A novel pleural-bladder pump for the management of recurrent malignant pleural effusions: a feasibility animal study.

BACKGROUND: Recurrent malignant pleural effusions (MPE) are common and associated with significant morbidity in cancer patients. A new pump connecting the pleural cavity and the bladder may have application for the management of recurrent MPE. In a pre-clinical study, we investigated the utility of this pump in healthy pigs. METHODS: A novel pump system (Pleurapump® system) was inserted into four pigs under general anaesthesia. A tunnelled-pleural catheter was connected to a subcutaneously implanted pump while the urinary bladder was connected by percutaneous technique. Animals were ventilated mechanically and pump functioning was tested using a range of ventilation parameters and spontaneous breathing. Fluid was added to the pleural space to mimic pleural effusion and to assess the effectiveness of the pump at removing fluid to the bladder. RESULTS: The 'pleurapump' system successfully transported fluid from the pleural cavity to the bladder. Pressure variations caused by respiration and variations in the amount of fluid in the pleural cavity had no impact on the pumping. Pumping stopped when the pleural cavity was drained. CONCLUSION: This pump can be implanted into pigs and successfully removed fluid from the pleural cavity to the bladder and may represent a new treatment for management of recurrent MPE. Evaluation in humans is planned.

Surgical technique for placement of the automated low flow ascites pump (Alfapump).

Approximately 10% of patients with ascites associated with cirrhosis fail to respond to dietary rules and diuretic treatment and therefore present with refractory ascites. In order to avoid iterative large-volume paracentesis in patients with contraindication to TIPS, the automated low flow ascites pump system (Alfapump) was developed to pump ascites from the peritoneal cavity into the urinary bladder, where it is eliminated spontaneously by normal micturition. This manuscript reports the surgical technique for placement of the Alfapump.

Treatment of refractory ascites with an automated low-flow ascites pump in patients with cirrhosis.

BACKGROUND: Refractory ascites (RA) is a frequent complication of cirrhosis, requiring large volume paracentesis or placement of a transjugular intrahepatic portosystemic shunt (TIPSS). The automated low-flow ascites pump (alfapump, Sequana Medical AG, Zurich, Switzerland) is an innovative treatment option for patients with RA. AIM: To assess safety and efficacy of this treatment in patients with a contraindication to TIPSS. METHODS: Fifty-six patients (43 males; mean age 62 years) from centres in Germany, Switzerland, UK and Spain were included and followed for up to 24 months. Complications, device deficiencies, paracentesis frequency and patient survival were recorded. RESULTS: At the time of this analysis, 3 patients completed the 24-month observation period, monitoring of 3 was ongoing, 9 underwent liver transplantation, 17 patients were withdrawn due to serious adverse events and 23 patients died. Most frequently observed technical complication was blocking of the peritoneal catheter. Twenty-three pump-related reinterventions (17 patients) and 12 pump exchanges (11 patients) were required during follow-up. The pump system was explanted in 48% of patients (in 17 patients due to serious adverse events, in 9 at the time of liver transplantation and in 1 due to recovery from RA). Median frequency of paracentesis dropped from 2.17 to 0.17 per month. CONCLUSIONS: The alfapump can expand therapeutic options for cirrhotic patients with RA. Continuous drainage of ascites in a closed loop automated system led to significant reduction in paracentesis frequency. Technical and procedural improvements are required to reduce the rate of adverse events and reinterventions. https://clinicaltrials.gov/ct2/show/NCT01532427.

[Prognostic value of the reception, attendance and classification of patients in the emergency department of a tertiary hospital]. / Estudio de la validez pronóstica de la recepción, acogida y clasificación de pacientes en el área de urgencias en un hospital terciario.

BACKGROUND: The objective of this investigation is to study the usefulness, validity and reproducibility of the reception, attendance and classification of emergency cases employed by nurses in the emergency department of a tertiary hospital (Hospital de Navarra, Pamplona, Spain) (RACHN). MATERIAL AND METHODS: By studying the proportion of emergencies classified according to our triage system (reception, attendance and classification [recepción, acogida y clasificación] at the Hospital de Navarra, RACHN), we carried out a transversal descriptive study between 3rd November and 17th November 2003 to evaluate the concordance between the level of severity assigned by a nurse using the RACHN triage system and that determined by a senior doctor. In addition, we evaluated the discrepancy between the degree of severity assigned by a nurse using the triage system and the level of severity determined by a senior doctor and that between the request for complementary tests by the nurse with the opinion of a senior doctor. RESULTS: During the study period, 85.3% of the emergency cases were assigned severity levels using the 4 level scale RACHN triage system. The kappa index for severity assigned by the nurse was 0.76 (95%CI: 0.66-0.86) and that of the senior doctor, 0.71 (95% CI: 0.60-0.81). The discrepancy in severity was 26.1%. The request for complementary tests by the nurse proved correct in 95.2% of the cases, as determined by the senior doctor. CONCLUSION: The RACHN triage system depends primarily on the protocol established by the nurse that carries out triage. We have found a good degree of concordance between triage carried out by the nurse using the RACHN and that determined by the doctor. We believe the discrepancy can be reduced by establishing a five level scale of severity and by carrying out periodic reviews of the RACHN.

Development and mapping of a codominant SCAR marker linked to the andromonoecious gene of melon.

Monoecy is an important goal for melon breeding because of the agronomic advantages it provides to parental lines in that they do not require hand emasculation to develop monoecious F1 hybrids, the latter producing fruits of higher quality. Monoecious phenotype is conferred by the dominant allele of the andromonoecious (a) gene, whereas recessive homozygous plants are andromonoecious. A bulked segregant analysis (BSA) approach performed in a set of 38 double-haploid lines has allowed us to identify an AFLP marker linked to the a gene at 3.3 cM. Following cloning and sequencing of the AFLP fragment, specific PCR primers were designed and used in the amplification of a codominant SCAR marker. Using a backcrossed mapping population of 530 plants, the SCAR marker could be mapped near the a locus (5.5 cM). Size difference between the two allelic SCAR fragments is 42 bp and might be due to a deletion/insertion. The SCAR marker is closest to the a gene identified to date, and can be useful in breeding programs, using marker-assisted selection procedures to screen for sexual types in melon.

Phototropin-related NPL1 controls chloroplast relocation induced by blue light.

In photosynthetic cells, chloroplasts migrate towards illuminated sites to optimize photosynthesis and move away from excessively illuminated areas to protect the photosynthetic machinery. Although this movement of chloroplasts in response to light has been known for over a century, the photoreceptor mediating this process has not been identified. The Arabidopsis gene NPL1 (ref. 2) is a paralogue of the NPH1 gene, which encodes phototropin, a photoreceptor for phototropic bending. Here we show that NPL1 is required for chloroplast relocation induced by blue light. A loss-of-function npl1 mutant showed no chloroplast avoidance response in strong blue light, whereas the accumulation of chloroplasts in weak light was normal. These results indicate that NPL1 may function as a photoreceptor mediating chloroplast relocation.

An Arabidopsis circadian clock component interacts with both CRY1 and phyB.

Most organisms, from cyanobacteria to mammals, use circadian clocks to coordinate their activities with the natural 24-h light/dark cycle. The clock proteins of Drosophila and mammals exhibit striking homology but do not show similarity with clock proteins found so far from either cyanobacteria or Neurospora. Each of these organisms uses a transcriptionally regulated negative feedback loop in which the messenger RNA levels of the clock components cycle over a 24-h period. Proteins containing PAS domains are invariably found in at least one component of the characterized eukaryotic clocks. Here we describe ADAGIO1 (ADO1), a gene of Arabidopsis thaliana that encodes a protein containing a PAS domain. We found that a loss-of-function ado1 mutant is altered in both gene expression and cotyledon movement in circadian rhythmicity. Under constant white or blue light, the ado1 mutant exhibits a longer period than that of wild-type Arabidopsis seedlings, whereas under red light cotyledon movement and stem elongation are arrhythmic. Both yeast two-hybrid and in vitro binding studies show that there is a physical interaction between ADO1 and the photoreceptors CRY1 and phyB. We propose that ADO1 is an important component of the Arabidopsis circadian system.

Stamenless, a tomato mutant with homeotic conversions in petals and stamens.

A tomato (Lycopersicon esculentum Mill.) monogenic semidominant mutation, stamenless (sl), which results in homeotic conversions in two adjacent floral whorls, was studied. When grown at standard temperature, flowers of sl/sl plants showed sepaloid petals in the second whorl and strong transformation of stamens to carpels in whorl three. These transformed carpels were fused with each other and with the genuine carpels in the fourth whorl to form a unique gynoecium. The mutation is semidominant since heterozygous plants showed a phenotype intermediate between that of the wild type (WT) and that of homozygous mutant plants, with nearly WT petals but with feminized stamens bearing naked ovules on the base of their adaxial face. The initiation and position of organ primordia in sl/sl flowers were not altered when compared with WT primordia although development of organ primordia in the second and third whorls deviated from WT at an early stage as observed by scanning electron microscopy. The mutant phenotype is temperature sensitive and when sl/sl plants were cultured at low temperature, the morphology of some flowers resembled that of the WT. This reversion of the mutant phenotype is also induced by treatment of young sl/sl plants with gibberellic acid, providing evidence that gibberellin synthesis or sensitivity could mediate the effect of low temperature on the mutant phenotype. Southern blot analyses using a Deficiens-homologous gene from Solanum tuberosum as a probe showed a restriction-fragment-length polymorphism (RFLP) linked to the sl mutation. This result indicates that the mutation affects a Deficiens-like gene that controls the identity of petals and stamens.

FALSIFLORA, the tomato orthologue of FLORICAULA and LEAFY, controls flowering time and floral meristem identity.

Characterization of the tomato falsiflora mutant shows that fa mutation mainly alters the development of the inflorescence resulting in the replacement of flowers by secondary shoots, but also produces a late-flowering phenotype with an increased number of leaves below first and successive inflorescences. This pattern suggests that the FALSIFLORA (FA) locus regulates both floral meristem identity and flowering time in tomato in a similar way to the floral identity genes FLORICAULA (FLO) of Antirrhinum and LEAFY (LFY) of Arabidopsis. To analyse whether the fa phenotype is the result of a mutation in the tomato FLO/LFY gene, we have cloned and analysed the tomato FLO/LFY homologue (TOFL) in both wild-type and fa plants following a candidate gene strategy. The wild-type gene is predicted to encode a protein sharing 90% identity with NFL1 and ALF, the FLO/LFY-like proteins in Nicotiana and Petunia, and about 80 and 70% identity with either FLO or LFY. In the fa mutant, however, the gene showed a 16 bp deletion that results in a frameshift mutation and in a truncated protein. The co-segregation of this deletion with the fa phenotype in a total of 240 F2 plants analysed supports the idea that FA is the tomato orthologue to FLO and LFY. The gene is expressed in both vegetative and floral meristems, in leaf primordia and leaves, and in the four floral organs. The function of this gene in comparison with other FLO/LFY orthologues is analysed in tomato, a plant with a sympodial growth habit and a cymose inflorescence development.

Low temperature regulates Arabidopsis Lhcb gene expression in a light-independent manner.

Low temperature treatment of dark-grown seedlings of Arabidopsis thaliana results in a rapid increase in the amount of mRNAs encoding for the major polypeptides of the light-harvesting complex of photosystem II (Lhcb1 genes). This increase is transient and seems to be due mainly to the accumulation of Lhcb1*3 transcripts, indicating that low temperature differentially regulates the expression of the Arabidopsis Lhcb1 gene family in the dark. A 1.34 kb fragment of the Lhcb1*3 promoter is sufficient to confer low temperature regulation to a reporter gene in transgenic Arabidopsis etiolated seedlings, suggesting that the regulation is occurring at the transcriptional level. The cold-induced accumulation of Lhcb1*3 mRNA is not part of a general response to stressful conditions since no accumulation is detected in response to water stress, anaerobiosis or salt stress. The amount of Lhcb1*3 mRNA decrease in response to exogenous abscisic acid (ABA) suggesting that this phytohormone acts as a negative regulator. Moreover, the accumulation of Lhcb1*3 mRNAs in cold-treated ABA deficient etiolated seedlings is higher than that of wild-type and ABA insensitive etiolated seedlings, indicating that low temperature regulation of Lhcb1*3 is not mediated by ABA.

Tomato flower abnormalities induced by low temperatures are associated with changes of expression of MADS-Box genes

Flower and fruit development in tomato (Lycopersicon esculentum Mill. ) were severely affected when plants were grown at low temperatures, displaying homeotic and meristic transformations and alterations in the fusion pattern of the organs. Most of these homeotic transformations modified the identity of stamens and carpels, giving rise to intermediate organs. Complete homeotic transformations were rarely found and always affected organs of the reproductive whorls. Meristic transformations were also commonly observed in the reproductive whorls, which developed with an excessive number of organs. Scanning electron microscopy revealed that meristic transformations take place very early in the development of the flower and are related to a significant increase in the floral meristem size. However, homeotic transformations should occur later during the development of the organ primordia. Steady-state levels of transcripts corresponding to tomato MADS-box genes TM4, TM5, TM6, and TAG1 were greatly increased by low temperatures and could be related to these flower abnormalities. Moreover, in situ hybridization analyses showed that low temperatures also altered the stage-specific expression of TM4.

Assembly of chimeric connexin-aequorin proteins into functional gap junction channels. Reporting intracellular and plasma membrane calcium environments.

Chimeric proteins comprising connexins 26, 32, and 43 and aequorin, a chemiluminescent calcium indicator, were made by fusing the amino terminus of aequorin to the carboxyl terminus of connexins. The retention of function by the chimeric partners was investigated. Connexin 32-aequorin and connexin 43-aequorin retained chemiluminescent activity whereas that of connexin 26-aequorin was negligible. Immunofluorescent staining of COS-7 cells expressing the chimerae showed they were targeted to the plasma membrane. Gap junction intercellular channel formation by the chimerae alone and in combination with wild-type connexins was investigated. Stable HeLa cells expressing connexin 43-aequorin were functional, as demonstrated by Lucifer yellow transfer. Paris of Xenopus oocytes expressing connexin 43-aequorin were electrophysiologically coupled, but those expressing chimeric connexin 26 or 32 showed no detectable levels of coupling. The formation of heteromeric channels constructed of chimeric connexin 32 or connexin 43 and the respective wild-type connexins was inferred from the novel voltage gating properties of the junctional conductance. The results show that the preservation of function by each partner of the chimeric protein is dictated mainly by the nature of the connexin, especially the length of the cytoplasmic carboxyl-terminal domain. The aequorin partner of the connexin 43 chimera reported calcium levels in COS-7 cells in at least two different calcium environments.

Two homologous low-temperature-inducible genes from Arabidopsis encode highly hydrophobic proteins.

We have characterized two related cDNAs (RCI2A and RCI2B) corresponding to genes from Arabidopsis thaliana, the expression of which is transiently induced by low, nonfreezing temperatures. RCI2A and RCI2B encode small (54 amino acids), highly hydrophobic proteins that bear two potential transmembrane domains. They show similarity to proteins encoded by genes from barley (Hordeum vulgare L.) and wheatgrass (Lophophyrum elongatum) that are regulated by different stress conditions. Their high level of sequence homology (78%) and their genomic location in a single restriction fragment suggest that both genes originated as a result of a tandem duplication. However, their regulatory sequences have diverged enough to confer on them different expression patterns. Like most of the cold-inducible plant genes characterized, the expression of RCI2A and RCI2B is also promoted by abscisic acid (ABA) and dehydration but is not a general response to stress conditions, since it is not induced by salt stress or by anaerobiosis. Furthermore, low temperatures are able to induce RCI2A and RCI2B expression in ABA-deficient and -insensitive genetic backgrounds, indicating that both ABA-dependent and -independent pathways regulate the low-temperature responsiveness of these two genes.

Species-specific voltage-gating properties of connexin-45 junctions expressed in Xenopus oocytes.

Gap junctions composed of connexin-45 (Cx45) homologs from four species, zebrafish, chicken, mouse, and human, were expressed in pairs of Xenopus oocytes. The macroscopic conductance (gj) of all Cx45 junctions was modulated by transjunctional voltage (Vj) and by the inside-outside voltage (Vm), and the modulation was species specific. Although their gating characteristics varied in voltage sensitivity and kinetics, the four Cx45 junctions shared 1) maximum conductance at Vj = 0 and symmetrical gj reduction in response to positive and negative Vj of low amplitude, with little residual conductance; and 2) gj increases in response to simultaneous depolarization of the paired cells. The formation of hybrid channels, comprising Cx45 hemichannels from different species, allowed us to infer that two separate gates exist, one in each hemichannel, and that each Cx45 hemichannel is closed by the negativity of Vj on its cytoplasmic side. Interestingly, the Vm dependence of hybrid channels also suggests the presence of two gates in series, one Vm gate in each hemichannel. Thus the Vj and Vm dependence provides evidence that two independent voltage gates in each Cx45 hemichannel exist, reacting through specific voltage sensors and operating by different mechanisms, properties that have evolved divergently among species.

Equilocality and heterogeneity of constitutive heterochromatin: in situ localization of two families of highly repetitive DNA in Dociostaurus genei (Orthoptera).

The chromosome complement of the grasshopper Dociostaurus genei is characterized by the presence of constitutive heterochromatin (C-bands) located in the centromeric regions of all the chromosomes and in the distal regions of some autosomes in the form of supernumerary segments. A sequence analysis was carried out to obtain information about the molecular characteristics of both heterochromatic regions. Two families of tandemly repetitive DNA (DgT2 and DgA3) from D. genei were cloned and characterized. Data obtained from in situ hybridization indicate that these families are located solely in the regions of constitutive heterochromatin. The DgT2 clone is representative of a family of sequences which mainly forms the centromeric C-bands in each chromosome of the complement. The DgA3 family is the major component of the distal C-bands (supernumerary segments) present in most of the autosomal pairs. These results show the existence in D. genei of two different families of repetitive DNA restricted to different chromosomal domains. We discuss these results in the light of the possible role of chromosomal disposition in the maintenance of the differences between heterochromatic DNA from different chromosomal regions and the homogenization of DNA sequences from equilocal chromosomal domains.

Two related low-temperature-inducible genes of Arabidopsis encode proteins showing high homology to 14-3-3 proteins, a family of putative kinase regulators.

We have isolated two Rare Cold-Inducible (RCI1 and RCI2) cDNAs by screening a cDNA library prepared from cold-acclimated etiolated seedlings of Arabidopsis thaliana with a subtracted probe. RNA-blot hybridizations revealed that the expression of both RCI1 and RCI2 genes is induced by low temperature independently of the plant organ or the developmental stage considered. However, RCI1 mRNA accumulates faster and at higher levels than the RCI2 one indicating that these genes have differential responsiveness to cold stress. Additionally, when plants are returned to room temperature, RCI1 mRNA decreases faster than RCI2. In contrast to most of the cold-inducible plant genes characterized, the expression of RCI1 and RCI2 is not induced by ABA or water stress. The nucleotide sequences of RCI1 and RCI2 cDNAs predict two acidic polypeptides of 255 and 251 amino acids with molecular weights of 29 and 28 kDa respectively. The alignment of these polypeptides indicates that they have 181 identical amino acids suggesting that the corresponding genes have a common origin. Sequence comparisons reveal no similarities between the RCI proteins and any other cold-regulated plant protein so far described. Instead, they demonstrate that the RCI proteins are highly homologous to a family of proteins, known as 14-3-3 proteins, which are thought to be involved in the regulation of multifunctional protein kinases.

Non-random distribution of transposable elements in the nuclear genome of plants.

We have studied the genomic distribution of five different families of plant transposable elements by analyzing their location in DNA fractions from maize and tobacco genomes fractionated according to base composition. The results show that each family of elements is preferentially integrated in one specific fraction of its respective host genome. This demonstrates that the distribution of transposable elements in the nuclear genome of plants is not random but compartmentalized, i.e., the elements are located in specific genomic compartments characterized by having a specific G+C content and representing a small proportion of the genomes. Furthermore, these compartments seem to correspond to the genomic regions where most of the plant genes are also located, suggesting a preferential integration of transposable elements in the transcriptionally active regions of the plant genome. The implications of these results on the current applications of transposon tagging techniques are discussed.

Mutations at the Arabidopsis CHM locus promote rearrangements of the mitochondrial genome.

Nuclear recessive mutations at the chloroplast mutator (CHM) locus of Arabidopsis produce a variegated phenotype that is inherited in a non-Mendelian fashion. Molecular analysis of the cytoplasmic genomes of variegated plants from two independent chm mutant lines, using specific chloroplast and mitochondrial probes, showed that the chm mutations reproducibly induce the appearance of specific new restriction fragments in the mitochondrial genome. The presence of these restriction fragments cosegregated with the variegated phenotype in the progeny of crosses between mutant and wild-type plants. Sequence analysis of one of the new restriction fragments found in the variegated plants suggested that it was the product of a rearrangement event involving regions of the mitochondrial genome. Thus, it appears that the CHM locus may encode a protein involved in the control of specific mitochondrial DNA reorganization events.

The distribution of 5-methylcytosine in the nuclear genome of plants.

We have determined the 5-methylcytosine (5mC) content in high molecular weight DNA, from two dicot (tobacco and pea) and two monocot (wheat and maize) plant species, fractionated according to base composition. The results show that the proportion of 5mC in the genomic fractions increases linearly with their guanine + cytosine (G + C) content while the proportion of non-methylated cytosine remains almost constant. This can be interpreted as a consequence of a difference in mutation pressure related to spontaneous deamination of 5mC to thymine between the different compartments of plant genomes.