RESUMO
Sialidosis is a rare autosomal recessive disease that presents with progressive lysosomal storage of sialylated glycopeptides and oligosaccharides caused by homozygous or compound heterozygous sequence variants in the neuraminidase 1 (NEU1) gene. These sequence variants can lead to sialidosis type I and II; the latter is the most severe and presents prenatally or at early age. However, sialidosis diagnosis is challenging, especially in those health systems with limited resources of developing countries. Consequently, it is necessary to dip into high-throughput molecular diagnostic tools to allow for an accurate diagnosis with better cost-effectiveness and turnaround time. We report a 4-member pedigree segregating an ultrarare missense variant, c.1109A>G; p.Tyr370Cys, in NEU1 as detected by whole-exome sequencing. Two short-lived siblings, who presented with previously unreported clinical features from such a homozygous sequence variant, were diagnosed with sialidosis type II. Additionally, we present a novel molecular model exhibiting the consequences of the variant in the sialidase-1 tridimensional structure. This study allowed us to provide a definitive diagnosis for our patients, increase our understanding of this pathogenic variant, and improve genetic counseling.
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Chaperone-usher (CU) fimbriae are surface organelles particularly prevalent among the Enterobacteriaceae. Mainly associated to their adhesive properties, CU fimbriae play key roles in biofilm formation and host cell interactions. Little is known about the fimbriome composition of the opportunistic human pathogen Serratia marcescens. Here, by using a search based on consensus fimbrial usher protein (FUP) sequences, we identified 421 FUPs across 39 S. marcescens genomes. Further analysis of the FUP-containing loci allowed us to classify them into 20 conserved CU operons, 6 of which form the S. marcescens core CU fimbriome. A new systematic nomenclature is proposed according to FUP sequence phylogeny. We also established an in vivo transcriptional assay comparing CU promoter expression between an environmental and a clinical isolate of S. marcescens, which revealed that promoters from 3 core CU operons (referred as fgov, fpo, and fps) are predominantly expressed in the two strains and might represent key core adhesion appendages contributing to S. marcescens pathogenesis.
Assuntos
Fímbrias Bacterianas , Serratia marcescens , Fímbrias Bacterianas/genética , Humanos , Chaperonas Moleculares/genética , Óperon , Filogenia , Serratia marcescens/genéticaRESUMO
The expansion of the habitat of mosquitoes belonging to the Aedes genus puts nearly half of the world's population at risk of contracting dengue fever, and a significant fraction will develop its serious hemorrhagic complication, which can be fatal if not diagnosed properly and treated in a timely fashion. Although several diagnostic methods have been approved for dengue diagnostics, their applicability is limited in rural areas of developing countries by sample preparation costs and methodological requirements, as well as cross-reactivity among the different serotypes of the Dengue virus and other flavivirus, such as the Zika virus. For these reasons, it is necessary to generate more specific antigens to improve serological methods that could be cheaper and used in field operations. Here, we describe a strategy for the inactivation of cross-reacting epitopes on the surface of the Dengue virus envelope protein through the synthetic generation of recombinant peptide sequences, where key amino acid residues from Dengue virus serotype 1 (DENV-1) and 2 (DENV-2) are substituted by alanine residues. The proteins thus generated are recognized by 88% of sera from Dengue NS1+ patients and show improved serotype specificity because they do not react with the antibodies present in seroconverted, PCR-serotyped DEN-4 infected patients.
Assuntos
Alanina/imunologia , Vírus da Dengue/genética , Vírus da Dengue/imunologia , Epitopos/genética , Epitopos/imunologia , Proteínas do Envelope Viral/imunologia , Substituição de Aminoácidos , Anticorpos Antivirais/sangue , Reações Cruzadas , Dengue/imunologia , Dengue/virologia , Vírus da Dengue/classificação , Mapeamento de Epitopos , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Sorogrupo , Proteínas do Envelope Viral/genéticaRESUMO
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes Chikungunya fever. CHIKV entered Mexico through the state of Chiapas in October 2014. To fully understand the Chikungunya fever outbreak that occurred in southern Chiapas during 2015, we evaluated 22 PCR-confirmed CHIKV-positive patients, identified CHIKV genetic variability, reconstructed viral dispersal, and assessed possible viral mutations. Viruses were isolated and E2, 6K, and E1 genes were sequenced. We applied phylogenetic and phylogeographic approaches, modeled mutations, and estimated selective pressure. Different CHIKV strains circulated in Chiapas during summer 2015. Three isolates grouped themselves in a well-supported clade. Estimates show that the outbreak started in Ciudad Hidalgo and posteriorly dispersed towards Tapachula and neighboring municipalities. We found six non-synonymous mutations in our isolates. Two mutations occurred in one isolate and the remaining mutations occurred in single isolates. Mutations E2 T116I and E2 K221R changed the protein surface in contact with the host cell receptors. We could not find positive selected sites in our CHIKV sequences from southern Chiapas. This is the first viral phylogeographic reconstruction in Mexico characterizing the CHIKV outbreak in southern Chiapas.
Assuntos
Febre de Chikungunya/epidemiologia , Febre de Chikungunya/virologia , Vírus Chikungunya/genética , Febre de Chikungunya/transmissão , Vírus Chikungunya/classificação , Vírus Chikungunya/isolamento & purificação , Surtos de Doenças , Variação Genética , Humanos , México/epidemiologia , Modelos Moleculares , Filogenia , Filogeografia , RNA Viral/sangue , RNA Viral/genética , Seleção Genética , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
Androgenetic alopecia (AGA) or male pattern baldness is the most common form of hair loss in humans. Despite being a very frequent dermatological entity, molecular pathophysiology remains unclear. Several authors relate the presentation of AGA with a premature apoptotic process during the anagen phase and with an inflammatory microenvironment in the hair follicle. We evaluated a panel of 30 genes associated with inflammation and apoptosis in 5 AGA patients by targeted RNA-Seq. WNT7A gene was highly expressed in patients in stages 3V to 5 on the Hamilton-Norwood scale compared to patients with 5A stage. CASP7 and TNF genes were overexpressed in stages 3V and 4 compared to stages 5 and 5A. Overexpression of these genes detected only at early stages of AGA proves the role of WNT pathway, apoptosis, and inflammation in the development of this disorder.
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The microenvironment plays a fundamental role in carcinogenesis: Acidity and hypoxia are actively involved in this process. It is important to have in vitro models to study these mechanisms. The models that are most commonly referred to are the hypoxia chamber and the chemical induction [Cobalt (II) chloride]. It is not yet defined if these models are interchangeable if the metabolic effect is the same, and if the results may be compared in these models. In the present study, the response to the effect of stress (hypoxia and acidity) in both models was evaluated. The results indicated that in the chemical model, the effect of hypoxia appeared in an early form at 6 h; whereas in the gas chamber the effect was slow and gradual and at 72 h there was an overexpression of erythropoietin (EPO), vascular endothelial growth factor (VEGF), carbonic anhydrase 9 (CA9) and hypoxia-inducible factor 1α (HIF1α). In addition to the genes analyzed by reverse transcription-quantitative polymerase chain reaction, the global expression analysis between both models revealed the 9 most affected genes in common. The present study additionally identified 3 potential genes (lysyl oxidase, ankyrin repeat domain 37, B-cell lymphoma 2 interacting protein 3 like) previously identified in other studies, which may be considered as universal hypoxia genes along with HIF1α, EPO, VEGF, glucose transporter 1 (GLUT1), CA9, and LDH. To the best of the author's knowledge, this is the first time that both hypoxia models have been compared, and it was demonstrated that the effect of hypoxia induction was time sensitive in each model. These observations must be considered prior to selecting one of these models to identify selective hypoxia genes and their effects in cancer.
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Orexins or hypocretins are neurotransmitters produced by a small population of neurons in the lateral hypothalamus. This family of peptides modulates sleepwake cycle, arousal and feeding behaviors; however, the mechanisms regulating their expression remain to be fully elucidated. There is an interest in defining the key molecular elements in orexin regulation, as these may serve to identify targets for generating novel therapies for sleep disorders, obesity and addiction. Our previous studies showed that the expression of orexin was decreased in mice carrying nullmutations of the transcription factor early Bcell factor 2 (ebf2) and that the promoter region of the preproorexin (Hcrt) gene contained two putative ebfbinding sites, termed olf1 sites. In the present study, a minimal promoter region of the murine Hcrt gene was identified, which was able to drive the expression of a luciferase reporter gene in the human 293 cell line. Deletion of the olf1site proximal to the transcription start site of the Hcrt gene increased reporter gene expression, whereas deletion of the distal olf1like site decreased its expression. The lentiviral transduction of murine transcription factor ebf2 cDNA into 293 cells increased the gene expression driven by this minimal Hcrtgene promoter and an electrophoretic mobility shift assays demonstrated that the distal olf1like sequence was a binding site for ebf2.
Assuntos
Orexinas/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular , Expressão Gênica , Genes Reporter , Humanos , Camundongos , TransfecçãoRESUMO
Halloysite is an aluminosilicate clay that has been widely used for controlled drug delivery, immobilization of enzymes, and for the capture of circulating tumor cells (CTCs). Surface modification of halloysite by organosilanes has been explored to improve their properties. In this study halloysite clay nanotubes (HNTs) were functionalized by two different organosilanes: Trimethoxy(propyl)silane (TMPS), and Triethoxy(octyl)silane (EOS). Untreated and modified samples were characterized by scanning electron microscopy (SEM), X-ray diffractometry (XRD), thermogravimetrical analysis (TGA), and Fourier transform infrared spectroscopy (FTIR). Results showed a strong interaction of organosilanes with the chemical groups present in HNTs. Biocompatibility and cytotoxicity of these nanomaterials were determined using C6 rat glioblastoma cells. Our results indicate that prior to functionalization, HNTs show a high biocompatibility and low cytotoxicity. However, HNTs functionalized with EOS and TMPS showed high cytotoxicity by inducing apoptosis. These results allow the identification of potential applications in biomedical areas for HNTs.
RESUMO
The olfactory system, particularly the olfactory epithelium, presents a unique opportunity to study the regenerative capabilities of the brain, because of its ability to recover after damage. In this study, we ablated olfactory sensory neurons with methimazole and followed the anatomical and functional recovery of circuits expressing genetic markers for I7 and M72 receptors (M72-IRES-tau-LacZ and I7-IRES-tau-GFP). Our results show that 45 days after methimazole-induced lesion, axonal projections to the bulb of M72 and I7 populations are largely reestablished. Furthermore, regenerated glomeruli are re-formed within the same areas as those of control, unexposed mice. This anatomical regeneration correlates with functional recovery of a previously learned odorant-discrimination task, dependent on the cognate ligands for M72 and I7. Following regeneration, mice also recover innate responsiveness to TMT and urine. Our findings show that regeneration of neuronal circuits in the olfactory system can be achieved with remarkable precision and underscore the importance of glomerular organization to evoke memory traces stored in the brain.
Assuntos
Memória , Neurônios/fisiologia , Odorantes , Bulbo Olfatório/fisiologia , Animais , Axônios , Metimazol/farmacologia , Camundongos , Neurônios/citologia , Bulbo Olfatório/citologiaRESUMO
It is generally accepted that spongiform encephalopathies result from the aggregation into amyloid of a ubiquitous protein, the so-called prion protein. As a consequence, the dynamics of amyloid formation should explain the characteristics of the prion diseases: infectivity as well as sporadic and genetic occurrence, long incubation time, species barriers and strain specificities. The success of this amyloid hypothesis is due to the good qualitative agreement of this hypothesis with the observations. However, a number of difficulties appeared when comparing quantitatively the in vitro experimental results with the theoretical models, suggesting that some differences should hide important discrepancies. We used well defined quantitative models to analyze the experimental results obtained by in vitro polymerization of the recombinant hamster prion protein. Although the dynamics of polymerization resembles a simple nucleus-dependent fibrillogenesis, neither the initial concentration dependence nor off-pathway hypothesis fit with experimental results. Furthermore, seeded polymerization starts after a long time delay suggesting the existence of a specific mechanism that takes place before nucleus formation. On the other hand, polymerization dynamics reveals a highly stochastic mechanism, the origin of which appears to be caused by nucleation heterogeneity. Moreover, the specific structures generated during nucleation are maintained during successive seeding although a clear improvement of the dynamics parameters (polymerization rate and lag time) is observed. We propose that an additional on-pathway reaction takes place before nucleation and it is responsible for the heterogeneity of structures produced during prion protein polymerization in vitro. These amyloid structures behave like prion strains. A model is proposed to explain the genesis of heterogeneity among prion amyloid.
Assuntos
Placa Amiloide/metabolismo , Príons/química , Príons/metabolismo , Multimerização Proteica/fisiologia , Animais , Soluções Tampão , Cricetinae , Cristalização , Humanos , Cinética , Modelos Biológicos , Simulação de Dinâmica Molecular , Placa Amiloide/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
Prion diseases are caused by an abnormal form of the prion protein (PrP(Sc)). We identified, with lectins, post-translational modifications of brain proteins due to glycosylation in a Gerstmann-Sträussler-Scheinker (GSS) patient. The lectin Amaranthus leucocarpus (ALL), specific for mucin type O-glycosylated structures (Galß1,3 GalNAcα1,0 Ser/Thr or GalNAcα1,0 Ser/Thr), and Sambucus nigra agglutinin (SNA), specific for Neu5Acα2,6 Gal/GalNAc, showed positive labeling in all the prion deposits and in the core of the PrP(Sc) deposits, respectively, indicating specific distribution of O-glycosylated and sialylated structures. Lectins from Maackia amurensis (MAA, Neu5Acα2,3), Macrobrachium rosenbergii (MrL, Neu5,9Ac2-specific) and Arachis hypogaea (PNA, Gal-specific) showed low staining of prion deposits. Immunohistochemistry colocalization with prion antibody indicated that all lectins stained prion protein deposits. These results show that specific modifications in the glycosylation pattern are closely related to the hallmark lesions and might be an early event in neuronal degeneration in GSS disease.
Assuntos
Doença de Gerstmann-Straussler-Scheinker/metabolismo , Polissacarídeos/metabolismo , Proteínas PrPSc/metabolismo , Doença de Gerstmann-Straussler-Scheinker/patologia , Humanos , Imuno-Histoquímica , Lectinas , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Processamento de Proteína Pós-TraducionalRESUMO
The prion protein (PrP) plays a key role in the pathogenesis of prion diseases. However, the normal function of the protein remains unclear. The cellular isoform (PrP(C)) is expressed most abundantly in the brain, but has also been detected in other non-neuronal tissues as diverse as lymphoid cells, lung, heart, kidney, gastrointestinal tract, muscle, and mammary glands. Cell biological studies of PrP contribute to our understanding of PrP(C) function. Like other membrane proteins, PrP(C) is post-translationally processed in the endoplasmic reticulum and Golgi on its way to the cell surface after synthesis. Cell surface PrP(C) constitutively cycles between the plasma membrane and early endosomes via a clathrin-dependent mechanism, a pathway consistent with a suggested role for PrP(C) in cellular trafficking of copper ions. Although PrP(-/-) mice have been reported to have only minor alterations in immune function, PrP(C) is up-regulated in T cell activation and may be expressed at higher levels by specialized classes of lymphocytes. Furthermore, antibody cross-linking of surface PrP(C) modulates T cell activation and leads to rearrangements of lipid raft constituents and increased phosphorylation of signaling proteins. These findings appear to indicate an important but, as yet, ill-defined role in T cell function. Recent work has suggested that PrP(C) is required for self-renewal of haematopoietic stem cells. PrP(C) is highly expressed in the central nervous system, and since this is the major site of prion pathology, most interest has focused on defining the role of PrP(C) in neurones. Although PrP(-/-) mice have a grossly normal neurological phenotype, even when neuronal PrP(C) is knocked out postnatally, they do have subtle abnormalities in synaptic transmission, hippocampal morphology, circadian rhythms, and cognition and seizure threshold. Other postulated neuronal roles for PrP(C) include copper-binding, as an anti- and conversely, pro-apoptotic protein, as a signaling molecule, and in supporting neuronal morphology and adhesion. The prion protein may also function as a metal binding protein such as copper, yielding cellular antioxidant capacity suggesting a role in the oxidative stress homeostasis. Finally, recent observations on the role of PrP(C) in long-term memory open a challenging field.
Assuntos
Príons/fisiologia , Animais , Membrana Celular/fisiologia , Cobre/metabolismo , Camundongos , Príons/genética , Isoformas de ProteínasRESUMO
Homodimeric triosephosphate isomerase (TIM) from Trypanosoma cruzi (TcTIM) and T. brucei (TbTIM) are markedly similar in amino acid sequence and three-dimensional structure. In their dimer interfaces, each monomer has a Cys15 that is surrounded by loop3 of the adjoining subunit. Perturbation of Cys15 by methylmethane thiosulfonate (MMTS) induces abolition of catalysis and structural changes. In the two TIMs, the structural arrangements of their Cys15 are almost identical. Nevertheless, the susceptibility of TcTIM to MMTS is nearly 100-fold higher than in TbTIM. To ascertain the extent to which the characteristics of the interface Cys depend on the dynamics of its own monomer or on those of the adjacent monomer, we studied MMTS action on mutants of TcTIM that had the interface residues of TbTIM, and hybrids that have only one interfacial Cys15 (C15ATcTIM-wild type TbTIM). We found that the solvent exposure of the interfacial Cys depends predominantly on the characteristics of the adjoining monomer. The maximal inhibition of activity induced by perturbation of the sole interface Cys in the C15ATcTIM-TbTIM hybrid is around 60%. Hybrids formed with C15ATcTIM monomers and catalytically inert TbTIM monomers (E168DTbTIM) were also studied. Their activity drops by nearly 50% when the only interfacial Cys is perturbed. These results in conjunction with those on C15ATcTIM-wild type TbTIM hybrid indicate that about half of the activity of each monomer depends on the integrity of each of the two Cys15-loop3 portions of the interface. This could be another reason of why TIM is an obligatory dimer.
Assuntos
Cisteína/química , Metanossulfonato de Metila/análogos & derivados , Triose-Fosfato Isomerase/química , Trypanosoma brucei brucei/enzimologia , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Dimerização , Cinética , Metanossulfonato de Metila/química , Metanossulfonato de Metila/farmacologia , Mutagênese Sítio-Dirigida , Mapeamento de Interação de Proteínas/métodos , Estrutura Quaternária de Proteína , Triose-Fosfato Isomerase/antagonistas & inibidores , Triose-Fosfato Isomerase/genética , Triose-Fosfato Isomerase/metabolismoRESUMO
Homodimeric triosephosphate isomerases from Trypanosoma cruzi (TcTIM) and Trypanosoma brucei (TbTIM) have markedly similar catalytic properties and 3-D structures; their overall amino acid sequence identity is 68% and 85% in their interface residues. Nonetheless, active dimer formation from guanidinium chloride unfolded monomers is faster and more efficient in TcTIM than in TbTIM. The enzymes thus provide a unique opportunity for exploring the factors that control the formation of active dimers. The kinetics of reactivation at different protein concentrations showed that the process involved three reactions: monomer folding, association of folded monomers, and a transition from inactive to active dimers. The rate constants of the reactions indicated that, at relatively low protein concentrations, the rate-limiting step of reactivation was the association reaction; at high protein concentrations the transition of inactive to active dimers was rate limiting. The rates of the latter two reactions were higher in TcTIM than in TbTIM. Studies with a mutant of TcTIM that had the interface residues of TbTIM showed that the association rate constant was similar to that of TbTIM. However, the rate of the transition from inactive to active dimers was close to that of TcTIM; thus, this transition depends on the noninterfacial portion of the enzymes. When unfolded monomers of TcTIM and TbTIM were allowed to reactivate together, TcTIM, the hybrid, and TbTIM were formed in a proportion of 1:0.9:0.2. This distribution suggests that, in the hybrid, the characteristics of the TcTIM monomers influence the properties of TbTIM monomers.