Search

Three-dimensional (3D) spheroid models aim to bridge the gap between traditional two-dimensional (2D) cultures and the complex in vivo tissue environment. These models, created by self-clustering cells to mimic a 3D environment with surrounding extracellular framework, provide a valuable research tool. The NSC-34 cell line, generated by fusing mouse spinal cord motor neurons and neuroblastoma cells, is essential for studying neurodegenerative diseases like amyotrophic lateral sclerosis (ALS), where abnormal protein accumulation, such as TAR-DNA-binding protein 43 (TDP-43), occurs in affected nerve cells. However, NSC-34 behavior in a 3D context remains underexplored, and this study represents the first attempt to create a 3D model to determine its suitability for studying pathology. We generated NSC-34 spheroids using a nonadhesive hydrogel-based template and characterized them for 6 days. Light microscopy revealed that NSC-34 cells in 3D maintained high viability, a distinct round shape, and forming stable membrane connections. Scanning electron microscopy identified multiple tunnel-like structures, while ultrastructural analysis highlighted nuclear bending and mitochondria alterations. Using inducible GFP-TDP-43-expressing NSC-34 spheroids, we explored whether 3D structure affected TDP-43 expression, localization, and aggregation. Spheroids displayed nuclear GFP-TDP-43 expression, albeit at a reduced level compared with 2D cultures and generated both TDP-35 fragments and TDP-43 aggregates. This study sheds light on the distinctive behavior of NSC-34 in 3D culture, suggesting caution in the use of the 3D model for ALS or TDP-43 pathologies. Yet, it underscores the spheroids' potential for investigating fundamental cellular mechanisms, cell adaptation in a 3D context, future bioreactor applications, and drug penetration studies. RESEARCH HIGHLIGHTS: 3D spheroid generation: NSC-34 spheroids, developed using a hydrogel-based template, showed high viability and distinct shapes for 6 days. Structural features: advanced microscopy identified tunnel-like structures and nuclear and mitochondrial changes in the spheroids. Protein dynamics: the study observed how 3D structures impact TDP-43 behavior, with altered expression but similar aggregation patterns to 2D cultures. Research implications: this study reveals the unique behavior of NSC-34 in 3D culture, suggests a careful approach to use this model for ALS or TDP-43 pathologies, and highlights its potential in cellular mechanism research and drug testing applications., (© 2024 Wiley Periodicals LLC.)

Background: In adults, the cerebellopontine angle (CPA) pilocytic astrocytoma (PA) is very rare. This tumor has radiological features similar to those of a vestibular schwannoma in the few cases reported in the literature., Methods: In this study, we conducted a systematic review in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses protocol and scrutinized all original studies pertaining to pontocerebellar angle PA in adult patients. We conducted an analysis of the clinical, radiological, and molecular components of all eligible articles. We have also reported a case involving a 67-year-old male individual in whom the PA exhibited radiological characteristics similar to an epidermoid cyst., Results: After the screening phase, we found four cases of PA of the pontocerebellar angle. Three cases were identified that resembled vestibular schwannoma; however, in our case, the tumor resembled an epidermoid cyst. These uncommon tumors exhibit distinctive histological patterns and molecular characteristics (adenosine triphosphate dependent helicase (ATP- dependent helicase)+, Isocitrate dehydrogenase 1-), rendering them a potential differential diagnosis for glioblastoma (GBM)., Conclusion: The CPA PA has rarely been found in adult patients and should be considered in the differential diagnosis of vestibular schwannoma and epidermoid cysts. In these rare cases, the histological characteristics of PA are significant for the differential diagnosis of GBM., Competing Interests: There are no conflicts of interest., (Copyright: © 2024 Surgical Neurology International.)

Background : Spinal cord lymphomas represent a minority of extranodal lymphomas and often pose diagnostic challenges by imitating primary spinal tumors or inflammatory/infective lesions. This paper presents a unique case of primary cauda equina lymphoma (PCEL) and conducts a comprehensive review to delineate the clinical and radiological characteristics of this rare entity. Case Report : A 74-year-old male presented with progressive paresthesia, motor weakness, and symptoms indicative of cauda equina syndrome. Neurological examination revealed paraparesis and sphincter dysfunction. Imaging studies initially suggested an intradural meningioma. However, surgical intervention revealed a diffuse large B-cell lymphoma infiltrating the cauda equina. Findings : A systematic review of the pertinent literature identified 18 primary cauda equina lymphoma cases. These cases exhibited diverse clinical presentations, treatments, and outcomes. The mean age at diagnosis was 61.25 years for women and 50 years for men, with an average follow-up of 16.2 months. Notably, 35% of patients were alive at 18 months, highlighting the challenging prognosis associated with PCEL. Discussion : Primary spinal cord lymphomas, especially within the cauda equina, remain rare and diagnostically complex due to their nonspecific clinical manifestations. The review highlights the need to consider spinal cord lymphoma in patients with neurological symptoms, even without a history of systemic lymphoma. Diagnostic Approaches : Magnetic resonance imaging (MRI) serves as the primary diagnostic tool but lacks specificity. Histopathological examination remains the gold standard for definitive diagnosis. The review underscores the importance of timely biopsy in suspected cases to facilitate accurate diagnosis and appropriate management. Management and Prognosis : Current management involves biopsy and chemotherapy; however, optimal treatment strategies remain ambiguous due to the rarity of PCEL. Despite aggressive therapeutic interventions, prognosis remains poor, emphasizing the urgency for enhanced diagnostic and treatment modalities. Conclusions : Primary cauda equina lymphoma poses diagnostic and therapeutic challenges, necessitating a high index of suspicion in patients with atypical spinal cord symptoms. Collaborative efforts between neurosurgical, oncological, and infectious diseases teams are imperative for timely diagnosis and management. Advancements in diagnostic precision and therapeutic options are crucial for improving patient outcomes.

The gut microbiota is an integral component of the colorectal cancer (CRC) microenvironment and is intimately associated with CRC initiation, progression, and therapeutic outcomes. We reviewed recent advancements in utilizing nanotechnology for modulating gut microbiota, discussing strategies and the mechanisms underlying their design. For future nanomedicine design, we propose a 5I principle for individualized nanomedicine in CRC management., Competing Interests: The authors declare no conflict of interest., (© 2024 The Authors. iMeta published by John Wiley & Sons Australia, Ltd on behalf of iMeta Science.)

Since the discovery of graphene, there has been a wide range of the literature dealing with its versatile structure and easy binding of biomolecules as well as its large loading capacity. In the emerging field of immunotherapy, graphene and its derivatives have potential uses as drug delivery platforms directly into tumour sites or as adjuvants in cancer vaccines, as they are internalized by monocytes which in turn may activate adaptive anti-tumoral immune responses. In this study, we expose cells of the innate immune system and a human acute monocytic leukemia cell line (THP-1) to low doses of small-sized GO nanosheets functionalized with bovine serum albumin (BSA) and fluorescein isothiocyanate (FITC), to study their acute response after internalization. We show by flow cytometry, uptake in cells of GO-BSA-FITC reaches 80% and cell viability and ROS production are both unaffected by exposure to nanoparticles. On the contrary, GO-BSA nanosheets seem to have an inhibitory effect on ROS production, probably due to their antioxidant properties. We also provided results on chemotaxis of macrophages derived from peripheral blood monocytes treated with GO-BSA. In conclusion, we showed the size of nanosheets, the concentration used and the degree of functionalization were important factors for biocompatibility of GO in immune cells. Its low cytotoxicity and high adaptability to the cells of the innate immune system make it a good candidate for deployment in immunotherapy, in particular for delivering protein antigens to monocytes which activate adaptive immunity., Competing Interests: The authors declare no conflict of interest.

Heart failure is a major side effect of doxorubicin (DOX) treatment in patients with cancer. However, the mechanisms underlying the development of DOX-induced heart failure need to be addressed. This study aims to test whether the serine/threonine kinase MST1, a major Hippo pathway component, contributes to the development of DOX-induced myocardial injury. C57BL/6J WT mice and mice with cardiomyocyte-specific dominant-negative MST1 (kinase-dead) overexpression received three weekly injections of DOX, reaching a final cumulative dose of 18 mg/kg. Echocardiographic, histological and biochemical analyses were performed six weeks after the first DOX administration. The effects of MST1 inhibition on DOX-induced cardiomyocyte injury were also tested in vitro. MST1 signaling was significantly activated in cardiomyocytes in response to DOX treatment in vitro and in vivo. Wild-type (WT) mice treated with DOX developed cardiac dysfunction and mitochondrial abnormalities. However, these detrimental effects were abolished in mice with cardiomyocyte-specific overexpression of dominant-negative MST1 (DN-MST1) or treated with XMU-MP-1, a specific MST1 inhibitor, indicating that MST1 inhibition attenuates DOX-induced cardiac dysfunction. DOX treatment led to a significant downregulation of cardiac levels of SIRT3, a deacetylase involved in mitochondrial protection, in WT mice, which was rescued by MST1 inhibition. Pharmacological inhibition of SIRT3 blunted the protective effects of MST1 inhibition, indicating that SIRT3 downregulation mediates the cytotoxic effects of MST1 activation in response to DOX treatment. Finally, we found a significant upregulation of MST1 and downregulation of SIRT3 levels in human myocardial tissue of cancer patients treated with DOX. In summary, MST1 contributes to DOX-induced cardiomyopathy through SIRT3 downregulation., (© 2023. The Author(s).)

Glioblastoma is an aggressive brain tumor with an average life expectancy between 14 and 16 months after diagnosis. The Ki-67 labeling index (LI), a measure of cellular proliferation, is emerging as a prognostic marker in GBM. In this study, we investigated the ultrastructure of glioblastoma tissue from 9 patients with the same molecular profile (adult IDH wild-type glioblastoma, wild-type ATRX, and positive for TP53 expression, GFAP expression, and EGFR overexpression) to find possible ultrastructural features to be used as biomarkers and correlated with the only parameter that differs among our samples, the Ki-67 LI. Our main results were the visualization of the anatomical basis of astrocyte-endothelial cells crosstalk; the ultrastructural in situ imaging of clusters of hyperactivated microglia cells (MsEVs); the ultrastructural in situ imaging of microglia cells storing lipid vesicles (MsLVs); the ultrastructural in situ imaging of neoplastic cells mitophagy (NCsM). The statistical analysis of our data indicated that MsEVs and MsLVs correlate with the Ki-67 LI value. We can thus assume they are good candidates to be considered morphological biomarkers correlating to Ki-67 LI. The role of NCsM instead must be further evaluated. Our study findings demonstrate that by combining ultrastructural characteristics with molecular information, we can discover biomarkers that have the potential to enhance diagnostic precision, aid in treatment decision-making, identify targets for therapy, and enable personalized treatment plans tailored to each patient. However, further research with larger sample sizes is needed to validate these findings and fully utilize the potential of ultrastructural analysis in managing glioblastoma.

Although therapeutic targets have been developed for colorectal cancer (CRC) therapy, the therapeutic effects are not ideal and the survival rate for CRC patients remains poor. Therefore, it is crucial to recognize a specific target and develop an efficacious delivery system for CRC therapy. Herein, we demonstrate that reduced ALKBH5 mediates aberrant m 6 A modification and tumor progression in CRC. Mechanically, histone deacetylase 2-mediated H3K27 deacetylation inhibits ALKBH5 transcription in CRC, whereas ectopic ALKBH5 expression decreases tumorigenesis of CRC cells and protects mice from colitis-associated tumor development. Further, METTL14/ALKBH5/IGF2BPs combine to modulate JMJD8 stability in an m 6 A-dependent manner, which increases glycolysis and accelerates the development of CRC by enhancing the enzymatic activity of PKM2. Moreover, ALKBH5 mRNA-loaded folic acid-modified exosome-liposome hybrid nanoparticles were synthesized and significantly inhibit the progression of CRC in preclinical tumor models by modulating the ALKBH5/JMJD8/PKM2 axis and inhibiting glycolysis. Overall, our research confirms the crucial function of ALKBH5 in regulating the m 6 A status in CRC and provides a direct preclinical approach for using ALKBH5 mRNA nanotherapeutics for CRC.

The aim of this in vitro study was to evaluate thermal effects on implant surfaces using a 445 nm diode laser (Eltech K-Laser Srl, Treviso, Italy) with different power settings and irradiation modalities. Fifteen new implants (Straumann, Basel, Switzerland) were irradiated to evaluate surface alteration. Each implant was divided into two zones: the anterior and posterior areas. The anterior coronal areas were irradiated with a distance of 1 mm between the optical fiber and the implant; the anterior apical ones were irradiated with the fiber in contact with the implant. Instead, the posterior surfaces of all of the implants were not irradiated and used as control surfaces. The protocol comprised two cycles of laser irradiation, lasting 30 s each, with a one-minute pause between them. Different power settings were tested: a 0.5 W pulsed beam (T-on 25 ms; T-off 25 ms), a 2 W continuous beam and a 3 W continuous beam. Lastly, through a scanning electron microscopy (SEM) analysis, dental implants' surfaces were evaluated to investigate surface alterations. No surface alterations were detected using a 0.5 W laser beam with a pulsed mode at a distance of 1 mm. Using powers of irradiation of 2 W and 3 W with a continuous mode at 1 mm from the implant caused damage on the titanium surfaces. After the irradiation protocol was changed to using the fiber in contact with the implant, the surface alterations increased highly compared to the non-contact irradiation modality. The SEM results suggest that a power of irradiation of 0.5 W with a pulsed laser light emission mode, using an inactivated optical fiber placed 1 mm away from the implant, could be used in the treatment of peri-implantitis, since no implant surface alterations were detected.

Background/aim: In the latest 2021 WHO classification of central nervous system tumours (CNS), gliomas that present isocitrate dehydrogenase (IDH) mutations are defined as diffuse low-grade gliomas (DLGGs). IDH mutations are commonly observed in this tumour type. The Extent of Resection (EOR) positively influence survival; however, it is still debated whether the predictive value of EOR is independent of the 1p/19q co-deletion. We carried out a retrospective analysis on patients operated on for DLGG at the Sant'Andrea University Hospital Sapienza University of Rome, correlating the outcome with the presence of 1p/19q co-deletion and EOR., Patients and Methods: The study examined 66 patients with DLGG who had undergone surgery for tumour resection between 2008 and 2018. Patients with DLGG were divided into two groups; diffuse astrocytoma (DA) in which 1p/19q codeletion is absent and oligodendroglioma (OG) in which 1p/19q codeletion is present. According to EOR, both groups were divided into two subgroups: subtotal resection (STR) and gross total resection (GTR). Three end-point variables were considered: overall survival (OS), progression-free survival (PFS) and time to malignant transformation (TMT)., Results: In the DA group, the GTR subgroup had an average OS of 81.6 months, an average PFS of 45.9 months and an average TMT of 63.6 months. After surgery, these patients had an average Karnofsky Performance Score (KPS) of 83.4. The STR subgroup had an average OS of 60.4 months, PFS was 38.7 months, and TMT was 46.4 months, post-operative KPS was 83.4. In contrast, in the OG group, the GTR averagely had 101.7 months of OS, 64.9 months of PFS, 80.3 months of TMT and an average post-operative KPS of 84.2, and the STR subgroup had an average of OS of 73.3 months, PFS of 48.2 months, TMT of 57.3 and an average postoperative KPS of 96.2., Conclusion: In patients affected by DLGGs, 1p/19q codeletion is significantly associated with prolonged survival and longer time-to-malignant transformation (TMT) compared to the absence of 1p/19q codeletion. Also, the extent of surgical resection (EOR) in DLGG patients has been confirmed as one of the main prognostic factors. However, its predictive value is substantially influenced by the presence of the 1p/19q codeletion., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background : The use of effective, low-cost, and easy-to-use products for early caries management will avoid loss of dental vitality and impairment in oral function. The ability of fluoride to re-mineralize dental surfaces has been widely reported as well as vitamin D demonstrated to have significant potential in improving the remineralization of early lesions on enamel surfaces. The aim of the present ex vivo study was to evaluate the effect of a fluoride and vitamin D solution in terms of formation of mineral crystals on the enamel of primary teeth, and their permanence over time on dental surfaces. Methods : Sixteen extracted deciduous teeth were cut to obtain 64 specimens that were divided into two groups. The first consisted of immersion of specimens for 4 days in a fluoride solution (T1); in the second group, the specimens were immersed for 4 days (T1) in fluoride and Vitamin D solution, and for a further 2 (T2) and 4 days (T3) in saline solution. Then, samples were morphologically analyzed by using Variable Pressure Scanning Electron Microscope (VPSEM) and underwent 3D surface reconstruction. Results : After a 4-day immersion in both solutions, octahedral-shaped crystals were formed on the enamel surface of primary teeth, demonstrating any statistically significant differences in terms of number, size, and shape. Moreover, the binding of the same crystals seemed to be strong enough to be maintained until 4 days in saline solution. However, a partial dissolution was observed in a time-dependent manner. Conclusions : A topical application of fluoride and Vitamin D promoted the formation of persistent mineral crystals on enamel surfaces of deciduous teeth and should be further studied to be potentially used as an alternative strategy in preventive dentistry.

Oncogenic-driven lipogenic metabolism is a common hallmark of colorectal cancer (CRC) progression. Therefore, there is an urgent need to develop novel therapeutic strategies for metabolic reprogramming. Herein, the metabolic profiles in the plasma between CRC patients and paired healthy controls were compared using metabolomics assays. Matairesinol downregulation was evident in CRC patients, and matairesinol supplementation significantly represses CRC tumorigenesis in azoxymethane/dextran sulfate sodium (AOM/DSS) colitis-associated CRC mice. Matairesinol rewired lipid metabolism to improve the therapeutic efficacy in CRC by inducing mitochondrial damage and oxidative damage and blunting ATP production. Finally, matairesinol-loaded liposomes significantly promoted the enhanced antitumor activity of 5-Fu/leucovorin combined with oxaliplatin (FOLFOX) in CDX and PDX mouse models by restoring chemosensitivity to the FOLFOX regimen. Collectively our findings highlight matairesinol-mediated lipid metabolism reprogramming as a novel druggable strategy to restore CRC chemosensitivity, and this nanoenabled approach for matairesinol will improve the chemotherapeutic efficacy with good biosafety.

Several theories have tried to elucidate the mechanisms behind the pathophysiology of chronic subdural hematoma (CSDH). However, this process is complex and remains mostly unknown. In this study we performed a retrospective randomised analysis comparing the cortical atrophy of 190 patients with unilateral CSDH, with 190 healthy controls. To evaluate the extent of cortical atrophy, CT scan images were utilised to develop an index that is the ratio of the maximum diameter sum of 3 cisterns divided by the maximum diameter of the skull at the temporal lobe level. Also, we reported, for the first time, the ultrastructural analyses of the CSDH using a combination of immunohistochemistry methods and transmission electron microscopy techniques. Internal validation was performed to confirm the assessment of the different degrees of cortical atrophy. Relative Cortical Atrophy Index (RCA index) refers to the sum of the maximum diameter of three cisterns (insular cistern, longitudinal cerebral fissure and cerebral sulci greatest) with the temporal bones' greatest internal distance. This index, strongly related to age in healthy controls, is positively correlated to the preoperative and post-operative maximum diameter of hematoma and the midline shift in CSDH patients. On the contrary, it negatively correlates to the Karnofsky Performance Status (KPS). The Area Under the Receiver Operating Characteristics (AUROC) showed that RCA index effectively differentiated cases from controls. Immunohistochemistry analysis showed that the newly formed CD-31 positive microvessels are higher in number than the CD34-positive microvessels in the CSDH inner membrane than in the outer membrane. Ultrastructural observations highlight the presence of a chronic inflammatory state mainly in the CSDH inner membrane. Integrating these results, we have obtained an etiopathogenetic model of CSDH. Cortical atrophy appears to be the triggering factor activating the cascade of transendothelial cellular filtration, inflammation, membrane formation and neovascularisation leading to the CSDH formation., (© 2023. The Author(s).)

The Epstein-Barr virus (EBV) genome encodes a cluster of 22 viral microRNAs, called miR-BamHI-A rightward transcripts (miR-BARTs), which are shown to promote the development of cancer. Here, this study reports that EBV-miR-BART18-3p is highly expressed in colorectal cancer (CRC) and is closely associated with the pathological and advanced clinical stages of CRC. Ectopic expression of EBV-miR-BART18-3p leads to increased migration and invasion capacities of CRC cells in vitro and causes tumor metastasis in vivo. Mechanistically, EBV-miR-BART18-3p activates the hypoxia inducible factor 1 subunit alpha/lactate dehydrogenase A axis by targeting Sirtuin, which promotes lactate accumulation and acetyl-CoA production in CRC cells under hypoxic condition. Increased acetyl-CoA utilization subsequently leads to histone acetylation of fatty acid synthase and fatty acid synthase-dependent fat synthesis, which in turn drives de novo lipogenesis. The oncogenic role of EBV-miR-BART18-3p is confirmed in the patient-derived tumor xenograft mouse model. Altogether, the findings define a novel mechanism of EBV-miR-BART18-3p in CRC development through the lipogenesis pathway and provide a potential clinical intervention target for CRC., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Infections caused by bacterial biofilms represent a global health problem, causing considerable patient morbidity and mortality in addition to an economic burden. Escherichia coli , Staphylococcus aureus, and other medically relevant bacterial strains colonize clinical surfaces and medical devices via biofilm in which bacterial cells are protected from the action of the immune system, disinfectants, and antibiotics. Several approaches have been investigated to inhibit and disperse bacterial biofilms, and the use of drug delivery could represent a fascinating strategy. Ciprofloxacin (CIP), which belongs to the class of fluoroquinolones, has been extensively used against various bacterial infections, and its loading in nanocarriers, such as niosomes, could support the CIP antibiofilm activity. Niosomes, composed of two surfactants (Tween 85 and Span 80) without the presence of cholesterol, are prepared and characterized considering the following features: hydrodynamic diameter, ζ-potential, morphology, vesicle bilayer characteristics, physical-chemical stability, and biological efficacy. The obtained results suggest that: (i) niosomes by surfactants in the absence of cholesterol are formed, can entrap CIP, and are stable over time and in artificial biological media; (ii) the CIP inclusion in nanocarriers increase its stability, with respect to free drug; (iii) niosomes preparations were able to induce a relevant inhibition of biofilm formation.

All cells are capable of secreting extracellular vesicles (EVs), which are not a means to eliminate unneeded cellular compounds but represent a process to exchange material (nucleic acids, lipids and proteins) between different cells. This also happens in the brain, where EVs permit the crosstalk between neuronal and non-neuronal cells, functional to homeostatic processes or cellular responses to pathological stimuli. In brain tumors, EVs are responsible for the bidirectional crosstalk between glioblastoma cells and healthy cells, and among them, astrocytes, that assume a pro-tumoral or antitumoral role depending on the stage of the tumor progression. In this work, we show that astrocyte-derived small EVs (sEVs) exert a defensive mechanism against tumor cell growth and invasion. The effect is mediated by astrocyte-derived EVs (ADEVs) through the transfer to tumor cells of factors that hinder glioma growth. We identified one of these factors, enriched in ADEVs, that is miR124. It reduced both the expression and function of the volume-regulated anion channel (VRAC), that, in turn, decreased the cell migration and invasion of murine glioma GL261 cells.

Pseudomonas aeruginosa is an opportunistic pathogen causing several chronic infections resistant to currently available antibiotics. Its pathogenicity is related to the production of different virulence factors such as biofilm and protease secretion. Pseudomonas communities can persist in biofilms that protect bacterial cells from antibiotics. Hence, there is a need for innovative approaches that are able to counteract these virulence factors, which play a pivotal role, especially in chronic infections. In this context, antimicrobial peptides are emerging drugs showing a broad spectrum of antibacterial activity. Here, we tested the anti-virulence activity of a chionodracine-derived peptide (KHS-Cnd) on five P. aeruginosa clinical isolates from cystic fibrosis patients. We demonstrated that KHS-Cnd impaired biofilm development and caused biofilm disaggregation without affecting bacterial viability in nearly all of the tested strains. Ultrastructural morphological analysis showed that the effect of KHS-Cnd on biofilm could be related to a different compactness of the matrix. KHS-Cnd was also able to reduce adhesion to pulmonary cell lines and to impair the invasion of host cells by P. aeruginosa . A cytotoxic effect of KHS-Cnd was observed only at the highest tested concentration. This study highlights the potential of KHS-Cnd as an anti-biofilm and anti-virulence molecule against P. aeruginosa clinical strains.

Up-to-date in vitro and in vivo preclinical models expressing the patient-specific cancer lineage responsible for CRC and its metastatic behavior and responsiveness to therapy are needed. Exosomes' role in tumorigenesis and the metastatic process was demonstrated, and the material content and size of the exosomes are associated with a poor prognosis of CRC. Exosomes are generally imagined after their recovery from blood serum as isolated entities, and our work aims to investigate them "in situ" in their native environment by scanning and transmission electron microscopy to understand their secretion modalities. We studied CRC stem cells in patient-derived multicellular tumor spheroids (MTSs) and in their mouse xenograft to find possible differences in terms of exosome amount, size, and secretion site between in vitro and in vivo models. We observed that MTSs' exosome secretion patterns depend on their structural complexity: few-layer MTSs show a lesser exosome secretion, limited to the apical domain of cancer cells, secretion increases in multilayered MTSs, and it develops from apical and basolateral cancer cells domains. In xenograft models, exosome secretion occurs from all cancer cell domains, and it is quantitatively greater than that observed in MTSs. This difference in exosome secretion pattern between MTSs and xenografts may be due to the influence of surrounding non-tumor cells.

Background/aim: The treatment of solitary brain metastasis is a challenging intervention since the incidence increases and prognosis is poor. This study investigated the role of perilesional edema in the overall mass effect of solitary brain metastasis., Patients and Methods: We conducted a retrospective analysis on 88 patients with single supratentorial brain metastasis and concomitant perilesional edema undergoing en bloc resection. Each patient was evaluated for perilesional brain edema grading. We stratified patients into three groups based on the size of the metastatic lesion and the extent of perilesional edema., Results: The grade of perilesional edema at 30 days after surgical removal did not correlate with the maximum diameter of the metastasis (Pearson's correlation 0.098, p=0.494). In patients with a maximal metastatic diameter ≤2 cm, the grade of perilesional edema before surgical treatment was 1.63 (STD 0.43), while 30 days after removal it was significantly reduced; 0.47 (STD 0.26), p<0.001., Conclusion: The overall mass effect of solitary supratentorial brain metastases is not correlated to the size of the lesion and the grade of the associated perilesional edema should be considered. Surgical en bloc resection can be considered the first choice of treatment in the presence of solitary metastasis ≤2 cm in maximal diameter but with high-grade edema, since this treatment reduces the overall mass effect., (Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Recent studies have identified that long noncoding RNA (lncRNA) might affect the responses to anticancer drug treatment, including colorectal cancer (CRC). However, the association between single-nucleotide polymorphisms (SNPs) in PVT1 and the chemotherapy response in metastatic colorectal cancer has yet to be clarified. In this study, the PVT1 rs2278176 CT/TT genotypes were found to be associated with an increased overall survival (OS) and progression-free survival (PFS) compared with the CC genotype. Furthermore, patients harboring the rs2278176 CT/TT genotypes had a greater chance of achieving clinical benefit from 5-Fluorouracil/leucovorin combined with oxaliplatin (FOLFOX). In vivo nude mice experiments demonstrated that the CRISPR/Cas9 mediated rs2278176 C to T mutation significantly inhibited the tumorigenesis of colorectal cancer cells treated with 5-Fu, but not control DMSO treated cells. Furthermore, the apoptotic rate was significantly enhanced by treatment with 5-Fu in the CRC cells carrying with the CT/TT genotypes. Functional studies demonstrated that the PVT1 rs2278176 C to T mutation altered the binding site for hsa-miR-297, and that hsa-miR-297 downregulated Glutathione S-Transferase Alpha 2(GSTA2), a member of phase II detoxification enzyme, in an Argonaute 2(Ago2)-dependent manner. Moreover, GSTA2 levels were downregulated in the cancer tissues of patients carrying rs2278176 CT/TT genotypes. High GSTA2 expression predicted poor clinical outcome in metastatic colorectal cancer treated with FOLFOX. In conclusion, this study provided that PVT1 with rs2278176 T allele altered the binding affinity with hsa-miR-297, leading to decreased GSTA2 expression and sensitized CRC cells to FOLFOX chemotherapy, suggesting rs2278176 CT/TT genotypes might serve as a predictive biomarker to improve prognosis in patients with metastatic CRC treated with FOLFOX., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wu, Yang, Tang, Familiari, Relucenti, Aschner, Li and Chen.)

Chimeric antigen receptor (CAR) - T cell therapy is a new class of cellular immunotherapies, which has made great achievements in the treatment of malignant tumors. Despite improvements in colorectal cancer (CRC) therapy, treatment of many patients fails because of metastasis and recurrence. The human epidermal growth factor receptor 2 (HER2) is a substantiated target for CAR-T therapy, and has been reported recently to be over-expressed in CRC, which may provide a potential therapeutic target for CRC treatment. Herein, HER2 was a promising target of metastatic colorectal cancer (mCRC) in CAR-T therapy as assessed by flow cytometry and tissue microarray (TMA) with 9-year survival follow-up data. Furthermore, HER2-specific CAR-T cells exhibited strong cytotoxicity and cytokine-secreting ability against CRC cells in vitro. Moreover, through the tumor-bearing model of the NOD-Prkdc em26cd52 Il2rg em26Cd22 /Nju (NCG) mice, HER2 CAR-T cells showed signs of effectively preventing CRC progression in three different xenograft models. Notably, HER2 CAR-T cells displayed greater aggressiveness in HER2 + CRC in the patient-derived tumor xenograft (PDX) models and had potent immunotherapeutic capacity for mCRC in the metastatic xenograft mouse models. In conclusion, our studies provide scientific evidence that HER2 CAR-T cells represent an emerging immunotherapy for the treatment of mCRC., (© 2021. The Author(s).)

Background and Aims: Liver metastasis is a frequent occurrence in patients with colorectal cancer (CRC), with 15%-25% of CRC patients having liver metastases at the time of initial diagnosis. Specifically, some regional-stage patients with mild symptoms (stage 1 or 2) will also advance to liver metastases rapidly, even if the CRC lesion in situ is resected in time. Nevertheless, the precise mechanism of liver metastasis is still unclear., Approach and Results: Fresh tumor tissues from patients with CRC, adjacent noncancerous tissues, and colorectal adenoma tissues were subjected to microarray analysis to identify differentially expressed microRNA. Exosomes from human serum and cell culture medium were separated, quantitated, and verified by transmission electronic microscopy and Zetasizer Nano. Luciferase reporter assay, real-time quantitative PCR, western blot, immunoprecipitation, chromatin and re-chromatin immunoprecipitation, migration and invasion assay, PDX mouse model, flow cytometry, immunohistochemistry, and immunofluorescence staining were employed to explore the regulation among CRC liver metastases, immunosuppression, and cell adhesion. In this study, we demonstrated that the hypoxic microenvironment in primary CRC lesions boosted exosome release, selectively initiated favorable premetastatic niche formation in the liver but not in other organs. Mechanistically, Kupffer cells (KCs) can phagocytose exosomes containing highly expressed miR-135a-5p from the blood circulation into the liver. Exosomal miR-135a-5p initiated the large tumor suppressor kinase 2-yes-associated protein-matrix metalloproteinase 7 axis to promote the occurrence of CRC liver metastasis, and cluster of differentiation 30-TNF receptor-associated factor 2-p65-mediated immunosuppression signaling also contributed to this process., Conclusions: Hypoxia-induced exosomal miR-135a-5p correlates with the development, clinical severity, and prognosis of CRC liver metastases through the premetastatic niche; and our findings revealed that miR-135a-5p might be a promising target in halting CRC liver metastases., (© 2021 by the American Association for the Study of Liver Diseases.)

Cerebrovascular disease, a frequent complication of hypertension, is a major public health issue for which novel therapeutic and preventive approaches are needed. Autophagy activation is emerging as a potential therapeutic and preventive strategy toward stroke. Among usual activators of autophagy, the natural disaccharide trehalose (TRE) has been reported to be beneficial in preclinical models of neurodegenerative diseases, atherosclerosis and myocardial infarction. In this study, we tested for the first time the effects of TRE in the stroke-prone spontaneously hypertensive rat (SHRSP) fed with a high-salt stroke permissive diet (JD). We found that TRE reduced stroke occurrence and renal damage in high salt-fed SHRSP. TRE was also able to decrease systolic blood pressure. Through ex-vivo studies, we assessed the beneficial effect of TRE on the vascular function of high salt-fed SHRSP. At the molecular level, TRE restored brain autophagy and reduced mitochondrial mass, along with the improvement of mitochondrial function. The beneficial effects of TRE were associated with increased nuclear translocation of TFEB, a transcriptional activator of autophagy. Our results suggest that TRE may be considered as a natural compound efficacious for the prevention of hypertension-related target organ damage, with particular regard to stroke and renal damage., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Pseudomonas aeruginosa is an opportunistic pathogen responsible for nosocomial infections, and is often involved in airway infections of cystic fibrosis (CF) patients. P. aeruginosa virulence is related to its ability to form biofilm, trigger different types of motilities, and produce toxins (for example, bacterial pigments). In this scenario, essential oils (EOs) have gained notoriety for their role in phenotype modulation, including virulence modulation. Among different EOs previously analyzed, herein we investigated the activity of Coridothymus capitatus EO (CCEO) against specific virulence factors produced by P. aeruginosa isolated from CF patients. CCEO showed inhibition of new biofilm formation and reduction in mature biofilm in about half of the tested strains. On selected strains, SEM analysis provided interesting information regarding CCEO action in a pre-adhesion assay. CCEO treatment showed a dramatic modification of the extracellular matrix (ECM) structure. Our results clearly showed a drastic reduction in pyocyanin production (between 84% and 100%) for all tested strains in the presence of CCEO. Finally, CCEO was also able to strongly affect P. aeruginosa swarming and swimming motility for almost all tested strains. In consideration of the novel results obtained on clinical strains isolated from CF patients, CCEO may be a potential candidate to limit P. aeruginosa virulence.

Background and Aim: Diarrhea, abdominal pain, and bloating are frequent in irritable bowel syndrome (IBS)-like disorders, although little is known about their intestinal ultrastructural alterations. The aim of the present study was to study duodenal biopsies from IBS-like patients to find ultrastructural alterations., Materials and Methods: Study design: descriptive comparative pilot study. Thirty outpatients (9 male and 21 female; median age 37.7 years; range, 20 to 65 years) complaining of IBS-like symptoms were enrolled between January 2015 to May 2019 and were divided into 6 groups, each equally consisting of 5 patients: (A) untreated celiac disease (uCD); (B) treated celiac disease (tCD); (C) wheat allergy (WA); (D) Non-celiac gluten sensitivity (NCGS); (E) Nickel allergic contact mucositis (Ni ACM); (F) controls affected by GERD. Transmission electron microscopy (TEM) morphological characteristics were: microvilli length, intermicrovillar distance, junctional complexes (JC) gap width, autophagic bodies, apoptosis, altered mitochondria, lipid/chylomicron droplets, and mast cells. Regarding JC, we focused on tight junctions (TJ), adherens junctions (AJ), and desmosomes., Results: Major alterations in microvilli length and intermicrovillar distance have been observed in the subjects affected by uCD. Microvilli of tCD patients showed marked recovery after adequate GFD, although not comparable to controls. Intermediate microvillar alterations were instead observed in NCGS and Ni ACM, while characteristics of WA subjects appeared more similar to tCD. Regarding JC, TJ did not show significant differences between all groups studied, including controls. The AJ were significantly more dilated in all groups compared to controls, while no significant differences were found between the pathological groups. The distance between desmosomes was greater in uCD, NCGS, and Ni ACM than in tCD, WA, and controls. Finally, intracellular alterations have been detected in most of the groups studied although they seemed more unspecific., Conclusions: TEM analysis confirmed damages to the intestinal barrier and defense mechanisms by enterocytes in IBS-like patients, probably linked to low-grade inflammation or adverse reactions triggered by food allergens, heavy metals, or other unknown. On the other hand, our study needs confirmation and further investigations with larger populations to facilitate diagnosis, therapy, and prevention of IBS-like disorders in the future.

Spheroids from primary colorectal cancer cells and their mice xenografts have emerged as useful preclinical models for cancer research as they replicate tumor features more faithfully as compared to cell lines. While 3D models provide a reliable system for drug discovery and testing, their structural complexity represents a challenge and their structure-function relationships are only partly understood. Here, we present a comparative ultrastructural and flow citometric analysis of patient colorectal cancer-derived spheroids and their mice xenografts. Ultrastructural observations highlighted that multicellular spheroids and their xenografts contain the same cancer cell types but with different ratios, specifically multicellular spheroids were enriched in cells with a stem-like phenotype, while xenografts had an increased amount of lipid droplets-containing cells. The flow cytometric analysis for stem cell marker and activity showed enrichment of stem-like cells presence and activity in spheroids while xenografts had the inverse response. Our results evidence the effects on cancer cells of different in vitro and in vivo microenvironments. Those differences have to be paid into account in designing innovative experimental models for personalized drug testing.

Brain homeostasis needs continuous exchange of intercellular information among neurons, glial cells, and immune cells, namely microglial cells. Extracellular vesicles (EVs) are active players of this process. All the cells of the body, including the brain, release at least two subtypes of EVs, the medium/large EVs (m/lEVs) and small EVs (sEVs). sEVs released by microglia play an important role in brain patrolling in physio-pathological processes. One of the most common and malignant forms of brain cancer is glioblastoma. Altered intercellular communications constitute a base for the onset and the development of the disease. In this work, we used microglia-derived sEVs to assay their effects in vitro on murine glioma cells and in vivo in a glioma model on C57BL6/N mice. Our findings indicated that sEVs carry messages to cancer cells that modify glioma cell metabolism, reducing lactate, nitric oxide (NO), and glutamate (Glu) release. sEVs affect Glu homeostasis, increasing the expression of Glu transporter Glt-1 on astrocytes. We demonstrated that these effects are mediated by miR-124 contained in microglia-released sEVs. The in vivo benefit of microglia-derived sEVs results in a significantly reduced tumor mass and an increased survival of glioma-bearing mice, depending on miR-124.

Pseudomonas aeruginosa is an opportunistic pathogen often involved in airway infections of cystic fibrosis (CF) patients. Its pathogenicity is related to several virulence factors, such as biofilm formation, motility and production of toxins and proteases. The expression of these virulence factors is controlled by quorum sensing (QS). Thus, QS inhibition is considered a novel strategy for the development of antipathogenic compounds acting on specific bacterial virulence programs without affecting bacterial vitality. In this context, cold-adapted marine bacteria living in polar regions represent an untapped reservoir of biodiversity endowed with an interesting chemical repertoire. In this paper, we investigated the biological activity of a supernatant derived from a novel Antarctic bacterium (SN&#95;TAE2020) against specific virulence factors produced by P. aeruginosa strains isolated from FC patients. Our results clearly show a reduction in pyocyanin and protease production in the presence of SN&#95;TAE2020. Finally, SN&#95;TAE2020 was also able to strongly affect swarming and swimming motility for almost all tested strains. Furthermore, the effect of SN&#95;TAE2020 was investigated on biofilm growth and texture, captured by SEM analysis. In consideration of the novel results obtained on clinical strains, polar bacteria might represent potential candidates for the discovery of new compounds limiting P. aeruginosa virulence in CF patients.

Today, environmental health research on toxicological adverse effects of metal-inorganic materials diffused by cigarettes represents a new challenge for assessing new health risks directly related to the critical chemical-size features of the particles. Therefore, morpho-chemical analyses of hazardous particles become critical in response to the distinctive assumptions about the origin, evolution, and coexisting phases. Here, we report a detailed investigation through direct microscopy imaging of metal-inorganic contaminants for one traditional and two heat-not-burn commercial cigarettes of three different brands. Chemical-size studies revealed the critical presence of heavy metal-inorganic nanostructured microparticles on both paper and filter components of the cigarette, before and after smoking. The direct experimental imaging evidenced on how hazardous particles evolved in mass-size forming coexisting multi-phases of large agglomerate because of the persistence and accumulative effect of the heating puffing. The estimated porosity of the unsuitable engineered filters validated the allowed migration of micrometric pollutants independently from their intrinsic size-shape property. Furthermore, the inappropriate design of the filters made it an adverse sponge reservoir capable of collecting all possible hazardous chemical agents potentially toxic. These substantial results strongly support experimentally the tremendous effect of the smoke capable of transporting and manipulating a high amount of elusive particles, as a particles heat carrier., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Calcium silicate-based cements have reached excellent levels of performance in endodontics, providing predictable and successful results. To better assess the properties of these bioactive materials, the present study aimed to compare the biocompatibility and antibiofilm properties of ProRoot MTA and Biodentine. Human osteogenic sarcoma (Saos-2) cells were cultured on ProRoot MTA and Biodentine samples or in the presence of both cement extracts. Cell viability assay, measurement of reactive oxygen species (ROS), immunofluorescence analysis, as well as morphological evaluations were conducted. Moreover, Streptococcus mutans was used to assess the biofilm forming ability on ProRoot MTA and Biodentine disks. Finally, both cements were applied in vivo to treat immature permanent teeth affected by reversible pulpitis. Results: Cell viability assay demonstrated that Saos-2 cells had a dose- and time-dependent cytotoxicity to both analyzed cements, although cells exposed to ProRoot MTA showed a better cell vitality than those exposed to Biodentine ( p < 0.001). Both cements demonstrated ROS production while this was greater in the case of Biodentine than ProRoot MTA ( p < 0.001). Immunofluorescence images of the cytoskeleton and focal adhesions showed no differences in Saos-2 cells grown in the presence of ProRoot MTA eluate; whereas in the Biodentine groups, cells showed a morphology and focal adhesions more similar to that of the control sample, as the eluate concentration decreased. Morphological analysis revealed that Saos-2 cells were more flattened and exhibited better spreading when attached to ProRoot MTA disks than to Biodentine ones. The antibiofilm properties showed a time-dependent powerful inhibition of S. mutans superficial colonization and an antibiofilm effect of both cements. Clinically, complete root formation of the treated elements was achieved using the two studied cements, showing stable results over time. ProRoot MTA and Biodentine was demonstrated to be biocompatible and to possess antibiofilm properties. Their clinical application in vital pulp therapy provided successful outcomes after 2 years of follow-up.

Several imaging methodologies have been used in biofilm studies, contributing to deepening the knowledge on their structure. This review illustrates the most widely used microscopy techniques in biofilm investigations, focusing on traditional and innovative scanning electron microscopy techniques such as scanning electron microscopy (SEM), variable pressure SEM (VP-SEM), environmental SEM (ESEM), and the more recent ambiental SEM (ASEM), ending with the cutting edge Cryo-SEM and focused ion beam SEM (FIB SEM), highlighting the pros and cons of several methods with particular emphasis on conventional SEM and VP-SEM. As each technique has its own advantages and disadvantages, the choice of the most appropriate method must be done carefully, based on the specific aim of the study. The evaluation of the drug effects on biofilm requires imaging methods that show the most detailed ultrastructural features of the biofilm. In this kind of research, the use of scanning electron microscopy with customized protocols such as osmium tetroxide (OsO 4 ), ruthenium red (RR), tannic acid (TA) staining, and ionic liquid (IL) treatment is unrivalled for its image quality, magnification, resolution, minimal sample loss, and actual sample structure preservation. The combined use of innovative SEM protocols and 3-D image analysis software will allow for quantitative data from SEM images to be extracted; in this way, data from images of samples that have undergone different antibiofilm treatments can be compared.

Bacterial biofilm plays a pivotal role in chronic Staphylococcus aureus ( S. aureus ) infection and its inhibition may represent an important strategy to develop novel therapeutic agents. The scientific community is continuously searching for natural and "green alternatives" to chemotherapeutic drugs, including essential oils (EOs), assuming the latter not able to select resistant strains, likely due to their multicomponent nature and, hence, multitarget action. Here it is reported the biofilm production modulation exerted by 61 EOs, also investigated for their antibacterial activity on S. aureus strains, including reference and cystic fibrosis patients' isolated strains. The EOs biofilm modulation was assessed by Christensen method on five S. aureus strains. Chemical composition, investigated by GC/MS analysis, of the tested EOs allowed a correlation between biofilm modulation potency and putative active components by means of machine learning algorithms application. Some EOs inhibited biofilm growth at 1.00% concentration, although lower concentrations revealed different biological profile. Experimental data led to select antibiofilm EOs based on their ability to inhibit S. aureus biofilm growth, which were characterized for their ability to alter the biofilm organization by means of SEM studies.

The identification of the mechanisms predisposing to stroke may improve its preventive and therapeutic strategies in patients with essential hypertension. The role of macroautophagy/autophagy in the development of hypertension-related stroke needs to be clarified. We hypothesized that a defective autophagy may favor hypertension-related spontaneous stroke by promoting mitochondrial dysfunction. We studied autophagy in the stroke-prone spontaneously hypertensive (SHRSP) rat, which represents a clinically relevant model of stroke associated with high blood pressure. We assessed autophagy, mitophagy and NAD + :NADH levels in brains of SHRSP and stroke-resistant SHR fed with high salt diet. Vascular smooth muscle cells silenced for the mitochondrial complex I subunit Ndufc2 gene (NADH:ubiquinone oxidoreductase subunit C2) and cerebral endothelial cells isolated from SHRSP were also used to assess autophagy/mitophagy and mitochondrial function in response to high salt levels. We found a reduction of autophagy in brains of high salt-fed SHRSP. Autophagy impairment was associated with NDUFC2 downregulation, mitochondrial dysfunction and NAD + depletion. Restoration of NAD + levels by nicotinamide administration reactivated autophagy and reduced stroke development in SHRSP. A selective reactivation of autophagy/mitophagy by Tat-Beclin 1 also reduced stroke occurrence, restored autophagy/mitophagy and improved mitochondrial function. Endothelial progenitor cells (EPCs) from subjects homozygous for the thymine allele variant at NDUFC2 /rs11237379, which is associated with NDUFC2 deficiency and increased stroke risk, displayed an impairment of autophagy and increased senescence in response to high salt levels. EPC senescence was rescued by Tat-Beclin 1. Pharmacological activation of autophagy may represent a novel therapeutic strategy to reduce stroke occurrence in hypertension., Abbreviations: 10 VSMCs: aortic vascular smooth muscle cells; COX4I1/COX IV: cytochrome c oxidase subunit 4I1; ECs: endothelial cells; EPCs: endothelial progenitor cells; JD: Japanese-style diet; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; NAD: nicotinamide adenine dinucleotide; NDUFC2: NADH:ubiquinone oxidoreductase subunit C2; NMN: nicotinamide mononucleotide; RD: regular diet; SHRSP: stroke-prone spontaneously hypertensive rat; SHRSR: stroke-resistant spontaneously hypertensive rat.

Since the application of nanotechnology to drug delivery, both polymer-based and lipid-based nanocarriers have demonstrated clinical benefits, improving both drug efficacy and safety. However, to further address the challenges of the drug delivery field, hybrid lipid-polymer nanocomposites have been designed to merge the beneficial features of both polymer-based and lipid-based delivery systems in a single nanocarrier. Within this scenario, this work is aimed at developing novel hybrid vesicles following the recent strategy of modifying the internal structure of liposomes. Specifically, polyethylene glycol-dimethacrylate (PEG-DMA, molecular weight 750 or 4000), was entrapped within unilamellar liposomes made of hydrogenated soybean phosphatidylcholine/cholesterol, and photo-crosslinked, in order to transform the aqueous inner core of liposomes into a soft and elastic hydrogel. After appropriate optimization of the preparation and gelation procedures, the primary objective of this work was to analyze the effect of the molecular weight of PEG-DMA on the main properties of these Gel-in-Liposome (GiL) systems. Indeed, by varying the molecular weight of PEG-DMA also its hydrophilic/lipophilic balance was modified and different arrangements of the polymer within the structure of liposomes as well as different interaction with their membrane were obtained. Both polymers were found in the inner core of the liposomes, however, the more hydrophobic PEG 750 -DMA also formed localized clusters within the liposome membrane, whereas the more hydrophilic PEG 4000 -DMA formed a polymeric corona on the vesicle surface. Preliminary cytotoxicity studies were also performed to evaluate the biological safety of these GiL systems and their suitability as innovative materials drug delivery application., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)