Mechanism and dynamics of fatty acid photodecarboxylase.

Fatty acid photodecarboxylase (FAP) is a photoenzyme with potential green chemistry applications. By combining static, time-resolved, and cryotrapping spectroscopy and crystallography as well as computation, we characterized Chlorella variabilis FAP reaction intermediates on time scales from subpicoseconds to milliseconds. High-resolution crystal structures from synchrotron and free electron laser x-ray sources highlighted an unusual bent shape of the oxidized flavin chromophore. We demonstrate that decarboxylation occurs directly upon reduction of the excited flavin by the fatty acid substrate. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved carbon dioxide unexpectedly transformed in 100 nanoseconds, most likely into bicarbonate. This reaction is orders of magnitude faster than in solution. Two strictly conserved residues, R451 and C432, are essential for substrate stabilization and functional charge transfer.

AXSIS: Exploring the frontiers in attosecond X-ray science, imaging and spectroscopy.

X-ray crystallography is one of the main methods to determine atomic-resolution 3D images of the whole spectrum of molecules ranging from small inorganic clusters to large protein complexes consisting of hundred-thousands of atoms that constitute the macromolecular machinery of life. Life is not static, and unravelling the structure and dynamics of the most important reactions in chemistry and biology is essential to uncover their mechanism. Many of these reactions, including photosynthesis which drives our biosphere, are light induced and occur on ultrafast timescales. These have been studied with high time resolution primarily by optical spectroscopy, enabled by ultrafast laser technology, but they reduce the vast complexity of the process to a few reaction coordinates. In the AXSIS project at CFEL in Hamburg, funded by the European Research Council, we develop the new method of attosecond serial X-ray crystallography and spectroscopy, to give a full description of ultrafast processes atomically resolved in real space and on the electronic energy landscape, from co-measurement of X-ray and optical spectra, and X-ray diffraction. This technique will revolutionize our understanding of structure and function at the atomic and molecular level and thereby unravel fundamental processes in chemistry and biology like energy conversion processes. For that purpose, we develop a compact, fully coherent, THz-driven atto-second X-ray source based on coherent inverse Compton scattering off a free-electron crystal, to outrun radiation damage effects due to the necessary high X-ray irradiance required to acquire diffraction signals. This highly synergistic project starts from a completely clean slate rather than conforming to the specifications of a large free-electron laser (FEL) user facility, to optimize the entire instrumentation towards fundamental measurements of the mechanism of light absorption and excitation energy transfer. A multidisciplinary team formed by laser-, accelerator,- X-ray scientists as well as spectroscopists and biochemists optimizes X-ray pulse parameters, in tandem with sample delivery, crystal size, and advanced X-ray detectors. Ultimately, the new capability, attosecond serial X-ray crystallography and spectroscopy, will be applied to one of the most important problems in structural biology, which is to elucidate the dynamics of light reactions, electron transfer and protein structure in photosynthesis.

[Concordance analysis of referrals from Primary Care to Mental Health]. / Análisis de concordancia de las derivaciones de atención primaria a salud mental.

APPROACH: Proper coordination between Primary Care (PC) and Mental Health Units (MHU) is needed, and referrals between the two systems are an important issue. We attempt to analyse various aspects on the variation between referrals from PC to MHU in the Burgos area. METHODOLOGY: Descriptive cross-sectional study, including all first-time referral patients from PC to MHU in Burgos in 2006: 1132 referrals. RESULTS: Waiting time for the first consultation with the specialist was less than 30 days in 38% of cases, and between 30 and 45 days in 36.1% of the case. The agreement between Family Doctors (FD) requests (psychiatry/clinical psychology) and the specialist performing first consultation was very high (Kappa Index 0.784). The most common diagnosis in PC were anxiety disorders (29%) and depression disorders (25%). The most common diagnosis in MHU were adaptation disorders (26%), anxiety disorders (21%), and depression disorders (19%). Diagnostic agreement between Primary care and Specialist care was low (Kappa Index 0.342). Specialists judged referrals appropriate or very appropriate in 72.4% of the cases, and deemed the information in the referral form to be adequate in 56.7% of the cases. As regards the referral type (ordinary, preferential or urgent), the agreement between PC and Mental Health specialists was very low (Kappa Index 0.179). In 75.9% of referrals, psychiatrist and clinical psychologist indicated not having had previous contact with the primary care physician. Almost half (47.7%) of referred patients indicated previous pharmacological treatment in PC. CONCLUSIONS: The degree of agreement between PC and MHU was very high in terms of specialist requested and attending specialist (psychiatry/clinical psychologist), was low in terms of diagnostic agreement, and it was very low in terms of referral type. This suggests a need to reinforce the relationship between both care systems.

Análisis de concordancia de las derivaciones de atención primaria a salud mental / Concordance analysis of referrals from Primary Care to Mental Health

Planteamiento. Es necesaria una correcta coordinación entre atención primaria (AP) y los equipos de salud mental (ESM), siendo el punto crítico la derivación entre ambos sistemas. Nos propusimos analizar la concordancia de las derivaciones realizadas desde AP a los ESM de Burgos. Metodología. Estudio descriptivo transversal, con inclusión de los pacientes nuevos derivados desde AP a ESM de Burgos durante 2006: 1.132 derivaciones. Resultados. El tiempo de demora para la primera consulta fue inferior a 30 días en el 38% de los casos, de 30-45 días en 36,1%. El índice de concordancia entre la demanda del médico de AP y el facultativo de salud mental que atendió en primera consulta fue muy alta (índice kappa 0,784). Los diagnósticos más indicados por AP fueron trastornos de ansiedad (29%) y trastornos afectivos (25%). Los diagnósticos más frecuentes en los ESM fueron los trastornos adaptativos (26%), de ansiedad (21%) y afectivos (19%). La concordancia diagnóstica entre AP y ESM fue baja (índice kappa 0,342). Los ESM consideraron adecuadas o muy adecuadas las derivaciones en el 72,4% de los casos, y correcta la información de derivación en el 56,7%. En cuanto al carácter de la derivación (ordinaria, preferente o urgente), el acuerdo entre la calificación otorgada por AP y por ESM fue muy bajo (índice kappa 0,179). En el 75,9% de las ocasiones, psiquiatras y psicólogos clínicos indicaron no haber mantenido ningún contacto previo con AP. El 47,7% de los pacientes acudían al ESM con tratamiento farmacológico pautado en AP. Conclusiones. El grado de acuerdo entre AP y ESM fue muy alto en cuanto al profesional demandado y el que finalmente atendió (psiquiatra/psicólogo clínico), bajo en la concordancia diagnóstica, y muy bajo en lo que al carácter de la derivación se refiere, lo cual indica la necesidad de reforzar los vínculos informativos entre ambos sistemas asistenciales (AU)

Approach. Proper coordination between Primary Care (PC) and Mental Health Units (MHU) is needed, and referrals between the two systems are an important issue. We attempt to analyse various aspects on the variation between referrals from PC to MHU in the Burgos area. Methodology. Descriptive cross-sectional study, including all first-time referral patients from PC to MHU in Burgos in 2006: 1132 referrals. Results. Waiting time for the first consultation with the specialist was less than 30 days in 38% of cases, and between 30 and 45 days in 36.1% of the case. The agreement between Family Doctors (FD) requests (psychiatry/clinical psychology) and the specialist performing first consultation was very high (Kappa Index 0.784). The most common diagnosis in PC were anxiety disorders (29%) and depression disorders (25%). The most common diagnosis in MHU were adaptation disorders (26%), anxiety disorders (21%), and depression disorders (19%). Diagnostic agreement between Primary care and Specialist care was low (Kappa Index 0.342). Specialists judged referrals appropriate or very appropriate in 72.4% of the cases, and deemed the information in the referral form to be adequate in 56.7% of the cases. As regards the referral type (ordinary, preferential or urgent), the agreement between PC and Mental Health specialists was very low (Kappa Index 0.179). In 75.9% of referrals, psychiatrist and clinical psychologist indicated not having had previous contact with the primary care physician. Almost half (47.7%) of referred patients indicated previous pharmacological treatment in PC. Conclusions. The degree of agreement between PC and MHU was very high in terms of specialist requested and attending specialist (psychiatry/clinical psychologist), was low in terms of diagnostic agreement, and it was very low in terms of referral type. This suggests a need to reinforce the relationship between both care systems (AU)

Sequential single-shot imaging of nanoscale dynamic interactions with a table-top soft x-ray laser.

We demonstrate the first real-space recording of nanoscale dynamic interactions using single-shot soft x-ray (SXR) full-field laser microscopy. A sequence of real-space flash images acquired with a table-top SXR laser was used to capture the motion of a rapidly oscillating magnetic nanoprobe. Changes of 30 nm in the oscillation amplitude were detected when the nanoprobe was made to interact with stray fields from a magnetic sample. The table-top visualization of nanoscale dynamics in real space can significantly contribute to the understanding of nanoscale processes and can accelerate the development of new nanodevices.

Pineal gliosis and gland ageing. The possible role of the glia in the transfer of melatonin from pinealocytes to the blood and cerebrospinal fluid

Using immunohistochemical approaches appliedto 6 ìm-thick sections from the pineal glands ofcows with ages between 1 and 7 years, we studiedthe expression of GFAP, vimentin and S-100and of â-tubulin. The latter compound appearedin both pinealocytes and in some nerve fibres. Inboth younger and older cows, vimentin wasexpressed in the somata of cells localised inperipheral zones of the glands and in fibres witha bead-like aspect that coursed deep into the centresof the glands, as well as in pericapillary cells,apparently in the endothelia of vessels and, likeGFAP and S-100, in a thick network of prolongationslocated between the basal side of theepithelial cells of the pineal recess and pinealocytes,sometimes reaching the ependymal lumen.The network of glial prolongations formed aspace with a reticulated aspect interposedbetween the pinealocytes and the ependymalepithelium. The thickness of this network wasespecially striking in the older cows. The expressionof GFAP and S-100 was also observed in thesomata and the cytoplasmic prolongations of cellsdistributed throughout the gland that surroundedthe pinealocytes. In cows with ages of 4 and 7years the pineal gland showed a pronounceddegree of gliosis that isolated the pinealocytesfrom one another and from the vascular bed.Additionally, all three glial markers wereexpressed in cells whose somata and cytoplasmicprolongations configured spaces with a trabecularaspect, very poor in connective tissue, whichcoursed throughout the glands to reach the reticulatedspace, in especially thin neighbouringzones of the epithelium of the pineal recess. Theresults suggest a decisive role of the pineal glia ingland ageing, in the secretion of melatonin, andin the configuration of the vascular and ependymalpathways that the hormone must follow frompinealocytes to its target organs (AU)

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Debut de linfoma Hodgkin con clínica de compresión medular / Spinal involvement as first presentation of Hodgkin´s disease

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Melatonin-like immunoreactivity in the pineal gland of the cow: an immunohistochemical study.

With a view to checking the presence of melatonin in the pineal gland of the cow, in the present work we used six adult animals, ranging in age from one to six years, which were sacrificed at dawn. Sections of 6 micro m thickness of Bouin-fixed and paraffin-embedded pineal glands were incubated in an anti-melatonin serum, which was provided by the Institute for Molecular and Cellular Recognition, Gunma University, Maebshi, Japan. After incubation and successive washings in PBS, some of the sections were treated with the avidin-biotin-peroxidase complex (ABC) technique using antisera from Sigma, and developed with the method of Graham and Karnovsky (which employs 3,3'-diaminobenzidine and H2O2 as developer). Other sections were incubated in a goat-anti-rabbit IgG (H+L) bound to fluorochrome Cy5 for immunofluorescence studies. An intense reaction for melatonin was observed in the cytoplasm but not in the nucleus of melatonin secreting pinealocytes located in peripheral and intermediate zones of the pineal gland. Immunoabsorption of the antimelatonin primary antibody with melatonin at a dilution of 10 mM per 0.1 ml of serum prevented the reaction, as happened when any of the antisera used in the procedure were used. Immunoabsorption of anti-melatonin serum with different amounts of bovine albumin (ranging between 1/5 to 1/50) failed to inhibit the immunoreactivity. When a bovine anti-albumin antibody was employed, working with the above methods, no immunoreaction was detected. Our data suggest that the pinealocytes of cows sacrificed at dawn contain immunoreactive melatonin.

[Osteonecrosis and HIV]. / Osteonecrosis y VIH.

An increase of the incidence of osteonecrosis in subjects infected with the HIV has been reported. It is unknown if osteonecrosis is an HIV infection consequence, or just the consequence of so many risk factors present among HIV infected subjects. We report five osteonecrosis cases found in our series of 534 HIV patients, and we review the related bibliography. All cases have some conventional risk factor different to the HIV or the antiviral treatment. We can't conclude antiviral treatments are not involved in osteonecrosis, but they have been the only risk factor in none of our patients.

Osteonecrosis y VIH / Osteonecrosis and hiv

En la literatura aparece un incremento de la incidencia de osteonecrosis de cabeza femoral entre sujetos infectados por el VIH, si bien se desconoce si ésto es consecuencia directa o indirecta de la enfermedad infecciosa o más bien se debe al acúmulo de factores que en estos pacientes se da por su pluripatología. En este trabajo describimos cinco casos de osteonecrosis ocurridos entre los 534 pacientes VIH vistos en nuestro hospital y repasamos la bibliografía relacionada. Todos los casos presentan alguno de los factores de riesgo convencional ya relacionados con el desarrollo de osteonecrosis. Si bien no puede descartarse el papel del tratamiento o del VIH en el desarrollo de osteonecrosis, no hemos detectado ningún caso entre nuestros pacientes en que éste haya sido el único factor (AU)

An increase of the incidence of osteonecrosis in subjects infected with the HIV has been reported. It is unknown if osteonecrosis is an HIV infection consequence, or just the consequence of so many risk factors present among HIV infected subjects. We report five osteonecrosis cases found in our series of 534 HIV patients, and we review the related bibliography. All cases have some conventional risk factor different to the HIV or the antiviral treatment. We can’t conclude antiviral treatments are not involved in osteonecrosis, but they have been the only risk factor in none of our patients (AU)

Derrame pericárdico, ¿tuberculoso? / Pericardial effusion, idiopatic?

El derrame pericárdico es un problema clínico, sobre todo por la dificultad que entraña su diagnóstico etiológico, sin llegar a determinarse su causa en gran número de casos. Presentamos el caso de una paciente con taponamiento cardíaco in causa filiada, con empeoramiento clínico que precisó pericardiocentesis, y a la que se le administró con tuberculostáticos, de forma empírica, con mejoría y resolución del cuadro. Revisamos la frecuencia del derrame pericárdico de origen tuberculoso y sus técnicas diagnósticas (AU)

Severe experimental uraemia does not decrease the population of rat pituitary somatotrophs.

BACKGROUND: Growth hormone (GH) secretion by the anterior pituitary has been shown to be depressed in severely uraemic rats. Changes in the population of pituitary somatotrophs might be partially responsible for this decrease. METHODS: To analyse the population of pituitary somatotrophs in severe uraemia, immunocytochemical detection and quantification of GH-producing cells were carried out on paraffin sections from young rats either 5/6 nephrectomized, sham-operated fed ad libitum or sham-operated pair-fed with the nephrectomized animals. RESULTS: Nephrectomized rats were severely uraemic and growth retarded. The overall cell density (total pituitary cells/mm2) was higher in 5/6 nephrectomized animals in comparison with the two sham-operated groups. Thus, although the percentage of GH cells was slightly lower in nephrectomized than in control rats, no difference in either the density (cells/mm2) or the cross-sectional area of GH cells was found among groups. CONCLUSIONS: These results suggest that severe experimental uraemia interferes with the maturation process of the pituitary gland and support the contention that differences in either the number or the size of pituitary somatotrophs cannot explain the reduced GH secretion previously reported in severely uraemic rats.

Cellular proliferation in the rat pineal gland during postnatal development.

To establish a possible correlation between the rate of cellular proliferation and already documented functional and morphological characteristics of the rat pineal gland during postnatal development, the bromodeoxyuridine labelling method was used to evaluate the fraction of cells at the S phase of the cell cycle in paraffin sections from 1-, 7-, 14- and 28-day-old rats. Numerical density, taken as an indirect measure of cell hypertrophy, was also evaluated. During the first week after birth the percentage of S phase-cells in the rat pineal gland sharply decreased from around 9% to 1.3%. A smaller but also significant decrease was found from the 7th to the 14th postnatal day where S phase cells were less than 0.5% of all pineal cells. A very low percentage was also seen in samples from 28-day-old rats. Numerical density, namely, the total number of cells per surface unit of pineal section, decreased from birth to the end of the first month. This decrease was also steeper from birth to the 7th postnatal day than at any other period of the study. These results support the idea that a strong expansion of the cellular population of the rat pineal gland precedes morphological and functional maturation and opens the way to further exploration of the relationship between functional and proliferative responses of the pineal gland.

Expression of argyrophilic nucleolar organizer regions (AgNORs) in the harderian gland of male and female hamsters during postnatal development.

The expression of argyrophilic nucleolar organizer regions (AgNORs) was studied in the different secretory cell types of the Harderian gland of male and female Syrian hamsters during postnatal development. Mean AgNOR area was calculated for each cell type in paraffin sections from 7-, 14-, 21-, 28-, 45- and 90-day-old animals. AgNOR content was similar in male type I-cells and in female cells, decreasing in both cell types from the 7th to the 14th day, increasing afterwards at the 21st day, and remaining at relatively stable levels from that point to the end of the study. AgNOR content of male type II-cells was greater than in other cell type studied, and was greater in 45- and 90-day-old animals than in 28-day-olds. Changes of AgNOR content in type I-cells of male and female hamsters during the first two weeks seem to be related to changes in proliferative activity while metabolic activity might be responsible for changes taking place later on. Our results also support that male type I- and type II-cells have a different biological behaviour and that type II-cells are far from being degenerating cells.

Comparison of Vindelov et al. and bromodeoxyuridine/DNA double-staining flow cytometry methods for analysis of cell cycle distribution in rat thymocytes.

This study compares the cell cycle distribution in rat thymocytes obtained by means of bromodeoxyuridine (BrdUrd) labeling of S-phase cells and the analysis of the S-phase fraction obtained according to the technique of Vindelov et al. (Cytometry 3:332-338, 1983). The proportion of BrdUrd-labeled cells was analyzed in single cell suspensions of adult rat thymocytes after in vivo injection of BrdUrd and the results then compared with those obtained after measuring the cell DNA contents according to the Vindelov et al. method. The percentage of BrdUrd-positive cells was greater than the S-phase fraction obtained using the Vindelov et al. technique. By contrast, no major differences were observed between the percentage of BrdUrd-positive cells and the S-phase fraction obtained after analyzing the DNA histograms of the same data files with the RFIT mathematical model. The elimination of trypsin treatment used in the Vindelov et al. method did not alter the results, whereas the use of DNA denaturation with 2N HCl was shown to increase the percentage of S-phase rat thymocytes (calculated from DNA histograms) independently of whether trypsin treatment was used or not. However, the value of the S-phase fraction was not as great as that obtained after BrdUrd labeling. Thus when comparing BrdUrd-labeling and the Vindelov et al. technique, important differences in the percentage of S-phase adult rat thymocytes were observed. Selective G0/G1 cell loss during washing and centrifugation steps performed after the DNA denaturation used for BrdUrd detection was the main reason for these differences.

New insights into the cytodynamics of the hamster Harderian gland as provided by the bromodeoxyuridine-labelling method.

The fourth week of postnatal life is a critical point in the development of the hamster Harderian gland. During this week, cells with large lipid vacuoles (type-II cells) appear in the male gland, marking a morphological sex difference that is notorious in adult animals. The origin and fate of type-II cells are controversial. To gain insight into the mechanisms by which type-II cells become a major cell type in the gland of adult male hamsters, bromodeoxyuridine (BrdU) labelling was used to assess the proliferative activity of both types of glandular cells in 28-day-old animals. To search for possible sex differences in the proliferative activity of this gland, female animals of the same age as the males were also studied. No difference was found in the overall labelling index (BrdU-labelled cells/100 cells) between males (1.8 +/- 0.1%) and females (1.5 +/- 0.1%). In the gland of the males, the specific labelling index of type-II cells (3.4 +/- 0.4%) was significantly higher than that of type-I cells (0.9 +/- 0.2%). Interestingly, the proportion of type-II cells present in the male glands at this age (36.6%) was significantly lower than that of type-I cells. Our results strongly suggest that the proliferation of type-II cells, rather than a continuous differentiation of these cells from preexisting type-I cells, is a major event in the achievement of the mature form of this gland. The results reported here counsel a reappraisal of current theories about the cytodynamics of the hamster Harderian gland.

In vitro bromodeoxyuridine-labelling of single cell suspensions: effects of time and temperature of sample storage.

The present study was aimed to explore how the in vitro BrdUrd-labelling of rat thymocytes might be affected by both the time elapsed between obtaining the sample and the beginning of the labelling (0, 15, 30 or 60 min) and the effect of the temperature of storage (4 degrees C versus room temperature). Single cell suspensions obtained after in vivo labelling with BrdUrd were used as controls. The S phase fraction was calculated by flow cytometry both according to BrdUrd-immunolabelling and DNA content. Immediate incubation with BrdUrd after the sample was obtained resulted in a slight decrease of the proportion of S phase cells analysed either according to DNA content or to BrdUrd-immunolabelling. Regardless of storage-temperature, the S phase fraction decreased in samples kept for 15 min or more before BrdUrd incubation. No BrdUrd-positive cells were detected in samples stored for 60 min at room temperature. This effect was related to temperature since positive cells were found when the samples were kept at 4 degrees C during the same time period. Our results suggest that during in vitro incubation a relative loss of S phase cells exists and that a delay beyond 15 min between obtaining the sample and the in vitro labelling seriously compromises the results of this technique.