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Article ; Online: MicroRNA Expression Profile Distinguishes Glioblastoma Stem Cells from Differentiated Tumor Cells

Sara Tomei / Andrea Volontè / Shilpa Ravindran / Stefania Mazzoleni / Ena Wang / Rossella Galli / Cristina Maccalli

Journal of Personalized Medicine, Vol 11, Iss 264, p

2021 Volume 264

Abstract: Glioblastoma (GBM) represents the most common and aggressive tumor of the brain. Despite the fact that several studies have recently addressed the molecular mechanisms underlying the disease, its etiology and pathogenesis are still poorly understood. GBM ...

Abstract	Glioblastoma (GBM) represents the most common and aggressive tumor of the brain. Despite the fact that several studies have recently addressed the molecular mechanisms underlying the disease, its etiology and pathogenesis are still poorly understood. GBM displays poor prognosis and its resistance to common therapeutic approaches makes it a highly recurrent tumor. Several studies have identified a subpopulation of tumor cells, known as GBM cancer stem cells (CSCs) characterized by the ability of self-renewal, tumor initiation and propagation. GBM CSCs have been shown to survive GBM chemotherapy and radiotherapy. Thus, targeting CSCs represents a promising approach to treat GBM. Recent evidence has shown that GBM is characterized by a dysregulated expression of microRNA (miRNAs). In this study we have investigated the difference between human GBM CSCs and their paired autologous differentiated tumor cells. Array-based profiling and quantitative Real-Time PCR (qRT-PCR) were performed to identify miRNAs differentially expressed in CSCs. The Cancer Genome Atlas (TCGA) data were also interrogated, and functional interpretation analysis was performed. We have identified 14 miRNAs significantly differentially expressed in GBM CSCs ( p < 0.005). MiR-21 and miR-95 were among the most significantly deregulated miRNAs, and their expression was also associated to patient survival. We believe that the data provided here carry important implications for future studies aiming at elucidating the molecular mechanisms underlying GBM.
Keywords	glioblastoma ; microRNAs ; cancer ; qPCR ; cancer stem cells ; Medicine ; R
Subject code	610
Language	English
Publishing date	2021-04-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Effective Multivalent Oriented Presentation of Meningococcal NadA Antigen Trimers by Self-Assembling Ferritin Nanoparticles

Daniele Veggi / Lucia Dello Iacono / Enrico Malito / Giulietta Maruggi / Fabiola Giusti / Panchali Goswami / Werner Pansegrau / Sara Marchi / Sara Tomei / Enrico Luzzi / Matthew James Bottomley / Federico Fontani / Ilaria Ferlenghi / Maria Scarselli

International Journal of Molecular Sciences, Vol 24, Iss 6183, p

2023 Volume 6183

Abstract: The presentation of viral antigens on nanoparticles in multivalent arrays has emerged as a valuable technology for vaccines. On the nanoparticle surface, highly ordered, repetitive arrays of antigens can mimic their geometric arrangement on virion ... ...

Abstract	The presentation of viral antigens on nanoparticles in multivalent arrays has emerged as a valuable technology for vaccines. On the nanoparticle surface, highly ordered, repetitive arrays of antigens can mimic their geometric arrangement on virion surfaces and elicit stronger humoral responses than soluble viral antigens. More recently, bacterial antigens have been presented on self-assembling protein nanoparticles and have elicited protective antibody and effective T-helper responses, further supporting the nanoparticle platform as a universal approach for stimulating potent immunogenicity. Here, we present the rational design, structural analysis, and immunogenicity of self-assembling ferritin nanoparticles displaying eight copies of the Neisseria meningitidis trimeric adhesin NadA. We engineered constructs consisting of two different NadA fragments, head only and head with stalk, that we fused to ferritin and expressed in Escherichia coli . Both fusion constructs self-assembled into the expected nanoparticles as determined by Cryo electron microscopy. In mice, the two nanoparticles elicited comparable NadA antibody levels that were 10- to 100-fold higher than those elicited by the corresponding NadA trimer subunits. Further, the NadAferritin nanoparticles potently induced complement-mediated serum bactericidal activity. These findings confirm the value of self-assembling nanoparticles for optimizing the immunogenicity of bacterial antigens and support the broad applicability of the approach to vaccine programs, especially for the presentation of trimeric antigens.
Keywords	nanoparticles ; ferritin ; bacterial antigen ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	620
Language	English
Publishing date	2023-03-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Human sample authentication in biomedical research

Harshitha Shobha Manjunath / Nicola James / Rebecca Mathew / Muna Al Hashmi / Lee Silcock / Ida Biunno / Pasquale De Blasio / Chidambaram Manickam / Sara Tomei

Scientific Reports, Vol 11, Iss 1, Pp 1-

comparison of two platforms

2021 Volume 9

Abstract: Abstract Samples used in biomedical research are often collected over years, in some cases from subjects that may have died and thus cannot be retrieved in any way. The value of these samples is priceless. Sample misidentification or mix-up are ... ...

Abstract	Abstract Samples used in biomedical research are often collected over years, in some cases from subjects that may have died and thus cannot be retrieved in any way. The value of these samples is priceless. Sample misidentification or mix-up are unfortunately common problems in biomedical research and can eventually result in the publication of incorrect data. Here we have compared the Fluidigm SNPtrace and the Agena iPLEX Sample ID panels for the authentication of human genomic DNA samples. We have tested 14 pure samples and simulated their cross-contamination at different percentages (2%, 5%, 10%, 25% and 50%). For both panels, we report call rate, allele intensity/probability score, performance in distinguishing pure samples and contaminated samples at different percentages, and sex typing. We show that both panels are reliable and efficient methods for sample authentication and we highlight their advantages and disadvantages. We believe that the data provided here is useful for sample authentication especially in biorepositories and core facility settings.
Keywords	Medicine ; R ; Science ; Q
Subject code	310
Language	English
Publishing date	2021-07-01T00:00:00Z
Publisher	Nature Portfolio
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Immunomodulatory Effects of Vitamin D Supplementation in a Deficient Population

Mathieu Garand / Mohammed Toufiq / Parul Singh / Susie Shih Yin Huang / Sara Tomei / Rebecca Mathew / Valentina Mattei / Mariam Al Wakeel / Elham Sharif / Souhaila Al Khodor

International Journal of Molecular Sciences, Vol 22, Iss 5041, p

2021 Volume 5041

Abstract: In addition to its canonical functions, vitamin D has been proposed to be an important mediator of the immune system. Despite ample sunshine, vitamin D deficiency is prevalent (>80%) in the Middle East, resulting in a high rate of supplementation. ... ...

Abstract	In addition to its canonical functions, vitamin D has been proposed to be an important mediator of the immune system. Despite ample sunshine, vitamin D deficiency is prevalent (>80%) in the Middle East, resulting in a high rate of supplementation. However, the underlying molecular mechanisms of the specific regimen prescribed and the potential factors affecting an individual’s response to vitamin D supplementation are not well characterized. Our objective is to describe the changes in the blood transcriptome and explore the potential mechanisms associated with vitamin D3 supplementation in one hundred vitamin D-deficient women who were given a weekly oral dose (50,000 IU) of vitamin D3 for three months. A high-throughput targeted PCR, composed of 264 genes representing the important blood transcriptomic fingerprints of health and disease states, was performed on pre and post-supplementation blood samples to profile the molecular response to vitamin D3. We identified 54 differentially expressed genes that were strongly modulated by vitamin D3 supplementation. Network analyses showed significant changes in the immune-related pathways such as TLR4/CD14 and IFN receptors, and catabolic processes related to NF-kB, which were subsequently confirmed by gene ontology enrichment analyses. We proposed a model for vitamin D3 response based on the expression changes of molecules involved in the receptor-mediated intra-cellular signaling pathways and the ensuing predicted effects on cytokine production. Overall, vitamin D3 has a strong effect on the immune system, G-coupled protein receptor signaling, and the ubiquitin system. We highlighted the major molecular changes and biological processes induced by vitamin D3, which will help to further investigate the effectiveness of vitamin D3 supplementation among individuals in the Middle East as well as other regions.
Keywords	transcriptomic ; 25-hydroxyvitamin D ; 25(OH)D ; immune system ; immune response ; vitamin D deficiency ; Biology (General) ; QH301-705.5 ; Chemistry ; QD1-999
Subject code	610
Language	English
Publishing date	2021-05-01T00:00:00Z
Publisher	MDPI AG
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Investigation of genetic variation and lifestyle determinants in vitamin D levels in Arab individuals

Massimo Mezzavilla / Sara Tomei / Fadi Alkayal / Motasem Melhem / Maisa M. Ali / Monira Al-Arouj / Abdullah Bennakhi / Osama Alsmadi / Naser Elkum

Journal of Translational Medicine, Vol 16, Iss 1, Pp 1-

2018 Volume 6

Abstract: Abstract Background Differences in the concentrations of circulating 25-hydroxyvitamin D [25(OH)D] are associated with a wide range of health outcomes; however, most studies on genetic variants that impact 25(OH)D levels have been conducted in European ... ...

Abstract	Abstract Background Differences in the concentrations of circulating 25-hydroxyvitamin D [25(OH)D] are associated with a wide range of health outcomes; however, most studies on genetic variants that impact 25(OH)D levels have been conducted in European populations. Here we aimed to identify common genetic variants that affect vitamin D concentrations in individuals of self-reported Arab ethnicity. Methods The study included 1151 Arab subjects living in Kuwait. Common variants of single-nucleotide polymorphisms and genes previously associated with vitamin D levels, such as GC, PDE3B, CYP2R1, and NADSYN1, were genotyped. Raw vitamin D level data were corrected for age, body mass index, and sex and then normalized. Regression tree analyses were performed to identify the impact of genetic variants on vitamin D levels. Results Compared with other gene variants, the GC gene variants exhibited the greatest impact on vitamin D levels in our study population, of which rs2298850 had the lowest p value (0.003). Individuals homozygous for the derived allele C had lower vitamin D levels. Analyses of the interaction between the number of years for which the subjects had lived in Kuwait and genetic variation in the GC gene showed that those with the CC genotype of rs2298850 who had lived in Kuwait for < 51 years had a mean 25(OH)D level of 10 ng/ml, whereas those who were homozygous for the ancestral allele had a mean 25(OH)D level of 17 ng/ml. Furthermore, subjects who had lived in Kuwait for > 51 years had higher vitamin D levels (mean 28 ng/ml) regardless of the genotype of their GC gene. Conclusions The GC gene may play a major role in determining vitamin D levels in Arab populations.
Keywords	Vitamin D ; Arab population ; GC gene ; Recursive partitioning analysis ; Medicine ; R
Subject code	333
Language	English
Publishing date	2018-01-01T00:00:00Z
Publisher	BMC
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Influence of storage conditions of small volumes of blood on immune transcriptomic profiles

Rebecca Mathew / Mohammed Toufiq / Valentina Mattei / Muna Al Hashmi / Harshitha Shobha Manjunath / Basirudeen Syed Ahamed Kabeer / Rita Calzone / Chiara Cugno / Damien Chaussabel / Sara Deola / Sara Tomei

BMC Research Notes, Vol 13, Iss 1, Pp 1-

2020 Volume 7

Abstract: Abstract Objective Transcriptome analysis of human whole blood is used to discover biomarkers of diseases and to assess phenotypic traits. Here we have collected small volumes of blood in Tempus solution and tested whether different storage conditions ... ...

Abstract	Abstract Objective Transcriptome analysis of human whole blood is used to discover biomarkers of diseases and to assess phenotypic traits. Here we have collected small volumes of blood in Tempus solution and tested whether different storage conditions have an impact on transcriptomic profiling. Fifty µl of blood were collected in 100µl of Tempus solutions, freezed at − 20 °C for 1 day and eventually thawed, stored and processed under five different conditions: (i) − 20 °C for 1 week; (ii) +4 °C for 1 week; (iii) room temperature for 1 week; (iv) room temperature for 1 day, − 20 °C for 1 day, room temperature until testing at day 7, (v) − 20 °C for 1 week, RNA was isolated and stored in GenTegra solution. We used 272 immune transcript specific assays to test the expression profiling using qPCR based Fluidigm BioMark HD dynamic array. Results RNA yield ranged between 0.17 and 1.39µg. Except for one sample, RIN values were > 7. Using Principal Component Analysis, we saw that the storage conditions did not drive sample distribution. The condition that showed larger variability was the RT-FR-RT (room temperature–freezing–room temperature), suggesting that freezing–thawing cycles may have a worse effect on data reproducibility than keeping the samples at room temperature.
Keywords	Storage ; Blood ; Trancriptome ; QPCR ; Immune profiling ; Tempus ; Medicine ; R ; Biology (General) ; QH301-705.5 ; Science (General) ; Q1-390
Subject code	630
Language	English
Publishing date	2020-03-01T00:00:00Z
Publisher	BMC
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Molecular characterization of low grade and high grade bladder cancer.

Alessandro Apollo / Valerio Ortenzi / Cristian Scatena / Katia Zavaglia / Paolo Aretini / Francesca Lessi / Sara Franceschi / Sara Tomei / Carlo Alberto Sepich / Paolo Viacava / Chiara Maria Mazzanti / Antonio Giuseppe Naccarato

PLoS ONE, Vol 14, Iss 1, p e

2019 Volume 0210635

Abstract: Background Bladder cancer (BC) is the 9th most common cancer diagnosis worldwide. Low grade (LG) represents 70% of all BCs, characterized by recurrence and rare ability (10-15%) to progress to high grade (HG) and invade. The remaining 30% is high grade ( ... ...

Abstract	Background Bladder cancer (BC) is the 9th most common cancer diagnosis worldwide. Low grade (LG) represents 70% of all BCs, characterized by recurrence and rare ability (10-15%) to progress to high grade (HG) and invade. The remaining 30% is high grade (HG), fast invasive BC, which is resistant to therapy. Identifying biomarkers for predicting those tumors able to progress is a key goal for patient outcome improvement. This study focuses on the most promising prognostic markers. Materials and methods TP53 and FGFR3 mutational status, Survivin, CK19, CK20, E-cadherin and CD44 gene expression analysis were performed on 66 BCs. Results Survivin was found associated to tumor grade (p<0.05). Moreover, Survivin correlated with CD44 in TP53 wild type (p = 0.0242) and FGFR3 wild type (p = 0.0036) tumors. In particular the Survivin-CD44 correlation was associated to HG FGFR3 wild type BCs (p = 0.0045). Unsupervised hierarchical clustering based on gene expression data identified four distinct molecular groups reflecting the patient histology (p = 0.038). Conclusion We suggest Survivin, both as a biomarker associated to G3 BCs but negatively related to TP53 mutational status, and as a potential novel therapeutic target.
Keywords	Medicine ; R ; Science ; Q
Subject code	616
Language	English
Publishing date	2019-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Differential responsiveness to BRAF inhibitors of melanoma cell lines BRAF V600E-mutated

Muna Al Hashmi / Konduru S. Sastry / Lee Silcock / Lotfi Chouchane / Valentina Mattei / Nicola James / Rebecca Mathew / Davide Bedognetti / Valeria De Giorgi / Daniela Murtas / Wei Liu / Aouatef Chouchane / Ramzi Temanni / Barbara Seliger / Ena Wang / Francesco M. Marincola / Sara Tomei

Journal of Translational Medicine, Vol 18, Iss 1, Pp 1-

2020 Volume 9

Abstract: Abstract Background Most mutations in melanoma affect one critical amino acid on BRAF gene, resulting in the V600E substitution. Patient management is often based on the use of specific inhibitors targeting this mutation. Methods DNA and RNA mutation ... ...

Abstract	Abstract Background Most mutations in melanoma affect one critical amino acid on BRAF gene, resulting in the V600E substitution. Patient management is often based on the use of specific inhibitors targeting this mutation. Methods DNA and RNA mutation status was assessed in 15 melanoma cell lines by Sanger sequencing and RNA-seq. We tested the cell lines responsiveness to BRAF inhibitors (vemurafenib and PLX4720, BRAF-specific and sorafenib, BRAF non-specific). Cell proliferation was assessed by MTT colorimetric assay. BRAF V600E RNA expression was assessed by qPCR. Expression level of phosphorylated-ERK protein was assessed by Western Blotting as marker of BRAF activation. Results Three cell lines were discordant in the mutation detection (BRAF V600E at DNA level/Sanger sequencing and BRAF WT on RNA-seq). We initially postulated that those cell lines may express only the WT allele at the RNA level although mutated at the DNA level. A more careful analysis showed that they express low level of BRAF RNA and the expression may be in favor of the WT allele. We tested whether the discordant cell lines responded differently to BRAF-specific inhibitors. Their proliferation rate decreased after treatment with vemurafenib and PLX4720 but was not affected by sorafenib, suggesting a BRAF V600E biological behavior. Yet, responsiveness to the BRAF specific inhibitors was lower as compared to the control. Western Blot analysis revealed a decreased expression of p-ERK protein in the BRAF V600E control cell line and in the discordant cell lines upon treatment with BRAF-specific inhibitors. The discordant cell lines showed a lower responsiveness to BRAF inhibitors when compared to the BRAF V600E control cell line. The results obtained from the inhibition experiment and molecular analyses were also confirmed in three additional cell lines. Conclusion Cell lines carrying V600E mutation at the DNA level may respond differently to BRAF targeted treatment potentially due to a lower V600E RNA expression.
Keywords	Next-generation sequencing ; QPCR ; Sanger sequencing ; BRAF mutation ; Medicine ; R
Subject code	616
Language	English
Publishing date	2020-05-01T00:00:00Z
Publisher	BMC
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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Article ; Online: Monoallelic expression in melanoma

Lee Silcock / Hakeem Almabrazi / Younes Mokrab / Puthen Jithesh / Muna Al-Hashmi / Nicola James / Rebecca Mathew / Valentina Mattei / Davide Bedognetti / Francesca Lessi / Ramzi Temanni / Barbara Seliger / Rashid Al-Ali / Francesco M. Marincola / Ena Wang / Sara Tomei

Journal of Translational Medicine, Vol 17, Iss 1, Pp 1-

2019 Volume 7

Abstract: Abstract Background Monoallelic expression (MAE) is a frequent genomic phenomenon in normal tissues, however its role in cancer is yet to be fully understood. MAE is defined as the expression of a gene that is restricted to one allele in the presence of ... ...

Abstract	Abstract Background Monoallelic expression (MAE) is a frequent genomic phenomenon in normal tissues, however its role in cancer is yet to be fully understood. MAE is defined as the expression of a gene that is restricted to one allele in the presence of a diploid heterozygous genome. Constitutive MAE occurs for imprinted genes, odorant receptors and random X inactivation. Several studies in normal tissues have showed MAE in approximately 5–20% of the cases. However, little information exists on the MAE rate in cancer. In this study we assessed the presence and rate of MAE in melanoma. The genetic basis of melanoma has been studied in depth over the past decades, leading to the identification of mutations/genetic alterations responsible for melanoma development. Methods To examine the role of MAE in melanoma we used 15 melanoma cell lines and compared their RNA-seq data with genotyping data obtained by the parental TIL (tumor infiltrating lymphocytes). Genotyping was performed using the Illumina HumanOmni1 beadchip. The RNA-seq library preparation and sequencing was performed using the Illumina TruSeq Stranded Total RNA Human Kit and subsequently sequenced using a HiSeq 2500 according to manufacturer’s guidelines. By comparing genotyping data with RNA-seq data, we identified SNPs in which DNA genotypes were heterozygous and corresponding RNA genotypes were homozygous. All homozygous DNA genotypes were removed prior to the analysis. To confirm the validity to detect MAE, we examined heterozygous DNA genotypes from X chromosome of female samples as well as for imprinted and olfactory receptor genes and confirmed MAE. Results MAE was detected in all 15 cell lines although to a different rate. When looking at the B-allele frequencies we found a preferential pattern of complete monoallelic expression rather then differential monoallelic expression across the 15 melanoma cell lines. As some samples showed high differences in the homozygous and heterozygous call rate, we looked at the single chromosomes and showed that ...
Keywords	Monoallelic expression ; Melanoma ; RNA-seq ; Medicine ; R
Subject code	570
Language	English
Publishing date	2019-04-01T00:00:00Z
Publisher	BMC
Document type	Article ; Online
Database	BASE - Bielefeld Academic Search Engine (life sciences selection)

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