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Gestational diabetes mellitus (GDM) is a common condition during pregnancy and is associated with an increased risk of pre-eclampsia. The methylenetetrahydrofolate reductase (MTHFR) gene plays a crucial role in folate metabolism and has been implicated in GDM. To investigate the relationship between the MTHFR C677T gene polymorphism and the conditions of GDM and gestational prediabetes in pregnant women. A case-control study was conducted in 114 pregnant women with GDM and 96 pregnant women without GDM, from the first trimester to the prenatal examination at Can Tho Obstetrics Hospital. The pregnant women underwent a 1-hour (G1) and 2-hour (G2) oral glucose tolerance test (OGTT) and genetic polymorphism analysis based on real-time PCR technique. In pregnant women with GDM, weight, concentrations of G0, G1, G2, and folic acid were higher than those in the non-GDM group, with Pâ <â .05. When analyzing the subgroup without gestational diabetes, we found that the rate of prediabetes was 16.6% (16/96 pregnant women). In this group, blood glucose levels at 1 hour and 2 hours during the OGTT were higher compared to the normal glucose group (Pâ <â .05). A 2-hour post-OGTT glucose level of 7.78 mmol/L had a sensitivity of 93.8%, a specificity of 100%, and an area under the curve of 0.987 for diagnosing gestational prediabetes (Pâ <â .001). However, there were no statistically significant differences in the CC, CT, and TT polymorphisms of the MTHFR C677T gene among pregnant women with or without pre-gestational and GDM. Both fasting blood glucose and 2-hour glucose concentrations during the OGTT, as well as folic acid concentrations, were higher in both the pre-gestational and GDM groups compared to the non-gestational diabetes cohort. However, the analysis of MTHFR C677T polymorphisms revealed no statistically significant differences among the groups, highlighting the necessity for more extensive investigations to gain deeper insights into this relationship.
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Diabetes Gestacional , Teste de Tolerância a Glucose , Metilenotetra-Hidrofolato Redutase (NADPH2) , Humanos , Feminino , Gravidez , Diabetes Gestacional/genética , Diabetes Gestacional/epidemiologia , Metilenotetra-Hidrofolato Redutase (NADPH2)/genética , Adulto , Estudos de Casos e Controles , Glicemia/análise , Glicemia/metabolismo , Estado Pré-Diabético/genética , Estado Pré-Diabético/epidemiologia , Polimorfismo Genético , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Small-cell neuroendocrine cervical carcinoma (NECC) is a rare histology, and diagnosis and treatment of this condition are challenging because of its rarity, non-specific abdominopelvic symptoms, and less favorable prognosis compared to other cervical cancers. Here, we present a case of a 20-year-old patient diagnosed with small-cell NECC, defined within a cervical polyp, initially mimicking a benign lesion. Because of the difficulty in diagnosis, the patient underwent thorough diagnostics and interventions, including imaging, histopathology, and immunohistochemistry. Initially, the patient underwent a distinctive treatment plan encompassing four cycles of cisplatin and etoposide chemotherapy with minimal side effects. Subsequently, she received comprehensive surgical interventions, including hysterectomy, lymphadenectomy, and bilateral salpingectomy. Ovarian preservation, justified by the patient's youth and small cervical lesions (<4 cm) without parametrial disease or metastatic signs, was pursued. For long-term outcomes, the patient demonstrated no metastasis or recurrence during a five-year follow-up. This case emphasizes the requirement for strengthened awareness of neuroendocrine tumors in cervical masses, particularly in young patients, and the importance of individualized treatment approaches for optimal clinical outcomes. Continued documentation of such cases increases our understanding of managing infrequent cervical malignancies.
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BACKGROUND: Advancing health equity requires more contextualised evidence. OBJECTIVES: To synthesise published evidence using an existing framework on the origins of health disparities and determine care-related outcome disparities for residents of long-term care, comparing minoritised populations to the context-specific dominant population. DESIGN: Systematic review. SUBJECTS: Residents of 24-hour long-term care homes. METHODS: The protocol was registered a priori with PROSPERO (CRD42021269489). Literature published between 1 January 2000 and 26 September 2021, was searched, including studies comparing baseline characteristics and outcomes in minoritised versus dominant populations. Dual screening, two-reviewer verification for extraction, and risk of bias assessments were conducted to ensure rigour. Studies were synthesized using a conceptual framework to contextualise evidence according to multi-level factors contributing to the development of care disparities. RESULTS: Twenty-one of 34 included studies demonstrated disparities in care outcomes for minoritised groups compared to majority groups. Thirty-one studies observed differences in individual-level characteristics (e.g. age, education, underlying conditions) upon entry to homes, with several outcome disparities (e.g. restraint use, number of medications) present at baseline and remaining or worsening over time. Significant gaps in evidence were identified, particularly an absence of literature on provider information and evidence on the experience of intersecting minority identities that contribute to care-related outcome disparities in long-term care. CONCLUSION: This review found differences in minoritised populations' care-related outcomes. The findings provide guidance for future health equity policy and research-supporting diverse and intersectional capacity building in long-term care.
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Equidade em Saúde , Disparidades em Assistência à Saúde , Instituição de Longa Permanência para Idosos , Assistência de Longa Duração , Casas de Saúde , Humanos , Idoso , Masculino , FemininoRESUMO
A catalog of transcription factor (TF) binding sites in the genome is critical for deciphering regulatory relationships. Here we present the culmination of the modERN (model organism Encyclopedia of Regulatory Networks) consortium that systematically assayed TF binding events in vivo in two major model organisms, Drosophila melanogaster (fly) and Caenorhabditis elegans (worm). We describe key features of these datasets, comprising 604 TFs identifying 3.6M sites in the fly and 350 TFs identifying 0.9 M sites in the worm. Applying a machine learning model to these data identifies sets of TFs with a prominent role in promoting target gene expression in specific cell types. TF binding data are available through the ENCODE Data Coordinating Center and at https://epic.gs.washington.edu/modERNresource, which provides access to processed and summary data, as well as widgets to probe cell type-specific TF-target relationships. These data are a rich resource that should fuel investigations into TF function during development.
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N-Acetylneuraminic acid and its α2,3/α2,6-glycosidic linkages with galactose (Neu5Ac-Gal) are major carbohydrate antigen epitopes expressed in various pathological processes, such as cancer, influenza, and SARS-CoV-2. We here report a strategy for the synthesis and binding investigation of molecularly imprinted polymers (MIPs) toward α2,3 and α2,6 conformations of Neu5Ac-Gal antigens. Hydrophilic imprinted monoliths were synthesized from melamine monomer in the presence of four different templates, namely, N-acetylneuraminic acid (Neu5Ac), N-acetylneuraminic acid methyl ester (Neu5Ac-M), 3'-sialyllactose (3SL), and 6'-sialyllactose (6SL), in a tertiary solvent mixture at temperatures varying from -20 to +80 °C. The MIPs prepared at cryotemperatures showed a preferential affinity for the α2,6 linkage sequence of 6SL, with an imprinting factor of 2.21, whereas the α2,3 linkage sequence of 3SL resulted in nonspecific binding to the polymer scaffold. The preferable affinity for the α2,6 conformation of Neu5Ac-Gal was evident also when challenged by a mixture of other mono- and disaccharides in an aqueous test mixture. The use of saturation transfer difference nuclear magnetic resonance (STD-NMR) on suspensions of crushed monoliths allowed for directional interactions between the α2,3/α2,6 linkage sequences on their corresponding MIPs to be revealed. The Neu5Ac epitope, containing acetyl and polyalcohol moieties, was the major contributor to the sequence recognition for Neu5Ac(α2,6)Gal(ß1,4)Glc, whereas contributions from the Gal and Glc segments were substantially lower.
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Background: Despite the demonstrated efficacy of approved COVID-19 vaccines, high levels of hesitancy were observed in the first few months of the COVID-19 vaccines' rollout. Factors contributing to vaccine hesitancy are well-described in the literature. Among the various strategies for promoting vaccine confidence, educational interventions provide a foundationally and widely implemented set of approaches for supporting individuals in their vaccine decisions. However, the evidence around the measurable impact of various educational strategies to improve vaccine confidence is limited. We conducted a scoping review with the aim of exploring and characterizing educational interventions delivered during the pandemic to support COVID-19 vaccine confidence in adults. Methods: We developed a search strategy with a medical information scientist and searched five databases, including Ovid MEDLINE and Web of Science, as well as grey literature. We considered all study designs and reports. Interventions delivered to children or adolescents, interventions on non-COVID-19 vaccines, as well as national or mass vaccination campaigns without documented interaction(s) between facilitator(s) and a specific audience were excluded. Articles were independently screened by three reviewers. After screening 4602 titles and abstracts and 174 full-text articles across two rounds of searches, 22 articles met our inclusion criteria. Ten additional studies were identified through hand searching. Data from included studies were charted and results were described narratively. Results: We included 32 studies and synthesized their educational delivery structure, participants (i.e., facilitators and priority audience), and content. Formal, group-based presentations were the most common type of educational intervention in the included studies (75%). A third of studies (34%) used multiple strategies, with many formal group-based presentations being coupled with additional individual-based interventions (29%). Given the novelty of the COVID-19 vaccines and the unique current context, studies reported personalized conversations, question periods, and addressing misinformation as important components of the educational approaches reviewed. Conclusions: Various educational interventions were delivered during the COVID-19 pandemic, with many initiatives involving multifaceted interventions utilizing both formal and informal approaches that leveraged community (cultural, religious) partnerships when developing and facilitating COVID-19 vaccine education. Train-the-trainer approaches with recognized community members could be of value as trust and personal connections were identified as strong enablers throughout the review.
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Background: The rate of unfavorable outcomes, such as recurrence and death, in women with invasive breast cancer varies widely across countries and populations. Identifying those with high-risk profiles is critical so that early detection, prediction, and intervention can be made to improve their survival rate. Therefore, our study evaluated the rate of unfavorable outcomes and its association with clinicopathological characteristics in Vietnamese women with primary invasive breast cancer. Methods: A retrospective open cohort study was conducted on Vietnamese women with invasive breast cancer who underwent a mastectomy and were regularly followed up by the hospitals. Kaplan-Meier method was used to estimate the rate of unfavorable outcomes to take into account the follow-up time of each patient. Univariate and multiple Cox regression analyses were conducted to examine the associations between unfavorable outcomes and clinicopathological characteristics. Results: Among 204 women included in the data analysis, the mean age was 54.4 ± 10.9 years. The majority of patients were diagnosed with early-stage (76.5%) or locally advanced (22.5%) breast cancer. The 5-year rate of unfavorable outcomes was 12.8%, and the 8-year rate was 31.7%. Patients with advanced stages had a higher risk of unfavorable outcomes compared to those with early stages (IA, IIA, T2N1). Patients with lymph node metastases and those with triple-negative molecular classification had significantly higher rates of unfavorable outcomes. Conclusion: Although Vietnamese women with breast cancer have a relatively low rate of unfavorable outcomes compared to other countries, findings from this study emphasize the importance of early detection and underscore the need for targeted interventions for patients with advanced stages, lymph node metastases, and triple-negative breast cancer to optimize their treatment, outcomes, and overall prognosis.
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Tumor-infiltrating lymphocytes (TILs) and programmed death ligand 1 (PD-L1) are promising new factors in the prognosis and prediction of breast cancer patients. Our study evaluated the prevalence of expression of TILs on hematoxylin and eosin (H&E) slides, PD-L1 expression on immunohistochemistry, and their association with clinicopathological characteristics in Vietnamese women with invasive breast cancer. This study was conducted on 216 women with primary invasive breast cancer. The evaluation of TILs on the HE slides was based on the International TILs Working Group 2014 recommendation. PD-L1 protein expression was determined using the Combined Positive Score, the number of tumor cells, lymphocytes, and macrophages stained by PD-L1 divided by the total viable tumor cells multiplied by 100. Based on the cutoff of 11%, the prevalence of TILs expression was 35.6%, of which highly expressed TILs (≥50%) accounted for 15.3%. Postmenopausal women and those who had a body mass index of 25 kg/m2 or greater had a higher odds of having TILs expression. However, patients who had the expression of Ki-67, HER-positive molecular subtype, and triple-negative subtype were more likely to have TILs expression. The prevalence of PD-L1 expression was 30.1%. A significantly higher odds of having PD-L1 was found in patients who had a history of benign breast disease, self-detected tumor and had TILs expression. The expression of TILs and PD-L1 is common in Vietnamese women with invasive breast cancer. Because of the importance of these expressions, routine evaluation to find women who had TILs and PD-L1 is needed so that treatment and prognosis can be optimized. Such routine evaluation can be targeted to those who had a high-risk profile found in this study.
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Neoplasias da Mama , Humanos , Feminino , Antígeno B7-H1 , Linfócitos do Interstício Tumoral , População do Sudeste AsiáticoRESUMO
Gene activity defines cell identity, drives intercellular communication, and underlies the functioning of multicellular organisms. We present the single-cell resolution atlas of gene activity of a fertile adult metazoan: Caenorhabditis elegans. This compendium comprises 180 distinct cell types and 19,657 expressed genes. We predict 7541 transcription factor expression profile associations likely responsible for defining cellular identity. We predict thousands of intercellular interactions across the C. elegans body and the ligand-receptor pairs that mediate them, some of which we experimentally validate. We identify 172 genes that show consistent expression across cell types, are involved in basic and essential functions, and are conserved across phyla; therefore, we present them as experimentally validated housekeeping genes. We developed the WormSeq application to explore these data. In addition to the integrated gene-to-systems biology, we present genome-scale single-cell resolution testable hypotheses that we anticipate will advance our understanding of the molecular mechanisms, underlying the functioning of a multicellular organism and the perturbations that lead to its malfunction.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica , Expressão GênicaRESUMO
The novel process reported here described the manufacture of monolithic molecularly imprinted polymers (MIPs) using a terminally functionalized block copolymer as the imprinting template and pore-forming agent. The MIPs were prepared through a step-growth polymerization process using a melamine-formaldehyde precondensate in a biphasic solvent system. Despite having a relatively low imprinting factor, the use of MIP monolith in liquid chromatography demonstrated the ability to selectively target desired analytes. An MIP capillary column was able to separate monophosphorylated peptides from a tryptic digest of bovine serum albumin. Multivariate data analysis and modeling of the phosphorylated and nonphosphorylated peptide retention times revealed that the number of phosphorylations was the strongest retention contributor for peptide retention on the monolithic MIP capillary column.
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Bacterial pathogens have evolved intricate mechanisms to evade the human immune system, including the production of immunomodulatory enzymes. Streptococcus pyogenes serotypes secrete two multi-modular endo-ß-N-acetylglucosaminidases, EndoS and EndoS2, that specifically deglycosylate the conserved N-glycan at Asn297 on IgG Fc, disabling antibody-mediated effector functions. Amongst thousands of known carbohydrate-active enzymes, EndoS and EndoS2 represent just a handful of enzymes that are specific to the protein portion of the glycoprotein substrate, not just the glycan component. Here, we present the cryoEM structure of EndoS in complex with the IgG1 Fc fragment. In combination with small-angle X-ray scattering, alanine scanning mutagenesis, hydrolytic activity measurements, enzyme kinetics, nuclear magnetic resonance and molecular dynamics analyses, we establish the mechanisms of recognition and specific deglycosylation of IgG antibodies by EndoS and EndoS2. Our results provide a rational basis from which to engineer novel enzymes with antibody and glycan selectivity for clinical and biotechnological applications.
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Glicosídeo Hidrolases , Evasão da Resposta Imune , Humanos , Glicosídeo Hidrolases/metabolismo , Streptococcus pyogenes , Imunoglobulina G , Polissacarídeos/metabolismoRESUMO
BACKGROUND: Many people with asthma and COPD remain undiagnosed. We developed and validated a new case-finding questionnaire to identify symptomatic adults with undiagnosed obstructive lung disease. METHODS: Adults in the community with no prior history of physician-diagnosed lung disease who self-reported respiratory symptoms were contacted via random-digit dialling. Pre- and post-bronchodilator spirometry was used to confirm asthma or COPD. Predictive questions were selected using multinomial logistic regression with backward elimination. Questionnaire performance was assessed using sensitivity, predictive values and area under the receiver operating characteristic curve (AUC). The questionnaire was assessed for test-retest reliability, acceptability and readability. External validation was prospectively conducted in an independent sample and predictive performance re-evaluated. RESULTS: A 13-item Undiagnosed COPD and Asthma Population Questionnaire (UCAP-Q) case-finding questionnaire to predict undiagnosed asthma or COPD was developed. The most appropriate risk cut-off was determined to be 6% for either disease. Applied to the derivation sample (n=1615), the questionnaire yielded a sensitivity of 92% for asthma and 97% for COPD; specificity of 17%; and an AUC of 0.69 (95% CI 0.64-0.74) for asthma and 0.82 (95% CI 0.78-0.86) for COPD. Prospective validation using an independent sample (n=471) showed sensitivities of 93% and 92% for asthma and COPD, respectively; specificity of 19%; with AUCs of 0.70 (95% CI 0.62-0.79) for asthma and 0.81 (95% CI 0.74-0.87) for COPD. AUCs for UCAP-Q were higher compared to AUCs for currently recommended case-finding questionnaires for asthma or COPD. CONCLUSIONS: The UCAP-Q demonstrated high sensitivities and AUCs for identifying undiagnosed asthma or COPD. A web-based calculator allows for easy calculation of risk probabilities for each disease.
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Asma , Doença Pulmonar Obstrutiva Crônica , Adulto , Asma/diagnóstico , Broncodilatadores/uso terapêutico , Volume Expiratório Forçado , Humanos , Reprodutibilidade dos Testes , Espirometria , Inquéritos e QuestionáriosRESUMO
Bacteria produce a remarkably diverse range of glycoside hydrolases to metabolize glycans from the environment as a primary source of nutrients, and to promote the colonization and infection of a host. Here we focus on EndoE, a multi-modular glycoside hydrolase secreted by Enterococcus faecalis, one of the leading causes of healthcare-associated infections. We provide X-ray crystal structures of EndoE, which show an architecture composed of four domains, including GH18 and GH20 glycoside hydrolases connected by two consecutive three α-helical bundles. We determine that the GH20 domain is an exo-ß-1,2-N-acetylglucosaminidase, whereas the GH18 domain is an endo-ß-1,4-N-acetylglucosaminidase that exclusively processes the central core of complex-type or high-mannose-type N-glycans. Both glycoside hydrolase domains act in a concerted manner to process diverse N-glycans on glycoproteins, including therapeutic IgG antibodies. EndoE combines two enzyme domains with distinct functions and glycan specificities to play a dual role in glycan metabolism and immune evasion.
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Acetilglucosaminidase , Glicosídeo Hidrolases , Acetilglucosaminidase/metabolismo , Enterococcus faecalis/metabolismo , Glicosídeo Hidrolases/metabolismo , Manose/metabolismo , Polissacarídeos/metabolismoRESUMO
Glycosylation plays an important role in various pathological processes such as cancer. One key alteration in the glycosylation pattern correlated with cancer progression is an increased level as well as changes in the type of sialylation. Developing molecularly-imprinted polymers (MIPs) with high affinity for sialic acid able to distinguish different glycoforms such as sialic acid linkages is an important task which can help in early cancer diagnosis. Sialyllactose with α2,6' vs. α2,3' sialic acid linkage served as a model trisaccharide template. Boronate chemistry was employed in combination with a library of imidazolium-based monomers targeting the carboxylate group of sialic acid. The influence of counterions of the cationic monomers and template on their interactions was investigated by means of 1H NMR titration studies. The highest affinities were afforded using a combination of Br- and Na+ counterions of the monomers and template, respectively. The boronate ester formation was confirmed by MS and 1H/11B NMR, indicating 1 : 2 stoichiometries between sialyllactoses and boronic acid monomer. Polymers were synthesized in the form of microparticles using boronate and imidazolium monomers. This combinatorial approach afforded MIPs selective for the sialic acid linkages and compatible with an aqueous environment. The molecular recognition properties with respect to saccharide templates and glycosylated targets were reported.
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Caenorhabditis elegans is an animal with few cells but a wide diversity of cell types. In this study, we characterize the molecular basis for their specification by profiling the transcriptomes of 86,024 single embryonic cells. We identify 502 terminal and preterminal cell types, mapping most single-cell transcriptomes to their exact position in C. elegans' invariant lineage. Using these annotations, we find that (i) the correlation between a cell's lineage and its transcriptome increases from middle to late gastrulation, then falls substantially as cells in the nervous system and pharynx adopt their terminal fates; (ii) multilineage priming contributes to the differentiation of sister cells at dozens of lineage branches; and (iii) most distinct lineages that produce the same anatomical cell type converge to a homogenous transcriptomic state.
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Caenorhabditis elegans/embriologia , Caenorhabditis elegans/genética , Linhagem da Célula , Desenvolvimento Embrionário , Animais , Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , RNA-Seq , Análise de Célula Única , TranscriptomaRESUMO
To resolve cellular heterogeneity, we developed a combinatorial indexing strategy to profile the transcriptomes of single cells or nuclei, termed sci-RNA-seq (single-cell combinatorial indexing RNA sequencing). We applied sci-RNA-seq to profile nearly 50,000 cells from the nematode Caenorhabditis elegans at the L2 larval stage, which provided >50-fold "shotgun" cellular coverage of its somatic cell composition. From these data, we defined consensus expression profiles for 27 cell types and recovered rare neuronal cell types corresponding to as few as one or two cells in the L2 worm. We integrated these profiles with whole-animal chromatin immunoprecipitation sequencing data to deconvolve the cell type-specific effects of transcription factors. The data generated by sci-RNA-seq constitute a powerful resource for nematode biology and foreshadow similar atlases for other organisms.
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Caenorhabditis elegans/citologia , Caenorhabditis elegans/genética , Núcleo Celular/genética , Análise de Célula Única/métodos , Transcriptoma , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Imunoprecipitação da Cromatina , Células HEK293 , Humanos , Larva/genética , Camundongos , Células NIH 3T3 , Neurônios/metabolismo , RNA/genética , Análise de Sequência de RNA , Fatores de Transcrição/genéticaRESUMO
We generated detailed RNA-seq data for the nematode Caenorhabditis elegans with high temporal resolution in the embryo as well as representative samples from post-embryonic stages across the life cycle. The data reveal that early and late embryogenesis is accompanied by large numbers of genes changing expression, whereas fewer genes are changing in mid-embryogenesis. This lull in genes changing expression correlates with a period during which histone mRNAs produce almost 40% of the RNA-seq reads. We find evidence for many more splice junctions than are annotated in WormBase, with many of these suggesting alternative splice forms, often with differential usage over the life cycle. We annotated internal promoter usage in operons using SL1 and SL2 data. We also uncovered correlated transcriptional programs that span >80 kb. These data provide detailed annotation of the C. elegans transcriptome.
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Caenorhabditis elegans/genética , Regulação da Expressão Gênica no Desenvolvimento , Transcriptoma , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Anotação de Sequência MolecularRESUMO
The transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly. Uniform processing and comprehensive annotation of these data allow comparison across metazoan phyla, extending beyond earlier within-phylum transcriptome comparisons and revealing ancient, conserved features. Specifically, we discover co-expression modules shared across animals, many of which are enriched in developmental genes. Moreover, we use expression patterns to align the stages in worm and fly development and find a novel pairing between worm embryo and fly pupae, in addition to the embryo-to-embryo and larvae-to-larvae pairings. Furthermore, we find that the extent of non-canonical, non-coding transcription is similar in each organism, per base pair. Finally, we find in all three organisms that the gene-expression levels, both coding and non-coding, can be quantitatively predicted from chromatin features at the promoter using a 'universal model' based on a single set of organism-independent parameters.
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Caenorhabditis elegans/genética , Drosophila melanogaster/genética , Perfilação da Expressão Gênica , Transcriptoma/genética , Animais , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/crescimento & desenvolvimento , Cromatina/genética , Análise por Conglomerados , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/genética , Histonas/metabolismo , Humanos , Larva/genética , Larva/crescimento & desenvolvimento , Modelos Genéticos , Anotação de Sequência Molecular , Regiões Promotoras Genéticas/genética , Pupa/genética , Pupa/crescimento & desenvolvimento , RNA não Traduzido/genética , Análise de Sequência de RNARESUMO
Trypanosomatid protozoan parasites lack a functional heme biosynthetic pathway, so must acquire heme from the environment to survive. However, the molecular pathway responsible for heme acquisition by these organisms is unknown. Here we show that L. amazonensis LHR1, a homolog of the C. elegans plasma membrane heme transporter HRG-4, functions in heme transport. Tagged LHR1 localized to the plasma membrane and to endocytic compartments, in both L. amazonensis and mammalian cells. Heme deprivation in L. amazonensis increased LHR1 transcript levels, promoted uptake of the fluorescent heme analog ZnMP, and increased the total intracellular heme content of promastigotes. Conversely, deletion of one LHR1 allele reduced ZnMP uptake and the intracellular heme pool by approximately 50%, indicating that LHR1 is a major heme importer in L. amazonensis. Viable parasites with correct replacement of both LHR1 alleles could not be obtained despite extensive attempts, suggesting that this gene is essential for the survival of promastigotes. Notably, LHR1 expression allowed Saccharomyces cerevisiae to import heme from the environment, and rescued growth of a strain deficient in heme biosynthesis. Syntenic genes with high sequence identity to LHR1 are present in the genomes of several species of Leishmania and also Trypanosoma cruzi and Trypanosoma brucei, indicating that therapeutic agents targeting this transporter could be effective against a broad group of trypanosomatid parasites that cause serious human disease.