Hip Labral Morphological Changes in Patients with Femoroacetabular Impingement Speed Up the Onset of Early Osteoarthritis.

Over the last decade, evidence has mounted for a prominent etiologic role of femoroacetabular impingement (FAI) in the development of early hip osteoarthritis (OA). The aim of this study was to compare the ultrastructure and tissue composition of the hip labrum in healthy and pathological conditions, as FAI and OA, to provide understanding of structural changes which might be helpful in the future to design targeted therapies and improve treatment indications. We analyzed labral tissue samples from five healthy multi-organ donors (MCDs) (median age, 38 years), five FAI patients (median age, 37 years) and five late-stage OA patients undergoing total hip replacement (median age, 56 years). We evaluated morpho-functional by histology and transmission electron microscopy. Extracellular matrix (ECM) structure changes were similar in specimens from FAI compared to those from patients with OA (more severe in the latter) showing disorganization of collagen fibers and increased proteoglycan content. In FAI and in OA nuclei the chromatin was condensed, organelle degenerated and cytoplasm vacuolized. Areas of calcification were mainly observed in FAI and OA labrum, as well as apoptotic-like features. We showed that labral tissue of patients with FAI had similar pathological alterations of tissue obtained from OA patients, suggesting that FAI patients might have high susceptibility to develop OA.

Multi-Organ Morphological Findings in a Humanized Murine Model of Sickle Cell Trait.

Sickle cell disease (SCD) is caused by the homozygous beta-globin gene mutation that can lead to ischemic multi-organ damage and consequently reduce life expectancy. On the other hand, sickle cell trait (SCT), the heterozygous beta-globin gene mutation, is still considered a benign condition. Although the mechanisms are not well understood, clinical evidence has recently shown that specific pathological symptoms can also be recognized in SCT carriers. So far, there are still scant data regarding the morphological modifications referable to possible multi-organ damage in the SCT condition. Therefore, after genotypic and hematological characterization, by conventional light microscopy and transmission electron microscopy (TEM), we investigated the presence of tissue alterations in 13 heterozygous Townes mice, one of the best-known animal models that, up to now, was used only for the study of the homozygous condition. We found that endothelial alterations, as among which the thickening of vessel basal lamina, are ubiquitous in the lung, liver, kidney, and spleen of SCT carrier mice. The lung shows the most significant alterations, with a distortion of the general tissue architecture, while the heart is the least affected. Collectively, our findings contribute novel data to the histopathological modifications at microscopic and ultrastructural levels, underlying the heterozygous beta-globin gene mutation, and indicate the translational suitability of the Townes model to characterize the features of multiple organ involvement in the SCT carriers.

Paradigm Shift in Gastric Cancer Prevention: Harnessing the Potential of Aristolochia olivieri Extract.

Gastric cancer, particularly adenocarcinoma, is a significant global health concern. Environmental risk factors, such as Helicobacter pylori infection and diet, play a role in its development. This study aimed to characterize the chemical composition and evaluate the in vitro antibacterial and antitumor activities of an Aristolochia olivieri Colleg. ex Boiss. Leaves' methanolic extract (AOME). Additionally, morphological changes in gastric cancer cell lines were analyzed. AOME was analyzed using HPLC-MS/MS, and its antibacterial activity against H. pylori was assessed using the broth microdilution method. MIC and MBC values were determined, and positive and negative controls were included in the evaluation. Anticancer effects were assessed through in vitro experiments using AGS, KATO-III, and SNU-1 cancer cell lines. The morphological changes were examined through SEM and TEM analyses. AOME contained several compounds, including caffeic acid, rutin, and hyperoside. The extract displayed significant antimicrobial effects against H. pylori, with consistent MIC and MBC values of 3.70 ± 0.09 mg/mL. AOME reduced cell viability in all gastric cancer cells in a dose- and time-dependent manner. Morphological analyses revealed significant ultrastructural changes in all tumor cell lines, suggesting the occurrence of cellular apoptosis. This study demonstrated that AOME possesses antimicrobial activity against H. pylori and potent antineoplastic properties in gastric cancer cell lines. AOME holds promise as a natural resource for innovative nutraceutical approaches in gastric cancer management. Further research and in vivo studies are warranted to validate its potential clinical applications.

Electron paramagnetic resonance and transmission electron microscopy study of the interactions between asbestiform zeolite fibers and model membranes.

Different asbestiform zeolite fibers of the erionite (termed GF1 and MD8, demonstrated carcinogenic) and offretite (termed BV12, suspected carcinogenic) families were investigated by analyzing the electron paramagnetic resonance (EPR) spectra of selected surfactant spin probes and transmission electron microscopy (TEM) images in the presence of model membranes-cetyltrimethylammonium (CTAB) micelles, egg-lecithin liposomes, and dimyristoylphosphatidylcholine (DMPC) liposomes. This was undertaken to obtain information on interactions occurring at a molecular level between fibers and membranes which correlate with entrance of fibers into the membrane model or location of the fibers at the external or internal membrane interfaces. For CTAB micelles, all fibers were able to enter the micelles, but the hair-like structure and chemical surface characteristics of GF1 modified the micelle structure toward a bilayer-like organization, while MD8 and BV12, being shorter fibers and with a high density of surface interacting groups, partially destroyed the micelles. For liposomes, GF1 fibers partially penetrated the core solution, but DMPC liposomes showed increasing rigidity and organization of the bilayer. Conversely, for MD8 and BV12, the fibers did not cross the membrane demonstrating a smaller membrane structure perturbation. Scolecite fibers (termed SC1), used for comparison, presented poor interactions with the model membranes. The carcinogenicity of the zeolites, as postulated in the series SC1

Extracellular Vesicles Released by Oxidatively Injured or Intact C2C12 Myotubes Promote Distinct Responses Converging toward Myogenesis.

Myogenic differentiation is triggered, among other situations, in response to muscle damage for regenerative purposes. It has been shown that during myogenic differentiation, myotubes release extracellular vesicles (EVs) which participate in the signalling pattern of the microenvironment. Here we investigated whether EVs released by myotubes exposed or not to mild oxidative stress modulate the behaviour of targeted differentiating myoblasts and macrophages to promote myogenesis. We found that EVs released by oxidatively challenged myotubes (H2O2-EVs) are characterized by an increased loading of nucleic acids, mainly DNA. In addition, incubation of myoblasts with H2O2-EVs resulted in a significant decrease of myotube diameter, myogenin mRNA levels and myosin heavy chain expression along with an upregulation of proliferating cell nuclear antigen: these effects collectively lead to an increase of recipient myoblast proliferation. Notably, the EVs from untreated myotubes induced an opposite trend in myoblasts, that is, a slight pro-differentiation effect. Finally, H2O2-EVs were capable of eliciting an increased interleukin 6 mRNA expression in RAW264.7 macrophages. Notably, this is the first demonstration that myotubes communicate with surrounding macrophages via EV release. Collectively, the data reported herein suggest that myotubes, depending on their conditions, release EVs carrying differential signals which could contribute to finely and coherently orchestrate the muscle regeneration process.

Multiparameter analysis of apoptosis in puromycin-treated Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, a typical apoptotic phenotype is induced by some stress factors such as sugars, acetic acid, hydrogen peroxide, aspirin and age. Nevertheless, no data have been reported for apoptosis induced by puromycin, a damaging agent known to induce apoptosis in mammalian cells. We treated S. cerevisiae with puromycin to induce apoptosis and evaluated the percentage of dead cells by using Hoechst 33342 staining, transmission electron microscopy (TEM) and Annexin V flow cytometry (FC) analysis. Hoechst 33342 fluorescence images were processed to acquire parameters to use for multiparameter analysis [and perform a principal component analysis, (PCA)]. Cell viability was evaluated by Rhodamine 123 (Rh 123) and Acridine Orange microscope fluorescence staining. The results show puromycin-induced apoptosis in S. cerevisiae, and the PCA analysis indicated that the increasing percentage of apoptotic cells delineated a well-defined graph profile. The results were supported by TEM and FC. This study gives new insights into yeast apoptosis using puromycin as inducer agent, and PCA analysis may complement molecular analysis facilitating further studies to its detection.

In vitro activity of Carvacrol against titanium-adherent oral biofilms and planktonic cultures.

OBJECTIVE: The aim of this study was to test the effect of Carvacrol against oral pathogens and their preformed biofilms on titanium disc surface. METHODS: Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and biofilm inhibitory concentration (BIC) were performed to evaluate Carvacrol antibacterial activity, while flow cytometry (FCM) was used to verify the Carvacrol effect on esterase activity and membrane permeability. Carvacrol was tested in vitro on single- and multi-species biofilms formed on titanium disc by Streptococcus mutans ATCC 25175, Porphyromonas gingivalis ATCC 33277 or Fusobacterium nucleatum ATCC 25586, in different combinations, comparing its effect to that of chlorhexidine. RESULTS: The pathogens were sensitive to Carvacrol with MICs and MBCs values of 0.25 % and 0.50 % and BICs of 0.5 % for S. mutans ATCC 25175 and 1 % for P. gingivalis ATCC 33277 and F. nucleatum ATCC 25586. FCM analysis showed that treatment of planktonic cultures with Carvacrol caused an increase of damaged cells and a decrement of bacteria with active esterase activity. Moreover, Carvacrol demonstrated greater biofilm formation preventive property compared to chlorhexidine against titanium-adherent single- and multi-specie biofilms, with statistically significant values. CONCLUSIONS: Carvacrol showed inhibitory activity against the tested oral pathogens and biofilm formation preventive property on their oral biofilm; then, it could be utilized to control and prevent the colonization of microorganisms with particular significance in human oral diseases. CLINICAL RELEVANCE: This natural compound may be proposed in daily hygiene formulations or as an alternative agent supporting traditional antimicrobial protocols to prevent periodontal diseases in implanted patients.

Melatonin prevents chemical-induced haemopoietic cell death.

Melatonin (MEL), a methoxyindole synthesized by the pineal gland, is a powerful antioxidant in tissues as well as within cells, with a fundamental role in ameliorating homeostasis in a number of specific pathologies. It acts both as a direct radical scavenger and by stimulating production/activity of intracellular antioxidant enzymes. In this work, some chemical triggers, with different mechanisms of action, have been chosen to induce cell death in U937 hematopoietic cell line. Cells were pre-treated with 100 µM MEL and then exposed to hydrogen peroxide or staurosporine. Morphological analyses, TUNEL reaction and Orange/PI double staining have been used to recognize ultrastructural apoptotic patterns and to evaluate DNA behavior. Chemical damage and potential MEL anti-apoptotic effects were quantified by means of Tali® Image-Based Cytometer, able to monitor cell viability and apoptotic events. After trigger exposure, chromatin condensation, micronuclei formation and DNA fragmentation have been observed, all suggesting apoptotic cell death. These events underwent a statistically significant decrease in samples pre-treated with MEL. After caspase inhibition and subsequent assessment of cell viability, we demonstrated that apoptosis occurs, at least in part, through the mitochondrial pathway and that MEL interacts at this level to rescue U937 cells from death.

Efficient and highly reproducible production of red blood cell-derived extracellular vesicle mimetics for the loading and delivery of RNA molecules.

Extracellular vesicles (EVs) are promising natural nanocarriers for the delivery of therapeutic agents. As with any other kind of cell, red blood cells (RBCs) produce a limited number of EVs under physiological and pathological conditions. Thus, RBC-derived extracellular vesicles (RBCEVs) have been recently suggested as next-generation delivery systems for therapeutic purposes. In this paper, we show that thanks to their unique biological and physicochemical features, RBCs can be efficiently pre-loaded with several kinds of molecules and further used to generate RBCEVs. A physical vesiculation method, based on "soft extrusion", was developed, producing an extremely high yield of cargo-loaded RBCEV mimetics. The RBCEVs population has been deeply characterized according to the new guidelines MISEV2023, showing great homogeneity in terms of size, biological features, membrane architecture and cargo. In vitro preliminary results demonstrated that RBCEVs are abundantly internalized by cells and exert peculiar biological effects. Indeed, efficient loading and delivery of miR-210 by RBCEVs to HUVEC has been proven, as well as the inhibition of a known mRNA target. Of note, the bench-scale process can be scaled-up and translated into clinics. In conclusion, this investigation could open the way to a new biomimetic platform for RNA-based therapies and/or other therapeutic cargoes useful in several diseases.

Ultraviolet B (UVB) irradiation-induced apoptosis in various cell lineages in vitro.

Ultraviolet B (UVB) radiation acts as a strong apoptotic trigger in many cell types, in tumor and normal cells. Several studies have demonstrated that UVB-induced cell death occurs through the generation of reactive oxygen species. The consequent oxidative stress includes the impairment of cellular antioxidants, the induction of DNA damage and the occurrence of apoptosis. In this review, we investigated UVB apoptotic action in various cell models by using ultrastructural, molecular and cytofluorimetric techniques. Myeloid leukemia HL-60, T-lymphoblastoid Molt-4 and myelomonocytic U937 human cells, generally affected by apoptotic stimuli, were studied. Human chondrocytes and C2C12 skeletal muscle cells, known to be more resistant to damage, were also considered. All of them, when exposed to UVB radiation, revealed a number of characteristic apoptotic markers. Membrane blebbing, cytoplasm shrinkage and chromatin condensation were detected by means of electron microscopy. DNA cleavage, investigated by using agarose gel electrophoresis and TUNEL reaction, was observed in suspended cells. Differently, in chondrocytes and in skeletal muscle cells, oligonucleosomic DNA fragmentation did not appear, even if a certain TUNEL positivity was detected. These findings demonstrate that UVB radiation appears to be an ideal tool to study the apoptotic behavior.

Morpho-functional analyses reveal that changes in the chemical structure of a marine bisindole alkaloid alter the cytotoxic effect of its derivatives.

2,2-bis(6-bromo-1H-indol-3-yl) ethanamine, a marine bisindole alkaloid, showed anticancer property in several tumor cell lines thanks to the presence of a 3,3'-diindolylmethane scaffold. Here, the modifications in its chemical structure into alkaloid-like derivatives, have been evaluated, to investigate changes in its biological activities. Three derivatives have been considered and their potential apoptotic action has been evaluated through morpho-functional analyses in a human cancer cell line. Apoptosis appears strongly decreased in the derivatives without the bromine atoms (1) and in those where the bromine atoms have been substituted with fluorine atoms (2). On the contrary, the methylation of indole NH (3) does not alter the alkaloid apoptotic activity that occurs through mitochondria involvement supported by cardiolipin peroxidation and dysfunctional mitochondria presence. This manuscript highlights the alkaloid derivative cytotoxic effect, which is strictly correlated to the presence of N-methylated bisindole alkaloid and bromine atoms, conditions which assure to maintain the pro-apoptotic activity. Since molecular therapies, by targeting mitochondria pathways, have shown positive outcomes against several cancer cells, the alkaloid with bisindole methylated scaffold and the two bromine atoms can be considered a promising candidate to develop new derivatives with strong anticancer property. RESEARCH HIGHLIGHTS: 2,2-bis(6-bromo-1H-indol-3-yl) ethanamine is an alkaloid known for its anticancer properties. Morpho-functional analyses evaluated cytotoxicity of its synthetic derivatives in tumor cells. Anticancer properties depend on the presence of bisindole scaffold and the two bromine units.

The discoid lateral meniscus in children: a narrative review of pathology, diagnosis and treatment.

Background and Objective: The discoid lateral meniscus (DLM) is a congenital abnormality of the meniscal shape, characterized by a typical central hypertrophy and a diameter larger than a regular meniscus, potentially leading to knee pain and symptoms, especially in children. The present study provides an update and a general review of this uncommon meniscal pathology. The incidence of discoid meniscus is about 0.4-17% for the lateral and 0.1-0.3% for the medial, although, being often asymptomatic, the true prevalence is unknown. We aim to enhance awareness on this subject to medical care provider. Methods: A literature search was performed on PubMed, including articles written in English until October 2021. Key Content and Findings: The articles regarding etiology, diagnosis and management of DLM in children or in patients younger than 18 years were reviewed using the narrative approach. Conclusions: Recent literature has shown that DLM is one of the most frequent congenital anomalies of the knee encountered during childhood. While asymptomatic children with incidental finding can be managed nonoperatively, symptomatic painful DLM should be addressed surgically, restoring typical anatomy using saucerization, tear repair, and stable fixation of the meniscus. The risk of osteoarthritis progression seems to be higher in children with operated DLM, imposing prolonged follow-up and cartilage preserving strategies for these patients.

Extracellular vesicle-derived miRNAs improve stem cell-based therapeutic approaches in muscle wasting conditions.

Skeletal muscle holds an intrinsic capability of growth and regeneration both in physiological conditions and in case of injury. Chronic muscle illnesses, generally caused by genetic and acquired factors, lead to deconditioning of the skeletal muscle structure and function, and are associated with a significant loss in muscle mass. At the same time, progressive muscle wasting is a hallmark of aging. Given the paracrine properties of myogenic stem cells, extracellular vesicle-derived signals have been studied for their potential implication in both the pathogenesis of degenerative neuromuscular diseases and as a possible therapeutic target. In this study, we screened the content of extracellular vesicles from animal models of muscle hypertrophy and muscle wasting associated with chronic disease and aging. Analysis of the transcriptome, protein cargo, and microRNAs (miRNAs) allowed us to identify a hypertrophic miRNA signature amenable for targeting muscle wasting, consisting of miR-1 and miR-208a. We tested this signature among others in vitro on mesoangioblasts (MABs), vessel-associated adult stem cells, and we observed an increase in the efficiency of myogenic differentiation. Furthermore, injections of miRNA-treated MABs in aged mice resulted in an improvement in skeletal muscle features, such as muscle weight, strength, cross-sectional area, and fibrosis compared to controls. Overall, we provide evidence that the extracellular vesicle-derived miRNA signature we identified enhances the myogenic potential of myogenic stem cells.

Preclinical testing of the Akt inhibitor triciribine in T-cell acute lymphoblastic leukemia.

Over the past 20 years, survival rates of T-cell acute lymphoblastic leukemia (T-ALL) patients have improved, mainly because of advances in polychemotherapy protocols. Despite these improvements, we still need novel and less toxic treatment strategies targeting aberrantly activated signaling networks which increase proliferation, survival, and drug resistance of T-ALL cells. One such network is represented by the phosphatidylinositol 3-kinase (PI3K)/Akt axis. PI3K inhibitors have displayed some promising effects in preclinical models of T-ALL. Here, we have analyzed the therapeutic potential of the Akt inhibitor, triciribine, in T-ALL cell lines. Triciribine caused cell cycle arrest and caspase-dependent apoptosis. Western blots demonstrated a dose-dependent dephosphorylation of Akt1/Akt2, and of mammalian target of rapamycin complex 1 downstream targets in response to triciribine. Triciribine induced autophagy, which could be interpreted as a defensive mechanism, because an autophagy inhibitor (chloroquine) increased triciribine-induced apoptosis. Triciribine synergized with vincristine, a chemotherapeutic drug employed for treating T-ALL patients, and targeted the side population of T-ALL cell lines, which might correspond to leukemia initiating cells. Our findings indicate that Akt inhibition, either alone or in combination with chemotherapeutic drugs, may serve as an efficient treatment towards T-ALL cells requiring upregulation of this signaling pathway for their proliferation and survival.

Matrix metalloproteinase 13 loss associated with impaired extracellular matrix remodeling disrupts chondrocyte differentiation by concerted effects on multiple regulatory factors.

OBJECTIVE: To link matrix metalloproteinase 13 (MMP-13) activity and extracellular matrix (ECM) remodeling to alterations in regulatory factors leading to a disruption in chondrocyte homeostasis. METHODS: MMP-13 expression was ablated in primary human chondrocytes by stable retrotransduction of short hairpin RNA. The effects of MMP-13 knockdown on key regulators of chondrocyte differentiation (SOX9, runt-related transcription factor 2 [RUNX-2], and beta-catenin) and angiogenesis (vascular endothelial growth factor [VEGF]) were scored at the protein level (by immunohistochemical or Western blot analysis) and RNA level (by real-time polymerase chain reaction) in high-density monolayer and micromass cultures under mineralizing conditions. Effects on cellular viability in conjunction with chondrocyte progression toward a hypertrophic-like state were assessed in micromass cultures. Alterations in SOX9 subcellular distribution were assessed using confocal microscopy in micromass cultures and also in osteoarthritic cartilage. RESULTS: Differentiation of control chondrocyte micromasses progressed up to a terminal phase, with calcium deposition in conjunction with reduced cell viability and scant ECM. MMP-13 knockdown impaired ECM remodeling and suppressed differentiation in conjunction with reduced levels of RUNX-2, beta-catenin, and VEGF. MMP-13 levels in vitro and ECM remodeling in vitro and in vivo were linked to changes in SOX9 subcellular localization. SOX9 was largely excluded from the nuclei of chondrocytes with MMP-13-remodeled or -degraded ECM, and exhibited an intranuclear staining pattern in chondrocytes with impaired MMP-13 activity in vitro or with more intact ECM in vivo. CONCLUSION: MMP-13 loss leads to a breakdown in primary human articular chondrocyte differentiation by altering the expression of multiple regulatory factors.

An ultracentrifugation - hollow-fiber flow field-flow fractionation orthogonal approach for the purification and mapping of extracellular vesicle subtypes.

In the course of their life span, cells release a multitude of different vesicles in the extracellular matrix (EVs), constitutively and/or upon stimulation, carrying signals either inside or on their membrane for intercellular communication. As a natural delivery tool, EVs present many desirable advantages, such as biocompatibility and low toxicity. However, due to the complex biogenesis of EVs and their high heterogeneity in size distribution and composition, the characterization and quantification of EVs and their subpopulations still represents an enticing analytical challenge. Centrifugation methods allow to obtain different subpopulations in an easy way from cell culture conditioned medium and biological fluids including plasma, amniotic fluid and urine, but they still present some drawbacks and limitations. An unsatisfactory isolation can limit their downstream analysis and lead to wrong conclusions regarding biological activities. Isolation and characterization of biologically relevant nanoparticles like EVs is crucial to investigate specific molecular and signaling patterns and requires new combined approaches. Our work was focused on HF5 (miniaturized, hollow-fiber flow field-flow fractionation), and its hyphenation to ultracentrifugation techniques, which are the most assessed techniques for vesicle isolation. We exploited model samples obtained from culture medium of murine myoblasts (C2C12), known to release different subsets of membrane-derived vesicles. Large and small EVs (LEVs and SEVs) were isolated by differential ultracentrifugation (UC). Through an HF5 method employing UV, fluorescence and multi-angle laser scattering as detectors, we characterized these subpopulations in terms of size, abundance and DNA/protein content; moreover, we showed that microvesicles tend to hyper-aggregate and partially release nucleic matter. The quali-quantitative information we obtained from the fractographic profiles was improved with respect to Nano Tracking Analysis (NTA) estimation. The SEV population was then further separated using density gradient centrifugation (DGC), and four fractions were submitted again to HF5-multidetection. This technique is based on a fully orthogonal principle, since F4 does not separate by density, and provided uncorrelated information for each of the fractions processed. The "second dimension" achieved with HF5 showed good promise in sorting particles with both different size and content, and allowed to identify the presence of fibrilloid nucleic matter. This analytical bidimensional approach proved to be effective for the characterization of highly complex biological samples such as mixtures of EVs and could provide purified fractions for further biological characterization.

How Inflammation Pathways Contribute to Cell Death in Neuro-Muscular Disorders.

Neuro-muscular disorders include a variety of diseases induced by genetic mutations resulting in muscle weakness and waste, swallowing and breathing difficulties. However, muscle alterations and nerve depletions involve specific molecular and cellular mechanisms which lead to the loss of motor-nerve or skeletal-muscle function, often due to an excessive cell death. Morphological and molecular studies demonstrated that a high number of these disorders seem characterized by an upregulated apoptosis which significantly contributes to the pathology. Cell death involvement is the consequence of some cellular processes that occur during diseases, including mitochondrial dysfunction, protein aggregation, free radical generation, excitotoxicity and inflammation. The latter represents an important mediator of disease progression, which, in the central nervous system, is known as neuroinflammation, characterized by reactive microglia and astroglia, as well the infiltration of peripheral monocytes and lymphocytes. Some of the mechanisms underlying inflammation have been linked to reactive oxygen species accumulation, which trigger mitochondrial genomic and respiratory chain instability, autophagy impairment and finally neuron or muscle cell death. This review discusses the main inflammatory pathways contributing to cell death in neuro-muscular disorders by highlighting the main mechanisms, the knowledge of which appears essential in developing therapeutic strategies to prevent the consequent neuron loss and muscle wasting.

Detection and Investigation of Extracellular Vesicles in Serum and Urine Supernatant of Prostate Cancer Patients.

Prostate Cancer (PCa) is one of the most frequently identified urological cancers. PCa patients are often over-diagnosed due to still not highly specific diagnostic methods. The need for more accurate diagnostic tools to prevent overestimated diagnosis and unnecessary treatment of patients with non-malignant conditions is clear, and new markers and methods are strongly desirable. Extracellular vesicles (EVs) hold great promises as liquid biopsy-based markers. Despite the biological and technical issues present in their detection and study, these particles can be found highly abundantly in the biofluid and encompass a wealth of macromolecules that have been reported to be related to many physiological and pathological processes, including cancer onset, metastasis spreading, and treatment resistance. The present study aims to perform a technical feasibility study to develop a new workflow for investigating EVs from several biological sources. Serum and urinary supernatant EVs of PCa, benign prostatic hyperplasia (BPH) patients, and healthy donors were isolated and investigated by a fast, easily performable, and cost-effective cytofluorimetric approach for a multiplex detection of 37 EV-antigens. We also observed significant alterations in serum and urinary supernatant EVs potentially related to BPH and PCa, suggesting a potential clinical application of this workflow.

Early Detection and Investigation of Extracellular Vesicles Biomarkers in Breast Cancer.

Breast cancer (BC) is the most commonly diagnosed malignant tumor in women worldwide, and the leading cause of cancer death in the female population. The percentage of patients experiencing poor prognosis along with the risk of developing metastasis remains high, also affecting the resistance to current main therapies. Cancer progression and metastatic development are no longer due entirely to their intrinsic characteristics, but also regulated by signals derived from cells of the tumor microenvironment. Extracellular vesicles (EVs) packed with DNA, RNA, and proteins, are the most attractive targets for both diagnostic and therapeutic applications, and represent a decisive challenge as liquid biopsy-based markers. Here we performed a study based on a multiplexed phenotyping flow cytometric approach to characterize BC-derived EVs from BC patients and cell lines, through the detection of multiple antigens. Our data reveal the expression of EVs-related biomarkers derived from BC patient plasma and cell line supernatants, suggesting that EVs could be exploited for characterizing and monitoring disease progression.

Dually Cross-Linked Core-Shell Structure Nanohydrogel with Redox-Responsive Degradability for Intracellular Delivery.

A redox-responsive nanocarrier is a promising strategy for the intracellular drug release because it protects the payload, prevents its undesirable leakage during extracellular transport, and favors site-specific drug delivery. In this study, we developed a novel redox responsive core-shell structure nanohydrogel prepared by a water in oil nanoemulsion method using two biocompatible synthetic polymers: vinyl sulfonated poly(N-(2-hydroxypropyl) methacrylamide mono/dilactate)-polyethylene glycol-poly(N-(2-hydroxypropyl) methacrylamide mono/dilactate) triblock copolymer, and thiolated hyaluronic acid. The influence on the nanohydrogel particle size and distribution of formulation parameters was investigated by a three-level full factorial design to optimize the preparation conditions. The surface and core-shell morphology of the nanohydrogel were observed by scanning electron microscope, transmission electron microscopy, and further confirmed by Fourier transform infrared spectroscopy and Raman spectroscopy from the standpoint of chemical composition. The redox-responsive biodegradability of the nanohydrogel in reducing environments was determined using glutathione as reducing agent. A nanohydrogel with particle size around 250 nm and polydispersity index around 0.1 is characterized by a thermosensitive shell which jellifies at body temperature and crosslinks at the interface of a redox-responsive hyaluronic acid core via the Michael addition reaction. The nanohydrogel showed good encapsulation efficiency for model macromolecules of different molecular weight (93% for cytochrome C, 47% for horseradish peroxidase, and 90% for bovine serum albumin), capacity to retain the peroxidase-like enzymatic activity (around 90%) of cytochrome C and horseradish peroxidase, and specific redox-responsive release behavior. Additionally, the nanohydrogel exhibited excellent cytocompatibility and internalization efficiency into macrophages. Therefore, the developed core-shell structure nanohydrogel can be considered a promising tool for the potential intracellular delivery of different pharmaceutical applications, including for cancer therapy.