Early reduction of the splicing factor2/alternative splicing factor: a cellular inhibitor of the JC polyomavirus in natalizumab-treated MS patients long before developing progressive multifocal leukoencephalopathy.

Natalizumab is effective against relapsing-remitting multiple sclerosis (MS) but increases the risk of progressive multifocal leukoencephalopathy (PML), which is caused by the activation of the JCV polyomavirus. SF2/ASF (splicing factor2/alternative splicing factor) is a potent cellular inhibitor of JCV replication and large T-antigen (T-Ag) expression. We reported that SF2/ASF levels in blood cells increase during the first year of natalizumab therapy and decrease thereafter, inversely related to T-Ag expression, and suggested a correlation with JCV reactivation. Here, we report SF2/ASF levels of longitudinal blood samples of two patients undergoing natalizumab therapy, who developed PML while monitored, in comparison to natalizumab-treated controls and to one-off PML samples. After 6 months of therapy, SF2/ASF levels of the two cases were reduced, instead of increased, and their overall SF2/ASF levels were lower than those from natalizumab controls. Since SF2/ASF inhibits JCV, its early reduction might have a role in subsequent PML. We are aware of the limitations of the study, but the uniqueness of serial blood samples collected before and after PML onset in natalizumab-treated patients must be stressed. If confirmed in other patients, SF2/ASF evaluation could be a new and early biomarker of natalizumab-associated PML risk, allowing an 18-24-month interval before PML onset (presently ~ 5 months), in which clinicians could evaluate other risk factors and change therapy.

MicroRNA-425-5p Expression Affects BRAF/RAS/MAPK Pathways In Colorectal Cancers.

Colorectal cancer (CRC) is a leading cause of cancer death worldwide and about 20% is metastatic at diagnosis and untreatable. The anti-EGFR therapy in metastatic patients is led by the presence of KRAS-mutations in tumor tissue. KRAS-wild-type CRC patients showed a positive response rate of about 70% to cetuximab or panitumumab combined with chemotherapy. MiRNAs are promising markers in oncology and could improve our knowledge on pathogenesis and drug resistance in CRC patients. This class of molecules represents an opportunity for the development of miRNA-based strategies to overcome the ineffectiveness of anti-EGFR therapy. We performed an integrative analysis of miRNA expression profile between KRAS-mutated CRC and KRAS-wildtype CRC and paired normal colic tissue (NCT). We revealed an overexpression of miR-425-5p in KRAS-mutated CRC compared to KRAS-wild type CRC and NCT and demonstrated that miR-425-5p exerts regulatory effects on target genes involved in cellular proliferation, migration, invasion, apoptosis molecular networks. These epigenetic mechanisms could be responsible of the strong aggressiveness of KRAS-mutated CRC compared to KRAS-wildtype CRC. We proved that some miR-425-5p targeted genes are involved in EGFR tyrosine kinase inhibitor resistance pathway, suggesting that therapies based on miR-425-5p may have strong potential in targeting KRAS-driven CRC. Moreover, we demonstrated a role in the oncogenesis of miR-31-5p, miR-625-5p and miR-579 by comparing CRC versus NCT. Our results underlined that miR-425-5p might act as an oncogene to participate in the pathogenesis of KRAS-mutated CRC and contribute to increase the aggressiveness of this subcategory of CRC, controlling a complex molecular network.

Expression of HERV Genes as Possible Biomarker and Target in Neurodegenerative Diseases.

Human endogenous retroviruses (HERVs) are genetic parasites, in-between genetics and environment. Few HERVs retain some coding capability. Sometimes, the host has the advantage of some HERV genes; conversely, HERVs may contribute to pathogenesis. The expression of HERVs depends on several factors, and is regulated epigenetically by stimuli such as inflammation, viral and microbial infections, etc. Increased expression of HERVs occurs in physiological and pathological conditions, in one or more body sites. Several diseases have been attributed to one or more HERVs, particularly neurological diseases. The key problem is to differentiate the expression of a HERV as cause or effect of a disease. To be used as a biomarker, a correlation between the expression of a certain HERV and the disease onset and/or behavior must be found. The greater challenge is to establish a pathogenic role. The criteria defining causal connections between HERVs and diseases include the development of animal models, and disease modulation in humans, by anti-HERV therapeutic antibody. So far, statistically significant correlations between HERVs and diseases have been achieved for HERV-W and multiple sclerosis; disease reproduction in transgenic animals was achieved for HERV-W and multiple sclerosis, and for HERV-K and amyotrophic lateral sclerosis. Clinical trials for both diseases are in progress.

Multiple Signatures of the JC Polyomavirus in Paired Normal and Altered Colorectal Mucosa Indicate a Link with Human Colorectal Cancer, but Not with Cancer Progression.

The JC polyomavirus (JCV) has been repeatedly but discordantly detected in healthy colonic mucosa, adenomatous polyps, and colorectal cancer (CRC), and proposed to contribute to oncogenesis. The controversies may derive from differences in JCV targets, patient's cohorts, and methods. Studies of simultaneous detection, quantification, and characterization of JCV presence/expression in paired samples of normal/altered tissues of the same patient are lacking. Therefore, we simultaneously quantified JCV presence (DNA) and expression (mRNA and protein) of T-antigen (T-Ag), Viral Protein 1 (Vp1), and miR-J1-5p in paired normal/altered tissues of CRC or polyps, and from controls. JCV signatures were found in most samples. They increased in patients, but were higher in normal mucosa than in corresponding polyp or CRC lesions. JCV non-coding control region (NCCR) DNA rearrangements increased in CRC patients, also in normal mucosa, thus before the onset of the lesion. A new ∆98bp NCCR DNA rearrangement was detected. T-Ag levels were higher in normal mucosa than in adenoma and adenocarcinoma lesions, but decreased to levels of controls in established CRC lesions. In CRC, miR-J1-5p expression decreased with CRC progression. Vp1 expression was not detected. The data indicate a JCV link with the disease, but possible JCV contributes to oncogenesis should occur at pre-polyp stages.

Integrated Analysis of miRNA and mRNA Endorses a Twenty miRNAs Signature for Colorectal Carcinoma.

Colorectal cancer (CRC) ranks as the most frequent carcinoma worldwide. CRC patients show strong prognostic differences and responses to treatment, and 20% have incurable metastatic disease at diagnosis. We considered it essential to investigate mechanisms that control cellular regulatory networks, such as the miRNA-mRNA interaction, known to be involved in cancer pathogenesis. We conducted a human miRNome analysis by TaqMan low density array, comparing CRC to normal colon tissue (NCT, and experimentally identified gene targets of miRNAs deregulated, by anti-correlation analysis, with the CRC whole-transcriptome profile obtained from RNASeq experiments. We identified an integrated signature of 20 deregulated miRNAs in CRC. Enrichment analyses of the gene targets controlled by these miRNAs brought to light 25 genes, members of pathways known to lead to cell growth and death (CCND1, NKD1, FZD3, MAD2L1, etc.), such as cell metabolism (ACSL6, PRPS1-2). A screening of prognosis-mediated miRNAs underlined that the overexpression of miR-224 promotes CRC metastasis, and is associated with high stage and poor survival. These findings suggest that the biology and progression of CRC depend on deregulation of multiple miRNAs that cause a complex dysfunction of cellular molecular networks. Our results have further established miRNA-mRNA interactions and defined multiple pathways involved in CRC pathogenesis.

The EGF epidermal growth factor counteracts Tat modulation of human endogenous retroviruses of the W family in astrocytes.

Human astrocyte cells were exposed to HIV-Tat and/or epidermal growth factor (EGF), to monitor the expression of the neuropathogenic MSRV and Syncytin-1 elements of the HERV-W family of endogenous retroviruses and of TNFα. The results indicate that EGF counteracts Tat regulation of HERV-W/MSRVenv/Syncytin-1, throughout EGFR activation; this effect occurs by interfering with the induction of TNFα production by Tat. The novel effect of EGF counteraction of Tat-mediated regulation of the neuropathogenic HERV-Ws could be neuro-protective, but its actual role in the brain remains to be elucidated.

JC polyomavirus expression and bell-shaped regulation of its SF2/ASF suppressor during the follow-up of multiple sclerosis patients treated with natalizumab.

Natalizumab is effective against multiple sclerosis (MS), but is associated with progressive multifocal leukoencephalopathy (PML), fatal disease caused by the JCV polyomavirus. The SF2/ASF (splicing factor2/alternative splicing factor) inhibits JCV in glial cells. We wondered about SF2/ASF modulation in the blood of natalizumab-treated patients and if this could influence JCV reactivation. Therefore, we performed a longitudinal study of MS patients under natalizumab, in comparison to patients under fingolimod and to healthy blood donors. Blood samples were collected at time intervals. The expression of SF2/ASF and the presence and expression of JCV in PBMC were analyzed. A bell-shaped regulation of SF2/ASF was observed in patients treated with natalizumab, increased in the first year of therapy, and reduced in the second one, while slightly changed, if any, in patients under fingolimod. Notably, SF2/ASF was up-regulated, during the first year, only in JCV DNA-positive patients, or with high anti-JCV antibody response; the expression of the JCV T-Ag protein in circulating B cells was inversely related to SF2/ASF protein expression. The SF2/ASF reduction, parallel to JCV activation, during the second year of therapy with natalizumab, but not with fingolimod, may help explain the increased risk of PML after the second year of treatment with natalizumab, but not with fingolimod. We propose that SF2/ASF has a protective role against JCV reactivation in MS patients. This study suggests new markers of disease behavior and, possibly, help in re-evaluations of therapy protocols.

The DNA damage response promotes polyomavirus JC infection by nucleus to cytoplasm NF- kappaB activation.

BACKGROUND: Infection of glial cells by human neurotropic polyomavirus JC (JCV), the causative agent of the CNS demyelinating disease progressive multifocal leukoencephalopathy (PML), rapidly inflicts damage to cellular DNA. This activates DNA damage response (DDR) signaling including induction of expression of DNA repair factor Rad51. We previously reported that Rad51 co-operates with the transcription factor NF-κB p65 to activate JCV early transcription. Thus Rad51 induction by JCV infection may provide positive feedback for viral activation early in JCV infection. DDR is also known to stimulate NF-κB activity, a phenomenon known as nucleus to cytoplasm or "inside-out" NF-κB signaling, which is initiated by Ataxia telangiectasia mutated (ATM) protein, a serine/threonine kinase recruited and activated by DNA double-strand breaks. Downstream of ATM, there occurs a series of post-translational modifications of NF-κB essential modulator (NEMO), the γ regulatory subunit of inhibitor of NF-κB (IκB) kinase (IKK), resulting in NF-κB activation. METHODS: We analyzed the effects of downstream pathways in the DDR by phosphospecific Western blots and analysis of the subcellular distribution of NEMO by cell fractionation and immunocytochemistry. The role of DDR in JCV infection was analyzed using a small molecule inhibitor of ATM (KU-55933). NEMO sumoylation was investigated by Western and association of ATM and NEMO by immunoprecipitation/Western blots. RESULTS: We show that JCV infection caused phosphorylation and activation of ATM while KU-55933 inhibited JCV replication. JCV infection caused a redistribution of NEMO from cytoplasm to nucleus. Co-expression of JCV large T-antigen and FLAG-tagged NEMO showed the occurrence of sumoylation of NEMO, while co-expression of ATM and FLAG-NEMO demonstrated physical association between ATM and NEMO. CONCLUSIONS: We propose a model where JCV infection induces both overexpression of Rad51 protein and activation of the nucleus to cytoplasm NF-κB signaling pathway, which then act together to enhance JCV gene expression.

An outbreak of COVID-19 after a pilgrimage to Medjugorje due to Delta sub-lineages.

INTRODUCTION: A COVID-19 outbreak occurred at the end of October 2021 among pilgrims returning from Medjugorje (Bosnia and Herzegovina). METHODOLOGY: Whole genome sequencing (WGS) of SARS-CoV-2, epidemiological data, and phylogenetic analysis were used to reconstruct outbreak dynamics. RESULTS: The results suggest that only in one case, associated with the SARS-CoV-2 sub-lineage AY.9.2, it is possible to trace back the place of contagion to Medjugorje, while the other cases were likely to be acquired in the country of origin. CONCLUSIONS: The combined use of phylogenetic data derived from WGS, and epidemiological data allowed us to study epidemic dynamics and to formulate a possible hypothesis on the place of exposure to SARS-CoV-2. The identification of different sub-lineages of the SARS-CoV-2 Delta variant also suggested that different chains of transmission contributed to the outbreak.

Genomic Snapshot of SARS-CoV-2 in Migrants Entering Through Mediterranean Sea Routes.

In December 2019, a novel coronavirus emerged in Wuhan, China, rapidly spreading into a global pandemic. Italy was the first European country to experience SARS-CoV-2 epidemic, and one of the most severely affected during the first wave of diffusion. In contrast to the general restriction of people movements in Europe, the number of migrants arriving at Italian borders via the Mediterranean Sea route in the summer of 2020 had increased dramatically, representing a possible, uncontrolled source for the introduction of novel SARS-CoV-2 variants. Importantly, most of the migrants came from African countries showing limited SARS-CoV-2 epidemiological surveillance. In this study, we characterized the SARS-CoV-2 genome isolated from an asymptomatic migrant arrived in Sardinia via the Mediterranean route in September 2020, in comparison with SARS-CoV-2 isolates arrived in Sicily through the Libyan migration route; with SARS-CoV-2 isolates circulating in Sardinia during 2020; and with viral genomes reported in African countries during the same summer. Results showed that our sequence is not phylogenetically related to isolates from migrants arriving in Sicily, nor to isolates circulating in Sardinia territory, having greater similarity to SARS-CoV-2 genomes reported in countries known for being sites of migrant embarkation to Italy. This is in line with the hypothesis that most SARS-CoV-2 infections among migrants have been acquired prior to embarking to Italy, possibly during the travel to or the stay in crowded Libyan immigrant camps. Overall, these observations underline the importance of dedicated SARS-CoV-2 surveillance of migrants arriving in Italy and in Europe through the Mediterranean routes.

A straightforward molecular strategy to retrospectively investigate the spread of SARS-CoV-2 VOC202012/01 B.1.1.7 variant.

The spread of new SARS-CoV-2 variants represents a serious threat worldwide, thus rapid and cost-effective methods are required for their identification. Since November 2020, the TaqPath COVID-19 assay (Thermo Fisher Scientific) has been used to identify viral strains of the new lineage B.1.1.7, since it fails to detect the S-gene with the ∆69/70 deletion. Here, we proposed S-gene mutations screening with the Allplex SARS-CoV-2 assay (Seegene), another widely used RT-PCR test that targets Sarbecovirus E, SARS-CoV-2 N, and RdRp/S genes. Accordingly, we evaluated the S gene amplification curve pattern compared to those of the other genes. Exploiting an Allplex assay-generated dataset, we screened 663 RT-PCR digital records, including all SARS-CoV-2 respiratory samples tested in our laboratory with the Allplex assay between January 1st and February 25th, 2021. This approach enabled us to detect 64 samples with peculiar non-sigmoidal amplification curves. Sequencing a selected group of 4 RNA viral genomes demonstrated that those curves were associated with B.1.1.7 variant strains. Our results strongly suggest that B.1.1.7 variant spread has begun in this area at least since January and imply the potential of these analytical methods to track and characterize the spread of B.1.1.7 strains in those areas where Allplex SARS-CoV-2 datasets have been previously recorded.

"Swab Team" in the SARS-CoV-2 outbreak containment among healthcare workers.

INTRODUCTION: To analyze the virus spread among Sassari Hospital staff in the first Covid-19 wave and the impact of the Swab Team, a multidisciplinary task force entitled of nasopharyngeal swab collection and testing. METHODOLOGY: Nasopharyngeal swabs from HCWs between March 6 and May 28 2020 are evaluated. RESULTS: 4919 SARS-CoV-2 tests were performed on 3521 operators. Nurses and doctors are the categories at highest risk. After the Swab Team institution, the average number of swabs raised from 47/day to 86/day (p = 0.007). Positive samples decreased from 18.6% to 1.7% (p < 0.0001). CONCLUSIONS: The Swab Team is effective in increasing the cases tested and in reducing the reporting time. Procedure standardization reduces the risk for all the subjects involved (no transmission among swab team members, nor during the sample collection).

The Inhibitory Role of miR-486-5p on CSC Phenotype Has Diagnostic and Prognostic Potential in Colorectal Cancer.

Colorectal cancer (CRC) is the third most frequent cancer worldwide and the second cause of cancer deaths. Increasing evidences supports the idea that the poor prognosis of patients is related to the presence of cancer stem cells (CSCs), a cell population able to drive cancer recurrence and metastasis. The deregulation of microRNAs (miRNAs) plays a role in the formation of CSC. We investigated the role of hsa-miR-486-5p (miR-486-5p) in CRC, CSCs, and metastasis, in order to reach a better understanding of the biomolecular and epigenetic mechanisms mir-486-5p-related. The expression of miR-486-5p was investigated in three different matrices from CRC patients and controls and in CSCs obtained from the CRC cell lines HCT-116, HT-29, and T-84. In the human study, miR-486-5p was up-regulated in serum and stool of CRC patients in comparison with healthy controls but down-regulated in tumor tissue when compared with normal mucosa. miR-486-5p was also down-regulated in the sera of metastatic patients. In vitro, miR-486-5p was down-regulated in CSC models and it induced an inhibitory effect on stem factors and oncogenes in the main pathways of CSCs. Our results provide a step forward in understanding the role of mir-486-5p in CRC and CSC, and suggest that further studies are needed to investigate its diagnostic and prognostic power, possibly in combination with other biomarkers.

Disruption by SaCas9 Endonuclease of HERV-Kenv, a Retroviral Gene with Oncogenic and Neuropathogenic Potential, Inhibits Molecules Involved in Cancer and Amyotrophic Lateral Sclerosis.

The human endogenous retrovirus (HERV)-K, human mouse mammary tumor virus like-2 (HML-2) subgroup of HERVs is activated in several tumors and has been related to prostate cancer progression and motor neuron diseases. The cellular splicing factor 2/alternative splicing factor (SF2/ASF) is a positive regulator of gene expression, coded by a potent proto-oncogene, amplified, and abnormally expressed in tumors. TAR DNA-binding protein-43 (TDP-43) is a DNA/RNA-binding protein, negative regulator of alternative splicing, known for causing neurodegeneration, and with complex roles in oncogenesis. We used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology, with the Cas9 system from Staphylococcus aureus (SaCas9), to disrupt the HERV-K(HML-2)env gene, and evaluated the effects on cultured cells. The tool was tested on human prostate cancer LNCaP cells, whose HERV-Kenv transcription profile is known. It caused HERV-K(HML-2)env disruption (the first reported of a HERV gene), as evaluated by DNA sequencing, and inhibition of env transcripts and proteins. The HERV-K(HML-2)env disruption was found to interfere with important regulators of cell expression and proliferation, involved in manaling, RNA-binding, and alternative splicing, such as epidermal growth factor receptor (EGF-R), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), SF2/ASF, and TDP-43. These novel findings suggest that HERV-K is not an innocent bystander, they reinforce its links to oncogenesis and motor neuron diseases, and they open potential innovative therapeutic options.

The aliens inside human DNA: HERV-W/MSRV/syncytin-1 endogenous retroviruses and neurodegeneration.

The human genome contains remnants of ancestral retroviruses now endogenously transmitted, called human endogenous retroviruses (HERVs). HERVs can be variably expressed, and both beneficial and detrimental effects have described. This review focuses on the MSRV and syncytin-1 HERV-W elements in relationship to neurodegeneration in view of their neuro-pathogenic and immune-pathogenic properties. Multiple sclerosis (MS) and a neurodegenerative disease (neuroAIDS) are reported in this review. In vivo studies in patients and controls for molecular epidemiology and follow-up studies are reviewed, along with in vitro cellular studies of the effects of treatments and of molecular mechanisms. HERV-W/MSRV has been repeatedly found in MS patients (in blood, spinal fluid, and brain samples), and MRSV presence/load strikingly parallels MS stages and active/remission phases, as well as therapy outcome. The DNA of MS patients has increased MSRVenv copies, while syncytin-1 copies are unchanged in controls. Presence of MSRV in the spinal fluid predicted the worst MS progression, ten years in advance. The Epstein-Barr virus (EBV) activates HERV-W/MSRV both in vitro and in vivo. With respect to neuroAIDS, the HIV transactivator of transcription (Tat) protein activates HERV-W/MSRV in monocytes/macrophages and astrocytes indirectly by interaction with TLR4 and induction of TNFa. HERV-W/MSRV can be considered a biomarker for MS behavior and therapy outcome. Regarding MS pathogenesis, we postulate the possibility for EBV of an initial trigger of future MS, years later, and for MSRV of a direct role of effector of neuropathogenesis during MS. Additionally, HERV-W/MSR/syncytin-1 activation by HIV Tat could contribute to the HIV-related neurodegeneration.