Extracellular Vesicles based STAT3 delivery as innovative therapeutic approach to restore STAT3 signaling deficiency.

Extracellular Vesicles (EVs) have been proposed as a promising tool for drug delivery because of their natural ability to cross biological barriers, protect their cargo, and target specific cells. Moreover, EVs are not recognized by the immune system as foreign, reducing the risk of an immune response and enhancing biocompatibility. Herein, we proposed an alternative therapeutic strategy to restore STAT3 signaling exploiting STAT3 loaded EVs. This approach could be useful in the treatment of Autosomal Dominant Hyper-IgE Syndrome (AD-HIES), a rare primary immunodeficiency and multisystem disorder due to the presence of mutations in STAT3 gene. These mutations alter the signal transduction of STAT3, thereby impeding Th17 CD4+ cell differentiation that leads to the failure of immune response. We set up a simple and versatile method in which EVs were loaded with fully functional STAT3 protein. Moreover, our method allows to follow the uptake of STAT3 loaded vesicles inside cells due to the presence of EGFP in the EGFP-STAT3 fusion protein construct. Taken together, the data presented in this study could provide the scientific background for the development of new therapeutic strategy aimed to restore STAT3 signaling in STAT3 misfunction associated diseases like AD-HIES. In the future, the administration of fully functional wild type STAT3 to CD4+ T cells of AD-HIES patients might compensate its loss of function and would be beneficial for these patients, lowering the risk of infections, the use of medications, and hospitalizations.

Link between Monkeypox Virus Genomes from Museum Specimens and 1965 Zoo Outbreak.

We used pathogen genomics to test orangutan specimens from a museum in Bonn, Germany, to identify the origin of the animals and the circumstances of their death. We found monkeypox virus genomes in the samples and determined that they represent cases from a 1965 outbreak at Rotterdam Zoo in Rotterdam, the Netherlands.

The Haves and Have-Nots: The Mitochondrial Permeability Transition Pore across Species.

The demonstration that F1FO (F)-ATP synthase and adenine nucleotide translocase (ANT) can form Ca2+-activated, high-conductance channels in the inner membrane of mitochondria from a variety of eukaryotes led to renewed interest in the permeability transition (PT), a permeability increase mediated by the PT pore (PTP). The PT is a Ca2+-dependent permeability increase in the inner mitochondrial membrane whose function and underlying molecular mechanisms have challenged scientists for the last 70 years. Although most of our knowledge about the PTP comes from studies in mammals, recent data obtained in other species highlighted substantial differences that could be perhaps attributed to specific features of F-ATP synthase and/or ANT. Strikingly, the anoxia and salt-tolerant brine shrimp Artemia franciscana does not undergo a PT in spite of its ability to take up and store Ca2+ in mitochondria, and the anoxia-resistant Drosophila melanogaster displays a low-conductance, selective Ca2+-induced Ca2+ release channel rather than a PTP. In mammals, the PT provides a mechanism for the release of cytochrome c and other proapoptotic proteins and mediates various forms of cell death. In this review, we cover the features of the PT (or lack thereof) in mammals, yeast, Drosophila melanogaster, Artemia franciscana and Caenorhabditis elegans, and we discuss the presence of the intrinsic pathway of apoptosis and of other forms of cell death. We hope that this exercise may help elucidate the function(s) of the PT and its possible role in evolution and inspire further tests to define its molecular nature.

Identity, structure, and function of the mitochondrial permeability transition pore: controversies, consensus, recent advances, and future directions.

The mitochondrial permeability transition (mPT) describes a Ca2+-dependent and cyclophilin D (CypD)-facilitated increase of inner mitochondrial membrane permeability that allows diffusion of molecules up to 1.5 kDa in size. It is mediated by a non-selective channel, the mitochondrial permeability transition pore (mPTP). Sustained mPTP opening causes mitochondrial swelling, which ruptures the outer mitochondrial membrane leading to subsequent apoptotic and necrotic cell death, and is implicated in a range of pathologies. However, transient mPTP opening at various sub-conductance states may contribute several physiological roles such as alterations in mitochondrial bioenergetics and rapid Ca2+ efflux. Since its discovery decades ago, intensive efforts have been made to identify the exact pore-forming structure of the mPT. Both the adenine nucleotide translocase (ANT) and, more recently, the mitochondrial F1FO (F)-ATP synthase dimers, monomers or c-subunit ring alone have been implicated. Here we share the insights of several key investigators with different perspectives who have pioneered mPT research. We critically assess proposed models for the molecular identity of the mPTP and the mechanisms underlying its opposing roles in the life and death of cells. We provide in-depth insights into current controversies, seeking to achieve a degree of consensus that will stimulate future innovative research into the nature and role of the mPTP.

An isoform of the giant protein titin is a master regulator of human T lymphocyte trafficking.

Response to multiple microenvironmental cues and resilience to mechanical stress are essential features of trafficking leukocytes. Here, we describe unexpected role of titin (TTN), the largest protein encoded by the human genome, in the regulation of mechanisms of lymphocyte trafficking. Human T and B lymphocytes express five TTN isoforms, exhibiting cell-specific expression, distinct localization to plasma membrane microdomains, and different distribution to cytosolic versus nuclear compartments. In T lymphocytes, the LTTN1 isoform governs the morphogenesis of plasma membrane microvilli independently of ERM protein phosphorylation status, thus allowing selectin-mediated capturing and rolling adhesions. Likewise, LTTN1 controls chemokine-triggered integrin activation. Accordingly, LTTN1 mediates rho and rap small GTPases activation, but not actin polymerization. In contrast, chemotaxis is facilitated by LTTN1 degradation. Finally, LTTN1 controls resilience to passive cell deformation and ensures T lymphocyte survival in the blood stream. LTTN1 is, thus, a critical and versatile housekeeping regulator of T lymphocyte trafficking.

Stamp Technique: An Explorative SEM Analysis.

BACKGROUND: Achieving correct tooth anatomy and saving time at the dental chair are some of the goals of modern restorative dentistry. Stamp technique has gained acceptance in clinical practice. The aim of this study was to evaluate the effectiveness of this technique in terms of microleakage, voids, overhangs and marginal adaptation of Class I restorations, and to analyse the operative times in comparison with traditional restorative procedures. METHODS: Twenty extracted teeth were divided into 2 groups. Ten teeth in the study group (SG) were Class I prepared and restored using stamp technique, and ten teeth in the control group (CG) were Class I restored traditionally. SEM analysis was performed to evaluate voids, microleakage, overhangs, and marginal adaptation, and operative times were recorded. A statistical analysis was performed. RESULTS: There were no significant differences in microleakage, marginal adaptation and filling defects between the two groups, however, the stamp technique seems to facilitate the formation of large overflowing margins that require a careful finishing phase. CONCLUSIONS: Stamp technique does not seem to have any critical aspects in terms of restoration durability and it can be performed in a short time.

The mitochondrial permeability transition pore in Ca2+ homeostasis.

The mitochondrial Permeability Transition Pore (PTP) can be defined as a Ca2+ activated mega-channel involved in mitochondrial damage and cell death, making its inhibition a hallmark for therapeutic purposes in many PTP-related paradigms. Although long-lasting PTP openings have been widely studied, the physiological implications of transient openings (also called "flickering" behavior) are still poorly understood. The flickering activity was suggested to play a role in the regulation of Ca2+ and ROS homeostasis, and yet this hypothesis did not reach general consensus. This state of affairs might arise from the lack of unquestionable experimental evidence, due to limitations of the available techniques for capturing transient PTP activity and to a still partial understanding of its molecular identity. In this review we will focus on possible implications of the PTP in physiology, in particular its role as a Ca2+ release pathway, discussing the consequences of its forced inhibition. We will also consider the recent hypothesis of the existence of more permeability pathways and their potential involvement in mitochondrial physiology.

TiO2-Ag-NP adhesive photocatalytic films able to disinfect living indoor spaces with a straightforward approach.

TiO2-Ag doped nanoparticulate (TiO2-Ag-NP) adhesive photocatalytic films were used to assess the ability in dropping down the burden of indoor microbial particles. The application of an easy-to use photocatalytic adhesive film to cleanse indoor living spaces from microbial pollution, represents a novelty in the field of photocatalytic devices. Reduction was attained by photocatalysis in selected spaces, usually with overcrowding (≥ 3 individuals) in the common working daily hours, and upon indoor microclimate monitoring. TiO2-Ag doped nanoparticulate (TiO2-Ag-NP) adhesive photocatalytic films were applied within five types of living spaces, including schools and job places. The microbial pollution was assessed at time 0 (far from routine clean, ≥ 9 h) and throughout 2-4 weeks following the photocatalyst application by relative light unit (RLU) luminometry and microbial indirect assessment (colony forming units per cubic meter, CFU/m3). TiO2-Ag-NP photocatalyst reduced RLU and CFU/m3 by rates higher than 70% leading to RLU ≤ 20 and microbial presence ≤ 35 CFU/m3. The described TiO2-Ag-NP is able to reduce microbial pollution to the lowest RLU threshold (≤ 20) within 60 min in open daylight in a standardized test room of 100 m2. The correlation between RLU and CFU/m3 was positive (r = 0.5545, p < 0.05), assessing that the microbial reduction of indoor areas by the TiO2-Ag-NP adhesive film was real. Titania photocatalysts represent promising tools to ensure air cleaning and sanitization in living indoor microclimates with a low cost, feasible and straightforward approach. This approach represents an easy to handle, cost effective, feasible and efficacious approach to reduce microbial pollution in indoor spaces, by simply attaching a TiO2-Ag-NP adhesive film on the wall.

Histology and Long-term Clinical Outcome of Crushed Cartilage with Double-layer Gelatin Sponge Membrane for Dorsum Refinement in Primary Rhinoplasty.

This article demonstrates the ability to use autologous crushed cartilage grafts in rhinoplasty with rapid recovery and optimal nasal functionality without any tissue damage and allows its rapid rejuvenation. Eligible patients underwent primary rhinoplasty using autologous crushed cartilage graft followed by microscopy imaging of the grafted tissue after recovery. Tissue and cytological analysis using optical microscopy, transmission electronic microscopy (TEM), and scanning electronic microscopy (SEM) showed complete viability of chondrocytes, formation of new collagen fibers, neo-perichondrium, neo-angiogenesis, and exhibiting optimal aesthetic outcome. The surgical approach is easy to perform, feasible, and less time-consuming, with excellent tissue rejuvenation and rapid recovery.

Mitochondrial Permeability Transition.

The mitochondrial permeability transition (PT) is a phenomenon that can be broadly defined as an increase in the permeability of the mitochondrial inner membrane [...].

Ultrastructural Characterization of Human Bronchial Epithelial Cells during SARS-CoV-2 Infection: Morphological Comparison of Wild-Type and CFTR-Modified Cells.

SARS-CoV-2 replicates in host cell cytoplasm. People with cystic fibrosis, considered at risk of developing severe symptoms of COVID-19, instead, tend to show mild symptoms. We, thus, analyzed at the ultrastructural level the morphological effects of SARS-CoV-2 infection on wild-type (WT) and F508del (ΔF) CFTR-expressing CFBE41o- cells at early and late time points post infection. We also investigated ACE2 expression through immune-electron microscopy. At early times of infection, WT cells exhibited double-membrane vesicles, representing typical replicative structures, with granular and vesicular content, while at late time points, they contained vesicles with viral particles. ∆F cells exhibited double-membrane vesicles with an irregular shape and degenerative changes and at late time of infection, showed vesicles containing viruses lacking a regular structure and a well-organized distribution. ACE2 was expressed at the plasma membrane and present in the cytoplasm only at early times in WT, while it persisted even at late times of infection in ΔF cells. The autophagosome content also differed between the cells: in WT cells, it comprised vesicles associated with virus-containing structures, while in ΔF cells, it comprised ingested material for lysosomal digestion. Our data suggest that CFTR-modified cells infected with SARS-CoV-2 have impaired organization of normo-conformed replicative structures.

Ozone at low concentration modulates microglial activity in vitro: A multimodal microscopy and biomolecular study.

Oxygen-ozone (O2 -O3 ) therapy is an adjuvant/complementary treatment based on the activation of antioxidant and cytoprotective pathways driven by the nuclear factor erythroid 2-related factor 2 (Nrf2). Many drugs, including dimethyl fumarate (DMF), that are used to reduce inflammation in oxidative-stress-related neurodegenerative diseases, act through the Nrf2-pathway. The scope of the present investigation was to get a deeper insight into the mechanisms responsible for the beneficial result of O2 -O3 treatment in some neurodegenerative diseases. To do this, we used an integrated approach of multimodal microscopy (bright-field and fluorescence microscopy, transmission and scanning electron microscopy) and biomolecular techniques to investigate the effects of the low O3 concentrations currently used in clinical practice in lipopolysaccharide (LPS)-activated microglial cells human microglial clone 3 (HMC3) and in DMF-treated LPS-activated (LPS + DMF) HMC3 cells. The results at light and electron microscopy showed that LPS-activation induced morphological modifications of HMC3 cells from elongated/branched to larger roundish shape, cytoplasmic accumulation of lipid droplets, decreased electron density of the cytoplasm and mitochondria, decreased amount of Nrf2 and increased migration rate, while biomolecular data demonstrated that Heme oxygenase 1 gene expression and the secretion of the pro-inflammatory cytokines, Interleukin-6, and tumor necrosis factor-α augmented. O3 treatment did not affect cell viability, proliferation, and morphological features of both LPS-activated and LPS + DMF cells, whereas the cell motility and the secretion of pro-inflammatory cytokines were significantly decreased. This evidence suggests that modulation of microglia activity may contribute to the beneficial effects of the O2 -O3 therapy in patients with neurodegenerative disorders characterized by chronic inflammation. HIGHLIGHTS: Low-dose ozone (O3 ) does not damage activated microglial cells in vitro Low-dose O3 decreases cell motility and pro-inflammatory cytokine secretion in activated microglial cells in vitro Low-dose O3 potentiates the effect of an anti-inflammatory drug on activated microglial cells.

Bi-allelic LETM1 variants perturb mitochondrial ion homeostasis leading to a clinical spectrum with predominant nervous system involvement.

Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) encodes an inner mitochondrial membrane protein with an osmoregulatory function controlling mitochondrial volume and ion homeostasis. The putative association of LETM1 with a human disease was initially suggested in Wolf-Hirschhorn syndrome, a disorder that results from de novo monoallelic deletion of chromosome 4p16.3, a region encompassing LETM1. Utilizing exome sequencing and international gene-matching efforts, we have identified 18 affected individuals from 11 unrelated families harboring ultra-rare bi-allelic missense and loss-of-function LETM1 variants and clinical presentations highly suggestive of mitochondrial disease. These manifested as a spectrum of predominantly infantile-onset (14/18, 78%) and variably progressive neurological, metabolic, and dysmorphic symptoms, plus multiple organ dysfunction associated with neurodegeneration. The common features included respiratory chain complex deficiencies (100%), global developmental delay (94%), optic atrophy (83%), sensorineural hearing loss (78%), and cerebellar ataxia (78%) followed by epilepsy (67%), spasticity (53%), and myopathy (50%). Other features included bilateral cataracts (42%), cardiomyopathy (36%), and diabetes (27%). To better understand the pathogenic mechanism of the identified LETM1 variants, we performed biochemical and morphological studies on mitochondrial K+/H+ exchange activity, proteins, and shape in proband-derived fibroblasts and muscles and in Saccharomyces cerevisiae, which is an important model organism for mitochondrial osmotic regulation. Our results demonstrate that bi-allelic LETM1 variants are associated with defective mitochondrial K+ efflux, swollen mitochondrial matrix structures, and loss of important mitochondrial oxidative phosphorylation protein components, thus highlighting the implication of perturbed mitochondrial osmoregulation caused by LETM1 variants in neurological and mitochondrial pathologies.

The mitochondrial chaperone TRAP1 regulates F-ATP synthase channel formation.

Binding of the mitochondrial chaperone TRAP1 to client proteins shapes bioenergetic and proteostatic adaptations of cells, but the panel of TRAP1 clients is only partially defined. Here we show that TRAP1 interacts with F-ATP synthase, the protein complex that provides most cellular ATP. TRAP1 competes with the peptidyl-prolyl cis-trans isomerase cyclophilin D (CyPD) for binding to the oligomycin sensitivity-conferring protein (OSCP) subunit of F-ATP synthase, increasing its catalytic activity and counteracting the inhibitory effect of CyPD. Electrophysiological measurements indicate that TRAP1 directly inhibits a channel activity of purified F-ATP synthase endowed with the features of the permeability transition pore (PTP) and that it reverses PTP induction by CyPD, antagonizing PTP-dependent mitochondrial depolarization and cell death. Conversely, CyPD outcompetes the TRAP1 inhibitory effect on the channel. Our data identify TRAP1 as an F-ATP synthase regulator that can influence cell bioenergetics and survival and can be targeted in pathological conditions where these processes are dysregulated, such as cancer.

CFTR Modulation Reduces SARS-CoV-2 Infection in Human Bronchial Epithelial Cells.

People with cystic fibrosis should be considered at increased risk of developing severe symptoms of COVID-19. Strikingly, a broad array of evidence shows reduced spread of SARS-CoV-2 in these subjects, suggesting a potential role for CFTR in the regulation of SARS-CoV-2 infection/replication. Here, we analyzed SARS-CoV-2 replication in wild-type and CFTR-modified human bronchial epithelial cell lines and primary cells to investigate SARS-CoV-2 infection in people with cystic fibrosis. Both immortalized and primary human bronchial epithelial cells expressing wt or F508del-CFTR along with CRISPR/Cas9 CFTR-ablated clones were infected with SARS-CoV-2 and samples were harvested before and from 24 to 72 h post-infection. CFTR function was also inhibited in wt-CFTR cells with the CFTR-specific inhibitor IOWH-032 and partially restored in F508del-CFTR cells with a combination of CFTR modulators (VX-661+VX-445). Viral load was evaluated by real-time RT-PCR in both supernatant and cell extracts, and ACE-2 expression was analyzed by both western blotting and flow cytometry. SARS-CoV-2 replication was reduced in CFTR-modified bronchial cells compared with wild-type cell lines. No major difference in ACE-2 expression was detected before infection between wild-type and CFTR-modified cells, while a higher expression in wild-type compared to CFTR-modified cells was detectable at 72 h post-infection. Furthermore, inhibition of CFTR channel function elicited significant inhibition of viral replication in cells with wt-CFTR, and correction of CFTR function in F508del-CFTR cells increased the release of SARS-CoV-2 viral particles. Our study provides evidence that CFTR expression/function is involved in the regulation of SARS-CoV-2 replication, thus providing novel insights into the role of CFTR in SARS-CoV-2 infection and the development of therapeutic strategies for COVID-19.

Measurement of mitochondrial respiratory chain enzymatic activities in Drosophila melanogaster samples.

Mitochondrial respiratory chain (MRC) dysfunction is linked to mitochondrial disease as well as other common conditions such as diabetes, neurodegeneration, cancer, and aging. Thus, the evaluation of MRC enzymatic activities is fundamental for diagnostics and research purposes on experimental models. Here, we provide a verified and reliable protocol for mitochondria isolation from various D. melanogaster samples and subsequent measurement of the activity of MRC complexes I-V plus citrate synthase (CS) through UV-VIS spectrophotometry. For complete details on the use and execution of this protocol, please refer to Brischigliaro et al. (2021).

Tissue-Material Integration and Biostimulation Study of Collagen Acellular Matrices.

BACKGROUND: Breast reconstruction after mastectomy using silicone implants is a surgical procedure that occasionally leads to capsular contracture formation. This phenomenon constitutes an important and persistent cause of morbidity, and no successful therapies are available to date. Recently, the use of acellular membranes as a protective material for silicone prostheses has been gaining attention due to their ability to prevent this adverse outcome. For this reason, the evaluation of the tissue-material integration and the induced biostimulation by acellular membranes results crucial. Evaluation of in vivo tissue integration and biostimulation induced by three different natural acellular collagen membranes. METHODS: Scanning electron microscopy was performed to analyse the membrane porosity and cells-biomaterial interaction in vitro, both in dry and wet conditions. Adipose-derived stem cells were cultured in the presence of membranes, and the colonisation capacity and differentiation potential of cells were assessed. In vivo tests and ex vivo analyses have been performed to evaluate dermal integration, absorption degree and biostimulation induced by the evaluated membrane. RESULTS: Analysis performed in vitro on the three different acellular dermal matrices evidenced that porosity and the morphological structure of membranes influence the liquid swelling ratio, affecting the cell mobility and the colonisation capacity. Moreover, the evaluated membranes influenced in different manner the adipose derived stem cells differentiation and their survival. In vivo investigation indicated that the absorption degree and the fluid accumulation surrounding the implant were membrane-dependent. Finally, ex vivo analysis confirmed the membrane-dependent behavior revealing different degree of tissue integration and biostimulation, such as adipogenic stimulation. CONCLUSION: The physico-chemical characteristics of the membranes play a key role in the biostimulation of the cellular environment inducing the development of well-organized adipose tissue.

Evaluation of biocompatibility, osteointegration and biomechanical properties of the new Calcemex® cement: An in vivo study.

The mixture of polymethylmethacrylate (PMMA) and ß-tricalciumphospate (ß-TCP) is the most widely used bone graft. Common features of bone cement are the biocompatibility, bioactivity, mechanical stability and ability to fuse with the host's bone tissue. However, there are still few studies that have evaluated these characteristics in vivo. Our study aims to acquire these parameters, using an animal model with functional characteristics similar to those of humans. The analyzed cement is Calcemex®, evaluated both in compact and fluid formulation. The chosen animal models were 5 pigs, treated with femoral and tibial implants of Calcemex® samples. After one year, the pigs were sacrificed and the specimens explanted for morphological, histological, ultrastructural and mechanical evaluations. For both formulations, the investigation highlighted the absence of foreign body reactions in the host, the histological integration with the surrounding tissues and the preservation of mechanical compression resistance.

The mitochondrial permeability transition: Recent progress and open questions.

Major progress has been made in defining the basis of the mitochondrial permeability transition, a Ca2+ -dependent permeability increase of the inner membrane that has puzzled mitochondrial research for almost 70 years. Initially considered an artefact of limited biological interest by most, over the years the permeability transition has raised to the status of regulator of mitochondrial ion homeostasis and of druggable effector mechanism of cell death. The permeability transition is mediated by opening of channel(s) modulated by matrix cyclophilin D, the permeability transition pore(s) (PTP). The field has received new impulse (a) from the hypothesis that the PTP may originate from a Ca2+ -dependent conformational change of F-ATP synthase and (b) from the reevaluation of the long-standing hypothesis that it originates from the adenine nucleotide translocator (ANT). Here, we provide a synthetic account of the structure of ANT and F-ATP synthase to discuss potential and controversial mechanisms through which they may form high-conductance channels; and review some intriguing findings from the wealth of early studies of PTP modulation that still await an explanation. We hope that this review will stimulate new experiments addressing the many outstanding problems, and thus contribute to the eventual solution of the puzzle of the permeability transition.