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The Antisecretory Factor (AF) is a protein that can reduce intestinal hypersecretion and various inflammation disorders in vivo. Discovered in many mammalian tissues and plasma, its mechanism of action remains unknown. Interestingly, its induction has been found to counteract vertigo in patients with Méniere's disease. This suggests an inherent ability to control body balance and posture, an activity that may play a role in cerebellar function. Therefore, it may be worthwhile to investigate whether this activity can inhibit neuronal cells involved in cerebellar circuitries and its potential action on enteric nervous system ganglia, which could explain its antisecretory effect in the intestine.Previously, we studied the role of AF on GABAA receptors in cerebellar granule cells, taking advantage of electrophysiology and evaluating the effects of the administration of AF-16, an AF peptide. Treatment with AF-16 increased GABAA receptor responses, especially those containing the α6 subunit. Here, we performed immunofluorescence experiments by staining α1 and α6 subunits before and after incubation with AF-16, analyzed super-resolved images comparing pre- and post-treatment maps and critically examined these experimental results with our previous electrophysiological data to shed light on the mechanisms of action of AF protein on GABAA receptor subpopulations, specifically the ''fast'' receptors of αn β2/3 γ2 composition that contain either the α1 or the α6 subunit.The results indicate that the α6 subunit is redistributed, with a decrease in neurites and an increase in soma. Conversely, the α1 subunit shows opposite results, with an increase in neurites and a decrease in soma.

Label-free super-resolution (LFSR) imaging relies on light-scattering processes in nanoscale objects without a need for fluorescent (FL) staining required in super-resolved FL microscopy. The objectives of this Roadmap are to present a comprehensive vision of the developments, the state-of-the-art in this field, and to discuss the resolution boundaries and hurdles which need to be overcome to break the classical diffraction limit of the LFSR imaging. The scope of this Roadmap spans from the advanced interference detection techniques, where the diffraction-limited lateral resolution is combined with unsurpassed axial and temporal resolution, to techniques with true lateral super-resolution capability which are based on understanding resolution as an information science problem, on using novel structured illumination, near-field scanning, and nonlinear optics approaches, and on designing superlenses based on nanoplasmonics, metamaterials, transformation optics, and microsphere-assisted approaches. To this end, this Roadmap brings under the same umbrella researchers from the physics and biomedical optics communities in which such studies have often been developing separately. The ultimate intent of this paper is to create a vision for the current and future developments of LFSR imaging based on its physical mechanisms and to create a great opening for the series of articles in this field.

Background: The aging process starts in the center of the face, in the periocular region and around the mouth, with a combination of volume loss, tissue descent, deepened wrinkles, and the loss of skin structure and quality. Recently, several studies have demonstrated the efficacy of therapies based on autologous adipose tissue grafting, which leverages the properties of stromal vascular fraction (SVF) and adipose-derived mesenchymal stem cells (ADSCs) to accelerate the regenerative processes of the skin. This study aims to verify the ability of guided superficial enhanced fluid fat injection (SEFFI) in the facial area to correct volume loss and skin aging, proving that this standardized procedure has a very low rate of complications. Methods: We retrospectively collected data from 2365 procedures performed in Italian centers between 2019 and 2021. Guided SEFFI was performed alone or combined with cosmetic treatments, including the use of hyaluronic acid filler, suspension threads, synthetic calcium hydroxylapatite, botulin toxin, and microneedling. Results: guided SEFFI was used alone in more than 60% of the patients and in all facial areas. In about one-tenth of the patients, guided SEFFI was combined with a botulin toxin treatment or hyaluronic acid filling. Other procedures were used more rarely. Ecchymosis in the donor or injection sites was the most frequent adverse event but was only observed in 14.2% and 38.6% of the patients, respectively. Conclusions: The guided SEFFI technique is standardized and minimally invasive, leading to very few complications. It constitutes a promising antiaging medical treatment that combines effectiveness, safety, and simplicity.

Super-resolution microscopy techniques can be challenging for live cells and thick samples. Here, the authors propose a method to reduce beam intensity and remove out-of-focus fluorescence background in image-scanning microscopy (ISM) and its combination with stimulated emission depletion (STED).

Deciphering the spatiotemporal coordination between nuclear functions is important to understand its role in the maintenance of human genome. In this context, super-resolution microscopy has gained considerable interest because it can be used to probe the spatial organization of functional sites in intact single-cell nuclei in the 20-250 nm range. Among the methods that quantify colocalization from multicolor images, image cross-correlation spectroscopy (ICCS) offers several advantages, namely it does not require a presegmentation of the image into objects and can be used to detect dynamic interactions. However, the combination of ICCS with super-resolution microscopy has not been explored yet. Here, we combine dual-color stimulated emission depletion (STED) nanoscopy with ICCS (STED-ICCS) to quantify the nanoscale distribution of functional nuclear sites. We show that super-resolved ICCS provides not only a value of the colocalized fraction but also the characteristic distances associated to correlated nuclear sites. As a validation, we quantify the nanoscale spatial distribution of three different pairs of functional nuclear sites in MCF10A cells. As expected, transcription foci and a transcriptionally repressive histone marker (H3K9me3) are not correlated. Conversely, nascent DNA replication foci and the proliferating cell nuclear antigen(PCNA) protein have a high level of proximity and are correlated at a nanometer distance scale that is close to the limit of our experimental approach. Finally, transcription foci are found at a distance of 130 nm from replication foci, indicating a spatial segregation at the nanoscale. Overall, our data demonstrate that STED-ICCS can be a powerful tool for the analysis of the nanoscale distribution of functional sites in the nucleus.

Abstract Studying biomolecules in their native environment represents the ideal sample condition for structural biology investigations. Here we present a novel protocol which allows to delivery proteins into eukaryotic cells through a mild thermal stimulation. The data presented herein show the efficacy of this approach for delivering proteins in the intracellular environment of mammalian cells reaching a concentration range suitable for successfully applying biophysical methods, such as double electron electron resonance (DEER) measurements for characterising protein conformations.

The study of the GABAA receptor itself and its pharmacology is of paramount importance for shedding light on the role of this receptor in the central nervous system. Caged compounds have emerged as powerful tools to support research in this field, as they allow to control, in space and time, the release of neurotransmitters enabling, for example, to map receptors’ distribution and dynamics. Here we focus on γ-aminobutyric acid (GABA)-caged compounds, particularly on a commercial complex called RuBi-GABA, which has high efficiency of uncaging upon irradiation at visible wavelengths. We characterized, by electrophysiological measurements, the effects of RuBi-GABA on GABAA receptors of rat cerebellar granule cells in vitro. In particular, we evaluated the effects of side products obtained after RuBi-GABA photolysis. For this purpose, we developed a procedure to separate the “RuBi-cage” from GABA after uncaging RuBi-GABA with a laser source; then, we compared electrophysiological measurements acquired with and without administering the RuBi-cage in the perfusing bath. In conclusion, to investigate the role of the “cage” molecules both near and far from the cell soma, we compared experiments performed changing the distance of the uncaging point from the cell.

Background: Family violence has been shown to have a dramatic impact on individual and family life in the United States and other countries. Numerous studies have assessed the influence that exposure to violence can have on family dynamics and parent-child relationships. However, less is known about the association between family violence and parent-child relationships with Mexican families. Objective: Guided by social interaction learning theory, the purpose of this study was to explore the link between family violence, PTSD, and mother-child interaction patterns. Methods: Eighty-seven mother-child dyads from Mexico completed assessments for exposure to family violence and PTSD symptoms. We coded and analyzed observational tasks to assess prosocial parent-child interactions, such as positive communication and problem solving. We conducted an actor-partner independence model (APIM) to examine the association between exposure to family violence, PTSD and mother-child relationship dynamics. Results: As expected, higher exposure to family violence was linked to higher PTSD symptoms for mothers. Unexpectedly, higher maternal PTSD symptoms were associated with better communication during dyadic interaction tasks with their children. Conclusions: The present study suggests that individuals from certain cultures (i.e., Mexico) may respond differently to experiencing family violence. The use of multiple measurement methods to assess the relational effects of trauma on family dynamics can advance the scientific understanding of trauma affected families.

Photodynamic therapy (PDT) is a clinically approved procedure that can exert a curative action against malignant cells. The treatment implies the administration of a photoactive molecular species that, upon absorption of visible or near infrared light, sensitizes the formation of reactive oxygen species. These species are cytotoxic and lead to tumor cell death, damage vasculature, and induce inflammation. Clinical investigations demonstrated that PDT is curative and does not compromise other treatment options. One of the major limitations of the original method was the low selectivity of the photoactive compounds for malignant over healthy tissues. The development of conjugates with antibodies has endowed photosensitizing molecules with targeting capability, so that the compounds are delivered with unprecedented precision to the site of action. Given their fluorescence emission capability, these supramolecular species are intrinsically theranostic agents.

The photodynamic treatment for antimicrobial applications or anticancer therapy relies on reactive oxygen species generated by photosensitizing molecules after absorption of visible or near-infrared light. If the photosensitizing molecule is in close vicinity of the microorganism or the malignant cell, a photocytotoxic action is exerted. Therefore, the effectiveness of photosensitizing compounds strongly depends on their capability to target microbial or cancer-specific proteins. In this study, we report on the preparation and preliminary characterization of human recombinant myoglobin fused to the vasoactive intestinal peptide to target vasoactive intestinal peptide receptor (VPAC) receptors. Fe-protoporphyrin IX was replaced by the photosensitizing compound Zn-protoporphyrin IX. Taking advantage of the fluorescence emission by Zn-protoporphyrin IX, we show that the construct can bind prostate cancer cells where the VPAC receptors are expressed.

Background:. Nose thread lifting is a relatively recent technique. It offers a chance to approach nose morphology defects without turning to surgery to obtain a temporary improvement. Nevertheless, it lacks standardization and it can therefore offer quite variable results and short durability. The authors’ experience is presented here along with a recommended methodological approach to deliver reliable techniques for predictable results. Thread lifting techniques for nose reshaping by means of insertion of poly-L-lactic/poly-caprolactone threads are presented, based on principles of graft-based techniques, to obtain temporary morphological correction of selected nose deformities. Methods:. A total of 553 patients underwent nose reshaping using poly-L-lactic/poly-caprolactone threads. Of the procedures, 471 were primary treatment, and 82 were secondary treatment after a previous rhinoplasty. The mean follow-up period was carried out through patient photographs and lasted 33.4 months (range 2–60 months). Clinical examinations and patient satisfaction surveys were done at 6 months and 1 year from thread lifting. Results:. The Freiburg questionnaire survey (subjective Global Aesthetic Improvement Scale) allowed the authors to confirm a 95% satisfaction rate after 6 months, and 62% at 1 year after treatment. A flow chart based on the recorded results is presented to support operators in selecting the proper correction method to adopt, based on the different listed indications. Conclusions:. Nose reshaping techniques with poly-L-lactic/poly-caprolactone threads are presented together with patients’ treatment satisfaction. Standardization is based upon the authors’ experience. Contraindications and encountered complications are discussed, so as to provide the readers with a complete “state-of-the art” presentation of these techniques. In the authors’ experience, this is a reliable and safe approach to obtain temporary amelioration of selected nose defects via a nonsurgical and minimally invasive method.

The availability of the Airyscan detector in the Zeiss LSM 880 has made possible the development of a new concept in fluctuation correlation spectroscopy using super-resolution. The Airyscan unit acquires data simultaneously on 32 detectors arranged in a hexagonal array. This detector opens up the possibility to use fluctuation methods based on time correlation at single points or at a number of points simultaneously, as well as methods based on spatial correlation in the area covered by the detector. Given the frame rate of this detector, millions of frames can be acquired in seconds, providing a robust statistical basis for fluctuation data. We apply the comprehensive analysis to the molecular fluctuations of free GFP diffusing in live cells at different subcellular compartments to show that at the nanoscale different cell environments can be distinguished by the comprehensive fluctuation analysis.

Abstract Quantifying the imaging performances in an unbiased way is of outmost importance in super-resolution microscopy. Here, we describe an algorithm based on image correlation spectroscopy (ICS) that can be used to assess the quality of super-resolution images. The algorithm is based on the calculation of an autocorrelation function and provides three different parameters: the width of the autocorrelation function, related to the spatial resolution; the brightness, related to the image contrast; the relative noise variance, related to the signal-to-noise ratio of the image. We use this algorithm to evaluate the quality of stimulated emission depletion (STED) images of DNA replication foci in U937 cells acquired under different imaging conditions. Increasing the STED depletion power improves the resolution but may reduce the image contrast. Increasing the number of line averages improves the signal-to-noise ratio but facilitates the onset of photobleaching and subsequent reduction of the image contrast. Finally, we evaluate the performances of two different separation of photons by lifetime tuning (SPLIT) approaches: the method of tunable STED depletion power and the commercially available Leica Tau-STED. We find that SPLIT provides an efficient way to improve the resolution and contrast in STED microscopy.