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We present an innovative approach allowing the identification, isolation, and molecular characterization of disease-related exosomes based on their different antigenic reactivities. The designed strategy could be immediately translated into any disease in which exosomes are involved. The identification of specific markers and their subsequent association with exosome subtypes, together with the possibility to engineer target-guided exosome-like particles, could represent the key for the effective adoption of exosomes in clinical practice.

Objectives: Major depressive disorder (MDD) is a psychiatric disorder with pathogenesis influenced by both genetic and environmental factors. To date, the molecular-level understanding of its aetiology remains unclear. Thus, we aimed to identify genetic variants and susceptibility genes for MDD with a genome-wide association study (GWAS) approach. Methods: We performed a meta-analysis of GWASs and a gene-based analysis on two Northern Italy isolated populations (cases/controls n = 166/472 and 33/320), followed by replication and polygenic risk score (PRS) analyses in Italian independent samples (cases n = 464, controls n = 339). Results: We identified two novel MDD-associated genes, KCNQ5 (lead SNP rs867262, p = 3.82 × 10-9) and CTNNA2 (rs6729523, p = 1.25 × 10-8). The gene-based analysis revealed another six genes (p < 2.703 × 10-6): GRM7, CTNT4, SNRK, SRGAP3, TRAPPC9, and FHIT. No replication of the genome-wide significant SNPs was found in the independent cohort, even if 14 SNPs around CTNNA2 showed association with MDD and related phenotypes at the nominal level of p (

Objectives Nearly three years into the pandemic, SARS-CoV-2 infections are occurring in vaccinated and naturally infected populations. While humoral and cellular responses in COVID-19 are being characterized, novel immune biomarkers also being identified. Recently, an increase in angiotensin-converting enzyme 2 expressing (aka, ACE2 positive) circulating exosomes (ExoACE2) were identified in the plasma of COVID-19 patients (El-Shennawy et al.). In this pilot study, we describe a method to characterize the exosome-associated microRNA (exo-miRNA) signature in ACE2-positive and ACE2-negative exosomal populations (non-ExoACE2). Methods We performed a sorting protocol using the recombinant biotin-conjugated SARS CoV-2 spike protein containing the receptor binding domain (RBD) on plasma samples from six patients. Following purification, exo-miRNA were characterized for ACE2-positive and ACE2-negative exosome subpopulations by RT-PCR. Results We identified differential expression of several miRNA. Specifically let-7g-5p and hsa-miR-4454+miR-7975 were upregulated, while hsa-miR-208a-3p and has-miR-323-3p were downregulated in ExoACE2 vs. non-ExoACE2. Conclusions The SARS CoV-2 spike-protein guided exosome isolation permits isolation of ExoACE2 exosomes. Such purification facilitates detailed characterization of potential biomarkers (e.g. exo-miRNA) for COVID-19 patients. This method could be used for future studies to further the understanding mechanisms of host response against SARS CoV-2.

Already used for the treatment of some allergic and inflammatory diseases, such as asthma or atopic dermatitis, dupilumab has also been approved as add-on therapy for patients with CRSwNP, and it could represent the keystone to reducing the remission time as well as to improve healing and quality of life. On the other hand, the role of miRNAs as potential biomarkers of immune modulation is emerging. We analyzed the effects of a short-time treatment with dupilumab in patients with CRSwNP, analyzing the immune response modification as well as miRNAs modulations. First, in this early observation stage, all patients experienced remarkable improvement and were clinically stable. Indeed, we observed a significant decrease in CD4+ T cells and a significant reduction in total IgE (p < 0.05) and serum IL-8 levels (p < 0.01), indicating a reduction in the general inflammatory condition. In addition, we analyzed a panel of about 200 circulating miRNAs. After treatment, we noted a significant downregulation of hsa-mir-25-3p (p-value = 0.02415) and hsa-mir-185-5p (p-value = 0.04547), two miRNAs involved in the proliferation, inflammation, and dug-resistance, in accordance with the clinical status of patients. All these preliminary data aimed to identify new biomarkers of prognosis, identifiable with non-invasive procedures for patients. Further, these patients are still under observation, and others with different levels of responsiveness to treatment need to be enrolled to increase the statistical data.

Background: Intracerebral accumulation of amyloid-b in the extracellular plaques of Alzheimer’s disease (AD) brains represents the main cause of reactive astrogliosis and neuroinflammatory response. Of relevance, Leucine-Rich-Repeat Kinase 2 (LRRK2), a kinase linked to genetic and sporadic Parkinson’s disease (PD), has been identified as a positive mediator of neuroinflammation upon different inflammatory stimuli, however its pathogenicity in AD remains mainly unexplored. Methods: In this study, by using pharmacological inhibition of LRRK2 and murine primary astrocytes, we explored whether LRRK2 regulates astrocytic activation in response to amyloid-b1-42 (Ab1-42) fibrils. Results: Our results show that primary astrocytes become activated and recruit LRRK2 upon Ab1-42 fibrils exposure. Moreover, we found that pharmacological inhibition of LRRK2, with two different kinase inhibitors, can attenuate Ab1-42-mediated inflammation and favor the clearance of Ab1-42 fibrils in astrocytes. Conclusions: Overall, our findings report that LRRK2 kinase activity modulates astrocytic reactivity and functions in the presence of Ab1-42 deposits and indicate that PD-linked LRRK2 might contribute to AD-related neuroinflammation and pathogenesis.

Purpose: Thoracic aortic aneurysm and dissection (TAAD) is a life-threatening disease with often unrecognized inherited forms. We sought to identify novel pathogenic variants associated with autosomal dominant inheritance of TAAD. Methods: We analyzed exome sequencing data from 35 French TAAD families and performed next-generation sequencing capture panel of genes in 1114 unrelated TAAD patients. Functional effects of pathogenic variants identified were validated in cell, tissue, and mouse models. Results: We identified five functional variants in THSD4 of which two heterozygous variants lead to a premature termination codon. THSD4 encodes ADAMTSL6 (member of the ADAMTS/L superfamily), a microfibril-associated protein that promotes fibrillin-1 matrix assembly. The THSD4 variants studied lead to haploinsufficiency or impaired assembly of fibrillin-1 microfibrils. Thsd4+/− mice showed progressive dilation of the thoracic aorta. Histologic examination of aortic samples from a patient carrying a THSD4 variant and from Thsd4+/− mice, revealed typical medial degeneration and diffuse disruption of extracellular matrix. Conclusion: These findings highlight the role of ADAMTSL6 in aortic physiology and TAAD pathogenesis. They will improve TAAD management and help develop new targeted therapies.

Chronic venous disease is a condition globally widespread, resulting in a disabling pathological disorder. The CD4 + Th17+ (Cluster Differentiation 4) lymphocytes represent a regulative factor for innate immunity related to the development of complex diseases. Recently, these mechanisms have been associated with vascular disease. The aim of this work is to validate whether the Th17 response correlates with the development of CVI (Chronic venous insufficiency)and CVLUs (chronic venous limbs ulcers) and whether Th17 markers can be used, both as intrinsic risk factors and diagnostic markers, for disease development. PBL derived from peripheral blood samples of patients and controls were subjected to gene expression analysis for IL23R, IL17, SGK1, TGFβ, RORγ, FOXO1, and RANBP1 by qRT-PCR and immunoblot. A post hoc correlation, the diagnostic performance of the target genes, and multivariable analyses were properly conducted. The main expression markers of the CD4 + Th17+ switch were strongly activated in chronic venous insufficiency and in advanced ulceration. The correlation analysis demonstrated the inter-dependence on Th17’s signature modulation. ROC (Receiver Operating Characteristic) analysis defined, for the examined genes, a clinical value as the potential diagnostic markers. Multi-logistic regression studies showed that Th17 markers behave as empirical risk factors for CVD (chronic venous disease) development. Taken together, the present data provide a new hypothesis for the TH17-dependent pathogenesis of CVD, favoring the possibility for the development of new diagnostic, preventive, and therapeutic approaches.

Background Major depressive disorder (MDD) is a common, chronic, debilitating mood disorder that causes serious functional impairment and significantly decreased quality of life. Pharmacotherapy represents the first-line treatment option; however, only approximately one third of patients respond to the first treatment because of the ineffectiveness or side effects of antidepressants. Precision medicine in psychiatry might offer clinicians the possibility to tailor treatment according to the best possible evidence of efficacy and tolerability for each subject. In this context, our study aims to carry out a clinical validation of a combinatorial pharmacogenomics (PGx) test in an Italian MDD patient cohort with advocacy license independence. Methods Our study is a prospective participant- and rater-blinded, randomized, controlled clinical observational trial enrolling 300 MDD patients who are referred to psychiatric services to receive a new antidepressant due to the failure of their current treatment and/or the onset of adverse effects. Eligible participants are randomized to the TGTG group (Treated with Genetic Test Guide) or TAU group (Treated as Usual). For all subjects, DNA is collected with a buccal brush. The primary outcome is the reduction in depressive symptomatology. The secondary outcomes involve a range of scales that assess MDD symptoms and social functioning outcomes. The assessment is performed at four timepoints: baseline and 4, 8, and 12 weeks. Discussion This project represents the first randomized controlled clinical trial to investigate whether a non-commercial PGx test improves outcomes in an MDD naturalistic cohort. Moreover, the identification of new genetic variants associated with non-response or side effects will improve the efficacy of the test, leading to further cost-saving. Trial registration number ClinicalTrials.gov NCT04615234. Registered on November 4, 2020.

Achondrogenesis type II (ACG2) is a lethal skeletal dysplasia caused by dominant pathogenic variants in COL2A1. Most of the variants found in patients with ACG2 affect the glycine residue included in the Gly-X-Y tripeptide repeat that characterizes the type II collagen helix. In this study, we reported a case of a novel splicing variant of COL2A1 in a fetus with ACG2. An NGS analysis of fetal DNA revealed a heterozygous variant c.1267-2_1269del located in intron 20/exon 21. The variant occurred de novo since it was not detected in DNA from the blood samples of parents. We generated an appropriate minigene construct to study the effect of the variant detected. The minigene expression resulted in the synthesis of a COL2A1 messenger RNA lacking exon 21, which generated a predicted in-frame deleted protein. Usually, in-frame deletion variants of COL2A1 cause a phenotype such as Kniest dysplasia, which is milder than ACG2. Therefore, we propose that the size and position of an in-frame deletion in COL2A1 may be relevant in determining the phenotype of skeletal dysplasia.

Achondrogenesis type II (ACG2) is a lethal skeletal dysplasia caused by dominant pathogenic variants in COL2A1. Most of the variants found in patients with ACG2 affect the glycine residue included in the Gly-X-Y tripeptide repeat that characterizes the type II collagen helix. In this study, we reported a case of a novel splicing variant of COL2A1 in a fetus with ACG2. An NGS analysis of fetal DNA revealed a heterozygous variant c.1267-2_1269del located in intron 20/exon 21. The variant occurred de novo since it was not detected in DNA from the blood samples of parents. We generated an appropriate minigene construct to study the effect of the variant detected. The minigene expression resulted in the synthesis of a COL2A1 messenger RNA lacking exon 21, which generated a predicted in-frame deleted protein. Usually, in-frame deletion variants of COL2A1 cause a phenotype such as Kniest dysplasia, which is milder than ACG2. Therefore, we propose that the size and position of an in-frame deletion in COL2A1 may be relevant in determining the phenotype of skeletal dysplasia.

The immunoglobulin B cell receptor (IgBCR) expressed by chronic lymphocytic leukemia (CLL) B cells plays a pivotal role in tumorigenesis, supporting neoplastic transformation, survival, and expansion of tumor clones. We demonstrated that in the same patient, two or more CLL clones could coexist, recognized by the expression of different variable regions of the heavy chain of IgBCR, composing the antigen-binding site. In this regard, phage display screening could be considered the easier and most advantageous methodology for the identification of small peptide molecules able to mimic the natural antigen of the tumor IgBCRs. These molecules, properly functionalized, could be used as a probe to specifically identify and isolate single CLL subpopulations, for a deeper analysis in terms of drug resistance, phenotype, and gene expression. Furthermore, CLL cells express another surface membrane receptor, the CD5, which is commonly expressed by normal T cells. Piece of evidence supports a possible contribution of CD5 to the selection and maintenance of autoreactivity in B cells and the constitutive expression of CD5 on CLL cells could induce pro-survival stimuli. In this brief research report, we describe a peptide-based single-cell sorting using as bait the IgBCR of tumor cells; in the next step, we performed a quantitative analysis of CD5 expression by qRT-PCR related to the expressed IgBCR. Our approach could open a new perspective for the identification, isolation, and investigation of all subsets of IgBCR-related CLL clones, with particular attention to the more aggressive clones.

Ovarian cancer is the second most common gynecological malignancy worldwide. Paclitaxel is particularly important in the therapy of ovarian carcinomas, but the treatment efficacy is counteracted by the development of resistance to chemotherapy. The identification of target molecules that can prevent or control the development of chemoresistance might provide important tools for the management of patients affected by ovarian cancer. Serum- and glucocorticoid-regulated kinase 1 (SGK1) appears to be a key determinant of resistance to chemo- and radiotherapy. Specifically, SGK1 affects paclitaxel sensitivity in RKO colon carcinoma cells by modulating the specificity protein 1 (SP1)–dependent expression of Ran-specific GTPase-activating protein (RANBP1), a member of the GTP-binding nuclear protein Ran (RAN) network that is required for the organization and function of the mitotic spindle. SGK1 inhibition might thus be useful for counteracting the development of paclitaxel resistance. Here, we present in vitro data obtained using ovarian carcinoma cell lines that indicate that the SGK1 inhibitor SI113 inhibits cancer cell proliferation, potentiates the effects of paclitaxel-based chemotherapy, counteracts the development of paclitaxel resistance, and restores paclitaxel sensitivity in paclitaxel-resistant A2780 ovarian cancer cells. The results were corroborated by preclinical studies of xenografts generated in nude mice through the implantation of paclitaxel-resistant human ovarian cancer cells. The SGK1 inhibitor SI113 synergizes with paclitaxel in the treatment of xenografted ovarian cancer cells. Taken together, these data suggest that SGK1 inhibition should be investigated in clinical trials for the treatment of paclitaxel-resistant ovarian cancer.

Chronic Lymphocytic Leukemia (CLL) is a heterogeneous disease characterized by variable clinical courses among different patients. This notion was supported by the possible coexistence of two or more independent CLL clones within the same patients, identified by the characterization of the B cell receptor immunoglobulin (BcR IG) idiotypic sequence. By using the antigen-binding site of the BcR IG as bait, the identification and isolation of aggressive and drug-resistance leukemic B-cell clones could allow a deeper biological and molecular investigation. Indeed, by the screening of phage display libraries, we previously selected a peptide binder of the idiotypic region of CLL BCR IGs expressing the unmutated rearrangement IGHV1-69 and used it as a probe to perform a peptide-based cell sorting by flow cytometry in peripheral blood samples from patients with CLL. Since the IGHV1-69 clones persisted during the follow-up time in both patients, we explored the possibility of these clones having acquired an evolutive advantage compared to the other coexisting clones in terms of a higher expression of genes involved in the survival and apoptosis escape processes. To this end, we studied the expression patterns of a panel of genes involved in apoptosis regulation and in NF-kB-dependent pro-survival signals by comparative qRT-PCR assays. According to the results, IGHV1-69 clones showed a higher expression of pro-survival and anti-apoptotic genes as compared to the other CLL clones with different immunogenetic characteristics. Moreover, these IGHV1-69 clones did not carry any characteristic genetic lesions, indicating the relevance of our approach in performing a comprehensive molecular characterization of single tumor clones, as well as for designing new personalized therapeutic approaches for the most aggressive and persistent tumor clones.

Major depressive disorder (MDD) is a heterogeneous psychiatric disease characterized by persistent low mood, diminished interests, and impaired cognitive and social functions. The multifactorial etiology of MDD is still largely unknown because of the complex genetic and environmental interactions involved. Therefore, no established mechanism can explain all the aspects of the disease. In this light, an extensive research about the pathophysiology of MDD has been carried out. Several pathogenic hypotheses, such as monoamines deficiency and neurobiological alterations in the stress-responsive system, including the hypothalamic-pituitary-adrenal (HPA) axis and the immune system, have been proposed for MDD. Over time, remarkable studies, mainly on preclinical rodent models, linked the serum- and glucocorticoid-regulated kinase 1 (SGK1) to the main features of MDD. SGK1 is a serine/threonine kinase belonging to the AGK Kinase family. SGK1 is ubiquitously expressed, which plays a pivotal role in the hormonal regulation of several ion channels, carriers, pumps, and transcription factors or regulators. SGK1 expression is modulated by cell stress and hormones, including gluco- and mineralocorticoids. Compelling evidence suggests that increased SGK1 expression or function is related to the pathogenic stress hypothesis of major depression. Therefore, the first part of the present review highlights the putative role of SGK1 as a critical mediator in the dysregulation of the HPA axis, observed under chronic stress conditions, and its controversial role in the neuroinflammation as well. The second part depicts the negative regulation exerted by SGK1 in the expression of both the brain-derived neurotrophic factor (BDNF) and the vascular endothelial growth factor (VEGF), resulting in an anti-neurogenic activity. Finally, the review focuses on the antidepressant-like effects of anti-oxidative nutraceuticals in several preclinical model of depression, resulting from the restoration of the physiological expression and/or activity of SGK1, which leads to an increase in neurogenesis. In summary, the purpose of this review is a systematic analysis of literature depicting SGK1 as molecular junction of the complex mechanisms underlying the MDD in an effort to suggest the kinase as a potential biomarker and strategic target in modern molecular antidepressant therapy.

The sensitivity of interferometric gravitational wave detectors is optimized, in part, by balanced finesse in the long Fabry&ndash, Perot arm cavities. The input test mass mirrors of Advanced Virgo feature parallel faces, which creates an etalon within the substrate, adding variability in the total mirror reflectivity, in order to correct imbalanced finesse due to manufacturing tolerances. Temperature variations in mirror substrate change the optical path length primarily through varying the index of refraction and are tuned to correct for a finesse imbalance of up to 2.8% by a full etalon fringe of 0.257 K. A negative feedback control system was designed to control the mirror temperature by using an electrical resistive heating belt actuator for a heat transfer process modeled as a two-pole plant. A zero controller filter was designed which achieves temperature control within 2.3% of the etalon fringe and recovers to within 10% of the working point within 32 hours after a step input of one etalon fringe. A preliminary unlock condition control designed to compensate when the interferometer unlocks shows that the control remains stable even after a drastic change in the plant due to the absence of the laser heating. Further improvements to the control must also consider the full heat transfer mechanisms by using modern control state space models.

Current knowledge on the role of exosomal microRNA (miRNA) in canine leishmaniasis (CL), with particular regards to the interaction between miR-122 and lipid alterations, is limited. The aim of this study was to isolate/characterize exosomes in canine serum and evaluate the expression of miR-122 in ten healthy and ten leishmaniotic dogs. Serum exosomes were isolated using a polymer-based kit, ExoQuick&reg, and characterized by flow cytometry and transmission electron microscopy, whereas miR-122-5p expression was evaluated by quantitative reverse-transcriptase polymerase chain reaction. A significant decreased expression of exosomal miR-122-5p, decreased serum levels of high-density lipoproteins, and increased serum levels of low-density lipoproteins were seen in leishmaniotic dogs when compared with healthy dogs. These results suggest that hepatic dysfunctions induced by the parasite interfere with lipoprotein status. The decreased expression of exosomal miR122 represents an additional effect of Leishmania infection in dogs as in people.

Canine atopic dermatitis (AD) is a very common inflammatory skin disease, but limited data are available on the genetic characterization (somatic mutations, microarrays, and genome-wide association study (GWAS)) of skin lesions in affected dogs. microRNAs are good biomarkers in inflammatory and neoplastic diseases in people. The aim of this study was to evaluate microRNA expression in the skin of atopic beagles, before and after exposure to Dermatophagoides farinae. Four atopic and four unrelated age-matched healthy beagle dogs were enrolled. Total RNA was extracted from flash-frozen skin biopsies of healthy and atopic dogs. For the atopic dogs, skin biopsies were taken from non-lesional (day 0) and lesional skin (day 28 of weekly environmental challenge with Dermatophagoides farinae). Small RNA libraries were constructed and sequenced. The microRNA sequences were aligned to CanFam3.1 genome. Differential expressed microRNAs were selected on the basis of fold-change and statistical significance (fold-change ≥ 1.5 and p ≤ 0.05 as thresholds. A total of 277 microRNAs were sequenced. One hundred and twenty-one differentially regulated microRNAs were identified between non-lesional and healthy skin. Among these, two were increased amount and 119 were decreased amount. A total of 45 differentially regulated microRNAs between lesional and healthy skin were identified, 44 were decreased amount and one was increased amount. Finally, only two increased amount microRNAs were present in lesional skin when compared with that of non-lesional skin. This is the first study in which dysregulation of microRNAs has been associated with lesional and non-lesional canine AD. Larger studies are needed to understand the role of microRNA in canine AD.

BACKGROUND Inflammatory bowel disease (IBD) is due to the interaction of genetic and environmental factors that trigger an unbalanced immune response ultimately resulting in the peculiar inflammatory reaction. Experimental models of IBD point to a role of T-cell-derived cytokines (Th17) and to SGK1 as mediator of the Th17 switch. We hypothesize that SGK1, a salt inducible kinase, directs lymphocytic behavior and tissue damage. METHODS Eleven controls and 32 ulcerative colitis (UC) patients were randomized according to endoscopic Mayo score. Mucosal biopsies from different intestinal tracts were analyzed by immunohistochemistry and quantitative real-time polymerase chain reaction to check the expression of disease markers including SGK1. Peripheral blood mononuclear cells (PBMCs) from patients and controls were analyzed by fluorescence-activated cell sorting. Finally, an in vitro cell model was developed to test the hypothesis. RESULTS SGK1 mRNA and protein expression in lesional areas of UC patients were lower than in normal peri-lesional areas of the same patients and in normal tissues of healthy controls. SGK1 expression was increased in PBMCs from UC patients, particularly in the CD4+ cell population, enriched in Th17 cells. IL17/IL13 was increased in patients and correlated with SGK1 expression. Genetically engineered Jurkat cells confirmed the effect of SGK1 overexpression on viability of RKO cells. CONCLUSIONS These observations suggest a pathogenic mechanism whereby SGK1 overexpression in CD4+ T cells induces the secretion of the inflammatory cytokines IL17 and IL13, which downregulate the expression of SGK1 in target tissues. Our data suggest a novel hypothesis in the pathogenesis of UC, integrating colonic epithelial cells and lymphocytes.

Pseudoxanthoma elasticum is an autosomal recessive heritable disorder caused by mutations in ABCC6. We describe two siblings showing typical skin lesions and a clinical diagnosis of pseudoxanthoma elasticum. Genetic analysis of ABCC6 revealed a novel homozygous c.4041G > A variant located in the last position of exon 28 that compromises the splicing donor site, resulting in a shorter messenger RNA. The deletion impairs the nucleotide-binding fold region, which is crucial for ABCC6 function.

Glioblastoma multiforme (GBM) is the most aggressive CNS tumor and is characterized by a very high frequency of clinical relapse after therapy and thus by a dismal prognosis, which strongly compromises patients survival. We have recently identified the small molecule SI113, as a potent and selective inhibitor of SGK1, a serine/threonine protein kinase, that modulates several oncogenic signaling cascades. The SI113-dependent SGK1 inhibition induces cell death, blocks proliferation and perturbs cell cycle progression by modulating SGK1-related substrates. SI113 is also able to strongly and consistently block, in vitro and in vivo, growth and survival of human hepatocellular-carcinomas, either used as a single agent or in combination with ionizing radiations. In the present paper we aim to study the effect of SI113 on human GBM cell lines with variable p53 expression. Cell viability, cell death, caspase activation and cell cycle progression were then analyzed by FACS and WB-based assays, after exposure to SI113, with or without oxidative stress and ionizing radiations. Moreover, autophagy and related reticulum stress response were evaluated. We show here, that i) SGK1 is over-expressed in highly malignant gliomas and that the treatment with SI113 leads to ii) significant increase in caspase-mediated apoptotic cell death in GBM cell lines but not in normal fibroblasts; iii)enhancement of the effects of ionizing radiations; iv) modulation of the response to oxidative reticulum stress; v) induction of cytotoxic autophagy. Evidence reported here underlines the therapeutic potential of SI113 in GBM, suggesting a new therapeutic strategy either alone or in combination with radiotherapy.

Resveratrol (trans-3,4’,5-trihydroxystilbene) is a polyphenolic natural product with a well-known polypharmacological profile that places it among the multi-target-directed ligands (MTDLs). Given its protective action against a wide number of chronic diseases, in this review, we introduce a general overview about the cardioprotective and antioxidant effects, the antidiabetic, neuroprotective and anti-inflammatory effects of this polyphenol. In the second part of the manuscript, we focused our attention on the anticancer activity of Resveratrol, given the alteration of many different signaling pathways, leading to suppression of tumor cell proliferation in numerous cancer types. Among the several anticancer targets involved in the mechanism of action of Resveratrol, here we introduce experimental and molecular modeling studies performed against the SGK1 protein as a novel anticancer target of Resveratrol. SGK1 inhibitors have been demonstrated to inhibit cell growth of different cancer cells. We demonstrated that resveratrol inhibits SGK1 in vitro and in intact cells, affecting proliferation and survival of HUH7 human hepatoma cells. Our findings demonstrate that resveratrol may function as a SGK1 inhibitor, suggesting possible applications in sodium retention and cancer.

Among the proteins putatively interacting with PTPRJ, a tyrosine phosphatase with tumor suppressor activity, we focused on the oncoprotein CD98hc as a very interesting candidate for the development of innovative targeted drugs. In fact, CD98hc, representing the heavy chain of a transmembrane aminoacid transporter, is overexpressed in several human cancers. Furthermore, CD98hc higher expression is associated with poor prognosis in lung cancer patients. CD98hc is linked to light chains (LATs, xCT) by disulfide bridge, polar and hydrophobic interactions. The light chain confers substrate specificity, and ERK, AKT, FAK and mTOR pathways are involved in CD98hc-LATs/xCT downstream signals. Moreover, CD98hc is a coreceptor of b-integrins and it is involved in cell proliferation, migration and invasion. We identified two peptides and 15 small molecules (putatively targeting the disulfide bridge) as candidate CD98hc inhibitors by phage display and in silico screenings, respectively, and validated in vitro the binding between peptides and transmembrane fraction of CD98hc by cytofluorimetric assay. Then, we tested the capability of both types of compounds to affect cell viability and proliferation through MTT and CFSE assays, respectively. We observed that the targeting of CD98hc through both peptides and small molecules reduced cell proliferation in A549 human lung cancer cells, strongly encouraging a deeper characterization of these candidate anticancer molecules. Indeed, our next goals will be the assessment of biochemical activity of CD98hc following to its inhibition, as well as the evaluation of compounds toxicity, both in vitro and in vivo. The final aim is to identify lead compounds inhibiting CD98hc, in order to develop novel molecules to be translated in the clinical setting and to be used as monotherapy and/or in a combinatorial approaches for the treatment of cancer patients. Citation Format: Delia Lanzillotta, Enrico Iaccino, Anna Artese, Selena Mimmi, Sabrina D'Agostino, Isabella Romeo, Patrizia Cantafio, Vincenzo Dattilo, Giosuè Costa, Carolina Brescia, Eugenio Gaudio, Stefano Alcaro, Francesco Trapasso. The CD98hc oncoprotein as a target of new anticancer therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B122. doi:10.1158/1535-7163.TARG-19-B122

PTPRJ, a receptor protein tyrosine phosphatase strongly downregulated in human cancer, displays tumor suppressor activity by negatively modulating several proteins involved in proliferating signals. Here, through a proteomic-based approach, we identified a list of potential PTPRJ-interacting proteins and among them we focused on CD98hc, a type II glycosylated integral membrane protein encoded by SLC3A2, corresponding to the heavy chain of a heterodimeric transmembrane amino-acid transporter, including LAT1. CD98hc is widely overexpressed in several types of cancers and contributes to the process of tumorigenesis by interfering with cell proliferation, adhesion, and migration. We first validated PTPRJ-CD98hc interaction, then demonstrated that PTPRJ overexpression dramatically reduces CD98hc protein levels in A549 lung cancer cells. In addition, following to the treatment of PTPRJ-transduced cells with MG132, a proteasome inhibitor, CD98hc levels did not decrease compared to controls, indicating that PTPRJ is involved in the regulation of CD98hc proteasomal degradation. Moreover, PTPRJ overexpression combined with CD98hc silencing consistently reduced cell proliferation and triggered apoptosis of lung cancer cells. Interestingly, by interrogating the can Evolve database, we observed an inverse correlation between PTPRJ and SLC3A2 gene expression. Indeed, the non-small cell lung cancers (NSCLCs) of patients showing a short survival rate express the lowest and the highest levels of PTPRJ and SLC3A2, respectively. Therefore, the results reported here contribute to shed lights on PTPRJ signaling in cancer cells: moreover, our findings also support the development of a novel anticancer therapeutic approach by targeting the pathway of PTPRJ that is usually downregulated in highly malignant human neoplasias.

Glioblastoma multiforme (GBM) is the deadliest brain tumor. State-of-art GBM therapy often fails to ensure control of a disease characterized by high frequency of recurrences and progression. In search for novel therapeutic approaches, we assayed the effect of compounds from a cancer drug library on the ADF GBM cell line, establishing their elevated sensitivity to mitotic spindle poisons. Our previous work showed that the effectiveness of the spindle poison paclitaxel in inhibiting cancer cell growth was dependent on the expression of RANBP1, a regulatory target of the serine/threonine kinase SGK1. Recently, we developed the small molecule SI113 to inhibit SGK1 activity. Therefore, we explored the outcome of the association between SI113 and selected spindle poisons, finding that these drugs generated a synergistic cytotoxic effect in GBM cells, drastically reducing their viability and clonogenic capabilities in vitro, as well as inhibiting tumor growth in vivo. We also defined the molecular bases of such a synergistic effect. Because SI113 displays low systemic toxicity, yet strong activity in potentiating the effect of radiotherapy in GBM cells, we believe that this drug could be a strong candidate for clinical trials, with the aim to add it to the current GBM therapeutic approaches.

Background/Aims: The importance of copper in the metabolism of cancer cells has been widely studied in the last 20 years and a clear-cut association between copper levels and cancer deregulation has been established. Copper-64, emitting positrons and β-radiations, is indicated for the labeling of a large number of molecules suitable for radionuclide imaging as well as radionuclide therapy. Glioblastoma multiforme (GBM) is the CNS tumor with the worse prognosis, characterized by high number of recurrences and strong resistance to chemo-radio therapy, strongly affecting patients survival. We have recently discovered and studied the small molecule SI113, as inhibitor of SGK1, a serine/threonine protein kinase, that affects several neoplastic phenotypes and signaling cascades. The SI113-dependent SGK1 inhibition induces cell death, blocks proliferation, perturbs cell cycle progression and restores chemo-radio sensibility by modulating SGK1-related substrates. In the present paper we aim to characterize the combined effects of 64CuCl2 and SI113 on human GBM cell lines with variable p53 expression. Methods: Cell viability, cell death and stress/authopagic related pathways were then analyzed by FACS and WB-based assays, after exposure to SI113 and/or 64CuCl2. Results: We demonstrate here, that i) 64CuCl2 is able to induce a time and dose dependent modulation of cell viability (with different IC50 values) in highly malignant gliomas and that the co-treatment with SI113 leads to ii) additive/synergistic effects in terms of cell death; iii) enhancement of the effects of ionizing radiations, probably by a TRC1 modulation; iv) modulation of the autophagic response. Conclusions: Evidence reported here underlines the therapeutic potential of the combined treatment with SI113 and 64CuCl2 in GBM cells.

// Anna Bilotta 1 , Vincenzo Dattilo 2 , Sabrina D'Agostino 1 , Stefania Belviso 1 , Stefania Scalise 1 , Mariaconcetta Bilotta 1 , Eugenio Gaudio 1, 3 , Francesco Paduano 1, 4 , Nicola Perrotti 2 , Tullio Florio 5 , Alfredo Fusco 6 , Rodolfo Iuliano 1 , Francesco Trapasso 1 1 Department of Medicina Sperimentale e Clinica, University Magna Graecia of Catanzaro, Catanzaro, Italy 2 Department of Scienze della Salute, University Magna Graecia of Catanzaro, Catanzaro, Italy 3 Lymphoma and Genomics Research Program, Institute of Oncology Research (IOR), Bellinzona, Switzerland 4 Tecnologica Research Institute, Biomedical Section, Crotone, Italy 5 Laboratory of Pharmacology, Dept. of Internal Medicine, and Center of Excellence for Biomedical Research (CEBR), University of Genova, Genova, Italy 6 Istituto di Endocrinologia e Oncologia Sperimentale - CNR c/o Dipartimento di Medicina Molecolare e Biotecnologie Mediche, University Federico II of Napoli, Napoli, Italy Correspondence to: Rodolfo Iuliano, email: iuliano@unicz.it Francesco Trapasso, email: trapasso@unicz.it Keywords: protein tyrosine phosphatase, soluble isoform, angiogenesis, glioblastoma, angiogenic factor Received: September 20, 2016 Accepted: December 13, 2016 Published: December 29, 2016 ABSTRACT PTPRJ is a receptor protein tyrosine phosphatase with tumor suppressor activity. Very little is known about the role of PTPRJ ectodomain, although recently both physiological and synthetic PTPRJ ligands have been identified. A putative shorter spliced variant, coding for a 539 aa protein corresponding to the extracellular N-terminus of PTPRJ, is reported in several databases but, currently, no further information is available. Here, we confirmed that the PTPRJ short isoform (named sPTPRJ) is a soluble protein secreted into the supernatant of both endothelial and tumor cells. Like PTPRJ, also sPTPRJ undergoes post-translational modifications such as glycosylation, as assessed by sPTPRJ immunoprecipitation. To characterize its functional activity, we performed an endothelial cell tube formation assay and a wound healing assay on HUVEC cells overexpressing sPTPRJ and we found that sPTPRJ has a proangiogenic activity. We also showed that sPTPRJ expression down-regulates endothelial adhesion molecules, that is a hallmark of proangiogenic activity. Moreover, sPTPRJ mRNA levels in human high-grade glioma, one of the most angiogenic tumors, are higher in tumor samples compared to controls. Further studies will be helpful not only to clarify the way sPTPRJ works but also to supply clues to circumvent its activity in cancer therapy.

The serum- and glucocorticoid-regulated kinase (SGK1) controls cell transformation and tumor progression. SGK1 affects mitotic stability by regulating the expression of RANBP1/RAN. Here, we demonstrate that SGK1 fluctuations indirectly modify the maturation of pre-miRNAs, by modulating the equilibrium of the RAN/RANBP1/RANGAP1 axis, the main regulator of nucleo-cytoplasmic transport. The levels of pre-miRNAs and mature miRNAs were assessed by qRT-PCR, in total extracts and after differential nuclear/cytoplasmic extraction. RANBP1 expression is the limiting step in the regulation of SGK1-SP1 dependent nuclear export. These results were validated in unrelated tumor models and primary human fibroblasts and corroborated in tumor-engrafted nude mice. The levels of pri-miRNAs, DROSHA, DICER and the compartmental distribution of XPO5 were documented. Experiments using RANGTP conformational antibodies confirmed that SGK1, through RANBP1, decreases the level of the GTP-bound state of RAN. This novel mechanism may play a role in the epigenomic regulation of cell physiology and fate.

Tumor-derived exosomes (TDEs) play a pivotal role in tumor establishment and progression, and are emerging biomarkers for tumor diagnosis in personalized medicine. To date, there is a lack of efficient technology platforms for exosome isolation and characterization. Multiple myeloma (MM) is an incurable B-cell malignancy due to the rapid development of drug-resistance. MM-released exosomes express the immunoglobulin B-cell receptor (Ig-BCR) of the tumor B-cells, which can be targeted by Idiotype-binding peptides (Id-peptides). In this study, we analyzed the production of MM-released exosomes in the murine 5T33MM multiple myeloma model as biomarkers of tumor growth. To this end, we selected Id-peptides by screening a phage display library using as bait the Ig-BCR expressed by 5T33MM cells. By FACS, the FITC-conjugated Id-peptides detected the MM-released exosomes in the serum of 5T33MM-engrafted mice, levels of which are correlated with tumor progression at an earlier time point compared to serum paraprotein. These results indicate that Id-peptide-based recognition of MM-released exosomes may represent a very sensitive diagnostic approach for clinical evaluation of disease progression. Electronic supplementary material The online version of this article (10.1186/s12943-017-0730-8) contains supplementary material, which is available to authorized users.

The mammalian target of rapamycin (mTOR) pathway has been recently indicated as a suitable drug target for the prevention of epileptogenesis. The mTOR pathway is known for its involvement in the control of the immune system. Since neuroinflammation is recognized as a major contributor to epileptogenesis, we wished to examine whether the neuroprotective effects of mTOR modulation could involve a suppression of the neuroinflammatory process in epileptic brain. We have investigated the early molecular mechanisms involved in the effects of intracerebral administration of the lipopolysaccharide (LPS) in the WAG/Rij rat model of absence epilepsy, in relation to seizure generation and depressive-like behavior; we also tested whether the effects of LPS could be modulated by treatment with rapamycin (RAP), a specific mTOR inhibitor. We determined, in specific rat brain areas, levels of p-mTOR/p-p70S6K and also p-AKT/p-AMPK as downstream or upstream indicators of mTOR activity and tested the effects of LPS and RAP co-administration. Changes in the brain levels of pro-inflammatory cytokines IL-1β and TNF-α and their relative mRNA expression levels were measured, and the involvement of nuclear factor-κB (NF-κB) was also examined in vitro. We confirmed that RAP inhibits the aggravation of absence seizures and depressive-like/sickness behavior induced by LPS in the WAG/Rij rats through the activation of mTOR and show that this effect is correlated with the ability of RAP to dampen and delay LPS increases in neuroinflammatory cytokines IL-1β and TNF-α, most likely through inhibition of the activation of NF-κB. Our results suggest that such a mechanism could contribute to the antiseizure, antiepileptogenic and behavioral effects of RAP and further highlight the potential therapeutic usefulness of mTOR inhibition in the management of human epilepsy and other neurological disorders. Furthermore, we show that LPS-dependent neuroinflammatory effects are also mediated by a complex interplay between AKT, AMPK and mTOR with specificity to selective brain areas. In conclusion, neuroinflammation appears to be a highly coordinated phenomenon, where timing of intervention may be carefully evaluated in order to identify the best suitable target.

The serum- and glucocorticoid-regulated kinase (SGK) family consists of three members, SGK1, SGK2 and SGK3, all displaying serine/threonine kinase activity and sharing structural and functional similarities with the AKT family of kinases. SGK1 was originally described as a key enzyme in the hormonal regulation of several ion channels and pumps. Over time, growing and impressive evidence has been accumulated, linking SGK1 to the cell survival, de-differentiation, cell cycle control, regulation of caspases, response to chemical, mechanical and oxidative injury in cancer models as well as to the control of mitotic stability. Much evidence shows that SGK1 is over-expressed in a variety of epithelial tumors. More recently, many contributions to the published literature demonstrate that SGK1 can mediate chemo-and radio-resistance during the treatment of various human tumors, both in vitro and in vivo. SGK1 appears therefore as a dirty player in the stress response to chemical and radio-agents, responsible of a selective advantage that favors the uncontrolled tumor progression and the selection of the most aggressive clones. The purpose of this review is the analysis of the literature describing SGK1 as central node of the cell resistance, and a summary of the possible strategies in the pharmacological targeting of SGK1.

Expression of PTPRJ, which is a ubiquitous receptor-type protein tyrosine phosphatase, is significantly reduced in a vast majority of human epithelial cancers and cancer cell lines (i.e. colon, lung, thyroid, mammary and pancreatic tumours). A possible role for microRNAs (miRNAs) in the negative regulation of PTPRJ expression has never been investigated. In this study, we show that overexpression of microRNA-328 (miR-328) decreases PTPRJ expression in HeLa and SKBr3 cells. Further investigations demonstrate that miR-328 acts directly on the 3′UTR of PTPRJ, resulting in reduced mRNA levels. Luciferase assay and site-specific mutagenesis were used to identify a functional miRNA response element in the 3′UTR of PTPRJ. Expression of miR-328 significantly enhances cell proliferation in HeLa and SKBr3 cells, similar to the effects of downregulation of PTPRJ with small interfering RNA. Additionally, in HeLa cells, the proliferative effect of miR-328 was not observed when PTPRJ was silenced with small interfering RNA; conversely, restoration of PTPRJ expression in miR-328-overexpressing cells abolished the proliferative activity of miR-328. In conclusion, we report the identification of miR-328 as an important player in the regulation of PTPRJ expression, and we propose that the interaction of miR-328 with PTPRJ is responsible for miR-328-dependent increase of epithelial cell proliferation.

// Cristina Talarico 1, * , Lucia D’Antona 1, * , Domenica Scumaci 2 , Agnese Barone 2 , Francesco Gigliotti 1 , Claudia Vincenza Fiumara 2 , Vincenzo Dattilo 1 , Enzo Gallo 3 , Paolo Visca 3 , Francesco Ortuso 1 , Claudia Abbruzzese 4 , Lorenzo Botta 5 , Silvia Schenone 6 , Giovanni Cuda 2 , Stefano Alcaro 1 , Cataldo Bianco 2 , Patrizia Lavia 7 , Marco G. Paggi 4 , Nicola Perrotti 1, * , Rosario Amato 1, * 1 Department of “Scienze della Salute”, University “Magna Graecia” of Catanzaro, Viale Europa, Catanzaro, Italy 2 Department of “Medicina Sperimentale e Clinica”, University “Magna Graecia” of Catanzaro, Viale Europa, Catanzaro, Italy 3 Section of Pathology, Regina Elena National Cancer Institute, IRCCS, Rome, Italy 4 Experimental Oncology, Regina Elena National Cancer Institute, IRCCS, Rome, Italy 5 Department of Biotecnologie, Chimica e Farmacia, University of Siena, Siena, Italy 6 Department of Farmacia, University of Genova, Genova, Italy 7 Institute of Molecular Biology and Pathology (IBPM), National Research Council of Italy (CNR), c/o University “La Sapienza”, Rome, Italy * These authors have contributed equally to this work Correspondence to: Rosario Amato, e-mail: rosario.amato@unicz.it Nicola Perrotti, e-mail: perrotti@unicz.it Keywords: SGK1, HCC, kinase inhibitor, radiotherapy, apoptosis Received: June 04, 2015 Accepted: September 28, 2015 Published: October 08, 2015 ABSTRACT The SGK1 kinase is pivotal in signal transduction pathways operating in cell transformation and tumor progression. Here, we characterize in depth a novel potent and selective pyrazolo[3,4-d]pyrimidine-based SGK1 inhibitor. This compound, named SI113, active in vitro in the sub-micromolar range, inhibits SGK1-dependent signaling in cell lines in a dose- and time-dependent manner. We recently showed that SI113 slows down tumor growth and induces cell death in colon carcinoma cells, when used in monotherapy or in combination with paclitaxel. We now demonstrate for the first time that SI113 inhibits tumour growth in hepatocarcinoma models in vitro and in vivo . SI113-dependent tumor inhibition is dose- and time-dependent. In vitro and in vivo SI113-dependent SGK1 inhibition determined a dramatic increase in apotosis/necrosis, inhibited cell proliferation and altered the cell cycle profile of treated cells. Proteome-wide biochemical studies confirmed that SI113 down-regulates the abundance of proteins downstream of SGK1 with established roles in neoplastic transformation, e.g. MDM2, NDRG1 and RAN network members. Consistent with knock-down and over-expressing cellular models for SGK1, SI113 potentiated and synergized with radiotherapy in tumor killing. No short-term toxicity was observed in treated animals during in vivo SI113 administration. These data show that direct SGK1 inhibition can be effective in hepatic cancer therapy, either alone or in combination with radiotherapy.

We report results from a search for gravitational waves produced by perturbed intermediate mass black holes (IMBH) in data collected by LIGO and Virgo between 2005 and 2010. The search was sensitive to astrophysical sources that produced damped sinusoid gravitational wave signals, also known as ringdowns, with frequency $50\le f_{0}/\mathrm{Hz} \le 2000$ and decay timescale $0.0001\lesssim \tau/\mathrm{s} \lesssim 0.1$ characteristic of those produced in mergers of IMBH pairs. No significant gravitational wave candidate was detected. We report upper limits on the astrophysical coalescence rates of IMBHs with total binary mass $50 \le M/\mathrm{M}_\odot \le 450$ and component mass ratios of either 1:1 or 4:1. For systems with total mass $100 \le M/\mathrm{M}_\odot \le 150$, we report a 90%-confidence upper limit on the rate of binary IMBH mergers with non-spinning and equal mass components of $6.9\times10^{-8}\,$Mpc$^{-3}$yr$^{-1}$. We also report a rate upper limit for ringdown waveforms from perturbed IMBHs, radiating 1% of their mass as gravitational waves in the fundamental, $\ell=m=2$, oscillation mode, that is nearly three orders of magnitude more stringent than previous results., Comment: for the science summary, see http://www.ligo.org/science/Publication-S5S6RD/index.php

PTPRJ is a receptor protein tyrosine phospha- tase involved in both physiological and oncogenic pathways. We previously reported that its expression is strongly reduced in the majority of explored cancer cell lines and tumor samples; moreover, its restoration blocks in vitro cancer cell proliferation and in vivo tumor formation. By means of a phage display library screening, we recently identified two peptides able to bind and activate PTPRJ, resulting in cell growth inhibition and apoptosis of both cancer and endothelial cells. Here, on a previously discovered PTPRJ agonist peptide, PTPRJ-pep19, we synthe- sized and assayed a panel of nonapeptide analogues with the aim to identify specific amino acid residues responsible for peptide activity. These second-generation nonapeptides were tested on both cancer and primary endothelial cells (HeLa and HUVEC, respectively); interestingly, one of them (PTPRJ-19.4) was able to both dramatically reduce cell proliferation and effectively trigger apoptosis of both HeLa and HUVECs compared to its first-generation counterpart. Moreover, PTPRJ-pep19.4 significantly inhibited in vitro tube formation on Matrigel. Intriguingly, while ERK1/2 phosphorylation and cell proliferation were both inhibited by PTPRJ-pep19.4 in breast cancer cells (MCF-7 and SKBr3), no effects were observed on primary normal human mammary endothelial cells (HMEC). We further characterized these peptides by molecular modeling and NMR experiments reporting, for the most active peptide, the possibility of self-aggregation states and highlighting new hints of structure−activity relationship. Thus, our results indicate that this nonapeptide might represent a great potential lead for the development of novel targeted anticancer drugs.

Expression of PTPRJ, which is a ubiquitous receptor-type protein tyrosine phosphatase, is significantly reduced in a vast majority of human epithelial cancers and cancer cell lines (i.e. colon, lung, thyroid, mammary and pancreatic tumours). A possible role for microRNAs (miRNAs) in the negative regulation of PTPRJ expression has never been investigated. In this study, we show that overexpression of microRNA-328 (miR-328) decreases PTPRJ expression in HeLa and SKBr3 cells. Further investigations demonstrate that miR-328 acts directly on the 3'UTR of PTPRJ, resulting in reduced mRNA levels. Luciferase assay and site-specific mutagenesis were used to identify a functional miRNA response element in the 3'UTR of PTPRJ. Expression of miR-328 significantly enhances cell proliferation in HeLa and SKBr3 cells, similar to the effects of downregulation of PTPRJ with small interfering RNA. Additionally, in HeLa cells, the proliferative effect of miR-328 was not observed when PTPRJ was silenced with small interfering RNA; conversely, restoration of PTPRJ expression in miR-328-overexpressing cells abolished the proliferative activity of miR-328. In conclusion, we report the identification of miR-328 as an important player in the regulation of PTPRJ expression, and we propose that the interaction of miR-328 with PTPRJ is responsible for miR-328-dependent increase of epithelial cell proliferation.

Single nucleotide polymorphisms (SNPs) are germline variations interspersed in the human genome. These subtle changes of DNA sequence can influence the susceptibility to various pathologies including cancer. The functional meaning of SNPs is not always clear, being, the majority of them, localized in noncoding regions. The discovery of microRNAs, tiny noncoding RNAs able to bind the 3′ untranslated region (UTR) of target genes and to consequently downregulate their expression, has provided a functional explanation of how some SNPs positioned in noncoding regions contribute to cancer susceptibility. In this paper we summarize the current knowledge of the effect on cancer susceptibility of SNPs included in regions related with miRNA-dependent pathways. Hereditary cancer comes up from mutations that occur in high-penetrant predisposing tumor genes. However, a considerable part of inherited cancers arises from multiple low-penetrant predisposing gene variants that influence the behavior of cancer insurgence. Despite the established significance of such polymorphic variants in cancer predisposition, sometimes their functional role remains unknown. The discovery of a new group of genes called microRNAs (miRNAs) opened an avenue for the functional interpretation of polymorphisms involved in cancer predisposition.