The control of endopolygalacturonase expression by the sugarcane RAV transcription factor during aerenchyma formation.

The development of lysigenous aerenchyma starts with cell expansion and degradation of pectin from the middle lamella, leading to cell wall modification, and culminating with cell separation. Here we report that nutritional starvation of sugarcane induced gene expression along sections of the first 5 cm of the root and between treatments. We selected two candidate genes: a RAV transcription factor, from the ethylene response factors superfamily, and an endopolygalacturonase (EPG), a glycosyl hydrolase related to homogalacturonan hydrolysis from the middle lamella. epg1 and rav1 transcriptional patterns suggest they are essential genes at the initial steps of pectin degradation during aerenchyma development in sugarcane. Due to the high complexity of the sugarcane genome, rav1 and epg1 were sequenced from 17 bacterial artificial chromosome clones containing hom(e)ologous genomic regions, and the sequences were compared with those of Sorghum bicolor. We used one hom(e)olog sequence from each gene for transactivation assays in tobacco. rav1 was shown to bind to the epg1 promoter, repressing ß-glucuronidase activity. RAV repression upon epg1 transcription is the first reported link between ethylene regulation and pectin hydrolysis during aerenchyma formation. Our findings may help to elucidate cell wall degradation in sugarcane and therefore contribute to second-generation bioethanol production.

The Eucalyptus linker histone variant EgH1.3 cooperates with the transcription factor EgMYB1 to control lignin biosynthesis during wood formation.

Wood, also called secondary xylem, is a specialized vascular tissue constituted by different cell types that undergo a differentiation process involving deposition of thick, lignified secondary cell walls. The mechanisms needed to control the extent of lignin deposition depending on the cell type and the differentiation stage are far from being fully understood. We found that the Eucalyptus transcription factor EgMYB1, which is known to repress lignin biosynthesis, interacts specifically with a linker histone variant, EgH1.3. This interaction enhances the repression of EgMYB1's target genes, strongly limiting the amount of lignin deposited in xylem cell walls. The expression profiles of EgMYB1 and EgH1.3 overlap in xylem cells at early stages of their differentiation as well as in mature parenchymatous xylem cells, which have no or only thin lignified secondary cell walls. This suggests that a complex between EgMYB1 and EgH1.3 integrates developmental signals to prevent premature or inappropriate lignification of secondary cell walls, providing a mechanism to fine-tune the differentiation of xylem cells in time and space. We also demonstrate a role for a linker histone variant in the regulation of a specific developmental process through interaction with a transcription factor, illustrating that plant linker histones have other functions beyond chromatin organization.

Eucalyptus hairy roots, a fast, efficient and versatile tool to explore function and expression of genes involved in wood formation.

Eucalyptus are of tremendous economic importance being the most planted hardwoods worldwide for pulp and paper, timber and bioenergy. The recent release of the Eucalyptus grandis genome sequence pointed out many new candidate genes potentially involved in secondary growth, wood formation or lineage-specific biosynthetic pathways. Their functional characterization is, however, hindered by the tedious, time-consuming and inefficient transformation systems available hitherto for eucalypts. To overcome this limitation, we developed a fast, reliable and efficient protocol to obtain and easily detect co-transformed E. grandis hairy roots using fluorescent markers, with an average efficiency of 62%. We set up conditions both to cultivate excised roots in vitro and to harden composite plants and verified that hairy root morphology and vascular system anatomy were similar to wild-type ones. We further demonstrated that co-transformed hairy roots are suitable for medium-throughput functional studies enabling, for instance, protein subcellular localization, gene expression patterns through RT-qPCR and promoter expression, as well as the modulation of endogenous gene expression. Down-regulation of the Eucalyptus cinnamoyl-CoA reductase1 (EgCCR1) gene, encoding a key enzyme in lignin biosynthesis, led to transgenic roots with reduced lignin levels and thinner cell walls. This gene was used as a proof of concept to demonstrate that the function of genes involved in secondary cell wall biosynthesis and wood formation can be elucidated in transgenic hairy roots using histochemical, transcriptomic and biochemical approaches. The method described here is timely because it will accelerate gene mining of the genome for both basic research and industry purposes.

Life-threatening diphenhydramine toxicity presenting with seizures and a wide complex tachycardia improved with intravenous fat emulsion.

Diphenhydramine toxicity manifests with signs of anticholinergic toxicity; therapy is generally supportive. In rare cases, patients can also present with a wide complex tachycardia due to sodium channel blockade. Treatment involves sodium bicarbonate. Lidocaine and hypertonic saline are used for arrhythmias refractory to sodium bicarbonate. Although intravenous fat emulsion (IFE) therapy is proposed as an adjunctive therapy due to the lipophilicity of diphenhydramine (octanol/water partition coefficient of 3.3), successful use of IFE after a confirmed sole ingestion of diphenhydramine is not previously reported. We present the case of a 30-year-old woman presenting with seizures, a wide complex tachycardia, and cardiovascular collapse after an ingestion of diphenhydramine refractory to other therapies with rapid improvement after IFE administration.

Benzodiazepine-associated atrioventricular block.

Dysrhythmias, although common in overdose situations, are not often seen after benzodiazepine exposures. We report two cases of transient atrioventricular block after benzodiazepine misuse. Case 1 is a 4-year-old boy who was found unresponsive after an ingestion of clonazepam. An electrocardiogram (EKG) performed on emergency department presentation demonstrated first-degree atrioventricular block (PR 206 ms). After flumazenil administration, he developed second-degree atrioventricular block (Mobitz Type 1). EKG abnormalities resolved by morning. Serum clonazepam was 478 ng/mL (laboratory clonazepam reference range, 10-75 ng/mL with a dose of up to 6 mg/day) 5 hours after being found unresponsive. Case 2 is a 23-year-old man who presented to the emergency department after ingesting risperidone, combination hydrocodone/acetaminophen, and alprazolam. On arrival, his EKG demonstrated sinus bradycardia with a PR interval of 182 msec. He subsequently developed second-degree atrioventricular block (Mobitz Type I). Sinus bradycardia with resolution of his atrioventricular block (PR 200 ms) was seen on a third EKG performed 5 hours after presentation. These two patients demonstrated transient first- and second-degree atrioventricular block after benzodiazepine exposure. Benzodiazepines have been shown to alter L-type Ca2+ channel function. This alteration in function may account for the dysrhythmias seen in our patients. Together, these cases serve to remind clinicians of this rare but potentially serious complication associated with benzodiazepine exposure.

61-year-old woman • nausea • paresthesia • cold allodynia • Dx?

► Nausea ►Paresthesia ► Cold allodynia.

Maintenance of previously uncultured freshwater archaea from anoxic waters under laboratory conditions.

Culture conditions for the maintenance of previously uncultured members of the Archaea thriving in anoxic water layers of stratified freshwater lakes are described. The proposed enrichment conditions, based on the use of defined medium composition and the maintenance of anoxia, have been proven effective for the maintenance of the archaeal community with virtually no changes over time for periods up to 6 months as revealed by a PCR-DGGE analysis. Phylotypes belonging to groups poorly represented in culture collections such as the Deep-Sea Hydrothermal Vent Euryarchaeota (DHVE) and the Miscellaneous Crenarchaeotic Group (MCG) were maintained and selectively enriched when compared to the correspondent indigenous planktonic archaeal community.

Vertical distribution of ammonia-oxidizing crenarchaeota and methanogens in the epipelagic waters of Lake Kivu (Rwanda-Democratic Republic of the Congo).

Four stratified basins in Lake Kivu (Rwanda-Democratic Republic of the Congo) were sampled in March 2007 to investigate the abundance, distribution, and potential biogeochemical role of planktonic archaea. We used fluorescence in situ hybridization with catalyzed-reported deposition microscopic counts (CARD-FISH), denaturing gradient gel electrophoresis (DGGE) fingerprinting, and quantitative PCR (qPCR) of signature genes for ammonia-oxidizing archaea (16S rRNA for marine Crenarchaeota group 1.1a [MCG1] and ammonia monooxygenase subunit A [amoA]). Abundance of archaea ranged from 1 to 4.5% of total DAPI (4',6-diamidino-2-phenylindole) counts with maximal concentrations at the oxic-anoxic transition zone (∼50-m depth). Phylogenetic analysis of the archaeal planktonic community revealed a higher level of richness of crenarchaeal 16S rRNA gene sequences (21 of the 28 operational taxonomic units [OTUs] identified [75%]) over euryarchaeotal ones (7 OTUs). Sequences affiliated with the kingdom Euryarchaeota were mainly recovered from the anoxic water compartment and mostly grouped into methanogenic lineages (Methanosarcinales and Methanocellales). In turn, crenarchaeal phylotypes were recovered throughout the sampled epipelagic waters (0- to 100-m depth), with clear phylogenetic segregation along the transition from oxic to anoxic water masses. Thus, whereas in the anoxic hypolimnion crenarchaeotal OTUs were mainly assigned to the miscellaneous crenarchaeotic group, the OTUs from the oxic-anoxic transition and above belonged to Crenarchaeota groups 1.1a and 1.1b, two lineages containing most of the ammonia-oxidizing representatives known so far. The concomitant vertical distribution of both nitrite and nitrate maxima and the copy numbers of both MCG1 16S rRNA and amoA genes suggest the potential implication of Crenarchaeota in nitrification processes occurring in the epilimnetic waters of the lake.

The Woody-Preferential Gene EgMYB88 Regulates the Biosynthesis of Phenylpropanoid-Derived Compounds in Wood.

Comparative phylogenetic analyses of the R2R3-MYB transcription factor family revealed that five subgroups were preferentially found in woody species and were totally absent from Brassicaceae and monocots (Soler et al., 2015). Here, we analyzed one of these subgroups (WPS-I) for which no gene had been yet characterized. Most Eucalyptus members of WPS-I are preferentially expressed in the vascular cambium, the secondary meristem responsible for tree radial growth. We focused on EgMYB88, which is the most specifically and highly expressed in vascular tissues, and showed that it behaves as a transcriptional activator in yeast. Then, we functionally characterized EgMYB88 in both transgenic Arabidopsis and poplar plants overexpressing either the native or the dominant repression form (fused to the Ethylene-responsive element binding factor-associated Amphiphilic Repression motif, EAR). The transgenic Arabidopsis lines had no phenotype whereas the poplar lines overexpressing EgMYB88 exhibited a substantial increase in the levels of the flavonoid catechin and of some salicinoid phenolic glycosides (salicortin, salireposide, and tremulacin), in agreement with the increase of the transcript levels of landmark biosynthetic genes. A change in the lignin structure (increase in the syringyl vs. guaiacyl, S/G ratio) was also observed. Poplar lines overexpressing the EgMYB88 dominant repression form did not show a strict opposite phenotype. The level of catechin was reduced, but the levels of the salicinoid phenolic glycosides and the S/G ratio remained unchanged. In addition, they showed a reduction in soluble oligolignols containing sinapyl p-hydroxybenzoate accompanied by a mild reduction of the insoluble lignin content. Altogether, these results suggest that EgMYB88, and more largely members of the WPS-I group, could control in cambium and in the first layers of differentiating xylem the biosynthesis of some phenylpropanoid-derived secondary metabolites including lignin.

The Use of Physostigmine by Toxicologists in Anticholinergic Toxicity.

The anticholinergic toxidrome is well described and relatively common. Despite controversy, studies have shown that physostigmine is relatively safe and effective in reversing this toxidrome. We would expect toxicologists would be liberal in its use. We retrospectively analyzed data in the Toxicology Investigators Consortium (ToxIC) registry, representing data from medical toxicologists in multiple institutions nationwide, searching for patients who exhibited an anticholinergic toxidrome, determining what treatment(s) they received, and classifying the treatments as physostigmine, benzodiazepines, physostigmine and benzodiazepines, antipsychotics, or no definitive treatment. The causal agents of the toxidrome were as reported by the treating toxicologist. Eight hundred fifteen consecutive patients with anticholinergic toxidromes were analyzed. Benzodiazepines alone were given in 28.7 %, 12.4 % were given physostigmine alone, 8.8 % received both physostigmine and benzodiazepines, 2.7 % were given antipsychotics, and 47.4 % were given no definitive treatment. In patients who received only physostigmine, there was a significant difference in the rate of intubation (1.9 vs. 8.4 %, OR 0.21, 95 % CI 0.05-0.87) versus other treatment groups. Physostigmine was given at varying rates based on causative agent with use in agents with mixed or unknown effects (15.1 %) being significantly lower than those with primarily anticholinergic effects (26.6 %) (p < 0.001). Patients with anticholinergic toxicity were more likely to receive benzodiazepines than physostigmine. Those patients who received only physostigmine had a significantly lower rate of intubation. Physostigmine was more likely to be used with agents exerting primarily anticholinergic toxicity than in those agents with multiple actions.

Phylogenetic characterization and quantification of ammonia-oxidizing archaea and bacteria from Lake Kivu in a long-term microcosm incubation.

A microcosm cultivation-based method was set up to investigate the growth of ammonia-oxidizing archaea (AOA), isolated from a water sample acquired at a depth of 50 m from the northern basin of Lake Kivu. For this purpose, both CARD-FISH and qPCR targeting of archaeal 16S rRNA and amoA genes were used. Archaeal cell growth at the end of the 246-day microcosm experiment accounted for 35% of the SybrGold-stained cells, which corresponded to 6.61 x 10(6) cells/ml and 1.76 +/- 0.09 x 10(6) archaeal 16S rRNA gene copies/ml. Clone libraries and DGGE fingerprinting confirmed the dominance of AOA phylotypes in the archaeal community microcosm. The majority of the identified archaeal 16S rRNA gene sequences in the clone libraries were affiliated with Thaumarchaeota Marine Group 1 .1a. Subsequent cultivation of the AOA community on deep-well microtiter plates in medium containing different carbon sources to stimulate archaeal growth failed to show significant differences in archaeal abundance (ANOVA t14 = -1.058, P = 0.308 and ANOVA t14= 1.584, P = 0.135 for yeast extract and simple organic acids, respectively). The lack of growth stimulation by organic compounds is in concordance with the oligotrophic status of Lake Kivu. Finally, the addition of antibiotics to the growth medium resulted in archaeal cell counts that were significantly lower than those obtained from cultures in antibiotic-free medium (ANOVA t14 = 12.12, P < 0.001).

Active bacteria and archaea cells fixing bicarbonate in the dark along the water column of a stratified eutrophic lagoon.

We studied the carbon dioxide fixation activity in a stratified hypereutrophic karstic lagoon using a combination of fingerprinting techniques targeting bacterial and archaeal 16S rRNA genes, functional gene cloning [the acetyl-CoA carboxylase (accC)], and isotopic labelling ((14)C-bicarbonate) coupled to single-cell analyses [microautoradiography combined with catalyzed reported deposition-FISH (MAR-CARD-FISH)]. The microbial planktonic community was dominated by bacteria with maximal abundances of archaea just below the oxic/anoxic transition zone (7% of total cells). In situ incubations with radiolabelled bicarbonate showed maximal photoassimilation activity in the oxic epilimnion, whereas dark CO(2) fixation was consistently observed throughout the water column, with a maximum at the oxic/anoxic interface (8.6 mg C m(-3) h(-1)). The contributions of light and dark carbon fixation activities in the whole water column were 69% and 31% of the total C incorporated, respectively. MAR-CARD-FISH incubations corroborated these results and revealed that the highest fraction of bacterial and archaeal cells actively uptaking bicarbonate in the light was found at the surface. The bacterial community was mainly composed of green sulfur bacteria (Chlorobi) and members of the Betaproteobacteria and the Bacteroidetes. The archaeal assemblage was composed of phylotypes of the Miscellaneous Crenarchaeotic Group and a few methanogens. Clone libraries of the accC gene showed an absolute dominance of bacterial carboxylases. Our results suggest that the dark carbon fixation activity measured was mainly related to CO(2) incorporation by heterotrophs rather than to the activity of true chemoautotrophs.

Phylogenetic characterization and quantification of ammonia-oxidizing archaea and bacteria from Lake Kivu in a long-term microcosm incubation

A microcosm cultivation-based method was set up to investigate the growth of ammonia-oxidizing archaea (AOA), isolated from a water sample acquired at a depth of 50 m from the northern basin of Lake Kivu. For this purpose, both CARD-FISH and qPCR targeting of archaeal 16S rRNA and amoA genes were used. Archaeal cell growth at the end of the 246-day microcosm experiment accounted for 35% of the SybrGold-stained cells, which corresponded to 6.61 x 10(6) cells/ml and 1.76 +/- 0.09 x 10(6) archaeal 16S rRNA gene copies/ml. Clone libraries and DGGE fingerprinting confirmed the dominance of AOA phylotypes in the archaeal community microcosm. The majority of the identified archaeal 16S rRNA gene sequences in the clone libraries were affiliated with Thaumarchaeota Marine Group 1 .1a. Subsequent cultivation of the AOA community on deep-well microtiter plates in medium containing different carbon sources to stimulate archaeal growth failed to show significant differences in archaeal abundance (ANOVA t14 = -1.058, P = 0.308 and ANOVA t14= 1.584, P = 0.135 for yeast extract and simple organic acids, respectively). The lack of growth stimulation by organic compounds is in concordance with the oligotrophic status of Lake Kivu. Finally, the addition of antibiotics to the growth medium resulted in archaeal cell counts that were significantly lower than those obtained from cultures in antibiotic-free medium (ANOVA t14 = 12.12, P < 0.001) (AU)

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