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

Abstract Background Stenodactylin is a highly toxic plant lectin purified from the caudex of Adenia stenodactyla , with molecular structure, intracellular routing and enzyme activity similar to those of ricin, a well-known type 2 ribosome-inactivating protein. However, in contrast with ricin, stenodactylin is retrogradely transported not only in peripheral nerves but also in the central nervous system. Purpose Stenodactylin properties make it a potential candidate for application in neurobiology and in experimental therapies against cancer. Thus, it is necessary to better clarify the toxic activity of this compound. Study design We investigated the mechanism of stenodactylin-induced cell death in the neuroblastoma-derived cell line, NB100, evaluating the implications of different death pathways and the involvement of oxidative stress. Methods Stenodactylin cytotoxicity was determined by evaluating protein synthesis and other viability parameters. Cell death pathways and oxidative stress were analysed through flow cytometry and microscopy. Inhibitors of apoptosis, oxidative stress and necroptosis were tested to evaluate their protective effect against stenodactylin cytotoxicity. Results Stenodactylin efficiently blocked protein synthesis and reduced the viability of neuroblastoma cells at an extremely low concentration and over a short time (1 pM, 24 h). Stenodactylin induced the strong and rapid activation of apoptosis and the production of free radicals. Here, for the first time, a complete and long lasting protection from the lethal effect induced by a toxic type 2 ribosome-inactivating protein has been obtained by combining the caspase inhibitor Z-VAD-fmk, to either the hydrogen peroxide scavenger catalase or the necroptotic inhibitor necrostatin-1. Conclusion In respect to stenodactylin cytotoxicity, our results: (i) confirm the high toxicity to nervous cells, (ii) indicate that multiple cell death pathways can be induced, (iii) show that apoptosis is the main death pathway, (iv) demonstrate the involvement of necroptosis and (v) oxidative stress

Primary sequence and 3D structure prediction of the plant toxin stenodactylin

Producción CientíficaStenodactylin 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.Universidad de Bolonia y Pallotti Legacies for Cancer Research; Fundación CARISBO - (Project 2019.0539)Junta de Castilla y León - (Project VA033G19

The usefulness of Moynihan questionnaire in the evaluation of knowledge on healthy diet of patients undergoing cardiology rehabilitation

Background. The aim of study was to test the usefulness of Moynihan questionnaire in the evaluation of knowledge on healthy diet of patients undergoing cardiology rehabilitation. Methods. We enrolled 51 patients (pts): 41 men and 10 women, mean age 67.97 + 11.2 years. The case study included: 21 pts that underwent coronary bypass surgery, 16 pts replaced plastic tube, 14 pts had surgery for the other reasons. All pts underwent nutritional investigation by a dietitian. Anthropometric and biochemical parameters were detected and, by the end, the Moynihan questionnaire was administrated. Pts underwent nutritional coaching, and questionnaire and dietary assessment were rechecked after 3 months. Results. At baseline, the mean Questionnaire score was 22.4 + 3.2 points, decreased to 20.6 + 3.1 points after 3 months (p<0.05). A detailed analysis of the questions showed that the major informations gaps were related to consumption of fruits and vegetables, consumption of fat and salt. In addition pts have acquired more general knowledge about food composition. Conclusions. The Moynihan questionnaire is an useful instrument of evaluation of dietary knowledge even in selected patients population. In the present study involving patients after cardiac surgery the main difficulties were related to high age of pts, the low cultural level and, mainly, to the post-surgery stress. However, an increase of correct answers as well as an increased knowledge about food composition were detected after educational intervention performed by the dietitian

Immunotoxins and Other Conjugates Containing Saporin-S6 for Cancer Therapy

Ribosome-inactivating proteins (RIPs) are a family of plant toxins that permanently damage ribosomes and possibly other cellular substrates, thus causing cell death. RIPs are mostly divided in two types: Type 1 RIPs that are single-chain enzymatic proteins, and type 2 RIPs that consist of an active A chain (similar to a type 1 RIP) linked to a B chain with lectin properties. RIP-containing conjugates have been used in many experimental strategies against cancer cells, often showing great efficacy in clinical trials. Saporin-S6, a type 1 RIP extracted from Saponaria officinalis L. seeds, has been extensively utilized to construct anti-cancer conjugates because of its high enzymatic activity, stability and resistance to conjugation procedures, resulting in the efficient killing of target cells. This review summarizes saporin-S6-containing conjugates and their application in cancer therapy, considering in-vitro and in-vivo studies both in animal models and in clinical trials. The review is structured on the basis of the targeting of hematological versus solid tumors and on the antigen recognized on the cell surface

Pathogenesis of cell toxicity by plant toxins

Ribosome inactivating proteins (RIPs) are a family of plant proteins that depurinate the major rRNA, inhibiting the protein synthesis. RIPs are divided into type 1, single chain proteins with enzymatic activity, and type 2 RIPs (toxic and non-toxic), with the enzymatic chain linked to a binding chain. RIPs have been used alone or as toxic component of immunotoxins for experimental therapy of many diseases. The knowledge of cell death pathway(s) induced by RIPs could be useful for clarifying the mechanisms induced by RIPs and for designing specific immunotherapy. The topic of the current study was (i) the determination of the amino acid sequence of the type 2 RIP stenodactylin. The comparison with other RIPs showed that the A chain is related to other toxic type 2 RIPs. whereas the B chain is more related to the non-toxic type 2 RIPs. This latter result is surprising because stenodactylin is actually the most toxic type 2 RIP known; (ii) the study of the cell death mechanisms induced by stenodactylin in human neuroblastoma cells (NB100). High doses of stenodactylin can activate the effector caspases (perhaps through the DNA damage and/or intrinsic/extrinsic pathways) and also cause ROS generation. Low doses cause a caspase-dependent apoptosis, mainly via extrinsic pathway. Moreover, the activation of caspases precedes the inhibition of protein synthesis; (iii) the investigation of the cell death pathway induced by the non-toxic type 2 RIPs ebulin l and nigrin b. These RIPs demonstrated high enzymatic activity in a cell-free system, but they lack high cytotoxicity. These preliminary studies demonstrate that the cell death mechanism induced by the two non-toxic RIPs is partially caspase-dependent apoptosis, but other mechanisms seem to be involve

Molecular Features Contributing to Virus-Independent Intracellular Localization and Dynamic Behavior of the Herpesvirus Transport Protein U_S9

<div><p>Reaching the right destination is of vital importance for molecules, proteins, organelles, and cargoes. Thus, intracellular traffic is continuously controlled and regulated by several proteins taking part in the process. Viruses exploit this machinery, and viral proteins regulating intracellular transport have been identified as they represent valuable tools to understand and possibly direct molecules targeting and delivery. Deciphering the molecular features of viral proteins contributing to (or determining) this dynamic phenotype can eventually lead to a virus-independent approach to control cellular transport and delivery. From this virus-independent perspective we looked at U_S9, a virion component of Herpes Simplex Virus involved in anterograde transport of the virus inside neurons of the infected host. As the natural cargo of U_S9-related vesicles is the virus (or its parts), defining its autonomous, virus-independent role in vesicles transport represents a prerequisite to make U_S9 a valuable molecular tool to study and possibly direct cellular transport. To assess the extent of this autonomous role in vesicles transport, we analyzed U_S9 behavior in the absence of viral infection. Based on our studies, Us9 behavior appears similar in different cell types; however, as expected, the data we obtained in neurons best represent the virus-independent properties of U_S9. In these primary cells, transfected U_S9 mostly recapitulates the behavior of U_S9 expressed from the viral genome. Additionally, ablation of two major phosphorylation sites (i.e. Y₃₂Y₃₃ and S₃₄ES₃₆) have no effect on protein incorporation on vesicles and on its localization on both proximal and distal regions of the cells. These results support the idea that, while U_S9 post-translational modification may be important to regulate cargo loading and, consequently, virion export and delivery, no additional viral functions are required for U_S9 role in intracellular transport.</p></div

Erratum: “Detecting Reconnection Events in Kinetic Vlasov Hybrid Simulations Using Clustering Techniques” (2021, ApJ, 908, 107)

International audienc

Detecting Reconnection Events in Kinetic Vlasov Hybrid Simulations Using Clustering Techniques

International audienceAbstract Kinetic turbulence in magnetized space plasmas has been extensively studied via in situ observations, numerical simulations, and theoretical models. In this context, a key point concerns the formation of coherent current structures and their disruption through magnetic reconnection. We present automatic techniques aimed at detecting reconnection events in a large data set of numerical simulations. We make use of clustering techniques known as K -means and DBscan (usually referred to in literature as unsupervised machine-learning approaches), and other methods based on thresholds of standard reconnection proxies. All our techniques also use a threshold on the aspect ratio of the regions selected. We test the performance of our algorithms. We propose an optimal aspect ratio to be used in the automated machine-learning algorithm: AR = 18. The performance of the unsupervised approach results in it being strongly competitive with respect to those of other methods based on thresholds of standard reconnection proxies

GFP-U_S9 localization.

<p>Cells shown here are representative of several experiments and all share the typical GFP-U_S9 pattern, i.e. a bright punctuate signal present throughout the cytoplasm and partially concentrated in a peri-nuclear region. Additionally, GFP-U_S9 can be seen at the plasma membrane, while no fluorescent signal is present in the nuclear region. In the panel labeled MDA hm (higher magnification), the microphotograph focuses on the extension tip protruding from one transfected cell and reaching the body of one untransfected cell. GFP-U_S9 can be detected in vesicles present in this distal region as well as on the plasma membrane. In green: GFP-U_S9 fluorescence; nuclei are stained in blue with Hoechst 33342. Bar = 10 µm.</p

Intracellular localization and membrane association of U_S9.

<p>A) MDA cells transfected with GFP, g9, g9-TM (GFP fused the U_S9 trans-membrane domain), and g9-ΔTM (GFP fused the U_S9 hydrophilic domain) were imaged under a fluorescence microscope. Nuclei were stained with Hoechst 33342 (blue) and images were merged in B). C) Proteins extracted from MDA cells expressing the same constructs described in A) were divided in post-nuclear soluble (S), and membrane (M) fractions, separated on SDS Page, and revealed with GFP antibody. Bar = 10 µm.</p