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Single-particle tracking is a powerful technique to investigate the motion of molecules or particles. Here, we review the methods for analyzing the reconstructed trajectories, a fundamental step for deciphering the underlying mechanisms driving the motion. First, we review the traditional analysis based on the mean squared displacement (MSD), highlighting the sometimes-neglected factors potentially affecting the accuracy of the results. We then report methods that exploit the distribution of parameters other than displacements, e.g., angles, velocities, and times and probabilities of reaching a target, discussing how they are more sensitive in characterizing heterogeneities and transient behaviors masked in the MSD analysis. Hidden Markov Models are also used for this purpose, and these allow for the identification of different states, their populations and the switching kinetics. Finally, we discuss a rapidly expanding field-trajectory analysis based on machine learning. Various approaches, from random forest to deep learning, are used to classify trajectory motions, which can be identified by motion models or by model-free sets of trajectory features, either previously defined or automatically identified by the algorithms. We also review free software available for some of the analysis methods. We emphasize that approaches based on a combination of the different methods, including classical statistics and machine learning, may be the way to obtain the most informative and accurate results.

Temporal resolution is a key parameter in the observation of dynamic processes, as in the case of single molecules motions visualized in real time in two-dimensions by wide field (fluorescence) microscopy, but a systematic investigation of its effects in all the single particle tracking analysis steps is still lacking. Here we present tools to quantify its impact on the estimation of diffusivity and of its distribution using one of the most popular tracking software for biological applications on simulated data and movies. We found important shifts and different widths for diffusivity distributions, depending on the interplay of temporal sampling conditions with various parameters, such as simulated diffusivity, density of spots, signal-to-noise ratio, lengths of trajectories, and kind of boundaries in the simulation. We examined conditions starting from the ones of experiments on the fluorescently labelled receptor p75 NTR , a relatively fast-diffusing membrane receptor (diffusivity around 0.5-1 µm 2 /s), visualized by TIRF microscopy on the basal membrane of living cells. From the analysis of the simulations, we identified the best conditions in cases similar to these ones; considering also the experiments, we could confirm a range of values of temporal resolution suitable for obtaining reliable diffusivity results. The procedure we present can be exploited in different single particle/molecule tracking applications to find an optimal temporal resolution., (© 2024. The Author(s).)

Background: Labeling efficiency is a crucial parameter in fluorescence applications, especially when studying biomolecular interactions. Current approaches for estimating the yield of fluorescent labeling have critical drawbacks that usually lead them to be inaccurate or not quantitative., Results: We present a method to quantify fluorescent-labeling efficiency that addresses the critical issues marring existing approaches. The method operates in the same conditions of the target experiments by exploiting a ratiometric evaluation with two fluorophores used in sequential reactions. We show the ability of the protocol to extract reliable quantification for different fluorescent probes, reagents concentrations, and reaction timing and to optimize labeling performance. As paradigm, we consider the labeling of the membrane-receptor TrkA through 4'-phosphopantetheinyl transferase Sfp in living cells, visualizing the results by TIRF microscopy. This investigation allows us to find conditions for demanding single and multi-color single-molecule studies requiring high degrees of labeling., Conclusions: The developed method allows the quantitative determination and the optimization of staining efficiency in any labeling strategy based on stable reactions., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Investigating biological mechanisms in ever greater detail requires continuous advances in microscopy techniques and setups. Total internal reflection fluorescence (TIRF) microscopy is a well-established technique for visualizing processes on the cell membrane. TIRF allows studies down to the single molecule level, mainly in single-colour applications. Instead, multicolour setups are still limited. Here, we describe our strategies for implementing a multi-channel TIRF microscopy system capable of simultaneous two-channel excitation and detection, starting from a single-colour commercial setup. First, we report some applications at high molecule density and then focus on the challenges we faced for achieving the single molecule level simultaneously in different channels, showing that rigorous optimizations on the setup are needed to increase its sensitivity up to this point, from camera setting to background minimization. We also discuss our strategies regarding crucial points of fluorescent labelling for this type of experiment: labelling strategy, kind of probe, efficiency, and orthogonality of the reaction, all of which are aspects that can influence the achievable results. This work may provide useful guidelines for setting up advanced single-molecule multi-channel TIRF experiments to obtain insights into interaction mechanisms on the cell membrane of living cells., (© 2023 the author(s), published by De Gruyter.)

Probe choice in single-molecule microscopy requires deeper evaluations than those adopted for less sensitive fluorescence microscopy studies. Indeed, fluorophore characteristics can alter or hide subtle phenomena observable at the single-molecule level, wasting the potential of the sophisticated instrumentation and algorithms developed for advanced single-molecule applications. There are different reasons for this, linked, e.g., to fluorophore aspecific interactions, brightness, photostability, blinking, and emission and excitation spectra. In particular, these spectra and the excitation source are interdependent, and the latter affects the autofluorescence of sample substrate, medium, and/or biological specimen. Here, we review these and other critical points for fluorophore selection in single-molecule microscopy. We also describe the possible kinds of fluorophores and the microscopy techniques based on single-molecule fluorescence. We explain the importance and impact of the various issues in fluorophore choice, and discuss how this can become more effective and decisive for increasingly demanding experiments in single- and multiple-color applications.

The p75 neurotrophin (NT) receptor (p75 NTR ) plays a crucial role in balancing survival-versus-death decisions in the nervous system. Yet, despite 2 decades of structural and biochemical studies, a comprehensive, accepted model for p75 NTR activation by NT ligands is still missing. Here, we present a single-molecule study of membrane p75 NTR in living cells, demonstrating that the vast majority of receptors are monomers before and after NT activation. Interestingly, the stoichiometry and diffusion properties of the wild-type (wt) p75 NTR are almost identical to those of a receptor mutant lacking residues previously believed to induce oligomerization. The wt p75 NTR and mutated (mut) p75 NTR differ in their partitioning in cholesterol-rich membrane regions upon nerve growth factor (NGF) stimulation: We argue that this is the origin of the ability of wt p75 NTR , but not of mut p75 NTR , to mediate immature NT (proNT)-induced apoptosis. Both p75 NTR forms support proNT-induced growth cone retraction: We show that receptor surface accumulation is the driving force for cone collapse. Overall, our data unveil the multifaceted activity of the p75 NTR monomer and let us provide a coherent interpretative frame of existing conflicting data in the literature., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The efficacy of SSRIs in relapse prevention in major depression has been extensively demonstrated. Considering published data, the relapse rate during a psychopharmacological continuation treatment ranges from 10% to 30%. Since the reasons of depressive relapses could be heterogeneous, we have tested the effect of clinical, psychosocial and genetic variables in sustained remission from an index depressive episode during continuation treatment with fluvoxamine over a 6-month follow-up period. 101 patients maintained the same full dosage treatment after remission from a depressive episode efficaciously treated with fluvoxamine. During the follow-up period, they were clinical assessed monthly by an experienced psychiatrist and SASS was administered, to assess their psychosocial adjustment. From a genetical point of view, SERTPR and CLOCK polymorphisms were analyzed for each patients, using PCR-based techniques. At the end of follow-up period, the 57.4% of the patients maintained remission during fluvoxamine continuation treatment; the 8.9% relapsed within the first 2 months of continuation; the 7.9% switched and the 25.8% dropped-out for poor compliance. Relapsed subjects presented a significantly longer mean duration of the index depressive episode than non-relapsed subjects and a subjective poor social adjustment than non-relapsed also in the euthymia period. None of the analyzed polymorphisms significantly appear to influence the outcome of the whole sample. The present data confirm that patients with severe depression and a long duration of the episode have a major risk of psychosocial disability. These patients could need a different psychopharmacological strategies and peculiar psychological intervention.

Controlled trials in clinical psychopharmacology may fail to provide reliable information about the benefit of treatment for the patient when considered in a real-life setting rather than as a part of a well-defined sampling procedure. Previously, we applied the mathematical model of an artificial neural network (ANN) to a pool of clinical information gathered through case descriptions provided by senior psychiatrists in clinical charts of patients receiving their first exposure to sertraline. In the present study, we applied the same mathematical model to a larger sample. The performance of the ANN model in forecasting successful and unsuccessful treatment showed an overall accuracy of classification of 97.12%. This result supports our previous finding about the potential application of this method as a reliable predictor of a given psychiatric patient's outcome during a specific psychopharmacological therapy.

Aim of the Study: Aims of the study were: 1. to evaluate the results of surgical treatment of type IV thoraco-abdominal aneurysms (TAA), with relationship to other types, 2. to evaluate results obtained with an approach different from the traditional thoraco-phreno laparatomy, with specific attention to postoperative respiratory function., Material and Methods: We have retrospectively compared type IV TAA with all other types of thoraco-abdominal aneurysms electively treated between January 1st, 1994 and May 31st, 2003. Data on perioperative mortality, spinal cord ischemia and renal failure (both temporary and permanent) occurring in the first 30 postoperative days were considered. Protection from spinal cord ischemia was accomplished through liquor drainage and prostaglandin E1 (PGE1) infusion. When the aneurysm extension was limited to the celiac axis an extrapleuric access with removal of XI rib was performed. In this subgroup of patients we have considered postoperative recovery time of respiratory function (intubation time, number of days in intensive care unit, postoperative pulmonary complications) postoperative renal failure, perioperative mortality and morbidity., Results: Seventy-eight TAA have been treated in the period of time of the study. Twenty cases were type IV TAA (25.6%) of which 2 due to chronic dissection. Cumulative postoperative mortality has been 19.2%. The single perioperative death in the group of type IV TAA (5%) occurred in post-operative day 15 for multiple organ failure. No spinal cord ischemia occurred in this group. Temporary renal failure occurred in 3 cases (15%) with one case requiring dialysis. In 10 cases (50%) an extrapleuric access with removal of XI rib was performed, with adequate control of the proximal aorta. Postoperative respiratory failure requiring and intubation time longer than 12 hours occurred in 2 cases (20%). In the remaining 8 cases the mean intubation time was 5.3 hours (range 4-8 hrs). Tracheostomy was not necessary in any case. Mean time of intensive care unit stay was 3.5 days (range 0-15 days)., Conclusion: The appropriate treatment of type IV TAA leads to low mortality and morbidity with results similar to those of pararenal aneurysms rather than those of other TAA forms. Left extrapleuric access when feasible allows faster recovery of a normal respiratory function.

Anticonvulsant properties of some 2,3-benzodiazepine derivatives acting as alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) antagonists have been examined in vivo in the genetically epilepsy-prone rats using an audiogenic seizures assay. 2,3-Benzodiazepin-4-ones (CFMs) are nonselective AMPA antagonists that have been found to be potent anticonvulsant compound is in acute models of epilepsy. Because very little is known about their actions in a chronic model of epilepsy, and no correlations exist between anticonvulsant potency and plasma levels of these derivatives, we planned to investigate such a relationship. Maximal anticonvulsant protection occurred 15-60 min after the IP administration of GYKI 52466, 30-90 min after CFM-2, and 45-120 min after CFM-3. In addition, maximal anticonvulsant effect was observed 60-120 min after the IP administration of CFM-4 and at 90 min after CFM-5. The therapeutic index revealed that GYKI 52466 was slightly more toxic than CFM-2 and CFM-3. The time course of plasma levels of rats treated showed that peak plasma concentration was observed 45 min after IP administration of CFM-2 and CFM-3 and 75 min after CFM-4 and CFM-5. Following IP administration of CFM-3 two curves were detected, one is referred to the injected compound, and the other to its demethylated metabolite, which corresponds to CFM-2. Also. for the nitroderivative CFM-4 two curves were detected: one of an injected compound and the second due to its reduced metabolite (CFM-2). Finally, three different metabolites were detected in rat plasma after IP administration of CFM-5. The present study demonstrated that CFMs showed a significant protection against auditory stimulation during the period of peak plasma concentrations, suggesting a marked inhibition of those brain structures involved in the initiation and/or spreading of the audiogenic seizures.

Gabapentin (1-50 mg/kg, intraperitoneally (i.p.)) was able to antagonize audiogenic seizures in Dilute Brown Agouti DBA2J (DBA/2) mice in a dose-dependent manner. Gabapentin at dose of 2.5 mg/kg i.p., which per se did not significantly affect the occurrence of audiogenic seizures in DBA/2 mice, potentiated the anticonvulsant activity of carbamazepine, diazepam, felbamate, lamotrigine, phenytoin, phenobarbital and valproate against sound-induced seizures in DBA/2 mice. The potentiation induced by gabapentin was greatest for diazepam, phenobarbital and valproate, less for felbamate and phenytoin and least for carbamazepine and lamotrigine. The increase in anticonvulsant activity was associated with a comparable increase in motor impairment. However, the therapeutic index of combined treatment of the above drugs + gabapentin was more favourable than that of the same drugs + saline. Since gabapentin did not significantly influence the total and free plasma levels of the anticonvulsant drugs studied, we suggest that pharmacokinetic interactions, in terms of total or free plasma levels, are not probable. However, the possibility that gabapentin can modify the clearance from the brain of the anticonvulsant drugs studied can not be excluded. In addition, gabapentin did not significantly affect the hypothermic effects of the anticonvulsants tested. In conclusion, gabapentin showed an additive effect when administered in combination with certain classical anticonvulsants, most notably diazepam, phenobarbital, felbamate, phenytoin and valproate.

The effects of the selective delta opioid receptor agonist, ala-deltorphin, microinfused into the dorsal hippocampus or into the locus coeruleus, on the electrocorticographic (ECoG) activity continuously quantified in its spectrum were studied in rats. The microinjection into the dorsal hippocampus of ala-deltorphin (0.03, 0.3 and 1.0 nmol) produced dose-dependent motor and ECoG epileptogenic disorders. The microinfusion of similar doses of ala-deltorphin into the locus coeruleus did not evoke motor and ECoG changes. Intrahippocampal injection of naltrindole (10 and 20 pmol) antagonized the epileptogenic effects induced by ala-deltorphin; also naloxone (20 and 50 pmol) was able to prevent the effects induced by ala-deltorphin, although with a minor potency. In conclusion, the present experiments show that ala-deltorphin is a useful tool to characterize central opioid effects mediated by delta receptors in the brain and suggest that the stimulation of this subtype of receptors in the hippocampus is responsible for epileptogenic effects of opiates.