Lentiviral Vectors Expressing Chimeric NEDD4 Ubiquitin Ligases: An Innovative Approach for Interfering with Alpha-Synuclein Accumulation.

One of the main pathological features of Parkinson's disease (PD) is a diffuse accumulation of alpha-synuclein (aS) aggregates in neurons. The NEDD4 E3 Ub ligase promotes aS degradation by the endosomal-lysosomal route. Interestingly, NEDD4, as well as being a small molecule able to trigger its functions, is protective against human aS toxicity in evolutionary distant models. While pharmacological activation of E3 enzymes is not easy to achieve, their flexibility and the lack of "consensus" motifs for Ub-conjugation allow the development of engineered Ub-ligases, able to target proteins of interest. We developed lentiviral vectors, encoding well-characterized anti-human aS scFvs fused in frame to the NEDD4 catalytic domain (ubiquibodies), in order to target ubiquitinate aS. We demonstrate that, while all generated ubiquibodies bind to and ubiquitinate aS, the one directed against the non-amyloid component (NAC) of aS (Nac32HECT) affects aS's intracellular levels. Furthermore, Nac32HECT expression partially rescues aS's overexpression or mutation toxicity in neural stem cells. Overall, our data suggest that ubiquibodies, and Nac32HECT in particular, represent a valid platform for interfering with the effects of aS's accumulation and aggregation in neurons.

Suppression of a SARS-CoV-2 outbreak in the Italian municipality of Vo'.

On 21 February 2020, a resident of the municipality of Vo', a small town near Padua (Italy), died of pneumonia due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection1. This was the first coronavirus disease 19 (COVID-19)-related death detected in Italy since the detection of SARS-CoV-2 in the Chinese city of Wuhan, Hubei province2. In response, the regional authorities imposed the lockdown of the whole municipality for 14 days3. Here we collected information on the demography, clinical presentation, hospitalization, contact network and the presence of SARS-CoV-2 infection in nasopharyngeal swabs for 85.9% and 71.5% of the population of Vo' at two consecutive time points. From the first survey, which was conducted around the time the town lockdown started, we found a prevalence of infection of 2.6% (95% confidence interval (CI): 2.1-3.3%). From the second survey, which was conducted at the end of the lockdown, we found a prevalence of 1.2% (95% CI: 0.8-1.8%). Notably, 42.5% (95% CI: 31.5-54.6%) of the confirmed SARS-CoV-2 infections detected across the two surveys were asymptomatic (that is, did not have symptoms at the time of swab testing and did not develop symptoms afterwards). The mean serial interval was 7.2 days (95% CI: 5.9-9.6). We found no statistically significant difference in the viral load of symptomatic versus asymptomatic infections (P = 0.62 and 0.74 for E and RdRp genes, respectively, exact Wilcoxon-Mann-Whitney test). This study sheds light on the frequency of asymptomatic SARS-CoV-2 infection, their infectivity (as measured by the viral load) and provides insights into its transmission dynamics and the efficacy of the implemented control measures.

Multifunctional, CD44v6-Targeted ORMOSIL Nanoparticles Enhance Drugs Toxicity in Cancer Cells.

Drug-loaded, PEGylated, organic-modified silica (ORMOSIL) nanoparticles prepared by microemulsion condensation of vinyltriethoxysilane (VTES) were investigated as potential nanovectors for cancer therapy. To target cancer stem cells, anti-CD44v6 antibody and hyaluronic acid (HA) were conjugated to amine-functionalized PEGylated ORMOSIL nanoparticles through thiol-maleimide and amide coupling chemistries, respectively. Specific binding and uptake of conjugated nanoparticles were studied on cells overexpressing the CD44v6 receptor. Cytotoxicity was subsequently evaluated in the same cells after the uptake of the nanoparticles. Internalization of nanocarriers loaded with the anticancer drug 3N-cyclopropylmethyl-7-phenyl-pyrrolo- quinolinone (MG2477) into cells resulted in a substantial increase of the cytotoxicity with respect to the free formulation. Targeting with anti-CD44v6 antibodies or HA yielded nanoparticles with similar effectiveness, in their optimized formulation.

Oral Mucosa-Derived Induced Pluripotent Stem Cells from Patients with Ectrodactyly-Ectodermal Dysplasia-Clefting Syndrome.

Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare monogenic disease with autosomal dominant inheritance caused by mutations in the TP63 gene, leading to progressive corneal keratinocyte loss, limbal stem cell deficiency (LSCD), and eventually blindness. Currently, there is no treatment available to cure or slow down the keratinocyte loss. Human oral mucosal epithelial stem cells (hOMESCs), which are a mixed population of keratinocyte precursor stem cells, are used as source of autologous tissue for treatment of bilateral LSCD. However, hOMESCs from EEC patients have a reduced life span due to TP63 mutations and cannot be used for autologous transplantation. Human induced pluripotent stem cells (hiPSCs) represent a potentially unlimited source of autologous limbal stem cell for EEC patients and can be genetically modified by genome editing technologies to correct the disease ex vivo before transplantation. In this study, we describe for the first time the generation of integration-free EEC-hiPSCs from hOMESCs of EEC patients by Sendai virus vector and episomal vector-based reprogramming. The generated hiPSC clones expressed pluripotency markers and were successfully differentiated into derivatives of the three germ layers, as well as toward corneal epithelium. These cells may be used for EEC disease modeling and open perspectives for applications in cell therapy of LSCD.

Generation of a transgene-free human induced pluripotent stem cell line (UNIPDi001-A) from oral mucosa epithelial stem cells.

Human oral mucosa epithelial stem cells (hOMESCs) were obtained from a fresh oral biopsy collected from a healthy subject at the Fondazione Banca degli Occhi del Veneto (FBOV). An integration-free reprogramming protocol was applied exploiting episomal plasmids transfected into cells using a Nucleofector device. Around day 20 post transfection, several human induced pluripotent stem cell (hiPSC) colonies were manually picked and expanded. One of these (UNIPDi001-A-hiPSCs) expressed undifferentiated state marker alkaline phosphatase along with a panel of pluripotency state markers and was able to differentiate into the derivatives of all the three germ layers.

Induced pluripotent stem cells line (UNIPDi003-A) from a patient affected by EEC syndrome carrying the R279H mutation in TP63 gene.

Oral mucosa epithelial stem cells from a patient affected by Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome carrying the R279H mutation in the TP63 gene were reprogrammed into human induced pluripotent stem cells (hiPSCs) with episomal vectors. The generated UNIPDi003-A-hPSC line retained the mutation of the parental cells and showed a normal karyotype upon long term culture. Analysis of residual transgenes expression showed that the episomal vectors were eliminated from the cell line. UNIPDi003-A-hiPSCs expressed the undifferentiated state marker alkaline phosphatase along with a panel of pluripotency markers, and formed embryoid bodies capable of expressing markers belonging to all the three germ layers.

Generation of a transgene-free induced pluripotent stem cells line (UNIPDi002-A) from oral mucosa epithelial stem cells carrying the R304Q mutation in TP63 gene.

Transgene free UNIPDi002-A-human induced pluripotent stem cell (hiPSC) line was generated by Sendai Virus Vectors reprogramming from human oral mucosal epithelial stem cells (hOMESCs) of a patient affected by ectrodactyly-ectodermal dysplasia-clefting (EEC)-syndrome, carrying a mutation in exon 8 of the TP63 gene (R304Q). The UNIPDi002-A-hiPSC line retained the mutation of the parental R304Q-hOMESCs and displayed a normal karyotype. No residual expression of transgenes nor Sendai virus vector sequences were detected in the line at passage 8. UNIPDi002-A-hiPSC expressed a panel of pluripotency-associated markers and could form embryoid bodies expressing markers belonging to the three germ layers ectoderm, endoderm and mesoderm.

HPV16 E6 and E7 upregulate the histone lysine demethylase KDM2B through the c-MYC/miR-146a-5p axys.

Persistent infection by high-risk human papillomaviruses (HPVs) is associated with the development of cervical cancer and a subset of anogenital and head and neck squamous cell carcinomas. Abnormal expression of cellular microRNAs (miRNAs) plays an important role in the development of cancer, including HPV-related tumors. In this study, we demonstrated that miR-146a-5p was down-regulated by E6 and, less efficiently, by E7 of high-risk HPV16 in keratinocytes and the presence of low levels of this miRNA in cervical carcinoma cell lines and in high-risk HPV-positive cervical specimens. Down-regulation of miR-146a-5p was mediated at least in part by the transcription repressor c-MYC, through binding sites in the miR-146a promoter. Overexpression of miR-146a-5p significantly inhibited proliferation and migration of keratinocytes and cervical cancer cells. The histone demethylase KDM2B was validated as a new direct target of miR-146a-5p and two putative binding sites for miR-146a-5p were identified in its 3'UTR sequence. Western blot analysis and immunohistochemistry showed that KDM2B was overexpressed in HPV16 E6/E7-positive keratinocytes, in cervical cancer cell lines, and in a subset of invasive cervical carcinomas and HPV-positive laryngeal squamous cell carcinomas. In these tumors, KDM2B overexpression was associated with c-MYC copy number gain. In vitro, silencing of KDM2B inhibited proliferation of cervical cancer cells. In conclusion, this study identified a novel player, the hystone demethylase KDM2B, in HPV-mediated tumorigenesis. E6 and, less efficiently, E7 of high-risk HPV16 up-regulated KDM2B expression in human keratinocytes through a pathway involving overexpression of c-MYC, which in turn downregulated miR-146a-5p.

Down-regulation of microRNA-146a is associated with high-risk human papillomavirus infection and epidermal growth factor receptor overexpression in penile squamous cell carcinoma.

Dysregulation of host microRNA expression has been involved in the development and progression of human papillomavirus (HPV)-related tumors. Analysis of miR-146a expression in a series of 59 penile squamous cell carcinomas (PSCCs) showed that its levels were lower in high-risk HPV-positive than in HPV-negative PSCCs and inversely correlated with expression of epidermal growth factor receptor (EGFR), a known target for miR-146a. Analysis of genotype distribution for rs2910164, a common functional polymorphism of miR-146a, did not identify correlations with miR-146a levels and EGFR expression in PSCCs. In vitro experiments demonstrated that E6 of HPV type 16, but not low-risk HPV-6, down-regulated miR-146a in human foreskin keratinocytes and up-regulated EGFR. Ectopic expression of miR-146a decreased expression of EGFR and inhibited proliferation of keratinocytes and cervical carcinoma cells. EGFR is commonly overexpressed in penile cancer and in other squamous cell carcinomas. Molecular mechanisms leading to EGFR overexpression and activation are known for HPV-negative cancers and include amplification or mutations of the EGFR gene. The results of this study indicate that down-regulation of miR-146a may represent another mechanism of EGFR overexpression in PSCCs, which can be mediated by high-risk HPV E6 in HPV-related tumors.

Characterization of Intra-Type Variants of Oncogenic Human Papillomaviruses by Next-Generation Deep Sequencing of the E6/E7 Region.

Different human papillomavirus (HPV) types are characterized by differences in tissue tropism and ability to promote cell proliferation and transformation. In addition, clinical and experimental studies have shown that some genetic variants/lineages of high-risk HPV (HR-HPV) types are characterized by increased oncogenic activity and probability to induce cancer. In this study, we designed and validated a new method based on multiplex PCR-deep sequencing of the E6/E7 region of HR-HPV types to characterize HPV intra-type variants in clinical specimens. Validation experiments demonstrated that this method allowed reliable identification of the different lineages of oncogenic HPV types. Advantages of this method over other published methods were represented by its ability to detect variants of all HR-HPV types in a single reaction, to detect variants of HR-HPV types in clinical specimens with multiple infections, and, being based on sequencing of the full E6/E7 region, to detect amino acid changes in these oncogenes potentially associated with increased transforming activity.

Changes in microRNA expression during disease progression in patients with chronic viral hepatitis.

BACKGROUND & AIMS: MicroRNAs (miRNAs) have been involved in hepatocarcinogenesis, but little is known on their role in the progression of chronic viral hepatitis. Aim of this study was to identify miRNA signatures associated with stages of disease progression in patients with chronic viral hepatitis. METHODS: MiRNA expression profile was investigated in liver biopsies from patients with chronic viral hepatitis and correlated with clinical, virological and histopathological features. Relevant miRNAs were further investigated. RESULTS: Most of the significant changes in miRNA expression were associated with liver fibrosis stages and included the significant up-regulation of a group of miRNAs that were demonstrated to target the master regulators of epithelial-mesenchymal transition ZEB1 and ZEB2 and involved in the preservation of epithelial cell differentiation, but also in cell proliferation and fibrogenesis. In agreement with miRNA data, immunostaining of liver biopsies showed that expression of the epithelial marker E-cadherin was maintained in severe fibrosis/cirrhosis while expression of ZEBs and other markers of epithelial-mesenchymal transition were low or absent. Severe liver fibrosis was also significantly associated with the down-regulation of miRNAs with antiproliferative and tumour suppressor activity. Similar changes in miRNA and target gene expression were demonstrated along with disease progression in a mouse model of carbon tetrachloride (CCl4)-induced liver fibrosis, suggesting that they might represent a general response to liver injury. CONCLUSION: Chronic viral hepatitis progression is associated with the activation of miRNA pathways that promote cell proliferation and fibrogenesis, but preserve the differentiated hepatocyte phenotype.

Microfluidic-driven viral infection on cell cultures: Theoretical and experimental study.

Advanced cell culture systems creating a controlled and predictable microenvironment together with computational modeling may be useful tools to optimize the efficiency of cell infections. In this paper, we will present a phenomenological study of a virus-host infection system, and the development of a multilayered microfluidic platform used to accurately tune the virus delivery from a diffusive-limited regime to a convective-dominated regime. Mathematical models predicted the convective-diffusive regimes developed within the system itself and determined the dominating mass transport phenomena. Adenoviral vectors carrying the enhanced green fluorescent protein (EGFP) transgene were used at different multiplicities of infection (MOI) to infect multiple cell types, both in standard static and in perfused conditions. Our results validate the mathematical models and demonstrate how the infection processes through perfusion via microfluidic platform led to an enhancement of adenoviral infection efficiency even at low MOIs. This was particularly evident at the longer time points, since the establishment of steady-state condition guaranteed a constant viral concentration close to cells, thus strengthening the efficiency of infection. Finally, we introduced the concept of effective MOI, a more appropriate variable for microfluidic infections that considers the number of adenoviruses in solution per cell at a certain time.

Human papillomavirus genotyping by 454 next generation sequencing technology.

BACKGROUND: An accurate tool for human papillomavirus (HPV) typing is important both for management of patients with HPV infection and for surveillance studies. OBJECTIVES: Design and evaluation of an HPV typing method based on 454 next generation sequencing (NGS) technology. STUDY DESIGN: Development of an HPV typing method based on 454 NGS of HPV L1 amplicons generated with MY09/11-based primers. Evaluation of the NGS method in control samples and in a panel of cervical cytological samples. Comparison of the NGS typing method with cycle sequencing and with the reverse hybridization-based INNO-LiPA HPV Genotyping Extra assay (LiPA). RESULTS: In control samples carrying mixtures of HPV16 and HPV18 DNA, the NGS method could reliably detect genotype sequences occurring at a frequency of 1% in multiple infections with a sensitivity of 100 genome equivalents/µL. In cervical cytology samples, comparison with cycle sequencing demonstrated accuracy of HPV typing by NGS. The NGS method had however lower sensitivity for some HPV types than LiPA, conceivably due to the poor sensitivity of the MY09/11-based primers. At variance, LiPA could not detect HPV types which were present in low proportion in multiple infections (<10% of HPV reads obtained by NGS). In addition, NGS allowed identifying the presence of different variants of the same HPV type in a specimen. CONCLUSIONS: NGS is a promising method for HPV typing because of its high sensitivity in multiple infection and its potential ability to detect a broad spectrum of HPV types, subtypes, and variants.

WU and KI polyomaviruses in the brains of HIV-positive patients with and without progressive multifocal leukoencephalopathy.

The polyomaviruses KI (KIPyV) and WU (WUPyV) were recently identified mainly in respiratory samples from children and immunosuppressed patients. Investigation of 54 autopsy brain tissue samples from 22 subjects demonstrated that WUPyV DNA and KIPyV DNA could be detected in 1 of 4 human immunodeficiency virus (HIV)-positive individuals with progressive multifocal leukoencephalopathy (PML) and in 3 of 10 HIV-positive individuals without PML, but not in 8 HIV-negative individuals. Viruses were localized in all regions of the central nervous system that were analyzed, that is, the cerebral hemispheres, cerebellum, pons, and medulla oblongata. No specific histopathological findings were found to be associated with the presence of WUPyV and KIPyV.

Human cytomegalovirus productively infects adrenocortical cells and induces an early cortisol response.

Following our recent findings on the presence of human cytomegalovirus (HCMV) in the normal human adrenal cortex and in adrenocortical tumors, especially in cortisol-secreting tumors, aim of the present study was to investigate the direct effects of HCMV infection on human adrenocortical cells. To this aim, both clinical isolates and laboratory strains of HCMV were used to assess the early effects of infection on human adrenocortical cell morphology, proliferation, gene expression, and steroidogenic function. Both clinical and laboratory HCMV strains could infect and replicate in primary human adrenocortical cell cultures and in adrenocortical carcinoma cell lines, leading to cytopathic changes. Most importantly, in the first hours post-infection (p.i.), adrenocortical cells showed a significant increase of cortisol and estrogen production, paralleled by up-regulation of steroidogenic acute regulatory protein and expression of steroidogenic enzymes involved in the last steps of adrenal steroidogenesis. This effect was probably due to HCMV immediate-early gene expression, since it was most evident in the early phases p.i. and UV-inactivated viral particles did not affect hormone production. Moreover, the effect on steroidogenesis was HCMV specific, since it was not observed after infection with herpes simplex virus. These data suggest that human adrenocortical cells are permissive to HCMV infection and acutely respond to infection with increased cortisol production. An acute glucocorticoid response is typically triggered by infections and is considered to be critical to host defense against pathogens, although, in the case of HCMV infection, it might also enhance viral replication and reactivation from latency.

Investigation on human adrenocortical cell response to adenovirus and adenoviral vector infection.

After systemic administration, adenoviral vectors (AdVs) are sequestered in the liver and adrenal glands. Adenoviral vector transduction has been shown to cause cytopathic effects on human hepatocytes and to induce an inflammatory response, whereas the effect of AdVs on human adrenocortical cells has never been investigated. In this study, human adrenocortical carcinoma cell lines and primary cell cultures were used to assess the effects of wild-type adenovirus (Ad5WT) and E1/E3-deleted AdVs on cell proliferation and steroidogenesis. Ad5WT could efficiently replicate in adrenocortical cells, leading to S phase induction, followed by cell death, whereas high titer AdVs transduction had only mild effects on adrenocortical cell proliferation, with accumulation of cells in G2/M. Both AdVs and Ad5WT induced expression of inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-alpha, but, most importantly, they led to a marked and dose-dependent increase of cortisol and other steroid hormone production and consistently modulated expression of key steroidogenic enzymes and regulators of steroidogenesis. This effect, which was already apparent at 6 h post-infection, probably represented a response to adenoviral entry and/or early phases of infection. The result of this study contribute to the understanding of host response to adenoviral vector administration.

Primary bilateral adrenal B-cell lymphoma associated with EBV and JCV infection.

Primary lymphoma of the adrenal gland is a rare and highly aggressive disease, with only a few reports in the literature. The pathogenesis is unknown, but detection of Epstein Barr virus (EBV) genome sequences and gene expression in some cases of primary adrenal lymphomas suggested the virus might be a causative agent of the malignancy. While investigating the presence of genome sequences of oncogenic viruses in a large series of adrenal tumors, both EBV and JC polyomavirus (JCV) DNA sequences were detected in a diffuse large primary bilateral B-cell non-Hodgkin lymphoma of the adrenal gland, which was diagnosed only at postmortem examination in a 77 year-old woman with incidentally discovered adrenal masses and primary adrenal insufficiency. The presence of both EBV and JCV genome sequences suggests the relevance of EBV and JCV coinfection in the pathogenesis of this rare form of B-cell lymphoma.

Expression of aromatase and estrogen receptors in human adrenocortical tumors.

We recently demonstrated that adrenocortical carcinoma cells express aromatase and estrogen receptors (ERs) and that 17beta-estradiol enhances adrenocortical cell proliferation. To provide a clue to the role of estrogens in adrenal tumorigenesis, we investigated the expression profile of genes involved in sex steroid hormone production and activity in a large series of normal and neoplastic human adrenocortical tissues. Quantitative reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemistry showed that ERalpha and ERbeta, androgen receptor (AR), and aromatase were expressed in the adrenal cortex and in adrenocortical tumors. ERbeta was the predominant ER subtype and was mainly expressed in the zona glomerulosa and fasciculata. Western blot analysis revealed the presence of a truncated form of AR in adrenocortical tissues. With respect to the normal adrenal cortex and adrenocortical adenomas, carcinomas were characterized by significantly lower ERbeta levels, ERalpha upregulation, and aromatase overexpression. ER expression correlated with expression of nuclear hormone receptors, suggesting they could be involved in ER modulation. In agreement with our in vitro findings, the results of this study suggest that estrogens, locally produced by aromatase, could enhance adrenocortical cell proliferation though autocrine/paracrine mechanisms. This study opens new perspectives on the potential use of antiestrogens and aromatase inhibitors as therapeutic agents against ACC.