Amino acid- vs. peptide-odorants: responses of individual olfactory receptor neurons in an aquatic species.

Amino acids are widely used waterborne olfactory stimuli proposed to serve as cues in the search for food. In natural waters the main source of amino acids is the decomposition of proteins. But this process also produces a variety of small peptides as intermediate cleavage products. In the present study we tested whether amino acids actually are the natural and adequate stimuli for the olfactory receptors they bind to. Alternatively, these olfactory receptors could be peptide receptors which also bind amino acids though at lower affinity. Employing calcium imaging in acute slices of the main olfactory epithelium of the fully aquatic larvae of Xenopus laevis we show that amino acids, and not peptides, are more effective waterborne odorants

Group I and group II peptides elicit significantly different [Ca²⁺]_i transients in individual olfactory receptor neurons.

<p>(A) The mean time points of amino acid- and peptide-evoked calcium transient maxima varied for individual stimuli. Transients evoked by group I peptides show a tendency to reach their maximum amplitude later if compared to amino acid stimulations (green, group I peptides, 1 mM; number of responses averaged: AA mix, 67; L-arginine (Arg), 10; L-methionine (Met), 11; L-lysine (Lys), 6; L-arginyl-L-methionine (Arg-Met), 3; L-arginyl-L-methionyl-L-arginine (Arg-Met-Arg), 4; L-methionyl-L-arginyl-L-methionine (Met-Arg-Met), 9; L-methionyl-L-arginine (Met-Arg), 9; L-arginyl-L-lysine (Arg-Lys), 4; L-arginyl-L-lysyl-L-arginine (Arg-Lys-Arg), 7; L-lysyl-L-arginyl-L-lysine (Lys-Arg-Lys), 7; L-lysyl-L-arginine (Lys-Arg), 2; out of 12 ORNs, four OE slices; orange, group II peptides, 200 µM; number of responses averaged: L-arginine (Arg), 7; L-methionine (Met), 3; glycine (Gly), 3; L-arginyl-glycine (Arg-Gly), 10; glycyl-L-arginine (Gly-Arg), 4; L-methionyl-glycine (Met-Gly), 4; glycyl-glycine (Gly-Gly), 4; glycyl-glycyl-glycine (Gly-Gly-Gly), 2; out of six ORNs, four OE slices). (B) A combined analysis reveals that calcium transients evoked by applications of group I peptides show a significant delay of their maximum amplitude if compared to responses to the mixture of amino acids. In contrast, response maxima evoked by group II petides are not significantly shifted in comparison to amino acid controls. Even more clearly, response maxima evoked by L-arginyl-glycine (Arg-Gly) are not shifted if compared to maxima evoked by L-arginine in L-arginine-specific ORNs (not responsive to the other two amino acids L-methionine and glycine). Bars indicate standard deviation and error bars represent the standard error of the mean (*, p<0.0001; unpaired t-test, number of evaluated responses for the first group, AA mix: 67 responses, Pep I: 45 responses, 12 cells, four OE slices; for the second group, AA: 12 responses, Pep II: 25 responses, six cells, four OE slices; and for exclusively L-Arginine positive ORNs, Arg: four responses, Arg-Gly: six responses). (C) Typical responses upon application of amino acids and group I peptides. The maximum amplitude of [Ca²⁺]_i transients induced by group I peptides is smaller and shows a significant delay in comparison to [Ca²⁺]_i transients induced by amino acids. Circles and dotted lines indicate the maximum amplitude of each response. AA mix (200 µM, blue), L-arginyl-L-lysine (Arg-Lys; 1 mM, dark green), L-methionyl-L-arginyl-L-methionine (Met-Arg-Met; 1 mM, green), L-methionyl-L-arginine (Met-Arg; 1 mM, light-green). The odorant application is marked by a grey bar. (D) Representative example of [Ca²⁺]_i transients of an ORN sensitive to L-arginine (200 µM, blue), L-arginyl-glycine (Arg-Gly; 200 µM, orange) and glycyl-L-arginine (Gly-Arg; 200 µM, light-orange). Calcium signals evoked by L-arginyl-glycine showed the highest mean maximum amplitude of all tested peptides. In both peptide responses, the maximum amplitude is not shifted in comparison to the arginine application. [AA mix: amino acid mixture, AA: amino acids, Arg: L-arginine, Met: L-methionine, Lys: L-lysine, Gly: glycine, Pep I: group I peptides, Pep II: group II peptides].</p

Amino acid- and peptide-induced changes in calcium-dependent fluorescence of individual ORNs in slices of the olfactory epithelium.

<p>(A) Slice preparation of the OE of larval <i>Xenopus laevis</i> stained with Fluo-4 AM. The colored ovals (#1–#8) indicate the eight ORNs that were responsive to the mixture of amino acids. (B) Time courses of [Ca²⁺]_i transients of the eight ORNs marked in A, elicited by application of amino acids (L-arginine, L-methionine and L-lysine as a mixture or singularly; each at a concentration of 200 µM) and peptides (consisting of L-arginine, L-methionine and L-lysine; 200 µM and 1 mM). Discernible peptide induced [Ca²⁺]_i transients are marked by an asterisk. To check for ORN viability, the mixture of amino acids was applied at the end of the experiment. (C) Examples of peptide induced calcium transients originating from different ORNs (group I peptides, green, L-arginyl-L-methionine (Arg-Met), 5 mM; L-arginyl-L-methionyl-L-arginine (Arg-Met-Arg), 1 mM; L-methionyl-L-arginyl-L-methionine (Met-Arg-Met), 1 mM; L-methionyl-L-arginine (Met-Arg), 5 mM; L-arginyl-L-lysine (Arg-Lys), 200 µM; L-lysyl-L-arginine (Lys-Arg), 1 mM; L-arginyl-L-lysyl-L-arginine (Arg-Lys-Arg), 1 mM; L-lysyl-L-arginyl-L-lysine (Lys-Arg-Lys), 1 mM;; group II peptides (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053097#s2" target="_blank">Material and Methods</a>), orange, all applied at 200 µM). As reference also the highest amino acid-induced (200 µM) calcium transient is depicted. [AA mix: amino acid mixture].</p

Response profiles of ORNs to amino acid and peptide stimulation.

<p>(A) Relative number of amino acid-sensitive ORNs reacting to individual amino acids (200 µM) or at least to one of the thirteen tested peptides. Only a fraction of amino acid-responsive ORNs also responded to group I peptides (1 mM, 12 of 42 ORNs in four slices) or group II peptides (200 µM, 6 of 28 ORNs in four slices). The fraction of ORNs sensitive to group I peptides did not differ from the fraction of ORNs sensitive to group II peptides. (B) Response matrix of all peptide-sensitive ORNs to the applied stimuli (green, response to applied stimulus; red, no response; grey, not tested; applied peptide concentration: ORN #1–#12, 1 mM; ORN #13–#21, 5 mM; ORN #22–#24, 10 mM; ORN #25–#31, 200 µM). [AA mix: amino acid mixture, AA: amino acids, Arg: L-arginine, Met: L-methionine, Lys: L-lysine, Gly: glycine, Pep I: group I peptides, Pep II: group II peptides].</p

Endovascular Thrombectomy for Anterior Circulation Large Vessel Occlusion Stroke: An Evolution of Trials.

The last decade's progress in demonstrating the clinical benefit of endovascular thrombectomy (EVT) in patients with large vessel occlusion stroke has transformed the paradigm of care for these patients. This review presents the milestones in implementing EVT as standard of care, demonstrates the current state of evidence, provides guidance for identifying the candidate patient for EVT, and highlights unsolved and controversial issues. Ongoing trials investigate broadening of EVT indications for patients who present with large core infarction, adjunctive intra-arterial thrombolysis, medium vessel occlusion, low NIHSS, and tandem occlusion

Early recurrence in paroxysmal versus sustained atrial fibrillation in patients with acute ischaemic stroke

Background The relationship between different patterns of atrial fibrillation and early recurrence after an acute ischaemic stroke is unclear. Purpose In a prospective cohort study, we evaluated the rates of early ischaemic recurrence after an acute ischaemic stroke in patients with paroxysmal atrial fibrillation or sustained atrial fibrillation which included persistent and permanent atrial fibrillation. Methods In patients with acute ischaemic stroke, atrial fibrillation was categorised as paroxysmal atrial fibrillation or sustained atrial fibrillation. Ischaemic recurrences were the composite of ischaemic stroke, transient ischaemic attack and symptomatic systemic embolism occurring within 90 days from acute index stroke. Results A total of 2150 patients (1155 females, 53.7%) were enrolled: 930 (43.3%) had paroxysmal atrial fibrillation and 1220 (56.7%) sustained atrial fibrillation. During the 90-day follow-up, 111 ischaemic recurrences were observed in 107 patients: 31 in patients with paroxysmal atrial fibrillation (3.3%) and 76 with sustained atrial fibrillation (6.2%) (hazard ratio (HR) 1.86 (95% CI 1.24-2.81)). Patients with sustained atrial fibrillation were on average older, more likely to have diabetes mellitus, hypertension, history of stroke/ transient ischaemic attack, congestive heart failure, atrial enlargement, high baseline NIHSS-score and implanted pacemaker. After adjustment by Cox proportional hazard model, sustained atrial fibrillation was not associated with early ischaemic recurrences (adjusted HR 1.23 (95% CI 0.74-2.04)). Conclusions After acute ischaemic stroke, patients with sustained atrial fibrillation had a higher rate of early ischaemic recurrence than patients with paroxysmal atrial fibrillation. After adjustment for relevant risk factors, sustained atrial fibrillation was not associated with a significantly higher risk of recurrence, thus suggesting that the risk profile associated with atrial fibrillation, rather than its pattern, is determinant for recurrence.Peer reviewe