Comparative genomics of the major parasitic worms

Parasitic nematodes (roundworms) and platyhelminths (flatworms) cause debilitating chronic infections of humans and animals, decimate crop production and are a major impediment to socioeconomic development. Here we report a broad comparative study of 81 genomes of parasitic and non-parasitic worms. We have identified gene family births and hundreds of expanded gene families at key nodes in the phylogeny that are relevant to parasitism. Examples include gene families that modulate host immune responses, enable parasite migration though host tissues or allow the parasite to feed. We reveal extensive lineage-specific differences in core metabolism and protein families historically targeted for drug development. From an in silico screen, we have identified and prioritized new potential drug targets and compounds for testing. This comparative genomics resource provides a much-needed boost for the research community to understand and combat parasitic worms

Epigenetic biomarkers in obesity, weight loss and inflammation: a role for circadian rhythm and methyl donors

Epigenetics refers to all the modifications that alter gene activity without nucleotide sequence modification, but including the chromatin structure alteration as a direct consequence. Indeed, the most widely studied epigenetic mechanism is DNA methylation, which involves the addition of a methyl group onto cytosine nucleotide. DNA methylation may be modified by environmental stimuli including dietary patterns and nutrients. The DNA methylation pattern alteration has been associated with the development of obesity, inflammation and metabolic disturbances (type 2 diabetes, hypercholesterolemia, hypertension, cardiovascular disease). In this context, obesity is considered a contributing factor to the onset and aggravation of the conditions that lead to metabolic syndrome. In the last years it has been reported that low-grade inflammation underlies the pathological processes that are tied to obesity and metabolic syndrome, meanwhile the disruption of the circadian system has also been associated with higher risk to develop obesity-related comorbidities. Furthermore, in the era of personalized nutrition , the DNA methylation pattern of each individual has emerged as a promising tool for the prediction, screening, diagnosis and prognosis of obesity and related pathologies. Likewise, the modulation of DNA methylation marks by different dietary compounds may be a target for newer therapeutic strategies concerning the prevention and treatment of these diseases. In this context, this research work has taken advantage of omics and high-throughput screening technologies in order to address the following aims: 1) to analyse the association between DNA methylation in white blood cells and the development of obesity in a pediatric population; 2) to investigate the influence of a weight loss intervention in the DNA methylation levels of genes involved in the circadian system, and the association between DNA methylation and changes in the lipid profile; 3) to identify potential epigenetic biomarkers for weight loss within a weight-loss program by integrating transcriptome and methylome microarray data; 4) to evaluate whether a low intake of folic acid is related to adverse metabolic features in obese subjects through changes in gene-specific DNA methylation pattern, and 5) to study whether folic acid and other dietary methyl donors can prevent the inflammatory response in an in vitro model through epigenetic mechanisms. In relation to the first objective, the results of the first chapter of this thesis suggest a role for DNA methylation, particularly in PTPRS and PER3 genes, in childhood obesity development. Concerning the second objective, we observe that DNA methylation in circadian genes, particularly in BMAL1, is dependent on dietary factors such as energy and carbohydrate intake, and could be used as a biomarker of the lipid profile response to the diet. The third chapter demonstrates that CD44 may have a role in body weight regulation, and its methylation levels can be used as a predictor of the success to a weight-loss intervention. The fourth chapter evidences that subjects with lower folate intake showed more adiposity and higher circulating levels of insulin, glucose, PAI-1, and cortisol, but lower CAMKK2 methylation levels. Moreover, CAMKK2 methylation was negatively associated with HOMA-IR index whereas CAMKK2 expression positively correlated with insulin resistance, suggesting that the methylation of this gene could be an epigenetic mechanism underlying low folic acid intake-mediated insulin resistance. Finally, in relation to the fifth objective, an in vitro study conducted in THP-1 monocytes and macrophages confirms that methyl donors, particularly folic acid, are able to decrease the expression and secretion of several pro-inflammatory mediators like IL-1β and TNF-α, which was accompanied by epigenetic modifications such as increased methylation of IL1B, SERPINE1 and IL18

Epigenetic biomarkers in obesity, weight loss and inflammation: a role for circadian rhythm and methyl donors

Epigenetics refers to all the modifications that alter gene activity without nucleotide sequence modification, but including the chromatin structure alteration as a direct consequence. Indeed, the most widely studied epigenetic mechanism is DNA methylation, which involves the addition of a methyl group onto cytosine nucleotide. DNA methylation may be modified by environmental stimuli including dietary patterns and nutrients. The DNA methylation pattern alteration has been associated with the development of obesity, inflammation and metabolic disturbances (type 2 diabetes, hypercholesterolemia, hypertension, cardiovascular disease). In this context, obesity is considered a contributing factor to the onset and aggravation of the conditions that lead to metabolic syndrome. In the last years it has been reported that low-grade inflammation underlies the pathological processes that are tied to obesity and metabolic syndrome, meanwhile the disruption of the circadian system has also been associated with higher risk to develop obesity-related comorbidities. Furthermore, in the era of personalized nutrition , the DNA methylation pattern of each individual has emerged as a promising tool for the prediction, screening, diagnosis and prognosis of obesity and related pathologies. Likewise, the modulation of DNA methylation marks by different dietary compounds may be a target for newer therapeutic strategies concerning the prevention and treatment of these diseases. In this context, this research work has taken advantage of omics and high-throughput screening technologies in order to address the following aims: 1) to analyse the association between DNA methylation in white blood cells and the development of obesity in a pediatric population; 2) to investigate the influence of a weight loss intervention in the DNA methylation levels of genes involved in the circadian system, and the association between DNA methylation and changes in the lipid profile; 3) to identify potential epigenetic biomarkers for weight loss within a weight-loss program by integrating transcriptome and methylome microarray data; 4) to evaluate whether a low intake of folic acid is related to adverse metabolic features in obese subjects through changes in gene-specific DNA methylation pattern, and 5) to study whether folic acid and other dietary methyl donors can prevent the inflammatory response in an in vitro model through epigenetic mechanisms. In relation to the first objective, the results of the first chapter of this thesis suggest a role for DNA methylation, particularly in PTPRS and PER3 genes, in childhood obesity development. Concerning the second objective, we observe that DNA methylation in circadian genes, particularly in BMAL1, is dependent on dietary factors such as energy and carbohydrate intake, and could be used as a biomarker of the lipid profile response to the diet. The third chapter demonstrates that CD44 may have a role in body weight regulation, and its methylation levels can be used as a predictor of the success to a weight-loss intervention. The fourth chapter evidences that subjects with lower folate intake showed more adiposity and higher circulating levels of insulin, glucose, PAI-1, and cortisol, but lower CAMKK2 methylation levels. Moreover, CAMKK2 methylation was negatively associated with HOMA-IR index whereas CAMKK2 expression positively correlated with insulin resistance, suggesting that the methylation of this gene could be an epigenetic mechanism underlying low folic acid intake-mediated insulin resistance. Finally, in relation to the fifth objective, an in vitro study conducted in THP-1 monocytes and macrophages confirms that methyl donors, particularly folic acid, are able to decrease the expression and secretion of several pro-inflammatory mediators like IL-1β and TNF-α, which was accompanied by epigenetic modifications such as increased methylation of IL1B, SERPINE1 and IL18

Intestinal permeability, gut inflammation, and gut immune system response are linked to aging-related changes in gut microbiota composition: A study in female mice

Aging entails changes at the cellular level that increase the risk of various pathologies. An association between gut microbiota and age-related diseases has also been attributed. This study aims to analyze changes in fecal microbiota composition and their association with genes related to immune response, gut inflammation, and intestinal barrier impairment. Fecal samples of female mice at different ages (2 months, 6 months, 12 months, and 18 months) and gene expression in colon tissue were analyzed. Results showed that the older mice group had a more diverse microbiota than the younger group. Additionally, the abundance of Cyanobacteria, Proteobacteria, Flavobacteriaceae, Bacteroides, Parabacteroides, Prevotellaceae_UCG-001, Akkermansia, and Parabacteroides goldsteinii increased with age. In contrast, there was a notable decline in Clostridiaceae, Lactobacillaceae, Monoglobaceae, Ligilactobacillus, Limosilactobacillus, Mucispirillum, and Bacteroides faecichinchillae. These bacteria imbalances were positively correlated with increased inflammation markers in the colon, including Tnf-α, Ccl2, and Ccl12, and negatively with the expression of tight junction genes (Jam2, Tjp1, and Tjp2), as well as immune response genes (Cd4, Cd72, Tlr7, Tlr12, and Lbp). In conclusion, high levels of diversity did not result in improved health in older mice; however, the imbalance in bacteria abundance that occurs with aging might contribute to immune senescence, inflammation, and leaky gut disease

Functionalized immunostimulating complexes with protein A via lipid vinyl sulfones to deliver cancer drugs to trastuzumab-resistant HER2-overexpressing breast cancer cells

Fernando Rodríguez-Serrano,1,* Nuria Mut-Salud,1,* Teresa Cruz-Bustos,2 Mercedes Gomez-Samblas,2 Esther Carrasco,1 Jose Manuel Garrido,3 F Javier López-Jaramillo,4 Francisco Santoyo-Gonzalez,4 Antonio Osuna2 1Institute of Biopathology and Regenerative Medicine, 2Molecular Biochemistry and Parasitology Research Group, Department of Parasitology, Faculty of Sciences, Institute of Biotechnology, University of Granada, 3Department of Cardiovascular Surgery, Virgen de las Nieves Hospital, 4Department of Organic Chemistry, Faculty of Sciences, Institute of Biotechnology, University of Granada, Granada, Spain *These authors contributed equally to this work Background: Around 20%–30% of breast cancers overexpress the proto-oncogene human epidermal growth receptor 2 (HER2), and they are characterized by being very invasive. Therefore, many current studies are focused on testing new therapies against tumors that overexpress this receptor. In particular, there exists major interest in new strategies to fight breast cancer resistant to trastuzumab (Tmab), a humanized antibody that binds specifically to HER2 interfering with its mitogenic signaling. Our team has previously developed immunostimulating complexes (ISCOMs) as nanocapsules functionalized with lipid vinyl sulfones, which can incorporate protein A and bind to G immunoglobulins that makes them very flexible nanocarriers.Methods and results: The aim of this in vitro study was to synthesize and evaluate a drug delivery system based on protein A-functionalized ISCOMs to target HER2-overexpressing cells. We describe the preparation of ISCOMs, the loading with the drugs doxorubicin and paclitaxel, the binding of ISCOMs to alkyl vinyl sulfone-protein A, the coupling of Tmab, and the evaluation in both HER2-overexpressing breast cancer cells (HCC1954) and non-overexpressing cells (MCF-7) by flow cytometry and fluorescence microscopy. Results show that the uptake is dependent on the level of overexpression of HER2, and the analysis of the cell viability reveals that targeted drugs are selective toward HCC1954, whereas MCF-7 cells remain unaffected.Conclusion: Protein A-functionalized ISCOMs are versatile carriers that can be coupled to antibodies that act as targeting agents to deliver drugs. When coupling to Tmab and loading with paclitaxel or doxorubicin, they become efficient vehicles for the selective delivery of the drug to Tmab-resistant HER2-overexpressing breast cancer cells. These nanoparticles may pave the way for the development of novel therapies for poor prognosis resistant patients. Keywords: targeted drug delivery, doxorubicin, HER2, nanoparticle, paclitaxel, protein A, trastuzuma

Use of sera cell free DNA (cfDNA) and exovesicle-DNA for the molecular diagnosis of chronic Chagas disease.

Chagas disease, a neglected tropical disease, is now considered a worldwide health concern as a result of migratory movements from Central and South America to other regions that were considered free of the disease, and where the epidemiological risk is limited to transplacental transmission or blood or organ donations from infected persons. Parasite detection in chronically ill patients is restricted to serological tests that only determine infection by previous infection and not the presence of the parasite, especially in patients undergoing treatment evaluation or in newborns. We have evaluated the use of nucleic acids from both circulating exovesicles and cell-free DNA (cfDNA) from 50 samples twice randomly selected from a total of 448 serum samples from immunologically diagnosed patients in whom the presence of the parasite was confirmed by nested PCR on amplicons resulting from amplification with kinetoplastid DNA-specific primers 121F-122R. Six samples were randomly selected to quantify the limit of detection by qPCR in serum exovesicles. When the nucleic acids thus purified were assayed as a template and amplified with kinetoplastid DNA and nuclear satellite DNA primers, a 100% positivity rate was obtained for all positive samples assayed with kDNA-specific primers and 96% when SAT primers were used. However, isolation of cfDNA for Trypanosoma cruzi and amplification with SAT also showed 100% positivity. The results demonstrate that serum exovesicles contain DNA of mitochondrial and nuclear origin, which can be considered a mixed population of exovesicles of parasitic origin. The results obtained with serum samples prove that both cfDNA and Exovesicle DNA can be used to confirm parasitaemia in chronically ill patients or in samples where it is necessary to demonstrate the active presence of the parasite. The results confirm for the first time the existence of exovesicles of mitochondrial origin of the parasite in the serum of those affected by Chagas disease

Noncoding RNAs, cytokines, and inflammation-related diseases

Chronic inflammation is involved in the onset and development of many diseases, including obesity, atherosclerosis, type 2 diabetes, osteoarthritis, autoimmune and degenerative diseases, asthma, periodontitis, and cirrhosis. The inflammation process is mediated by chemokines, cytokines, and different inflammatory cells. Although the molecules and mechanisms that regulate this primary defense mechanism are not fully understood, recent findings offer a putative role of noncoding RNAs, especially microRNAs (miRNAs), in the progression and management of the inflammatory response. These noncoding RNAs are crucial for the stability and maintenance of gene expression patterns that characterize some cell types, tissues, and biologic responses. Several miRNAs, such as miR-126, miR-132, miR-146, miR-155, and miR-221, have emerged as important transcriptional regulators of some inflammation-related mediators. Additionally, little is known about the involvement of long noncoding RNAs, long intergenic noncoding RNAs, and circular RNAs in inflammation-mediated processes and the homeostatic imbalance associated with metabolic disorders. These noncoding RNAs are emerging as biomarkers with diagnosis value, in prognosis protocols, or in the personalized treatment of inflammation-related alterations. In this context, this review summarizes findings in the field, highlighting those noncoding RNAs that regulate inflammation, with emphasis on recognized mediators such as TNF-α, IL-1, IL-6, IL-18, intercellular adhesion molecule 1, VCAM-1, and plasminogen activator inhibitor 1. The down-regulation or antagonism of the noncoding RNAs and the administration of exogenous miRNAs could be, in the near future, a promising therapeutic strategy in the treatment of inflammation-related diseases.—Marques-Rocha, J. L., Samblas, M., Milagro, F. I., Bressan, J., Martínez, J. A., Marti, A. Noncoding RNAs, cytokines, and inflammation-related diseases. Inflammation is a complex protective process that requires a cross-talk between different types of immune cells to remove or neutralize harmful stimuli (1). In the classic view, the inflammatory process is induced by an invasion of foreign pathogens of biologic origin or by tissue damage. Neutrophils, dendritic cells, and macrophages express almost all types of TLRs participating in the transmission of a signal from the plasma membrane through a multistep cascade to responsive transcription factors. Members of the TLR family have emerged as the primary evolutionarily conserved sensors of pathogen-associated molecular patterns (1). Binding of the TLRs to their respective ligands initiates a wide spectrum of responses, from phagocytosis to production of a variety of cytokines, which in turn shape and enhance the inflammatory and adaptive immune responses. Typical transcription factors that activate inflammatory mediators are NF-κB (2), activator protein 1 (3), signal transducer and activator of transcription (STAT) (4), CCAAT enhancer binding protein (C/EBP) (5), and Ets-like gene 1 (6). The interactions between transcription factors that compete for binding sites in the promoter regions of specific target genes are highly complex. Usually the multistep signaling leads to a prompt transcription of genes resulting in accumulation of specific mRNAs coding for TNF-α, IL-1, IL-6, IL-8, monocyte chemotactic protein 1 (MCP-1), and other cytokines involved in inflammation (7). Some cytokines may elicit a broad inflammatory response, while others act on specific cell types. The activation, proliferation, and recruitment phenomena of specific differentiated immune cells are involved in resolving the nonhomeostatic state [for a review, see Shi (8)]. Thus, macrophages stimulate the inflammatory responses of neutrophils, fibroblasts, and endothelial cells (2). Other sentinel cells may present antigens to the T-helper cells, which play a central role in coordinating immune responses, such as clonal expansion of T cells and B cell responses (9). Acute inflammation is an important part of the immune response, but chronic inappropriate inflammation can cause metabolic disorders (10). For example, chronic low-grade inflammation has been repeatedly associated with the onset and prevalence of metabolic syndrome (11, 12). The International Diabetes Federation estimates that one quarter of the world’s adult population has metabolic syndrome (13). This phenomenon is defined by a combination of interconnected cardiometabolic alterations that include the presence of arterial hypertension, insulin resistance, dyslipidemia, cardiovascular disease, and abdominal obesity (11). With the pathologic enlargement of the adipose tissue in obesity, the blood supply to adipocytes may be reduced, with subsequent hypoxia leading to a an elevated production of proinflammatory mediators [TNF-α and IL-6, plasminogen activator inhibitor-1 (PAI-1), and C-reactive protein (CRP)] and increased infiltration of immune cells, particularly adipose tissue macrophages (14, 15). These altered signals mediate multiple processes, including insulin sensitivity (16), oxidative stress (17), energy metabolism, blood coagulation, and inflammatory responses (12). These pathologic conditions predispose to diabetes mellitus, hepatic steatosis, atherosclerosis, plaque rupture, and atherothrombosis (11). However, to date, the available information is controversial and does not necessarily imply an unequivocal causal role. The data obtained by functional genomics techniques indicate that several hundreds of genes participate in the inflammatory response and that their coordinated expression is tightly regulated [reviewed in Jura and Koj (18)]. Nevertheless, the involved pathways and the regulatory mechanisms are not completely understood. In the last few years, there has been a growing interest in the role of microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) in the development of several inflammation-related diseases (Table 1). These noncoding RNAs have emerged as important transcriptional regulators in both physiologic and pathophysiological conditions (19–21). In physiologic homeostasis, these nucleic acids may participate in cell differentiation, proliferation, apoptosis, hematopoiesis, limb morphogenesis, and important metabolic pathways, such as insulin secretion, triglyceride and cholesterol biosynthesis, and oxidative stress (20, 22, 23). Given their fundamental biologic roles, it is not surprising that miRNAs expression is tightly controlled and that its dysregulation can lead to disease onset