EDF-1, a novel gene product down-regulated in human endothelial cell differentiation.

Abstract Endothelial cell differentiation is a crucial step in angiogenesis. Here we report the identification of EDF-1, a novel gene product that is down-regulated when endothelial cells are induced to differentiate in vitro. The cDNA encodingEDF-1 was isolated by RNA fingerprinting from human endothelial cells exposed to human immunodeficiency virus type 1 Tat, a viral protein known to be angiogenic. The deduced amino acid sequence of EDF-1 encodes a basic intracellular protein of 148 amino acids that is homologous to MBF1 (multiprotein-bridgingfactor 1) of the silkworm Bombyx mori and to H7, which is implicated in the early developmental events of Dictyostelium discoideum. Interestingly, human immunodeficiency virus type 1 Tat, which affects endothelial functions, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate and culture on fibrin gels, which promote endothelial differentiationin vitro, all down-regulate EDF-1 expression both at the RNA and protein levels. In addition, the inhibition of EDF-1 translation by an antisense anti-EDF-1 construct results in the inhibition of endothelial cell growth and in the transition from a nonpolar cobblestone phenotype to a polar fibroblast-like phenotype. These data suggest that EDF-1 may play a role in the regulation of human endothelial cell differentiation

Development of a Cell-Based Immunodetection Assay for Simultaneous Screening of Antiviral Compounds Inhibiting Zika and Dengue Virus Replication:

Practical cell-based assays can accelerate anti-Zika (ZIKV) and anti-dengue (DENV) virus drug discovery. We developed an immunodetection assay (IA), using a pan-flaviviral monoclonal antibody recognizing a conserved envelope domain. The final protocol includes a direct virus yield reduction assay (YRA) carried out in the human Huh7 cell line, followed by transfer of the supernatant to a secondary Huh7 culture to characterize late antiviral effects. Sofosbuvir and ribavirin were used to validate the assay, while celgosivir was used to evaluate the ability to discriminate between early and late antiviral activity. In the direct YRA, at 100, 50, and 25 TCID50, sofosbuvir IC50 values were 5.0 ± 1.5, 2.7 ± 0.5, 2.5 ± 1.1 µM against ZIKV and 16.6 ± 2.8, 4.6 ± 1.4, 2.6 ± 2.2 µM against DENV; ribavirin IC50 values were 6.8 ± 4.0, 3.8 ± 0.6, 4.5 ± 1.4 µM against ZIKV and 17.3 ± 4.6, 7.6 ± 1.2, 4.1 ± 2.3 µM against DENV. Sofosbuvir and ribavirin IC50 values determined in the secondary YRA were reproducible and comparable with those obtained by direct YRA and plaque reduction assay (PRA). In agreement with the proposed mechanism of late action, celgosivir was active against DENV only in the secondary YRA (IC50 11.0 ± 1.0 µM) and in PRA (IC50 10.1 ± 1.1 µM). The assay format overcomes relevant limitations of the gold standard PRA, allowing concurrent analysis of candidate antiviral compounds against different viruses and providing preliminary information about early versus late antiviral activity

Cellular and Molecular Mechanisms of In Vivo and In Vitro SARS-CoV-2 Infection: A Lesson from Human Sperm

Despite the major target of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, being the respiratory system, clinical evidence suggests that the male reproductive system may represent another viral target organ. Revealing the effect of SARS-CoV-2 infection on testis and sperm is a priority for reproductive biology, as well as for reproductive medicine. Here, we confirmed that the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) is highly expressed on human testis and ejaculated sperm; moreover, we provide evidence for the expression of the co-receptors transmembrane protease/serine (TMPRSS2), Basigin (BSG), and Catepsin L (CTSL). Human sperm were readily infected, both in vivo and in vitro, by SARS-CoV-2, as demonstrated by confocal and electron microscopy. The demonstration that the seminiferous epithelium and sperm support SARS-CoV-2 viral replication suggests the possibility that the spermatogenetic process may be detrimentally affected by the virus, and at the same time, supports the need to implement safety measures and guidelines to ensure specific care in reproductive medicine

PAK4 regulates stemness and progression in endocrine resistant ER-positive metastatic breast cancer

Despite the effectiveness of endocrine therapies to treat estrogen receptor-positive (ER+) breast tumours, two thirds of patients will eventually relapse due to de novo or acquired resistance to these agents. Cancer Stem-like Cells (CSCs), a rare cell population within the tumour, accumulate after anti-estrogen treatments and are likely to contribute to their failure. Here we studied the role of p21-activated kinase 4 (PAK4) as a promising target to overcome endocrine resistance and disease progression in ER+ breast cancers. PAK4 predicts for resistance to tamoxifen and poor prognosis in 2 independent cohorts of ER+ tumours. We observed that PAK4 strongly correlates with CSC activity in metastatic patient-derived samples irrespective of breast cancer subtype. However, PAK4-driven mammosphere-forming CSC activity increases alongside progression only in ER+ metastatic samples. PAK4 activity increases in ER+ models during acquired resistance to endocrine therapies. Targeting PAK4 with either CRT PAKi, a small molecule inhibitor of PAK4, or with specific siRNAs abrogates CSC activity/self-renewal in clinical samples and endocrine-resistant cells. Together, our findings establish that PAK4 regulates stemness during disease progression and that its inhibition reverses endocrine resistance in ER+ breast cancers

External quality assessment of HIV-1 DNA quantification assays used in the clinical setting in Italy

none18no: Total cell-associated HIV-1 DNA is a surrogate marker of the HIV-1 reservoir, however, certified systems for its quantification are not available. The Italian HIV DNA Network was launched to validate HIV-1 DNA quantification methods in use at University and Hospital labs. A quality control panel including HIV-1 DNA standards, reconstructed blood samples (RBSs) and DNA from different HIV-1 subtypes was blindly tested by 12 participating labs by quantitative real-time PCR (n = 6), droplet digital PCR (n = 3) or both (n = 3). The median 95% hit rate was 4.6 (3.7-5.5) copies per test and linearity in the tested range was excellent (R2 = 1.000 [1.000-1.000]). The median values obtained across labs were 3,370 (2,287-4,245), 445 (299-498), 59 (40-81) and 7 (6-11) HIV-1 DNA copies, for the 3,584, 448, 56 and 7-copy standards, respectively. With RBSs, measured values were within twofold with respect to the median in two thirds of cases. HIV-1 subtypes were missed (CRF01_AE by 3 labs) or underestimated by > 1 log (subtypes A, C, D, F by one lab; CRF01_AE by one lab; CRF02_AG by one lab). The overall performance was excellent with HIV-1 DNA standards, however detection of different HIV-1 subtypes must be improved.openVicenti, Ilaria; Dragoni, Filippo; Giannini, Alessia; Casabianca, Anna; Lombardi, Francesca; Di Sante, Laura; Turriziani, Ombretta; Racca, Sara; Paolucci, Stefania; Lai, Alessia; Bon, Isabella; Abbate, Isabella; Rozera, Gabriella; Belmonti, Simone; Scutari, Rossana; Alteri, Claudia; Saladini, Francesco; Zazzi, MaurizioVicenti, Ilaria; Dragoni, Filippo; Giannini, Alessia; Casabianca, Anna; Lombardi, Francesca; Di Sante, Laura; Turriziani, Ombretta; Racca, Sara; Paolucci, Stefania; Lai, Alessia; Bon, Isabella; Abbate, Isabella; Rozera, Gabriella; Belmonti, Simone; Scutari, Rossana; Alteri, Claudia; Saladini, Francesco; Zazzi, Maurizi

The nucleolar RNA methyltransferase Misu (NSun2) is required for mitotic spindle stability

Myc-induced SUN domain–containing protein (Misu or NSun2) is a nucleolar RNA methyltransferase important for c-Myc–induced proliferation in skin, but the mechanisms by which Misu contributes to cell cycle progression are unknown. In this study, we demonstrate that Misu translocates from the nucleoli in interphase to the spindle in mitosis as an RNA–protein complex that includes 18S ribosomal RNA. Functionally, depletion of Misu caused multiple mitotic defects, including formation of unstructured spindles, multipolar spindles, and chromosome missegregation, leading to aneuploidy and cell death. The presence of both RNA and Misu is required for correct spindle assembly, and this process is independent of active translation. Misu might mediate its function at the spindle by recruiting nucleolar and spindle-associated protein (NuSAP), an essential microtubule-stabilizing and bundling protein. We further identify NuSAP as a novel direct target gene of c-Myc. Collectively, our results suggest a novel mechanism by which c-Myc promotes proliferation by stabilizing the mitotic spindle in fast-dividing cells via Misu and NuSAP

Cloning and characterization of murine EDF-1.

Murine endothelial differentiation-related factor (mEDF-1) encodes a basic intracellular protein of 148 amino acids which is highly homologous to the human and rat polypeptides. mEDF-1 is expressed in most murine tissues tested and is evolutionary conserved. mEDF-1 expression is modulated in mouse development, since its expression is high early in development and decreases thereafter. Because EDF-1 has been isolated as a gene differentially expressed by exposure of endothelial cells to the Tat protein of HIV, we evaluated mEDF-1 expression in different cell lines derived from tumors which spontaneously develop in Tat transgenic mice. Cells isolated from adenocarcinomas and leiomyosarcomas express very high amounts of EDF-1, independently from their capability to secrete Tat. Tat transgenic mice also develop skin lesions which closely resemble human Kaposi's sarcoma. Since Kaposi spindle cells, which are the proliferative component of the sarcoma, differentiate from an endothelial precursor, it is noteworthy that spindle cells derived from Kaposi-like lesions of the Tat transgenic mice downregulate EDF-1 when compared to microvascular endothelial cells isolated from the same tissue

Extending Security-by-Contract with Quantitative Trust on Mobile Devices

Security-by-Contract (S?C) is a novel paradigm providing security assurances for mobile applications. In this work, we present an extension of S?C enriched with an au- tomatic trust management infrastructure. Indeed, we enhance the already existing architecture by adding new modules and configurations for contracts managing. At deploy-time, our system decides the run-time configuration depending on the credentials of the contract provider. Roughly, the run-time environment can both enforce a security policy and monitor the declared contract. According to the actual behaviour of the running program our architecture updates the trust level associated with the contract provider. The main advantage of this method is an automatic management of the level of trust of software and contract releaser

Comparative analysis of different cell systems for Zika virus (ZIKV) propagation and evaluation of anti-ZIKV compounds in vitro

A strong correlation between Zika virus (ZIKV) infection and severe neurological disease in newborns and occasionally adults has emerged in the Brazilian outbreak. Efficient human cell-based assays are required to test candidate inhibitors of ZIKV replication. The aim of this work was to investigate ZIKV propagation and quantification in different cell lines. The human (U87, A549, Huh7), mosquito (C6/36) and monkey (VERO E6) cell lines tested were all permissive to ZIKV infection. When assessed by plaque forming units (PFU) in three different target cell lines, the maximal production of ZIKV was achieved in Huh7 at day 3 post-infection (6.38 ± 0.44 log10PFU/ml). The C6/36 cell line showed a low and slow production of virus when compared with other cell lines. A549 readout cells generated a larger number of plaques compared to Huh7 but not to VERO E6 cells. ZIKV PFU and RNA titers showed the highest correlation when Huh7 and A549 were used as the producer and readout cells, respectively. Also, U87 cells produced ZIKV RNA titers which were highly correlated with PFU independently from the readout cell line. Using the best virus-cell system, sofosbuvir and ribavirin EC50were 1.2 μM and 1.1 μM when measured through plaque assay, and 4.2 μM and 5.2 μM when measured by quantitative real time PCR (qRT-PCR), respectively. In summary, ZIKV can efficiently infect different human cell lines and rapidly reach peak viral titers. Overall, A549 cells appear to be as efficient as the VERO E6 gold standard for plaque assay allowing the use of human, rather than simian, cells for evaluating candidate anti-ZIKV compounds by the reference assay. The possibility to replace the labor-intensive plaque assay with the more rapid and easy-to-perform qRT-PCR is appealing and warrants further investigation