A very luminous magnetar-powered supernova associated with an ultra-long γ-ray burst.

A new class of ultra-long-duration (more than 10,000 seconds) γ-ray bursts has recently been suggested. They may originate in the explosion of stars with much larger radii than those producing normal long-duration γ-ray bursts or in the tidal disruption of a star. No clear supernova has yet been associated with an ultra-long-duration γ-ray burst. Here we report that a supernova (SN 2011kl) was associated with the ultra-long-duration γ-ray burst GRB 111209A, at a redshift z of 0.677. This supernova is more than three times more luminous than type Ic supernovae associated with long-duration γ-ray bursts, and its spectrum is distinctly different. The slope of the continuum resembles those of super-luminous supernovae, but extends further down into the rest-frame ultraviolet implying a low metal content. The light curve evolves much more rapidly than those of super-luminous supernovae. This combination of high luminosity and low metal-line opacity cannot be reconciled with typical type Ic supernovae, but can be reproduced by a model where extra energy is injected by a strongly magnetized neutron star (a magnetar), which has also been proposed as the explanation for super-luminous supernovae.

A simple method to evaluate groundwater vulnerability in urbanizing agricultural regions.

A methodology to evaluate groundwater vulnerability was developed and tested in a case study in the Central Valleys of the state of Oaxaca, Mexico, a region known for intensive agricultural activities and poor water management policies. An analysis was conducted to create and evaluate scenarios reflecting anthropogenic and natural stressors on groundwater using an analytical hierarchy process (AHP) and geographic information systems. Uncertainty in the vulnerability model was assessed using a Monte Carlo analysis. Five indices (abstraction (Abs), pollution (Po), runoff (Ru), groundwater recharge (Re), and marginalization (Ma)) were selected after an evaluation of the effects of population growth, climatology, hydrogeological features, and social marginalization on access to groundwater. Abstraction, pollution, and recharge rates are the main drivers of groundwater vulnerability, accounting for 87% of the vulnerability. The analysis revealed that the proposed model generates consistent results and contains low uncertainty. It also showed that more than 50% of the region's groundwater is moderately, and the vulnerability has become increasingly with abstraction, reduced recharge, and pollution (the most sensitive indices), indicating that groundwater in the Central Valleys is under great stress. Pollution and abstraction of groundwater resources are expected to rise in the more vulnerable areas, which will increase water crises and reduce access to water in rural communities. The approach and the indicators establish a baseline for the management and protection of water resources in developing countries where high-resolution data are lacking.

Human decidual stromal cells secrete soluble pro-apoptotic factors during decidualization in a cAMP-dependent manner.

STUDY QUESTION: Is there a relationship between decidualization and apoptosis of decidual stromal cells (DSC)? SUMMARY ANSWER: Decidualization triggers the secretion of soluble factors that induce apoptosis in DSC. WHAT IS KNOWN ALREADY: The differentiation and apoptosis of DSC during decidualization of the receptive decidua are crucial processes for the controlled invasion of trophoblasts in normal pregnancy. Most DSC regress in a time-dependent manner, and their removal is important to provide space for the embryo to grow. However, the mechanism that controls DSC death is poorly understood. STUDY DESIGN, SIZE, DURATION: The apoptotic response of DSC was analyzed after exposure to different exogenous agents and during decidualization. The apoptotic potential of decidualized DSC supernatants and prolactin (PRL) was also evaluated. PARTICIPANTS/MATERIALS, SETTING, METHODS: DSC lines were established from samples of decidua from first trimester pregnancies. Apoptosis was assayed by flow cytometry. PRL production, as a marker of decidualization, was determined by enzyme-linked immunosorbent assay. MAIN RESULTS AND THE ROLE OF CHANCE: DSCs were resistant to a variety of apoptosis-inducing substances. Nevertheless, DSC underwent apoptosis during decidualization in culture, with cAMP being essential for both apoptosis and differentiation. In addition, culture supernatants from decidualized DSC induced apoptosis in undifferentiated DSC, although paradoxically these supernatants decreased the spontaneous apoptosis of decidual lymphocytes. Exogenously added PRL did not induce apoptosis in DSC and an antibody that neutralized the PRL receptor did not decrease the apoptosis induced by supernatants. LIMITATIONS, REASONS FOR CAUTIONS: Further studies are needed to examine the involvement of other soluble factors secreted by decidualized DSC in the induction of apoptosis. WIDER IMPLICATIONS OF THE FINDINGS: The present results indicate that apoptosis of DSC occurs in parallel to differentiation, in response to decidualization signals, with soluble factors secreted by decidualized DSC being responsible for triggering cell death. These studies are relevant in the understanding of how the regression of decidua, a crucial process for successful pregnancy, takes place. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the Consejería de Economía, Innovación y Ciencia, Junta de Andalucía (Grant CTS-6183, Proyectos de Investigación de Excelencia 2010 to C.R.-R.) and the Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Spain (Grants PS09/00339 and PI12/01085 to E.G.O.). E.L.-D. was supported by fellowships from the Ministerio de Educación y Ciencia, Spain and the University of Granada. The authors have no conflict of interest.

Human decidual stromal cells secrete C-X-C motif chemokine 13, express B cell-activating factor and rescue B lymphocytes from apoptosis: distinctive characteristics of follicular dendritic cells.

BACKGROUND: Decidual stromal cells (DSCs) have classically been considered fibroblastic cells, although their function, cell lineage and origin are not fully understood. We previously demonstrated that human DSCs showed similarities with follicular dendritic cells (FDCs): DSCs expressed FDC-associated antigens, both types of cells are contractile and both are related to mesenchymal stem cells (MSCs). To further characterize DSCs, we investigated whether DSCs and FDCs share any distinctive phenotypical and functional characteristics. METHODS: Human FDC lines were obtained from tonsillectomy samples, human DSC lines from elective termination of pregnancy samples and human MSC lines from bone marrow aspirates. We isolated DSC, FDC and MSC lines and compared their characteristics with flow cytometry and enzyme-linked immunosorbent assay. Cell lines were cultured with tumour necrosis factor (TNF) and lymphotoxin (LT)α(1)ß(2), cytokines involved in FDC differentiation. Cell lines were also differentiated in culture after exposure to progesterone and cAMP, factors involved in the differentiation (decidualization) of DSC. RESULTS: Like MSCs, DSCs and FDCs expressed MSC-associated antigens (CD10, CD29, CD54, CD73, CD106, α-smooth muscle actin and STRO-1) and lacked CD45 expression, and all three types of cell line showed increased expression of CD54 (ICAM-1) and CD106 (VCAM-1) when cultured TNF and LTα(1)ß(2). DSCs and FDCs, however, exhibited characteristics not observed in MSCs: DSCs expressed FDC-associated antigens CD14, CD21 and CD23, B cell-activating factor and secreted C-X-C motif chemokine 13. Moreover, DSC lines but not MSC lines inhibited the spontaneous apoptosis of B lymphocytes, a typical functional attribute of FDC. During culture with progesterone and cAMP, FDCs, like DSCs but in contrast to MSCs, changed their morphology from a fibroblastic to a rounder shape, and cells secreted prolactin. CONCLUSIONS: Our results suggest that DSCs and FDCs share a common precursor in MSCs but this precursor acquires new capacities when it homes to peripheral tissues. We discuss these shared properties in the context of immune-endocrine regulation during pregnancy.

Bioinformatic analysis of the genome of infectious salmon anemia virus associated with outbreaks with high mortality in Chile.

The infectious salmon anemia virus (ISAV), an orthomyxovirus, is the major cause of outbreaks of high mortality rates in salmon in Chile. It has been proposed that the virulence of ISAV isolates lies mainly in hemagglutinin-esterase and fusion glycoproteins. However, based on current information, the contribution of other viral genes cannot be ruled out. To study this, we isolated and determined the complete coding sequence of two high-prevalence Chilean isolates associated with outbreaks of high mortality rates: ISAV752_09 and ISAV901_09. These isolates were compared to 15 Norwegian isolates that exhibit differences in their virulence. For this purpose, we performed bioinformatic analyses of (i) functional domains, (ii) specific mutations, (iii) Bayesian phylogenetics, and (iv) structural comparisons between ISAV and influenza virus glycoproteins by using molecular modeling. Phylogenetic analysis shows two genogroups for each protein, one of them containing the Chilean isolates. The gene sequence of the polymerase complex and nucleoprotein indicated that they are closely related to homologues from highly pathogenic Norwegian viruses. Notably, seven of the eight mutations that are present only in the Chilean isolates are on the polymerase complex and nucleoprotein. Structural modeling of hemagglutinin-esterase shows patches of variable residues on its surface. Fusion protein modeling shows that insertions are flexible regions that could affect proteolytic processing, increasing either the accessibility or the number of recognition sites for specific proteases. We found antigenic drift processes related to insertion into the isolated segment 5 of the ISAV752_09. Our results confirm the European origin of Chilean isolates to be the result of reassortments from Norwegian ancestors.

Inhibition of brain renin-angiotensin system improves diastolic cardiac function following myocardial infarction in rats.

1. Recently, we demonstrated that oral captopril treatment improved diastolic function and attenuated cardiac remodelling after myocardial infarction (MI) in rats. Considering the feasible role of the brain renin-angiotensin system (RAS) in heart failure, in the present study we investigated the role of the captopril injected intracerebroventricularly (i.c.v.) on the progression of cardiac dysfunction. 2. Male Wistar rats underwent experimental MI or sham operation. Infarcted animals received daily i.c.v. injections of captopril (approximately 200 mg/kg; MI + Cap) or saline (MI) from 11 to 18 days after infarction. Electro- and echocardiogram assessments were performed before and after i.c.v. treatment (10 and 18 days after MI, respectively). Water and hypertonic saline ingestion were determined daily between 12 and 16 days after MI. 3. Electrocardiograms from the MI and MI + Cap groups showed signs that resembled large MI before and after i.c.v. treatment. However, despite similar systolic dysfunction observed in both groups, only captopril-treated rats exhibited reduced left ventricular (LV) dilatation and improved LV filling, as assessed by echocardiograms, and low levels of water ingestion compared with the saline-treated control group. 4. The results of the present study suggest that the brain RAS may participate in the development of cardiac dysfunction induced by ischaemia and that inhibition of the brain RAS may provide a new strategy for the prevention of diastolic dysfunction.

Propranolol inhibits myocardial infarction-induced brown adipose tissue D2 activation and maintains a low thyroid hormone state in rats.

Considering the recognized role of thyroid hormones on the cardiovascular system during health and disease, we hypothesized that type 2 deiodinase (D2) activity, the main activation pathway of thyroxine (T4)-to-triiodothyronine (T3), could be an important site to modulate thyroid hormone status, which would then constitute a possible target for ß-adrenergic blocking agents in a myocardial infarction (MI) model induced by left coronary occlusion in rats. Despite a sustained and dramatic fall in serum T4 concentrations (60-70%), the serum T3 concentration fell only transiently in the first week post-infarction (53%) and returned to control levels at 8 and 12 weeks after surgery compared to the Sham group (P<0.05). Brown adipose tissue (BAT) D2 activity (fmol T4·min-1·mg ptn-1) was significantly increased by approximately 77% in the 8th week and approximately 100% in the 12th week in the MI group compared to that of the Sham group (P<0.05). Beta-blocker treatment (0.5 g/L propranolol given in the drinking water) maintained a low T3 state in MI animals, dampening both BAT D2 activity (44% reduction) and serum T3 (66% reduction in serum T3) compared to that of the non-treated MI group 12 weeks after surgery (P<0.05). Propranolol improved cardiac function (assessed by echocardiogram) in the MI group compared to the non-treated MI group by 40 and 57%, 1 and 12 weeks after treatment, respectively (P<0.05). Our data suggested that the beta-adrenergic pathway may contribute to BAT D2 hyperactivity and T3 normalization after MI in rats. Propranolol treatment maintained low T3 state and improved cardiac function additionally.

How to Improve Speaking Skills in Plastic Surgery Training? Survey in Residents Participants in Pecha Kucha Contest.

BACKGROUND: Little is known about educational games in Plastic Surgery training. Pecha kucha game has proved to be helpful tool to improve communicative skills. This study survey in resident participants in Pecha Kucha contest assessed how to improve speaking skills in plastic surgery training. METHODS: In the second edition of Pecha Kucha contest of the Mexican Society of Plastic Surgery, a survey was conducted with the residents to know the utility of this educational game. RESULTS: Twenty-six residents participated in the survey. Most of them from the Universidad Nacional Autonoma de México. Most of the residents considered it to be a good tool in order to improve communication skills and helpful for their future practice. The amount of time to present an idea was considered enough to express an idea. The most common proportion between words and images was 20-80% in the presentation. CONCLUSION: Pecha Kucha helped to improve communication skills during residents' training. We encourage other plastic surgery societies to incorporate educational games in their national and international meetings.

Site-specific genomic integration in mammalian cells mediated by phage phiC31 integrase.

We previously established that the phage phiC31 integrase, a site-specific recombinase, mediates efficient integration in the human cell environment at attB and attP phage attachment sites on extrachromosomal vectors. We show here that phage attP sites inserted at various locations in human and mouse chromosomes serve as efficient targets for precise site-specific integration. Moreover, we characterize native "pseudo" attP sites in the human and mouse genomes that also mediate efficient integrase-mediated integration. These sites have partial sequence identity to attP. Such sites form naturally occurring targets for integration. This phage integrase-mediated reaction represents an effective site-specific integration system for higher cells and may be of value in gene therapy and other chromosome engineering strategies.

Behavioral and neuropharmacological evidence that serotonin crosses the blood-brain barrier in Coturnix japonica (Galliformes; Aves).

This study was carried out aiming to reach behavioral and neuropharmacological evidence of the permeability of the blood-brain barrier (BBB) to serotonin systemically administered in quails. Serotonin injected by a parenteral route (250-1000 microg x kg(-1), sc) elicited a sequence of behavioral events concerned with a sleeping-like state. Sleeping-like behaviors began with feather bristling, rapid oral movements, blinking and finally crouching and closure of the eyes. Previous administration of 5-HT2C antagonist, LY53857 (3 mg x kg(-1), sc) reduced the episodes of feather bristling and rapid oral movements significantly but without altering the frequency of blinking and closure of the eyes. Treatment with the 5-HT2A/2C antagonist, ketanserin (3 mg x kg(-1), sc) did not affect any of the responses evoked by the serotonin. Quipazine (5 mg x kg(-1), sc) a 5-HT2A/2C/3 agonist induced intense hypomotility, long periods of yawning-like and sleeping-like states. Previous ketanserin suppressed gaping responses and reduced hypomotility, rapid oral movements and bristling but was ineffective for remaining responses induced by quipazine. Results showed that unlike mammals, serotonin permeates the BBB and activates hypnogenic mechanisms in quails. Studies using serotoninergic agonist and antagonists have disclosed that among the actions of the serotonin, feather bristling, rapid oral movements and yawning-like state originated from activation of 5-HT2 receptors while blinking and closure of the eyes possibly require other subtypes of receptors.

Nocturnal plasma levels of melatonin in quails (Coturnix japonica) injected with l-5-hydroxy-tryptophan.

This study aimed to demonstrate the influence of the systemic administration of l-5-hydroxy-tryptophan (L-HTP) on the plasma levels of melatonin during the dark period in quails. Throughout daylight, the plasma levels of melatonin did not differ significantly, oscillating between 110.2 +/- 15.8 pg.mL(-1) and 157.4 +/- 34.8 pg.mL(-1), from 8 to 16 hours. L-HTP (25 mg.kg(-1), through the intracelomic route) administered at 18 hours lessened significantly the nocturnal increase of the plasma levels of melatonin (controls, 327.3 +/- 20.1 and 315.8 +/- 20.9 pg.mL(-1) vs. 242.1 +/- 24.8 and 217.5 +/- 21 pg.mL(-1), respectively, at 20 and 24 hours, P < 0.05). The results obtained showed that the administration of LHTP reduced the nocturnal melatonin release, possibly by bringing about an increase in serotonin synthesis and synaptic release in the pineal. Therefore, the serotoninergic transmission from the raphe towards the pineal would constitute a mechanism of modulation of the synthesis and melatonin release in quails.

Effect of L-5-Hydroxytryptophan on drinking behavior in Coturnix japonica (Temminck and Schlegel, 1849) (Galliformes: Aves): involvement of renin-angiotensin system.

The purpose of this study was to explore the role of L-5-hydroxytryptophan (L-HTP) and its relationship with the renin-angiotensin system (RAS) on the drinking behavior in Japanese quails. Normally-hydrated quails that received injections of L-HTP (12.5; 25 and 50 mg.kg-1) by the intracoelomic route (ic) expressed an increase in water intake, which was inhibited by captopril, an angiotensin converting enzyme (ACE) inhibitor. In addition, captopril also induced such a response in birds under previous fluid deprivation. High doses of captopril (35-70 mg.kg-1, sc) in normally-hydrated quails decreased the spontaneous water intake while low doses of captopril (2-5 mg.kg-1, sc) did not prompt water intake after L-HTP administration. Losartan, an AT1 receptor antagonist in mammals, did not change the water intake levels in normally-hydrated or water-deprivated birds. Serotonin (5-HT) injections did not provoke its known dipsogenic response.

Astronomy. ASASSN-15lh: A highly super-luminous supernova.

We report the discovery of ASASSN-15lh (SN 2015L), which we interpret as the most luminous supernova yet found. At redshift z = 0.2326, ASASSN-15lh reached an absolute magnitude of Mu ,AB = -23.5 ± 0.1 and bolometric luminosity Lbol = (2.2 ± 0.2) × 10(45) ergs s(-1), which is more than twice as luminous as any previously known supernova. It has several major features characteristic of the hydrogen-poor super-luminous supernovae (SLSNe-I), whose energy sources and progenitors are currently poorly understood. In contrast to most previously known SLSNe-I that reside in star-forming dwarf galaxies, ASASSN-15lh appears to be hosted by a luminous galaxy (MK ≈ -25.5) with little star formation. In the 4 months since first detection, ASASSN-15lh radiated (1.1 ± 0.2) × 10(52) ergs, challenging the magnetar model for its engine.

Cardiac effects of oxytocin: is there a role for this peptide in cardiovascular homeostasis?

Oxytocin is well known for its role in reproduction. However, evidence has emerged suggesting a role in cardiovascular and hydroelectrolytic homeostasis. Although its renal effects have been characterized, the cardiac ones have not been much studied. Therefore, we aimed to investigate the cardiac effects of oxytocin both in vivo and in vitro. In unanesthetized rats (n=6) intravenous oxytocin (1 mug) decreased dP/dt(max) by 15% (P<0.05) and heart rate by 20% (P<0.001), at the first minute after injection. dP/dt(max) was still lower in OT-treated rats than in controls (n=8) after 15 min (P<0.05), while heart rate returned to control values after 5 min. In isolated hearts, oxytocin was able to promote negative inotropic and chronotropic effects. Perfusion with 10(-5), 10(-6) and 10(-7)M oxytocin resulted in approximately 60% (P<0.01), 25% (P<0.01) and 10% (P<0.05) reduction of left ventricle developed pressure, without effect in lower concentrations (10(-10) to 10(-8) M). Also, dP/dt(max) was reduced by 45 and 20% (10(-5) e 10(-6) M; P<0.01), while diastolic pressure raised and heart rate fell only with 10(-5)M oxytocin (P<0.05). Intravenous oxytocin (1 mug; n=6) increased arterial pressure by 22% at the first minute (+23+/-3 mm Hg; P<0.001), returning to control value thereafter. Thus, oxytocin is able to promote directly negative inotropic and chronotropic effects, but its in vivo effect also involves a reflex mechanism, originated from its pressor effect.

Chronic excitotoxic lesion of the dorsal raphe nucleus induces sodium appetite.

We determined if the dorsal raphe nucleus (DRN) exerts tonic control of basal and stimulated sodium and water intake. Male Wistar rats weighing 300-350 g were microinjected with phosphate buffer (PB-DRN, N = 11) or 1 microg/0.2 microl, in a single dose, ibotenic acid (IBO-DRN, N = 9 to 10) through a guide cannula into the DRN and were observed for 21 days in order to measure basal sodium appetite and water intake and in the following situations: furosemide-induced sodium depletion (20 mg/kg, sc, 24 h before the experiment) and a low dose of dietary captopril (1 mg/g chow). From the 6th day after ibotenic acid injection IBO-DRN rats showed an increase in sodium appetite (12.0 +/- 2.3 to 22.3 +/- 4.6 ml 0.3 M NaCl intake) whereas PB-DRN did not exceed 2 ml (P < 0.001). Water intake was comparable in both groups. In addition to a higher dipsogenic response, sodium-depleted IBO-DRN animals displayed an increase of 0.3 M NaCl intake compared to PB-DRN (37.4 +/- 3.8 vs 21.6 +/- 3.9 ml 300 min after fluid offer, P < 0.001). Captopril added to chow caused an increase of 0.3 M NaCl intake during the first 2 days (IBO-DRN, 33.8 +/- 4.3 and 32.5 +/- 3.4 ml on day 1 and day 2, respectively, vs 20.2 +/- 2.8 ml on day 0, P < 0.001). These data support the view that DRN, probably via ascending serotonergic system, tonically modulates sodium appetite under basal and sodium depletion conditions and/or after an increase in peripheral or brain angiotensin II.

Evidence indicating participation of the serotonergic system in controlling feeding behavior in Coturnix japonica (Galliformes: Aves).

We investigated participation of the brain serotonergic system in food intake control by using oral and systemic administration of serotonin precursors in quails (Coturnix japonica). Dietary supplemental tryptophan (0.1-50.0 g/kg) provoked a dose-dependent inhibition of food intake during a 5-h observation period, which persisted up to 24 h for doses of 30.0 and 50.0 g/kg. Normally fed and fasted animals treated with hydroxytryptophan (12.5-50.0 mg/kg) by the intracoelomic route showed an acute inhibition of food intake. Hypophagia in fasted birds was only effective when the precursor was administered immediately before food presentation. A similar response was obtained by administering serotonin (0.125-2.5 mg/kg, sc), with animals showing a hypnogenic response within the first ten minutes after administration, suggesting that, in contrast to mammals, the amine crosses the blood-brain barrier in quails. Administration of hydroxytryptophan at all doses tested induced significant dipsogenic behavior despite the concomitant hypnogenic response. The results suggest the involvement of serotonergic pathways in food intake control in quails and also show, for the first time, hypnogenic action induced by serotonin and a hyperdipsic effect elicited by hydroxytryptophan.

Influence of serotonergic transmission anD postsynaptic 5-HT2C action on the feeding behavior of Coturnix japonica (Galliformes: Aves).

We investigated the role of 5-HT2C receptors and serotonergic transmission in the feeding behavior control of quails. Administration of serotonin releaser, fenfluramine (FEN) and 5-HT2C agonists, mCPP and MK212, 1.0 and 3.3 mg/Kg induced significant inhibition of food intake in previously fasted fowls (0.71 +/- 0.18 g and 0.47 +/- 0.2 g; 0.49 +/- 0.22 g and 0.48 +/- 0.29 g; 0.82 +/- 0.13 g and 0.71 +/- 0.16 g, respectively). Control groups ranged from 2.89 +/- 0.21 g to 2.97 +/- 0.22 g, 60 min after reintroduction of food, P < 0.0001). Similar results were obtained with normally fed quails. Both serotonin releaser and 5-HT2C agonists, in a 3.3 mg/Kg dose, induced hypophagy (FEN, 0.78 +/- 0.08 g; mCPP, 0.89 +/- 0.07 g; MK212, 1.25 +/- 0.17 g vs. controls, 2.05 +/- 0.12 g, 120 min after food was presented, P < 0.0001 to P < 0.01). Previous administration of 5-HT2C antagonist, LY53857 (5.0 mg/Kg) blocked the hypophagic response induced by 5-HT2C agonists 60 min after food was reintroduced. Current data show a modulatory role of serotonin release and postsynaptic 5-HT2C receptors in the feeding behavior of quails.

Simultaneous measurement of cerebral blood flow and energy metabolites in piglets using deuterium and phosphorus nuclear magnetic resonance.

This report demonstrates the feasibility of using deuterium (2H) and phosphorus (31P) nuclear magnetic resonance (NMR) spectroscopy to make multiple simultaneous determinations of changes in cerebral blood flow, brain intracellular pH, and phosphorylated metabolites for individual animals. In vivo spectra were obtained from the brains of newborn piglets immediately following an intracarotid bolus injection of deuterium oxide. Experiments were performed at magnetic field strengths of 1.9 T (2H NMR only) or 4.7 T (interleaved 2H and 31P NMR). The rate of clearance of deuterium signal was used to calculate cerebral perfusion rates (CBFdeuterium) during a stable control physiologic state and conditions known to alter blood flow. CBFdeuterium values measured at 1.9 T under conditions of control (normocarbia, normotension), hypercarbia, hypocarbia, and varying degrees of ischemia induced by hypotension showed a significant positive correlation with values measured simultaneously using radiolabeled microspheres (CBFdeuterium = 0.4 x CBFmicrospheres + 8; r = 0.8). Simultaneous interleaved 2H and 31P NMR measurements under control conditions indicate that brain energy metabolites and intracellular pH remained at constant levels during the time course of the administration and clearance of deuterium oxide. Also, brain phosphorylated metabolites and intracellular pH did not differ significantly from their preinjection levels. Under control physiologic conditions, CBFdeuterium varied by +/- 6% and phosphorylated metabolite levels did not show a significant change with time, as measured from 15 blood flow determinations collected over 4 h. The results indicate that CBFdeuterium determinations have excellent reproducibility and do not affect brain energy metabolite levels. The procedures described here have the potential to bring a novel methodology to bear on investigating the relationship between cerebral perfusion and energy status during conditions such as ischemia or asphyxia.

Phage R4 integrase mediates site-specific integration in human cells.

The R4 integrase is a site-specific, unidirectional recombinase derived from the genome of phage R4 of Streptomyces parvulus. Here we define compact attB and attP recognition sites for the R4 integrase and express the enzyme in mammalian cells. We demonstrate that R4 integrase functions in human cells, performing efficient and precise recombination between R4 attB and attP sites cloned on an extrachromosomal vector. We also provide evidence that the enzyme can mediate integration of an incoming plasmid bearing an attB or attP site into endogenous sequences in the human genome. Furthermore, when R4 attB and attP sites are placed into the human genome, either by random integration or at a specific sequence by using the phi C31 integrase, they act as targets for integration of incoming plasmids bearing R4 att sites. The R4 integrase has immediate utility as a site-specific integration tool for genome engineering, as well as potential for further development.

Routine HLA DRB/DQB oligonucleotide typing by a non-radioactive dot-blot micromethod.

In the last few years the clinical need for HLA genotyping has become evident. However, the routine use of PCR-based DNA typing techniques has been hampered by economical and/or technical considerations. The classical PCR-SSO (product-dot) method has been widely tested and proven to be useful for large-scale HLA DNA typing. However, it is not a suitable method for routine typing of single samples because it takes several days. Using primers and probes for sequences identical to those compiled by the Eleventh International Histocompatibility Workshop, we designed a non-radioactive dot-blot technique in which each hybridization reaction is performed in a microtiter plate well containing PCR-amplified DNA that has been previously dotted on a small nylon membrane, so that a large number of oligonucleotide probes tailed with biotin-14-dATP can be simultaneously tested against the same sample. We studied 23 B-lymphoblastoid cell lines of known HLA genotype to test the method and, so far, it has been validated on more than 100 patients and healthy relatives typed prospectively. This simple, rapid, inexpensive PCR-SSO dot-blot micromethod makes DRB/DQB DNA typing of single samples possible in a short period of time, and is therefore an attractive alternative to serological typing in routine medical practice.