Giulia Riolo

Since 2001, human Metapneumovirus has been a significant cause of human respiratory disease worldwide, and no vaccine or preventive treatment is currently available. The ELISA-based live virus microneutralization assay is a method to detect neutralizing antibodies against a target pathogen. The aim of this study was to demonstrate the suitability of this approach to quantifying neutralizing antibodies against A1 and B1 virus subtypes in human serum samples. To standardize and validate this microneutralization assay, we carried out analytical procedures according to the International Council of Harmonization guidelines; these procedures are described in detail. In addition, we compared the validated method with the indirect ELISA, and confirmed that the ELISA-based microneutralization assay provides reliable, accurate and reproducible results. The use of this high-throughput method for large-scale serological studies could effectively support the evaluation of the immunogenicity of new vaccines, thereby improving therapeutical strategies against human Metapneumovirus.

Amyotrophic Lateral Sclerosis (ALS) is characterized by the progressive degeneration of upper or lower motor neurons, leading to muscle wasting and paralysis, resulting in respiratory failure and death. The precise ALS aetiology is poorly understood, mainly due to clinical and genetic heterogeneity. Thus, the identification of reliable biomarkers of disease could be helpful in clinical practice. In this study, we investigated whether the levels of brain-derived neurotrophic factor (BDNF) and its precursor Pro-BDNF in serum and cerebrospinal fluid (CSF) may reflect the pathological changes related to ALS. We found higher BDNF and lower Pro-BDNF levels in ALS sera compared to healthy controls. BDNF/Pro-BDNF ratio turned out to be accurate in distinguishing ALS patients from controls. Then, the correlations of these markers with several ALS clinical variables were evaluated. This analysis revealed three statistically significant associations: (1) Patients carrying the C9orf72 expansion significantly differed from non-carrier patients and showed serum BDNF levels comparable to control subjects; (2) BDNF levels in CSF were significantly higher in ALS patients with faster disease progression; (3) lower serum levels of Pro-BDNF were associated with a shorter survival. Therefore, we suggest that BDNF and Pro-BDNF, alone or in combination, might be used as ALS prognostic biomarkers.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal disorder characterized by degeneration of motor neurons in the cerebral cortex, brain stem, and spinal cord. Most cases of ALS appear sporadically, but 5–10% of patients have a family history of disease. Mutations in the superoxide dismutase 1 gene (SOD1) have been found in 12–23% of familial cases and in 1–2% of sporadic cases. Currently, more than 180 different SOD1 gene variants have been identified in ALS patients. Here, we describe two apparently sporadic ALS patients carrying the same SOD1 c.355G>A variant, leading to the p.V119M substitution, not previously described. Both the patients showed pure lower motor neuron phenotype. The former presented with the flail leg syndrome, a rare ALS variant, characterized by progressive distal onset weakness and atrophy of lower limbs, slow progression and better survival than typical ALS. The latter exhibited rapidly progressive weakness of upper and lower limbs, neither upper motor neuron nor bulbar involvement, and shorter survival than typical ALS. We provide an accurate description of the phenotype, and a bioinformatics analysis of the p.V119M variant on protein structure. This study may increase the knowledge about genotype-phenotype correlations in ALS and improve the approach to ALS patients.

The Ramazzottius varieornatus tardigrade is an extremotolerant terrestrial invertebrate with a length of 0.1–1.0 mm. These small animals show an extraordinary tolerance to extreme conditions such as high pressure, irradiation, chemicals and dehydration. These abilities are linked to a recently discovered damage suppressor protein (Dsup). Dsup is a nucleosome-binding protein that avoids DNA damage after X-ray and oxidative stress exposure without impairing cell life in Dsup-transfected animal and plant cells. The exact “protective” role of this protein is still under study. In human cells, we confirmed that Dsup confers resistance to UV-C and H₂O₂ exposure compared to untransfected cells. A different transcription factor activation was also observed. In addition, a different expression of endogenous genes involved in apoptosis, cell survival and DNA repair was found in Dsup+ cells after H₂O₂ and UV-C. In UV-C exposed cells, Dsup efficiently upregulates DNA damage repair genes, while H₂O₂ treatment only marginally involves the activation of pathways responsible for DNA repair in Dsup+ cells. These data are in agreement with the idea of a direct protective effect of the protein on DNA after oxidative stress. In conclusion, our data may help to outline the different mechanisms by which the Dsup protein works in response to different insults.

Alternative splicing (AS) is a crucial process to enhance gene expression driving organism development. Interestingly, more than 95% of human genes undergo AS, producing multiple protein isoforms from the same transcript. Any alteration (e.g., nucleotide substitutions, insertions, and deletions) involving consensus splicing regulatory sequences in a specific gene may result in the production of aberrant and not properly working proteins. In this review, we introduce the key steps of splicing mechanism and describe all different types of genomic variants affecting this process (splicing variants in acceptor/donor sites or branch point or polypyrimidine tract, exonic, and deep intronic changes). Then, we provide an updated approach to improve splice variants detection. First, we review the main computational tools, including the recent Machine Learning-based algorithms, for the prediction of splice site variants, in order to characterize how a genomic variant interferes with splicing process. Next, we report the experimental methods to validate the predictive analyses are defined, distinguishing between methods testing RNA (transcriptomics analysis) or proteins (proteomics experiments). For both prediction and validation steps, benefits and weaknesses of each tool/procedure are accurately reported, as well as suggestions on which approaches are more suitable in diagnostic rather than in clinical research.

The Ramazzottius varieornatus tardigrade is an extremotolerant terrestrial invertebrate belonging to the phylum of Tardigrada. At a length of 0.1-1.0 mm, tardigrades are small animals with an exceptional tolerance to extreme conditions such as high pressure, chemicals and irradia-tion. These properties have been attributed to the recently-discovered Dsup protein. Dsup is a nucleosome-binding protein that prevents DNA damage against X-ray and oxidative stress without impairing cell life, also in Dsup-transfected animal and plant cells. However, the precise “protective” role of this protein is still under study. We performed experiments on human cells and shows that, as compared to control cells, Dsup+ cells are more resistant to UV-C exposure and H2O2. Real-time PCR identified different expression patterns of endogenous genes involved in apoptosis, cell survival and DNA damage repair in Dsup+ cells in response to H2O2 and UV-C. While H2O2 treatment in Dsup+ cells only marginally involved the activation of pathways responsible for DNA repair reinforcing the idea of a direct protective effect of the protein on DNA, in UV-C exposed cells, Dsup efficiently upregulates DNA damage repair genes. In conclusion, our data may help to delineate the different mechanisms by which the Dsup protein operates in response to different insults.

Cerebral cavernous malformations (CCMs) are vascular lesions that affect predominantly microvasculature in the brain and spinal cord. CCM can occur either in sporadic or familial form, characterized by autosomal dominant inheritance and development of multiple lesions throughout the patient’s life. Three genes associated with CCM are known: CCM1/KRIT1 (krev interaction trapped 1), CCM2/MGC4607 (encoding a protein named malcavernin), and CCM3/PDCD10 (programmed cell death 10). All the mutations identified in these genes cause a loss of function and compromise the protein functions needed for maintaining the vascular barrier integrity. Loss of function of CCM proteins causes molecular disorganization and dysfunction of endothelial adherens junctions. In this review, we provide an overall vision of the CCM pathology, starting with the genetic bases of the disease, describing the role of the proteins, until we reach the cellular level. Thus, we summarize the genetics of CCM, providing a description of CCM genes and mutation features, provided an updated knowledge of the CCM protein structure and function, and discuss the molecular mechanisms through which CCM proteins may act within endothelial cells, particularly in endothelial barrier maintenance/regulation and in cellular signaling.

Cerebral cavernous malformations (CCMs) are vascular malformations that may result in headaches, seizures, focal neurological deficits, and hemorrhage. CCMs occur sporadically (80%) or in familial form (20%), with autosomal dominant inheritance. Among the three CCM-related genes, mutations in KRIT1 account for 53–65% of familial cases and more than 100 different mutations have been identified so far. In the present work, we describe the clinical, neuroradiological, and genetic findings of sixteen CCM Italian patients, 13 belonging to 4 unrelated families and 3 sporadic cases. Six distinct KRIT1 gene variants, two novel (c.1730+1_1730+3del, c.1664 C>T) and four previously described (c.966G>A, c.1255-1G>A c.1197_1200del, c.1255-1_1256del), were identified, including a possible de novo mutation. All the variants resulted in a premature stop codon. Cerebral 1.5 T magnetic resonance imaging showed multiple CCMs in all the mutation carriers for whom it was available, including sporadic cases. One patient had also cutaneous angiomas. Among the mutation carriers, symptomatic patients constituted 66% and a variable phenotypic expression was observed. Our data confirms phenotypic variability and incomplete penetrance of neurological symptoms in KRIT1-positive families, expands the mutational spectrum of this gene, and highlights how sporadic cases with multiple lesions need an approach similar to individuals with familial CCM.

MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression in both animals and plants. By pairing to microRNA responsive elements (mREs) on target mRNAs, miRNAs play gene-regulatory roles, producing remarkable changes in several physiological and pathological processes. Thus, the identification of miRNA-mRNA target interactions is fundamental for discovering the regulatory network governed by miRNAs. The best way to achieve this goal is usually by computational prediction followed by experimental validation of these miRNA-mRNA interactions. This review summarizes the key strategies for miRNA target identification. Several tools for computational analysis exist, each with different approaches to predict miRNA targets, and their number is constantly increasing. The major algorithms available for this aim, including Machine Learning methods, are discussed, to provide practical tips for familiarizing with their assumptions and understanding how to interpret the results. Then, all the experimental procedures for verifying the authenticity of the identified miRNA-mRNA target pairs are described, including High-Throughput technologies, in order to find the best approach for miRNA validation. For each strategy, strengths and weaknesses are discussed, to enable users to evaluate and select the right approach for their interests.