Tuberculosis-Associated MicroRNAs: From Pathogenesis to Disease Biomarkers.

Tuberculosis (TB) caused by Mycobacterium tuberculosis is one of the most lethal infectious diseases with estimates of approximately 1.4 million human deaths in 2018. M. tuberculosis has a well-established ability to circumvent the host immune system to ensure its intracellular survival and persistence in the host. Mechanisms include subversion of expression of key microRNAs (miRNAs) involved in the regulation of host innate and adaptive immune response against M. tuberculosis. Several studies have reported differential expression of miRNAs during active TB and latent tuberculosis infection (LTBI), suggesting their potential use as biomarkers of disease progression and response to anti-TB therapy. This review focused on the miRNAs involved in TB pathogenesis and on the mechanism through which miRNAs induced during TB modulate cell antimicrobial responses. An attentive study of the recent literature identifies a group of miRNAs, which are differentially expressed in active TB vs. LTBI or vs. treated TB and can be proposed as candidate biomarkers.

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.

New avenues for therapeutic discovery against West Nile virus.

Introduction: West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus, which is endemic in many countries, especially in Europe and in North America, where the virus has increased its activity in the recent years. No vaccines nor antiviral drugs are available for the prevention and treatment of WNV infection in humans.Areas covered: This review article describes viral and host targets that have been addressed by anti-WNV drug discovery studies and summarizes the most relevant anti-WNV candidate compounds identified so far, focusing on those showing antiviral efficacy in in vivo models and broad-spectrum anti-flavivirus activity.Expert opinion: The most promising anti-WNV drug candidates target conserved enzymatic motifs in viral NS3 protease and NS5 polymerase and are effective against different flaviviruses. Targeting host factors required for viral infection and replication and modulation of host innate antiviral response are also promising approaches, which may lead to the development of compounds with broad-spectrum antiviral activity, a desirable feature for an antiviral drug.

Clinical and virological findings in patients with Usutu virus infection, northern Italy, 2018.

BackgroundUsutu virus (USUV) is a mosquito-borne flavivirus, which shares its transmission cycle with the phylogenetically related West Nile virus (WNV). USUV circulates in several European countries and its activity has increased over the last 5 years.AimTo describe human cases of USUV infection identified by surveillance for WNV and USUV infection in the Veneto Region of northern Italy in 2018.MethodsFrom 1 June to 30 November 2018, all cases of suspected autochthonous arbovirus infection and blood donors who had a reactive WNV nucleic acid test were investigated for both WNV and USUV infection by in-house molecular methods. Anti-WNV and anti-USUV IgM and IgG antibodies were detected by ELISA and in-house immunofluorescence assay, respectively; positive serum samples were further tested by WNV and USUV neutralisation assays run in parallel.ResultsEight cases of USUV infection (one with neuroinvasive disease, six with fever and one viraemic blood donor who developed arthralgia and myalgia) and 427 cases of WNV infection were identified. A remarkable finding of this study was the persistence of USUV RNA in the blood and urine of three patients during follow-up. USUV genome sequences from two patients shared over 99% nt identity with USUV sequences detected in mosquito pools from the same area and clustered within lineage Europe 2.ConclusionsClinical presentation and laboratory findings in patients with USUV infection were similar to those found in patients with WNV infection. Cross-reactivity of serology and molecular tests challenged the differential diagnosis.

Human papillomavirus infection is not involved in esophageal verrucous carcinoma.

Verrucous carcinoma of the esophagus (VCE) is a rare variant of squamous cell cancer, with a puzzling clinical, etiological, and molecular profile. The etiological involvement of human papillomavirus (HPV) in the cancer's natural history is controversial. This study considers 9 cases of VCE, focusing on patients' clinical history before surgery, histologic phenotype, immunophenotype (epidermal growth factor receptor [EGFR], E-cadherin, cyclin D1, p16, and p53 expression), HPV infection, and TP53 gene mutational status (exons 5-8). Using 3 different molecular test methods, not one of these cases of VCE featured HPV infection. The only case with synchronous nodal metastasis was characterized by a TP53 missense point mutation in association with high EGFR and low E-cadherin expression levels. In conclusion, HPV infection is probably not involved with VCE, while TP53 gene mutation, EGFR overexpression, and E-cadherin loss might fuel the tumor's proliferation and lend it a metastatic potential.

Optimizing PCR primers targeting the bacterial 16S ribosomal RNA gene.

BACKGROUND: Targeted amplicon sequencing of the 16S ribosomal RNA gene is one of the key tools for studying microbial diversity. The accuracy of this approach strongly depends on the choice of primer pairs and, in particular, on the balance between efficiency, specificity and sensitivity in the amplification of the different bacterial 16S sequences contained in a sample. There is thus the need for computational methods to design optimal bacterial 16S primers able to take into account the knowledge provided by the new sequencing technologies. RESULTS: We propose here a computational method for optimizing the choice of primer sets, based on multi-objective optimization, which simultaneously: 1) maximizes efficiency and specificity of target amplification; 2) maximizes the number of different bacterial 16S sequences matched by at least one primer; 3) minimizes the differences in the number of primers matching each bacterial 16S sequence. Our algorithm can be applied to any desired amplicon length without affecting computational performance. The source code of the developed algorithm is released as the mopo16S software tool (Multi-Objective Primer Optimization for 16S experiments) under the GNU General Public License and is available at http://sysbiobig.dei.unipd.it/?q=Software#mopo16S . CONCLUSIONS: Results show that our strategy is able to find better primer pairs than the ones available in the literature according to all three optimization criteria. We also experimentally validated three of the primer pairs identified by our method on multiple bacterial species, belonging to different genera and phyla. Results confirm the predicted efficiency and the ability to maximize the number of different bacterial 16S sequences matched by primers.

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.

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.

Profiling of expression of human papillomavirus-related cancer miRNAs in penile squamous cell carcinomas.

Penile squamous cell carcinoma (PSCC) is a rare tumor associated with high-risk human papillomavirus (HR-HPV) infection in 30% to 60% of cases. Altered expression of miRNAs has been reported in HPV-related cervical and head and neck cancers, but such data have not been available for PSCC. We analyzed a series of 59 PSCCs and 8 condylomata for presence of HPV infection, for p16(INK4a), Ki-67, and p53 immunohistochemical expression, and for expression of a panel of cellular miRNAs (let-7c, miR-23b, miR-34a, miR-145, miR-146a, miR-196a, and miR-218) involved in HPV-related cancer. HR-HPV DNA (HPV16 in most cases) was detected in 17/59 (29%) PSCCs; all penile condylomata (8/8) were positive for low-risk HPV6 or HPV11. HR-HPV(+) PSCCs overexpressed p16(INK4a) in 88% cases and p53 in 35% of cases, whereas HR-HPV(-) PSCCs were positive for p16(INK4a) and p53 immunostaining in 9% and 44% of cases, respectively. Among the miRNAs investigated, expression of miR-218 was lower in PSCCs with HR-HPV infection and in p53(-) cancers. Hypermethylation of the promoter of the SLIT2 gene, which contains miR-218-1 in its intronic region, was frequently observed in PSCCs, mainly in those with low miR-218 expression. Epigenetic silencing of miR-218 is a common feature in HR-HPV(+) PSCCs and in HR-HPV(-) PSCCs without immunohistochemical detection of p53.

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.