Mechanisms of malignancy in glioblastoma cells are linked to mitochondrial Ca2+ uniporter upregulation and higher intracellular Ca2+ levels.

Glioblastoma (GBM) is one of the most malignant brain tumours and, despite advances in treatment modalities, it remains largely incurable. Ca2+ regulation and dynamics play crucial roles in different aspects of cancer, but they have never been investigated in detail in GBM. Here, we report that spontaneous Ca2+ waves in GBM cells cause unusual intracellular Ca2+ ([Ca2+]i) elevations (>1 µM), often propagating through tumour microtubes (TMs) connecting adjacent cells. This unusual [Ca2+]i elevation is not associated with the induction of cell death and is concomitant with overexpression of mitochondrial Ca2+ uniporter (MCU). We show that MCU silencing decreases proliferation and alters [Ca2+]i dynamics in U87 GBM cells, while MCU overexpression increases [Ca2+]i elevation in human astrocytes (HAs). These results suggest that changes in the expression level of MCU, a protein involved in intracellular Ca2+ regulation, influences GBM cell proliferation, contributing to GBM malignancy.This article has an associated First Person interview with the first author of the paper.

Role of Microenvironment in Glioma Invasion: What We Learned from In Vitro Models.

The invasion properties of glioblastoma hamper a radical surgery and are responsible for its recurrence. Understanding the invasion mechanisms is thus critical to devise new therapeutic strategies. Therefore, the creation of in vitro models that enable these mechanisms to be studied represents a crucial step. Since in vitro models represent an over-simplification of the in vivo system, in these years it has been attempted to increase the level of complexity of in vitro assays to create models that could better mimic the behaviour of the cells in vivo. These levels of complexity involved: 1. The dimension of the system, moving from two-dimensional to three-dimensional models; 2. The use of microfluidic systems; 3. The use of mixed cultures of tumour cells and cells of the tumour micro-environment in order to mimic the complex cross-talk between tumour cells and their micro-environment; 4. And the source of cells used in an attempt to move from commercial lines to patient-based models. In this review, we will summarize the evidence obtained exploring these different levels of complexity and highlighting advantages and limitations of each system used.

Prevalence of hemostatic alterations in patients with recurrent spontaneous subconjunctival hemorrhage.

BACKGROUND: Subconjunctival hemorrage (SCH) is a frequent, mild bleeding manifestation and a common cause of consultation. Hemostatic alterations are possible causes of SCH but their role and prevalence is unknown. We assessed the prevalence of hemostatic abnormalities in patients with spontaneous, recurrent SCH to clarify the role of the hemostasis laboratory in this clinical setting. METHODS: A total of 105 SCH patients (21-78 years, 65 females) with no identifiable cause (hypertension-trauma-conjunctivitis) or concomitant treatments (NSAIDs- aspirin-oral anticoagulants-antiplatelet agents) and 53 age and sex-matched healthy controls (HCs) (22-72 years, 29 females) were evaluated for skin bleeding time, PFA-100®, blood clotting screening, platelet count, light transmission aggregomery, VWF:Ag, VWF:RCo, RIPA, FVIII activity, FXIII antigen and activity and ISTH Bleeding Severity Score (BSS). RESULTS: Prevalence of hemostatic abnormalities was not higher in the SCH population than in HCs BSS was 0.83 (95% CI 0.62-1.06) in SCH and 0.66 (0.37-0.95) in HC (p=NS). Type I Von Willebrand disease was diagnosed in one SCH and none HC patients, a prevalence not significantly different (p=NS by χ2). CONCLUSIONS: The prevalence of hemostatic alterations in patients with recurrent, spontaneous SCH is not different from the general population; hemostatic screening or second level tests are of no use in patients with recurrent SCH and no other bleedings.

Study of alpha-lipoic acid penetration in the human aqueous after topical administration.

PURPOSE: To determine the concentration of alpha-lipoic acid in the aqueous and investigate if its topical instillation can increase quantities. METHODS: A total of 70 patients selected to undergo cataract surgery were randomly divided into two groups. Group 1 was used as a control group; for the patients in Group 2, a single instillation of alpha-lipoic acid eye drops (1%) was administered. Immediately before surgery, an aliquot of 40-120 microL of aqueous was aspirated. The individual aspirations were combined to constitute pools representing time intervals with respect to administration. The levels of alpha-lipoic acid in the aqueous were measured using gas chromatography/mass spectrometry. RESULTS: Pool 0 was created by combining the samples of aqueous obtained from the patients in Group 1, the control group; and the level of alpha-lipoic acid was 27.5 + 2.6 ng/mL. In the other pools, the time interval between the administration of the eye drops and sampling was, respectively, 23 min, 53 min, 72 min, 93 min and 114 min; and the level of alpha-lipoic acid was 33.0 + 10.8 ng/mL, 52.0 + 2.5 ng/mL, 86.7 + 2.5 ng/mL, 91.2 + 2.5 ng/mL and 80.3 + 2.5 ng/mL. CONCLUSION: Our study demonstrates the presence of alpha-lipoic acid in the aqueous and indicates that its concentration increases after it is administered in the form of eye drops, reaching maximum values after around 93 min. The concentrations that are achieved in the anterior chamber allow us to theorize the possibility of exploiting the antioxidant properties of alpha-lipoic acid.

Heterogeneity Matters: Different Regions of Glioblastoma Are Characterized by Distinctive Tumor-Supporting Pathways.

The glioblastoma microenvironment plays a substantial role in glioma biology. However, few studies have investigated its spatial heterogeneity. Exploiting 5-ALA Fluorescence Guided Surgery (FGS), we were able to distinguish between the tumor core (ALA+), infiltrating area (ALA-PALE) and healthy tissue (ALA-) of the glioblastoma, based on the level of accumulated fluorescence. The aim of this study was to investigate the properties of the microenvironments associated with these regions. For this purpose, we isolated glioma-associated stem cells (GASC), resident in the glioma microenvironment, from ALA+, ALA-PALE and ALA- samples and compared them in terms of growth kinetic, phenotype and for the expression of 84 genes associated with cancer inflammation and immunity. Differentially expressed genes were correlated with transcriptomic datasets from TCGA/GTEX. Our results show that GASC derived from the three distinct regions, despite a similar phenotype, were characterized by different transcriptomic profiles. Moreover, we identified a GASC-based genetic signature predictive of overall survival and disease-free survival. This signature, highly expressed in ALA+ GASC, was also well represented in ALA PALE GASC. 5-ALA FGS allowed to underline the heterogeneity of the glioma microenvironments. Deepening knowledge of these differences can contribute to develop new adjuvant therapies targeting the crosstalk between tumor and its supporting microenvironment.

Semaphorin-7A on Exosomes: A Promigratory Signal in the Glioma Microenvironment.

Exosomes are one of the most important mediators of the cross talk occurring between glioma stem cells (GSCs) and the surrounding microenvironment. We have previously shown that exosomes released by patient-derived glioma-associated stem cells (GASC) are able to increase, in vitro, the aggressiveness of both GSC and glioblastoma cell lines. To understand which molecules are responsible for this tumour-supporting function, we performed a descriptive proteomic analysis of GASC-exosomes and identified, among the others, Semaphorin7A (SEMA7A). SEMA7A was described as a promigratory cue in physiological and pathological conditions, and we hypothesised that it could modulate GSC migratory properties. Here, we described that SEMA7A is exposed on GASC-exosomes' surface and signals to GSC through Integrin ß1. This interaction activates focal adhesion kinase into GSC and increases their motility, in our patient-based in vitro model. Our findings suggest SEMA7A-ß1-integrin as a new target to disrupt the communication between GSCs and the supporting microenvironment.

Reasons for visits to an emergency center and hemostatic alterations in patients with recurrent spontaneous subconjunctival hemorrhage.

PURPOSE: To evaluate causes of visits to the Eye Emergency Department, determine the prevalence of subconjunctival hemorrhage (SCH), and assess the role of hemostatic abnormalities among patients with spontaneous recurrent SCH (SRSCH). METHODS: In a prospective study conducted over 2 years, hemostatic function was studied in a subgroup of 105 consecutive patients (39 male) with SRSCH free of systemic risk factors and in 53 age- and sex-matched healthy controls (HC) (24 male). RESULTS: A total of 10,090 patients (mean age 57.2 ± 16.7 years, range 0-94, median 58.4) were evaluated. A total of 39.3% had ocular trauma, 34.9% inflammatory ocular surface disorder, 5.7% floaters, 3.3% visual symptoms of neurologic origin, 1.6% uveitis, 1.5% ocular hypertension, 0.8% retinal tear or detachment, 0.7% retinal vascular disease, and 0.5% other causes. A total of 1.6% of the patients were hospitalized. A total of 11.7% of patients had SCH: in 86.7% it was spontaneous, in 13.3% consequent to trauma or to ocular surface disorders. A total of 105 patients had SRSCH, and the prevalence of hemostatic abnormalities among them was not different from HC. Type I von Willebrand disease was diagnosed in 1 patient with SCH and in none of the HC (χ² = 0.13, p = 0.72). CONCLUSIONS: Most patients had ocular infection or trauma and were treated on an outpatient basis; SCH was the third cause of access. The large majority of SCH were unprovoked, and the prevalence of hemostatic alterations in patients with SRSCH and no systemic causes was not different from the general population. Hemostatic screening or second level blood clotting tests were of no use in these patients.

Altered expression of neuropeptides in FoxG1-null heterozygous mutant mice.

Foxg1 gene encodes for a transcription factor essential for telencephalon development in the embryonic mammalian forebrain. Its complete absence is embryonic lethal while Foxg1 heterozygous mice are viable but display microcephaly, altered hippocampal neurogenesis and behavioral and cognitive deficiencies. In order to evaluate the effects of Foxg1 alteration in adult brain, we performed expression profiling in total brains from Foxg1+/- heterozygous mutants and wild-type littermates. We identified statistically significant differences in expression levels for 466 transcripts (P<0.001), 29 of which showed a fold change ≥ 1.5. Among the differentially expressed genes was found a group of genes expressed in the basal ganglia and involved in the control of movements. A relevant (three to sevenfold changes) and statistically significant increase of expression, confirmed by qRT-PCR, was found in two highly correlated genes with expression restricted to the hypothalamus: Oxytocin (Oxt) and Arginine vasopressin (Avp). These neuropeptides have an important role in maternal and social behavior, and their alteration is associated with impaired social interaction and autistic behavior. In addition, Neuronatin (Nnat) levels appear significantly higher both in Foxg1+/- whole brain and in hippocampal neurons after silencing Foxg1, strongly suggesting that it is directly or indirectly repressed by Foxg1. During fetal and neonatal brain development, Nnat may regulate neuronal excitability, receptor trafficking and calcium-dependent signaling and, in the adult brain, it is predominantly expressed in parvalbumin-positive GABAergic interneurons. Overall, these results implicate the overexpression of a group of neuropeptides in the basal ganglia, hypothalamus, cortex and hippocampus in the pathogenesis FOXG1 behavioral impairments.