Do-not-resuscitate order. What happens after hospital discharge?

We observed 55 inpatients with "do-not-resuscitate" (DNR) orders to determine what happened to their DNR status after hospital discharge. All were admitted to the medical service of a Department of Veterans Affairs hospital. Of the 55 patients, 16 died in the hospital, 10 were discharged to inpatient hospice units, and 1 was transferred to an acute care hospital. An additional 19 patients were discharged to nursing homes. The other 9 patients (16% of the total) survived their hospital stays; 6 successful contacts were made with patients'' spouses. In 1 case the spouse thought a DNR order was no longer desirable. In the other 5 cases the spouse said the DNR status was "probably" or "definitely" still warranted, but only 1 spouse had a written DNR order at home. We contacted 9 of the 14 house officers who had cared for the patients in hospital. Only 2 had ever written a DNR order after hospital discharge. Two house officers said they routinely discussed with family members a patient''s expected dying process at home. Unwanted resuscitation is as undesirable at home as in the hospital. Physicians should discuss future resuscitation procedures with patients who have DNR orders at the time of hospital discharge. Physicians, paramedic service directors, and policymakers also should develop protocols and standardized home DNR orders so that paramedics can honor the wishes of patients in the prehospital setting.     

Profiling the phospho-status of the BKCa channel alpha subunit in rat brain reveals unexpected patterns and complexity

Molecular diversity of ion channel structure and function underlies variability in electrical signaling in nerve, muscle, and non-excitable cells. Protein phosphorylation and alternative splicing of pre-mRNA are two important mechanisms to generate structural and functional diversity of ion channels. However, systematic mass spectrometric analyses of in vivo phosphorylation and splice variants of ion channels in native tissues are largely lacking. Mammalian large-conductance calcium-activated potassium (BK(Ca)) channels are tetramers of alpha subunits (BKalpha) either alone or together with beta subunits, exhibit exceptionally large single channel conductance, and are dually activated by membrane depolarization and intracellular Ca(2+). The cytoplasmic C terminus of BKalpha is subjected to extensive pre-mRNA splicing and, as predicted by several algorithms, offers numerous phospho-acceptor amino acids. Here we use nanoflow liquid chromatography tandem mass spectrometry on BK(Ca) channels affinity-purified from rat brain to analyze in vivo BKalpha phosphorylation and splicing. We found 7 splice variations and identified as many as 30 Ser/Thr in vivo phosphorylation sites; most of which were not predicted by commonly used algorithms. Of the identified phosphosites 23 are located in the C terminus, four were found on splice insertions. Electrophysiological analyses of phospho- and dephosphomimetic mutants transiently expressed in HEK-293 cells suggest that phosphorylation of BKalpha differentially modulates the voltage- and Ca(2+)-dependence of channel activation. These results demonstrate that the pore-forming subunit of BK(Ca) channels is extensively phosphorylated in the mammalian brain providing a molecular basis for the regulation of firing pattern and excitability through dynamic modification of BKalpha structure and function.     

Arsenic upregulates MMP-9 and inhibits wound repair in human airway epithelial cells

As part of the innate immune defense, the polarized conducting lung epithelium acts as a barrier to keep particulates carried in respiration from underlying tissue. Arsenic is a metalloid toxicant that can affect the lung via inhalation or ingestion. We have recently shown that chronic exposure of mice or humans to arsenic (10-50 ppb) in drinking water alters bronchiolar lavage or sputum proteins consistent with reduced epithelial cell migration and wound repair in the airway. In this report, we used an in vitro model to examine effects of acute exposure of arsenic (15-290 ppb) on conducting airway lung epithelium. We found that arsenic at concentrations as low as 30 ppb inhibits reformation of the epithelial monolayer following scrape wounds of monolayer cultures. In an effort to understand functional contributions to epithelial wound repair altered by arsenic, we showed that acute arsenic exposure increases activity and expression of matrix metalloproteinase (MMP)-9, an important protease in lung function. Furthermore, inhibition of MMP-9 in arsenic-treated cells improved wound repair. We propose that arsenic in the airway can alter the airway epithelial barrier by restricting proper wound repair in part through the upregulation of MMP-9 by lung epithelial cells.     

Testing different 3D techniques using geometric morphometrics: Implications for cranial fluctuating asymmetry in humans

This study aimed to test the performance of 3D digitizer, CT scanner, and surface scanner in detecting cranial fluctuating asymmetry. Sets of 32 landmarks (6 in the midline and 13 bilateral) were acquired from 14 archeological crania using a 3D digitizer, and from 3D models generated from a CT scanner and surface scanner using Viewbox 4. Levels of shape variation were analyzed in MorphoJ using Procrustes analysis of variance and Principal component analysis. Intra-observer error accounted for 1.7%, 1.8%, and 4.5% of total shape variation for 3D digitizer, CT scanner, and surface scanner respectively. Fluctuating asymmetry accounted for 15%–16% of total shape variation. Variation between techniques accounted for 18% of total shape variation. We found a higher level of missing landmarks in our surface scan data than for both 3D digitizer and CT scanner data, and both 3D model-based techniques sometimes obscured taphonomic damage. All three 3D techniques are appropriate for measuring cranial fluctuating asymmetry. We advise against combining data collected with different techniques.     

Nucleotide sequence analysis of genes encoding a toluene/benzene-2-monooxygenase from Pseudomonas sp. strain JS150.

It was previously shown by others that Pseudomonas sp. strain JS150 metabolizes benzene and alkyl- and chloro-substituted benzenes by using dioxygenase-initiated pathways coupled with multiple downstream metabolic pathways to accommodate catechol metabolism. By cloning genes encoding benzene-degradative enzymes, we found that strain JS150 also carries genes for a toluene/benzene-2-monooxygenase. The gene cluster encoding a 2-monooxygenase and its cognate regulator was cloned from a plasmid carried by strain JS150. Oxygen (18O2) incorporation experiments using Pseudomonas aeruginosa strains that carried the cloned genes confirmed that toluene hydroxylation was catalyzed through an authentic monooxygenase reaction to yield ortho-cresol. Regions encoding the toluene-2-monooxygenase and regulatory gene product were localized in two regions of the cloned fragment. The nucleotide sequence of the toluene/benzene-2-monooxygenase locus was determined. Analysis of this sequence revealed six open reading frames that were then designated tbmA, tbmB, tbmC, tbmD, tbmE, and tbmF. The deduced amino acid sequences for these genes showed the presence of motifs similar to well-conserved functional domains of multicomponent oxygenases. This analysis allowed the tentative identification of two terminal oxygenase subunits (TbmB and TbmD) and an electron transport protein (TbmF) for the monooxygenase enzyme. In addition to these gene products, all the tbm polypeptides shared significant homology with protein components from other bacterial multicomponent monooxygenases. Overall, the tbm gene products shared greater similarity with polypeptides from the phenol hydroxylases of Pseudomonas putida CF600, P35X, and BH than with those from the toluene monooxygenases of Pseudomonas mendocina KR1 and Burkholderia (Pseudomonas) pickettii PKO1. The relationship found between the phenol hydroxylases and a toluene-2-monooxygenase, characterized in this study for the first time at the nucleotide sequence level, suggested that DNA probes used for surveys of environmental populations should be carefully selected to reflect DNA sequences corresponding to the metabolic pathway of interest.     

Differentiation between Actinobacillus (Haemophilus) actinomycetemcomitans, Haemophilus aphrophilus and Haemophilus paraphrophilus by multilocus enzyme electrophoresis

Genetic relationships among isolates assigned to Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus and H. paraphrophilus were determined by analysis of electrophoretically demonstrable allelic variation in 14 structural genes encoding metabolic enzymes. Among the 51 isolates analysed there were 25 electrophoretic types (ETs), among which mean genetic diversity per locus was 0.753. Cluster analysis of ETs demonstrated one well-defined group of 11 ETs representing solely the genotypes of all 17 isolates assigned to A. actinomycetemcomitans. The remaining 14 ETs represented the genotypes of the 34 isolates of H. aphrophilus and H. paraphrophilus. With the exception of ATCC 13252, all strains of H. aphrophilus were closely related, whereas strains assigned to H. paraphrophilus included distantly related lineages, some of which were similar to those of H. aphrophilus and should be assigned to this species. Thus, the study showed that there is no significant overall genetic similarity between A. actinomycetemcomitans and the two Haemophilus spp.     

The SH2 Domain Interaction Landscape

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Highlights? The recognition specificity of 70 SH2 domains is probed ? Recognition specificity diverges faster than sequence ? PepspotDB is a database of protein interactions mediated by SH2 domains     

Soil microfloral and microfaunal response to Salicornia bigelovii planting density and soil residue amendment

Seawater-irrigated halophyte systems have been proposed as sites for carbon storage, and therefore the fate of halophyte-derived carbon in the soil needs to be determined. To evaluate the role of the microfloral and microfaunal communities in soil carbon cycling of a halophyte agroecosystem, the response to various agronomic practices was investigated. Biomass and activity of the soil microflora and the abundance and trophic composition of the soil microfauna were determined under three planting densities of the halophyte Salicornia bigelovii (Chenopodiaceae) in plots with and without incorporated post-harvest halophyte residues. Microbial biomass and activity, as well as the abundance of nematode grazers, increased in response to the amendment of soil with halophyte residues. The microbial response to the density and presence of halophyte plants was, however, limited. Microbial activity increased in response to the presence of plants only after Salicornia had entered senescence, a result suggesting that in the mineral soil where halophytes were cropped, only dead root material provided a significant amount of microbially available organic matter. Success of halophyte agroecosystems in storing plant-derived carbon will depend primarily on the management of post-harvest residues and secondarily on the growing practices used prior to plant senescence. This revised version was published online in June 2006 with corrections to the Cover Date.     

Acclimation and photoprotection of young gametophytes of Acrostichum danaeifolium to UV-B stress

The effect of ultraviolet B radiation (UV-B) on cellular ultrastructure, chlorophyll (Chl), carotenoids, and total phenolics of Acrostichum danaeifolium gametophytes was analyzed. The control group of spores was germinated under standard conditions, while the test group of spores was germinated with additional UV-B for 30 min every day for 34 d. The cell characteristics were preserved in gametophytes irradiated with UV-B, but the number of starch grains increased in the chloroplasts and the more developed grana organization in contrast to the chloroplasts of the control group. Chl a content decreased, while Chl b content increased in the gametophytes cultivated with UV-B for 34 d. Contents of lutein and zeaxanthin decreased and trans-β-carotene concentration was enhanced in the gametophytes irradiated with UV-B. The content of total phenolic compounds increased in the gametophytes cultivated with UV-B. Therefore our data suggest that the gametophytes of A. danaeifolium, a fern endemic to the mangrove biome, were sensitive to enhancement of UV-B radiation at the beginning of their development and they exhibited alterations in their ultrastructure, pigment contents, and protective mechanisms of the photosynthetic apparatus, when exposed to this radiation.