Résumé— Le réponse immunitaire humorale et cellulaire a une infection expérimentale àT. verrucosum a été appréciée par des biospsies cutanées successives, des dosages d'anticorps et la recherche microscopique de champignons. L'examen histopathologique a montré un afflux de lymphocytes et d'autres cellules inflammatoires dane le derme des sites infectés. Les marquages en immunopéroxydase par un anticorps monoclonal de coupes congelées a montré un influx de macrophages, lymphocytes BoCD4+ et BoCD8+ et des cellules T γδ, du 5e au 33e jour de l'infection. Un marquage par une protéine G—or colloidal a révélé d'immunogolglobulines dans le derme et les couches supéerficielles de l'épiderme. Les anticorps spécifiques de Trichophyton sont apparus entre 33 et 55 jours. L'examen microscopique des tissus infectés a révélé une augmentation du nombre d'éléments de T. verrucosum (mycelium et spores ectothrix) du 19e au 55e jours. Les éléments fongiques dans les zones infectées n'ont pas diminué avant que les réponses humorales et cellulaires ne solent établies. Ces résponses impliquent qu'une coopération des réponses humorales et cellulaires étaient associées dans l'immunité et la défense contre T. verrucosum. Zusammenfassung— Die zellvermittelten und humoralen Immunantworten auf die experimentelle Infektion mit T. verrucosum wurden durch eine Serie von Hautbiopsien, Antikörperuntersuchungen und mikroskopischer Untersuchung auf das Vorhandensein von Pilzen ausgewertet. Die histopathologische Untersuchung zeigte eine Ansammlung von Lymphozyten und anderen Entzündungszellen in der Dermis der infizierten Stellen. Die Immunperoxidasefärbung der Gefrierschnitte mit monoklonalen Antikörperzubereitungen zeigte einen Influx von Makrophagen, BoCd4-und BoCD8-Lymphozyten und gamma-delta-T-Zellen vom 5. bis zum 33. Tag der Infektion. Es wurde auch ein mäßiger Influx von B-Zellen beobachtet. Die Protein-G-kolloidale Goldfärbung zeigte die Anwesenheit von Immunglobulinen in der Dermis und den oberflächlichen epidermalen Schichten. Trichophyton-spezifische Serumantikörper traten zwischen Tag 33 und 55 auf. Die mikroskopische Untersuchung infizierter Gewebe zeigte eine Zunahme von T. verrucosum-Bestandteilen (Myzel und exktothrixe Sporen) vom Tag 19 bis 55. Pilzteile in infizierten Bereichen verminderten sich weder, nachdem humorale, noch nachdem zellvermittelte Elemente der Immunantwort auftraten. Diese Reaktionen deuten an, daß eine Kombination von zellvermittelten und humoralen Vorgängen im Zusammenhang mit T. verrucosum-Immumtät und Abheilung beim Kalb vorliegt. Resumen Por medio de biposias cutáneas secuenciales, medida de anticuerpos y exámen microscópico de presencia de hongos, se estudió la respuesta inmunitaria de tipo humoral y celular producida por la infección experimental con T. verrucosum. El exámen histopatológico reveló la presencia de agregación de linfocitos y otras células inflamatorias procedentes de la dermis de los puntos infectados. La tintura por medio de inmunoperoxidasa de las secciones congeladas, con preparaciones monoclonales de anticuerpos, demostró un aflujo de macrófagos BoCD4 + y BoCD8 + linfocitos y linfocitos, Tαδ, desde el quinto hasta el día 33 la infección. También se observó un aflugo moderado de linfocitos B. La tintura aúrica de proteina coloidal G reveló la presencia de inmunoglobulinas en al dermis y capas superficiales de la epidermis. Los anticuerpos específicos para la especie Trichophyton aparecieron entre los días 33 y 55. El exámen microscópico de los tejidos afectados demostró un incremento, de los elementos füngicos T. verrucosum (micelio y esporas exótricas) desde los días 19 al 55. Los elementos fúngicos en áreas infectadas no disminuyeron hasta después del establecimiento de ambos tipos de respuesta inmunitaria, humoral y celular. Estas respuestas implican que la combinación de ciertos fenómenos de inmunidad celular y humoral, están relacionados con la desaparición y la inmunidad de la infección producia por T. verrucosum en el ternero.     

The effect of some Frescon analogs on the aquatic snail,Lymnaea stagnalis: II. Correlation of neurotoxicity to Molluscicidal effectiveness

Although the molluscicide Frescon is a strong neurotoxin to the Lymnaea stagnalis central nervous system in vitro, it is probable that the exposure of the whole animal to this molluscicide fails to result in central nervous system abnormalities: Frescon does not appear to reach the brain in sufficient quantity to disrupt its normal activity. However, only those Frescon analogs found to be neurotoxic were molluscicidal, suggesting some related mode, if not site, of action. Frescon and its analogs may act by affecting excitable tissues other than the nervous system (e.g., the snail musculature) by altering certain functional and/or structural membrane properties.     

吡啶衍生物研究(IX):N-(1-甲氧羰基)乙基-N-[5-(2-氯吡啶-4-基)-1,3,4-噻二唑-2-基]酰胺的合成及除草活性

为了进一步研究前期发现的除草先导化合物2-仲丁氨基-5-(2-氯吡啶-4-基)-1，3，4-噻二唑(BCPT)的结构-活性关系并提高其除草活性，设计并合成了一系列N-(1-甲氧羰基)乙基-N-[5-(2-氯吡啶-4-基)-1，3，4-噻二唑-2-基]酰胺类化合物。其苗后除草活性测定结果表明，所有化合物的活性都远低于BCPT本身。说明BCPT可能具有与传统酰胺类除草剂不同的作用机制。     

The Inhibitors of CDK4/6 from a Library of Marine Compound Database: A Pharmacophore,ADMET, Molecular Docking and Molecular Dynamics Study

Background: CDK4/6 (Cyclin-dependent kinases 4/6) are the key promoters of cell cycle transition from G1 phase to S phase. Thus, selective inhibition of CDK4/6 is a promising cancer treatment. Methods: A total of 52,765 marine natural products were screened for CDK4/6. To screen out better natural compounds, pharmacophore models were first generated, then the absorption, distribution, metabolism, elimination, and toxicity (ADMET) were tested, followed by molecular docking. Finally, molecular dynamics simulation was carried out to verify the binding characteristics of the selected compounds. Results: Eighty-seven marine small molecules were screened based on the pharmacophore model. Then, compounds 41369 and 50843 were selected according to the ADMET and molecular docking score for further kinetic simulation evaluation. Finally, through molecular dynamics analysis, it was confirmed that compound 50843 maintained a stable conformation with the target protein, so it has the opportunity to become an inhibitor of CDK4/6. Conclusion: Through structure-based pharmacophore modeling, ADMET, the molecular docking method and molecular dynamics (MD) simulation, marine natural compound 50843 was proposed as a promising marine inhibitor of CDK4/6.     

Computationally Assisted Structural Elucidation of Cembranoids from the Soft Coral Sarcophyton tortuosum

A persistent study on soft coral Sarcophyton tortuosum resulted in the characterization of two new cembranolides, tortuolides A and B (1 and 2), and a new related diterpene, epi-sarcophytonolide Q. Their structures were determined not only by extensive spectroscopic analysis but also by DFT calculations of ECD and NMR data, the latter of which was combined with statistical analysis methods, e.g., DP4+ and J-DP4 approaches. Anti-inflammatory and cytotoxicity activities were evaluated in this study.     

喷锌对蕹菜锌含量及产量的影响

不同喷施浓度及喷施次数对蕹菜产量及茎叶锌含量的影响结果表明：喷施０．０５％及０．１％的硫酸锌液均能提高蕹菜的产量,但以０．０５％的处理对产量的提高作用最明显,浓度超过０．１％时,对蕹菜生长有抑制作用提高喷锌浓度,蕹菜锌含量的增加为叶片大于茎;喷施１～２次,有明显的增产效果,喷施超过２次反而减产;随着喷施次数的增加,蕹菜锌含量也增加。     

洋葱AcSCL4的克隆及其功能分析

【目的】初步探讨AcSCL4在洋葱成花中的功能,为解析洋葱成花分子调控机制提供理论基础。【方法】以长日生态型洋葱品系SB2为试验材料,根据已有的洋葱转录组数据,通过qRT-PCR筛选目的基因AcSCL4后进行基因克隆,并对其进行生物信息学分析;构建AcSCL4基因的过表达载体pB221-3300-AcSCL4并转化农杆菌,采用花序浸染法浸染拟南芥,并对T₃代纯系转基因植株进行表型分析和开花相关基因表达量分析。【结果】洋葱AcSCL4基因CDS长1 515 bp,编码504个氨基酸;AcSCL4蛋白N端高度变异,C端有GRAS结构域。聚类分析发现,AcSCL4与石刁柏和棉花的SCL4蛋白亲缘关系较近。与野生型拟南芥相比,AcSCL4过表达植株表现晚花表型。荧光定量PCR结果表明,与野生型拟南芥相比,AcSCL4过表达植株开花调控基因CO和FT的表达量极显著下降。【结论】AcSCL4通过调控CO及FT基因表达来抑制洋葱开花。     

禽腺病毒流行株FAdV-4-AH-F41的分子特征及对细胞因子的诱导作用

【目的】分析1株血清4型禽腺病毒（FAdV-4）流行株的分子特征及其感染对细胞因子的影响。【方法】采集安徽省某禽类养殖场疑似心包积水 肝炎综合症的鸡肝脏组织样本,用鸡肝癌细胞（LMH）对病原进行分离,采用PCR、透射电镜（TEM）观察和间接免疫荧光试验（IFA）对分离的毒株进行鉴定。采用分段扩增后拼接的方法克隆分离毒株的全基因组,对其进行遗传进化分析和序列重组分析。基于柯赫法则,用分离毒株接种无特定病原体（SPF）鸡,检验其致病性。采用实时荧光定量PCR法,检测分离毒株对LMH细胞天然免疫相关细胞因子环鸟苷酸-腺苷酸（cGAS）、干扰素刺激因子（STING）、白介素-1β（IL-1β）、IL-6、IL-8、干扰素-α（IFN-α）、IFN-β、干扰素调节因子7（IRF7）、线粒体抗病毒信号蛋白（MAVS）、主要组织相容性复合体（MHC）Ⅰ-α、MHCⅡ-β等11种细胞因子的诱导作用。【结果】分离鉴定到1株FAdV,其在LMH细胞中培养未出现明显细胞病变效应;病毒粒子直径为70~90 nm,符合FAdV-4的结构特征;免疫荧光试验结果显示,在接种分离毒株的LMH细胞内可观察到亮红色荧光,而对照细胞没有荧光;将该毒株命名为FAdV-4-AH-F41。采用分段扩增后拼接的策略获得了FAdV-4-AH-F41全基因组序列（43 708 bp）;遗传进化分析和序列重组分析发现,FAdV-4-AH-F41与FAdV-4株核苷酸相似性为99.6%;存在3个重组事件,第1个重组事件发生在30 453-30 674 bp,第2个发生在36 884-36 988 bp,第3个发生在43 401-43 715 bp。致病性试验显示,FAdV-4-AH-F41是导致鸡心包积水-肝炎综合症的病原。实时荧光定量PCR检测结果显示,与对照（DMEM/F12处理）相比,用FAdV-4-AH-F41处理后LMH细胞中11种免疫因子的表达量水平均有提升,其中cGAS、IL-6、IRF7、MHCⅡ β差异达极显著水平（P<0.01）,IL-1β、IFN-α、MAVS差异达显著水平（P<0.05）,STING、IL-8、IFN-β、MHCⅠ-α差异不显著。【结论】成功分离1株重组血清4型禽腺病毒,感染LMH细胞会诱导强烈的天然免疫反应。     

副溶血弧菌VP-5的分离鉴定及其与鲈鱼免疫信号通路相互关系研究

为探讨福建省漳州市盛明养殖场鲈鱼的致病菌及其与鲈鱼免疫信号通路的相互关系,从而可以预防和治疗该疾病。解剖患病鲈鱼肝脏组织后划线培养分离纯化了致病菌VP-5。显微镜观察该致病菌VP-5形态为球杆状,弧形,有单鞭毛,运动活跃,革兰阴性菌。在VITEK-32全自动微生物分析仪鉴定后,该致病菌对酪氨酸、葡萄糖、甘露醇等反应阳性,对蔗糖,阿拉伯糖和棉子糖等反应阴性。分析了该致病菌16SrRNA和tdh1基因序列,发现其与副溶血弧菌的亲缘关系非常接近。综合后鉴定致病菌VP-5为副溶血弧菌。并研究了副溶血弧菌VP-5侵染鲈鱼后寄主免疫信号通路基因cd63/cxcr4/il-8的表达水平差异。鲈鱼腹腔注射感染副溶血弧菌VP-5,感染后0-96h解剖鲈鱼肝脏组织和表皮组织。采用Trizol试剂盒抽提鲈鱼总RNA,反转录后生成cDNA。利用实时荧光定量PCR技术对鲈鱼免疫信号通路基因cd63/cxcr4/il-8表达水平进行测定。鲈鱼受到副溶血弧菌VP-5感染后,免疫信号通路基因cd63/cxcr4/il-8在鲈鱼肝脏组织的表达量12h和24h时与对照组相比较差异显著(P<0.05);在表皮组织的表达量在24h和36h时与对照组相比较差异显著(P<0.05)。证实鲈鱼肝脏组织免疫信号通路基因的表达与副溶血弧菌VP-5的感染正相关,相对于表皮组织反应更快。因此鲈鱼的免疫信号通路基因cd63/cxcr4/il-8积极反应于致病菌,这些基因的快速表达会引发鲈鱼自身免疫应答,增强其抵抗致病菌的能力。     

Analysis of the gacS-gacA regulatory genes of spontaneous mutants of Pseudomonas fluorescens biocontrol strain PfG32R

Pseudomonas fluorescens strain PfG32R, a potential biocontrol agent against soilborne pathogens, frequently loses its antifungal activity and ability to produce enzymes. To characterize genetically the instability of these bacterial functions, we analyzed gacS and gacA genes of PfG32R and three spontaneous mutants of PfG32R (NR1, NR9, and ASW6), which had lost their ability to produce proteases and antifungal activities. The gacS and gacA sequences of PfG32R had 77%–89% and 78%–87% homology, respectively, with several known gacS and gacA homologues. All three spontaneous mutants were subjected to complementation analysis. Introduction of clones containing an intact gacS of PfG32R and another P. fluorescens strain Pf-5 as well as strain tea632 of P. syringae pv. theae complemented all three mutants restoring protease and antifungal activities, indicating a mutation in gacS. In sequencing analysis, the mutants had a deletion or change in amino acids in the conserved sensor kinase domains of GacS. The three mutants maintained both their antibacterial activity against Ralstonia solanacearum and Clavibacter michiganensis ssp. michiganensis and siderophore production, indicating that they are not controlled by the GacS/GacA system. The sequences reported in this article have been deposited in the DDBJ database under accession numbers AB219364 and AB219365.