MYCN Amplification, along with Wild-Type RB1 Expression, Enhances CDK4/6 Inhibitors’ Efficacy in Neuroblastoma Cells

Neuroblastoma (NB) is one of the primary causes of death for pediatric malignancies. Given the high heterogeneity in NB's mutation landscape, optimizing individualized therapies is still challenging. In the context of genomic alterations, MYCN amplification is the most correlated event with poor outcomes. MYCN is involved in the regulation of several cellular mechanisms, including cell cycle. Thus, studying the influence of MYCN overexpression in the G1/S transition checkpoint of the cell cycle may unveil novel druggable targets for the development of personalized therapeutical approaches. Here, we show that high expression of E2F3 and MYCN correlate with poor prognosis in NB despite the RB1 mRNA levels. Moreover, we demonstrate through luciferase reporter assays that MYCN bypasses RB function by incrementing E2F3-responsive promoter activity. We showed that MYCN overexpression leads to RB inactivation by inducing RB hyperphosphorylation during the G1 phase through cell cycle synchronization experiments. Moreover, we generated two MYCN-amplified NB cell lines conditionally knockdown (cKD) for the RB1 gene through a CRISPRi approach. Indeed, RB KD did not affect cell proliferation, whereas cell proliferation was strongly influenced when a non-phosphorylatable RB mutant was expressed. This finding revealed the dispensable role of RB in regulating MYCN-amplified NB's cell cycle. The described genetic interaction between MYCN and RB1 provides the rationale for using cyclin/CDK complexes inhibitors in NBs carrying MYCN amplification and relatively high levels of RB1 expression

Selected pomegranate germplasm from Afghanistan: morphological variability and relationship among collected accessions

During the period 2008-09 and in the frame of PHDP 58 Afghan and 18 foreign pomegranate accessions have been collected and grown under homogeneous environmental and cultivation practice conditions in ex situ collections. Standardized procedures were adopted to describe mature trees, leaves, flowers and fruits for a total of 30 phenotypic traits. Within the National Collection of Pomegranate of Afghanistan, coefficients of variability ranged from 8.8 to 31.7% for fruit diameter and weight of non edible part, respectively. Principal component analysis revealed the absence of correlated variables among different organs. The whole set of accessions resulted discriminated on the basis of the studied morphological parameters and all the accessions were grouped into 3 sub-sets by hierarchical cluster analysis. Local accessions resulted distributed in the 3 clusters, nevertheless the largest one held all the foreign varieties while the second one included all the accessions collected under the putative name of ‘Bedana’. The adopted morphological studies allowed to identify one true-to-type ‘Bedana’ accession, considered the best Afghan variety for fresh consume due to its very soft seed, and to solve the cases of homonymy. Analogously, various accessions originally collected from different regions of Afghanistan under the name of ‘Kandahari’ were identified and renamed

Distinct Regulatory Functions of Calpain 1 and 2 during Neural Stem Cell Self-Renewal and Differentiation

Calpains are calcium regulated cysteine proteases that have been described in a wide range of cellular processes, including apoptosis, migration and cell cycle regulation. In addition, calpains have been implicated in differentiation, but their impact on neural differentiation requires further investigation. Here, we addressed the role of calpain 1 and calpain 2 in neural stem cell (NSC) self-renewal and differentiation. We found that calpain inhibition using either the chemical inhibitor calpeptin or the endogenous calpain inhibitor calpastatin favored differentiation of NSCs. This effect was associated with significant changes in cell cycle-related proteins and may be regulated by calcium. Interestingly, calpain 1 and calpain 2 were found to play distinct roles in NSC fate decision. Calpain 1 expression levels were higher in self-renewing NSC and decreased with differentiation, while calpain 2 increased throughout differentiation. In addition, calpain 1 silencing resulted in increased levels of both neuronal and glial markers, β-III Tubulin and glial fibrillary acidic protein (GFAP). Calpain 2 silencing elicited decreased levels of GFAP. These results support a role for calpain 1 in repressing differentiation, thus maintaining a proliferative NSC pool, and suggest that calpain 2 is involved in glial differentiation

Microfluidic chip with molecular beacons detects miRNAs in Human CSF to reliably characterize CNS-specific disorders

peer reviewe

miRNA and mRNA cancer signatures determined by analysis of expression levels in large cohorts of patients

Toward identifying a cancer-specific gene signature we applied surprisal analysis to the RNAs expression behavior for a large cohort of breast, lung, ovarian, and prostate carcinoma patients. We characterize the cancer phenotypic state as a shared response of a set of mRNA or microRNAs (miRNAs) in cancer patients versus noncancer controls. The resulting signature is robust with respect to individual patient variability and distinguishes with high fidelity between cancer and noncancer patients. The mRNAs and miRNAs that are implicated in the signature are correlated and are known to contribute to the regulation of cancer-signaling pathways. The miRNA and mRNA networks are common to the noncancer and cancer patients, but the disease modulates the strength of the connectivities. Furthermore, we experimentally assessed the cancer-specific signatures as possible therapeutic targets. Specifically we restructured a single dominant connectivity in the cancer-specific gene network in vitro. We find a deflection from the cancer phenotype, significantly reducing cancer cell proliferation and altering cancer cellular physiology. Our approach is grounded in thermodynamics augmented by information theory. The thermodynamic reasoning is demonstrated to ensure that the derived signature is bias-free and shows that the most significant redistribution of free energy occurs in programming a system between the noncancer and cancer states. This paper introduces a platform that can elucidate miRNA and mRNA behavior on a systems level and provides a comprehensive systematic view of both the energetics of the expression levels of RNAs and of their changes during tumorigenicity

17-β-Estradiol increases neuronal excitability through MAP kinase-induced calpain activation

17-β-Estradiol (E2) is a steroid hormone involved in numerous brain functions. E2 regulates synaptic plasticity in part by enhancing NMDA receptor function and spine density in the hippocampus, resulting in increased long-term potentiation and facilitation of learning and memory. As the calcium-dependent neutral protease, calpain, is also involved in these processes, we tested whether E2 could activate calpain and examined the functional consequences of E2-mediated calpain activation in hippocampus. Calpain activity was analyzed by a fluorescence resonance energy transfer (FRET)-based assay that allows both quantitative determination and spatial resolution. E2 rapidly activated calpain in cultured cortical and hippocampal neurons, prominently in dendrites and dendritic spines. E2-induced calpain activation was mediated through mitogen-activated protein kinase (MAPK), as it was completely blocked by MEK inhibitors. It was also calcium-independent, as it was still evident in presence of the calcium chelator, BAPTA-AM. Activation of ERα and ERβ receptors by specific agonists stimulated calpain activity. Finally, the rapid E2-mediated increase in excitability in acute hippocampal slices was prevented by a membrane-permeable calpain inhibitor. Furthermore, E2 treatment of acute hippocampal slices resulted in increased actin polymerization and membrane levels of GluR1 but not GluR2/3 subunits of AMPA receptors; both effects were also blocked by a calpain inhibitor. Our results indicate that E2 rapidly stimulates calpain activity through MAP kinase-mediated phosphorylation, resulting in increased membrane levels of AMPA receptors. These effects could be responsible for E2-mediated increase in neuronal excitability and facilitation of cognitive processes

Angiotropism, Pericytic Mimicry and Extravascular Migratory Metastasis in Melanoma: An Alternative to Intravascular Cancer Dissemination

For more than 15 years, angiotropism in melanoma has been emphasized as a marker of extravascular migration of tumor cells along the abluminal vascular surface, unveiling an alternative mechanism of tumor spread distinct from intravascular dissemination. This mechanism has been termed extravascular migratory metastasis (EVMM). During EVMM, angiotropic tumor cells migrate in a 'pericytic-like' manner (pericytic mimicry) along the external surfaces of vascular channels, without intravasation. Through this pathway, melanoma cells may spread to nearby or more distant sites. Angiotropism is a prognostic factor predicting risk for metastasis in human melanoma, and a marker of EVMM in several experimental models. Importantly, analogies of EVMM and pericytic mimicry include neural crest cell migration, vasculogenesis and angiogenesis, and recent studies have suggested that the interaction between melanoma cells and the abluminal vascular surface induce differential expression of genes reminiscent of cancer migration and embryonic/stem cell state transitions. A recent work revealed that repetitive UV exposure of primary cutaneous melanomas in a genetically engineered mouse model promotes metastatic progression via angiotropism and migration along the abluminal vascular surface. Finally, recent data using imaging of melanoma cells in a murine model have shown the progression of tumor cells along the vascular surfaces. Taken together, these data provide support for the biological phenomenon of angiotropism and EVMM, which may open promising new strategies for reducing or preventing melanoma metastasis