The Continuous Adaptive Challenge Played by Arboviruses: An In Silico Approach to Identify a Possible Interplay between Conserved Viral RNA Sequences and Host RNA Binding Proteins (RBPs).

Climate change and globalization have raised the risk of vector-borne disease (VBD) introduction and spread in various European nations in recent years. In Italy, viruses carried by tropical vectors have been shown to cause viral encephalitis, one of the symptoms of arboviruses, a spectrum of viral disorders spread by arthropods such as mosquitoes and ticks. Arboviruses are currently causing alarm and attention, and the World Health Organization (WHO) has released recommendations to adopt essential measures, particularly during the hot season, to restrict the spreading of the infectious agents among breeding stocks. In this scenario, rapid analysis systems are required, because they can quickly provide information on potential virus-host interactions, the evolution of the infection, and the onset of disabling clinical symptoms, or serious illnesses. Such systems include bioinformatics approaches integrated with molecular evaluation. Viruses have co-evolved different strategies to transcribe their own genetic material, by changing the host's transcriptional machinery, even in short periods of time. The introduction of genetic alterations, particularly in RNA viruses, results in a continuous adaptive fight against the host's immune system. We propose an in silico pipeline method for performing a comprehensive motif analysis (including motif discovery) on entire genome sequences to uncover viral sequences that may interact with host RNA binding proteins (RBPs) by interrogating the database of known RNA binding proteins, which play important roles in RNA metabolism and biological processes. Indeed, viral RNA sequences, able to bind host RBPs, may compete with cellular RNAs, altering important metabolic processes. Our findings suggest that the proposed in silico approach could be a useful and promising tool to investigate the complex and multiform clinical manifestations of viral encephalitis, and possibly identify altered metabolic pathways as targets of pharmacological treatments and innovative therapeutic protocols.

Novel Germline PHD2 Variant in a Metastatic Pheochromocytoma and Chronic Myeloid Leukemia, but in the Absence of Polycythemia.

Background: Pheochromocytoma (Pheo) and paraganglioma (PGL) are rare tumors, mostly resulting from pathogenic variants of predisposing genes, with a genetic contribution that now stands at around 70%. Germline variants account for approximately 40%, while the remaining 30% is attributable to somatic variants. Objective: This study aimed to describe a new PHD2 (EGLN1) variant in a patient affected by metastatic Pheo and chronic myeloid leukemia (CML) without polycythemia and to emphasize the need to adopt a comprehensive next-generation sequencing (NGS) panel. Methods: Genetic analysis was carried out by NGS. This analysis was initially performed using a panel of genes known for tumor predisposition (EGLN1, EPAS1, FH, KIF1Bß, MAX, NF1, RET, SDHA, SDHAF2, SDHB, SDHC, SDHD, TMEM127, and VHL), followed initially by SNP-CGH array, to exclude the presence of the pathogenic Copy Number Variants (CNVs) and the loss of heterozygosity (LOH) and subsequently by whole exome sequencing (WES) comparative sequence analysis of the DNA extracted from tumor fragments and peripheral blood. Results: We found a novel germline PHD2 (EGLN1) gene variant, c.153G>A, p.W51*, in a patient affected by metastatic Pheo and chronic myeloid leukemia (CML) in the absence of polycythemia. Conclusions: According to the latest guidelines, it is mandatory to perform genetic analysis in all Pheo/PGL cases regardless of phenotype. In patients with metastatic disease and no evidence of polycythemia, we propose testing for PHD2 (EGLN1) gene variants. A possible correlation between PHD2 (EGLN1) pathogenic variants and CML clinical course should be considered.

An emerging role for BAG3 in gynaecological malignancies.

BAG3, a member of the BAG family of co-chaperones, is a multidomain protein with a role in several cellular processes, including the control of apoptosis, autophagy and cytoskeletal dynamics. The expression of bag3 is negligible in most cells but can be induced by stress stimuli or malignant transformation. In some tumours, BAG3 has been reported to promote cell survival and resistance to therapy. The expression of BAG3 has been documented in ovarian, endometrial and cervical cancers, and studies have revealed biochemical and functional connections of BAG3 with proteins involved in the survival, invasion and resistance to therapy of these malignancies. BAG3 expression has also been shown to correlate with the grade of dysplasia in squamous intraepithelial lesions of the uterine cervix. Some aspects of BAG3 activity, such as its biochemical and functional interaction with the human papillomavirus proteins, could help in our understanding of the mechanisms of oncogenesis induced by the virus. This review aims to highlight the potential value of BAG3 studies in the field of gynaecological tumours.

A HS6ST2 gene variant associated with X-linked intellectual disability and severe myopia in two male twins.

X-linked intellectual disability (XLID) refers to a clinically and genetically heterogeneous neurodevelopmental disorder, in which males are more heavily affected than females. Among the syndromic forms of XLID, identified by additional clinical signs as part of the disease spectrum, the association between XLID and severe myopia has been poorly characterized. We used whole exome sequencing (WES) to study two Italian male twins presenting impaired intellectual function and adaptive behavior, in association with severe myopia and mild facial dysmorphisms. WES analysis detected the novel, maternally inherited, mutation c.916G > C (G306R) in the X-linked heparan sulfate 6-O-sulfotransferase 2 (HS6ST2) gene. HS6ST2 transfers sulfate from adenosine 3'-phosphate, 5'-phosphosulfate to the sixth position of the N-sulphoglucosamine residue in heparan sulfate (HS) proteoglycans. Low HS sulfation levels are associated with defective optic disc and stalk morphogenesis during mammalian visual system development. The c.916G>C variant affects the HS6ST2 substrate binding site, and its effect was considered "deleterious" by in-silico tools. An in-vitro enzymatic assay showed that the HS6ST2 mutant isoform had significantly reduced sulphotransferase activity. Taken together, the results suggest that mutant HS6ST2 is possibly involved in the development of myopia and cognitive impairment, characteristics of the probands reported here.

BAG3 Protein Is Over-Expressed in Endometrioid Endometrial Adenocarcinomas.

Endometrioid endometrial cancer is the most common gynaecological tumor in developed countries, and its incidence is increasing. The definition of subtypes, based on clinical and endocrine features or on histopathological characteristics, correlate to some extent with patient's prognosis, but there is substantial heterogeneity within tumor types. The search for molecules and mechanisms implied in determining the progression and the response to therapy for this cancer is still ongoing. BAG3 protein, a member of BAG family of co-chaperones, has a pro-survival role in several tumor types. BAG3 anti-apoptotic properties rely on its characteristic to bind several intracellular partners, thereby, modulating crucial events such as apoptosis, differentiation, cell motility, and autophagy. BAG3 expression in human endometrial cancer tissues was not investigated so far. Here, we show that BAG3 protein levels are elevated in tumoral and hyperplastic cells in respect to normal glands. Furthermore, BAG3 subcellular localization appears to be changed in tumoral compared to normal cells. Our results indicate a possible role for BAG3 protein in the maintenance of cell survival in endometrioid endometrial cancer and suggest that this field of studies is worthy of further investigations. J. Cell. Physiol. 232: 309-311, 2017. © 2016 Wiley Periodicals, Inc.

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.

Targeted Next Generation Sequencing molecular profiling and its clinical application in adrenocortical cancer.

OBJECTIVE: Adrenal cortical carcinoma (ACC) is a rare malignancy with a generally poor but heterogeneous prognosis, especially depending on the tumour stage at diagnosis. Identification of somatic gene alterations combined with clinical/histopathological evaluation of the tumour can help improve prognostication. We applied a simplified targeted-Next-Generation Sequencing (NGS) panel to characterise the mutational profiles of ACCs, providing potentially relevant information for better patient management. DESIGN AND METHODS: Thirty frozen tumour specimens from a local ACC series were retrospectively analysed by a custom-NGS panel (CDKN2A, CTNNB1, DAXX, MED12, NF1, PRKAR1A, RB1, TERT, TP53, ZNRF3) to detect somatic prioritised single-nucleotide variants. This cohort was integrated with 86 patients from the ACC-TCGA series bearing point-mutations in the same genes and their combinations identified by our panel. Primary endpoints of the analysis on the total cohort (113 patients) were overall survival (OS) and progression-free survival (PFS), and hazard ratio (HR) for the different alterations grouped by the signalling pathways/combinations affected. RESULTS: Different PFS, OS, and HR were associated to the different pathways/combinations, being NF1 + TP53 and Wnt/ß-catenin + Rb/p53 combined mutations the most deleterious, with a statistical significance for progression HR which is retained only in low-(I/II) stages-NF1 + TP53 combination: HR = 2.96[1.01-8.69] and HR = 13.23[3.15-55.61], all and low stages, respectively; Wnt/ß-catenin + Rb/p53 combined pathways: HR = 6.47[2.54-16.49] and HR = 16.24[3.87-68.00], all and low-stages, respectively. CONCLUSIONS: A simplified targeted-NGS approach seems the best routinely applicable first step towards somatic genetic characterisation of ACC for prognostic assessment. This approach proved to be particularly promising in low-stage cases, suggesting the need for more stringent surveillance and personalised treatment.

The Target Therapy Hyperbole: "KRAS (p.G12C)"-The Simplification of a Complex Biological Problem.

Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) gene variations are linked to the development of numerous cancers, including non-small cell lung cancer (NSCLC), colorectal cancer (CRC), and pancreatic ductal adenocarcinoma (PDAC). The lack of typical drug-binding sites has long hampered the discovery of therapeutic drugs targeting KRAS. Since "CodeBreaK 100" demonstrated Sotorasib's early safety and efficacy and led to its approval, especially in the treatment of non-small cell lung cancer (NSCLC), the subsequent identification of specific inhibitors for the p.G12C mutation has offered hope. However, the CodeBreaK 200 study found no significant difference in overall survival (OS) between patients treated with Docetaxel and Sotorasib (AMG 510), adding another degree of complexity to this ongoing challenge. The current study compares the three-dimensional structures of the two major KRAS isoforms, KRAS4A and KRAS4B. It also investigates the probable structural changes caused by the three major mutations (p.G12C, p.G12D, and p.G12V) within Sotorasib's pocket domain. The computational analysis demonstrates that the wild-type and mutant isoforms have distinct aggregation propensities, resulting in the creation of alternate oligomeric configurations. This study highlights the increased complexity of the biological issue of using KRAS as a therapeutic target. The present study stresses the need for a better understanding of the structural dynamics of KRAS and its mutations to design more effective therapeutic approaches. It also emphasizes the potential of computational approaches to shed light on the complicated molecular pathways that drive KRAS-mediated oncogenesis. This study adds to the ongoing efforts to address the therapeutic hurdles presented by KRAS in cancer treatment.

De Novo Pathogenic Variant in FBRSL1, Non OMIM Gene Paralogue AUTS2, Causes a Novel Recognizable Syndromic Manifestation with Intellectual Disability; An Additional Patient and Review of the Literature.

FBRSL1, together with FBRS and AUTS2 (Activator of Transcription and Developmental Regulator; OMIM 607270), constitutes a tripartite AUTS2 gene family. AUTS2 and FBRSL1 are evolutionarily more closely related to each other than to FBRS (Fibrosin 1; OMIM 608601). Despite its paralogous relation to AUTS2, FBRSL1's precise role remains unclear, though it likely shares functions in neurogenesis and transcriptional regulation. Herein, we report the clinical presentation with therapeutic approaches and the molecular etiology of a patient harboring a de novo truncating variant (c.371dupC) in FBRSL1, leading to a premature stop codon (p.Cys125Leufs*7). Our study extends previous knowledge by highlighting potential interactions and implications of this variant, alongside maternal and paternal duplications, for the patient's phenotype. Using sequence conservation data and in silico analysis of the truncated protein, we generated a predicted domain structure. Furthermore, our in silico analysis was extended by taking into account SNP array results. The extension of in silico analysis was performed due to the possibility that the coexistence of FBRSL1 truncating variant contemporary with maternal and paternal duplication could be a modifier of proband's phenotype and/or influence the novel syndrome clinical characteristics. FBRSL1 protein may be involved in neurodevelopment due to its homology with AUTS2, together with distinctive neuronal expression profiles, and thus should be considered as a potential modulation of clinical characteristics in a novel syndrome. Finally, considering that FBRSL1 is apparently involved in neurogenesis and in transcriptional regulatory networks that orchestrate gene expression, together with the observation that different genetic syndromes are associated with distinct genomic DNA methylation patterns, the specific episignature has been explored.

Pancreatic Cancer-Secreted Proteins: Targeting Their Functions in Tumor Microenvironment.

Pancreatic Ductal Adenocarcinoma (PDAC) is a ravaging disease with a poor prognosis, requiring a more detailed understanding of its biology to foster the development of effective therapies. The unsatisfactory results of treatments targeting cell proliferation and its related mechanisms suggest a shift in focus towards the inflammatory tumor microenvironment (TME). Here, we discuss the role of cancer-secreted proteins in the complex TME tumor-stroma crosstalk, shedding lights on druggable molecular targets for the development of innovative, safer and more efficient therapeutic strategies.