Engineering paralog-specific PSD-95 recombinant binders as minimally interfering multimodal probes for advanced imaging techniques.

Despite the constant advances in fluorescence imaging techniques, monitoring endogenous proteins still constitutes a major challenge in particular when considering dynamics studies or super-resolution imaging. We have recently evolved specific protein-based binders for PSD-95, the main postsynaptic scaffold proteins at excitatory synapses. Since the synthetic recombinant binders recognize epitopes not directly involved in the target protein activity, we consider them here as tools to develop endogenous PSD-95 imaging probes. After confirming their lack of impact on PSD-95 function, we validated their use as intrabody fluorescent probes. We further engineered the probes and demonstrated their usefulness in different super-resolution imaging modalities (STED, PALM, and DNA-PAINT) in both live and fixed neurons. Finally, we exploited the binders to enrich at the synapse genetically encoded calcium reporters. Overall, we demonstrate that these evolved binders constitute a robust and efficient platform to selectively target and monitor endogenous PSD-95 using various fluorescence imaging techniques.

Compressive forces stabilize microtubules in living cells.

Microtubules are cytoskeleton components with unique mechanical and dynamic properties. They are rigid polymers that alternate phases of growth and shrinkage. Nonetheless, the cells can display a subset of stable microtubules, but it is unclear whether microtubule dynamics and mechanical properties are related. Recent in vitro studies suggest that microtubules have mechano-responsive properties, being able to stabilize their lattice by self-repair on physical damage. Here we study how microtubules respond to cycles of compressive forces in living cells and find that microtubules become distorted, less dynamic and more stable. This mechano-stabilization depends on CLASP2, which relocates from the end to the deformed shaft of microtubules. This process seems to be instrumental for cell migration in confined spaces. Overall, these results demonstrate that microtubules in living cells have mechano-responsive properties that allow them to resist and even counteract the forces to which they are subjected, being a central mediator of cellular mechano-responses.

Novel imaging methods and force probes for molecular mechanobiology of cytoskeleton and adhesion.

Detection and conversion of mechanical forces into biochemical signals is known as mechanotransduction. From cells to tissues, mechanotransduction regulates migration, proliferation, and differentiation in processes such as immune responses, development, and cancer progression. Mechanosensitive structures such as integrin adhesions, the actin cortex, ion channels, caveolae, and the nucleus sense and transmit forces. In vitro approaches showed that mechanosensing is based on force-dependent protein deformations and reorganizations. However, the mechanisms in cells remained unclear since cell imaging techniques lacked molecular resolution. Thanks to recent developments in super-resolution microscopy (SRM) and molecular force sensors, it is possible to obtain molecular insight of mechanosensing in live cells. We discuss how understanding of molecular mechanotransduction was revolutionized by these innovative approaches, focusing on integrin adhesions, actin structures, and the plasma membrane.

Molecular organization and mechanics of single vimentin filaments revealed by super-resolution imaging.

Intermediate filaments (IFs) are involved in key cellular functions including polarization, migration, and protection against large deformations. These functions are related to their remarkable ability to extend without breaking, a capacity that should be determined by the molecular organization of subunits within filaments. However, this structure-mechanics relationship remains poorly understood at the molecular level. Here, using super-resolution microscopy (SRM), we show that vimentin filaments exhibit a ~49-nanometer axial repeat both in cells and in vitro. As unit-length filaments (ULFs) were measured at ~59 nanometers, this demonstrates a partial overlap of ULFs during filament assembly. Using an SRM-compatible stretching device, we also provide evidence that the extensibility of vimentin is due to the unfolding of its subunits and not to their sliding, thus establishing a direct link between the structural organization and its mechanical properties. Overall, our results pave the way for future studies of IF assembly, mechanical, and structural properties in cells.

Cell stretching is amplified by active actin remodelling to deform and recruit proteins in mechanosensitive structures.

Detection and conversion of mechanical forces into biochemical signals controls cell functions during physiological and pathological processes. Mechanosensing is based on protein deformations and reorganizations, yet the molecular mechanisms are still unclear. Using a cell-stretching device compatible with super-resolution microscopy and single-protein tracking, we explored the nanoscale deformations and reorganizations of individual proteins inside mechanosensitive structures. We achieved super-resolution microscopy after live stretching on intermediate filaments, microtubules and integrin adhesions. Simultaneous single-protein tracking and stretching showed that while integrins followed the elastic deformation of the substrate, actin filaments and talin also displayed lagged and transient inelastic responses associated with active acto-myosin remodelling and talin deformations. Capturing acute reorganizations of single molecules during stretching showed that force-dependent vinculin recruitment is delayed and depends on the maturation of integrin adhesions. Thus, cells respond to external forces by amplifying transiently and locally cytoskeleton displacements, enabling protein deformation and recruitment in mechanosensitive structures.

Compulsory psychiatric treatment checklist: Instrument development and clinical application.

Instruments designed to evaluate the necessity of compulsory psychiatric treatment (CPT) are scarce to non-existent. We developed a 25-item Checklist (scoring 0 to 50) with four clusters (Legal, Danger, Historic and Cognitive), based on variables identified as relevant to compulsory treatment. The Compulsory Treatment Checklist (CTC) was filled with information on case (n=324) and control (n=251) subjects, evaluated under the Portuguese Mental Health Act (Law 36/98), in three hospitals. For internal validation, we used Confirmatory Factor Analysis (CFA), testing unidimensional and bifactor models. Multilevel logistic regression model (MLL) was used to predict the odds ratio (OR) for compulsory treatment based on the total scale score. Receiver Operating Characteristic analysis (ROC) was performed to predict compulsory treatment. CFA revealed the best fit indexes for the bifactor model, with all items loading on one General factor and the residual loading in the a priori predicted four specific factors. Reliability indexes were high for the General factor (88.4%), and low for specific factors (<5%), which demonstrate that CTC should not be performed in the subscales to access compulsory treatment. MLL reveals that for each item scored in the scale, it increases the OR by 1.26 for compulsory treatment (95%CI 1.21-1.31, p<0.001). Based on the total score, accuracy was 90%, and the best cut-off point of 23.5 detects compulsory treatment with a sensitivity of 75% and specificity of 93.6%. The CTC presents robust internal structure with a strong unidimensional characteristic, and a cut-off point for compulsory treatment of 23.5. The improved 20-item version of the CTC could represent an important instrument to improve clinical decision regarding CPT, and ultimately to improve mental health care of patients with severe psychiatric disorders.

Reliability and validity of the Portuguese version of the Generalized Anxiety Disorder (GAD-7) scale.

BACKGROUND: Generalized anxiety disorder has a strong impact on health-related quality of life. For this reason, it seems relevant to develop strategies allowing early diagnoses in order to promote appropriate treatments. The objective of this study was to culturally adapt and validate the GAD-7 for the Portuguese patients with generalized anxiety disorder. METHODS: For the cultural adaptation of the Portuguese version of the GAD-7 scale we started with a previous translation made by Mapi Institute and decided to perform a clinical review followed by a cognitive debriefing with patients. Once piloted, this version was then tested in a larger sample for feasibility and reliability (1-week test-retest). Construct validity was assessed by the relationship between GAD-7 and socio-demographic and clinical variables. Its unidimensionality was tested by principal component factor analysis. Criterion validity was assessed by comparing GAD-7 scores with those obtained by HADS, and EQ-5D. STAI was mainly used as a screening indicator for patient inclusion. RESULTS: GAD-7 was considered feasible with a mean completion time of 2.3 minutes and no major floor or ceiling effects. We found an excellent Cronbach's alpha internal consistency score (0.880) and the test-retest and interclass correlation coefficients were also very good. Regarding the construct validity, younger patients, those with higher education, employed and without anxiety symptoms revealed lower GAD-7 scores, meaning better health. The unidimensionality of GAD-7 index was also confirmed by principal component factor analysis. At last, GAD-7 was significantly correlated with other health outcome indices and the classification levels created by it and by HADS showed to be dependent. CONCLUSION: The excellent metric properties confirmed the cultural adaptation and validity of GAD-7 into Portuguese population, allowing the clinicians an early detection and treatment of these patients.

Considerations on the taxonomy of the Phylum Tardigrada.

An analysis of the taxonomy of the Tardigrada is offered, based on the latest checklist version. A total of 1167 species from 113 genera were counted, but marine species are misrepresented on account of being understudied. Moreover, many poor descriptions and synonyms remain in this constantly growing list. We advocate more accurate future taxonomic work, aiming for an official list of species that better represents true values of biodiversity.

Integrative taxonomy allows the identification of synonymous species and the erection of a new genus of Echiniscidae (Tardigrada, Heterotardigrada).

The taxonomy of tardigrades is challenging as these animals demonstrate a limited number of useful morphological characters, therefore several species descriptions are supported by only minor differences. For example, Echiniscus oihonnae and Echiniscus multispinosus are separated exclusively by the absence or presence of dorsal spines at position Bd. Doubts were raised on the validity of these two species, which were often sampled together. Using an integrative approach, based on genetic and morphological investigations, we studied two new Portuguese populations, and compared these with archived collections. We have determined that the two species must be considered synonymous with Echiniscus oihonnae the senior synonym. Our study showed generally low genetic distances of cox1 gene (with a maximum of 4.1%), with specimens displaying both morphologies sharing the same haplotype, and revealed character Bd to be variable. Addition-ally, a more detailed morphological and phylogenetic study based on the 18S gene uncovered a new evolutionary line within the Echiniscidae, which justified the erection of Diploechiniscus gen. nov. The new genus is in a sister group relationship with Echiniscus and is, for the moment, composed of a single species.

Observations on Pyxidium tardigradum (Ciliophora), a protozoan living on Eutardigrada: infestation, morphology and feeding behaviour.

Pyxidium tardigradum is a protozoan that has been reported on a few occasions as an epizoan symphoriont living on eutardigrades. We report here the first records of this species from Kirghizia (the first Asian record), Poland and Portugal. The Portuguese population revealed the largest P. tardigradum infestation ever described in terms of both the whole tardigrade population, with 60% affected animals, as well as a single host, with 35 attached protozoan. The first ever Scanning Electron Microscope (SEM) photomicrographs and pictures of live P. tardigradum are also given. No considerable ultrastructural variability was detected within or between the populations, suggesting that P. tardigradum may be a true cosmopolitan species. Given that the ciliate imposed significant extra volumes on infested tardigrades (from 1% to as much as 136%), we also discuss possible negative effects of the protozoan on the fitness of the host and suggest that P. tardigradum should probably be considered as a eutardigrade parasite. Furthermore, some hypotheses about the life history strategies of the ciliate are proposed.