Lack of Direct Effects of Neurotrophic Factors in an In Vitro Model of Neuroinflammation.

Neuroinflammation is associated with several neurological disorders including temporal lobe epilepsy. Seizures themselves can induce neuroinflammation. In an in vivo model of epilepsy, the supplementation of brain-derived neurotropic factor (BDNF) and fibroblast growth factor-2 (FGF-2) using a Herpes-based vector reduced epileptogenesis-associated neuroinflammation. The aim of this study was to test whether the attenuation of the neuroinflammation obtained in vivo with BDNF and FGF-2 was direct or secondary to other effects, for example, the reduction in the severity and frequency of spontaneous recurrent seizures. An in vitro model of neuroinflammation induced by lipopolysaccharide (LPS, 100 ng/mL) in a mouse primary mixed glial culture was used. The releases of cytokines and NO were analyzed via ELISA and Griess assay, respectively. The effects of LPS and neurotrophic factors on cell viability were determined by performing an MTT assay. BDNF and FGF-2 were tested alone and co-administered. LPS induced a significant increase in pro-inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and NO. BDNF, FGF-2, and their co-administration did not counteract these LPS effects. Our study suggests that the anti-inflammatory effect of BDNF and FGF-2 in vivo in the epilepsy model was indirect and likely due to a reduction in seizure frequency and severity.

A multiparameter liquid biopsy approach allows to track melanoma dynamics and identify early treatment resistance.

Melanoma heterogeneity is a hurdle in metastatic disease management. Although the advent of targeted therapy has significantly improved patient outcomes, the occurrence of resistance makes monitoring of the tumor genetic landscape mandatory. Liquid biopsy could represent an important biomarker for the real-time tracing of disease evolution. Thus, we aimed to correlate liquid biopsy dynamics with treatment response and progression by devising a multiplatform approach applied to longitudinal melanoma patient monitoring. We conceived an approach that exploits Next Generation Sequencing (NGS) and droplet digital PCR, as well as the FDA-cleared platform CellSearch, to analyze circulating tumor DNA (ctDNA) trend and circulating melanoma cell (CMC) count, together with their customized genetic and copy number variation analysis. The approach was applied to 17 stage IV melanoma patients treated with BRAF/MEK inhibitors, followed for up to 28 months. BRAF mutations were detected in the plasma of 82% of patients. Single nucleotide variants known or suspected to confer resistance were identified in 70% of patients. Moreover, the amount of ctDNA, both at baseline and during response, correlated with the type and duration of the response itself, and the CMC count was confirmed to be a prognostic biomarker. This work provides proof of principle of the power of this approach and paves the way for a validation study aimed at evaluating early ctDNA-guided treatment decisions in stage IV melanoma. The NGS-based molecular profile complemented the analysis of ctDNA trend and, together with CMC analysis, revealed to be useful in capturing tumor evolution.

Prioritization of putatively detrimental variants in euploid miscarriages.

Miscarriage is the spontaneous termination of a pregnancy before 24 weeks of gestation. We studied the genome of euploid miscarried embryos from mothers in the range of healthy adult individuals to understand genetic susceptibility to miscarriage not caused by chromosomal aneuploidies. We developed GP , a pipeline that we used to prioritize 439 unique variants in 399 genes, including genes known to be associated with miscarriages. Among the prioritized genes we found STAG2 coding for the cohesin complex subunit, for which inactivation in mouse is lethal, and TLE4 a target of Notch and Wnt, physically interacting with a region on chromosome 9 associated to miscarriages.

Methylation Markers in Cutaneous Melanoma: Unravelling the Potential Utility of Their Tracking by Liquid Biopsy.

Malignant melanoma is the most serious, life-threatening form of all dermatologic diseases, with a poor prognosis in the presence of metastases and advanced disease. Despite recent advances in targeted therapy and immunotherapy, there is still a critical need for a better understanding of the fundamental mechanisms behind melanoma progression and resistance onset. Recent advances in genome-wide methylation methods have revealed that aberrant changes in the pattern of DNA methylation play an important role in many aspects of cancer progression, including cell proliferation and migration, evasion of cell death, invasion, and metastasization. The purpose of the current review was to gather evidence regarding the usefulness of DNA methylation tracking in liquid biopsy as a potential biomarker in melanoma. We investigated the key genes and signal transduction pathways that have been found to be altered epigenetically in melanoma. We then highlighted the circulating tumor components present in blood, including circulating melanoma cells (CMC), circulating tumor DNA (ctDNA), and tumor-derived extracellular vesicles (EVs), as a valuable source for identifying relevant aberrations in DNA methylation. Finally, we focused on DNA methylation signatures as a marker for tracking response to therapy and resistance, thus facilitating personalized medicine and decision-making in the treatment of melanoma patients.

LINE-1 methylation in cleft lip tissues: Influence of infant MTHFR c.677C>T genotype.

OBJECTIVE: To investigate the influence of MTHFR c.677C>T genotype on LINE-1 methylation in lateral and medial tissues from cleft lip (CL). METHODS: Forty-five consecutive non-syndromic cleft lip with or without cleft palate (nsCL/P) cases were included in the study. Genomic DNA was extracted from tissues at both sides of cleft lip, and LINE-1 methylation was detected by bisulfite conversion and pyrosequencing. MTHFR c.677C>T genotyping was carried out using the TaqMan genotyping assay. RESULTS: LINE-1 methylation level was significantly higher on medial side of cleft lip compared with lateral side (p = 0.001). This difference was not significantly influenced by the case's sex or cleft type. However, MTHFR c.677C>T genotyping revealed that the difference in LINE-1 methylation across cleft lip was restricted to carriers of C allele of MTHFR c.677C>T and was not apparent in TT homozygous cases (p = 0.027). CONCLUSION: This integrated analysis supports the previous finding of differences in DNA methylation across the two sides of cleft lip and further suggests a possible role of MTHFR c.677C>T genotype in establishing this difference.