Molecular evidence for convergent evolution and allopolyploid speciation within the Physcomitrium-Physcomitrella species complex.

BACKGROUND: The moss Physcomitrella patens (Hedw.) Bruch & Schimp. is an important experimental model system for evolutionary-developmental studies. In order to shed light on the evolutionary history of Physcomitrella and related species within the Funariaceae, we analyzed the natural genetic diversity of the Physcomitrium-Physcomitrella species complex. RESULTS: Molecular analysis of the nuclear single copy gene BRK1 reveals that three Physcomitrium species feature larger genome sizes than Physcomitrella patens and encode two expressed BRK1 homeologs (polyploidization-derived paralogs), indicating that they may be allopolyploid hybrids. Phylogenetic analyses of BRK1 as well as microsatellite simple sequence repeat (SSR) data confirm a polyphyletic origin for three Physcomitrella lineages. Differences in the conservation of mitochondrial editing sites further support hybridization and cryptic speciation within the Physcomitrium-Physcomitrella species complex. CONCLUSIONS: We propose a revised classification of the previously described four subspecies of Physcomitrella patens into three distinct species, namely Physcomitrella patens, Physcomitrella readeri and Physcomitrella magdalenae. We argue that secondary reduction of sporophyte complexity in these species is due to the establishment of an ecological niche, namely spores resting in mud and possible spore dispersal by migratory birds. Besides the Physcomitrium-Physcomitrella species complex, the Funariaceae are host to their type species, Funaria hygrometrica, featuring a sporophyte morphology which is more complex. Their considerable developmental variation among closely related lineages and remarkable trait evolution render the Funariaceae an interesting group for evolutionary and genetic research.

FI on-line chemiluminescence reaction for determination of MCPA in water samples.

This paper reports an economic, simple, and rapid FI-CL method for the determination of MCPA. This method requires simple instrumentation and it is fast enough to be used in routine analyses. A chemiluminescence signal is generated by reaction between photodegraded MCPA and ferricyanide solution in alkaline medium. All physical and chemical parameters in the flow injection chemiluminescence system were optimized in the experimental setting. To eliminate interference, a solid-phase extraction stage with SDB-1 cartridges and ethanol elution is applied. The signal-MCPA concentration relation is linear in concentration intervals between 0.0015 and 0.6 µg·mL(-1). The calibration lines are statistically similar in different working conditions: standards with ethanol without extraction and standards with ethanol and extraction, allowing standards to be excluded from the extraction step, which simplifies the process. The detection limit (DL) is 0.5 ng·mL(-1), which is the same order as the maximum limit established in legislation regarding pesticide limits in water destined for human consumption. A DL of 0.13 ng·mL(-1) can be reached if a sample of 100 mL is preconcentrated. The interday variance coefficient is 3% and the sample throughput is 90 h(-1). The water analysis method is efficient with relative error percentages lower than 5% with respect to the added concentration.

Correction of distal limb prolapse of a diverting colostomy by stapling under sedation.

AIM: To describe a surgical technique to treat colostomy prolapse as a day case procedure with the patient under sedation and analgesia. METHOD: A 60-mm GIA Universal Stapler is inserted into the lumen of the prolapsed colon at right angles to the contour of the abdominal wall. Several firings are then made to completely divide the prolapsed colon. The instrument is then placed parallel to the skin to remove the prolapsed portion leaving 1-2 cm of bowel above the level of the skin. RESULTS: Two patients underwent the procedure. The operation times were 30 and 13 min. Both took oral liquids 2 h after surgery and solids 2 h later. They were discharged at 24 and 4 h after surgery, respectively. No postoperative pain was reported in either case. At 14 and 6 months of follow-up there has been no recurrence. CONCLUSION: Stapling treatment of prolapsed colostomies has the advantage of being an extra-abdominal procedure. It is performed under sedation and analgesia, the operation time is very short, recovery to normal life is rapid and there is less likelihood of complications by avoiding a laparotomy.

The rye transcription factor ScSTOP1 regulates the tolerance to aluminum by activating the ALMT1 transporter.

Plants have evolved different mechanisms to increase their tolerance to aluminum (Al) toxicity and low pH in the soil. The Zn finger transcription factor SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) plays an essential role in the adaptation of plants to Al and low pH stresses. In this work, we isolated the ScSTOP1 gene from rye (Secale cereale L.), which is located on chromosome 3RS. The ectopic expression of ScSTOP1 complements the Arabidopsis stop1 mutation in terms of root growth inhibition due to Al and pH stress, as well as phosphate starvation tolerance, suggesting that rye ScSTOP1 is a functional ortholog of AtSTOP1. A putative STOP1 binding motif was identified in the promoter of a well-known STOP1 target from rye and Arabidopsis and was later corroborated by genomic DAP-seq analyses. Coexpression analyses verified that ScSTOP1 activated the promoter of ScALMT1. We have also identified a putative phosphorylatable serine in STOP1 that is phylogenetically conserved and critical for such activation. Our data indicated that ScSTOP1 also regulated Al and pH tolerance in rye.

Isolation and characterization of a new MATE gene located in the same chromosome arm of the aluminium tolerance (Alt1) rye locus.

Aluminium (Al) toxicity is the major constraint for crop productivity in acid soils. Wild rye species (Secale spp.) exhibit high Al tolerance, being a good source of genes related to this trait. The Alt1 locus located on the 6RS chromosome arm is one of the four main loci controlling Al tolerance in rye and is known to harbour major genes but, so far, none have been found. Through synteny among the short arm of the rye chromosome 6R and the main grass species, we found a candidate MATE gene for the Atl1 locus, later named ScMATE3, which was isolated and characterized in different Secale species. The sequence comparisons revealed both intraspecific and interspecific variability, with high sequence conservation in the Secale genus. SNP with replacement substitution that changed the structure of the protein and can be involved in the Al tolerance trait were found in ScMATE3 gene. The predicted subcellular localization of ScMATE3 is the vacuolar membrane which, together with the phylogenetic relationships performed with other MATE genes of the Poaceae related to Al detoxification, suggest involvement of ScMATE3 in an internal tolerance mechanism. Moreover, expression studies of this gene in rye corroborate its contribution in some Al resistance mechanisms. The ScMATE3 gene is located on the 6RS chromosome arm between the same markers in which the Alt1 locus is involved in Al resistance mechanisms in rye, thus being a good candidate gene for this function.

[Combined regional-general anesthesia with use of the Proseal laryngeal mask during prolonged peripheral plastic surgery]. / Anestesia regional combinada con mascarilla laríngea proseal en cirugía plástica periférica prolongada.

The ProSeal laryngeal mask offers a better interface for controlled ventilation than does a traditional mask because of the ProSeal's improved airway seal and the possibility of draining the digestive tract. Limb reconstructive surgery is normally a long procedure involving the use of grafts or flaps located at a distance from the lesion. Regional anesthesia is therefore of limited use. We report a series of 24 adult patients who underwent peripheral plastic surgery under combined regional and general intravenous anesthesia and in whom the ProSeal mask was used. Data recorded included assessment of the airway and intubation difficulty, disease and duration of the procedure, type of regional anesthesia, respiratory and hemodynamic variables, patient satisfaction, laryngeal complications, and postoperative analgesia. All patients were adequately ventilated. One episode of bradycardia was controlled with atropine. In the immediate postoperative period, no patient presented laryngeal complications and only 1 patient reported significant pain (8 on a visual analog scale). The ProSeal mask was a good choice for use with combined regional-general anesthesia, ensuring control of the airway without causing complications. Analgesia was adequate in most cases.

Effectiveness of commercial face masks to reduce personal PM exposure.

Cycling and walking are promoted as means of transportation which can contribute to the reduction of traffic pollution in urban areas. However, cyclists and pedestrians may be exposed to high concentrations of air pollutants due to their proximity to vehicle emissions. Commercial face mask respirators are widely used, in both developing and developed countries, as an individual protective measure against particle pollution. However scientific data on the efficacy of face mask respirators in reducing airborne particle exposure is limited. In this study, a custom experimental set-up was developed in order to measure the effectiveness of nine different respirators under real environmental conditions in terms of particle mass concentration below 2.5 µm (PM2.5), particle number concentration (PNC), Lung Deposited Surface Area (LDSA) and Black Carbon concentration (BC). Face mask performances were assessed in a typical traffic affected urban background environment in the city of Barcelona under three different simulated breathing rates to investigate the influence of flow rate. Results showed a median face mask effectiveness for PM2.5 equal to 48% in a range of 14-96%, 19% in a range of 6%-61% for BC concentration, 19% in a range of 4%-63% for PNC and 22% in a range of 5%-65% for LDSA. For each pollutant under investigation, the best performance was found always with the same mask (N7) although it is not the most expensive (in a range of price of 1 to 44, its cost was 20 euros), which has a filter on the entire surface except for the 2 exhalation valves where air cannot enter but just exit and shows a good fit on the dummy head.

Glial expression of cannabinoid CB(2) receptors and fatty acid amide hydrolase are beta amyloid-linked events in Down's syndrome.

Recent data suggest that the endocannabinoid system (ECS) may be involved in the glial response in different types of brain injury. Both acute and chronic insults seem to trigger a shift in the pattern of expression of some elements of this system from neuronal to glial. Specifically, data obtained in human brain tissue sections from Alzheimer's disease patients showed that the expression of cannabinoid receptors of the CB(2) type is induced in activated microglial cells while fatty acid amide hydrolase (FAAH) expression is increased in reactive astrocytes. The present study was designed to determine the time-course of the shift from neuronal to glial induction in the expression of these proteins in Down's syndrome, sometimes referred to as a human model of Alzheimer-like beta-amyloid (Abeta) deposition. Here we present immunohistochemical evidence that both CB(2) receptors and FAAH enzyme are induced in Abeta plaque-associated microglia and astroglia, respectively, in Down's syndrome. These results suggest that the induction of these elements of the ECS contributes to, or is a result of, amyloid deposition and subsequent plaque formation. In addition, they confirm a striking differential pattern of distribution of FAAH and CB(2) receptors.

Cannabinoid CB2 receptors in human brain inflammation.

The presence of functional cannabinoid CB2 receptors in the CNS has provoked considerable controversy over the past few years. Formerly considered as an exclusively peripheral receptor, it is now accepted that it is also present in limited amounts and distinct locations in the brain of several animal species, including humans. Furthermore, the inducible nature of these receptors under neuroinflammatory conditions, in contrast to CB1, makes them attractive targets for the development of novel therapeutic approaches. In fact, the undesired psychoactive effects caused by CB1 activation have largely limited the clinical use of cannabinoid-related compounds that act on these receptors. In this review some recent findings on the antiinflammatory properties of CB2 receptors are presented, as well as new perspectives that have been obtained based on studies of human postmortem brain samples. In addition, various working hypotheses are also proposed and discussed.

Differential clinical, structural and P300 parameters in schizophrenia patients resistant to conventional neuroleptics.

Schizophrenia is a heterogeneous clinical condition that may reflect a variety of biological processes. In particular, treatment-resistant (TR) schizophrenia may have a distinct neurobiological substrate. Within the context of clinical data, a simultaneous study with different imaging techniques could help to elucidate differences in cerebral substrates among schizophrenia patients with different responses to treatment. In the present work we used a set of biological data (basal and longitudinal volumetry, and P300 event-related potential measurements) to compare TR and treatment-responsive chronic schizophrenia patients with healthy controls. The TR patients showed higher baseline clinical scores, a more severe basal profile of brain alterations, as well as a different outcome as regards to volume deficits. These data support the notion that biological substrates vary among groups of different psychotic patients, even when they have the same diagnosis, and that those substrates may be related to the response to treatment.

Molecular cloning of TaMATE2 homoeologues potentially related to aluminium tolerance in bread wheat (Triticum aestivum L.).

Recently, members of the MATE family have been implicated in aluminium (Al) tolerance by facilitating citrate efflux in plants. The aim of the present work was to perform a molecular characterisation of the MATE2 gene in bread wheat. Here, we cloned a member of the MATE gene family in bread wheat and named it TaMATE2, which showed the typical secondary structure of MATE-type transporters maintaining all the 12 transmembrane domains. Amplification in Chinese Spring nulli-tetrasomic and ditelosomic lines revealed that TaMATE2 is located on the long arm of homoeologous group 1 chromosomes. The transcript expression of TaMATE2 homoeologues in two diverse bread wheat genotypes, Barbela 7/72/92 (Al-tolerant) and Anahuac (Al-sensitive), suggested that TaMATE2 is expressed in both root and shoot tissues of bread wheat, but mainly confined to root rather than shoot tissues. A time-course analysis of TaMATE2 homoeologue transcript expression revealed the Al responsive expression of TaMATE2 in root apices of the Al-tolerant genotype, Barbela 7/72/92. Considering the high similarity of TaMATE2 together with similar Al responsive expression pattern as of ScFRDL2 from rye and OsFRDL2 from rice, it is likely that TaMATE2 also encodes a citrate transporter. Furthermore, the TaMATE2-D homoeologue appears to be near the previously reported locus (wPt0077) on chromosome 1D for Al tolerance. In conclusion, molecular cloning of TaMATE2 homoeologues, particularly TaMATE2-D, provides a plausible candidate for Al tolerance in bread wheat that can be used for the development of more Al-tolerant cultivars in this staple crop.

Dorsolateral prefrontal N-acetyl-aspartate concentration in male patients with chronic schizophrenia and with chronic bipolar disorder.

OBJECTIVES: A study of N-acetyl-aspartate (NAA) can provide data of interest about cortical alterations in psychotic illnesses. Although a decreased NAA level in the cerebral cortex is a replicated finding in chronic schizophrenia, the data are less consistent for bipolar disease. On the other hand, it is likely that NAA values in schizophrenia may differ in men and women. METHODS: We used proton magnetic resonance spectroscopy ((1)H MRS) to examine NAA levels in the prefrontal cortex in two groups of male patients, one with schizophrenia (n=11) and the other with bipolar disorder (n=13) of similar duration, and compared them to a sample of healthy control males (n=10). Additionally, we compared the degree of structural deviations from normal volumes of gray matter (GM) and cerebrospinal fluid (CSF) in the dorsolateral prefrontal cortex. RESULTS: Compared to controls, schizophrenia and bipolar patients presented decreased NAA to creatine ratios, while only the schizophrenia group showed an increase in CSF in the dorsolateral prefrontal region. There were no differences in choline to creatine ratios among the groups. CONCLUSIONS: These data suggest that the decrease in NAA in the prefrontal region may be similar in schizophrenia and bipolar disorder, at least in the chronic state. However, cortical CSF may be markedly increased in schizophrenia patients.

Preparation of organic monolithic columns in polytetrafluoroethylene tubes for reversed-phase liquid chromatography.

In this work, a method for the preparation and anchoring of polymeric monoliths in a polytetrafluoroethylene (PTFE) tubing as a column housing for microbore HPLC is described. In order to assure a covalent attachment of the monolith to the inner wall of the PTFE tube, a two-step procedure was developed. Two surface etching reagents, a commercial sodium naphthalene solution (Fluoroetch®), or mixtures of H2O2 and H2SO4, were tried and compared. Then, the obtained hydroxyl groups on the PTFE surface were modified by methacryloylation. Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy and scanning electron microscopy (SEM) confirmed the successful modification of the tubing wall and the stable anchorage of monolith to the wall, respectively. Special emphasis was also put on the reduction of the unwanted effects of shrinking of monolith during polymerization, by using an external proper mold and by selecting the adequate monomers in order to increase the flexibility of the polymer. Poly(glycidyl methacrylate-co-divinylbenzene) monoliths were in situ synthesized by thermal polymerization within the confines of surface-vinylized PTFE tubes. The modified PTFE tubing tightly held the monolith, and the monolithic column exhibited good pressure resistance up to 20 MPa. The column performance was also evaluated via the isocratic separation of a series of alkylbenzenes in the reversed-phase mode. The optimized monolithic columns gave plate heights ranged between 70 and 80 µm. The resulting monoliths were also satisfactorily applied to the separation of proteins.

Liver transplantation: experience with our first 100 cases.

OBJECTIVES: Our goal was to evaluate the results of the first 100 liver transplants performed in our institution. METHODS: We retrospectively analyzed the first 100 liver transplants undertaken in adults from November 2001 to August 2005. RESULTS: The mean age of the recipients was 50 years (20 to 69) and 73% were men. The mean waiting time was 35 days. The mean age of the donors was 60 years (15 to 87), and 60% were men. One-year patient and graft survival rates were 93% and 90%, respectively. Three-year patient and graft survival rates were 85% and 82%, respectively. The need for retransplantation was 3%. Surgical complications included hepatic artery stenoses, 2%; hepatic artery thromboses, 2%; biliary leaks, 6%; and biliary stenoses, 14%. CONCLUSIONS: These results are no different from the overall results for liver transplantation in Spain during the same period.

Molecular characterization of Trichinella genotypes by inter-simple sequence repeat polymerase chain reaction (ISSR-PCR).

A bulk analysis of inter-simple sequence repeat-polymerase chain reaction (ISSR-PCR) provides a quick, reliable, and highly informative system for DNA banding patterns that permit species identification. The present study evaluates the applicability of this system to Trichinella species identification. After a single amplification carried out on a single larva with the primer 816([CA]nRY) under high stringency conditions, which provide high reproducibility, we were able to identify by consistent banding patterns 5 sibling species: Trichinella spiralis (ISS48), 2 Trichinella britovi isolates (ISS11 and ISS86), Trichinella murrelli (ISS35), Trichinella nativa (ISS71), Trichinella nelsoni (ISS29); 3 additional Trichinella genotypes: T8 (ISS149), T9 (ISS408 and ISS409), and T6 (ISS34); and the nonencapsulated species Trichinella pseudospiralis (ISS13). Moreover, 33 new Trichinella isolates from 2 zoogeographical regions were unequivocally identified. All Trichinella isolates have shown an identical pattern with those produced by the reference strain. According to these data, we have demonstrated that ISSR-PCR is a robust technique that emerges as a useful new application for the molecular identification of Trichinella isolates in epidemiological studies.

Molecular diversity and genetic relationships in Secale.

The objective of this study was to quantify the molecular diversity and to determine the genetic relationships among Secale spp. and among cultivars of Secale cereale using RAPDs, ISSRs and sequence analysis of six exons of ScMATE1 gene. Thirteen ryes (cultivated and wild) were genotyped using 21 RAPD and 16 ISSR primers. A total of 435 markers (242 RAPDs and 193 ISSRs) were obtained, with 293 being polymorphic (146 RAPDs and 147 ISSRs). Two RAPD and nine ISSR primers generated more than 80% of polymorphism. The ISSR markers were more polymorphic and informative than RAPDs. Further, 69% of the ISSR primers selected achieved at least 70% of DNA polymorphism. The study of six exons of the ScMATE1 gene also demonstrated a high genetic variability that subsists in Secale genus. One difference observed in exon 1 sequences from S. vavilovii seems to be correlated with Al sensitivity in this species. The genetic relationships obtained using RAPDs, ISSRs and exons of ScMATE1 gene were similar. S. ancestrale, S. kuprijanovii and S. cereale were grouped in the same cluster and S. segetale was in another cluster. S. vavilovii showed evidences of not being clearly an isolate species and having great intraspecific differences.

Pancreatic islet GLUT2 glucose transporter mRNA and protein expression in humans with and without NIDDM.

GLUT2 glucose transporter mRNA has been shown to be underexpressed in pancreatic islets of numerous animal models of non-insulin-dependent diabetes mellitus (NIDDM). It has been proposed that this molecular defect contributes to the pathogenesis of diabetes, although information concerning the expression of GLUT2 in human pancreatic islet tissue is lacking. In contrast to the high abundance of GLUT2 in rat islets, human islets were found to express distinctly low levels of this glucose transporter mRNA and protein. Thus, a sensitive competitive reverse transcription-polymerase chain reaction assay was developed to quantify human GLUT2 mRNA. We obtained pancreases from 4 human organ donors with previously diagnosed NIDDM and 11 nondiabetic donors and found no significant differences in GLUT2 mRNA between the two groups. GLUT2 mRNA was 0.24 +/- 0.08 amol/micrograms RNA (mean +/- SE) in pancreases from humans with diabetes and 0.27 +/- 0.06 amol/microgram RNA in those without this diagnosis. Similarly, human pancreatic islet GLUT2 protein was measured by immunoblot and found to be present at similar levels in two individuals with diabetes relative to six control samples. These results thus demonstrate the existence of species differences in the abundance of islet GLUT2 mRNA and protein. Furthermore, the analysis of islet GLUT2 in a small sample of human organ donors with and without diabetes raises the possibility that decreased beta-cell GLUT2 may not represent a widespread feature of humans with NIDDM.

Association of Isozymes with a Reciprocal Translocation in Cultivated Rye (SECALE CEREALE L.).

In rye (Secale cereale L. cv. "Ailés") the progeny of a cross between a structural heterozygote for a reciprocal translocation (involving the 1R chromosome) and a homozygote for the standard chromosome arrangement were analyzed for the electrophoretic patterns of eight different leaf isozymes and also for their meiotic configuration at metaphase I.--The Got-3 and Mdh-2b loci are linked to each other and also to the reciprocal translocation. The Mdh-2b locus is located in the interstitial segment of the 3Rq chromosome arm, with an estimated distance of 8 cM to the breakpoint. Therefore, the reciprocal translocation involves the 1R and 3R chromosomes.--Also, the Mdh-1 and 6-Pgd-2 loci are linked (16 +/- 3 cM) and have been located on the 2Rq arm. Finally, the Per-3 and Per-4 loci are located on the 2Rp chromosome arm at an estimated distance of 26 +/- 4 cM.

On the consequences of aluminium stress in rye: repression of two mitochondrial malate dehydrogenase mRNAs.

Plants have developed several external and internal aluminium (Al) tolerance mechanisms. The external mechanism best characterised is the exudation of organic acids induced by Al. Rye (Secale cereale L.), one of the most Al-tolerant cereal crops, secretes both citrate and malate from its roots in response to Al. However, the role of malate dehydrogenase (MDH) genes in Al-induced stress has not been studied in rye. We have isolated the ScMDH1 and ScMDH2 genes, encoding two different mitochondrial MDH isozymes, in three Al-tolerant rye cultivars (Ailés, Imperial and Petkus) and one sensitive inbred rye line (Riodeva). These genes, which have seven exons and six introns, were located on the 1R (ScMDH1) and 3RL (ScMDH2) chromosomes. Exon 1 of ScMDH1 and exon 7 of ScMDH2 were the most variable among the different ryes. The hypothetical proteins encoded by these genes were classified as putative mitochondrial MDH isoforms. The phylogenetic relationships obtained using both cDNA and protein sequences indicated that the ScMDH1 and ScMDH2 proteins are orthologous to mitochondrial MDH1 and MDH2 proteins of different Poaceae species. The expression studies of the ScMDH1 and ScMDH2 genes indicate that it is more intense in roots than in leaves. Moreover, the amount of their corresponding mRNAs in roots from plants treated and not treated with Al was higher in the tolerant cultivar Petkus than in the sensitive inbred line Riodeva. In addition, ScMDH1 and ScMDH2 mRNA levels decreased in response to Al stress (repressive behaviour) in the roots of both the tolerant Petkus and the sensitive line Riodeva.

The role of two superoxide dismutase mRNAs in rye aluminium tolerance.

Aluminium (Al) is the main factor that limits crop production in acidic soils. There is evidence that antioxidant enzymes such as superoxide dismutase (SOD) play a key role against Al-induced oxidative stress in several plant species. Rye is one of the most Al-tolerant cereals and exudes both citrate and malate from the roots in response to Al. The role of SOD against Al-induced oxidative stress has not been studied in rye. Al accumulation, lipid peroxidation, H2O2 production and cell death were significantly higher in sensitive than in tolerant rye cultivars. Also, we characterised two genes for rye SOD: ScCu/ZnSOD and ScMnSOD. These genes were located on the chromosome arms of 2RS and 3RL, respectively, and their corresponding hypothetical proteins were putatively classified as cytosolic and mitochondrial, respectively. The phylogenetic relationships indicate that the two rye genes are orthologous to the corresponding genes of other Poaceae species. In addition, we studied Al-induced changes in the expression profiles of mRNAs from ScCu/ZnSOD and ScMnSOD in the roots and leaves of tolerant Petkus and sensitive Riodeva rye. These genes are mainly expressed in roots in both ryes, their repression being induced by Al. The tolerant cultivar has more of both mRNAs than the sensitive line, indicating that they are probably involved in Al tolerance.