Correlated TKD/EDS - TEM - APT analysis on selected interfaces of CoSi2 thin films.

Correlative analysis is a powerful way to relate crystallographic and chemical information to the properties of materials. In this work, a procedure is proposed to select and analyze interfaces of polycrystalline thin film materials through correlative transmission Kikuchi diffraction/energy dispersive X-ray spectroscopy (TKD/EDS), transmission electron microscopy (TEM) and atom probe tomography (APT). TKD provides information on the crystallographic orientation. The EDS analysis performed together with TKD in the scanning electron microscope (SEM) makes chemical information available allowing phases of similar crystal structure, but with a different composition to be distinguished. The information of TKD/EDS can be correlated to successive TEM and APT analysis on selected interfaces for structural and chemical analysis at the atomic scale. An interface of an epitaxial orientated grain of a polycrystalline CoSi2 thin film on (111)Si is selected and analyzed. The selected interface has a twin character and shows facets of different orientation and area. Site-specific segregation of Ge to junctions of the facets is evidenced. The correlation between local strain from misfit (defects) at the interface and segregation is discussed.

Micro-arc oxidation as a tool to develop multifunctional calcium-rich surfaces for dental implant applications.

Titanium (Ti) is commonly used in dental implant applications. Surface modification strategies are being followed in last years in order to build Ti oxide-based surfaces that can fulfill, simultaneously, the following requirements: induced cell attachment and adhesion, while providing a superior corrosion and tribocorrosion performance. In this work micro-arc oxidation (MAO) was used as a tool for the growth of a nanostructured bioactive titanium oxide layer aimed to enhance cell attachment and adhesion for dental implant applications. Characterization of the surfaces was performed, in terms of morphology, topography, chemical composition and crystalline structure. Primary human osteoblast adhesion on the developed surfaces was investigated in detail by electronic and atomic force microscopy as well as immunocytochemistry. Also an investigation on the early cytokine production was performed. Results show that a relatively thick hybrid and graded oxide layer was produced on the Ti surface, being constituted by a mixture of anatase, rutile and amorphous phases where calcium (Ca) and phosphorous (P) were incorporated. An outermost nanometric-thick amorphous oxide layer rich in Ca was present in the film. This amorphous layer, rich in Ca, improved fibroblast viability and metabolic activity as well as osteoblast adhesion. High-resolution techniques allowed to understand that osteoblasts adhered less in the crystalline-rich regions while they preferentially adhere and spread over in the Ca-rich amorphous oxide layer. Also, these surfaces induce higher amounts of IFN-γ cytokine secretion, which is known to regulate inflammatory responses, bone microarchitecture as well as cytoskeleton reorganization and cellular spreading. These surfaces are promising in the context of dental implants, since they might lead to faster osseointegration.

Ultrastructural and mineral phase characterization of the bone-like matrix assembled in F-OST osteoblast cultures.

Cell cultures are often used to study bone mineralization; however, not all systems achieve a bone-like matrix formation. In this study, the mineralized matrix assembled in F-OST osteoblast cultures was analyzed, with the aim of establishing a novel model for bone mineralization. The ultrastructure of the cultures was investigated using scanning electron microscopy, atomic force microscopy, and transmission electron microscopy (TEM). The mineral phase was characterized using conventional and high-resolution TEM, energy-dispersive X-ray spectroscopy, X-ray diffraction, Fourier transform infrared spectroscopy, and solid-state (31)P and (1)H nuclear magnetic resonance. F-OST osteoblast cultures presented a clear nodular mineralization pattern. The chief features of the mineralizing nodules were globular accretions ranging from about 100 nm to 1.5 µm in diameter, loaded with needle-shaped crystallites. Accretions seemed to bud from the cell membrane, increase in size, and coalesce into larger ones. Arrays of loosely packed, randomly oriented collagen fibrils were seen along with the accretions. Mineralized fibrils were often observed, sometimes in close association with accretions. The mineral phase was characterized as a poorly crystalline hydroxyapatite. The Ca/P atomic ratio was 1.49 ± 0.06. The presence of OH was evident. The lattice parameters were a = 9.435 Å and c = 6.860 Å. The average crystallite size was 20 nm long and 10 nm wide. Carbonate substitutions were seen in phosphate and OH sites. Water was also found within the apatitic core. In conclusion, F-OST osteoblast cultures produce a bone-like matrix and may provide a good model for bone mineralization studies.

Congenital hypothyroidism: etiology.

UNLABELLED: The etiology of congenital hypothyroidism (CH) is important in determining its severity, prognosis, genetic counseling and clinical management. AIMS: investigate the causes of CH and their severity using serum levels of FreeT4 and TSH. PATIENTS AND METHODS: 243 neonates with CH (61% were girls) diagnosed by the Neonatal Screening Program of Minas Gerais between 1996 and 2003. The thyroid function was assessed through serum FreeT4 and TSH by chemilumiscence. CH etiology was evaluated by ultrasonography, scintigraphy, potassium perchlorate discharge test and serum thyroglobulin levels. RESULTS: Out of 243 patients, dysgenesis was found in 114 (47%): 3.3% had athyreosis; 0.4% eutopic dysgenetic gland due to maternal use of 131I; 22% ectopic glands (8.6% an isolated ectopic gland and 13% also an eutopic dysgenetic thyroid); 9% eutopic dysgenesis, 8.6% hypoplasia and 3.7% hemiagenesis. Thyroid in situ was found in 129 (52%): 23.5% had iodide organification defect; 3.7% thyroglobulin synthesis defect; 6.2% other 0.4% dyshomonogenesis; iodide transport defect; 1.2% transient CH and 18% a normal gland. Patients with dysgenesis had a more severe CH than those with thyroid in situ (TSH 248.08 vs. 18.17 microIU/mL and FT4 0.32 vs. 0.95 ng/dL, p < 0.001). CONCLUSIONS: Some cases had more complex dysgenesis, presenting ectopia associated to a dysgenetic eutopic gland. The ultrasound was the best tool to detect the dysgenetic tissue, but the scintigraphy was the most effective in identifying the functioning tissue. The thyroid hormone synthesis defects were found more frequently than expected, but in some cases they could not be defined.

Não-preferência para oviposição e alimentação de Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae) por cultivares de amendoim / Non-oviposition and non-feeding preference of Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae) for peanut cultivars

Este trabalho buscou selecionar cultivares de amendoim resistentes a Spodoptera frugiperda, dos tipos não-preferência para oviposição e alimentação, em testes com e sem chance de escolha. Utilizaram-se cultivares de amendoim de hábitos de crescimento ereto (IAC 5, IAC 8112, IAC 22 e IAC Tatu ST) e rasteiro (IAC 503, IAC 505, IAC 147, IAC 125, IAC Caiapó e IAC Runner 886). Os testes de não-preferência para oviposição foram realizados com adultos recém-emergidos, os quais foram alimentados com solução de mel a 10%, permanecendo nas gaiolas por quatro dias até o início das avaliações. Estas consistiram na quantificação do número de ovos e posturas, com posterior cálculo do número de ovos por postura. Para os testes de não-preferência para alimentação foram colocados discos foliares das cultivares em placas de Petri, liberando uma lagarta de terceiro ínstar por cultivar. Avaliou-se a atratividade a 1, 5, 10, 15, 30, 60, 120, 360, 720; 1.440 minutos, contando-se o número de lagartas que se alimentavam nas cultivares e, ao término do experimento, quantificou-se a área foliar consumida por cultivar. Nos testes de não-preferência para oviposição ou alimentação, com ou sem chance, nenhuma cultivar de hábitos de crescimento ereto ou rasteiro influenciou a oviposição ou alimentação de S. frugiperda. Dessa maneira, as cultivares de amendoim não apresentaram resistência dos tipos não-preferência para oviposição e alimentação.

This study aimed to select peanut cultivars resistant to Spodoptera frugiperda, selecting non-oviposition and non-feeding preference in choice and no-choice tests. Peanut cultivars with upright growth habit (IAC 5, IAC 8112, IAC 22 and IAC Tatu ST) and runner growth habit (IAC 503, IAC 505, IAC 147, IAC 125, IAC Caiapó and IAC Runner 886) were evaluated. Tests of non-oviposition preference were conducted with newly emerged adults. Adults were fed a 10% honey solution, remaining in cages for 4 days until the start of the evaluations. The evaluations consisted of quantifying the number of eggs and egg masses, followed by calculating the number of eggs per egg mass. To perform non-feeding preference tests, leaf discs from different peanut cultivars were placed in disposable Petri dishes followed by releasing of a third-instar caterpillar for each cultivar. The attractiveness at 1, 5; 10, 15, 30, 60, 120, 360, 720 and 1,440 minutes was evaluated by counting the caterpillars that were feeding on cultivars. The leaf area consumed was evaluated at the end of the experiment for each cultivar. In the tests of non-oviposition and non-feeding preference, with choice or no-choice, none of the cultivars of the upright growth and runner growth habit influenced the oviposition or food of S. frugiperda. Thus, the peanut cultivars showed no resistance of the non-feeding and non-oviposition types.

Investigation of aerobic and anaerobic ammonium-oxidising bacteria presence in a small full-scale wastewater treatment system comprised by UASB reactor and three polishing ponds.

This work applied PCR amplification method and Fluorescence in situ hybridisation (FISH) with primers and probes specific for the anammox organisms and aerobic ammonia-oxidising beta-Proteobacteria in order to detect these groups in different samples from a wastewater treatment system comprised by UASB reactor and three polishing (maturation) ponds in series. Seven primer pairs were used in order to detect Anammox bacteria. Positive results were obtained with three of them, suggesting that Anammox could be present in polishing pond sediments. However, Anammox bacteria were not detected by FISH, indicating that they were not present in sediment samples, or they could be present but below FISH detection limit. Aerobic ammonia- and nitrite-oxidising bacteria were verified in water column samples through Most Probable Number (MPN) analysis, but they were not detected in sediment samples by FISH. Ammonia removal efficiencies occurred systematically along the ponds (24, 32, and 34% for polishing pond 1, 2, and 3, respectively) but the major reaction responsible for this removal is still unclear. Some nitrification might have occurred in water samples because some nitrifying bacteria were present. Also Anammox reaction might have occurred because Anammox genes were detected in the sediments, but probably this reaction was too low to be noticed. It is important also to consider that some of the ammonia removal observed might be related to NH(3) stripping, associated with the pH increase resulting from the intensive photosynthetic activity in the ponds (mechanism under investigation). Therefore, it can be concluded that more than one mechanism (or reaction) might be involved in the ammonia removal in the polishing ponds investigated in this study.

Galactosemia: una enfermedad neurometabólica / Galactosemia: a neurometabolic disease

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Clinical features and long-term outcome of obesity-associated focal segmental glomerulosclerosis.

BACKGROUND: Several cases of obesity-associated focal segmental glomerulosclerosis (OB-FSG) have been reported but little is known about the clinico-pathological features of this entity and its long-term outcomes. METHODS: We studied 15 obese patients (BMI 35+/-5.2 kg/m(2)) with biopsy-proven FSG. They were compared with a control group of 15 non-obese patients with idiopathic FSG (I-FSG). RESULTS: Mean proteinuria at the time of renal biopsy was 3.1+/-2 g/24 h in OB-FSG; it reached the nephrotic range (> or =3.5 g/24 h) during follow-up in 12 patients (80%), but none of them had oedema, hypoproteinaemia, or hypoalbuminaemia. Proteinuria was more marked amongst I-FSG (6.5+/-4.2 g/24 h) and most of them developed oedema and biochemical nephrotic syndrome. Glomerulomegaly was observed in all renal biopsies from OB-FSG patients (mean glomerular diameter 256+/-24 microm in OB-FSG vs 199+/-26 microm in I-FSG, P<0.001). Twelve OB-FSG patients (80%) were treated with ACE inhibitors (ACEI) and proteinuria significantly decreased within the first 6 months of treatment but showed a later increase. None of the obese patients achieved a sustained weight loss. Seven (46%) patients with OB-FSG experienced a progressive renal insufficiency and five of them started intermittent dialysis. Kaplan-Meier estimated probabilities of renal survival after 5 and 10 years were 77 and 51%, respectively, in OB-FSG patients, and 52 and 30% in I-FSG (P<0.05). The risk of developing progressive renal failure among OB-FSG patients was statistically correlated with serum creatinine and creatinine clearance at presentation. CONCLUSIONS: OB-FSG indicates a poor prognosis with almost one-half of patients developing advanced renal failure. Knowledge of the clinico-pathological features of this entity (obesity, FSG lesions with glomerulomegaly, absence of nephrotic syndrome despite nephrotic-range proteinuria) should be helpful in establishing an accurate and early diagnosis.

A seven-iron ferredoxin from the thermoacidophilic archaeon Desulfurolobus ambivalens.

A seven-iron ferredoxin was isolated from aerobically grown cells of the hyperthermoacidophilic archaeon Desulfurolobus ambivalens (DSM 3772). The protein is monomeric, with an apparent molecular mass of 15 kDa and contains 7 iron atoms/molecule. The N-terminal sequence shows a large similarity (70% identity) with that of the ferredoxin isolated from the archaeon Sulfolobus acidocaldarius. The EPR characteristics in both the native (oxidized) and dithionite-reduced states of this protein allowed an unequivocal identification of a [3Fe-4S]1+/0 center, with a reduction potential of -270 +/- 20 mV, at pH 7.5. The protein also contains a [4Fe-4S]2+/1+ center with a very low reduction potential (Eo = -540 mV, pH 7.0), which yields a rhombic EPR spectrum upon reduction with sodium dithionite at high pH. The reduction potentials of both centers are slightly pH dependent between pH 6 and 9. The [3Fe-4S] ferredoxin center is able to accept electrons from pyruvate oxidase and NADH oxidase isolated from D. ambivalens. This ferredoxin is present in large amounts (at least 130 mg/kg wet cells), which allowed the unequivocal observation of oxidized [3Fe-4S] clusters in intact D. ambivalens cells.

Characterization of mutant Met100Lys of cytochrome c-550 from Thiobacillus versutus with lysine-histidine heme ligation.

The heme iron in cytochrome c-550 from Thiobacillus versutus has a methionine and a histidine as axial ligands. In order to study the characteristics of a possible lysine-histidine ligation in a heme protein, the methionine has been replaced by a lysine. This residue acts as a ligand between pH 3 and 12. The midpoint potential of the mutant has shifted -329 mV compared to wild type, but apart from this shift the pH dependence of the midpoint potential is unchanged, suggesting that the large drop is caused by specific ligand effects and not by protein refolding. While the EPR spectrum of wild-type cytochrome c-550 shows one species with gz = 3.35, in the spectrum of the mutant two species occur with gz values of 3.53 and 3.30. The intensity ratio of both species depends on the presence of organic cosolvents. In the low frequency region (-4 to -1 ppm) of the 1H NMR spectrum of mutant ferrocytochrome c-550, four one-proton peaks replace the resonances of the ligand methionine side chain protons. Using two-dimensional NMR spectroscopy (COSY and NOESY), these protons and five others have been assigned to the lysine ligand. The spectroscopic results obtained for this mutant show similarities with those observed for the alkaline form of cytochrome c, supporting the Lys-His ligation proposed for this protein. The data are consistent with the evidence for amine ligation in cytochrome f: the EPR spectrum of M100K cytc-550 is similar to that of cytochrome f. However, the NMR spectra show significant differences.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of lysine 99 of Thiobacillus versutus cytochrome c-550 in the alkaline transition.

The methionine ligand of the heme iron in ferricytochrome c-550 from Thiobacillus versutus is replaced by another residue at high pH. This transition is similar to the alkaline transition in mitochondrial cytochrome c. To investigate the possible role of lysine 99 in this process, this residue has been mutated to a glutamate. The mutation causes the apparent pKa of the transition to decrease from 11.2 in wild type to 10.8 in Lys99Glu cytochrome c-550. This destabilization of the native form is ascribed to the absence of the hydrogen bond between the epsilon-amine group of Lys99 and the carbonyl of Lys54 in the mutant protein. The 1H-NMR spectrum of Lys99Glu ferricytochrome c-550 at alkaline pH still shows resonance positions of the heme methyl peaks that are characteristic of the alkaline form. These results strongly suggest that Lys99 does not act as a ligand in the high-pH form, contrary to the case of yeast iso-1-cytochrome c. Evidence has been presented that in the latter protein the homologous Lys79 can act as a ligand in the alkaline form [1993, J. Am. Chem. Soc. 115, 7507-7508]. In the EPR spectrum of Lys99Glu cytochrome c-550 the species with Met-His coordination (gz = 3.27) is replaced by two forms with gz = 3.45 and 3.20 in the alkaline form (pH > or = 10.6). At pH > 11 yet another form is observed with g-values 2.87, 2.18 and 1.60, tentatively identified as a species with a lysine-histidinate coordination of the heme iron.

Cytochrome c6 from Monoraphidium braunii. A cytochrome with an unusual heme axial coordination.

A soluble monoheme c-type cytochrome (cytochrome c6) has been isolated from the green alga Monoraphidium braunii. It has a molecular mass of 9.3 kDa, an isoelectric point of 3.6 and a reduction potential of 358 mV at pH 7. The determined amino acid sequence allows its classification as a class-I c-type cytochrome. The ferric and ferrous cytochrome forms and their pH equilibria have been studied using 1H-NMR, ultraviolet/visible, EPR and Mössbauer spectroscopies. The pH equilibria are complex, several pKa values and pH-dependent forms being observed. The amino acid sequence, the reduction-potential value and the visible and NMR spectroscopies data in the pH range 4-9 indicate that the heme iron has a methionine-histidine axial coordination. However, the EPR and Mössbauer data obtained for the ferricytochrome show that in this pH range two distinct forms are present: form I, gz = 3.27, gy = 2.05 and gx = 1.05; form II, gz = 2.95, gy = 2.29 and gx = 1.43. While form I has crystal-field parameters typical of a methionine-histidine coordination, those associated with form II would suggest a histidine-histidine axial ligation. This possibility was extensively analyzed by spectroscopic methods and by chemical modification of a histidine residue. It was concluded that form II actually corresponds to an unusual type of methionine-histidine axial coordination. Straightforward examples of this type of coordination have recently been found in other c-type hemeproteins [Teixeira, M., Campos, A. P., Aguiar, A. P., Costa, H. S., Santos, H., Turner, D. L. & Xavier, A. V. (1993) FEBS Lett. 317, 233-236], corroborating our proposal. Since both forms, with very distinct crystal-field parameters, are shown to have the same reduction potential, it may be concluded that the axial and rhombic distortions of the heme-iron ligand field cannot be directly correlated with the heme-reduction potential. The pH-dependence studies have also shown that the form I and form II are interconvertible, with pKa approximately 5. To establish a possible physiological significance for this process, in particular for the interaction of the cytochrome with the membrane-bound electron-transfer complexes b6f and photosystem I, the effect of surfactants on the spectroscopic characteristics of cytochrome c6 has been studied.

Pitfalls in assigning heme axial coordination by EPR. c-Type cytochromes with atypical Met-His ligation.

Different monohemic c-type cytochromes were analyzed by visible, EPR and 1H NMR spectroscopies. While the visible and NMR data show unambiguously that the heme iron has a Met-His heme axial coordination, the EPR data indicate an axial ligand field typical of that for a bis-histidinyl ligation. The validity of the widely used EPR methods for the determination of the heme iron axial coordination, based on the crystal field parameters (tetragonality and rhombicity), is questioned.