A multi-omic approach to study an interesting case of type VI osteogenesis imperfecta

Background: Osteogenesis imperfecta (OI) also known as brittle bone disease, is a genetic pathology in which bones do not form properly and therefore are fragile and break easily. Is a heterogeneous congenital heritable disease that mainly affects connective tissues. Nowadays we number 18 types of OI, distinguished into autosomal dominant, recessive and X-linked inheritance. OI type VI is caused by loss-of-function mutations in SERPINF1, which shows recessive inheritance. Lack of the gene product, PEDF, causes an atypical bone mineralization defect determining a unique clinical phenotype. Aim: the aim of the study is to identify genomic, epigenomic and metabolomic variations that are associated to the disease status in individuals that belong to a nuclear Pakistan family in which is supposed to be segregate type VI osteogenesis imperfecta. Results: exome sequencing confirmed the consanguinity between the parents and shared regions of homozygosity between affected were observed in chr7, chr12 and chr22. In the hypothesis of Autosomal Recessive disease, any compatible mutation was found, and no clear pathogenic variant have been detected. Thus, we explore compound heterozygosis model, identifying and suggesting as potential candidate CERCAM gene, but it is role in bone homeostasis it is still unknown. Epigenetic investigation highlights some interesting genes, known to be involved in bone metabolism, such as RXRA (Retinoid X Receptor Alpha), ELK3 (ETS Transcription Factor) and GLI2 (GLI Family Zinc Finger 2). Metabolomic profiling found 4 modulated pathways: phenylalanine, tyrosine and tryptophan biosynthesis, tryptophan metabolism, pyrimidine metabolism, and Vitamin B6 metabolism. Conclusions: the future investigation will try to enhance and integrate the results from the present omics (transcriptomic analysis is ongoing) into a context of system biology aimed to depict and clarify the defects and biological processes associated to the disease

New Insights into the Runt Domain of RUNX2 in Melanoma Cell Proliferation and Migration

The mortality rate for malignant melanoma (MM) is very high, since it is highly invasive and resistant to chemotherapeutic treatments. The modulation of some transcription factors affects cellular processes in MM. In particular, a higher expression of the osteogenic master gene RUNX2 has been reported in melanoma cells, compared to normal melanocytes. By analyzing public databases for recurrent RUNX2 genetic and epigenetic modifications in melanoma, we found that the most common RUNX2 genetic alteration that exists in transcription upregulation is, followed by genomic amplification, nucleotide substitution and multiple changes. Additionally, altered RUNX2 is involved in unchecked pathways promoting tumor progression, Epithelial Mesenchymal Transition (EMT), and metastasis. In order to investigate further the role of RUNX2 in melanoma development and to identify a therapeutic target, we applied the CRISPR/Cas9 technique to explore the role of the RUNT domain of RUNX2 in a melanoma cell line. RUNT-deleted cells showed reduced proliferation, increased apoptosis, and reduced EMT features, suggesting the involvement of the RUNT domain in different pathways. In addition, del-RUNT cells showed a downregulation of genes involved in migration ability. In an in vivo zebrafish model, we observed that wild-type melanoma cells migrated in 81% of transplanted fishes, while del-RUNT cells migrated in 58%. All these findings strongly suggest the involvement of the RUNT domain in melanoma metastasis and cell migration and indicate RUNX2 as a prospective target in MM therapy

A potential role of RUNX2- RUNT domain in modulating the expression of genes involved in bone metastases: an in vitro study with melanoma cells

Ectopic expression of RUNX2 has been reported in several tumors. In melanoma cells, the RUNT domain of RUNX2 increases cell proliferation and migration. Due to the strong link between RUNX2 and skeletal development, we hypothesized that the RUNT domain may be involved in the modulation of mechanisms associated with melanoma bone metastasis. Therefore, we evaluated the expression of metastatic targets in wild type (WT) and RUNT KO melanoma cells by array and real-time PCR analyses. Western blot, ELISA, immunofluorescence, migration and invasion ability assays were also performed. Our findings showed that the expression levels of bone sialoprotein (BSP) and osteopontin (SPP1) genes, which are involved in malignancy-induced hypercalcemia, were reduced in RUNT KO cells. In addition, released PTHrP levels were lower in RUNT KO cells than in WT cells. The RUNT domain also contributes to increased osteotropism and bone invasion in melanoma cells. Importantly, we found that the ERK/p-ERK and AKT/p-AKT pathways are involved in RUNT-promoted bone metastases. On the basis of our findings, we concluded that the RUNX2 RUNT domain is involved in the mechanisms promoting bone metastasis of melanoma cells via complex interactions between multiple players involved in bone remodeling

Physical Exercise Modulates miR-21-5p, miR-129-5p, miR-378-5p, and miR-188-5p Expression in Progenitor Cells Promoting Osteogenesis

Physical exercise is known to promote beneficial effects on overall health, counteracting risks related to degenerative diseases. MicroRNAs (miRNAs), short non-coding RNAs affecting the expression of a cell's transcriptome, can be modulated by different stimuli. Yet, the molecular effects on osteogenic differentiation triggered by miRNAs upon physical exercise are not completely understood. In this study, we recruited 20 male amateur runners participating in a half marathon. Runners' sera, collected before (PRE RUN) and after (POST RUN) the run, were added to cultured human mesenchymal stromal cells. We then investigated their effects on the modulation of selected miRNAs and the consequential effects on osteogenic differentiation. Our results showed an increased expression of miRNAs promoting osteogenic differentiation (miR-21-5p, miR-129-5p, and miR-378-5p) and a reduced expression of miRNAs involved in the adipogenic differentiation of progenitor cells (miR-188-5p). In addition, we observed the downregulation of PTEN and SMAD7 expression along with increased AKT/pAKT and SMAD4 protein levels in MSCs treated with POST RUN sera. The consequent upregulation of RUNX2 expression was also proven, highlighting the molecular mechanisms by which miR-21-5p promotes osteogenic differentiation. In conclusion, our work proposes novel data, which demonstrate how miRNAs may regulate the osteogenic commitment of progenitor cells in response to physical exercise

Sphingomyelin and medullary sponge kidney disease: a biological link identified by omics approach

Background: Molecular biology has recently added new insights into the comprehension of the physiopathology of the medullary sponge kidney disease (MSK), a rare kidney malformation featuring nephrocalcinosis and recurrent renal stones. Pathogenesis and metabolic alterations associated to this disorder have been only partially elucidated.Methods: Plasma and urine samples were collected from 15 MSK patients and 15 controls affected by idiopathic calcium nephrolithiasis (ICN). Plasma metabolomic profile of 7 MSK and 8 ICN patients was performed by liquid chromatography combined with electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS). Subsequently, we reinterrogated proteomic raw data previously obtained from urinary microvesicles of MSK and ICN focusing on proteins associated with sphingomyelin metabolism. Omics results were validated by ELISA in the entire patients' cohort.Results: Thirteen metabolites were able to discriminate MSK from ICN (7 increased and 6 decreased in MSK vs. ICN). Sphingomyelin reached the top level of discrimination between the two study groups (FC: -1.8, p < 0.001). Ectonucleotide pyrophophatase phosphodiesterase 6 (ENPP6) and osteopontin (SPP1) resulted the most significant deregulated urinary proteins in MSK vs. ICN (p < 0.001). ENPP6 resulted up-regulated also in plasma of MSK by ELISA.Conclusion: Our data revealed a specific high-throughput metabolomics signature of MSK and indicated a pivotal biological role of sphingomyelin in this disease

Analyzing BioRad-Illumina Single Cell RNA-Seq data with open source tools

Single cell RNA-Seq is a powerful technique that is becoming more popular since it enables to sequence the transcriptome of each cell within a population of different cell types in a single experiment. Currently, there are a few different technologies, like BioRad-Illumina ddSeq and 10X Chromium

Runx2 stimulates neoangiogenesis through the Runt domain in melanoma

Runx2 is a transcription factor involved in melanoma cell migration and proliferation. Here, we extended the analysis of Runt domain of Runx2 in melanoma cells to deepen understanding of the underlying mechanisms. By the CRISPR/Cas9 system we generated the Runt KO melanoma cells 3G8. Interestingly, the proteome analysis showed a specific protein signature of 3G8 cells related to apoptosis and migration, and pointed out the involvement of Runt domain in the neoangiogenesis process. Among the proteins implicated in angiogenesis we identified fatty acid synthase, chloride intracellular channel protein-4, heat shock protein beta-1, Rho guanine nucleotide exchange factor 1, D-3-phosphoglycerate dehydrogenase, myosin-1c and caveolin-1. Upon querying the TCGA provisional database for melanoma, the genes related to these proteins were found altered in 51.36% of total patients. In addition, VEGF gene expression was reduced in 3G8 as compared to A375 cells; and HUVEC co-cultured with 3G8 cells expressed lower levels of CD105 and CD31 neoangiogenetic markers. Furthermore, the tube formation assay revealed down-regulation of capillary-like structures in HUVEC co-cultured with 3G8 in comparison to those with A375 cells. These findings provide new insight into Runx2 molecular details which can be crucial to possibly propose it as an oncotarget of melanoma

Identification of miRNAs of Strongyloides stercoralis L1 and iL3 larvae isolated from human stool

Strongyloidiasis is a neglected tropical disease caused by the soil-transmitted nematode by Strongyloides stercoralis, that affects approximately 600 million people worldwide. In immunosuppressed individuals disseminated strongyloidiasis can rapidly lead to fatal outcomes. There is no gold standard for diagnosing strongyloidiasis, and infections are frequently misdiagnosed. A better understanding of the molecular biology of this parasite can be useful for example for the discovery of potential new biomarkers. Interestingly, recent evidence showed the presence of small RNAs in Strongyloididae, but no data was provided for S. stercoralis. In this study, we present the first identification of miRNAs of both L1 and iL3 larval stages of S. stercoralis. For our purpose, the aims were: (i) to analyse the miRNome of L1 and iL3 S. stercoralis and to identify potential miRNAs of this nematode, (ii) to obtain the mRNAs profiles in these two larval stages and (iii) to predict potential miRNA target sites in mRNA sequences. Total RNA was isolated from L1 and iL3 collected from the stool of 5 infected individuals. For the miRNAs analysis, we used miRDeep2 software and a pipeline of bio-informatic tools to construct a catalog of a total of 385 sequences. Among these, 53% were common to S. ratti, 19% to S. papillosus, 1% to Caenorhabditis elegans and 44% were novel. Using a differential analysis between the larval stages, we observed 6 suggestive modulated miRNAs (STR-MIR-34A-3P, STR-MIR-8397-3P, STR-MIR-34B-3P and STR-MIR-34C-3P expressed more in iL3, and STR-MIR-7880H-5P and STR-MIR-7880M-5P expressed more in L1). Along with this analysis, we obtained also the mRNAs profiles in the same samples of larvae. Multiple testing found 81 statistically significant mRNAs of the total 1553 obtained (FDR < 0.05; 32 genes expressed more in L1 than iL3; 49 genes expressed more in L3 than iL1). Finally, we found 33 predicted mRNA targets of the modulated miRNAs, providing relevant data for a further validation to better understand the role of these small molecules in the larval stages and their valuein clinical diagnostics

Physical activity modulates miRNAs levels and enhances MYOD expression in myoblasts

Stem cells functions are regulated by different factors and non-conding RNAs, such as microRNA. MiRNAsplay an important role in modulating the expression of genes involved in the commitment and differentiation of progenitor cells. MiRNAs are post transcriptional regulators which may be modulated by physical exercise. MiRNAs, by regulating different signaling pathways, play an important role in myogenesis as well as in muscle activity. MiRNAs quantification may be considered for evaluating physical performance or muscle recovery. With the aim to identify specific miRNAs potentially involved in myogenesis and modulated by physical activity, we investigated miRNAs expression following physical performance in Peripheral Blood Mononuclear Cells (PBMCs) and in sera of half marathon (HM) runnners. The effect of runners sera on Myogenesis in in vitro cellular models was also explored. Therefore, we performed Microarray Analysis and Real Time PCR assays, as well as in vitro cell cultures analysis to investigate myogenic differentiation. Our data demonstrated gender-specific expression patterns of PBMC miRNAs before physical performance. In particular, miR223-3p, miR26b-5p, miR150-5p and miR15-5p expression was higher, while miR7a-5p and miR7i-5p expression was lower in females compared to males. After HM, miR152-3p, miR143-3p, miR27a-3p levels increased while miR30b-3p decreased in both females and males: circulating miRNAs mirrored these modulations. Furthermore, we also observed that the addition of post-HM participants sera to cell cultures exerted a positive effect in stimulating myogenesis. In conclusion, our data suggest that physical activity induces the modulation of myogenesis-associated miRNAs in bothfemales and males, despite the gender-associated different expression of certain miRNAs, Noteworthy, these findings might be useful for evaluating potential targets for microRNA based-therapies in diseases affecting the myogenic stem cells population

Physical activity prevents cartilage degradation: a metabolomics study pinpoints the involvement of vitamin B6

Osteoarthritis (OA) is predominantly characterized by the progressive degradation of articular cartilage, the connective tissue produced by chondrocytes, due to an imbalance between anabolic and catabolic processes. In addition, physical activity (PA) is recognized as an important tool for counteracting OA. To evaluate PA effects on the chondrocyte lineage, we analyzed the expression of SOX9, COL2A1, and COMP in circulating progenitor cells following a half marathon (HM) performance. Therefore, we studied in-depth the involvement of metabolites affecting chondrocyte lineage, and we compared the metabolomic profile associated with PA by analyzing runners' sera before and after HM performance. Interestingly, this study highlighted that metabolites involved in vitamin B6 salvage, such as pyridoxal 5'-phosphate and pyridoxamine 5'-phosphate, were highly modulated. To evaluate the effects of vitamin B6 in cartilage cells, we treated differentiated mesenchymal stem cells and the SW1353 chondrosarcoma cell line with vitamin B6 in the presence of IL1\u3b2, the inflammatory cytokine involved in OA. Our study describes, for the first time, the modulation of the vitamin B6 salvage pathway following PA and suggests a protective role of PA in OA through modulation of this pathway