Departamento de Genética Humana
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- Acute venous thromboembolism plasma and red blood cell metabolomic profiling reveals potential new early diagnostic biomarkers: observational clinical studyPublication . Febra, Claúdia; Saraiva, Joana; Vaz, Fátima; Soares, Nelson; Penque, PenqueBackground: Venous thromboembolism (VTE) is a leading cause of cardiovascular mortality. The diagnosis of acute VTE is based on complex imaging exams due to the lack of biomarkers. Recent multi-omics based research has contributed to the development of novel biomarkers in cardiovascular diseases. Our aim was to determine whether patients with acute VTE have differences in the metabolomic profile compared to non-acute VTE. Methods: This observational trial included 62 patients with clinical suspicion of acute deep vein thrombosis or pulmonary embolism, admitted to the emergency room. There were 50 patients diagnosed with acute VTE and 12 with non-acute VTE conditions and no significant differences were found between the two groups for clinical and demographic characteristics. Metabolomics assays identified and quantified a final number of 91 metabolites in plasma and 55 metabolites in red blood cells (RBCs). Plasma from acute VTE patients expressed tendency to a specific metabolomic signature, with univariate analyses revealing 23 significantly different molecules between acute VTE patients and controls (p < 0.05). The most relevant metabolic pathway with the strongest impact on the acute VTE phenotype was D-glutamine and D-glutamate (p = 0.001, false discovery rate = 0.06). RBCs revealed a specific metabolomic signature in patients with a confirmed diagnosis of DVT or PE that distinguished them from other acutely diseased patients, represented by 20 significantly higher metabolites and four lower metabolites. Three of those metabolites revealed high performant ROC curves, including adenosine 3',5'-diphosphate (AUC 0.983), glutathione (AUC 0.923), and adenine (AUC 0.91). Overall, the metabolic pathway most impacting to the differences observed in the RBCs was the purine metabolism (p = 0.000354, false discovery rate = 0.68). Conclusions: Our findings show that metabolite differences exist between acute VTE and nonacute VTE patients admitted to the ER in the early phases. Three potential biomarkers obtained from RBCs showed high performance for acute VTE diagnosis. Further studies should investigate accessible laboratory methods for the future daily practice usefulness of these metabolites for the early diagnosis of acute VTE in the ER.
- Comparative analysis of hybrid‑SNP microarray and nanopore sequencing for detection of large‑sized copy number variants in the human genomePublication . Silva, Catarina; Ferrão, José; Marques, Bárbara; Pedro, Sónia; Correia, Hildeberto; Valente, Ana; Rodrigues, António Sebastião; Vieira, LuísBackground: Nanopore sequencing is a technology that holds great promise for identifying all types of human genome variations, particularly structural variations. In this work, we used nanopore sequencing technology to sequence 2 human cell lines at low depth of coverage to call copy number variations (CNV), and compared the results variant by variant with chromosomal microarray (CMA) results. Results: We analysed sequencing data using CuteSV and Sniffles2 variant callers, compared breakpoints based on hybrid-SNP microarray, nanopore sequencing and Sanger sequencing, and analysed CNV coverage. From a total of 48 high confidence variants (truth set), variant calling detected 79% of the truth set variants, increasing to 86% for interstitial CNV. Simultaneous use of the 2 callers slightly increased variant calling. Both callers performed better when calling CNV losses than gains. Variant sizes from CMA and nanopore sequencing showed an excellent correlation, with breakpoints determined by nanopore sequencing differing by only 20 base pairs on average from Sanger sequencing. Nanopore sequencing also revealed that four variants concealed genomic inversions undetectable by CMA. In the 10 CNV not called in nanopore sequencing, 8 showed coverage evidence of genomic loss or gain, highlighting the need to improve SV calling algorithms performance. Conclusions: Nanopore sequencing offers advantages over CMA for structural variant detection, including the identification of multiple variant types and their breakpoints with increased precision. However, further improvements in variant calling algorithms are still needed for nanopore sequencing to become a highly robust and standardized approach for a comprehensive analysis of genomic structural variation.
- A Comparative Overview of the Role of Human Ribonucleases in Nonsense-Mediated mRNA DecayPublication . da Costa, Paulo J.; Menezes, Juliane; Guedes, Raquel; Reis, Filipa P.; Teixeira, Alexandre; Saramago, Margarida; Viegas, Sandra C.; Arraiano, Cecília M.; Romão, LuísaEukaryotic cells possess surveillance mechanisms that detect and degrade defective transcripts. Aberrant transcripts include mRNAs with a premature termination codon (PTC), targeted by the nonsense-mediated decay (NMD) pathway, and mRNAs lacking a termination codon, targeted by the nonstop decay (NSD) pathway. The eukaryotic exosome, a ribonucleolytic complex, plays a crucial role in mRNA processing and turnover through its catalytic subunits PM/Scl100 (Rrp6 in yeast), DIS3 (Rrp44 in yeast), and DIS3L1. Additionally, eukaryotic cells have other ribonucleases, such as SMG6 and XRN1, that participate in RNA surveillance. However, the specific pathways through which ribonucleases recognize and degrade mRNAs remain elusive. In this study, we characterized the involvement of human ribonucleases, both nuclear and cytoplasmic, in the mRNA surveillance mechanisms of NMD and NSD. We performed knockdowns of SMG6, PM/Scl100, XRN1, DIS3, and DIS3L1, analyzing the resulting changes in mRNA levels of selected natural NMD targets by RT-qPCR. Additionally, we examined the levels of different human β-globin variants under the same conditions: wild-type, NMD-resistant, NMD-sensitive, and NSD-sensitive. Our results demonstrate that all the studied ribonucleases are involved in the decay of certain endogenous NMD targets. Furthermore, we observed that the ribonucleases SMG6 and DIS3 contribute to the degradation of all β-globin variants, with an exception for βNS in the former case. This is also the case for PM/Scl100, which affects all β-globin variants except the NMD-sensitive variants. In contrast, DIS3L1 and XRN1 show specificity for β-globin WT and NMD-resistant variants. These findings suggest that eukaryotic ribonucleases are target-specific rather than pathway-specific. In addition, our data suggest that ribonucleases play broader roles in mRNA surveillance and degradation mechanisms beyond just NMD and NSD.
- Exploring BPA alternatives - Environmental levels and toxicity reviewPublication . Adamovsky, Ondrej; Groh, Ksenia J.; Białk-Bielińska, Anna; Escher, Beate I.; Beaudouin, R.; Mora Lagares, Liadys; Tollefsen, Knut Erik; Fenske, Martina; Mulkiewicz, Ewa; Creusot, Nicolas; Sosnowska, Anita; Loureiro, Susana; Beyer, Jonny; Repetto, Guillermo; Štern, Alja; Lopes, Isabel; Monteiro, Marta; Zikova-Kloas, Andrea; Eleršek, Tina; Vračko, Marjan; Zdybel, Szymon; Puzyn, Tomasz; Koczur, Weronika; Ebsen Morthorst, Jane; Holbech, Henrik; Carlsson, Gunnar; Örn, Stefan; Herrero, Óscar; Siddique, Ayesha; Liess, Matthias; Braun, Georg; Srebny, Vanessa; Žegura, Bojana; Hinfray, Nathalie; Brion, François; Knapen, Dries; Vandeputte, Ellen; Stinckens, Evelyn; Vergauwen, Lucia; Behrendt, Lars; Silva, Maria João; Blaha, Ludek; Kyriakopoulou, KaterinaBisphenol A alternatives are manufactured as potentially less harmful substitutes of bisphenol A (BPA) that offer similar functionality. These alternatives are already in the market, entering the environment and thus raising ecological concerns. However, it can be expected that levels of BPA alternatives will dominate in the future, they are limited information on their environmental safety. The EU PARC project highlights BPA alternatives as priority chemicals and consolidates information on BPA alternatives, with a focus on environmental relevance and on the identification of the research gaps. The review highlighted aspects and future perspectives. In brief, an extension of environmental monitoring is crucial, extending it to cover BPA alternatives to track their levels and facilitate the timely implementation of mitigation measures. The biological activity has been studied for BPA alternatives, but in a non-systematic way and prioritized a limited number of chemicals. For several BPA alternatives, the data has already provided substantial evidence regarding their potential harm to the environment. We stress the importance of conducting more comprehensive assessments that go beyond the traditional reproductive studies and focus on overlooked relevant endpoints. Future research should also consider mixture effects, realistic environmental concentrations, and the long-term consequences on biota and ecosystems.
- Impact of PPP1R1A Knockdown on the Proteomic Landscape of INS-1 Cells: A Focus on Significant Modulated PathwaysPublication . Taneera, Jalal; Giddey, Alexander D.; Soares, Nelson C.; Khalique, Anila; Mohammed, Abdul Khader; Mahgoub, Mohamed Omer; Mahgoub, EglalPPP1R1A (protein phosphatase 1 regulatory inhibitor subunit 1A) is a cAMP/PKA-responsive inhibitor of protein phosphatase 1 (PP1) with a pivotal role in pancreatic β-cell physiology. To investigate its functional impact, Ppp1r1a was silenced in INS-1 (832/13) rat β-cells, and proteomic alterations were profiled using label-free DIA mass spectrometry (Orbitrap Exploris 480) with a rat spectral library. Quantitative analysis (n = 4/group) identified ∼2846 proteins with >2-fold change, revealing extensive proteome reprogramming. Key biological processes affected included vesicle trafficking and exocytosis, insulin biosynthesis and processing, organelle organization, mRNA processing, and autophagy. Pathway enrichment highlighted disruptions in insulin secretion, insulin resistance, and mTOR signaling. Crucial β-cell proteins, including INS2, Cacna1a, CPEB2, PCSK2, SNAP25, SYT5, and VAMP7, were significantly downregulated. Validation confirmed reduced phosphorylated AKT levels and p-AKT/T-AKT ratio, consistent with impaired mTOR signaling. Collectively, these findings demonstrate that PPP1R1A is essential for maintaining β-cell function and insulin secretion, and its depletion triggers broad proteomic and signaling alterations. Thus, PPP1R1A emerges as a regulatory node with potential therapeutic relevance in modulating β-cell activity and insulin dynamics in diabetes.
- Medwakh smoking induces alterations in salivary proteins and cytokine expression: a clinical exploratory proteomics investigationPublication . Aghila Rani, K.G.; Soares, Nelson C.; Rahman, Betul; Giddey, Alexander D.; Al-Hroub, Hamza M.; Semreen, Mohammad H.; Al Kawas, SausanBackground: Medwakh smoking has radically expanded among youth in the Middle East and around the world. The rising popularity of medwakh/dokha usage is linked to the onset of several chronic illnesses including cardiovascular diseases and cancers. Medwakh smoking is reported to increase the risk of inflammation in the lower respiratory tract owing to oxidative burden. To date, there are no reported studies investigating the impact of medwakh smoking on salivary protein profile. The current study aims to elucidate alterations in the salivary proteome profile of medwakh smokers. Methods: Saliva samples collected from 33 medwakh smokers and 30 non-smokers were subjected to proteomic analysis using UHPLC-ESI-QTOF-MS. Saliva samples were further subjected to validatory experiments involving analysis of inflammatory cytokine profile using LEGENDplex™ Human Essential Immune Response Panel. Results: Statistical analysis revealed alterations in the abundance of 74 key proteins including immune mediators and inflammatory markers in medwakh smokers (Accession: PXD045901). Proteins involved in building oxidative stress, alterations in cell anchorage, and cell metabolic processes were enhanced in medwakh smokers. Salivary immune response evaluation further validated the proteome findings, revealing significantly higher levels of IL-1β, IL-12p70, IL-23, IFN-γ (Th1 cytokines), IL-6 (Th2 cytokine), and MCP-1 (chemokine) in medwakh smokers. In addition, a substantial increase in abundance of involucrin suggesting a plausible stratified squamous cell differentiation and increased cell lysis in the oral cavity of medwakh smokers akin to chronic obstructive pulmonary diseases (COPD). The protein-metabolite joint pathway analysis further showed significantly enriched differentially expressed proteins and metabolites of glycolysis/gluconeogenesis, pentose phosphate, fructose and mannose, nicotinate and nicotinamide, and glutathione metabolism pathways among medwakh smokers. Conclusions: The findings of the study provide valuable insights on potential perturbations in various key immune molecules, cytokines, and signaling pathways among medwakh smokers. Medwakh smokers displayed elevated inflammation, increased oxidative stress and defective antioxidant responses, dysregulated energy metabolism, and alterations in proteins related to cell adhesion, migration, differentiation, and proliferation. The findings of study underscore the urgent need for comprehensive public health interventions among youth by raising awareness, implementing effective smoking cessation programs, and promoting healthy lifestyle to safeguard the well-being of individuals and communities worldwide.
- Metabolomics insights into doxorubicin and 5-fluorouracil combination therapy in triple-negative breast cancer: a xenograft mouse model studyPublication . Hassanein, Mai M.; Hagyousif, Yousra A.; Zenati, Ruba A.; Al-Hroub, Hamza M.; Khan, Farman Matloob; Abuhelwa, Ahmad Y.; Alzoubi, Karem H.; Soares, Nelson C.; El-Huneidi, Waseem; Abu-Gharbieh, Eman; Omar, Hany; Zaher, Dana M.; Bustanji, Yasser; Semreen, Mohammad H.Background: Breast cancer is one of the most prevalent malignancies and a leading cause of death among women worldwide. Among its subtypes, triple-negative breast cancer (TNBC) poses significant clinical challenges due to its aggressive behavior and limited treatment options. This study aimed to investigate the effects of doxorubicin (DOX) and 5-fluorouracil (5-FU) as monotherapies and in combination using an established MDA-MB-231 xenograft model in female BALB/C nude mice employing advanced metabolomics analysis to identify molecular alterations induced by these treatments. Methods: We conducted comprehensive plasma and tumor tissue sample profiling using ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS). Results: Each treatment group exhibited unique metabolic profiles in plasma and tumor analysis. Univariate and enrichment analyses identified alterations in metabolic pathways. The combination treatment of DOX + 5-FU induced the most extensive metabolic alterations disrupting key pathways including purine, pyrimidine, beta-alanine, and sphingolipid metabolism. It significantly reduced critical metabolites such as guanine, xanthine, inosine, L-fucose, and sphinganine, demonstrating enhanced cytotoxic effects compared to individual treatments. The DOX treatment uniquely increased ornithine levels, while 5-FU altered sphingolipid metabolism, promoting apoptosis. Significance: This in vivo study highlights TNBC's metabolic alterations to chemotherapeutics, identifying potential biomarkers like L-fucose and beta-alanine, and provides insights for improving treatment strategies.
- Methicillin-Resistant Staphylococcus aureus: The Shifting Landscape in the United Arab EmiratesPublication . Boucherabine, Syrine; Nassar, Rania; Mohamed, Lobna; Habous, Maya; Nabi, Anju; Husain, Riyaz Amirali; Alfaresi, Mubarak; Oommen, Seema; Khansaheb, Hamda Hassan; Al Sharhan, Mouza; Celiloglu, Handan; Raja, Mubarak Hussain; Abdelkarim, Eman; Ali, Nishi; Tausif, Salman; Olowoyeye, Victory; Soares, Nelson Cruz; Hachim, Mahmood; Moradigaravand, Danesh; Everett, Dean; Mueller, Elke; Monecke, Stefan; Ehricht, Ralf; Senok, AbiolaBackground: Methicillin-resistant Staphylococcus aureus (MRSA) is a significant burden globally, particularly in the Arabian Gulf region. The United Arab Emirates (UAE) has experienced rising MRSA prevalence, with increasing diversity in the clonal complexes (CCs) identified. The COVID-19 pandemic, with its increased hospitalization rates and antibiotic use, may have further influenced MRSA's genetic evolution and epidemiology in the country. Methods: To investigate this influence, genomic profiling of 310 MRSA clinical isolates collected between February and November 2022 was performed using a DNA microarray-based assay. Results: Isolates were assigned to 22 clonal complexes and 72 distinct strain assignments. The predominant clonal complexes were CC5, CC6, CC361, CC22, CC1, and CC8. Community-acquired MRSA lineages were dominant, with only one healthcare-associated MRSA lineage isolate identified. Upward trends of CC1153 were observed along with rare CCs, such as CC121-MRSA and CC7-MRSA, with the latter being reported for the first time in the Arabian Gulf region. The presence of pandemic strains USA300 CC8-MRSA-[IVa + ACME1] and CC8-MRSA-IV strains were also observed, including variants lacking Panton-Valentine leukocidin (pvl) genes and missing tst1 or enterotoxin genes. The PVL-negative CC772-MRSA-V/VT was identified, representing its first report in the UAE. A novel variant, CC361-MRSA-IV (tst1+/PVL+), was identified. Pvl genes were observed in 36% of the isolates, primarily from skin and soft tissue infections, while fusC (SCC-borne fusidic acid resistance) was identified in 13% of the isolates. Conclusions: The findings highlight the ongoing evolution of MRSA in the UAE, with the persistence and emergence of diverse and rare clonal complexes, driving the need for continuous genomic surveillance.
- Molecular signatures of xenograft colorectal cancer in mice treated with topotecan: A mass spectrometry-based studyPublication . Hagyousif, Yousra A.; Zenati, Ruba A.; Soares, Nelson C.; Al-Hroub, Hamza M.; Khan, Farman Matloob; Qaisar, Rizwan; Hamoudi, Rifat; El-Awady, Raafat; Abuhelwa, Ahmad Y.; Ramadan, Wafaa; El-Huneidi, Waseem; Abu-Gharbieh, Eman; Alzoubi, Karem H.; Bustanji, Yasser; Semreen, Mohammad H.Background: Colorectal cancer (CRC) is one of the most common cancers worldwide, yet it continues to have a low survival rate, largely due to the lack of effective treatments. Metabolomics offers new insight into disease diagnosis and biomarkers discovery. The aim of the study is to identify serum biomarkers in a CRC xenograft mouse model treated with topotecan using advanced metabolomics techniques to enhance our understanding and management of the disease. Methods: The therapeutic potentials of the anticancer drug topotecan on metabolomic alterations in CRC were explored using the UHPLC-ESI-QTOF-MS platform. A comprehensive metabolomic analysis was conducted to compare four different animal groups: HCT-116 CRC xenograft mice treated with topotecan (treated group), vehicle-control HCT-116 xenograft mice (untreated CRC xenograft mice), positive controls (healthy mice injected with topotecan), and negative controls (healthy mice). Results: The study identified 53 altered metabolites across all four groups (p-value < 0.05). Independent T-test revealed that 15 metabolites were statistically significant among vehicle controls and negative controls. Additionally, 20 metabolites showed significant differences between the potential responders to topotecan and the vehicle controls. Moreover, only one metabolite was statistically significant between the positive and negative controls. Conclusion: The findings provide a detailed characterization of metabolic alterations associated with topotecan treatment in CRC. These insights contribute to a better understanding of the drug's mechanism of action, which may help predict CRC patients' response to topotecan and guide the development of personalized therapeutic strategies.
- Occupational second-hand smoke exposure: A comparative shotgun proteomics study on nasal epithelia from healthy restaurant workersPublication . Neves, Sofia; Pacheco, Solange; Vaz, Fátima; James, Peter; Simões, Tânia; Penque, Deborah; NevesNon-smokers exposed to second-hand smoke (SHS) present risk of developing tobacco smoke-associated pathologies. To investigate the airway molecular response to SHS exposure that could be used in health risk assessment, comparative shotgun proteomics was performed on nasal epithelium from a group of healthy restaurant workers, non-smokers (never and former) exposed and not exposed to SHS in the workplace. HIF1α-glycolytic targets (GAPDH, TPI) and proteins related to xenobiotic metabolism, cell proliferation and differentiation leading to cancer (ADH1C, TUBB4B, EEF2) showed significant modulation in non-smokers exposed. In never smokers exposed, enrichment of glutathione metabolism pathway and EEF2-regulating protein synthesis in genotoxic response were increased, while in former smokers exposed, proteins (LYZ, ATP1A1, SERPINB3) associated with tissue damage/regeneration, apoptosis inhibition and inflammation that may lead to asthma, COPD or cancer, were upregulated. The identified proteins are potential response and susceptibility/risk biomarkers for SHS exposure.