Nasopharyngeal virome analysis of COVID-19 patients during three different waves in Campania region of Italy.

From December 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has spread rapidly, leading to a global pandemic. Little is known about possible relationships between SARS-CoV-2 and other viruses in the respiratory system affecting patient prognosis and outcomes. This study aims to characterize respiratory virome profiles in association with SARS-CoV-2 infection and disease severity, through the analysis in 89 nasopharyngeal swabs collected in a patient's cohort from the Campania region (Southern Italy). Results show coinfections with viral species belonging to Coronaviridae, Retroviridae, Herpesviridae, Poxviridae, Pneumoviridae, Pandoraviridae, and Anelloviridae families and only 2% of the cases (2/89) identified respiratory viruses.

NGS analysis of nasopharyngeal microbiota in SARS-CoV-2 positive patients during the first year of the pandemic in the Campania Region of Italy.

Since its first appearance, the SARS-CoV-2 has spread rapidly in the human population, reaching the pandemic scale with >280 million confirmed infections and more than 5 million deaths to date (https://covid19.who.int/). These data justify the urgent need to enhance our understanding of SARS-CoV-2 effects in the respiratory system, including those linked to co-infections. The principal aim of our study is to investigate existing correlations in the nasopharynx between the bacterial community, potential pathogens, and SARS-CoV-2 infection. The main aim of this study was to provide evidence pointing to possible relationships between components of the bacterial community and SARS-CoV-2 in the nasopharynx. Meta-transcriptomic profiling of the nasopharyngeal microbial community was carried out in 89 SARS-Cov-2 positive subjects from the Campania Region in Italy. To this end, RNA extracted from nasopharyngeal swabs collected at different times during the initial phases of the pandemic was analyzed by Next-Generation Sequencing (NGS). Results show a consistently high presence of members of the Proteobacteria (41.85%), Firmicutes (28.54%), and Actinobacteria (16.10%) phyla, and an inverted correlation between the host microbiome, co-infectious bacteria, and super-potential pathogens such as Staphylococcus aureus, Klebsiella pneumoniae, Streptococcus pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, and Neisseria gonorrhoeae. In depth characterization of microbiota composition in the nasopharynx can provide clues to understand its potential contribution to the clinical phenotype of Covid-19, clarifying the interaction between SARS-Cov-2 and the bacterial flora of the host, and highlighting its dysbiosis and the presence of pathogens that could affect the patient's disease progression and outcome.

Identification of a novel truncating mutation in PALB2 gene by a multigene sequencing panel for mutational screening of breast cancer risk-associated and related genes.

BACKGROUND: Breast cancer (BC) is the most common neoplasm in women, with 5%-10% patients showing a familial predisposition, where germline mutations in BRCA1/BRCA2 genes are found in -20% of cases. Next-generation sequencing (NGS) is among the best available options for genetic screening, providing several benefits that include enhanced sensitivity and unbiased mutation detection. PALB2 (partner and localizer of BRCA2) is a cancer predisposing gene recently described that encodes a protein partner of BRCA2 involved in DNA double-strand break repair and cell cycle control. The DNA damage response represents a key cellular event, targeted by innovative anticancer therapies, including those based on poly (ADP-ribose) polymerase (PARP) inhibitors targeting PARP1 and PARP2 enzymes, activated by DNA damage and involved in single-strand break and base excision repair. METHODS: Genomic DNA was isolated from 34 patient samples and four BC cell lines, as controls, and 27 breast cancer predisposing genes belonging to the BRCA1/BRCA2 and PARP pathways were sequenced by NGS. RESULTS: The panel described here allowed identification of several sequence variations in most investigated genes, among which we found a novel truncating mutation in PALB2. CONCLUSIONS: The NGS-based strategy designed here for molecular analysis of a customized panel of BC predisposing and related genes was found to perform effectively, providing a comprehensive exploration of all genomic sequences of the investigated genes. It is thus useful for BC molecular diagnosis, in particular for familiar cases where alterations in routinely investigated genes, such as BRCAs, result to be absent.

Characterization of two de novoKCNT1 mutations in children with malignant migrating partial seizures in infancy.

The KCNT1 gene encodes for subunits contributing to the Na(+)-activated K(+) current (KNa), expressed in many cell types. Mutations in KCNT1 have been found in patients affected with a wide spectrum of early-onset epilepsies, including Malignant Migrating Partial Seizures in Infancy (MMPSI), a severe early-onset epileptic encephalopathy characterized by pharmacoresistant focal seizures migrating from one brain region or hemisphere to another and neurodevelopment arrest or regression, resulting in profound disability. In the present study we report identification by whole exome sequencing (WES) of two de novo, heterozygous KCNT1 mutations (G288S and, not previously reported, M516V) in two unrelated MMPSI probands. Functional studies in a heterologous expression system revealed that channels formed by mutant KCNT1 subunits carried larger currents when compared to wild-type KCNT1 channels, both as homo- and heteromers with these last. Both mutations induced a marked leftward shift in homomeric channel activation gating. Interestingly, the KCNT1 blockers quinidine (3-1000µM) and bepridil (0.03-10µM) inhibited both wild-type and mutant KCNT1 currents in a concentration-dependent manner, with mutant channels showing higher sensitivity to blockade. This latter result suggests two genotype-tailored pharmacological strategies to specifically counteract the dysfunction of KCNT1 activating mutations in MMPSI patients.

Host nasopharyngeal transcriptome dataset of a SARS-CoV-2 positive Italian cohort.

The ongoing COVID-19 pandemic caused by SARS-CoV-2 has affected millions of people worldwide and has significant implications for public health. Host transcriptomics profiling provides comprehensive understanding of how the virus interacts with host cells and how the host responds to the virus. COVID-19 disease alters the host transcriptome, affecting cellular pathways and key molecular functions. To contribute to the global effort to understand the virus's effect on host cell transcriptome, we have generated a dataset from nasopharyngeal swabs of 35 individuals infected with SARS-CoV-2 from the Campania region in Italy during the three outbreaks, with different clinical conditions. This dataset will help to elucidate the complex interactions among genes and can be useful in the development of effective therapeutic pathways.

Rapid and sensitive detection of SARS-CoV-2 variants in nasopharyngeal swabs and wastewaters.

The SARS-CoV-2 virus is continuously evolving, with appearance of new variants characterized by multiple genomic mutations, some of which can affect functional properties, including infectivity, interactions with host immunity, and disease severity. The rapid spread of new SARS-CoV-2 variants has highlighted the urgency to trace the virus evolution, to help limit its diffusion, and to assess effectiveness of containment strategies. We propose here a PCR-based rapid, sensitive and low-cost allelic discrimination assay panel for the identification of SARS-CoV-2 genotypes, useful for detection in different sample types, such as nasopharyngeal swabs and wastewater. The tests carried out demonstrate that this in-house assay, whose results were confirmed by SARS-CoV-2 whole-genome sequencing, can detect variations in up to 10 viral genome positions at once and is specific and highly sensitive for identification of all tested SARS-CoV-2 clades, even in the case of samples very diluted and of poor quality, particularly difficult to analyze.

Epidemiological and Clinical Features of SARS-CoV-2 Variants Circulating between April-December 2021 in Italy.

SARS-CoV-2 is constantly evolving, leading to new variants. We analysed data from 4400 SARS-CoV-2-positive samples in order to pursue epidemiological variant surveillance and to evaluate their impact on public health in Italy in the period of April-December 2021. The main circulating strain (76.2%) was the Delta variant, followed by the Alpha (13.3%), the Omicron (5.3%), and the Gamma variants (2.9%). The B.1.1 lineages, Eta, Beta, Iota, Mu, and Kappa variants, represented around 1% of cases. There were 48.2% of subjects who had not been vaccinated, and they had a lower median age compared to the vaccinated subjects (47 vs. 61 years). An increasing number of infections in the vaccinated subjects were observed over time, with the highest proportion in November (85.2%). The variants correlated with clinical status; the largest proportion of symptomatic patients (59.6%) was observed with the Delta variant, while subjects harbouring the Gamma variant showed the highest proportion of asymptomatic infection (21.6%), albeit also deaths (5.4%). The Omicron variant was only found in the vaccinated subjects, of which 47% had been hospitalised. The diffusivity and pathogenicity associated with the different SARS-CoV-2 variants are likely to have relevant public health implications, both at the national and international levels. Our study provides data on the rapid changes in the epidemiological landscape of the SARS-CoV-2 variants in Italy.

An Overview of Candidate Therapeutic Target Genes in Ovarian Cancer.

Ovarian cancer (OC) shows the highest mortality rate among gynecological malignancies and, because of the absence of specific symptoms, it is frequently diagnosed at an advanced stage, mainly due to the lack of specific and early biomarkers, such as those based on cancer molecular signature identification. Indeed, although significant progress has been made toward improving the clinical outcome of other cancers, rates of mortality for OC are essentially unchanged since 1980, suggesting the need of new approaches to identify and characterize the molecular mechanisms underlying pathogenesis and progression of these malignancies. In addition, due to the low response rate and the high frequency of resistance to current treatments, emerging therapeutic strategies against OC focus on targeting single factors and pathways specifically involved in tumor growth and metastasis. To date, loss-of-function screenings are extensively applied to identify key drug targets in cancer, seeking for more effective, disease-tailored treatments to overcome lack of response or resistance to current therapies. We review here the information relative to essential genes and functional pathways recently discovered in OC, often strictly interconnected with each other and representing promising biomarkers and molecular targets to treat these malignancies.

The nuclear receptor ERß engages AGO2 in regulation of gene transcription, RNA splicing and RISC loading.

BACKGROUND: The RNA-binding protein Argonaute 2 (AGO2) is a key effector of RNA-silencing pathways It exerts a pivotal role in microRNA maturation and activity and can modulate chromatin remodeling, transcriptional gene regulation and RNA splicing. Estrogen receptor beta (ERß) is endowed with oncosuppressive activities, antagonizing hormone-induced carcinogenesis and inhibiting growth and oncogenic functions in luminal-like breast cancers (BCs), where its expression correlates with a better prognosis of the disease. RESULTS: Applying interaction proteomics coupled to mass spectrometry to characterize nuclear factors cooperating with ERß in gene regulation, we identify AGO2 as a novel partner of ERß in human BC cells. ERß-AGO2 association was confirmed in vitro and in vivo in both the nucleus and cytoplasm and is shown to be RNA-mediated. ChIP-Seq demonstrates AGO2 association with a large number of ERß binding sites, and total and nascent RNA-Seq in ERß + vs ERß - cells, and before and after AGO2 knock-down in ERß + cells, reveals a widespread involvement of this factor in ERß-mediated regulation of gene transcription rate and RNA splicing. Moreover, isolation and sequencing by RIP-Seq of ERß-associated long and small RNAs in the cytoplasm suggests involvement of the nuclear receptor in RISC loading, indicating that it may also be able to directly control mRNA translation efficiency and stability. CONCLUSIONS: These results demonstrate that AGO2 can act as a pleiotropic functional partner of ERß, indicating that both factors are endowed with multiple roles in the control of key cellular functions.

Thymidine kinase-mediated shut down of bone morphogenetic protein-4 expression allows regulated bone production.

Bone morphogenetic Proteins (BMPs) are growth factors also involved in ossification and chondrogenesis that have generated interest for their efficiency in inducing bone neo-synthesis. BMPs expression in engineered cells has been successful in stimulating osteoblastic differentiation and ectopic and orthotopic bone formation in vivo. We have previously shown that an adenoviral vector expressing bone morphogenetic protein type-4 (BMP-4) is able to efficiently drive bone formation in a rabbit model of discontinuous bone lesions. However, unregulated secretion of BMPs has also been implicated in bone overproduction and exostosis. We have constructed a replication-defective first generation adenoviral (FG-Ad) vector containing a cassette for the expression of BMP-4 associated with the Herpes Simplex virus thymidine kinase (TK) gene (FG-B4TK) in order to shut down BMP-4 expression and, therefore, regulate bone production. TK expression does not interfere with BMP-4 ability to induce ectopic bone formation in athymic nude mice. Administration of ganciclovir blocks ectopic bone production in quadriceps muscle transduced with the FG-B4TK with no effect on the contralateral muscle transduced with a vector expressing only BMP-4. Histological findings confirmed the pro-apoptotic activity of TK and the reduction of mineralized areas in the quadriceps transduced with FG-B4TK in mice treated with ganciclovir. We have generated a system to block BMP-4 secretion by inducing apoptosis in transduced cells therefore blocking unwanted bone formation. This system is an additional tool to generate regulated amount of bone in discontinuous bone lesions and can be easily coupled with biomaterials capable of recruiting cells and generating a local bioreactor.