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Alzheimer's disease (AD), the most prevalent neurodegenerative disease in the growing population of elderly people, is still lacking minimally-invasive circulating biomarkers that could facilitate the diagnosis and the monitoring of disease progression. MicroRNAs (miRNAs) are emerging as tissue-specific and/or circulating biomarkers of several age-related diseases, but evidence on AD is still not conclusive. Since a systemic pro-inflammatory status was associated with an increased risk of AD development and progression, we focused our investigation on a subset of miRNAs modulating the inflammatory process, namely inflamma-miRNAs. The expression of inflamma-miR-17-5p, -21-5p, -126-3p, and -146a-5p was analyzed in plasma samples from 116 patients with AD compared with 41 age-matched healthy control (HC) subjects. MiR-17-5p, miR-21-5p, and miR-126-3p plasma levels were significantly increased in AD patients compared to HC. Importantly, a strong inverse relationship was observed between miR-21-5p and miR-126-3p, and the cognitive impairment, assessed by Mini-Mental State Examination (MMSE). Notably, miR-126-3p was able to discriminate between mild and severe cognitive impairment. Overall, our results reinforce the hypothesis that circulating inflamma-miRNAs could be assessed as minimally invasive tools associated with the development and progression of cognitive impairment in AD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Giuliani, Gaetani, Sorgentoni, Agarbati, Laggetta, Matacchione, Gobbi, Rossi, Galeazzi, Piccinini, Pelliccioni, Bonfigli, Procopio, Albertini, Sabbatinelli, Olivieri and Fazioli.)

Background: The diagnosis of heart failure (HF) is often difficult and underestimated in very elderly comorbid patients, especially when an echocardiographic evaluation is not available or feasible., Aim: to evaluate NT-proBNP values and their correlation with in-hospital mortality in a population of very elderly hospitalized for medical conditions other than HF., Methods: We performed a prospective observational study on 403 very elderly admitted to an Internal Medicine and Geriatrics Department. Exclusion criterion was an admission diagnosis of HF. Patients with at least one symptom or sign compatible with HF were tested for NT-proBNP. NT-proBNP values < 300 pg/ml were considered as an age-independent exclusion criterion for HF (high negative predictive value), while NT-proBNP values ≥ 1800 pg/ml were considered as a diagnostic criterion. Main comorbidities and laboratory parameters were considered to adjust regression analyses between NT-proBNP and in-hospital mortality., Results: NT-proBNP values ≥ 1800 pg/ml were present in 61.0% of patients and 32.8% of patients laid between 300 ≤ NT-proBNP < 1800 pg/ml values. NT-proBNP values were associated with the main indices of disease severity/organ failure considered such as reduced eGFR, reduced albumin and elevated CRP. NT-proBNP values ≥ 1800 pg/ml and ln(NT-proBNP) values were significantly associated with in-hospital mortality independently from the main comorbidities and lab parameters considered. The patients, who were already taking ACE inhibitors/Angiotensin Receptor Blockers before admission, showed lower in-hospital mortality., Conclusions: Testing for NT-proBNP should be strongly recommended in the hospitalized very elderly, because of the very high prevalence of underlying HF and its impact on in-hospital mortality, to identify an underlying cardiac involvement that requires appropriate treatment.

Objective: To study how to identify patients with "triple negative" sporadic breast cancers (BCs) having BRCA1 silenced or down-regulated due to epigenetic BRCA1 inactivation., Study Design: We selected, from our database, patients diagnosed with BC between 1995 and 2001 with tumors exhibiting the "triple negative" phenotype. "Triple positive" tumors were used as controls. BRCA1 protein expression was determined by immunohistochemistry. Methylation specific polymerase chain reaction (PCR) and bisulfite sequencing on genomic DNA were used to assess BRCA1 promoter methylation. BRCA1 m-RNA expression analysis was conducted by real-time PCR., Results: Forty-four triple negative and 68 controls (triple positive) were eligible for our analysis. BRCA1 promoter methylation was present in 31.8% of triple negative and in 20.6% of triple positive cases. BRCA1 was inactivated (absent BRCA1 m-RNA expression and lack of BRCA1 protein) in 21.4% of tumors with BRCA1 promoter methylation, as compared with 6% of non-methylated ones (p = 0.0453)., Conclusion: BRCA1 inactivation due to promoter methylation could play an important role in some sporadic BC cases. Patients with this signature could represent the basis for prospective studies aiming to compare clinical response to different drugs.

Background: A novel Cys-Ser Ret germline point mutation in a 58-year-old woman with bilateral medullary thyroid carcinoma (MTC) prompted us to perform genetic analysis of the family and evaluate the biological consequences of such a mutation., Methods: Ret analysis by direct sequencing was performed in five family members. The biological activity and biochemical properties of the Ret- Cys515Ser mutant were analyzed in NIH-3T3 cells., Results: The proband's son, age 35, had the Ret- Cys515Ser mutation and the L769 CTT/CTG exon 13 polymorphic variant, which was also found in his father. Clinical evaluation of the son also revealed bilateral multifocal microscopic MTC and papillary thyroid carcinoma (PTC). In vitro and in vivo analysis indicated ligand-independent activation of the Ret-Cys515Ser mutant due to aberrant disulfide homodimerization, increased mitogenic activity, and ability to induce anchorage-independent growth in NIH-3T3 cells in comparison to wild-type Ret, suggesting a possible role of Cys515Ser in tumor development., Conclusions: The Cys515Ser mutation adds to cysteine substitution groups that have been described in association with MTC. Our data also highlight the importance of performing a complete genetic analysis in patients who present with MTC.

Ligand-induced membrane trafficking of the anaplastic lymphoma kinase (ALK) was studied using a chimeric receptor in which the extracellular and transmembrane domain of ALK was substituted for the corresponding regions of epidermal growth factor receptor (EGFR). Wild-type EGFR, EGFR/ALK and an EGFR/ALK kinase negative mutant were independently expressed in mouse NR6 fibroblasts. The capacity of EGFR/ALK to mediate [125I]-EGF internalization, receptor degradation and downregulation, which has never been previously described, was assayed. The rate of [125I]-EGF-induced internalization mediated by the cytoplasmic domain of ALK was reduced several fold compared with the wild-type EGFR. The low rate of EGF internalization promoted by EGFR/ALK correlated with an impaired degradation and downregulation of the receptor and indicate that ALK is not subject to traditional mechanisms used to regulate receptor tyrosine kinase function. Accordingly, ALK-activated intracellular domain does not associate in vivo with c-cbl and does not undergo ligand-mediated ubiquitination. The current study provides new insight into the function and regulation of ALK suggesting that the relative long membrane residence of activated ALK might confers a more potent and prolonged signaling activity. Indeed NR6-EGFR/ALK cells exhibited a approximately 3-fold increase in a maximal mitogenic response than NR6-EGFR.

Oncogenic rearrangements of the anaplastic lymphoma kinase (ALK) gene, encoding a receptor type tyrosine kinase, are frequently associated with anaplastic large cell lymphomas. Such rearrangements juxtapose the intracellular domain of ALK to 5'-end sequences belonging to different genes and create transforming fusion proteins. To understand how the oncogenic versions of ALK contribute to lymphomagenesis, it is important to analyze the biological effects and the biochemical properties of this receptor under controlled conditions of activation. To this aim, we constructed chimeric receptor molecules in which the extracellular domain of the ALK kinase is replaced by the extracellular, ligand-binding domain of the epidermal growth factor receptor (EGFR). Upon transfection in NIH 3T3 fibroblasts, the EGFR/ALK chimera was correctly synthesized and transported to the cell surface, where it was fully functional in forming high versus low affinity EGF-binding sites and transducing an EGF-dependent signal intracellularly. Overexpression of the EGFR/ALK chimera in NIH 3T3 was sufficient to induce the malignant phenotype; the appearance of the transformed phenotype was, however, conditionally dependent on the administration of EGF. Moreover, the EGFR/ALK chimera was significantly more active in inducing transformation and DNA synthesis than the wild type EGFR when either was expressed at similar levels in NIH 3T3 cells. Comparative analysis of the biochemical pathways implicated in the transduction of mitogenic signals did not show any increased ability of the EGFR/ALK to phosphorylate PLC-gamma and MAPK compared with the EGFR. On the contrary, EGFR/ALK showed to have a consistently greater effect on phosphatidylinositol 3-kinase activity compared with the EGFR, indicating that this enzyme plays a major role in mediating the mitogenic effects of ALK in NIH 3T3 cells.