RESUMO
The most abundant N6-methyladenosine (m6A) modification on mRNAs is installed non-stoichiometrically across transcripts, with 5' untranslated regions (5' UTRs) being the least conductive. 5' UTRs are essential for translation initiation, yet the molecular mechanisms orchestrated by m6A remain poorly understood. Here, we combined structural, biochemical, and single-molecule approaches and show that at the most common position, a single m6A does not affect translation yields, the kinetics of translation initiation complex assembly, or start codon recognition both under permissive growth and following exposure to oxidative stress. Cryoelectron microscopy (cryo-EM) structures of the late preinitiation complex reveal that m6A purine ring established stacking interactions with an arginine side chain of the initiation factor eIF2α, although with only a marginal energy contribution, as estimated computationally. These findings provide molecular insights into m6A interactions with the initiation complex and suggest that the subtle stabilization is unlikely to affect the translation dynamics under homeostatic conditions or stress.
Assuntos
Adenosina/análogos & derivados , Iniciação Traducional da Cadeia Peptídica , Biossíntese de Proteínas , Regiões 5' não Traduzidas , Microscopia Crioeletrônica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Códon de Iniciação/genéticaRESUMO
The conformational changes in a sugar moiety along the hydrolytic pathway are key to understand the mechanism of glycoside hydrolases (GHs) and to design new inhibitors. The two predominant itineraries for mannosidases go via O S2 âB2,5 â1 S5 and 3 S1 â3 H4 â1 C4 . For the CAZy family 92, the conformational itinerary was unknown. Published complexes of Bacteroides thetaiotaomicron GH92 catalyst with a S-glycoside and mannoimidazole indicate a 4 C1 â4 H5 /1 S5 â1 S5 mechanism. However, as observed with the GH125 family, S-glycosides may not act always as good mimics of GH's natural substrate. Here we present a cooperative study between computations and experiments where our results predict the E5 âB2,5 /1 S5 â1 S5 pathway for GH92 enzymes. Furthermore, we demonstrate the Michaelis complex mimicry of a new kind of C-disaccharides, whose biochemical applicability was still a chimera.
Assuntos
Glicosídeos , Manosidases , Glicosídeo Hidrolases/metabolismo , Glicosídeos/química , Manosidases/química , Conformação MolecularRESUMO
MonteCarbo is an open-source software to construct simple 5-, 6-, and 7-membered ring multifunctionalized monosaccharides and nucleobases and dock them into the active site of carbohydrate-active enzymes. The core bash script executes simple orders to generate the Z-matrix of the neutral molecule of interest. After that, a Fortran90 code based on a pseudo-random number generator (Monte Carlo method) is executed to assign dihedral angles to the different rotamers present in the structure (ring and rotating functional groups). The program also has a generalized internal coordinates (GIC) implementation of the Cremer and Pople puckering coordinates ring. Once the structures are generated and optimized, a second code is ready to execute in serial the docking of multiple conformers in the active site of a wide family of enzymes.
Assuntos
Adesinas de Escherichia coli/química , Carboidratos/química , Proteínas de Fímbrias/química , Simulação de Acoplamento Molecular , Monossacarídeos/química , Software , Adesinas de Escherichia coli/metabolismo , Proteínas de Fímbrias/metabolismo , Método de Monte CarloRESUMO
OBJECTIVE: this study aimed to determine the epidemiological, technical and clinical data of transjugular intrahepatic portosystemic shunt (TIPS) performed by Interventional Radiology departments in Spain. Furthermore, the total number of TIPS carried out in Spain was determined and compared with other countries. MATERIAL AND METHODS: a retrospective study was performed with the approval of the Ethical Committee of the Spanish Society of Interventional Radiology (SERVEI). A survey was performed with 31 items (demographic, technical and clinical data) for data acquisition on the current status of TIPS in Spain. The survey was sent to the 49 hospitals that SERVEI included in a previous registry with data of TIPS performed in Spain in 2016. RESULTS: of the 49 centers surveyed, 33 (67.35 %) replied to the survey. These centers had completed 265 of the 415 TIPS that year in Spain. The most frequent indication was upper GI bleeding from gastroesophageal varices, which accounted for 144 (54.33 %); 62.26 % of the TIPS were performed urgently and 37.7 % on a scheduled basis. The technical success was 89.16 ± 20.9 %, with a rebleeding rate of 17.9 %. Sixty-nine patients (26.03 %) presented complications, 19.62 % of them minor and 6.41 % major. The 30-day mortality related to the disease was 14.33 %, while mortality at one year was 18.49 %. CONCLUSION: notably in our study, the complications of TIPS did not show a clear relationship with the number of procedures performed. With regard to other countries like the United States and France, the number of TIPS in Spain per million inhabitants is currently substantially lower. There were no significant changes compared to the number completed in 2013.
Assuntos
Varizes Esofágicas e Gástricas , Derivação Portossistêmica Transjugular Intra-Hepática , Varizes Esofágicas e Gástricas/epidemiologia , Varizes Esofágicas e Gástricas/cirurgia , Hemorragia Gastrointestinal/epidemiologia , Hemorragia Gastrointestinal/etiologia , Hemorragia Gastrointestinal/cirurgia , Humanos , Derivação Portossistêmica Transjugular Intra-Hepática/efeitos adversos , Sistema de Registros , Estudos Retrospectivos , Espanha/epidemiologia , Resultado do TratamentoRESUMO
STUDY OBJECTIVE: To evaluate the incidence of leiomyosarcoma (LMS) at surgery for presumed uterine myomas according to different age groups. DESIGN: A retrospective cohort study. SETTING: A tertiary referral hospital. PATIENTS: All women undergoing surgery for presumed uterine myomas between January 1, 2006, and December 31, 2016. INTERVENTIONS: Laparoscopic myomectomy, laparotomic myomectomy, total hysterectomy, or hysteroscopic myomectomy. MEASUREMENTS AND MAIN RESULTS: A total of 1398 patients underwent surgery for presumed uterine myomas. The incidence of LMS was 2.15 per 1000 surgeries (nâ¯=â¯3, 1/466, 0.2%). In women under 40 years old, the incidence of occult LMS was 0 (0/561). In women between 40 and 49 years old, 190 myomectomies were performed (28% of the surgeries), and the rate of LMS was 1.49 per 1000 (nâ¯=â¯1, 1/673, 0.15%). In women over 49 years old, a total hysterectomy was performed in 82.3% of the cases, and the incidence of LMS was 12.2 per 1000 surgeries (nâ¯=â¯2, 1/82, 1.2%). CONCLUSION: The incidence of occult LMS in patients under 40 years old undergoing surgery for presumed uterine myomas was 0. These findings are suggestive that the use of power morcellation in this population may be safe.
Assuntos
Laparoscopia , Leiomioma , Leiomiossarcoma , Morcelação , Mioma , Miomectomia Uterina , Neoplasias Uterinas , Adulto , Feminino , Humanos , Histerectomia , Incidência , Leiomioma/epidemiologia , Leiomioma/cirurgia , Leiomiossarcoma/epidemiologia , Leiomiossarcoma/cirurgia , Pessoa de Meia-Idade , Morcelação/efeitos adversos , Mioma/cirurgia , Estudos Retrospectivos , Neoplasias Uterinas/epidemiologia , Neoplasias Uterinas/cirurgiaRESUMO
Conformational analysis of enzyme-catalyzed mannoside hydrolysis has revealed two predominant conformational itineraries through B2,5 or 3H4 transition-state (TS) conformations. A prominent unassigned catalytic itinerary is that of exo-1,6-α-mannosidases belonging to CAZy family 125. A published complex of Clostridium perfringens GH125 enzyme with a nonhydrolyzable 1,6-α-thiomannoside substrate mimic bound across the active site revealed an undistorted 4C1 conformation and provided no insight into the catalytic pathway of this enzyme. We show through a purely computational approach (QM/MM metadynamics) that sulfur-for-oxygen substitution in the glycosidic linkage fundamentally alters the energetically accessible conformational space of a thiomannoside when bound within the GH125 active site. Modeling of the conformational free energy landscape (FEL) of a thioglycoside strongly favors a mechanistically uninformative 4C1 conformation within the GH125 enzyme active site, but the FEL of corresponding O-glycoside substrate reveals a preference for a Michaelis complex in an OS2 conformation (consistent with catalysis through a B2,5 TS). This prediction was tested experimentally by determination of the 3D X-ray structure of the pseudo-Michaelis complex of an inactive (D220N) variant of C. perfringens GH125 enzyme in complex with 1,6-α-mannobiose. This complex revealed unambiguous distortion of the -1 subsite mannoside to an OS2 conformation, matching that predicted by theory and supporting an OS2 â B2,5 â 1S5 conformational itinerary for GH125 α-mannosidases. This work highlights the power of the QM/MM approach and identified shortcomings in the use of nonhydrolyzable substrate analogues for conformational analysis of enzyme-bound species.
Assuntos
Manose/química , Simulação de Dinâmica Molecular , Teoria Quântica , alfa-Manosidase/química , Clostridium perfringens/enzimologia , Manose/análogos & derivados , Manose/metabolismo , Estrutura Molecular , Conformação Proteica , alfa-Manosidase/metabolismoRESUMO
Previous studies have shown the reproducibility of the 2008 World Health Organization (WHO) classification in myelodysplastic syndromes (MDS), especially when multilineage dysplasia or excess of blasts are present. However, there are few data regarding the reproducibility of MDS with unilineage dysplasia. The revised International Prognostic Scoring System R-IPSS described two new morphological categories, distinguishing bone marrow (BM) blast cell count between 0-2 % and >2- < 5 %. This distinction is critical for establishing prognosis, but the reproducibility of this threshold is still not demonstrated. The objectives of our study were to explore the reliability of the 2008 WHO classification, regarding unilineage vs. multilineage dysplasia, by reviewing 110 cases previously diagnosed with MDS, and to study whether the threshold of ≤2 % BM blasts is reproducible among different observers. We used the same methodology as in our previous paper [Font et al. (2013) Ann Hematol 92:19-24], by encouraging investigators to include patients with <5 % BM blasts. Samples were collected from 11 hospitals and were evaluated by 11 morphologists. Each observer evaluated 20 samples, and each sample was analyzed independently by two morphologists. Discordance was observed in 36/108 suitable cases (33 %, kappa test 0.503). Diagnosis of MDS with unilineage dysplasia (refractory cytopenia with unilineage dysplasia (RCUD), refractory anemia with ring sideroblasts (RARS) or unclassifiable MDS) was assessed in 33 patients, by either of the two observers. We combined this series with the cases with RCUD or RARS included in our 2013 paper, thus obtaining 50 cases with unilineage dysplasia by at least one of the observers. The whole series showed very low agreement regarding RCUD (5/23, 21 %) and RARS (5/28, 18 %). Regarding BM blast count, the threshold of ≤2 % was not reproducible (discordance rate 32/108 cases, kappa test 0.277). Our study shows that among MDS WHO 2008 categories, interobserver discordance seems to be high in cases with unilineage dysplasia. We also illustrate that the threshold of ≤2 % BM blasts as settled by the R-IPSS may be not easy to reproduce by morphologists in real practice.
Assuntos
Crise Blástica/patologia , Medula Óssea/patologia , Síndromes Mielodisplásicas/diagnóstico , Síndromes Mielodisplásicas/patologia , Contagem de Células/estatística & dados numéricos , Linhagem da Célula , Citodiagnóstico/estatística & dados numéricos , Feminino , Humanos , Masculino , Variações Dependentes do Observador , Prognóstico , Reprodutibilidade dos TestesRESUMO
BACKGROUND & AIMS: Two new classification systems for the severity of acute pancreatitis (AP) have been proposed, the determinant-based classification (DBC) and a revision of the Atlanta classification (RAC). Our aim was to validate and compare these classification systems. METHODS: We analyzed data from adult patients with AP (543 episodes of AP in 459 patients) who were admitted to Hospital General Universitario de Alicante from December 2007 to February 2013. Imaging results were reviewed, and the classification systems were validated and compared in terms of outcomes. RESULTS: Pancreatic necrosis was present in 66 of the patients (12%), peripancreatic necrosis in 109 (20%), walled-off necrosis in 61 (11%), acute peripancreatic fluid collections in 98 (18%), and pseudocysts in 19 (4%). Transient and persistent organ failures were present in 31 patients (6%) and 21 patients (4%), respectively. Sixteen patients (3%) died. On the basis of the DBC, 386 (71%), 131 (24%), 23 (4%), and 3 (0.6%) patients were determined to have mild, moderate, severe, or critical AP, respectively. On the basis of the RAC, 363 patients (67%), 160 patients (30%), and 20 patients (4%) were determined to have mild, moderately severe, or severe AP, respectively. The different categories of severity for each classification system were associated with statistically significant and clinically relevant differences in length of hospital stay, need for admission to the intensive care unit, nutritional support, invasive treatment, and in-hospital mortality. In comparing similar categories between the classification systems, no significant differences were found. CONCLUSION: The DBC and the RAC accurately classify the severity of AP in subgroups of patients.
Assuntos
Pancreatite/classificação , Índice de Gravidade de Doença , Doença Aguda , Idoso , Feminino , Humanos , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Necrose , Apoio Nutricional , Pâncreas/patologiaRESUMO
Glycoside hydrolase family 31 (GH31) is a diversified family of anomer-retaining α-glycoside hydrolases, such as α-glucosidase and α-xylosidase, among others. Recently, GH31 α-N-acetylgalactosaminidases (Nag31s) have been identified to hydrolyze the core of mucin-type O-glycans and the crystal structure of a gut bacterium Enterococcus faecalis Nag31 has been reported. However, the mechanisms of substrate specificity and hydrolysis of Nag31s are not well investigated. Herein, we show that E. faecalis Nag31 has the ability to release N-acetylgalactosamine (GalNAc) from O-glycoproteins, such as fetuin and mucin, but has low activity against Tn antigen. Mutational analysis and crystal structures of the Michaelis complexes reveal that residues of the active site work in concert with their conformational changes to act on only α-N-acetylgalactosaminides. Docking simulations using GalNAc-attached peptides suggest that the enzyme mainly recognizes GalNAc and side chains of Ser/Thr, but not strictly other peptide residues. Moreover, quantum mechanics calculations indicate that the enzyme preferred p-nitrophenyl α-N-acetylgalactosaminide to Tn antigen and that the hydrolysis progresses through a conformational itinerary, 4C1 â 1S3 â 4C1, in GalNAc of substrates. Our results provide novel insights into the diversification of the sugar recognition and hydrolytic mechanisms of GH31 enzymes.
Assuntos
Glicosídeo Hidrolases , Domínio Catalítico , Glicosídeo Hidrolases/química , Hidrólise , Especificidade por Substrato , alfa-N-Acetilgalactosaminidase/química , alfa-N-Acetilgalactosaminidase/metabolismoRESUMO
Covalent modifications of standard DNA/RNA nucleobases affect epigenetic regulation of gene expression by modulating interactions between nucleic acids and protein readers. We derive here the absolute binding free energies and analyze the binding modalities between key modified nucleobases 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and N6-methyladenine (m6A) and all non-prolyl/non-glycyl protein side chains using molecular dynamics simulations and umbrella sampling in both water and methanol, the latter mimicking the low dielectric environment at the dehydrated nucleic-acid/protein interfaces. We verify the derived affinities by comparing against a comprehensive set of high-resolution structures of nucleic-protein complexes involving 5mC. Our analysis identifies protein side chains that are highly tuned for detecting cytosine methylation as a function of the environment and can thus serve as microscopic readers of epigenetic marks. Conversely, we show that the relative ordering of sidechain affinities for 5hmC and m6A does not differ significantly from those for their precursor bases, cytosine and adenine, respectively, especially in the low dielectric environment. For those two modified bases, the effect is more nuanced and manifests itself primarily at the level of absolute changes in the binding free energy. Our results contribute towards establishing a quantitative foundation for understanding, predicting and modulating the interactions between modified nucleic acids and proteins at the atomistic level.
Assuntos
5-Metilcitosina , Epigênese Genética , 5-Metilcitosina/metabolismo , Citosina/metabolismo , Proteínas/metabolismo , Metilação de DNA , DNA/químicaRESUMO
Despite the large body of research on sex differences in pain, there is a lack of translation to real-world pain management. Our aim was to analyse the sex differences in the analgesic response to oxycodone/naloxone (OXN) and tapentadol (TAP), in comparison with other opioids (OPO) commonly prescribed for chronic non-cancer pain (CNCP). An observational and cross-sectional study was conducted on ambulatory CNCP patients (n = 571). Sociodemographic, clinical (pain intensity, relief, and quality of life), safety (adverse events (AEs), adverse drug reactions), hospital frequentations and pharmacological (morphine equivalent daily dose (MEDD)) variables were collected. Multiple linear regressions were carried out to assess the association between sex and outcomes. Sex differences were observed, with lower female tolerability and higher hospital frequentation, especially in the OXN group (OR AEs report = 2.8 [1.8−4.4], p < 0.001). Here, females showed higher hospital use (23% hospital admission, 30% prescription change, p < 0.05), requiring a higher MEDD (127 ± 103 mg/day, p < 0.05), compared to OXN men. Regardless of the opioid group, CNCP women were significantly older than men (three years), with significantly higher benzodiazepine use (OR = 1.6 [1.1−2.3]), more constipation (OR = 1.34 [0.93−1.90]) and headache (OR = 1.45 [0.99−2.13]) AEs, than men who were more likely to refer sexual dysfunction (OR = 2.77 [1.53−5.01]), and loss of libido (OR = 1.93 [1.22−3.04]). Sex-differences were found related to poorer female drug tolerability and higher hospital resources, even worst in OXN female users. Other differences related to older female ages and benzodiazepine prescription, need to be further analysed from a gender perspective.
RESUMO
In the barley ß-D-glucan glucohydrolase, a glycoside hydrolase family 3 (GH3) enzyme, the Trp286/Trp434 clamp ensures ß-D-glucosides binding, which is fundamental for substrate hydrolysis during plant growth and development. We employ mutagenesis, high-resolution X-ray crystallography, and multi-scale molecular modelling methods to examine the binding and conformational behaviour of isomeric ß-D-glucosides during substrate-product assisted processive catalysis that operates in GH3 hydrolases. Enzyme kinetics reveals that the W434H mutant retains broad specificity, while W434A behaves as a strict (1,3)-ß-D-glucosidase. Investigations of reactant movements on the nanoscale reveal that processivity is sensitive to mutation-specific alterations of the tryptophan clamp. While wild-type and W434H utilise a lateral cavity for glucose displacement and sliding of (1,3)-linked hydrolytic products through the catalytic site without dissociation, consistent with their high hydrolytic rates, W434A does not adopt processive catalysis. Phylogenomic analyses of GH3 hydrolases disclose the evolutionary advantage of the tryptophan clamp that confers broad specificity, high catalytic efficiency, and processivity.
Assuntos
Glicosídeo Hidrolases , Triptofano , Cristalografia por Raios X , Glucose , Glucosidases/química , Glucosídeos , Glicosídeo Hidrolases/metabolismo , Glicosídeos , Cinética , Plantas/metabolismo , Especificidade por SubstratoRESUMO
Tapentadol (TAP) and oxycodone/naloxone (OXN) potentially offer an improved opioid tolerability. However, real-world studies in chronic non-cancer pain (CNCP) remain scarce. Our aim was to compare effectiveness and security in daily pain practice, together with the influence of pharmacogenetic markers. An observational study was developed with ambulatory test cases under TAP (n = 194) or OXN (n = 175) prescription with controls (prescribed with other opioids (control), n = 216) CNCP patients. Pain intensity and relief, quality of life, morphine equivalent daily doses (MEDD), concomitant analgesic drugs, adverse events (AEs), hospital frequentation and genetic variants of OPRM1 (rs1799971, A118G) and COMT (rs4680, G472A) genes, were analysed. Test CNCP cases evidenced a significantly higher pain relief predictable due to pain intensity and quality of life (R2 = 0.3), in front of controls. Here, OXN achieved the greatest pain relief under a 28% higher MEDD, 8-13% higher use of pregabalin and duloxetine, and 23% more prescription change due to pain, compared to TAP. Whilst, TAP yielded a better tolerability due the lower number of 4 [0-6] AEs/patient, in front of OXN. Furthermore, OXN COMT-AA homozygotes evidenced higher rates of erythema and vomiting, especially in females. CNCP real-world patients achieved higher pain relief than other traditional opioids with a better tolerability for TAP. Further research is necessary to clarify the potential influence of COMT and sex on OXN side-effects.
Assuntos
Dor do Câncer , Dor Crônica , Analgésicos Opioides/efeitos adversos , Dor do Câncer/tratamento farmacológico , Dor do Câncer/genética , Dor Crônica/induzido quimicamente , Dor Crônica/tratamento farmacológico , Dor Crônica/genética , Constipação Intestinal/tratamento farmacológico , Preparações de Ação Retardada , Combinação de Medicamentos , Feminino , Humanos , Morfina/efeitos adversos , Naloxona/efeitos adversos , Oxicodona/efeitos adversos , Testes Farmacogenômicos , Qualidade de Vida , TapentadolRESUMO
During menopausal transition, mild clinical signs of hyperandrogenism may appear as part of the normal aging process, but the development of frank virilization suggests a specific source of androgen excess. In this context, androgen-secreting tumors at both adrenal and ovarian levels should be ruled out. We present the case of a 51-year-old postmenopausal woman with signs of 12 month period virilization, associated with personal history of type 2 diabetes and arterial hypertension, poorly managed in the past year. Laboratory tests showed elevation of serum androgen levels and hyperinsulinemia. Images were requested, revealing both enlarged homogeneous and solid ovaries, with preserved adrenal glands, which led to suspicion of a possible thecal hyperplasia of the ovarian stroma. Laparoscopic bilateral adnexectomy was performed and the pathological report confirmed the presumptive diagnosis. One month later after surgery, serum testosterone levels returned to values ââclose to spected for a postmenopausal woman. Finding the source of virilization in postmenopausal women is challenging, and they are usually associated with rare pathologies. A detailed medical history is essential to differentiate the progressive development of virilization that characterizes benign causes from the rapid progression that characterizes malignant tumors. The adequate interpretation of laboratory tests with complementary images, as well as looking for the association of pathologies causing elevated cardiovascular risk such as diabetes and hypertension are essential to establish a right diagnosis and treatment.
Durante la transición menopáusica pueden aparecer signos clínicos leves de hiperandrogenismo, como parte del proceso de envejecimiento normal, pero el desarrollo de virilización franca sugiere una fuente específica de exceso de andrógenos debiendo descartar la presencia de tumores secretores de andrógenos tanto a nivel adrenal como ovárico. Se presenta un caso de una mujer de 51 años postmenopáusica con signos de virilización de 12 meses de evolución, asociado a antecedente personal de diabetes tipo 2 e hipertensión arterial, de mal manejo en el último año. Las pruebas de laboratorio mostraron una franca elevación de los niveles de andrógeno sérico e hiperinsulinemia asociada. Las imágenes solicitadas evidenciaron ambos ovarios aumentados de tamaño de aspecto homogéneo y sólido, con glándulas adrenales de aspecto conservado, lo que hizo sospechar de una posible hiperplasia tecal del estroma ovárico. Se realizó una anexectomía bilateral por laparoscopia, cuya anatomía patológica confirmó la presunción diagnóstica. Los dosajes de testosterona sérica al mes de la cirugía retornaron a valores cercanos a la normalidad para una mujer postmenopáusica. El diagnóstico causal de virilización en mujeres posmenopáusicas es un desafío, y por lo general están asociadas con patologías poco frecuentes. Una historia clínica detallada es fundamental para diferenciar el desarrollo progresivo de virilización que caracteriza las causas benignas de la rápida progresión que caracteriza a los tumores malignos. La interpretación de pruebas correctas de laboratorio con imágenes complementarias, así como la búsqueda de antecedentes de riesgo cardiovascular como la diabetes y la hipertensión asociadas son fundamentales para establecer un correcto diagnóstico y tratamiento.
Assuntos
Doenças Metabólicas , Pós-Menopausa , Feminino , Humanos , Hiperplasia , Estudos Retrospectivos , VirilismoRESUMO
By employing the computational protocol for calculation of reduction potentials of the Fe4 S4 -containing species validated using a representative series of well-defined synthetic complexes, we focused on redox properties of two prototypical radical SAM enzymes to reveal how they transform SAM into the reactive 5'-deoxyadenosyl radical, and how they tune this radical for its proper biological function. We found the reduction potential of SAM is indeed elevated by 0.3-0.4â V upon coordination to Fe4 S4 , which was previously speculated in the literature. This makes a generation of 5'-deoxyadenosyl radical from SAM less endergonic (by ca. 7-9â kcal mol-1 ) and hence more feasible in both enzymes as compared to the identical process in water. Furthermore, our calculations indicate that the enzyme-bound 5'-deoxyadenosyl radical has a significantly lower reduction potential than in referential aqueous solution, which may help the enzymes to suppress potential side redox reactions and simultaneously elevate its proton-philic character, which may, in turn, promote the radical hydrogen-atom abstraction ability.
Assuntos
Proteínas Ferro-Enxofre/metabolismo , S-Adenosilmetionina/metabolismo , Radicais Livres/química , Radicais Livres/metabolismo , Proteínas Ferro-Enxofre/química , Oxirredução , S-Adenosilmetionina/químicaRESUMO
BACKGROUND AIMS: Cord blood (CB) transplants with co-infusion of third-party donor (TPD) mobilized hematopoietic stem cells (MHSC) have been shown to result in 'bridge' engraftment with prompt neutrophil recovery and high final rates of CB engraftment and full chimerism. This strategy overcomes the limitation posed by low cellularity of CB units for unrelated transplants in adults. Enhancement of adaptive immunity reconstitution without increasing risks of graft-versus-host disease (GvHD) is required to optimize results further. Our objectives were to evaluate co-infusion of mesenchymal stromal cells (MSC) from the same TPD regarding tolerance, CB engraftment and effects on acute (a)GvHD, both preventive and therapeutic. METHODS: Ex vivo-expanded bone marrow MSC were infused at the time of the transplant or the in case of refractory aGvHD. RESULTS: Nine patients received 1.04 - 2.15 x 10(6)/kg (median 1.20) MSC immediately after CB and TPD MHSC. Neither immediate adverse side-effects nor significant differences regarding CB engraftment or aGvHD development were observed. Four patients developed grade II aGvHD, refractory to steroids in two. These reached complete remission after therapeutic infusions of MSC. CONCLUSIONS: In recipients of 'dual CB/TPD MHSC transplants', MSC infusions were therapeutically effective for severe aGvHD but no significant differences in CB engraftment and incidence of severe aGvHD were observed following their prophylactic use. Although results of this study alone cannot conclusively determine the application of MSC in CB transplantation, we believe that, in this setting, the best use of MSC could be as pre-emptive treatment for aGvHD.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Neoplasias Hematológicas/terapia , Células-Tronco Mesenquimais/metabolismo , Indução de Remissão , Células Estromais/metabolismo , Doença Aguda , Adulto , Medula Óssea/metabolismo , Contagem de Células , Quimerismo , Feminino , Sangue Fetal/citologia , Doença Enxerto-Hospedeiro/patologia , Doença Enxerto-Hospedeiro/prevenção & controle , Neoplasias Hematológicas/imunologia , Mobilização de Células-Tronco Hematopoéticas , Transplante de Células-Tronco Hematopoéticas , Humanos , Masculino , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/imunologia , Pessoa de Meia-Idade , Neutrófilos/imunologia , Neutrófilos/patologia , Projetos Piloto , Células Estromais/citologia , Células Estromais/imunologia , Doadores de Tecidos , Transplante HomólogoRESUMO
Random networks of coupled phase oscillators with phase shifts in the interaction functions are considered. In such systems, extensive chaos (turbulence) is observed in a wide range of parameters. We show that, by introducing global feedback, the turbulence can be suppressed and a transition to synchronous oscillations can be induced. Our attention is focused on the transition scenario and the properties of patterns, including intermittent turbulence, which are found at the edge of chaos. The emerging coherent patterns represent various self-organized active (sub)networks whose size and behavior can be controlled.
RESUMO
By means of QM(DFT)/MM metadynamics we have unraveled the hydrolytic reaction mechanism of Neisseria polysaccharea amylosucrase (NpAS), a member of GH13 family. Our results provide an atomistic picture of the active site reorganization along the catalytic double-displacement reaction, clarifying whether the glycosyl-enzyme reaction intermediate features an α-glucosyl unit in an undistorted 4 C 1 conformation, as inferred from structural studies, or a distorted 1 S 3-like conformation, as expected from mechanistic analysis of glycoside hydrolases (GHs). We show that, even though the first step of the reaction (glycosylation) results in a 4 C 1 conformation, the α-glucosyl unit undergoes an easy conformational change toward a distorted conformation as the active site preorganizes for the forthcoming reaction step (deglycosylation), in which an acceptor molecule, i.e., a water molecule for the hydrolytic reaction, performs a nucleophilic attack on the anomeric carbon. The two conformations (4 C 1 ad E 3) can be viewed as two different states of the glycosyl-enzyme intermediate (GEI), but only the E 3 state is preactivated for catalysis. These results are consistent with the general conformational itinerary observed for α-glucosidases.
RESUMO
This work presents experimental and computational studies on ZnO formation after decomposition of a sol-gel precursor containing ethanolamine and Zn(II) acetate. The structural modifications suffered during decomposition of the monomeric and dimeric Zn(II) complexes formed, containing bidentate deprotonated ethanolamine and acetato ligands, have been described experimentally and explained via Car-Parrinello Molecular Dynamics. Additional metadynamics simulations provide an overview of the dimer evolution by the cleavage of the Zn-N bond, the structural changes produced and their effects on the Zn(II) environment. The results provide conclusive evidence of the relevance of ethanolamine used as a stabilizer in the formation of ZnO.
RESUMO
BACKGROUND: Trypanosoma cruzi, the agent of Chagas disease, is a protozoan parasite transmitted to humans by blood-sucking triatomine vectors. However, and despite its utmost biological and epidemiological relevance, T. cruzi development inside the digestive tract of the insect remains a poorly understood process. METHODS/PRINCIPLE FINDINGS: Here we showed that Gp35/50 kDa mucins, the major surface glycoproteins from T. cruzi insect-dwelling forms, are involved in parasite attachment to the internal cuticle of the triatomine rectal ampoule, a critical step leading to its differentiation into mammal-infective forms. Experimental evidence supporting this conclusion could be summarized as follows: i) native and recombinant Gp35/50 kDa mucins directly interacted with hindgut tissues from Triatoma infestans, as assessed by indirect immunofluorescence assays; ii) transgenic epimastigotes over-expressing Gp35/50 kDa mucins on their surface coat exhibited improved attachment rates (~2-3 fold) to such tissues as compared to appropriate transgenic controls and/or wild-type counterparts; and iii) certain chemically synthesized compounds derived from Gp35/50 kDa mucins were able to specifically interfere with epimastigote attachment to the inner lining of T. infestans rectal ampoules in ex vivo binding assays, most likely by competing with or directly blocking insect receptor(s). A solvent-exposed peptide (smugS peptide) from the Gp35/50 kDa mucins protein scaffolds and a branched, Galf-containing trisaccharide (Galfß1-4[Galpß1-6]GlcNAcα) from their O-linked glycans were identified as main adhesion determinants for these molecules. Interestingly, exogenous addition of a synthetic Galfß1-4[Galpß1-6]GlcNAcα derivative or of oligosaccharides containing this structure impaired the attachment of Dm28c but not of CL Brener epimastigotes to triatomine hindgut tissues; which correlates with the presence of Galf residues on the Gp35/50 kDa mucins' O-glycans on the former but not the latter parasite clone. CONCLUSION/SIGNIFICANCE: These results provide novel insights into the mechanisms underlying T. cruzi-triatomine interplay, and indicate that inter-strain variations in the O-glycosylation of Gp35/50 kDa mucins may lead to differences in parasite differentiation and hence, in parasite transmissibility to the mammalian host. Most importantly, our findings point to Gp35/50 kDa mucins and/or the Galf biosynthetic pathway, which is absent in mammals and insects, as appealing targets for the development of T. cruzi transmission-blocking strategies.