In vitro and in vivo efficacy of anti‐chikungunya virus monoclonal antibodies produced in wild‐type and glycoengineered Nicotiana benthamiana plants

Chikungunya virus (CHIKV) is a mosquito‐transmitted alphavirus, and its infection can cause long‐term debilitating arthritis in humans. Currently, there are no licensed vaccines or therapeutics for human use to combat CHIKV infections. In this study, we explored the feasibility of using an anti‐CHIKV monoclonal antibody (mAb) produced in wild‐type (WT) and glycoengineered (?XFT) Nicotiana benthamiana plants in treating CHIKV infection in a mouse model. CHIKV mAb was efficiently expressed and assembled in plant leaves and enriched to homogeneity by a simple purification scheme. While mAb produced in ?XFT carried a single N‐glycan species at the Fc domain, namely GnGn structures, WT produced mAb exhibited a mixture of N‐glycans including the typical plant GnGnXF₃ glycans, accompanied by incompletely processed and oligomannosidic structures. Both WT and ?XFT plant‐produced mAbs demonstrated potent in vitro neutralization activity against CHIKV. Notably, both mAb glycoforms showed in vivo efficacy in a mouse model, with a slight increased efficacy by the ?XFT‐produced mAbs. This is the first report of the efficacy of plant‐produced mAbs against CHIKV, which demonstrates the ability of using plants as an effective platform for production of functionally active CHIKV mAbs and implies optimization of in vivo activity by controlling Fc glycosylation.     

Stain Solubilities Part III

Ethylenediaminetetraacetic acid (EDTA) solution is used to decalcify bone specimens for histological examination. Sodium hydroxide (NaOH) has been used to dissolve EDTA and to bring EDTA solutions to neutral pH. This solution, however, requires several weeks to decalcify bone specimens. We investigated a new de-calcification fluid using concentrated ammonium hydroxide (NH₄OH) to dissolve EDTA and to adjust the pH to neutral. Decalcification was performed using a magnetic stirrer with and without vacuum, or with a sonic cleaner. Decalcification end point was confirmed using both the weight loss and X-ray methods. After decalcification, specimens were processed through paraffin and sections were stained with hematoxylin and eosin. Decalcification employing NH₄OH required an average of six days. Light microscopy indicated good retention of cellular detail.     

Increasing the potency of neutralizing single-domain antibodies by functionalization with a CD11b/CD18 binding domain

Recombinant single domain antibodies (nanobodies) constitute an attractive alternative for the production of neutralizing therapeutic agents. Their small size warrants rapid bioavailability and fast penetration to sites of toxin uptake, but also rapid renal clearance, which negatively affects their performance. In this work, we present a new strategy to drastically improve the neutralizing potency of single domain antibodies based on their fusion to a second nanobody specific for the complement receptor CD11b/CD18 (Mac-1). These bispecific antibodies retain a small size (?30 kDa), but acquire effector functions that promote the elimination of the toxin-immunocomplexes. The principle was demonstrated in a mouse model of lethal toxicity with tetanus toxin. Three anti-tetanus toxin nanobodies were selected and characterized in terms of overlapping epitopes and inhibition of toxin binding to neuron gangliosides. Bispecific constructs of the most promising monodomain antibodies were built using anti Mac-1, CD45 and MHC II nanobodies. When co-administered with the toxin, all bispecific antibodies showed higher toxin-neutralizing capacity than the monomeric ones, but only their fusion to the anti-endocytic receptor Mac-1 nanobody allowed the mice to survive a 10-fold lethal dose. In a model of delayed neutralization of the toxin, the anti- Mac-1 bispecific antibodies outperformed a sheep anti-toxin polyclonal IgG that had shown similar neutralization potency in the co-administration experiments. This strategy should have widespread application in the development of nanobody-based neutralizing therapeutics, which can be produced economically and more safely than conventional antisera.     

New insights into the binding mode of pyridine-3-carboxamide inhibitors of E. coli DNA gyrase

Previously we have reported on a series of pyridine-3-carboxamide inhibitors of DNA gyrase and DNA topoisomerase IV that were designed using a computational de novo design approach and which showed promising antibacterial properties. Herein we describe the synthesis of additional examples from this series aimed specifically at DNA gyrase, along with crystal structures confirming the predicted mode of binding and in vitro ADME data which describe the drug-likeness of these compounds.     

泥炭藓: 一类具有重要生态、经济和科学价值的碳封存植物

全球气候变暖是人类面临最严峻的环境挑战。有效控制碳排放, 充分发挥生态系统的固碳能力是实现碳中和目标的重要手段。作为碳封存能力最强的一种湿地类型, 泥炭地是加快实现碳中和目标的关键陆地生态系统。作为泥炭地“有效的生态系统工程师”, 泥炭藓(Sphagnum)在泥炭地的碳汇功能、过滤淡水及保护土地免受洪水侵袭等方面具有极其重要的作用。100多年来, 泥炭藓广泛应用于医药保健、污染监测和废水处理等领域, 尤其是作为一类最值得信赖的土壤介质和保湿材料一直被广泛用于园艺产业。在全球气候变暖和“双碳”目标的大背景下, 泥炭藓已经成为生命科学和生态学研究的热点。该文主要从泥炭藓的形态、物种多样性和起源、生境与分布、繁殖和保护、培养与种植、环境指示和监测、用途和应用, 以及碳封存、储水和酸化能力等方面进行综述, 旨在为泥炭藓研究、泥炭地的保护和恢复以及泥炭藓开发利用和产业发展提供借鉴与参考。     

樟子松固沙林林水关系研究进展及对营林实践的指导

中国有着世界上最大面积的人工林, 如何维持人工林的可持续性已成为气候变化背景下需要面对的重大挑战。樟子松(Pinus sylvestris var. mongolica)以其抗旱、抗寒、耐贫瘠等优良特性成为中国北方生态治理中最主要的常绿针叶树种之一, 近70年来发挥了巨大的防风固沙与生态固碳功能。然而, 随着林分的生长与气候变化, 樟子松人工固沙林正经历着越来越严峻的环境胁迫, 部分地区出现了林分“早衰”或死亡的现象, 引起了人们对樟子松固沙林适应与应对气候变化能力的担忧。该文在回溯樟子松基本生物学特征与引种推广历史, 系统总结近年来樟子松林林水关系研究新成果的基础上, 全面分析了樟子松固沙林林水关系存在的主要矛盾, 并提出了基于林水关系相协调的林分经营措施的调整: 由倡导防护功能为主的单一目标向包含林分稳定性、生态固碳功能、可持续发展等多目标平衡方向调整; 由以沙地森林景观培育为主向以良好土壤生境培育为主的方向调整; 由倡导天然更新为主向以人工造林与天然更新相结合的世代更新方向调整。在立足于北方沙地脆弱生境与气候变化客观现实的基础背景下, 应坚持樟子松在固沙林生态系统演化过程中的先锋种与建群种地位, 基于“以水定绿”原则, 采取“隔行带伐+再造林”等方式开展林分密度动态调控, 促进林分向异龄林结构演化, 促进樟子松固沙林生态服务的优质化和生态固碳功能的最大化。     

Cloning of anti-LPS factor cDNA from Tachypleus tridentatus, expression in Bombyx mori larvae and its biological activity in vitro

In this article we report the cloning and expression of a cDNA encoding Tachypleus anti-lipopolysaccharide (LPS) factor, which is of interest for use as a potential inhibitor of the common core subunit of Gramnegative bacterial endotoxins. First, two degenerate primers were designed based on the sequence homology of anti-LPS factors purified from different species of horseshoe crab. The total RNA was extracted from amebocytes of Tachypleus tridentatus. The cDNA was then obtained by using the RT-PCR methods. Second, the cDNA of Tachypleus anti-LPS factor (TALF) was expressed in Bombyx mori larvae using baculovirus expression system, which showed a yield of up to 600 mg/L. Last, we determined the biological activity of the recombinant proteins by LPS neutralization assay and bacteriostatic assay in vitro.     

GC32细胞对三种虫媒病毒的敏感性研究

用胃癌细胞株ＧＣ３２,对乙型脑炎、基孔肯雅、兰加特病毒进行敏感性研究。并用ＢＨＫ２１细胞作对照,发现ＧＣ３２细胞对两种病毒的敏感性与对照细胞ＢＨＫ２１极为接近。ＢＨＫ２１和ＧＣ３２细胞对基孔肯雅、乙型脑炎和兰加特的免疫荧光实验,于感染后４８ｈ或７２ｈ都出现＋＋＋～＋＋＋＋的阳性结果。两种细胞对基孔肯雅和乙型脑炎病毒都形成空斑,ＢＨＫ２１细胞对基孔肯雅和乙型脑炎病毒的空斑形成单位分别为５．６５和５．３６;ＧＣ３２对基孔肯雅和乙型脑炎病毒的空斑形成单位分别为６．４８和５．６１。实验表明,ＧＣ３２细胞可以作为有关病毒实验的理想细胞株。     

Conventional and PCR Detection of Aphelenchoides fragariae in Diverse Ornamental Host Plant Species

A PCR-based diagnostic assay was developed for early detection and identification of Aphelenchoides fragariae directly in host plant tissues using the species-specific primers AFragFl and AFragRl that amplify a 169-bp fragment in the internal transcribed spacer (ITS1) region of ribosomal DNA. These species-specific primers did not amplify DNA from Aphelenchoides besseyi or Aphelenchoides ritzemabosi. The PCR assay was sensitive, detecting a single nematode in a background of plant tissue extract. The assay accurately detected A. fragariae in more than 100 naturally infected, ornamental plant samples collected in North Carolina nurseries, garden centers and landscapes, including 50 plant species not previously reported as hosts of Aphelenchoides spp. The detection sensitivity of the PCR-based assay was higher for infected yet asymptomatic plants when compared to the traditional, water extraction method for Aphelenchoides spp. detection. The utility of using NaOH extraction for rapid preparation of total DNA from plant samples infected with A. fragariae was demonstrated.