Bioquímica e Bioloxía Molecular

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Novel therapeutic strategies for atopic dermatitis: Biomarker modulation and clinical implications. A systematic review
(Springer Nature, 2026-01-29) Moreiras Arias, Noelia; Nieto Fontarigo, Juan José; Salgado Castro, Francisco Javier; González Vilas, Daniel; Paredes Suárez, Carmen; Combo García, Enma; Rodríguez Otero, Carmen; Flórez, Ángeles; Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular
Advances in the understanding of atopic dermatitis (AD) pathogenesis have driven the development of innovative systemic therapies targeting key immunologic pathways. This systematic review summarizes current evidence on the impact of biologic agents, Janus kinase (JAK) inhibitors, and other emerging treatments on AD-related biomarkers and their correlation with clinical outcomes. A comprehensive literature search was conducted across PubMed, Embase, Scopus, and Web of Science for studies published between 2014 and 2024. Eighty studies met the inclusion criteria. Dupilumab was the most extensively investigated therapy, followed by tralokinumab, JAK inhibitors, and novel agents such as amlitelimab, stapokibart, and tezepelumab. Across drug classes, consistent reductions in CCL17/TARC, LDH, and total IgE levels were observed, generally paralleling clinical improvement in EASI and SCORAD scores. Transcriptomic and proteomic analyses revealed normalization of Th2/Th22 inflammatory signatures and restoration of barrier-related gene expression, while microbiome studies showed a reduction in Staphylococcus aureus colonization. Despite these advances, the heterogeneity of study designs and analytical techniques limits the comparability of results. CCL17 and LDH currently represent the most reliable biomarkers associated with disease severity and treatment response, although their limited specificity restricts clinical applicability. Future research should aim to validate integrated biomarker panels combining immunologic, transcriptomic, and microbiomic data to enable precision medicine approaches in atopic dermatitis management.
Trajectory analysis of hepatic stellate cell differentiation reveals metabolic regulation of cell commitment and fibrosis
(Nature Research, 2025-02-10) Martínez García de la Torre, Raquel A.; Ayuso García, Paula; Varela Rey, Marta María; Woodhoo, Ashwin; Sancho-Bru, Pau; Universidade de Santiago de Compostela. Centro de Investigación en Medicina Molecular e Enfermidades Crónicas (CiMUS); Universidade de Santiago de Compostela. Departamento de Bioloxía Funcional; Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular
Defining the trajectory of cells during differentiation and disease is key for uncovering the mechanisms driving cell fate and identity. However, trajectories of human cells remain largely unexplored due to the challenges of studying them with human samples. In this study, we investigate the proteome trajectory of iPSCs differentiation to hepatic stellate cells (diHSCs) and identify RORA as a key transcription factor governing the metabolic reprogramming of HSCs necessary for diHSCs’ commitment, identity, and activation. Using RORA deficient iPSCs and pharmacologic interventions, we show that RORA is required for early differentiation and prevents diHSCs activation by reducing the high energetic state of the cells. While RORA knockout mice have enhanced fibrosis, RORA agonists rescue multi-organ fibrosis in in vivo models. Notably, RORA expression correlates negatively with liver fibrosis and HSCs activation markers in patients with liver disease. This study reveals that RORA regulates cell metabolic plasticity, important for mesoderm differentiation, pericyte quiescence, and fibrosis, influencing cell commitment and disease.
Parameters of glycemic variability in continuous glucose monitoring as predictors of diabetes: a prospective evaluation in a non-diabetic general population
(De Gruyter, 2025) Rodríguez García, Javier; Camiña Darriba, Manuel Félix; Ortolá Devesa, Juan B.; Rodríguez-Segade Villamarín, Santiago; Valle Rodríguez, Andrea; Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular
Objectives: To prospectively examine the ability of some glycemic variability metrics from continuous glucose monitoring (CGM) to predict the development of diabetes in a non-diabetic population. Methods: A total of 497 non-diabetic patients from the AEGIS study were included. Participants used a CGM system (iPro2®) over a six-day period. The following parameters were analyzed: standard deviation (SD), coefficient of variation (CV) and mean amplitude of glucose excursion (MAGE). Six-years follow-up was performed. ROC curves were constructed to determine the predictive value of glycemic variability metrics. Sensitivity and specificity were calculated. Results: Of the 497 participants, 16 women (4.9 %) and 9 men (5.2 %) developed diabetes. Initial HbA1c and fasting glucose levels were significantly higher in the participants who ultimately developed diabetes. Glycemic variability metrics were also significantly higher in these subjects (SD: 18 vs. 13 mg/dL; CV: 17 vs. 14 %; MAGE: 36 vs. 27 mg/dL; p<0.001 in all cases). SD showed the highest AUC (0.81), with a sensitivity of 80 % and a specificity of 72 % for a cut-off of 14.9 mg/dL. AUCs were higher in men for all metrics. Conclusions: The metrics obtained by MCG, especially SD, are effective predictors of progression to type 2 diabetes in a non-diabetic population. These findings suggest that glycemic variability is useful for the early identification of subjects at a higher risk of developing diabetes.
Deletion of pcnB affects antibiotic susceptibility in resistant Escherichia coli by reducing copy number of ColE1-family plasmids
(Nature Research, 2025-03-11) Wellner, Sandra marina; Fei, Xiao; Fresno Herrero, Ana; Olsen, John Elmerdahl; Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular
Plasmids play a major role in the spread of antibiotic resistance genes in bacteria. Plasmid copy number (PCN) is often tightly regulated. In plasmids of the ColE1-type, this regulation happens by a negative feedback mechanism using an antisense RNA. Here, we employed a sequencing-based method for determining PCN to demonstrate that copy number of different ColE1-family plasmids harboring antibiotic resistance genes increases during antibiotic treatment. Further, we show that deletion of the gene pcnB reduces the copy number of ColE1-family plasmids in E. coli MG1655, which in turn results in a reduced resistance to antimicrobials of the classes aminoglycosides, β-lactams and tetracyclines. In the absence of antibiotic selection, the deletion of pcnB also decreased the number of ColE1-type plasmids in a bacterial population. Hence, PcnB, which polyadenylates RNA, marking it for decay, represents a potential drug and helper-drug target that could be used to reduce PCN to re-sensitize bacteria with multi-copy-number resistance-plasmids to treatment with different antimicrobials.
Identification of Asthma Phenotypes in the Spanish MEGA Cohort Study Using Cluster Analysis
(Elsevier, 2023-01-18) Matabuena, Marcos; Salgado Castro, Francisco Javier; Nieto Fontarigo, Juan José; Álvarez-Puebla, María J.; Arismendi, Ebymar; Barranco, Pilar; Bobolea, Irina; Caballero, María L.; Cañas, José Antonio; Cárdaba, Blanca; Cruz, María Jesús; Curto, Elena; Domínguez-Ortega, Javier; Luna, Juan Alberto; Martínez-Rivera, Carlos; Mullol, Joaquim; Muñoz, Xavier; Rodríguez-García, Javier; Olaguibel, José María; Picado, César; Plaza, Vicente; Quirce, Santiago; Rial, Manuel J.; Romero-Mesones, Christian; Sastre, Beatriz; Soto-Retes, Lorena; Valero, Antonio; Valverde-Monge, Marcela; Pozo, Victoria del; Sastre, Joaquín; González Barcala, Francisco Javier; Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular; Universidade de Santiago de Compostela. Centro de Investigación en Tecnoloxías Intelixentes da USC (CiTIUS); Universidade de Santiago de Compostela. Departamento de Psiquiatría, Radioloxía, Saúde Pública, Enfermaría e Medicina
Introduction The definition of asthma phenotypes has not been fully established, neither there are cluster studies showing homogeneous results to solidly establish clear phenotypes. The purpose of this study was to develop a classification algorithm based on unsupervised cluster analysis, identifying clusters that represent clinically relevant asthma phenotypes that may share asthma-related outcomes. Methods We performed a multicentre prospective cohort study, including adult patients with asthma (N=512) from the MEGA study (Mechanisms underlying the Genesis and evolution of Asthma). A standardised clinical history was completed for each patient. Cluster analysis was performed using the kernel k-groups algorithm. Results Four clusters were identified. Cluster 1 (31.5% of subjects) includes adult-onset atopic patients with better lung function, lower BMI, good asthma control, low ICS dose, and few exacerbations. Cluster 2 (23.6%) is made of adolescent-onset atopic asthma patients with normal lung function, but low adherence to treatment (59% well-controlled) and smokers (48%). Cluster 3 (17.1%) includes adult-onset patients, mostly severe non-atopic, with overweight, the worse lung function and asthma control, and receiving combination of treatments. Cluster 4 (26.7%) consists of the elderly-onset patients, mostly female, atopic (64%), with high BMI and normal lung function, prevalence of smokers and comorbidities. Conclusion We defined four phenotypes of asthma using unsupervised cluster analysis. These clusters are clinically relevant and differ from each other as regards FEV1, age of onset, age, BMI, atopy, asthma severity, exacerbations, control, social class, smoking and nasal polyps.
Mepolizumab treatment alters the functional phenotype of eosinophils in severe eosinophilic asthma
(Frontiers Media, 2025-11-27) Miguéns Suárez, Pablo; Martelo Vidal, Laura; Vázquez Mera, Sara; Nieto Fontarigo, Juan José; Salgado Castro, Francisco Javier; González Barcala, Francisco Javier; Universidade de Santiago de Compostela. Departamento de Medicina
Background: Blood eosinophil count is usually employed as a predictive and response biomarker for mepolizumab treatment. However, its decrease is not always associated with an improvement in asthma symptoms. The aim of this work is to study the effect of mepolizumab in the activation status and functional phenotype of circulating eosinophils. Methods: Samples from healthy controls (N = 15) and patients with severe eosinophilic asthma (N = 15) before and after 4, 16 and 32 weeks of mepolizumab treatment (anti-IL5 mAb; 100 mg s.c./4 weeks) were screened. Demographic, clinical, hematological and biochemical characteristics were collected. Activation and functional phenotype of peripheral blood eosinophils was analyzed by flow cytometry. sCD62L in serum was measured by ELISA. Target mRNAs were quantified by Nanostring. Results: Eosinophils from severe eosinophilic asthma patients showed a higher activation profile (CD11b, CD44 and IL-5Rα expression) compared to healthy subjects. Mepolizumab treatment reduced the number of basophils and eosinophils in peripheral blood. We also found a clear downmodulation on the surface expression (% and MFI) of CD44 and IL-3Rα on eosinophils at week 4, which was maintained through all treatment period (4–32 weeks). The functional phenotype of the remaining eosinophils was also modified with the treatment, showing a reduction of inflammatory eosinophils (iEOS; CD62Llo) percentage without affecting the balance of regulatory subpopulations (CD16dim/hi eosinophils). This was accompanied by a decrease in serum sCD62L levels. mRNA and protein levels showed similar trends for some targets (e.g., IL-5Rα) but not for others (e.g., CD62L). Conclusions: Mepolizumab treatment modifies the functional phenotype of eosinophils resulting in a lower percentage of iEOS and reduced activation status. These changes occur at an early time point (4 weeks) and are maintained throughout all treatment period.
Urinary Proteome and Exosome Analysis Protocol for the Discovery of Respiratory Diseases Biomarkers
(Multidisciplinary Digital Publishing Institute (MDPI), 2025-01-01) Martelo Vidal, Laura; Vázquez Mera, Sara; Miguéns Suárez, Pablo; Domínguez Arca, Vicente; Gómez Carballa, Alberto; Salas Ellacuriaga, Antonio; González Barcala, Francisco Javier; Salgado Castro, Francisco Javier; Nieto Fontarigo, Juan José; Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular; Universidade de Santiago de Compostela. Instituto de Ciencias Forenses "Luís Concheiro" (INCIFOR); Universidade de Santiago de Compostela. Departamento de Psiquiatría, Radioloxía, Saúde Pública, Enfermaría e Medicina
This study aims to develop a protocol for respiratory disease-associated biomarker discovery by combining urine proteome studies with urinary exosome components analysis (i.e., miRNAs). To achieve this, urine was DTT treated to decrease uromodulin, then concentrated and ultracentrifuged. Proteomic analyses of exosome-free urine were performed using LC-MS/MS. Simultaneously, miRNA expression from urine exosomes was measured using either RTqPCR (pre-amplification) or nCounter Nanostring (non-amplication) analyses. We detected 548 different proteins in exosome-free urine samples (N = 5) with high confidence (FDR < 1%), many of them being expressed in different non-renal tissues. Specifically, lung-related proteins were overrepresented (Fold enrichment = 1.31; FDR = 0.0335) compared to whole human proteome, and 10–15% were already described as protein biomarkers for several pulmonary diseases. Urine proteins identified belong to several functional categories important in respiratory pathology. We could confirm the expression of miRNAs previously connected to respiratory diseases (i.e., miR-16-5p, miR-21-5p, miR-146a-5p, and miR-215-5p) in urine exosomes by RTqPCR. Finally, we detected 333 miRNAs using Nanostring, 15 of them up-regulated in T2high asthma (N = 4) compared to T2low asthma (N = 4) and healthy subjects (N = 4). Therefore, this protocol combining the urinary proteome (exosome free) with the study of urinary exosome components (i.e., miRNAs) holds great potential for molecular biomarker discovery of non-renal and particularly respiratory pathologies.
Exploring the Biological Properties of Zn(II) Bisthiosemicarbazone Helicates
(MDPI, 2023-01-23) Fernández-Fariña, Sandra; Velo Heleno, Isabel; Carballido, Rocío; Martínez-Calvo, Miguel; Barcia Vieitez, Ramiro; Palacios, Òscar; Capdevila, Mercè; González Noya, Ana María; Pedrido Castiñeiras, Rosa; Universidade de Santiago de Compostela. Departamento de Química Inorgánica; Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular
The design of artificial helicoidal molecules derived from metal ions with biological properties is one of the objectives within metallosupramolecular chemistry. Herein, we report three zinc helicates derived from a family of bisthiosemicarbazone ligands with different terminal groups, Zn2(LMe)2∙2H2O 1, Zn2(LPh)2∙2H2O 2 and Zn2(LPhNO2)2 3, obtained by an electrochemical methodology. These helicates have been fully characterized by different techniques, including X-ray diffraction. Biological studies of the zinc(II) helicates such as toxicity assays with erythrocytes and interaction studies with proteins and oligonucleotides were performed, demonstrating in all cases low toxicity and an absence of covalent interaction with the proteins and oligonucleotides. The in vitro cytotoxicity of the helicates was tested against MCF-7 (human breast carcinoma), A2780 (human ovarian carcinoma cells), NCI-H460 (human lung carcinoma cells) and MRC-5 (normal human lung fibroblasts), comparing the IC50 values with cisplatin. We will try to demonstrate if the terminal substituent of the ligand precursor exerts any effect in toxicity or in the antitumor activity of the zinc helicates
RNA-binding proteins in pluripotency, differentiation, and reprogramming
(Higher Education Press, 2014-08-25) Guallar Artal, Diana; Wang, Jianlong; Universidade de Santiago de Compostela. Centro de Investigación en Medicina Molecular e Enfermidades Crónicas (CiMUS); Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular
Embryonic stem cell maintenance, differentiation, and somatic cell reprogramming require the interplay of multiple pluripotency factors, epigenetic remodelers, and extracellular signaling pathways. RNA-binding proteins (RBPs) are involved in a wide range of regulatory pathways, from RNA metabolism to epigenetic modifications. In recent years we have witnessed more and more studies on the discovery of new RBPs and the assessment of their functions in a variety of biological systems, including stem cells. We review the current studies on RBPs and focus on those that have functional implications in pluripotency, differentiation, and/or reprogramming in both the human and mouse systems
Measuring quantitative proteomic distance between Spanish beef breeds
(Elsevier, 2020-06-15) Rodríguez Vázquez, Raquel; Mato Montero, Ariadna; López-Pedrouso, María; Franco Ruiz, Daniel; Sentandreu Vicente, Miguel Ángel; Zapata Babío, José Carlos; Universidade de Santiago de Compostela. Departamento de Zooloxía, Xenética e Antropoloxía Física; Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular; Universidade de Santiago de Compostela. Departamento de Enxeñaría Química
Estimates of quantitative proteomic distance between populations have not been reported to date. Here, quantitative proteomic distances between three Spanish bovine breeds (Asturiana de los Valles, AV; Retinta, RE; and Rubia Gallega, RG) were estimated from two-dimensional electrophoresis profiles of meat samples of longissimus thoracis muscle at 2 h post-mortem. Statistically significant distances were detected between AV/RG and the most genetically different RE breed, using the novel QD measure of quantitative proteomic distance. In total, 18 differentially abundant myofibrillar and sarcoplasmic proteins/isoforms contributing to proteomic distances between breeds were confidently identified by tandem mass spectrometry. The fast skeletal myosin regulatory light chain 2 followed by other five interacting proteins exhibited the most pronounced relative change between breeds. In addition, most differentially represented proteins could be associated with variations in meat tenderness. Therefore, they could be candidate biomarkers for molecular breeding programs and authentication of the three Spanish beef breeds
High diversity and variability of pipolins among a wide range of pathogenic Escherichia coli strains
(Nature Research, 2020-07-27) Flament Simon, Saskia Camille; Toro, María de; Chuprikova, Liubov; Blanco Álvarez, Miguel; Moreno González, Juan; Salas Falgueras, Margarita; Blanco Álvarez, Jorge; Redrejo Rodríguez, Modesto; Universidade de Santiago de Compostela. Facultade de Veterinaria; Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular; Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía
Self-synthesizing transposons are integrative mobile genetic elements (MGEs) that encode their own B-family DNA polymerase (PolB). Discovered a few years ago, they are proposed as key players in the evolution of several groups of DNA viruses and virus–host interaction machinery. Pipolins are the most recent addition to the group, are integrated in the genomes of bacteria from diverse phyla and also present as circular plasmids in mitochondria. Remarkably, pipolins-encoded PolBs are proficient DNA polymerases endowed with DNA priming capacity, hence the name, primer-independent PolB (piPolB). We have now surveyed the presence of pipolins in a collection of 2,238 human and animal pathogenic Escherichia coli strains and found that, although detected in only 25 positive isolates (1.1%), they are present in E. coli strains from a wide variety of pathotypes, serotypes, phylogenetic groups and sequence types. Overall, the pangenome of strains carrying pipolins is highly diverse, despite the fact that a considerable number of strains belong to only three clonal complexes (CC10, CC23 and CC32). Comparative analysis with a set of 67 additional pipolin-harboring genomes from GenBank database spanning strains from diverse origin, further confirmed these results. The genetic structure of pipolins shows great flexibility and variability, with the piPolB gene and the attachment sites being the only common features. Most pipolins contain one or more recombinases that would be involved in excision/integration of the element in the same conserved tRNA gene. This mobilization mechanism might explain the apparent incompatibility of pipolins with other integrative MGEs such as integrons. In addition, analysis of cophylogeny between pipolins and pipolin-harboring strains showed a lack of congruence between several pipolins and their host strains, in agreement with horizontal transfer between hosts. Overall, these results indicate that pipolins can serve as a vehicle for genetic transfer among circulating E. coli and possibly also among other pathogenic bacteria
Outer Membrane Proteins as Vaccine Targets Against Lawsonia intracellularis in Piglets
(MDPI, 2025) Aves, Kara L.; Fresno Herrero, Ana; Nisar, Sajid; Saraiva, Mauro M.; Goecke, Nicole B.; Sander, Adam F.; Nielsen, Morten A.; Olsen, John E.; Guerra, Priscila R.; Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular
Background: Lawsonia intracellularis (LI) is the agent of proliferative enteropathy in swine, a common disease that affects pigs for up to eight weeks after weaning. Aim: To evaluate the effectiveness of two novel subunit vaccines targeting outer membrane proteins on LI. Methods: The two vaccines included OMP2c.cVLP, where the OMP2c antigen was anchored on the surface of capsid virus-like particles (cVLP); and MBP.INVASc, where antigens were anchored to an MBP fusion protein. Groups of six mice, as proof of concept, and six piglets were immunized with either OMP2c.cVLP, MBP.INVASc., or PBS as a control using a prime-boost regime. Results: Both OMP2c.cVLP and MBP.INVASc subunit vaccines induced strong antigen-specific serum IgG and IgA responses. There were no significant differences in weight gain among the groups. Mild-to-moderate clinical signs of LI infection were observed, but vaccinated groups showed lower inflammatory scores and fewer animals tested positive for bacteria by immunohistochemistry. Although neither vaccine completely prevented clinical signs of LI infection, both effectively reduced inflammation and lowered the pathogen load, thereby mitigating the severity of the disease, particularly the MBP.INVASc vaccine. Conclusions: These findings suggest that both vaccines have the potential for further development and optimization to enhance their protective efficacy against LI infections.
Tex10 coordinates epigenetic control of super-enhancer activity in pluripotency and reprogramming
(Cell Press, 2015-06-04) Ding, Junjun; Huang, Xin; Shao, Ningyi; Zhou, Hongwei; Lee, Dung-Fan; Faiola, Francesco; Fidalgo Pérez, Miguel Ángel; Guallar Artal, Diana; Saunders, Arven; Shliaha, Pavel V.; Wang, Hailong; Waghray, Avinash; Papatsenko, Dmitri; Sánchez Priego, Carlos; Li, Dan; Yuan, Ye; Lemischka, Ihor R.; Shen, Li; Kelley, Kevin; Deng, Haiteng; Shen, Xiaohua; Wang, Jianlong; Universidade de Santiago de Compostela. Centro de Investigación en Medicina Molecular e Enfermidades Crónicas (CiMUS); Universidade de Santiago de Compostela. Departamento de Fisioloxía
Super-enhancers (SEs) are large clusters of transcriptional enhancers that are co-occupied by multiple lineage-specific transcription factors driving expression of genes that define cell identity. In embryonic stem cells (ESCs), SEs are highly enriched for the core pluripotency factors Oct4, Sox2, and Nanog. In this study, we sought to dissect the molecular control mechanism of SE activity in pluripotency and reprogramming. Starting from a protein interaction network surrounding Sox2, we identified Tex10 as a key pluripotency factor that plays a functionally significant role in ESC self-renewal, early embryo development, and reprogramming. Tex10 is enriched at SEs in a Sox2-dependent manner and coordinates histone acetylation and DNA demethylation at SEs. Tex10 activity is also important for pluripotency and reprogramming in human cells. Our study therefore highlights Tex10 as a core component of the pluripotency network and sheds light on its role in epigenetic control of SE activity for cell fate determination
Zfp281 Coordinates Opposing Functions of Tet1 and Tet2 in Pluripotent States
(Cell Press, 2016-09-01) Fidalgo Pérez, Miguel Ángel; Huang, Xin; Guallar Artal, Diana; Sánchez Priego, Carlos; Valdés, Víctor Julián; Saunders, Arven; Ding, Junjun; Wu, Wen-Shu; Clavel, Carlos; Wang, Jianlong; Universidade de Santiago de Compostela. Centro de Investigación en Medicina Molecular e Enfermidades Crónicas (CiMUS); Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular; Universidade de Santiago de Compostela. Departamento de Fisioloxía
Pluripotency is increasingly recognized as a spectrum of cell states defined by their growth conditions. Although naive and primed pluripotency states have been characterized molecularly, our understanding of events regulating state acquisition is wanting. Here, we performed comparative RNA sequencing of mouse embryonic stem cells (ESCs) and defined a pluripotent cell fate (PCF) gene signature associated with acquisition of naive and primed pluripotency. We identify Zfp281 as a key transcriptional regulator for primed pluripotency that also functions as a barrier toward achieving naive pluripotency in both mouse and human ESCs. Mechanistically, Zfp281 interacts with Tet1, but not Tet2, and its direct transcriptional target, miR-302/367, to negatively regulate Tet2 expression to establish and maintain primed pluripotency. Conversely, ectopic Tet2 alone, but not Tet1, efficiently reprograms primed cells toward naive pluripotency. Our study reveals a molecular circuitry in which opposing functions of Tet1 and Tet2 control acquisition of alternative pluripotent states
A genome-wide RNAi screen identifies opposing functions of Snai1 and Snai2 on the Nanog dependency in reprogramming
(Cell Press, 2014-10-02) Gingold, Julian A.; Fidalgo Pérez, Miguel Ángel; Guallar Artal, Diana; Lau, Zerlina; Sun, Zhen; Zhou, Hongwei; Faiola, Francesco; Huang, Xin; Lee, Dung-Fang; Waghray, Avinash; Schaniel, Christoph; Felsenfeld, Dan P; Lemischka, Ihor R; Wang, Jianlong; Universidade de Santiago de Compostela. Centro de Investigación en Medicina Molecular e Enfermidades Crónicas (CiMUS); Universidade de Santiago de Compostela. Departamento de Fisioloxía
Nanog facilitates embryonic stem cell self-renewal and induced pluripotent stem cell generation during the final stage of reprogramming. From a genome-wide small interfering RNA screen using a Nanog-GFP reporter line, we discovered opposing effects of Snai1 and Snai2 depletion on Nanog promoter activity. We further discovered mutually repressive expression profiles and opposing functions of Snai1 and Snai2 during Nanog-driven reprogramming. We found that Snai1, but not Snai2, is both a transcriptional target and protein partner of Nanog in reprogramming. Ectopic expression of Snai1 or depletion of Snai2 greatly facilitates Nanog-driven reprogramming. Snai1 (but not Snai2) and Nanog cobind to and transcriptionally activate pluripotency-associated genes including Lin28 and miR-290-295. Ectopic expression of miR-290-295 cluster genes partially rescues reprogramming inefficiency caused by Snai1 depletion. Our study thus uncovers the interplay between Nanog and mesenchymal factors Snai1 and Snai2 in the transcriptional regulation of pluripotency-associated genes and miRNAs during the Nanog-driven reprogramming process
RNA-dependent chromatin targeting of TET2 for endogenous retrovirus control in pluripotent stem cells
(Springer Nature, 2018-02-26) Guallar Artal, Diana; Bi, Xianju; Pardavila Paz, Jose Ángel; Huang, Xin; Saenz, Carmen; Shi, Xianle; Zhou, Hongwei; Faiola, Francesco; Ding, Junjun; Haruehanroengra, Phensinee; Yang, Fan; Li, Dan; Sánchez Priego, Carlos; Saunders, Arven; Pan, Feng; Valdés, Víctor Julián; Kelley, Kevin; González Blanco, Miguel; Chen, Lingyi; Wang, Huayan; Sheng, Jia; Xu, Mingjiang; Fidalgo Pérez, Miguel Ángel; Shen, Xiaohua; Wang, Jianlong; Universidade de Santiago de Compostela. Centro de Investigación en Medicina Molecular e Enfermidades Crónicas (CiMUS); Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular; Universidade de Santiago de Compostela. Departamento de Fisioloxía
Ten-eleven translocation (TET) proteins play key roles in the regulation of DNA-methylation status by oxidizing 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC), which can both serve as a stable epigenetic mark and participate in active demethylation. Unlike the other members of the TET family, TET2 does not contain a DNA-binding domain, and it remains unclear how it is recruited to chromatin. Here we show that TET2 is recruited by the RNA-binding protein Paraspeckle component 1 (PSPC1) through transcriptionally active loci, including endogenous retroviruses (ERVs) whose long terminal repeats (LTRs) have been co-opted by mammalian genomes as stage- and tissue-specific transcriptional regulatory modules. We found that PSPC1 and TET2 contribute to ERVL and ERVL-associated gene regulation by both transcriptional repression via histone deacetylases and post-transcriptional destabilization of RNAs through 5hmC modification. Our findings provide evidence for a functional role of transcriptionally active ERVs as specific docking sites for RNA epigenetic modulation and gene regulation
Detection of Salmonella enterica serotype Typhimurium with pUO-StVR2-like virulence-resistance hybrid plasmids in the United Kingdom
(Springer, 2009-05-12) Fresno Herrero, Ana; Mendoza Fernández, M. Carmen; Threlfall, E. J.; Rodicio, M. R.; Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular
The aim of this study was to investigate the presence in the United Kingdom (UK) of Salmonella enterica serovar Typhimurium isolates carrying pUO-StVR2-like virulence-resistance hybrid plasmids that originated from pSLT. One hundred and fifty ampicillin-resistant isolates of S. Typhimurium, collected in different regions of the UK during 2006, were screened for the presence of bla OXA-1 carried by an InH-like integron (2000 bp/bla OXA-1-aadA1) characteristic of pUO-StVR2. Positive isolates were tested for the presence of a large plasmid that hybridised with probes specific for the bla OXA-1 and spvC genes, used as resistance and virulence markers of the hybrid plasmid, respectively. Eleven out of the 150 isolates fulfilled both criteria and were assigned to the S. Typhimurium pUO-StVR2 group. Nine were resistant to ampicillin, chloramphenicol, streptomycin/spectinomycin, sulfonamides and tetracycline, encoded by bla OXA-1, catA1, aadA1-like, sul1 and tet(B), respectively, and carried a pUO-StVR2-like plasmid of ca. 130 kb. Two contained hybrid plasmids of smaller size and lacked resistance(s) to chloramphenicol or chloramphenicol and tetracycline. The eleven isolates, which showed five and six closely related XbaI and BlnI profiles, respectively, were resistant to nitrofurantoin. In conclusion, multidrug-resistant S. Typhimurium isolates of the pUO-StVR2 group, which are endemic in Spain, were also detected in the UK, albeit with a low frequency (7.3%)
qPCR as a powerful tool for microbial food spoilage quantification: significance for food quality
(Elsevier, 2011-07) Martínez Álvarez, Noelia; Martín, María Cruz; Fresno Herrero, Ana; Fernández García, María; Álvarez González, Miguel Ángel; Ladero Losada, Víctor; Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular
The use of real time quantitative PCR (qPCR) has recently been extended to food science. The literature has mainly focused on its use in ensuring food safety. However, it offers a number of advantages with respect to the quantification of non-pathogenic food spoilage microorganisms. Indeed, qPCR may have a promising future in improving the quality of food products. The present review examines the use of qPCR in this area, the basis of the technique, the requirements that must be met for optimal qPCR assays to be performed, and the advantages it offers over other techniques
Molecular epidemiology of emergent multidrug-resistant Salmonella enterica serotype Typhimurium strains carrying the virulence resistance plasmid pUO-StVR2
(Oxford University Press, 2006-01) Fresno Herrero, Ana; Rodicio, M. R.; González Hevia, M. A.; Mendoza, M. C.; Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular
Objectives: To evaluate the incidence of a distinct multidrug-resistant (MDR) grouping of Salmonella serotype Typhimurium strains carrying the hybrid virulence resistance plasmid pUO-StVR2, and its possible evolution in the region where it was first detected [Principality of Asturias (PA), Spain]. Methods: pUO-StVR2-containing isolates were tentatively identified by two genetic markers: the blaOXA-30 gene and the class 1 integron InH:2000 bp/blaOXA-30-aadA1a. Positive isolates were examined for resistance profile (RP), plasmid content, virulence profile (VP) and genomic polymorphisms using macrorestriction–PFGE. Results: A total of 182 out of 248 Typhimurium clinical isolates recorded in the PA over 2001–02 were ampicillin-resistant and could be distributed into several MDR groupings. A MDR grouping carrying pUO-StVR2, with a defined RP (AMP/blaOXA-30, CHL/catA1, [STR-SPT]/[strA/B,aadA1a], SUL/[sul1,sul2], TET/tet(B), qacEΔ1, merA, ±TMP/dfrA12, and containing InH), was represented by 49 isolates. The VPs of these isolates (24 genes screened) differed from that of the type strain LT2 by the absence of the sopE1 and pef genes. Macrorestriction analysis established six combined XbaI/BlnI PFGE profiles, and supported a clonal relationship among most of the isolates. Conclusions: During 2001–02, the isolates carrying pUO-StVR2 constituted the second most frequent S. Typhimurium MDR grouping recorded in the PA, preceded only by the pandemic pentaresistant DT104. Polymorphisms on the genomic DNA, different phage types, different plasmid profiles and the detection of trimethoprim resistance in one isolate encoded by an additional plasmid, were consistent with both intra-cluster evolution and horizontal transfer of the hybrid plasmid
Genomics, biofilm formation and infection of bladder epithelial cells in potentially uropathogenic Escherichia coli (UPEC) from animal sources and human urinary tract infections (UTIs) further support food-borne transmission
(Elsevier, 2023-05-03) García Menéndez, Vanesa; Lestón Cambeiro, Luz; Parga Martínez, Ana; García Meniño, Isidro; Fernández Domínguez, Javier; Otero Casal, Ana María; Olsen, John Elmerdahl; Fresno Herrero, Ana; Mora Gutiérrez, Azucena; Universidade de Santiago de Compostela. Departamento de Microbioloxía e Parasitoloxía; Universidade de Santiago de Compostela. Departamento de Bioquímica e Bioloxía Molecular; Universidade de Santiago de Compostela. Instituto de Investigación do Medio Acuático para Unha Saúde Global (iARCUS)
Escherichia coli is the main cause of urinary tract infections (UTI). While genomic comparison of specific clones recovered from animals, and human extraintestinal infections show high identity, studies demonstrating the uropathogenicity are lacking. In this study, comparative genomics combined with bladder-cell and biofilm formation assays, were performed for 31 E. coli of different origins: 7 from meat (poultry, beef, and pork); 2 from avian-farm environment; 12 from human uncomplicated UTI, uUTI; and 10 from human complicated UTI, cUTI. These isolates were selected based on their genetic uropathogenic (UPEC) status and phylogenetic background. In silico analysis revealed similar virulence-gene profiles, with flagella, type 1 and curli fimbriae, outer-membrane proteins (agn43, ompT, iha), and iron-uptake (iutA, entA, and fyuA) associated-traits as the most prevalent (>65%). In bladder-cell assays, moderate to strong values of association (83%, 60%, 77.8%) and invasion (0%, 70%, 55.5%) were exhibited by uUTI, cUTI, and animal-derived isolates, respectively. Of interest, uUTI isolates exhibited a significantly lower invasive capacity than cUTI isolates (p < 0.05). All isolates but one produced measurable biofilm. Notably, 1 turkey meat isolate O11:H6-F-ST457, and 2 cUTI isolates of the pandemic lineages O83:H42-F-ST1485-CC648 and O25b:H4-B2-ST131, showed strong association, invasion and biofilm formation. These isolates showed common carriage of type 1 fimbriae and csg operons, toxins (hlyF, tsh), iron uptake systems (iutA, entA, iroN), colicins, protectins (cvaC, iss, kpsM, traT), ompT, and malX. In summary, the similar in vitro behaviour found here for certain E. coli clones of animal origin would further reinforce the role of food-producing animals as a potential source of UPEC. Bladder-cell infection assays, combined with genomics, might be an alternative to in vivo virulence models to assess uropathogenicity

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