[Temporal analysis of the prevalence of congenital hypospadias in the municipality of Gela]. / Analisi temporale della prevalenza di ipospadia congenita nel comune di Gela.

OBJECTIVES: to evaluate the risk profile of hypospadias in Gela, an Italian National Priority Contaminated Site (NPCS) located in Sicily Region (Southern Italy), characterized by a significant excess of hypospadias in newborn residents compared to data from reference on regional, national, and international basis and, until 2014, by the presence of a petrochemical plant. DESIGN: geographical analyses were conducted by comparing the prevalence of the Gela municipality to prevalence found in Sicily, in a territorial area bordering Gela (ALG), and in the NPCSs of Milazzo and Priolo. The geographical comparisons were conducted for the period 2010-2020, the trend within the Gela NPCS was evaluated by comparing two subperiods (2010-2014 and 2015-2020). SETTING AND PARTICIPANTS: children up to 1 year of age with hypospadias resident in the municipality of Gela in the period 2010-2020. MAIN OUTCOMES MEASURES: crude odds ratios (OR) and respective 95% confidence intervals (95%CI) were used to compare the prevalence observed in Gela and that detected in the comparison areas. RESULTS: excess risk for hypospadias was highlighted in 2010-2020 in Gela vs Sicily (OR 4.45; 95%CI 3.45-5.75), vs ALG (OR 4.29; 95%CI 3.02-6.10), and vs the NPCSs of Milazzo (OR 2.32; 95%CI 1.32-4.07) and Priolo (OR 2.37; 95%CI 1.55-3.62). The between-period comparisons in Gela did not show an important difference between 2010-2014 and 2015-2020 (OR 1.37; 95%CI 0.83-2.24), with a prevalence of 98.9 and 72.4 per 10,000, respectively. CONCLUSIONS: the prevalence of hypospadias in 2015-2020 remains very high, although decreasing when compared to 2010-2014 period. The Gela data, despite the refinery being closed after 2014, suggest a complex situation in which multiple risk factors may play a role.

Gastric ghrelin cells in obese patients are hyperactive.

BACKGROUND/OBJECTIVES: Distribution and activity of ghrelin cells in the stomach of obese subjects are controversial. SUBJECTS/METHODS: We examined samples from stomachs removed by sleeve gastrectomy in 49 obese subjects (normoglycemic, hyperglycemic and diabetic) and quantified the density of ghrelin/chromogranin endocrine cells by immunohistochemistry. Data were compared with those from 13 lean subjects evaluated by gastroscopy. In 44 cases (11 controls and 33 obese patients) a gene expression analysis of ghrelin and its activating enzyme ghrelin O-acyl transferase (GOAT) was performed. In 21 cases (4 controls and 17 obese patients) the protein levels of unacylated and acylated-ghrelin were measured by ELISA tests. In 18 cases (4 controls and 14 obese patients) the morphology of ghrelin-producing cells was evaluated by electron microscopy. RESULTS: The obese group, either considered as total population or divided into subgroups, did not show any significant difference in ghrelin cell density when compared with control subjects. Inter-glandular smooth muscle fibres were increased in obese patients. In line with a positive trend of the desacylated form found by ELISA, Ghrelin and GOAT mRNA expression in obese patients was significantly increased. The unique ghrelin cell ultrastructure was maintained in all obese groups. In the hyperglycemic obese patients, the higher ghrelin expression matched with ultrastructural signs of endocrine hyperactivity, including expanded rough endoplasmic reticulum and reduced density, size and electron-density of endocrine granules. A positive correlation between ghrelin gene expression and glycemic values, body mass index and GOAT was also found. All obese patients with type 2 diabetes recovered from diabetes at follow-up after 5 months with a 16.5% of weight loss. CONCLUSIONS: Given the known inhibitory role on insulin secretion of ghrelin, these results suggest a possible role for gastric ghrelin overproduction in the complex architecture that takes part in the pathogenesis of type 2 diabetes.

Fusion Transcripts of Adjacent Genes: New Insights into the World of Human Complex Transcripts in Cancer.

The awareness of genome complexity brought a radical approach to the study of transcriptome, opening eyes to single RNAs generated from two or more adjacent genes according to the present consensus. This kind of transcript was thought to originate only from chromosomal rearrangements, but the discovery of readthrough transcription opens the doors to a new world of fusion RNAs. In the last years many possible intergenic cis-splicing mechanisms have been proposed, unveiling the origins of transcripts that contain some exons of both the upstream and downstream genes. In some cases, alternative mechanisms, such as trans-splicing and transcriptional slippage, have been proposed. Five databases, containing validated and predicted Fusion Transcripts of Adjacent Genes (FuTAGs), are available for the scientific community. A comparative analysis revealed that two of them contain the majority of the results. A complete analysis of the more widely characterized FuTAGs is provided in this review, including their expression pattern in normal tissues and in cancer. Gene structure, intergenic splicing patterns and exon junction sequences have been determined and here reported for well-characterized FuTAGs. The available functional data and the possible roles in cancer progression are discussed.

Measuring cancer driving force of chromosomal aberrations through multi-layer Boolean implication networks.

Multi-layer Complex networks are commonly used for modeling and analysing biological entities. This paper presents the advantage of using COMBO (Combining Multi Bio Omics) to suggest a new role of the chromosomal aberration as a cancer driver factor. Exploiting the heterogeneous multi-layer networks, COMBO integrates gene expression and DNA-methylation data in order to identify complex bilateral relationships between transcriptome and epigenome. We evaluated the multi-layer networks generated by COMBO on different TCGA cancer datasets (COAD, BLCA, BRCA, CESC, STAD) focusing on the effect of a specific chromosomal numerical aberration, broad gain in chromosome 20, on different cancer histotypes. In addition, the effect of chromosome 8q amplification was tested in the same TCGA cancer dataset. The results demonstrate the ability of COMBO to identify the chromosome 20 amplification cancer driver force in the different TCGA Pan Cancer project datasets.

KDM2B is required for ribosome biogenesis and its depletion unequally affects mRNA translation.

KDM2B is a JmjC domain lysine demethylase, which promotes cell immortalization, stem cell self-renewal and tumorigenesis. Here we employed a multi-omics strategy to address its role in ribosome biogenesis and mRNA translation. These processes are required to sustain cell proliferation, an important cancer hallmark. Contrary to earlier observations, KDM2B promotes ribosome biogenesis by stimulating the transcription of genes encoding ribosome biogenesis factors and ribosomal proteins, particularly those involved in the biogenesis of the 40S ribosomal subunits. Knockdown of KDM2B impaired the assembly of the small and large subunit processomes, as evidenced by specific defects in pre-ribosomal RNA processing. The final outcome was a decrease in the rate of ribosome assembly and in the abundance of ribosomes, and inhibition of mRNA translation. The inhibition of translation was distributed unequally among mRNAs with different features, suggesting that mRNA-embedded properties influence how mRNAs interpret ribosome abundance. This study identified a novel mechanism contributing to the regulation of translation and provided evidence for a rich biology elicited by a pathway that depends on KDM2B, and perhaps other regulators of translation.

Transcriptional regulation of amino acid metabolism by KDM2B, in the context of ncPRC1.1 and in concert with MYC and ATF4.

INTRODUCTION: KDM2B encodes a JmjC domain-containing histone lysine demethylase, which functions as an oncogene in several types of tumors, including TNBC. This study was initiated to address the cancer relevance of the results of our earlier work, which had shown that overexpression of KDM2B renders mouse embryonic fibroblasts (MEFs) resistant to oxidative stress by regulating antioxidant mechanisms. METHODS: We mainly employed a multi-omics strategy consisting of RNA-Seq, quantitative TMT proteomics, Mass-spectrometry-based global metabolomics, ATAC-Seq and ChIP-seq, to explore the role of KDM2B in the resistance to oxidative stress and intermediary metabolism. These data and data from existing patient datasets were analyzed using bioinformatic tools, including exon-intron-split analysis (EISA), FLUFF and clustering analyses. The main genetic strategy we employed was gene silencing with shRNAs. ROS were measured by flow cytometry, following staining with CellROX and various metabolites were measured with biochemical assays, using commercially available kits. Gene expression was monitored with qRT-PCR and immunoblotting, as indicated. RESULTS: The knockdown of KDM2B in basal-like breast cancer cell lines lowers the levels of GSH and sensitizes the cells to ROS inducers, GSH targeting molecules, and DUB inhibitors. To address the mechanism of GSH regulation, we knocked down KDM2B in MDA-MB-231 cells and we examined the effects of the knockdown, using a multi-omics strategy. The results showed that KDM2B, functioning in the context of ncPRC1.1, regulates a network of epigenetic and transcription factors, which control a host of metabolic enzymes, including those involved in the SGOC, glutamate, and GSH metabolism. They also showed that KDM2B enhances the chromatin accessibility and expression of MYC and ATF4, and that it binds in concert with MYC and ATF4, the promoters of a large number of transcriptionally active genes, including many, encoding metabolic enzymes. Additionally, MYC and ATF4 binding sites were enriched in genes whose accessibility depends on KDM2B, and analysis of a cohort of TNBCs expressing high or low levels of KDM2B, but similar levels of MYC and ATF4 identified a subset of MYC targets, whose expression correlates with the expression of KDM2B. Further analyses of basal-like TNBCs in the same cohort, revealed that tumors expressing high levels of all three regulators exhibit a distinct metabolic signature that carries a poor prognosis. CONCLUSIONS: The present study links KDM2B, ATF4, and MYC in a transcriptional network that regulates the expression of multiple metabolic enzymes, including those that control the interconnected SGOC, glutamate, and GSH metabolic pathways. The co-occupancy of the promoters of many transcriptionally active genes, by all three factors, the enrichment of MYC binding sites in genes whose chromatin accessibility depends on KDM2B, and the correlation of the levels of KDM2B with the expression of a subset of MYC target genes in tumors that express similar levels of MYC, suggest that KDM2B regulates both the expression and the transcriptional activity of MYC. Importantly, the concerted expression of all three factors also defines a distinct metabolic subset of TNBCs with poor prognosis. Overall, this study identifies novel mechanisms of SGOC regulation, suggests novel KDM2B-dependent metabolic vulnerabilities in TNBC, and provides new insights into the role of KDM2B in the epigenetic regulation of transcription.

Nucleolin acute degradation reveals novel functions in cell cycle progression and division in TNBC.

Nucleoli are large nuclear sub-compartments where vital processes, such as ribosome assembly, take place. Technical obstacles still limit our understanding of the biological functions of nucleolar proteins in cell homeostasis and cancer pathogenesis. Since most nucleolar proteins are essential, their abrogation cannot be achieved through conventional approaches. Additionally, the biological activities of many nucleolar proteins are connected to their physiological concentration. Thus, artificial overexpression might not fully recapitulate their endogenous functions. Proteolysis-based approaches, such as the Auxin Inducible Degron (AID) system paired with CRISPR/Cas9 knock-in gene-editing, have the potential to overcome these limitations, providing unprecedented characterization of the biological activities of endogenous nucleolar proteins. We applied this system to endogenous nucleolin (NCL), one of the most abundant nucleolar proteins, and characterized the impact of its acute depletion on Triple-Negative Breast Cancer (TNBC) cell behavior. Abrogation of endogenous NCL reduced proliferation and caused defective cytokinesis, resulting in bi-nucleated tetraploid cells. Bioinformatic analysis of patient data, and quantitative proteomics using our experimental NCL-depleted model, indicated that NCL levels are correlated with the abundance of proteins involved in chromosomal segregation. In conjunction with its effects on sister chromatid dynamics, NCL abrogation enhanced the anti-proliferative effects of chemical inhibitors of mitotic modulators such as the Anaphase Promoting Complex. In summary, using the AID system in combination with CRISPR/Cas9 for endogenous gene editing, our findings indicate a novel role for NCL in supporting the completion of the cell division in TNBC models, and that its abrogation could enhance the therapeutic activity of mitotic-progression inhibitors.

Transcriptional regulation of amino acid metabolism by KDM2B, in the context of ncPRC1.1 and in concert with MYC and ATF4.

Introduction: KDM2B encodes a JmjC domain-containing histone lysine demethylase, which functions as an oncogene in several types of tumors, including TNBC. This study was initiated to address the cancer relevance of the results of our earlier work, which had shown that overexpression of KDM2B renders mouse embryonic fibroblasts (MEFs) resistant to oxidative stress by regulating antioxidant mechanisms. Methods: We mainly employed a multi-omics strategy consisting of RNA-Seq, quantitative TMT proteomics, Mass-spectrometry-based global metabolomics, ATAC-Seq and ChIP-seq, to explore the role of KDM2B in the resistance to oxidative stress and intermediary metabolism. These data and data from existing patient datasets were analyzed using bioinformatic tools, including exon-intron-split analysis (EISA), FLUFF and clustering analyses. The main genetic strategy we employed was gene silencing with shRNAs. ROS were measured by flow cytometry, following staining with CellROX and various metabolites were measured with biochemical assays, using commercially available kits. Gene expression was monitored with qRT-PCR and immunoblotting, as indicated. Results: The knockdown of KDM2B in basal-like breast cancer cell lines lowers the levels of GSH and sensitizes the cells to ROS inducers, GSH targeting molecules, and DUB inhibitors. To address the mechanism of GSH regulation, we knocked down KDM2B in MDA-MB-231 cells and we examined the effects of the knockdown, using a multi-omics strategy. The results showed that KDM2B, functioning in the context of ncPRC1.1, regulates a network of epigenetic and transcription factors, which control a host of metabolic enzymes, including those involved in the SGOC, glutamate, and GSH metabolism. They also showed that KDM2B enhances the chromatin accessibility and expression of MYC and ATF4, and that it binds in concert with MYC and ATF4, the promoters of a large number of transcriptionally active genes, including many, encoding metabolic enzymes. Additionally, MYC and ATF4 binding sites were enriched in genes whose accessibility depends on KDM2B, and analysis of a cohort of TNBCs expressing high or low levels of KDM2B, but similar levels of MYC and ATF4 identified a subset of MYC targets, whose expression correlates with the expression of KDM2B. Further analyses of basal-like TNBCs in the same cohort, revealed that tumors expressing high levels of all three regulators exhibit a distinct metabolic signature that carries a poor prognosis. Conclusions: The present study links KDM2B, ATF4, and MYC in a transcriptional network that regulates the expression of multiple metabolic enzymes, including those that control the interconnected SGOC, glutamate, and GSH metabolic pathways. The co-occupancy of the promoters of many transcriptionally active genes, by all three factors, the enrichment of MYC binding sites in genes whose chromatin accessibility depends on KDM2B, and the correlation of the levels of KDM2B with the expression of a subset of MYC target genes in tumors that express similar levels of MYC, suggest that KDM2B regulates both the expression and the transcriptional activity of MYC. Importantly, the concerted expression of all three factors also defines a distinct metabolic subset of TNBCs with poor prognosis. Overall, this study identifies novel mechanisms of SGOC regulation, suggests novel KDM2B-dependent metabolic vulnerabilities in TNBC, and provides new insights into the role of KDM2B in the epigenetic regulation of transcription.

Olfactory Ensheathing Cells express both Ghrelin and Ghrelin Receptor in vitro: a new hypothesis in favor of a neurotrophic effect.

Olfactory Ensheathing Cells (OECs) are glial cells able to secrete different neurotrophic growth factors and thus promote axonal growth, also acting as a mechanical support. In the olfactory system, during development, they drive the non-myelinated axons of the Olfactory Receptor Neurons (ORNs) towards the Olfactory Bulb (OB). Ghrelin (Ghre), a gut-brain peptide hormone, and its receptor (GHS-R 1a) are expressed in different parts of the central nervous system. In the last few years, this peptide has stimulated particular interest as results show it to be a neuroprotective factor with antioxidant, anti-inflammatory and anti-apoptotic properties. Our previous studies showed that OB mitral cells express Ghre, thus being able to play an important role in regulating food behavior in response to odors. In this study, we investigated the presence of Ghre and GHS-R 1a in primary mouse OECs. The expression of both Ghre and its receptor was assessed by an immunocytochemical technique, Western Blot and Polymerase Chain Reaction (PCR) analysis. Our results demonstrated that OECs are able to express both Ghre and GHS-R 1a and that these proteins are detectable after extensive passages in vitro; in addition, PCR analysis further confirmed these data. Therefore, we can hypothesize that Ghre and GHS-R 1a interact with a reinforcement function, in the peripheral olfactory circuit, providing a neurotrophic support to the synaptic interaction between ORNs and mitral cells.

Working-for-Food Behaviors: A Preclinical Study in Prader-Willi Mutant Mice.

Abnormal feeding behavior is one of the main symptoms of Prader-Willi syndrome (PWS). By studying a PWS mouse mutant line, which carries a paternally inherited deletion of the small nucleolar RNA 116 (Snord116), we observed significant changes in working-for-food behavioral responses at various timescales. In particular, we report that PWS mutant mice show a significant delay compared to wild-type littermate controls in responding to both hour-scale and seconds-to-minutes-scale time intervals. This timing shift in mutant mice is associated with better performance in the working-for-food task, and results in better decision making in these mutant mice. The results of our study reveal a novel aspect of the organization of feeding behavior, and advance the understanding of the interplay between the metabolic functions and cognitive mechanisms of PWS.