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The activity of dental caries, combined with its multifactorial etiology, alters salivary molecule composition. The present systematic review was developed to answer the following question: "Are salivary biomarkers reliable for diagnosis of dental caries?". Following the "Preferred Reporting Item for Systematic Reviews and Meta-analysis" (PRISMA) guidelines, the review was conducted using multiple database research (Medline, Web of Science, and Scopus). Studies performed on healthy subjects with and without dental caries and providing detailed information concerning the clinical diagnosis of caries (Decayed, Missing, Filled Teeth-DMFT and International Caries Detection and Assessment System-ICDAS criteria) were included. The quality assessment was performed following a modified version of the Joanna Briggs Institute Prevalence Critical Appraisal Checklist. The protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO, ID: CRD42022304505). Sixteen papers were included in the review. All studies reported statistically significant differences in the concentration of salivary molecules between subjects with and without caries (p < 0.05). Proteins were the most investigated molecules, in particular alpha-amylase and mucins. Some studies present a risk of bias, such as identifying confounding factors and clearly defining the source population. Nevertheless, the 16 papers were judged to be of moderate to high quality. There is evidence that some salivary compounds studied in this review could play an important diagnostic role for dental caries, such as salivary mucins, glycoproteins (sCD14), interleukins (IL-2RA, 4,-13), urease, carbonic anhydrase VI, and urea.
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Periodontal diseases, including gingivitis and periodontitis, are among the most prevalent diseases in humans. Gingivitis is the mildest form of periodontal disease, characterized by inflammation of the gingiva caused by the accumulation of dental plaque. Salivary diagnostics are becoming increasingly popular due to the variation in saliva composition in response to pathological processes. We used a metabolomics approach to investigate whether a specific saliva metabolic composition could indicate preclinical stage of gingivitis. 1H-NMR spectroscopy was used to obtain the salivary metabolite profiles of 20 healthy subjects. Univariate/multivariate statistical analysis evaluated the whole saliva metabolite composition, and the Full-Mouth Bleeding Score (FMBS) was employed as a classification parameter. Identifying a signature of specific salivary metabolites could distinguish the subjects with high FMBS scores but still within the normal range. This set of metabolites may be due to the enzymatic activities of oral bacteria and be associated with the early stages of gingival inflammation. Although this analysis is to be considered exploratory, it seems feasible to establish an FMBS threshold that distinguishes between the absence and presence of early inflammatory alterations at the salivary level.
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Gengivite , Voluntários Saudáveis , Saliva , Humanos , Saliva/metabolismo , Feminino , Masculino , Projetos Piloto , Adulto , Gengivite/metabolismo , Gengivite/diagnóstico , Metabolômica/métodos , Hemorragia Gengival/metabolismo , Metaboloma , Adulto Jovem , Pessoa de Meia-Idade , Biomarcadores/metabolismoRESUMO
Saliva, which contains molecular information that may reflect an individual's health status, has become a valuable tool for discovering biomarkers of oral and general diseases. Due to the high vascularization of the salivary glands, there is a molecular exchange between blood and saliva. However, the composition of saliva is complex and influenced by multiple factors. This study aimed to investigate the possible relationships between the salivary and serum metabolomes to gain a comprehensive view of the metabolic phenotype under physiological conditions. Using 1H-NMR spectroscopy, we obtained the serum metabolite profiles of 20 healthy young individuals and compared them with the metabolomes of parotid, submandibular/sublingual, and whole-saliva samples collected concurrently from the same individuals using multivariate and univariate statistical analysis. Our results show that serum is more concentrated and less variable for most of the shared metabolites than the three saliva types. While we found moderate to strong correlations between serum and saliva concentrations of specific metabolites, saliva is not simply an ultrafiltrate of blood. The intense oral metabolism prevents very strong correlations between serum and salivary concentrations. This study contributes to a better understanding of salivary metabolic composition, which is crucial for utilizing saliva in laboratory diagnostics.
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Saliva , Glândulas Salivares , Humanos , Glândulas Salivares/metabolismo , Saliva/metabolismo , Glândula Parótida/metabolismo , MetabolomaRESUMO
Transglutaminase 2 (TG2), which mediates post-translational modifications of multiple intracellular enzymes, is involved in the pathogenesis and progression of cancer. We used 1 H-NMR metabolomics to study the effects of AA9, a novel TG2 inhibitor, on two breast cancer cell lines with distinct phenotypes, MCF-7 and MDA-MB-231. AA9 can promote apoptosis in both cell lines, but it is particularly effective in MD-MB-231, inhibiting transamidation reactions and decreasing cell migration and invasiveness. This metabolomics study provides evidence of a major effect of AA9 on MDA-MB-231 cells, impacting glutamate and aspartate metabolism, rather than on MCF-7 cells, characterised by choline and O-phosphocholine decrease. Interestingly, AA9 treatment induces myo-inositol alteration in both cell lines, indicating action on phosphatidylinositol metabolism, likely modulated by the G protein activity of TG2 on phospholipase C. Considering the metabolic deregulations that characterise various breast cancer subtypes, the existence of a metabolic pathway affected by AA9 further points to TG2 as a promising hot spot. The metabolomics approach provides a powerful tool to monitor the effectiveness of inhibitors and better understand the role of TG2 in cancer.
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Neoplasias da Mama , Proteína 2 Glutamina gama-Glutamiltransferase , Humanos , Feminino , Neoplasias da Mama/metabolismo , Células MCF-7 , Apoptose , Metabolômica , Linhagem Celular Tumoral , Transglutaminases/metabolismoRESUMO
Cerato-ulmin (CU) is a 75-amino-acid-long protein that belongs to the hydrophobin family. It self-assembles at hydrophobic-hydrophilic interfaces, forming films that reverse the wettability properties of the bound surface: a capability that may confer selective advantages to the fungus in colonizing and infecting elm trees. Here, we show for the first time that CU can elicit a defense reaction (induction of phytoalexin synthesis and ROS production) in non-host plants (Arabidopsis) and exerts its eliciting capacity more efficiently when in its soluble monomeric form. We identified two hydrophobic clusters on the protein's loops endowed with dynamical and physical properties compatible with the possibility of reversibly interconverting between a disordered conformation and a ß-strand-rich conformation when interacting with hydrophilic or hydrophobic surfaces. We propose that the plasticity of those loops may be part of the molecular mechanism that governs the protein defense elicitation capability.
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Plumbaginaceae , Plumbaginaceae/metabolismo , Proteínas Fúngicas/metabolismo , Fungos/metabolismo , Molhabilidade , Interações Hidrofóbicas e HidrofílicasRESUMO
We recently described C18 fatty acid acylated peptides as a new class of potent long-lasting single-chain RXFP1 agonists that displayed relaxin-like activities in vivo. Early pharmacokinetics and toxicological studies of these stearic acid acylated peptides revealed a relevant oxidative metabolism occurring in dog and minipig, and also seen at a lower extent in monkey and rat. Mass spectrometry combined to NMR spectroscopy studies revealed that the oxidation occurred, unexpectedly, on the stearic acid chain at ω-1, ω-2 and ω-3 positions. Structure-metabolism relationship studies on acylated analogues with different fatty acids lengths (C15-C20) showed that the extent of oxidation was higher with longer chains. The oxidized metabolites could be generated in vitro using liver microsomes and engineered bacterial CYPs. These systems were correlating poorly with in vivo metabolism observed across species; however, the results suggest that this biotransformation pathway might be catalyzed by some unknown CYP enzymes.
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Sistema Enzimático do Citocromo P-450 , Ácidos Graxos , Animais , Cães , Ratos , Sistema Enzimático do Citocromo P-450/metabolismo , Ácidos Graxos/metabolismo , Redes e Vias Metabólicas , Microssomos Hepáticos/metabolismo , Oxirredução , Ácidos Esteáricos , Suínos , Porco Miniatura/metabolismo , HaplorrinosRESUMO
Of-Pis1 is a potent piscidin antimicrobial peptide (AMP), recently isolated from rock bream (Oplegnathus fasciatus). This rich in histidines and glycines 24-amino acid peptide displays high and broad antimicrobial activity and no significant hemolytic toxicity against human erythrocytes, suggesting low toxicity. To better understand the mechanism of action of Of-Pis1 and its potential selectivity, using NMR and CD spectroscopies, we studied the interaction with eukaryotic and procaryotic membranes and membrane models. Anionic sodium dodecyl sulfate (SDS) and lipopolysaccharide (LPS) micelles were used to mimic procaryotic membranes, while zwitterionic dodecyl phosphocholine (DPC) was used as eukaryotic membrane surrogate. In an aqueous environment, Of-Pis1 adopts a flexible random coil conformation. In DPC and SDS instead, the N-terminal region of Of-Pis1 forms an amphipathic α-helix with the non-polar face in close contact with the micelles. Slower solvent exchange and higher pKas of the histidine residues in SDS than in DPC suggest that Of-Pis1 interacts more tightly with SDS. Of-Pis1 also binds tightly and structurally perturbs LPS micelles. Of-Pis1 interacts with both Escherichia coli and mammalian cell membranes, but only in the presence of Escherichia coli membranes it populates the helical conformation. Furthermore, ligand-based NMR experiments support a tighter and more specific interaction with bacterial than with eukaryotic membranes. Overall, these data clearly show the selective interaction of this broadly active AMP with bacterial over eukaryotic membranes. The conformational information is discussed in terms of Of-Pis1 amino acid sequence and composition to provide insights useful to design more potent and selective AMPs.
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Peptídeos Catiônicos Antimicrobianos , Histidina , Animais , Humanos , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Antimicrobianos , Escherichia coli/metabolismo , Lipopolissacarídeos/farmacologia , Mamíferos , MicelasRESUMO
The ubiquitous commensal Candida albicans, part of the human microbiota, is an opportunistic pathogen able to cause a wide range of diseases, from cutaneous mycoses to life-threatening infections in immunocompromised patients. Candida albicans adapts to different environments and survives long-time starvation. The ability to switch from yeast to hyphal morphology under specific environmental conditions is associated with its virulence. Using hydrogen nuclear magnetic resonance spectroscopy, we profiled the intracellular and extracellular metabolome of C. albicans kept in water, yeast extract-peptone-dextrose (YPD), and M199 media, at selected temperatures. Experiments were carried out in hypoxia to mimic a condition present in most colonized niches and fungal infection sites. Comparison of the intracellular metabolites measured in YPD and M199 at 37 °C highlighted differences in specific metabolic pathways: (i) alanine, aspartate, glutamate metabolism, (ii) arginine and proline metabolism, (iii) glycerolipid metabolism, attributable to the diverse composition of the media. Moreover, we hypothesized that the subtle differences in the M199 metabolome, observed at 30 °C and 37 °C, are suggestive of modifications propaedeutic to a subsequent transition from yeast to hyphal form. The analysis of the metabolites' profiles of C. albicans allows envisaging a molecular model to better describe its ability to sense and adapt to environmental conditions.
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Covid-19 is an infective respiratory illness caused by a novel virus, which might present different degrees of severity: from mild or even asymptomatic carriers to severe pneumonia, requiring intubation and intensive care unit (ICU) management. SARS-CoV-2 may cause also central nervous system involvement, including psychiatric manifestations. Some cases of psychosis apparently covid-related have been reported since the start of the pandemic; we will briefly review some of them here, then we will report a case concerning a patient with emerging psychosis during the disease caused by the virus. Our case describes a man with no prior personal or familiar psychiatric history, who developed delusion and a post-traumatic stress symptoms (PTSS) which required hospitalization in a psychiatric unit. The patient was treated with antipsychotic medications and underwent a brief follow-up.
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COVID-19 , Transtornos Psicóticos , Hospitalização , Humanos , Masculino , Pandemias , Transtornos Psicóticos/tratamento farmacológico , Transtornos Psicóticos/etiologia , SARS-CoV-2RESUMO
Lipidation, a common strategy to improve half-life of therapeutic peptides, affects their tendency to oligomerize, their interaction with plasmatic proteins, and their catabolism. In this work, we have leveraged the use of NMR and SPR spectroscopy to elucidate oligomerization propensity and albumin interaction of different analogs of the two marketed lipidated GLP-1 agonists liraglutide and semaglutide. As most lipidated therapeutic peptides are administered by subcutaneous injection, we have also assessed in vitro their catabolism in the SC tissue using the LC-HRMS-based SCiMetPep assay. We observed that oligomerization had a shielding effect against catabolism. At the same time, binding to albumin may provide only limited protection from proteolysis due to the higher unbound peptide fraction present in the subcutaneous compartment with respect to the plasma. Finally, identification of catabolites in rat plasma after SC dosing of semaglutide showed a good correlation with the in vitro data, with Tyr19-Leu20 being the major cleavage site. Early characterization of the complex interplay between oligomerization, albumin binding, and catabolism at the injection site is essential for the synthesis of lipidated peptides with good pharmacokinetic profiles.
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Diabetes Mellitus Tipo 2 , Peptídeo 1 Semelhante ao Glucagon , Albuminas , Animais , Meia-Vida , Hipoglicemiantes , Liraglutida , Peptídeos , RatosRESUMO
Since the multifunctionality of transglutaminase 2 (TG2) includes extra- and intracellular functions, we investigated the effects of intracellular administration of TG2 inhibitors in three breast cancer cell lines, MDA-MB-231, MDA-MB-436 and MDA-MB-468, which are representative of different triple-negative phenotypes, using a patch-clamp technique. The first cell line has a highly voltage-dependent a membrane current, which is low in the second and almost absent in the third one. While applying a voltage protocol to responsive single cells, injection of TG2 inhibitors triggered a significant decrease of the current in MDA-MB-231 that we attributed to voltage-dependent K+ channels using the specific inhibitors 4-aminopyridine and astemizole. Since the Kv10.1 channel plays a dominant role as a marker of cell migration and survival in breast cancer, we investigated its relationship with TG2 by immunoprecipitation. Our data reveal their physical interaction affects membrane currents in MDA-MB-231 but not in the less sensitive MDA-MB-436 cells. We further correlated the efficacy of TG2 inhibition with metabolic changes in the supernatants of treated cells, resulting in increased concentration of methyl- and dimethylamines, representing possible response markers. In conclusion, our findings highlight the interference of TG2 inhibitors with the Kv10.1 channel as a potential therapeutic tool depending on the specific features of cancer cells.
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Nuclear Magnetic Resonance (NMR) based metabolomic analysis of whole saliva has provided potential diagnostic biomarkers for numerous human diseases contributing to a better understanding of their mechanisms. However, a comprehensive interpretation of the significance of metabolites in whole, parotid, and submandibular/sublingual saliva subtypes is still missing. Precision and reproducibility of sample preparation is an essential step. Here, we present a simple and efficient protocol for saliva 1H-NMR metabolic profiling. This procedure has been specifically designed and optimized for the identification and quantification of low concentration metabolites (as low as 1.1 µM) and is suitable for all the saliva subtypes.
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Saliva , Espectroscopia de Prótons por Ressonância MagnéticaRESUMO
The insulin-like peptide human relaxin-2 was identified as a hormone that, among other biological functions, mediates the hemodynamic changes occurring during pregnancy. Recombinant relaxin-2 (serelaxin) has shown beneficial effects in acute heart failure, but its full therapeutic potential has been hampered by its short half-life and the need for intravenous administration limiting its use to intensive care units. In this study, we report the development of long-acting potent single-chain relaxin peptide mimetics. Modifications in the B-chain of relaxin, such as the introduction of specific mutations and the trimming of the sequence to an optimal size, resulted in potent, structurally simplified peptide agonists of the relaxin receptor Relaxin Family Peptide Receptor 1 (RXFP1) (e.g., 54). Introduction of suitable spacers and fatty acids led to the identification of single-chain lipidated peptide agonists of RXFP1, with sub-nanomolar activity, high subcutaneous bioavailability, extended half-lives, and in vivo efficacy (e.g., 64).
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Lipopeptídeos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores de Peptídeos/agonistas , Relaxina/análogos & derivados , Relaxina/farmacologia , Sequência de Aminoácidos , Animais , Doenças Cardiovasculares , Linhagem Celular Tumoral , Células HEK293 , Meia-Vida , Humanos , Lipopeptídeos/genética , Lipopeptídeos/farmacocinética , Masculino , Simulação de Dinâmica Molecular , Estrutura Molecular , Mutação , Subunidades Proteicas , Ratos Sprague-Dawley , Relaxina/genética , Relação Estrutura-AtividadeRESUMO
Among their various functions, the members of the cerato-platanin family can stimulate plants' defense responses and induce resistance against microbial pathogens. Recent results suggest that conserved loops, also involved in chitin binding, might be a structural motif central for their eliciting activity. Here, we focus on cerato-platanin and its orthologous cerato-populin, searching for a rationale of their diverse efficiency to elicit plants' defense and to interact with oligosaccharides. A 3D model of cerato-populin has been generated by homology modeling using the NMR-derived cerato-platanin structure as template, and it has been validated by fitting with residual dipolar couplings. Loops ß1-ß2 and ß2-ß3 have been indicated as important for some CPPs members to express their biological function. When compared to cerato-platanin, in cerato-populin they present two mutations and an insertion that significantly modify their electrostatic surface. NMR relaxation experiments point to a reduced conformational plasticity of cerato-populin loops with respect to the ones of cerato-platanin. The different electrostatic surface of the loops combined with a distinct network of intra-molecular interactions are expected to be factors that, by leading to a diverse spatial organization and dissimilar collective motions, can regulate the eliciting efficacy of the two proteins and their affinity for oligosaccharides.
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Ceratocystis/metabolismo , Proteínas Fúngicas/metabolismo , Oligossacarídeos/metabolismo , Doenças das Plantas/microbiologia , Ceratocystis/química , Proteínas Fúngicas/química , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Acid-sensing ion channels (ASICs) are a family of ion channels permeable to cations and largely responsible for the onset of acid-evoked ion currents both in neurons and in different types of cancer cells, thus representing a potential target for drug discovery. Owing to the limited attention ASIC2 has received so far, an exploratory program was initiated to identify ASIC2 inhibitors using diminazene, a known pan-ASIC inhibitor, as a chemical starting point for structural elaboration. The performed exploration enabled the identification of a novel series of ASIC2 inhibitors. In particular, compound 2u is a brain penetrant ASIC2 inhibitor endowed with an optimal pharmacokinetic profile. This compound may represent a useful tool to validate in animal models in vivo the role of ASIC2 in different neurodegenerative central nervous system pathologies.
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A new methodology based on Nuclear Magnetic Resonance (NMR) was developed to determine plasma protein binding (PPB) of drug candidates in drug discovery programs. A strong correlation was found between the attenuation of NMR signals of diverse drugs in the presence of different plasma concentrations and their fraction bound (fb) reported in the literature. Based on these results, a protocol for a rapid calculation of fb of small molecules was established. The advantage of using plasma instead of purified recombinant proteins and the possibility of pool analysis to increase throughput were also evaluated. This novel methodology proved to be very versatile, cost-effective, fast and suitable for automation. As a plus, it contemporarily provides a quality check and solubility of the compound.
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Proteínas Sanguíneas/química , Descoberta de Drogas/métodos , Ressonância Magnética Nuclear Biomolecular , Preparações Farmacêuticas/sangue , Descoberta de Drogas/instrumentação , Avaliação Pré-Clínica de Medicamentos , Humanos , Técnicas In Vitro , Preparações Farmacêuticas/química , Ligação Proteica , Proteínas Recombinantes/química , Albumina Sérica Humana/química , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Mammalian frataxin is a small mitochondrial protein involved in iron sulfur cluster assembly. Frataxin deficiency causes the neurodegenerative disease Friedreich's Ataxia. Valuable knowledge has been gained on the structural dynamics of frataxin, metal-ion-protein interactions, as well as on the effect of mutations on protein conformation, stability and internal motions. Additionally, laborious studies concerning the enzymatic reactions involved have allowed for understanding the capability of frataxin to modulate Fe-S cluster assembly function. Remarkably, frataxin biological function depends on its interaction with some proteins to form a supercomplex, among them NFS1 desulfurase and ISCU, the scaffolding protein. By combining multiple experimental tools including high resolution techniques like NMR and X-ray, but also SAXS, crosslinking and mass-spectrometry, it was possible to build a reliable model of the structure of the desulfurase supercomplex NFS1/ACP-ISD11/ISCU/frataxin. In this chapter, we explore these issues showing how the scientific view concerning frataxin structure-function relationships has evolved over the last years.
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Proteínas de Ligação ao Ferro/química , Proteínas de Ligação ao Ferro/metabolismo , Ataxia de Friedreich/genética , Humanos , Proteínas de Ligação ao Ferro/genética , Espalhamento a Baixo Ângulo , Relação Estrutura-Atividade , Difração de Raios X , FrataxinaRESUMO
Introduction: Deficiency of growth hormone (GH) in absence of pituitary injuries is one of the causes of short stature and of the non organic failure to thrive (NOFTT) condition. Advances in developmental psychology have highlighted the role of emotions and caregiving behaviors in the organization of child's personality and psychobiology, with the mother-son attachment bond being considered a fundamental developmental experience. The objective of the present preliminary study was to assess whether there are significant correlations between attachment patterns and GH levels in a sample of subjects with NOFTT. Methods: Overall, 27 children (mean age 9.49±2.63 years) with NOFTT were enrolled. Perceived attachment security was assessed through the Security Scale (SS) and its subscales focused on maternal and paternal security. Pearson partial correlation was used to test associations between GH levels and SS measures adjusting for confounding factors (i.e. age, gender and body mass index). Results: Across all subjects, GH was significantly positively correlated with general security (r=0.425; p=0.038) and maternal security (r=0.451; p=0.027) and not significantly correlated with paternal security (r=0.237; p=0.264). Discussion: These findings preliminarily suggest that the association between GH levels and perceived attachment security may play a role in the pathophysiology of NOFTT and add to the accumulating evidence that attachment patterns may be related with specific psychoendocrine underpinnings.
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Insuficiência de Crescimento/sangue , Insuficiência de Crescimento/psicologia , Hormônio do Crescimento Humano/sangue , Apego ao Objeto , Criança , Feminino , Humanos , MasculinoRESUMO
Human frataxin (FXN) is a highly conserved mitochondrial protein involved in iron homeostasis and activation of the iron-sulfur cluster assembly. FXN deficiency causes the neurodegenerative disease Friedreich's Ataxia. Here, we investigated the effect of alterations in loop-1, a stretch presumably essential for FXN function, on the conformational stability and dynamics of the native state. We generated four loop-1 variants, carrying substitutions, insertions and deletions. All of them were stable and well-folded proteins. Fast local motions (ps-ns) and slower long-range conformational dynamics (µs-ms) were altered in some mutants as judged by NMR. Particularly, loop-1 modifications impact on the dynamics of a distant region that includes residues from the ß-sheet, helix α1 and the C-terminal. Remarkably, all the mutants retain the ability to activate cysteine desulfurase, even when two of them exhibit a strong decrease in iron binding, revealing a differential sensitivity of these functional features to loop-1 perturbation. Consequently, we found that even for a small and relatively rigid protein, engineering a loop segment enables to alter conformational dynamics through a long-range effect, preserving the native-state structure and important aspects of function.
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Proteínas de Ligação ao Ferro/química , Simulação de Dinâmica Molecular , Humanos , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Mutação , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , FrataxinaRESUMO
Parasites are often a public health hazard, especially among schoolchildren. The transmission of these parasites is associated with poor hygiene and sanitation, as well as close contact between people. This study aimed to evaluate the occurrence of intestinal parasitic infections in schoolchildren from the rural and urban areas of the Palmeira das Missões county, a major town in northwestern Rio Grande do Sul State. Fecal samples from 209 schoolchildren aged three to fourteen were analyzed. The Hoffman, Pons and Janer, Faust, Ritchie, and Kinyoun's stain for coccidea parasite detection techniques were applied. 59.3% of the patients were positive for pathogenic or commensal parasites. The most common parasites were Ascaris lumbricoides, Entamoeba coli, Giardia duodenalis and Cryptosporidium spp., respectively. There were no significant differences in positivity among schoolchildren from rural and urban areas. Low family income and the mothers' schooling (up to 8 years of education) were statistically significant for the presence of parasites. This is the first parasite study carried out in the Palmeira das Missões county