Ricin and Abrin in Biosecurity: Detection Technologies and Strategic Responses

When a disaster occurs, the rapid gathering and sharing of crucial information among public safety agencies, emergency response units, and the public can save lives and reduce the scope of the problem; yet, this is seldom achieved. The lack of interoperability hinders effective collaboration across organizational and jurisdictional boundaries. In this article, we propose a general architecture for emergency communications that incorporates (1) an information broker, (2) events and event-driven processes, and (3) interoperability. This general architecture addresses the question of how an information broker can overcome obstacles, breach boundaries for seamless communication, and empower the public to become active participants in emergency communications. Our research is based on qualitative case studies on emergency communications, workshops with public safety agencies, and a comparative analysis of interoperability issues in the European public sector. This article features a conceptual approach toward proposing a way in which public safety agencies can achieve optimal interoperability and thereby enable seamless communication and crowdsourcing in emergency prevention and response.

Ribosome inactivating proteins (RIPs) are a family of plant proteins that irreversibly arrest protein synthesis through depurination of rRNA. The anticancer, antiviral and neurotoxic properties allow RIPs to have useful applications in agriculture and in many biomedical fields, including clinical drug development. Galactose–binding lectins contained in caudices of four Adenia species were purified by affinity chromatography on CL Sepharose 6B. All lectins agglutinate human erythrocytes. The lectin from A. kirkii, called kirkiin, had the highest toxicity, inhibiting protein synthesis both by cell-free system and by whole cells, and deadenylates mammalian and yeast ribosomes. Kirkiin is highly toxic to cells, in which it induces apoptosis with EC50 (concentration reducing viability by 50%) of 10-14M after 72h. Analysis of the genomic clones encoding kirkiin reveals a high degree of sequence similarity to other type 2 RIPs. Molecular modeling confirmed that kirkiin have a similar structure to ricin. Thus, kirkiin shows all the properties of a toxic type 2 RIP and is amongst the most potent plant toxins. The pathogenesis of cell intoxication induced by the non-toxic type 2 RIPs from Sambucus, ebulin l and nigrin b, was investigated in a neuroblastoma-derived cell line (NB100), which showed to be very sensitive to RIPs. Despite the high translational inhibition activity in cell-free systems, both RIPs showed a low cytotoxicity in NB100 cells with respect to both type 1 and type 2 RIPs. The results revealed that the mechanism of cell death induced by Sambucus RIPs (10-8M) is partly due to caspase-dependent apoptosis and suggest the existence of a further cell death pathway that would be prevalent at the early 24h of intoxication and that is different from necrosis, necroptosis and autophagy. A block of cell cycle can be excluded. After 24h, the apoptotic pathway becomes the main route of cell death.

Stenodactylin is one of the most potent type 2 ribosome-inactivating proteins (RIPs); its high toxicity has been demonstrated in several models both in vitro and in vivo. Due to its peculiarities, stenodactylin could have several medical and biotechnological applications in neuroscience and cancer treatment. In this work, we report the complete amino acid sequence of stenodactylin and 3D structure prediction. The comparison between the primary sequence of stenodactylin and other RIPs allowed us to identify homologies/differences and the amino acids involved in RIP toxic activity. Stenodactylin RNA was isolated from plant caudex, reverse transcribed through PCR and the cDNA was amplificated and cloned into a plasmid vector and further analyzed by sequencing. Nucleotide sequence analysis showed that stenodactylin A and B chains contain 251 and 258 amino acids, respectively. The key amino acids of the active site described for ricin and most other RIPs are also conserved in the stenodactylin A chain. Stenodactylin amino acid sequence shows a high identity degree with volkensin (81.7% for A chain, 90.3% for B chain), whilst when compared with other type 2 RIPs the identity degree ranges from 27.7 to 33.0% for the A chain and from 42.1 to 47.7% for the B chain.

The level of toxicity of aqueous extract from Ganoderma sp. fruiting body isolated from different plant media was determined in this experiment using Brine shrimp lethality test (BST) on Artemia salina Leach. The influence of media on the toxicity level of aqueous extract from Ganoderma sp. fruiting body was determined in this experiment. Ganoderma sp. and G. Lucidum were grown on three flamboyant, ( Delonix regia Bojer ex Hook Rafin), sengon ( Paraserianthes falcataria (L) Nielsen) and coconut( Cocos nucifera Linn.) plant media. The aqueous extracts were prepared from fruiting body using 0.02 M phosphate buffer pH 7.2. The toxicity level of these aqueous extract were then screened using the BST method. The level of the toxicity was determined by LC50. The results indicated that the aqueous extracts of Ganoderma sp. and G . lucidum isolated from flamboyant, sengon and coconut plant media were influenced by plant media. Ganoderma sp. was isolated from flamboyant plant media possesed the highest toxicity (LC50 480 g aqueous extract/ml), then followed by Ganoderma sp. isolated from sengon plant media (LC50 770 g aqueous extract /ml) and coconut (LC50 1040 g aqueous extract /ml). G. lucidum from coconut plant media possesed the highest toxicity (LC50 660 g aqueous extract /ml), followed by G. lucidum were isolated from sengon plant media (LC50 1100 g aqueous extract /ml) and flamboyant (LC50 1970 g aqueous extract /ml). It was suggested that G.lucidum having highest toxicity might produce compounds having antitumor activity. Key words: Ganoderma sp., aqueous extracts, toxicity .

Ricin is a plant-derived toxin with a history as a biothreat agent. The toxin's enzymatic subunit, ricin toxin A chain (RTA), is a ribosome-inactivating protein that, when delivered into the cytoplasm of mammalian cells, arrests protein synthesis with extraordinary efficiency. Once within the cytoplasm, RTA is shielded from circulating toxin-neutralizing antibodies. Here, we describe methods we developed to neutralize RTA within the cytoplasm of Vero cells using DNA-based delivery of alpaca-derived single-domain antibodies (VHHs) targeting RTA's active site. We describe the design of the VHH expression vectors, assessment of transient expression of VHHs in Vero cells by enzyme-linked immunosorbent assay and western blotting, and cytotoxicity studies. While the protocols here are specific to ricin, they are easily modified for other toxins or even intracellular pathogens such as viruses.

Ⅱ型核糖体失活蛋白(RIPs)是一类重要的蛋白毒素,该类毒素大都具有一对二硫键连接的A-B链结构特征,B链具有半乳糖结合特性,能够与真核细胞膜表面受体特异性结合,将具有N-糖苷酶活性的A链导入细胞,与核糖体特定位点发生脱嘌呤作用使核糖体失活,最终通过抑制蛋白质合成而展现出细胞毒性。Ⅱ型RIPs毒素毒性极强,来源于植物的蓖麻毒素(ricin)和相思子毒素(abrin)的毒性分别是神经性毒剂维埃克斯(Vx)的385倍和2885倍。同时,该类毒素来源广泛、易于制备、稳定性好,成为一类潜在化生恐怖战剂,受到国内外广泛关注,其中蓖麻毒素作为唯一的蛋白毒素被收录于禁止化学武器公约禁控清单。近年来发生的多次蓖麻毒素邮件恐怖事件,进一步促进了有关Ⅱ型RIPs毒素的准确、灵敏、快速的检测鉴定技术的发展。剧毒性Ⅱ型RIPs毒素的检测鉴定方法主要涉及免疫分析法为代表的特异性识别和生物质谱分析为主的定性定量检测方法,以及基于脱嘌呤反应活性和细胞毒性的毒素活性检测方法。基于抗原-抗体作用的免疫检测法及基于寡核苷酸适配体的特异性识别检测法具有速度快、灵敏度高的优势,但对于复杂样品中高度同源蛋白的检测,易产生假阳性结果。随着生物质谱技术的快速发展,电喷雾离化(ESI)或基质辅助激光解吸离化(MALDI)等技术广泛应用于蛋白质的准确鉴定,不仅能够提供蛋白毒素的准确分子量和结构序列信息,而且能够实现准确定量。酶解质谱法是应用最为广泛的检测鉴定方法,通过酶解肽指纹谱分析,实现蛋白毒素的准确鉴定;对于复杂样品中蛋白毒素的分析,通过多种蛋白酶酶解策略获得丰富的特异性肽段标志物,然后进行肽段标志物的靶向质谱分析从而获得准确的定性及定量信息,方法有效提升了Ⅱ型RIPs毒素鉴定的准确度和灵敏度。免疫分析法和生物质谱法能够准确鉴定Ⅱ型RIPs毒素,但无法识别毒素是否还保持毒性。对于Ⅱ型RIPs毒素的活性分析,主要包括基于N-糖苷酶活性的脱嘌呤反应测定法和细胞毒性测定法,两种方法均可实现毒素毒性的简便、快速、灵敏的分析检测,是Ⅱ型RIPs毒素检测方法的有效补充。由于该类毒素的高度敏感性,国际禁止化学武器组织(OPCW)对相关样品中Ⅱ型RIPs毒素的分析提出了唯一性鉴定的技术要求。该文引用了Ⅱ型RIPs毒素及其检测方法相关的70篇文献,综述了以上Ⅱ型RIPs毒素的结构性质、中毒机理及典型剧毒性Ⅱ型RIPs毒素检测方法的研究进展,对不同检测方法的特点和应用潜力进行了总结,并结合OPCW对Ⅱ型RIPs毒素唯一性鉴定的技术需求,展望了未来Ⅱ型RIPs毒素检测技术研究的发展趋势。

Apoptosis and necroptosis induced by stenodactylin in neuroblastoma cells can be completely prevented through caspase inhibition plus catalase or necrostatin-1

A battery of assays for chasing ricin and its activity.

The real-time transient stability detection and emergency control technology based on wide area response has become a hot research area in power system stability studies. Several different technologies have been proposed, but lots of problems remain to be solved before they can be applied in practice. A wide area measurement system (WAMS) based test platform is developed for transient stability detection and control. The design as well as main function modules of the platform are introduced. In addition, three generator power angle prediction methods and six response based transient instability detection technologies are given. Results of engineering application demonstrate that the developed test platform can provide a real-time operation environment, which can effectively compare and analyze the validity and practicability of these transient stability detection technologies. Based on the measured perturbed trajectories from actual power systems or the Real-Time Digital Simulators (RTDS), the platform can realize the assessment and visual result presentation of various responses from different transient instability detection technologies. The test platform can be applied to different power systems and it is convenient to embed new transient instability detection modules. Meanwhile some deficiencies and shortcomings in engineering application are pointed out and corresponding suggestions are given. In conclusion, the hardware and software structure, function modulus and engineering applications are presented. The application in actual power systems shows that it has a good application perspective.

Production of Abrus pulchellus ribosome-inactivating protein from seeds callus culture

Kirkiin is a new type 2 ribosome-inactivating protein (RIP) purified from the caudex of Adenia kirkii with a cytotoxicity compared to that of stenodactylin. The high toxicity of RIPs from Adenia genus plants makes them interesting tools for biotechnology and therapeutic applications, particularly in cancer therapy. The complete amino acid sequence and 3D structure prediction of kirkiin are here reported. Gene sequence analysis revealed that kirkiin is encoded by a 1572 bp open reading frame, corresponding to 524 amino acid residues, without introns. The amino acid sequence analysis showed a high degree of identity with other Adenia RIPs. The 3D structure of kirkiin preserves the overall folding of type 2 RIPs. The key amino acids of the active site, described for ricin and other RIPs, are also conserved in the kirkiin A chain. Sugar affinity studies and docking experiments revealed that both the 1α and 2γ sites of the kirkiin B chain exhibit binding activity toward lactose and D-galactose, being lower than ricin. The replacement of His246 in the kirkiin 2γ site instead of Tyr248 in ricin causes a different structure arrangement that could explain the lower sugar affinity of kirkiin with respect to ricin.

DaimonDNA: A portable, low-cost loop-mediated isothermal amplification platform for naked-eye detection of genetically modified organisms in resource-limited settings

Polychlorinated biphenyls (PCBs) are the organic pollutants that of persistence, fat soluble and character such as biological toxicity, widely distributed. It also showed a highly persistent in the environment, strong enrichment and high biological toxicity, having the serious harm to human health and ecological environment. The final destination of PCBs in the environmental system is soil and sediment due to its highly distribution coefficient between sediment and water. PCBs in soil is difficult to decompose and has strong adsorption. Therefore, the detection and remediation of PCBs in soil is very critical. PCBSin soil detection technologies becoming matured at present. Such as dual column gas chromatography analysis method, GC – MS, enzyme immunoassay for PCBs detecting have been widely used. Low cost, high resolution and high accuracy detection technology has been more and more attention. For the remediation technologies of contaminated soils, chemical remediation, bioremediation technologies developed fast. Efficient, economic and environmental multiple remediation method, such as elution pretreatment on contaminated soil, combination of multiple remediation techniques are the development direction of the future. Based on the analysis on detection and remediation technologies of PCBs in soil. The principle and suitable condition of different detection methods and remediation technologies of PCBs in soil was studied in this paper. To provide technical support to remediation the PCB-contaminated soils.

Fruticulosin: A novel type 2 ribosome-inactivating protein from Abrus fruticulosus seeds that exhibits toxic and antileishmanial activity

Plant ribosome-inactivating proteins (RIPs) are inhibitors with RNA-N-glycosidase activity that irreversibly inactivate eukaryotic ribosomes, thereby impairing protein synthesis. In recent years, more than 40 RIPs and lectins belonging to the Sambucus genus have been isolated and characterized to varying degrees. The type 2 RIPs isolated from Sambucus have the peculiarity that although they are enzymatically more active than ricin, they lack the high toxicity of ricin to intact cells and animals. The presence in the same tissue of heterodimeric and tetrameric type 2 RIPs, structurally related monomeric, and homodimeric lectins together with unrelated type 1 RIPs make Sambucus an ideal model for studying these special proteins whose biological role is unknown at present. In the light of the accumulated results on the Sambucus RIPs and lectins, we present here the main findings about structural features and biological activities of these proteins as well as the evolutionary relationship between them and some of their potential uses.