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.

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.

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.

Circadian variation in the distribution of cells throughout the different phases of the cell cycle in the anterior pituitary gland of adult male rats as analysed by flow cytometry.

Flow cytometric analysis of nuclei stained with propidium iodide (PI) has been used to study the distribution of cells throughout the different phases of the cell cycle in the anterior pituitary gland of adult male Sprague-Dawley rats at different times of the day. According to PI fluorescence intensity the relative numbers of cells in S phase (cells with a DNA content between that of somatic cells in interphase (2n) and that of somatic cells after duplication of the DNA prior to cell division (4n] and G2/M phase (4n) were calculated. A significant circadian rhythm was found for cells in both the S phase (P less than 0.05) and the G2/M phase (P less than 0.01). The wave of cells in S phase with a peak at the middle of the light period (14.00 h) precedes by about 6 h the wave of cells in G2/M phase (peak at 20.00 h). Most of the DNA-replicating cells were found during the early S phase at 11.00 h, advancing further up to the middle of this phase at 14.00 h. Cells were distributed homogeneously throughout the S phase at 17.00 h. These data strongly suggest that the beginning of the light period triggers a wave of cells to leave G0/G1 into S phase.

Influence of luteinizing hormone-releasing hormone (LHRH) treatment on cellular proliferation in the rat anterior pituitary.

This study was designed to gain insight into the action of LHRH on the control of cellular proliferation in the anterior pituitary. The fraction of cells labelled with bromodeoxyuridine (S-phase cells) was studied in cytospin preparations of anterior pituitary cells taken from control male and female rats and from rats treated with daily doses of 2 micrograms/100 g body weight of LHRH (7 days), with doses of 40 micrograms of LHRH given on alternate days for 14 days (7 doses) or, finally, treated with daily doses of 50 ng of busereline acetate (14 days). Treatment with LHRH for 14 days resulted in a significant increase in the fraction of S-phase cells. However, neither the blockade of gonadotrophin secretion with busereline acetate nor its stimulation with LHRH for seven days resulted in a significant change in the proliferative activity of anterior pituitary cells. This action was independent of sex. No significant changes were seen in the proportions of pituitary gonadotrops of the different study groups. Regardless of the treatment-group very few cells doubly-immunostained for BrdU and LH were found. It is concluded that LHRH may stimulate cellular proliferation in the anterior pituitary, but further studies are needed to define which cells are involved in this action.

Cellular proliferation and/or differentiation can condition modifications in the expression of AgNORs in rat anterior pituitary cells during growth.

The expression of argyrophilic nucleolar organizer regions (AgNORs) was studied in the anterior lobe of the hypophysis of 1-, 7-, 14-, 28- and 80-day-old rats. Mean area and numbers of AgNORs per nucleus decreased during this period with a simultaneous increase of both nuclear area and mean AgNOR-particle area. Comparison of our data on AgNOR expression with previously reported data on the proliferative activity and cell maturation and differentiation of anterior pituitary cells during a similar period led us to conclude that, at least in our experimental model, variations in the expression of AgNORs are more related to phenomena of cellular maturation than to proliferative activity.

Morphological study of the rat pituitary follicle stimulating hormone cells after alternate day treatment with luteinizing hormone-releasing hormone (LHRH).

The action of the luteinizing hormone-releasing hormone (LHRH) on the hypophyseal gonadotrophins has either an activatory or inhibitory effect depending on the doses administered or on the treatment followed. Both factors can induce a different response in the two hormones. In this work, the effect after the administration of 8 doses (40 micrograms/day) of LHRH at intervals of 48 hours, on the serum levels of follicle stimulating hormone (FSH), as well as the numerical density, distribution, intensity of staining and morphometrical parameters of the cells which react against the anti-FSH serum, are assessed. It has been found that with the treatment an increase of FSH serum levels, without modification in the number of immunoreactive cells, but a clear increase in the lightly stained cells, is produced. The distribution of the reactive cells, uniform in normal animals, shows a large numerical density in the dorsal and posterior hypophyseal areas in the treated animals. No change was observed in the nuclear and cellular areas between the different groups.

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.

Serotonin immunoreactivity in the intermediate lobe of the rat pituitary.

Immunocytochemical staining for serotonin (5-HT) in paraffin-embedded sections of rat pituitary resulted in the localization of reactive nerve fibres and cell bodies in the intermediate lobe. Immunostaining was also found in the anterior and posterior lobes. Labelled nerve fibres appear to enter the intermediate lobe from the neural lobe through the interlobular spaces. These fibres are relatively scarce and lightly stained. Neuroglandular contacts were identified between varicose nerve endings containing serotonin and immunoreactive perykarion. It is not clear whether intermediate lobe cells produced 5-HT themselves or, alternatively, these cells take in 5-HT from serotoninergic nerve terminals.

Immunocytochemical study of the epithelial lining of naturally occurring cysts in the rat intermediate lobe.

An immunocytochemical study of the epithelial lining of naturally occurring cysts in the rat intermediate lobe (IL) has been carried out. Paraffin-embedded sections, in which cysts were identifiable, were treated either with anti-serotonin or anti-S-100 protein sera. S-100-positive cells were intermingled with glandular cells surrounding the cyst lumen. These S-100-positive cells sent slender cytoplasmic processes as if to cover the apical surface of neighbouring cells. Rarely were 5-HT-immunopositive cells seen in the cyst epithelial lining. Most cells of the marginal layer of the IL were found reactive either to an S-100 or a-5-HT serum. The presence of an epithelial lining positive to S-100 protein sera is in keeping with the notion that cysts in the IL might form as evaginations of the epithelial lining of the pituitary cleft. The lack of correspondence between 5-HT-positive cells in the marginal layer and the cyst lining is controversial. A peculiar spatial relationship of 5-HT cells with the vascular network of the IL is suggested.

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.

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.

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.

Proliferative activity of cells of the intermediate lobe of the rat pituitary during the postnatal period.

Cellular proliferation was studied in the intermediate lobe (IL) of the pituitary gland of developing rats by labelling cells at the S-phase of the cell cycle with bromodeoxyuridine (BrdU). The number of BrdU-labelled cells in the IL decreased from birth until the 14th postnatal day and was low from that day until the end of the first month after birth. Throughout the postnatal period a large proportion of BrdU-labelled cells was found in the marginal layer (ML) of the IL, suggesting for the ML a role as a germinative layer of the IL during postnatal growth. Double immunostaining with anti-BrdU and anti-MSH showed that MSH cells actively proliferate as from the day of birth. Cells doubly immunostained with anti-BrdU and anti-S100 protein were first seen on the 14th postnatal day. From then onwards, most proliferating cells were labelled with either anti MSH or anti S-100 protein. This, together with the high proportion of proliferating cells found in the ML marks a clear difference with the pattern of cellular proliferation previously reported during a similar period in the anterior lobe of the rat pituitary.

[Adult respiratory distress syndrome secondary to varicella pneumonia in an immunocompetent male]. / Síndrome de distress respiratorio del adulto secundario a neumonía varicelosa en varón inmunocompetente.

We present a case of varicellous pneumonia in a 40-year-old patient without immunitary compromise, who quickly evolved to respiratory distress in the adult and required sustained ventilatory support. We review the literature, with special mention to the indications of the antivirical treatment with Aciclovir.

[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.

[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.