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Close proximities between organelles have been described for decades. However, only recently a specific field dealing with organelle communication at membrane contact sites has gained wide acceptance, attracting scientists from multiple areas of cell biology. The diversity of approaches warrants a unified vocabulary for the field. Such definitions would facilitate laying the foundations of this field, streamlining communication and resolving semantic controversies. This opinion, written by a panel of experts in the field, aims to provide this burgeoning area with guidelines for the experimental definition and analysis of contact sites. It also includes suggestions on how to operationally and tractably measure and analyze them with the hope of ultimately facilitating knowledge production and dissemination within and outside the field of contact-site research. Given the recent growing interest in interorganelle membrane contact sites, the field will benefit from clear rules to define and study them. In this Perspective, a panel of experts aims to provide this growing field with guidelines for experimental definition and analysis.

Background Breast cancer is the second leading cause of death among women after lung cancer. Despite the improvement in prevention and in therapy, breast cancer still remains a threat, both for pre- and postmenopausal women, due to the development of drug resistance. To counteract that, novel agents regulating gene expression have been studied in both hematologic and solid tumors. The Histone Deacetylase (HDAC) inhibitor Valproic Acid (VA), used for epilepsy and other neuropsychiatric diseases, has been demonstrated a strong antitumoral and cytostatic activity. In this study, we tested the effects of Valproic Acid on the signaling pathways involved in breast cancer cells viability, apoptosis and in Reactive Oxygen Species (ROS) production using ER-α positive MCF-7 and triple negative MDA-MB-231 cells. Methods Cell proliferation assay was performed by MTT Cell cycle, ROS levels and apoptosis were analyzed by flow cytometry, protein levels were detected by Western Blotting. Results Cell treatment with Valproic Acid reduced cell proliferation and induced G0/G1 cell cycle arrest in MCF-7 and G2/M block in MDA-MB-231 cells. In addition, in both cells the drug enhanced the generation of ROS by the mitochondria. In MCF-7 treated cells, it has been observed a reduction in mitochondrial membrane potential, a down regulation of the anti-apoptotic marker Bcl-2 and an increase of Bax and Bad, leading to release of cytochrome C and PARP cleavage. Less consistent effects are recorded in MDA-MB-231 cells, in which the greater production of ROS, compared to MCF-7cells, involves an inflammatory response (activation of p-STAT3, increased levels of COX2). Conclusions Our results have demonstrated that in MCF-7 cells the Valproic Acid is a suitable drug to arrest cell growth, to address apoptosis and mitochondrial perturbations, all factors that are important in determining cell fate and health. In a triple negative MDA-MB 231 cells, valproate directs the cells towards the inflammatory response with a sustained expression of antioxidant enzymes. Overall, the not always unequivocal data between the two cellular phenotypes indicate that further studies are needed to better define the use of the drug, also in combination with other chemotherapy, in the treatment of breast tumors.

Glioblastoma multiforme (GBM) is one of the most aggressive types of cancer characterized by poor patient outcomes. To date, it is believed that the major cause of its recurrence and chemoresistance is represented by the enrichment of GBM stem cells (GSCs) sustained by the abnormal activation of a number of signaling pathways. In this study, we found that in GBM cells, treatment with low toxicity doses of the γ-secretase inhibitor RO4929097 (GSI), blocking the Notch pathway activity, in combination with resveratrol (RSV) was able to reverse the basal mesenchymal phenotype to an epithelial-like phenotype, affecting invasion and stemness interplay. The mechanism was dependent on cyclin D1 and cyclin-dependent kinase (CDK4), leading to a reduction of paxillin (Pxn) phosphorylation. Consequently, we discovered the reduced interaction of Pxn with vinculin (Vcl), which, during cell migration, transmits the intracellular forces to the extracellular matrix. The exogenous expression of a constitutively active Cdk4 mutant prevented the RSV + GSI inhibitory effects in GBM cell motility/invasion and augmented the expression of stemness-specific markers, as well as the neurosphere sizes/forming abilities in untreated cells. In conclusion, we propose that Cdk4 is an important regulator of GBM stem-like phenotypes and invasive capacity, highlighting how the combined treatment of Notch inhibitors and RSV could be prospectively implemented in the novel therapeutic strategies to target Cdk4 for these aggressive brain tumors.

The ongoing war in Syria has led to the displacement of 12 million people since 2011, with minors representing 40% of all refugees. Syrian children living in refugee camps are at risk of developing a wide range of mental health problems, given their previous and ongoing exposure to episodes of violence, disruption of family ties, and discontinuous access to education. In this study, we drew on the salutogenic paradigm to investigate whether, and to what extent, high/low levels of resilience were associated with other indicators of mental health and post-traumatic response in Syrian children living in refugee camps. The sample was composed of 311 Syrian children living in Jordanian refugee camps as a consequence of the war in Syria. We administered quantitative self-report measures to assess participants’ exposure to trauma, individual levels of resilience, and mental health, performing discriminant analysis to examine the association between resilience and trauma/mental health. Syrian children living in Jordanian refugee camps reported intense exposure to traumatic events. The linear discriminant equation supported adoption of the function [Wilk’s Lambda (Λ = 0.827)]: lower levels of resilience were associated with trauma symptoms (re-experiencing, avoidance, and hyperarousal) and emotional problems, while higher levels of resilience were associated with pro-social behaviours. The findings of the present study suggest that resilience acts as a protective factor buffering children from the consequences of trauma and challenging life conditions. We discuss the implications for interventions designed to promote the wellbeing and mental health of children living in refugee camps. Highlights Syrian children living in refugee camps are at risk of developing psychological sequelae. Children with different levels of resilience may display differential patterns of trauma symptoms, emotional problems, and prosocial behaviours. We performed discriminant analysis to verify whether trauma symptoms, emotional adjustment, and prosocial competence distinguished highly resilient children from peers with low resilience. Prosocial behaviours were found to predict membership of the highly resilient cohort, while trauma and emotional distress predicted membership of the low resilience group. Clinical intervention designed to boost social resilience may help to reduce symptoms in children.

Long-read sequencing technologies such as Pacific Biosciences and Oxford Nanopore MinION are capable of producing long sequencing reads with average fragment lengths of over 10,000 base-pairs and maximum lengths reaching 100,000 base- pairs. Compared with short reads, the assemblies obtained from long-read sequencing platforms have much higher contig continuity and genome completeness as long fragments are able to extend paths into problematic or repetitive regions. Many successful assembly applications of the Pacific Biosciences technology have been reported ranging from small bacterial genomes to large plant and animal genomes. Recently, genome assemblies using Oxford Nanopore MinION data have attracted much attention due to the portability and low cost of this novel sequencing instrument. In this paper, we re-sequenced a well characterized genome, the Saccharomyces cerevisiae S288C strain using three different platforms: MinION, PacBio and MiSeq. We present a comprehensive metric comparison of assemblies generated by various pipelines and discuss how the platform associated data characteristics affect the assembly quality. With a given read depth of 31X, the assemblies from both Pacific Biosciences and Oxford Nanopore MinION show excellent continuity and completeness for the 16 nuclear chromosomes, but not for the mitochondrial genome, whose reconstruction still represents a significant challenge.

Background Metabolic disorders are associated with increased incidence, aggressive phenotype and poor outcome of breast cancer (BC) patients. For instance, hyperinsulinemia is an independent risk factor for BC and the insulin/insulin receptor (IR) axis is involved in BC growth and metastasis. Of note, the anti-diabetic metformin may be considered in comprehensive therapeutic approaches in BC on the basis of its antiproliferative effects obtained in diverse pre-clinical and clinical studies. Methods Bioinformatics analysis were performed using the information provided by The Invasive Breast Cancer Cohort of The Cancer Genome Atlas (TCGA) project. The naturally immortalized BC cell line, named BCAHC-1, as well as cancer-associated fibroblasts (CAFs) derived from BC patients were used as model systems. In order to identify further mechanisms that characterize the anticancer action of metformin in BC, we performed gene expression and promoter studies as well as western blotting experiments. Moreover, cell cycle analysis, colony and spheroid formation, actin cytoskeleton reorganization, cell migration and matrigel drops evasion assays were carried out to provide novel insights on the anticancer properties of metformin. Results We first assessed that elevated expression and activation of IR correlate with a worse prognostic outcome in estrogen receptor (ER)-positive BC. Thereafter, we established that metformin inhibits the insulin/IR-mediated activation of transduction pathways, gene changes and proliferative responses in BCAHC-1 cells. Then, we found that metformin interferes with the insulin-induced expression of the metastatic gene CXC chemokine receptor 4 (CXCR4), which we found to be associated with poor disease-free survival in BC patients exhibiting high levels of IR. Next, we ascertained that metformin prevents a motile phenotype of BCAHC-1 cells triggered by the paracrine liaison between tumor cells and CAFs upon insulin activated CXCL12/CXCR4 axis. Conclusions Our findings provide novel mechanistic insights regarding the anti-proliferative and anti-migratory effects of metformin in both BC cells and important components of the tumor microenvironment like CAFs. Further investigations are warranted to corroborate the anticancer action of metformin on the tumor mass toward the assessment of more comprehensive strategies halting BC progression, in particular in patients exhibiting metabolic disorders and altered insulin/IR functions.

Background The incidence of obesity, a known risk factor for several metabolic and chronic diseases, including numerous malignancies, has risen sharply in the world. Various clinical studies demonstrate that excessive Body Mass Index (BMI) may worsen the incidence, prognosis, and mortality rates of breast cancer. Thus, understanding the link tying up obesity and breast cancer onset and progression is critically important, as it can impact patients’ survival and quality of life. Recently, circulating extracellular vesicle (EV) derived miRNAs have attracted much attention for their diagnostic, prognostic and therapeutic potential in oncology research. Although the potential role of EV-derived miRNAs in the early detection of breast cancer has been repeatedly mentioned, screening of miRNAs packaged within serum EVs has not yet been reported in patients with obesity. Methods Circulating EVs were isolated from normal weight (NW), and overweight/obese (OW/Ob) breast cancer patients and characterized by Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA), and protein marker expression. Evaluation of EV-associated miRNAs was conducted in a screening (RNA-seq) and a validation (qRT-PCR) cohort. Bioinformatic analysis was performed to uncover significantly enriched biological processes, molecular functions and pathways. ROC and Kaplain-Meier survival analyses were used for clinical significance. Results Comparison of serum EV-derived miRNAs from NW and OW/Ob patients detected seven differentially expressed miRNAs (let-7a-5p, miR-122-5p, miR-30d-5p, miR-126-3p, miR-27b-3p, miR-4772-3p, and miR-10a-5p) in the screening cohort. GO analysis revealed the enrichment of protein phosphorylation, intracellular signal transduction, signal transduction, and vesicle-mediated transport among the top biological processes. In addition, the target genes were significantly enriched in pathways related to PI3K/Akt, growth hormones, and insulin signalings, which are all involved in obesity-related diseases and/or breast cancer progression. In the validation cohort, qRT-PCR confirmed a significant down-regulation of EV-derived let-7a in the serum of OW/Ob breast cancer patients compared to NW patients. Let-7a levels also exhibited a negative correlation with BMI values. Importantly, decreased let-7a miRNA expression was associated with higher tumor grade and poor survival in patients with breast cancer. Conclusion These results suggest that serum-EV derived miRNAs may reflect a differential profile in relation to a patient’s BMI, which, once validated in larger cohorts of patients, could provide insights into novel specific biomarkers and innovative targets to prevent the progression of obesity-mediated breast cancer.

(1) Background: Patients with hypertension face a relevant reduction in health-related quality of life. Specifically, the vitality domain is significantly impaired. This research aims to explore the association between quality of life and mental distress in patients with hypertension, explicitly emphasizing the mediating factor of social functioning above vitality. (2) Methods: This observational study consecutively recruited a hundred hypertensive patients (49% were males and 51% were females) aged between 23 and 82 years old (Mage = 56.04, SDage = 12.04). The Symptom Checklist-90-Revised (SCL-90-R) and the 36-Item Short Form Health Survey (SF-36) were administered to assess mental distress and quality of life, respectively. Two biological measures (i.e., cortisol levels and heart rate) were also collected. (3) Results: In total, 50% of participants indicated higher mental distress and reduced quality of life. Correlation analyses demonstrate various negative relationships between clinical features. Moreover, positive associations were found between mental distress and vitality and between vitality and physical and social functioning along with heart rate. Notably, it was determined that vitality negatively predicted mental distress directly and indirectly by mediating social functioning. (4) Conclusions: Despite these promising findings, this study’s cross-sectional nature does not allow for the definition of the causal relationship between the investigated variables. These results emphasize the importance of a comprehensive and multidisciplinary evaluation in understanding hypertensive patients’ psychophysical well-being and lifestyles, which social support may significantly modulate.

4,5-Dihydropyrrolo [1,2-a]quinoxalines are interesting druggable scaffolds, with multifaceted biological properties, including anticancer properties targeting the G protein-coupled estrogen receptor 1 (GPER). In this work, the synthesis and preliminary antiproliferative activity of a small set of new 4,5-dihydropyrrolo[1,2-a]quinoxalines (18-20) and pyrrolo[1,2-a]quinoxalines (21, 22) has been reported, inspired by known antiproliferative agents (G-1, G-15, and G-36). The synthesis of the pyrroloquinoxalinic core was employed following the Pictet–Spengler reaction, using the surfactant p-dodecylbenzene sulphonic acid (p-DBSA), as catalyst. It demonstrated efficiency in the catalysis of the 4-phenylpyrrole [1,2-a] quinoxaline type compound formation in mild solvents such as water, ethanol, and hydroalcoholic solutions. In addition, the reactions proceeded in a short time (between 15 and 120 minutes) at room temperature and with high yields. The in vitro MTT assays showed that the presence of isopropyl groups furnished promising antiproliferative compounds. Although, the acetyl group provided also antiproliferative effects, breaking down its responsibility in the GPER transactivation. Nevertheless, it is possible to conclude that the 4,5-dihydropyrrolo[1,2-a]quinoxalines remain a feasible scaffold to develop anticancer agents against GPER-expressing cells.

The present study aimed to verify whether psychopathological symptoms, such as anxiety and depression, among hypertensive patients mediate anger expression and mental health. To this purpose, one hundred hypertensive patients completed the State and Trait Anger Inventory-2, the Symptom Checklist-90-Revised, and the 36-Item Short Form Health Survey. Fifty percent of the participants reported higher levels of psychopathological symptoms and lower levels of quality of life. A correlation analysis described positive associations between most of the scales of anger and psychopathological symptoms, including anxiety and depression. In contrast, mental health was negatively associated with anger expression and trait anger. A serial mediation model revealed that anxiety and depression fully mediated the relationship between anger expression and mental health. Our findings highlight the importance of conducting a multidimensional and multidisciplinary assessment to describe the mental health of hypertensives and prevent their psychological distress through the management of their emotional states. Taking care of the mental health of these patients may help them reduce psychological factors that influence their medical conditions.