Current learning strategies in fire evacuation for seniors and people with disabilities in private seniors' residences and long-term care homes: a scoping review.

Current strategies for teaching evacuation methods in private seniors' residences (PSR) and long-term care (LTCH) homes may pose risks to people with disabilities (PWD) and seniors' physical and psychological health. This study aimed to address the following questions: (1) Which are the current fire evacuation learning strategies used with PWD or seniors? (2) What are the barriers and facilitators for PWD and seniors' during fire evacuation and learning strategies in PSR and LTCH? (3) What is the existing equipment that could be used with PWD seniors?. A scoping review of grey and scientific literature was done in six databases and Google scholar. Additional information was found on Québec government websites. This review identified 13 scientific papers and 22 documents. Twenty barriers (personal = 9, environmental = 11), and 14 facilitators (personal = 4, environmental = 10) were extracted. The current fire evacuation learning strategies currently used can be grouped into three categories: drills; training; promotion of a fire safety plan. Six types of evacuation equipment were found; however, their use has been scarcely documented. Safety for seniors during fire evacuation is still an important issue to be improved. Increasing awareness and creating new practices and tools that consider the strengths and difficulties of seniors seems to be a promising avenue for improving evacuation.

MT9, a natural peptide from black mamba venom antagonizes the muscarinic type 2 receptor and reverses the M2R-agonist-induced relaxation in rat and human arteries.

All five muscarinic receptors have important physiological roles. The endothelial M2 and M3 subtypes regulate arterial tone through direct coupling to Gq or Gi/o proteins. Yet, we lack selective pharmacological drugs to assess the respective contribution of muscarinic receptors to a given function. We used mamba snake venoms to identify a selective M2R ligand to investigate its contribution to arterial contractions. Using a bio-guided screening binding assay, we isolated MT9 from the black mamba venom, a three-finger toxin active on the M2R subtype. After sequencing and chemical synthesis of MT9, we characterized its structure by X-ray diffraction and determined its pharmacological characteristics by binding assays, functional tests, and ex vivo experiments on rat and human arteries. Although MT9 belongs to the three-finger fold toxins family, it is phylogenetically apart from the previously discovered muscarinic toxins, suggesting that two groups of peptides evolved independently and in a convergent way to target muscarinic receptors. The affinity of MT9 for the M2R is 100 times stronger than that for the four other muscarinic receptors. It also antagonizes the M2R/Gi pathways in cell-based assays. MT9 acts as a non-competitive antagonist against acetylcholine or arecaine, with low nM potency, for the activation of isolated rat mesenteric arteries. These results were confirmed on human internal mammary arteries. In conclusion, MT9 is the first fully characterized M2R-specific natural toxin. It should provide a tool for further understanding of the effect of M2R in various arteries and may position itself as a new drug candidate in cardio-vascular diseases.

A new Kunitz-type snake toxin family associated with an original mode of interaction with the vasopressin 2 receptor.

BACKGROUND AND PURPOSE: Venomous animals express numerous Kunitz-type peptides. The mambaquaretin-1 (MQ1) peptide identified from the Dendroaspis angusticeps venom is the most selective antagonist of the arginine-vasopressin V2 receptor (V2R) and the only unique Kunitz-type peptide active on a GPCR. We aimed to exploit other mamba venoms to enlarge the V2R-Kunitz peptide family and gain insight into the MQ1 molecular mode of action. EXPERIMENTAL APPROACH: We used a bio-guided screening assay to identify novel MQs and placed them phylogenetically. MQs were produced by solid-phase peptide synthesis and characterized in vitro by binding and functional tests and in vivo by diuresis measurement in rats. KEY RESULTS: Eight additional MQs were identified with nanomolar affinities for the V2R, all antagonists. MQs form a new subgroup in the Kunitz family, close to the V2R non-active dendrotoxins and to two V2R-active cobra toxins. Sequence comparison between active and non-active V2R Kunitz peptides highlighted five positions, among which four are involved in V2R interaction and belong to the two large MQ1 loops. We finally determined that eight positions, part of these two loops, interact with the V2R. The variant MQ1-K39A showed a higher affinity for the hV2R, but not for the rat V2R. CONCLUSIONS AND IMPLICATIONS: A new function and mode of action is associated with the Kunitz peptides. The number of MQ1 residues involved in V2R binding is large and may explain its absolute selectivity. MQ1-K39A represents the first step in the improvement of the MQ1 design from a medicinal perspective.

Ancestral protein resurrection and engineering opportunities of the mamba aminergic toxins.

Mamba venoms contain a multiplicity of three-finger fold aminergic toxins known to interact with various α-adrenergic, muscarinic and dopaminergic receptors with different pharmacological profiles. In order to generate novel functions on this structural scaffold and to avoid the daunting task of producing and screening an overwhelming number of variants generated by a classical protein engineering strategy, we accepted the challenge of resurrecting ancestral proteins, likely to have possessed functional properties. This innovative approach that exploits molecular evolution models to efficiently guide protein engineering, has allowed us to generate a small library of six ancestral toxin (AncTx) variants and associate their pharmacological profiles to key functional substitutions. Among these variants, we identified AncTx1 as the most α1A-adrenoceptor selective peptide known to date and AncTx5 as the most potent inhibitor of the three α2 adrenoceptor subtypes. Three positions in the ρ-Da1a evolutionary pathway, positions 28, 38 and 43 have been identified as key modulators of the affinities for the α1 and α2C adrenoceptor subtypes. Here, we present a first attempt at rational engineering of the aminergic toxins, revealing an epistasis phenomenon.

Expression and characterization of haemathrins, madanin-like thrombin inhibitors, isolated from the salivary gland of tick Haemaphysalis bispinosa (Acari: Ixodidae).

Saliva of hematophagous animals, such as ticks, is an excellent source of anticoagulant proteins and polypeptides. Here we describe the identification and characterization of two thrombin inhibitors named as haemathrin 1 and 2 from the salivary gland of tick Haemaphysalis bispinosa using genomic approach. Haemathrins are cysteine-less peptide anticoagulants, which share about 65-70% identity with madanins, and belong to inhibitor I53 superfamily of inhibitors of the MEROPS database. Haemathrins were overexpressed in E. coli and characterized to understand its mechanism of anticoagulant activity. Recombinant haemathrins (rHaemathrins) delayed the thrombin time, prothrombin time, activated partial thromboplastin time and fibrinogen clotting time. Selectivity screening against serine proteases of coagulation cascade reveals that rHaemathrins 1 and 2 specifically inhibit thrombin with an IC50 of 46.13±0.04µM and 40.05±0.05µM respectively. Similar to madanin, rHaemathrin 1 and 2 were cleaved by thrombin and consequently lost their inhibitory function over time. Analyses of the cleavage products revealed that the first cleavage, which occurs at the C-terminal end of rHaemathrins, drastically reduced their inhibitory activity. The synthetic peptides corresponding to the cleaved fragments showed significant loss in their ability to prolong plasma clotting times and to inhibit the amidolytic activity of thrombin. Thus haemathrins are the first cleavable thrombin inhibitors characterized from the salivary glands of H. bispinosa.

Impacts of regional climatic fluctuations on radial growth of Siberian and Scots pine at Mukhrino mire (central-western Siberia).

Ring width (TRW) chronologies from Siberian (Pinus sibirica) and Scots (Pinus sylvestris) pine trees were sampled at Mukhrino - a large mire complex in central-western Siberia - to evaluate the impacts of hydroclimatic variability on tree growth over the last three centuries. For this purpose, we compared climate-growth correlation profiles from trees growing on peat soils with those growing on adjacent mineral soils. Tree growth at both peat and mineral soils was positively correlated to air temperature during the vegetation period. This finding can be explained by (i) the positive influence of temperature on plant physiological processes (i.e. growth control) during the growing season and (ii) the indirect impact of air temperatures on water table fluctuations. We observe also a strong link between TRW and the winter Palmer Drought Severity Index (PDSI), especially in Siberian pine, reflecting the isolating effect of snow and limited freezing damage in roots. Significant negative relations were, by contrast, observed between bog TRW chronologies and hydroclimatic indices during spring and summer; they are considered an expression of the negative impacts of high water levels and moist peat soils on root development. Some unusually old bog pines - exhibiting >500 growth rings - apparently colonized the site at the beginning of the Little Ice Age, and therefore seem to confirm that (i) peat conditions may have been drier in Siberia than in most other regions of western Europe during this period. At the same time, the bog trees also point to (ii) their strong dependence on surface conditions.

Regulation of expression of venom toxins: silencing of prothrombin activator trocarin D by AG-rich motifs.

Trocarin D (TroD), a venom prothrombin activator from Tropidechis carinatus, shares similar structure and function with blood coagulation factor Xa [Tropidechis carinatus FX (TrFX) a]. Their distinct physiologic roles are due to their distinct expression patterns. The genes of TroD and TrFX are highly similar, except for promoter and intron 1, indicating that TroD has probably evolved by duplication of FX, the plasma counterpart. The promoter insertion in TroD accounts for the elevated but not venom gland-specific expression. Here we examined the roles of 3 insertions and 2 deletions in intron 1 of TroD in the regulation of expression using luciferase as a reporter. By systematic deletions, we showed that a 209 bp region within the second insertion silences expression in mammalian and unmilked venom gland cells. Through bioinformatics analysis, we identified 5 AG-rich motifs in this region. All except the 5th motif are important for silencing function. YY1, Sp3 and HMGB2 were identified to bind these AG-rich motifs and silence gene expression in mammalian cells. Similar AG-rich motif clusters are also found in other toxin genes but not in their physiologic counterparts. Thus, AG-rich motifs contribute to regulation of expression of TroD, and probably other toxin genes.-Han, S. X., Kwong, S., Ge, R., Kolatkar, P. R., Woods, A. E., Blanchet, G., Kini, R. M. Regulation of expression of venom toxins: silencing of prothrombin activator trocarin D by AG-rich motifs.

Molecular exploration of the α(1A)-adrenoceptor orthosteric site: binding site definition for epinephrine, HEAT and prazosin.

Despite the physiological and pharmacological importance of the α1A-adrenoreceptor, the mode of interactions of classical agonists and radioactive ligands with this receptor is not yet clearly defined. Here, we used mutagenesis studies and binding experiments to evaluate the importance of 11 receptor sites for the binding of (125)I-HEAT, (3)H-prazosin and epinephrine. Only one residue (F312) commonly interacts with the three molecules, and, surprisingly, D106 interacts only with epinephrine in a moderate way. Our docking model shows that prazosin and HEAT are almost superimposed into the orthosteric pocket with their tetralone and quinazoline rings close to the phenyl ring of the agonist.

Polypharmacology profiles and phylogenetic analysis of three-finger toxins from mamba venom: case of aminergic toxins.

Composition of mamba's venom is quite atypical and characterized by the presence of a large diversity of three-finger fold toxins (3FTx) interacting with various enzymes, receptors and ion channels. In particular, 3FTx from mambas display the unique property to interact with class A GPCRs, sometimes with a high affinity and selectivity. A screening of five of these toxins (MT1, MT3, MT7, ρ-Da1a and ρ-Da1b) on 29 different subtypes of bioaminergic receptors, using competition binding experiments, highlights the diversity of their pharmacological profiles. These toxins may display either absolute selectivity for one receptor subtype or a polypharmacological property for various bioaminergic receptors. Nevertheless, adrenoceptor is the main receptor family targeted by these toxins. Furthermore, a new receptor target was identified for 3FTx and toxins in general, the ρ-Da1b interacting competitively with the human dopamine D3 receptor in the micromolar range. This result expands the diversity of GPCRs targeted by toxins and more generally highlights the multipotent interacting property of 3FTx. Phylogenic analyzes of these toxins show that muscarinic, adrenergic and dopaminergic toxins may be pooled in one family called aminergic toxins, this family coming probably from a specific radiation of ligands present in mamba venoms.

Atractaspis aterrima toxins: the first insight into the molecular evolution of venom in side-stabbers.

Although snake venoms have been the subject of intense research, primarily because of their tremendous potential as a bioresource for design and development of therapeutic compounds, some specific groups of snakes, such as the genus Atractaspis, have been completely neglected. To date only limited number of toxins, such as sarafotoxins have been well characterized from this lineage. In order to investigate the molecular diversity of venom from Atractaspis aterrima-the slender burrowing asp, we utilized a high-throughput transcriptomic approach completed with an original bioinformatics analysis pipeline. Surprisingly, we found that Sarafotoxins do not constitute the major ingredient of the transcriptomic cocktail; rather a large number of previously well-characterized snake venom-components were identified. Notably, we recovered a large diversity of three-finger toxins (3FTxs), which were found to have evolved under the significant influence of positive selection. From the normalized and non-normalized transcriptome libraries, we were able to evaluate the relative abundance of the different toxin groups, uncover rare transcripts, and gain new insight into the transcriptomic machinery. In addition to previously characterized toxin families, we were able to detect numerous highly-transcribed compounds that possess all the key features of venom-components and may constitute new classes of toxins.

New α-adrenergic property for synthetic MTß and CM-3 three-finger fold toxins from black mamba.

Despite their isolation more than fifteen years ago from the venom of the African mamba Dendroaspis polylepis, very few data are known on the functional activity of MTß and CM-3 toxins. MTß was initially classified as a muscarinic toxin interacting non-selectively and with low affinity with the five muscarinic receptor subtypes while no biological function was determined for CM-3. Recent results highlight the multifunctional activity of three-finger fold toxins for muscarinic and adrenergic receptors and reveal some discrepancies in the pharmacological profiles of their venom-purified and synthetic forms. Here, we report the pharmacological characterization of chemically-synthesized MTß and CM-3 toxins on nine subtypes of muscarinic and adrenergic receptors and demonstrate their high potency for α-adrenoceptors and in particular a sub-nanomolar affinity for the α1A-subtype. Strikingly, no or very weak affinity were found for muscarinic receptors, highlighting that pharmacological characterizations of venom-purified peptides may be risky due to possible contaminations. The biological profile of these two homologous toxins looks like that one previously reported for the Dendroaspis angusticeps ρ-Da1a toxin. Nevertheless, MTß and CM-3 interact more potently than ρ-Da1a with α1B- and α1D-AR subtypes. A computational analysis of the stability of the MTß structure suggests that mutation S38I, could be involved in this gain in function.

Engineering of three-finger fold toxins creates ligands with original pharmacological profiles for muscarinic and adrenergic receptors.

Protein engineering approaches are often a combination of rational design and directed evolution using display technologies. Here, we test "loop grafting," a rational design method, on three-finger fold proteins. These small reticulated proteins have exceptional affinity and specificity for their diverse molecular targets, display protease-resistance, and are highly stable and poorly immunogenic. The wealth of structural knowledge makes them good candidates for protein engineering of new functionality. Our goal is to enhance the efficacy of these mini-proteins by modifying their pharmacological properties in order to extend their use in imaging, diagnostics and therapeutic applications. Using the interaction of three-finger fold toxins with muscarinic and adrenergic receptors as a model, chimeric toxins have been engineered by substituting loops on toxin MT7 by those from toxin MT1. The pharmacological impact of these grafts was examined using binding experiments on muscarinic receptors M1 and M4 and on the α(1A)-adrenoceptor. Some of the designed chimeric proteins have impressive gain of function on certain receptor subtypes achieving an original selectivity profile with high affinity for muscarinic receptor M1 and α(1A)-adrenoceptor. Structure-function analysis supported by crystallographic data for MT1 and two chimeras permits a molecular based interpretation of these gains and details the merits of this protein engineering technique. The results obtained shed light on how loop permutation can be used to design new three-finger proteins with original pharmacological profiles.

Muscarinic toxins.

Muscarinic toxins isolated from the venom of Dendroaspis snakes may interact with a high affinity, large selectivity and various functional properties with muscarinic receptors. Therefore, these toxins are invaluable tools for studying the physiological role, molecular functioning and structural organization of the five subtypes of these G-Protein Coupled Receptors. We review the data on the most relevant results dealing with the isolation/identification, mode of action, structure/function and exploitation of these toxins and finally highlight the unresolved issues related to their pharmacological studies.