Mammalian tissues display a remarkable complexity of splicing patterns. Nevertheless, only few examples of tissue-specific splicing regulators are known. Herein, we characterize a novel splicing regulator named RBM11, which contains an RNA Recognition Motif (RRM) at the amino terminus and a region lacking known homology at the carboxyl terminus. RBM11 is selectively expressed in brain, cerebellum and testis, and to a lower extent in kidney. RBM11 mRNA levels fluctuate in a developmentally regulated manner, peaking perinatally in brain and cerebellum, and at puberty in testis, in concomitance with differentiation events occurring in neurons and germ cells. Deletion analysis indicated that the RRM of RBM11 is required for RNA binding, whereas the carboxyl terminal region permits nuclear localization and homodimerization. RBM11 is localized in the nucleoplasm and enriched in SRSF2-containing splicing speckles. Transcription inhibition/release experiments and exposure of cells to stress revealed a dynamic movement of RBM11 between nucleoplasm and speckles, suggesting that its localization is affected by the transcriptional status of the cell. Splicing assays revealed a role for RBM11 in the modulation of alternative splicing. In particular, RBM11 affected the choice of alternative 5' splice sites in BCL-X by binding to specific sequences in exon 2 and antagonizing the SR protein SRSF1. Thus, our findings identify RBM11 as a novel tissue-specific splicing factor with potential implication in the regulation of alternative splicing during neuron and germ cell differentiation.

West Nile virus (WNV) is a neurovirulent single stranded RNA mosquito-borne flavivirus, whose main natural hosts are birds, but it also infects humans and horses. Nowadays, no human vaccine is commercially available and clinical treatment is only supportive. Recently, it has been shown that RNA transcripts, mimicking structural domains in the non-coding regions (NCRs) of the foot-and mouth disease virus (FMDV) induce a potent IFN response and antiviral activity in transfected cultured cells, and also reduced mice susceptibility to FMDV. By using different transcripts combinations, administration schedules, and infecting routes and doses, we have demonstrated that these FMDV RNA transcripts protect suckling and adult mice against lethal challenge with WNV. The protective activity induced by the transcripts was systemic and dependent on the infection route and dose. These results confirm the antiviral potential of these synthetic RNAs for fighting viruses of different families relevant for human and animal health.

Riboflavin is essential in numerous cellular oxidation/reduction reactions but is not synthesized by mammalian cells. Riboflavin absorption occurs through the human riboflavin transporters RFVT1 and RFVT3 in the intestine and RFVT2 in the brain. Mutations in these genes are causative for the Brown-Vialetto-Van Laere (BVVL), childhood-onset syndrome characterized by a variety of cranial nerve palsies as well as by spinal cord motor neuron (MN) degeneration. Why mutations in RFVTs result in a neural cell-selective disorder is unclear. As a novel tool to gain insights into the pathomechanisms underlying the disease, we generated MNs from induced pluripotent stem cells (iPSCs) derived from BVVL patients as an in vitro disease model. BVVL-MNs explained a reduction in axon elongation, partially improved by riboflavin supplementation. RNA sequencing profiles and protein studies of the cytoskeletal structures showed a perturbation in the neurofilament composition in BVVL-MNs. Furthermore, exploring the autophagy-lysosome pathway, we observed a reduced autophagic/mitophagic flux in patient MNs. These features represent emerging pathogenetic mechanisms in BVVL-associated neurodegeneration, partially rescued by riboflavin supplementation. Our data showed that this therapeutic strategy could have some limits in rescuing all of the disease features, suggesting the need to develop complementary novel therapeutic strategies.

Numerous infectious diseases impacting livestock impose an important economic burden and in some cases also represent a threat to humans and are classified as zoonoses. Some zoonotic diseases are transmitted by vectors and, due to complex environmental and socio-economic factors, the distribution of many of these pathogens is changing, with increasing numbers being found in previously unaffected countries. Here, we developed a multiplex assay, based on a suspension microarray, able to detect specific antibodies to five important pathogens of livestock (three of them zoonotic) that are currently emerging in new geographical locations: Rift Valley fever virus (RVFV), Crimean-Congo haemorrhagic fever virus (CCHFV), Schmallenberg virus (SBV), Bluetongue virus (BTV) and the bacteria complex Mycobacterium tuberculosis. Using the Luminex platform, polystyrene microspheres were coated with recombinant proteins from each of the five pathogens. The mix of microspheres was used for the simultaneous detection of antibodies against the five corresponding diseases affecting ruminants. The following panel of sera was included in the study: 50 sera from sheep experimentally infected with RVFV, 74 sera from calves and lambs vaccinated with SBV, 26 sera from cattle vaccinated with Mycobacterium bovis, 30 field sera from different species of ruminants infected with CCHFV and 88 calf sera infected with BTV. Finally, to determine its diagnostic specificity 220 field sera from Spanish farms free of the five diseases were assessed. All the sera were classified using commercial ELISAs specific for each disease, used in this study as the reference technique. The results showed the multiplex assay exhibited good performance characteristics with values of sensitivity ranging from 93% to 100% and of specificity ranging from 96% to 99% depending on the pathogen. This new tool allows the simultaneous detection of antibodies against five important pathogens, reducing the volume of sample needed and the time of analysis where these pathogens are usually tested individually.

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that causes an important disease in ruminants, with great economic losses. The infection can be also transmitted to humans; therefore, it is considered a major threat to both human and animal health. In a previous work, we described a novel RVFV variant selected in cell culture in the presence of the antiviral agent favipiravir that was highly attenuated in vivo. This variant displayed 24 amino acid substitutions in different viral proteins when compared to its parental viral strain, two of them located in the NSs protein that is known to be the major virulence factor of RVFV. By means of a reverse genetics system, in this work we have analyzed the effect that one of these substitutions, P82L, has in viral attenuation in vivo. Rescued viruses carrying this single amino acid change were clearly attenuated in BALB/c mice while their growth in an interferon (IFN)-competent cell line as well as the production of interferon beta (IFN-β) did not seem to be affected. However, the pattern of nuclear NSs accumulation was modified in cells infected with the mutant viruses. These results highlight the key role of the NSs protein in the modulation of viral infectivity.

Schmallenberg virus (SBV), an arthropod-transmitted pathogenic bunyavirus, continues to be a threat to the European livestock industry, causing morbidity and mortality among young ruminant livestock. Here, we describe a novel SBV subunit vaccine, based on bacterially expressed SBV nucleoprotein (SBV-N) administered with a veterinary-grade Saponin adjuvant. When assayed in an IFNAR-/- mouse model, SBV-N with Saponin induced strong non-neutralizing broadly virus-reactive antibodies, decreased clinical signs, as well as significantly reduced viremia. Vaccination assays also suggest that this level of immune protection is cell mediated, as evidenced by the lack of neutralizing antibodies, as well as interferon-γ secretion observed in vitro. Therefore, based on these results, bacterially expressed SBV-N, co-administered with veterinary-grade Saponin adjuvant may serve as a promising economical alternative to current SBV vaccines, and warrant further evaluation in large ruminant animal models. Moreover, we propose that this strategy may be applicable to other bunyaviruses.

The existence of individuals who remain cognitively intact despite presenting histopathological signs of Alzheimer's disease (AD), here referred to as "Nondemented with AD neuropathology" (NDAN), suggests that some mechanisms are triggered to resist cognitive impairment. Exposed phosphatidylserine (ePS) represents a neuronal "eat-me" signal involved in microglial-mediated phagocytosis of damaged synapses. A possible mediator of this process is TREM2, a microglial surface receptor activated by ligands including PS. Based on TREM2 role in the scavenging function of microglia, we hypothesize that an efficient microglial phagocytosis of damaged synapses underlies synaptic resilience in NDAN, thus protecting from memory deficits. Using immunofluorescence microscopy, we performed a comparative study of human post-mortem frontal cortices of aged-matched, AD and NDAN individuals. We studied the distribution of activated microglia (IBA1, IBA1+ /CD68+ cells) and phagocytic microglia-related proteins (TREM2, DAP12), demonstrating higher microglial activation and TREM2 expression in NDAN versus AD. A study of the preservation of synapses around plaques, assessed using MAP2 and βIII tubulin as dendritic and axonal markers, respectively, and PSD95 as a postsynaptic marker, revealed preserved axonal/dendritic structure around plaques in NDAN versus AD. Moreover, high levels of PSD95 around NDAN plaques and the colocalization of PSD95 with CD68 indicated a prompt removal of damaged synapses by phagocytic microglia. Furthermore, Annexin V assay on aged-matched, AD and NDAN individuals synaptosomes revealed increased levels of ePS in NDAN, confirming damaged synapses engulfment. Our results suggest a higher efficiency of TREM2-induced phagocytic microglia in removing damaged synapses, underlying synaptic resilience in NDAN individuals.

Several approaches have been developed to analyze the entry of highly pathogenic viruses. In this study, we report the implementation of a Bimolecular Multicellular Complementation (BiMuC) assay to safely and efficiently monitor SARS-CoV-2 S-mediated membrane fusion without the need for microscopy-based equipment. Using BiMuC, we screened a library of approved drugs and identified compounds that enhance S protein-mediated cell-cell membrane fusion. Among them, ethynylestradiol promotes the growth of SARS-CoV-2 and Influenza A virus in vitro. Our findings demonstrate the potential of BiMuC for identifying small molecules that modulate the life cycle of enveloped viruses, including SARS-CoV-2.

The innate immune system is the first line of defense against viral infections. Exploiting innate responses for antiviral, therapeutic and vaccine adjuvation strategies is being extensively explored. We have previously described, the ability of small in vitro RNA transcripts, mimicking the sequence and structure of different domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome (ncRNAs), to trigger a potent and rapid innate immune response. These synthetic non-infectious molecules have proved to have a broad-range antiviral activity and to enhance the immunogenicity of an FMD inactivated vaccine in mice. Here, we have studied the involvement of pattern-recognition receptors (PRRs) in the ncRNA-induced innate response and analyzed the antiviral and cytokine profiles elicited in swine cultured cells, as well as peripheral blood mononuclear cells (PBMCs).

Tubulinopathies constitute a family of neurodevelopmental/neurodegenerative disorders caused by mutations in several genes encoding tubulin isoforms. Loss-of-function mutations in TBCE, encoding one of the five tubulin-specific chaperones involved in tubulin folding and polymerization, cause two rare neurodevelopmental syndromes, hypoparathyroidism-retardation-dysmorphism and Kenny-Caffey syndrome. Although a missense mutation in Tbce has been associated with progressive distal motor neuronopathy in the pmn/pmn mice, no similar degenerative phenotype has been recognized in humans. We report on the identification of an early-onset and progressive neurodegenerative encephalopathy with distal spinal muscular atrophy resembling the phenotype of pmn/pmn mice and caused by biallelic TBCE mutations, with the c.464T>A (p.Ile155Asn) change occurring at the heterozygous/homozygous state in six affected subjects from four unrelated families originated from the same geographical area in Southern Italy. Western blot analysis of patient fibroblasts documented a reduced amount of TBCE, suggestive of rapid degradation of the mutant protein, similarly to what was observed in pmn/pmn fibroblasts. The impact of TBCE mutations on microtubule polymerization was determined using biochemical fractionation and analyzing the nucleation and growth of microtubules at the centrosome and extracentrosomal sites after treatment with nocodazole. Primary fibroblasts obtained from affected subjects displayed a reduced level of polymerized α-tubulin, similarly to tail fibroblasts of pmn/pmn mice. Moreover, markedly delayed microtubule re-polymerization and abnormal mitotic spindles with disorganized microtubule arrangement were also documented. Although loss of function of TBCE has been documented to impact multiple developmental processes, the present findings provide evidence that hypomorphic TBCE mutations primarily drive neurodegeneration.

Spinal muscular atrophy (SMA) is a motor neuron disease caused by loss of function mutations in the Survival Motor Neuron 1 (SMN1) gene and reduced expression of the SMN protein, leading to spinal motor neuron death, muscle weakness and atrophy. Although humans harbour the highly homologous SMN2 gene, its defective splicing regulation yields a truncated and unstable SMN protein. The first therapy for SMA was recently approved by the Food and Drug Administration and consists of an antisense oligonucleotide (Nusinersen) rendering SMN2 functional and thus improving patients' motor activity and quality of life. Nevertheless, not all patients equally respond to this therapy and the long-term tolerability and safety of Nusinersen are still unknown. Herein, in vivo splicing assays indicated that the HDAC inhibitor LBH589 is particularly efficient in rescuing the SMN2 splicing defect in SMA fibroblasts and SMA type-I mice-derived neural stem cells. Western blot analyses showed that LBH589 also causes a significant increase in SMN protein expression in SMA cells. Moreover chromatin immunoprecipitation analyses revealed that LBH589 treatment induces widespread H4 acetylation of the entire SMN2 locus and selectively favors the inclusion of the disease-linked exon 7 in SMN2 mature mRNA. The combined treatment of SMA cells with sub-optimal doses of LBH589 and of an antisense oligonucleotide that mimic Nusinersen (ASO_ISSN1) elicits additive effects on SMN2 splicing and SMN protein expression. These findings suggest that HDAC inhibitors can potentiate the activity of Nusinersen and support the notion that 'SMN-plus' combinatorial therapeutic approaches might represent an enhanced opportunity in the scenario of SMA therapy.

In vitro neutralizing antibodies have been often correlated with protection against Rift Valley fever virus (RVFV) infection. We have reported previously that a single inoculation of sucrose-purified modified vaccinia Ankara (MVA) encoding RVFV glycoproteins (rMVAGnGc) was sufficient to induce a protective immune response in mice after a lethal RVFV challenge. Protection was related to the presence of glycoprotein specific CD8+ cells, with a low-level detection of in vitro neutralizing antibodies. In this work we extended those observations aimed to explore the role of humoral responses after MVA vaccination and to study the contribution of each glycoprotein antigen to the protective efficacy. Thus, we tested the efficacy and immune responses in BALB/c mice of recombinant MVA viruses expressing either glycoprotein Gn (rMVAGn) or Gc (rMVAGc). In the absence of serum neutralizing antibodies, our data strongly suggest that protection of vaccinated mice upon the RVFV challenge can be achieved by the activation of cellular responses mainly directed against Gc epitopes. The involvement of cellular immunity was stressed by the fact that protection of mice was strain dependent. Furthermore, our data suggest that the rMVA based single dose vaccination elicits suboptimal humoral immune responses against Gn antigen since disease in mice was exacerbated upon virus challenge in the presence of rMVAGnGc or rMVAGn immune serum. Thus, Gc-specific cellular immunity could be an important component in the protection after the challenge observed in BALB/c mice, contributing to the elimination of infected cells reducing morbidity and mortality and counteracting the deleterious effect of a subneutralizing antibody immune response.

Riboflavin transporter deficiency (RTD) is a childhood-onset neurodegenerative disorder characterized by progressive pontobulbar palsy, sensory and motor neuron degeneration, sensorineural hearing loss, and optic atrophy. As riboflavin (RF) is the precursor of FAD and FMN, we hypothesize that both mitochondrial and peroxisomal energy metabolism pathways involving flavoproteins could be directly affected in RTD, thus impacting cellular redox status. In the present work, we used induced pluripotent stem cells (iPSCs) from RTD patients to investigate morphofunctional features, focusing on mitochondrial and peroxisomal compartments. Using this model, we document the following RTD-associated alterations: (i) abnormal colony-forming ability and loss of cell-cell contacts, revealed by light, electron, and confocal microscopy, using tight junction marker ZO-1; (ii) mitochondrial ultrastructural abnormalities, involving shape, number, and intracellular distribution of the organelles, as assessed by focused ion beam/scanning electron microscopy (FIB/SEM); (iii) redox imbalance, with high levels of superoxide anion, as assessed by MitoSOX assay accompanied by abnormal mitochondrial polarization state, evaluated by JC-1 staining; (iv) altered immunofluorescence expression of antioxidant systems, namely, glutathione, superoxide dismutase 1 and 2, and catalase, as assessed by quantitatively evaluated confocal microscopy; and (v) peroxisomal downregulation, as demonstrated by levels and distribution of fatty acyl β-oxidation enzymes. RF supplementation results in amelioration of cell phenotype and rescue of redox status, which was associated to improved ultrastructural features of mitochondria, thus strongly supporting patient treatment with RF, to restore mitochondrial- and peroxisomal-related aspects of energy dysmetabolism and oxidative stress in RTD syndrome.

Nuclear integrity and mechanical stability of the nuclear envelope (NE) are conferred by the nuclear lamina, a meshwork of intermediate filaments composed of A- and B-type lamins, supporting the inner nuclear membrane and playing a pivotal role in chromatin organization and epigenetic regulation. During cell senescence, nuclear alterations also involving NE architecture are widely described. In the present study, we utilized induced pluripotent stem cells (iPSCs) upon prolonged in vitro culture as a model to study aging and investigated the organization and expression pattern of NE major constituents. Confocal and four-dimensional imaging combined with molecular analyses, showed that aged iPSCs are characterized by nuclear dysmorphisms, nucleoskeletal components (lamin A/C-prelamin isoforms, lamin B1, emerin, and nesprin-2) imbalance, leading to impaired nucleo-cytoplasmic MKL1 shuttling, actin polymerization defects, mitochondrial dysfunctions, SIRT7 downregulation and NF-kBp65 hyperactivation. The observed age-related NE features of iPSCs closely resemble those reported for premature aging syndromes (e.g., Hutchinson-Gilford progeria syndrome) and for somatic cell senescence. These findings validate the use of aged iPSCs as a suitable cellular model to study senescence and for investigating therapeutic strategies aimed to treat premature aging.

Inorganic materials can provide a set of tools to decontaminate solid, liquid or air containing viral particles. The use of disinfectants can be limited or not practical in scenarios where continuous cleaning is not feasible. Physicochemical differences between viruses raise the need for effective formulations for all kind of viruses. In the present work we describe two types of antimicrobial inorganic materials: i) a novel soda-lime glass (G3), and ii) kaolin containing metals nanoparticles (Ag or CuO), as materials to disable virus infectivity. Strong antiviral properties can be observed in G3 glass, and kaolin-containing nanoparticle materials showing a reduction of viral infectivity close to 99%. in the first 10 ​min of contact of vesicular stomatitis virus (VSV). A potent virucidal activity is also present in G3 and kaolin containing Ag or CuO nanoparticles against all kinds of viruses tested, reducing more than 99% the amount of HSV-1, Adenovirus, VSV, Influenza virus and SARS-CoV-2 exposed to them. Virucidal properties could be explained by a direct interaction of materials with viruses as well as inactivation by the presence of virucidal elements in the material lixiviates. Kaolin-based materials guarantee a controlled release of active nanoparticles with antiviral activity. Current coronavirus crisis highlights the need for new strategies to remove viruses from contaminated areas. We propose these low-cost inorganic materials as useful disinfecting antivirals in the actual or future pandemic threats.

Bluetongue, caused by bluetongue virus (BTV), is a widespread arthropod-borne disease of ruminants that entails a recurrent threat to the primary sector of developed and developing countries. In this work, we report modified vaccinia virus Ankara (MVA) and ChAdOx1-vectored vaccines designed to simultaneously express the immunogenic NS1 protein and/or NS2-Nt, the N-terminal half of protein NS2 (NS21-180). A single dose of MVA or ChAdOx1 expressing NS1-NS2-Nt improved the protection conferred by NS1 alone in IFNAR(-/-) mice. Moreover, mice immunized with ChAdOx1/MVA-NS1, ChAdOx1/MVA-NS2-Nt, or ChAdOx1/MVA-NS1-NS2-Nt developed strong cytotoxic CD8+ T-cell responses against NS1, NS2-Nt, or both proteins and were fully protected against a lethal infection with BTV serotypes 1, 4, and 8. Furthermore, although a single immunization with ChAdOx1-NS1-NS2-Nt partially protected sheep against BTV-4, the administration of a booster dose of MVA-NS1-NS2-Nt promoted a faster viral clearance, reduction of the period and level of viremia and also protected from the pathology produced by BTV infection. IMPORTANCE Current BTV vaccines are effective but they do not allow to distinguish between vaccinated and infected animals (DIVA strategy) and are serotype specific. In this work we have develop a DIVA multiserotype vaccination strategy based on adenoviral (ChAdOx1) and MVA vaccine vectors, the most widely used in current phase I and II clinical trials, and the conserved nonstructural BTV proteins NS1 and NS2. This immunization strategy solves the major drawbacks of the current marketed vaccines.

Telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase holoenzyme, which adds telomeric DNA repeats on chromosome ends to counteract telomere shortening. In addition, there is evidence of TERT non-canonical functions, among which is an antioxidant role. In order to better investigate this role, we tested the response to X-rays and H2O2 treatment in hTERT-overexpressing human fibroblasts (HF-TERT). We observed in HF-TERT a reduced induction of reactive oxygen species and an increased expression of the proteins involved in the antioxidant defense. Therefore, we also tested a possible role of TERT inside mitochondria. We confirmed TERT mitochondrial localization, which increases after oxidative stress (OS) induced by H2O2 treatment. We next evaluated some mitochondrial markers. The basal mitochondria quantity appeared reduced in HF-TERT compared to normal fibroblasts and an additional reduction was observed after OS; nevertheless, the mitochondrial membrane potential and morphology were better conserved in HF-TERT. Our results suggest a protective function of TERT against OS, also preserving mitochondrial functionality.

The COVID-19 pandemic has underscored the importance of swift responses and the necessity of dependable technologies for vaccine development. Our team previously developed a fast cloning system for the modified vaccinia virus Ankara (MVA) vaccine platform. In this study, we reported on the construction and preclinical testing of a recombinant MVA vaccine obtained using this system. We obtained recombinant MVA expressing the unmodified full-length SARS-CoV-2 spike (S) protein containing the D614G amino-acid substitution (MVA-Sdg) and a version expressing a modified S protein containing amino-acid substitutions designed to stabilize the protein a in a pre-fusion conformation (MVA-Spf). S protein expressed by MVA-Sdg was found to be expressed and was correctly processed and transported to the cell surface, where it efficiently produced cell-cell fusion. Version Spf, however, was not proteolytically processed, and despite being transported to the plasma membrane, it failed to induce cell-cell fusion. We assessed both vaccine candidates in prime-boost regimens in the susceptible transgenic K18-human angiotensin-converting enzyme 2 (K18-hACE2) in mice and in golden Syrian hamsters. Robust immunity and protection from disease was induced with either vaccine in both animal models. Remarkably, the MVA-Spf vaccine candidate produced higher levels of antibodies, a stronger T cell response, and a higher degree of protection from challenge. In addition, the level of SARS-CoV-2 in the brain of MVA-Spf inoculated mice was decreased to undetectable levels. Those results add to our current experience and range of vaccine vectors and technologies for developing a safe and effective COVID-19 vaccine.

Coronavirus disease 2019 (COVID-19) is an infectious zoonotic disease caused by SARS-CoV-2. Monitoring the infection in pets is recommended for human disease surveillance, prevention, and control since the virus can spread from people to animals during close contact. Several diagnostic tests have been adapted from humans to animals, but limited data on the validation process are available.

PCDH19-related epilepsy is a rare genetic disease caused by defective function of PCDH19, a calcium-dependent cell-cell adhesion protein of the cadherin superfamily. This disorder is characterized by a heterogeneous phenotypic spectrum, with partial and generalized febrile convulsions that are gradually increasing in frequency. Developmental regression may occur during disease progression. Patients may present with intellectual disability (ID), behavioral problems, motor and language delay, and a low motor tone. In most cases, seizures are resistant to treatment, but their frequency decreases with age, and some patients may even become seizure-free. ID generally persists after seizure remission, making neurological abnormalities the main clinical issue in affected individuals. An effective treatment is lacking. In vitro studies using patient-derived induced pluripotent stem cells (iPSCs) reported accelerated neural differentiation as a major endophenotype associated with PCDH19 mutations. By using this in vitro model system, we show that accelerated in vitro neurogenesis is associated with a defect in the cell division plane at the neural progenitors stage. We also provide evidence that altered PCDH19 function affects proper mitotic spindle orientation. Our findings identify an altered equilibrium between symmetric versus asymmetric cell division as a previously unrecognized mechanism contributing to the pathogenesis of this rare epileptic encephalopathy.