Mechanisms by which nuclear A-type lamins are downregulated in neuroblastoma

Lamin A/C are essential components of the nuclear lamina. Together with B-type lamins and other structural components, they form the network which is located in the inner side of the nuclear membrane. It is now clear that lamins exert many different functions within the cell, most of them still largely unknown. Lamin A/C are absent in embryonic stem cells but are expressed in the majority of adult cell types, thereby suggesting their crucial role in differentiation. In addition, their expression is reduced or absent in several human malignancies, even though their role in the tumorigenesis has not been completely characterized yet. A down regulation of Lamin A/C could result in increased nuclear deformability thereby facilitating transit of cells through the capillaries and favouring cell invasive capacity. In a previous paper we demonstrated that LMNA gene knock-down inhibits differentiation in a cellular model of neuroblastoma and increases tumor progression. In this project I investigated some of the possible mechanisms by which Lamin A/C is down-regulated in neuroblastoma cells. As cellular models I employed two neuroblastoma cell lines showing different level of Lamin A/C expression (LAN-5 and SH-SY5Y cells). The analysis of the nascent transcripts of LMNA gene in both cell lines demonstrated that the transcriptional rate of this gene was reduced in the LAN-5 cells, indicating that the protein is regulated at transcriptional level. In addition, I observed that there is a minor recruitment of Sp1 transcription factor on the LMNA promoter of LAN-5 cells, as evaluated by ChIP assay. Moreover, even though the chromatin configuration upstream the LMNA gene promoter is open in both cell lines, as evidenced by a similar enrichment of the H3K4me3 histone marker in both cell lines, we found a different configuration in the coding sequence region (CDS), as shown by a reduced enrichment in LAN-5 cells of the histone marker H3K36me3 which is associated with transcriptional activation. We then tried to exogenously express the LMNA gene in LAN-5 cells, in which the protein is not detectable. In spite of a very high upregulation of LMNA gene, I did not observed any detectable expression of Lamin A/C protein. I hypothesized that the protein could be regulated by a miRNA-mediated post-transcriptional mechanism. An in silico analysis of the miRNAs predicted by miRWalk database to significantly target LMNA transcripts, have evidenced a unique miRNA, the has-miR-539-5p. However, the inhibition of the expression of such miRNA was not able to rescue the expression of Lamin A/C protein in LAN-5 cells. At the moment I can conclude that the regulation of Lamin A/C protein in neuroblastoma cells is mainly to be ascribed to a transcriptional mechanism. However, I cannot exclude the existence of an additional regulation mechanism occurring at post-transcriptional and/or post-translation level that have to be further investigated

Down-regulation of the Lamin A/C in neuroblastoma triggers the expansion of tumor initiating cells

Tumor-initiating cells constitute a population within a tumor mass that shares properties with normal stem cells and is considered responsible for therapy failure in many cancers. We have previously demonstrated that knockdown of the nuclear envelope component Lamin A/C in human neuroblastoma cells inhibits retinoic acid-mediated differentiation and results in a more aggressive phenotype. In addition, Lamin A/C is often lost in advanced tumors and changes in the nuclear envelope composition occur during tumor progression. Based on our previous data and considering that Lamin A/C is expressed in differentiated tissues, we hypothesize that the lack of Lamin A/C could predispose cells toward a stem-like phenotype, thus influencing the development of tumor-initiating cells in neuroblastoma. This paper demonstrates that knockdown of Lamin A/C triggers the development of a tumor-initiating cell population with self-renewing features in human neuroblastoma cells. We also demonstrates that the development of TICs is due to an increased expression of MYCN gene and that in neuroblastoma exists an inverse relationship between LMNA and MYCN expression

Confocal Analysis of Nuclear Lamina Behavior during Male Meiosis and Spermatogenesis in <i>Drosophila melanogaster</i>

<div><p>Lamin family proteins are structural components of a filamentous framework, the nuclear lamina (NL), underlying the inner membrane of nuclear envelope. The NL not only plays a role in nucleus mechanical support and nuclear shaping, but is also involved in many cellular processes including DNA replication, gene expression and chromatin positioning. Spermatogenesis is a very complex differentiation process in which each stage is characterized by nuclear architecture dramatic changes, from the early mitotic stage to the sperm differentiation final stage. Nevertheless, very few data are present in the literature on the NL behavior during this process. Here we show the first and complete description of NL behavior during meiosis and spermatogenesis in <i>Drosophila melanogaster</i>. By confocal imaging, we characterized the NL modifications from mitotic stages, through meiotic divisions to sperm differentiation with an anti-laminDm0 antibody against the major component of the <i>Drosophila</i> NL. We observed that continuous changes in the NL structure occurred in parallel with chromatin reorganization throughout the whole process and that meiotic divisions occurred in a closed context. Finally, we analyzed NL in <i>solofuso</i> meiotic mutant, where chromatin segregation is severely affected, and found the strict correlation between the presence of chromatin and that of NL.</p></div

A Survey to Assess Food Safety Knowledge and Habits Among High School Students, Their Parents and Teachers in Pescara and Chieti Provinces, Abruzzo Region (Italy)

Food safety is a topic of increasing relevance in the European Union (EU). Ensuring safe food from farm to fork is one of the main objectives of EU policies. In our study, we conducted an online survey to assess the food safety knowledge and eating habits in various categories. For young people, we chose school students aged 15–19, based on their ability to cook autonomously after school. For adults, we choose a representative sample consisting of students’ parents and teachers in Chieti and Pescara Provinces of Abruzzo Region (Italy). The survey was structured in eight sections with particular attention to questions regarding risks associated with microorganisms (e.g., Salmonella spp.) and bacterial cross-contamination. Students represented 60.7% of all respondents (n = 290). Risk perception associated with microorganisms in food was significantly different among students and teachers (p < 0.05). Students indicated microorganisms as a food risk in 20.2% of cases, while environmental pollutants were considered the greatest problem for 71.7% of participants. Among teachers, results indicated that 55.1% considered microorganisms a food-associated risk. An adequate theoretical knowledge regarding safe cooking practices was demonstrated by the majority of participants. Acceptable knowledge regarding microbiologically safe cooking practices was also demonstrated by participants. Based on these results, we provide suggestions to improve food safety education programs including the use of social media, as well as by adjusting educational strategies according to the background of learners

Passive Immunity to SARS-CoV-2 at Birth Induced by Vaccination in the First Trimester of Pregnancy

As is well known, the COVID-19 infection is affecting the whole world, causing a serious health, social and economic crisis. The viral infection can cause a mild or severe illness, depending on how effectively the virus is countered by the immune system. In this context, the position of pregnant women remains rather unknown. The case described here reports the immune response in a woman in good health and in her newborn son, having undergone complete vaccination during the first trimester of her pregnancy. We performed a serological assay, measuring IgG antibodies to SARS-CoV-2, by a fully automated solid phase DELFIA (time-resolved fluorescence) immunoassay in a few drops of blood, collected by a finger-prick and spotted on filter paper. The dried blood spot (DBS) sample we used is the same type of sample routinely used in a newborn screening program test. Such a simple and minimally invasive approach allowed us to monitor both the mother and the newborn soon after birth for their anti-SARS-CoV-2 IgG levels. The serological test on the DBS carried out on both mother and newborn revealed the presence of anti-SARS-CoV-2 IgG antibodies up to 7 months after vaccination in the mother, and already at 48 h of life in the newborn

Nuclear lamina distribution during the spermatid elongation.

<p>DNA in red (DAPI staining), nuclear lamina in green (anti Lam-Dm0). Note the “half moon” configuration of NL that localizes from the same nuclear side of the elongating Nebenkern (arrows). Scale bar 20 μm.</p

Nuclear lamina behavior during <i>solofuso</i> mutant meiotic and postmeiotic stages.

<p>DNA in red (DAPI staining), nuclear lamina in green (anti Lam-Dm0). <i>solofuso</i> mutant males show chromatin bridges at (a) Anaphase I (arrowheads) and (b) Telophase I (arrowheads). NL shows an irregular signal at nuclear rim and a punctuated pattern over the chromatin bridges (a and b, arrowheads). Note that NL encircles the micronucleus (b, arrows). (c) Spermatids at onion stage evidencing a micronucleus surrounded by NL. (d) Onion stages showing a Nebenkern (Nb) associated to a nucleus (N) and two Nebenkern not associated with nuclei (d, DNA, arrows). The NL signal is detected around the nucleus associated with Nebenkern while no structured NL is present close to the Nebenkern lacking nuclei (d, LAMINA). Scale bar 20 μm.</p

Nuclear lamina behavior in meiotic prophase and prometaphase cells.

<p>DNA in red (DAPI staining), nuclear lamina in green (anti Lam-Dm0). In young primary spermatocytes at S2a, S2b and S3 stages (a) and mature primary spermatocytes at S5 stage (b) the NL uniformly depicts the nuclear rim. In primary spermatocytes at S6 stage (c) the NL shows an irregular shape and invaginations (arrows). In prometaphase cells at M1b stage (d), the three chromatin clumps corresponding to the three major bivalents move to the equatorial plate, the nuclear envelope breaks down and the NL signal becomes discontinous (arrows). In a, b, c panels, the heavily lamin-stained cell in the bottom represents a cyst cell. Scale bar 20 μm.</p

Nuclear lamina distribution during spermatids differentiation.

<p>DNA in red (DAPI staining), nuclear lamina in green (anti Lam-Dm0). (a) T1 and T2 stages are characterized by the progressive aggregation of mitochondria into masses of different shapes. NL exhibits a punctuated pattern at the nuclear rim. (b) At onion stage nucleus and Nebenkern (DAPI staining, short and long arrows, respectively), in a 1:1 ratio, have the same round shape and size. NL shows a thick appearance at nuclear periphery, with interruptions (LAMINA staining, arrowheads). (c) T5 spermatid stage is characterized by an oval shaped Nebenkern (DAPI staining, arrows) with NL signal localized also inside the nuclei (c, LAMINA staining and merge). Scale bar 20 μm.</p

Circulating miRNAs in Small Extracellular Vesicles Secreted by a Human Melanoma Xenograft in Mouse Brains

The identification of liquid biomarkers remains a major challenge to improve the diagnosis of melanoma patients with brain metastases. Circulating miRNAs packaged into tumor-secreted small extracellular vesicles (sEVs) contribute to tumor progression. To investigate the release of tumor-secreted miRNAs by brain metastasis, we developed a xenograft model where human metastatic melanoma cells were injected intracranially in nude mice. The comprehensive profiles of both free miRNAs and those packaged in sEVs secreted by the melanoma cells in the plasma demonstrated that most (80%) of the sEV-associated miRNAs were also present in serum EVs from a cohort of metastatic melanomas, included in a publicly available dataset. Remarkably, among them, we found three miRNAs (miR-224-5p, miR-130a-3p and miR-21-5p) in sEVs showing a trend of upregulation during melanoma progression. Our model is proven to be valuable for identifying miRNAs in EVs that are unequivocally secreted by melanoma cells in the brain and could be associated to disease progression