bluuebunny/crossref_metadata_2025 huggingface dataset:
Nonperturbative Imaging of Nucleoid Morphology in Live Bacterial Cells during an Antimicrobial Peptide Attack
Authors: Bakshi Somenath , Choi Heejun , Rangarajan Nambirajan , Barns Kenneth J. , Bratton Benjamin P. , Weisshaar James C.
Published: June 2014

Studies of time-dependent drug and environmental effects on single, live bacterial cells would benefit significantly from a permeable, nonperturbative, long-lived fluorescent stain specific to the nucleoids (chromosomal DNA). The ideal stain would not affect cell growth rate or nucleoid morphology and dynamics, even during laser illumination for hundreds of camera frames. In this study, time-dependent, single-cell fluorescence imaging with laser excitation and a sensitive electron-multiplying charge-coupled-device (EMCCD) camera critically tested the utility of “dead-cell stains” (SYTOX orange and SYTOX green) and “live-cell stains” (DRAQ5 and SYTO 61) and also 4′,6-diamidino-2-phenylindole (DAPI). Surprisingly, the dead-cell stains were nearly ideal for imaging live
Escherichia coli
, while the live-cell stains and DAPI caused nucleoid expansion and, in some cases, cell permeabilization and the halting of growth. SYTOX orange performed well for both the Gram-negative
E. coli
and the Gram-positive
Bacillus subtilis
. In an initial application, we used two-color fluorescence imaging to show that the antimicrobial peptide cecropin A destroyed nucleoid-ribosome segregation over 20 min after permeabilization of the
E. coli
cytoplasmic membrane, reminiscent of the long-term effects of the drug rifampin. In contrast, the human cathelicidin LL-37, while similar to cecropin A in structure, length, charge, and the ability to permeabilize bacterial membranes, had no observable effect on nucleoid-ribosome segregation. Possible underlying causes are suggested.
Profile of Secreted Hydrolases, Associated Proteins, and SlpA in Thermoanaerobacterium saccharolyticum during the Degradation of Hemicellulose
Authors: Currie D. H. , Guss A. M. , Herring C. D. , Giannone R. J. , Johnson C. M. , Lankford P. K. , Brown S. D. , Hettich R. L. , Lynd L. R.
Published: June 2014

Thermoanaerobacterium saccharolyticum
, a Gram-positive thermophilic anaerobic bacterium, grows robustly on insoluble hemicellulose, which requires a specialized suite of secreted and transmembrane proteins. We report here the characterization of proteins secreted by this organism. Cultures were grown on hemicellulose, glucose, xylose, starch, and xylan in pH-controlled bioreactors, and samples were analyzed via spotted microarrays and liquid chromatography-mass spectrometry. Key hydrolases and transporters employed by
T. saccharolyticum
for growth on hemicellulose were, for the most part, hitherto uncharacterized and existed in two clusters (
Tsac_1445
through
Tsac_1464
for xylan/xylose and
Tsac_1344
through
Tsac_1349
for starch). A phosphotransferase system subunit, Tsac_0032, also appeared to be exclusive to growth on glucose. Previously identified hydrolases that showed strong conditional expression changes included XynA (Tsac_1459), XynC (Tsac_0897), and a pullulanase, Apu (Tsac_1342). An omnipresent transcript and protein making up a large percentage of the overall secretome, Tsac_0361, was tentatively identified as the primary S-layer component in
T. saccharolyticum
, and deletion of the
Tsac_0361
gene resulted in gross morphological changes to the cells. The view of hemicellulose degradation revealed here will be enabling for metabolic engineering efforts in biofuel-producing organisms that degrade cellulose well but lack the ability to catabolize C
5
sugars.
Distribution of Virulence-Associated Genes and Genetic Relationships in Non-O1/O139 Vibrio cholerae Aquatic Isolates from China
Authors: Li Fengjuan , Du Pengcheng , Li Baisheng , Ke Changwen , Chen Aiping , Chen Jie , Zhou Haijian , Li Jie , Morris J. Glenn , Kan Biao , Wang Duochun
Published: June 2014

Non-O1/O139
Vibrio cholerae
is naturally present in aquatic ecosystems and has been linked with cholera-like diarrhea and local outbreaks. The distribution of virulence-associated genes and genetic relationships among aquatic isolates from China are largely unknown. In this study, 295 aquatic isolates of
V. cholerae
non-O1/O139 serogroups from different regions in China were investigated. Only one isolate was positive for
ctxB
and harbored a rare genotype; 10 (3.4%) isolates carried several types of
rstR
sequences, eight of which carried rare types of toxin-coregulated pili (
tcpA
). Furthermore, 16 (5.4%) isolates carried incomplete (with partial open reading frames [ORFs]) vibrio seventh pandemic island I (VSP-I) or VSP-II clusters, which were further classified as 11 novel types. PCR-based analyses revealed remarkable variations in the distribution of putative virulence genes, including
mshA
(95.6%),
hlyA
(95.3%),
rtxC
(89.8%),
rtxA
(82.7%), IS1004 (52.9%),
chxA
(30.2%), SXT (15.3%), type III secretion system (18.0%), and NAG-ST (3.7%) genes. There was no correlation between the prevalence of putative virulence genes and that of CTX prophage or TCP genes, whereas there were correlations among the putative virulence genes. Further multilocus sequence typing (MLST) placed selected isolates (
n
= 70) into 69 unique sequence types (STs), which were different from those of the toxigenic O1 and O139 counterparts, and each isolate occupied a different position in the MLST tree. The
V. cholerae
non-O1/O139 aquatic isolates predominant in China have high genotypic diversity; these strains constitute a reservoir of potential virulence genes, which may contribute to evolution of pathogenic isolates.
Extracellular Enzyme Activity and Microbial Diversity Measured on Seafloor Exposed Basalts from Loihi Seamount Indicate the Importance of Basalts to Global Biogeochemical Cycling
Authors: Jacobson Meyers Myrna E. , Sylvan Jason B. , Edwards Katrina J.
Published: June 2014

Seafloor basalts are widely distributed and host diverse prokaryotic communities, but no data exist concerning the metabolic rates of the resident microbial communities. We present here potential extracellular enzyme activities of leucine aminopeptidase (LAP) and alkaline phosphatase (AP) measured on basalt samples from different locations on Loihi Seamount, HI, coupled with analysis of prokaryotic biomass and pyrosequencing of the bacterial 16S rRNA gene. The community maximum potential enzyme activity (
V
max
) of LAP ranged from 0.47 to 0.90 nmol (g rock)
−1
h
−1
; the
V
max
for AP was 28 to 60 nmol (g rock)
−1
h
−1
. The

K
m

of LAP ranged from 26 to 33 μM, while the

K
m

for AP was 2 to 7 μM. Bacterial communities on Loihi basalts were comprised primarily of
Alpha
-,
Delta
-, and
Gammaproteobacteria
,
Bacteroidetes
, and
Planctomycetes
. The putative ability to produce LAP is evenly distributed across the most commonly detected bacterial orders, but the ability to produce AP is likely dominated by bacteria in the orders
Xanthomonadales
,
Flavobacteriales
, and
Planctomycetales
. The enzyme activities on Loihi basalts were compared to those of other marine environments that have been studied and were found to be similar in magnitude to those from continental shelf sediments and orders of magnitude higher than any measured in the water column, demonstrating that the potential for exposed basalts to transform organic matter is substantial. We propose that microbial communities on basaltic rock play a significant, quantifiable role in benthic biogeochemical processes.
To Be or Not To Be a Poly(3-Hydroxybutyrate) (PHB) Depolymerase: PhaZd1 (PhaZ6) and PhaZd2 (PhaZ7) of Ralstonia eutropha, Highly Active PHB Depolymerases with No Detectable Role in Mobilization of Accumulated PHB
Authors: Sznajder Anna , Jendrossek Dieter
Published: June 2014

The putative physiological functions of two related intracellular poly(3-hydroxybutyrate) (PHB) depolymerases, PhaZd1 and PhaZd2, of
Ralstonia eutropha
H16 were investigated. Purified PhaZd1 and PhaZd2 were active with native PHB granules
in vitro
. Partial removal of the proteinaceous surface layer of native PHB granules by trypsin treatment or the use of PHB granules isolated from Δ
phaP1
or Δ
phaP1-phaP5
mutant strains resulted in increased specific PHB depolymerase activity, especially for PhaZd2. Constitutive expression of PhaZd1 or PhaZd2 reduced or even prevented the accumulation of PHB under PHB-permissive conditions
in vivo
. Expression of translational fusions of enhanced yellow fluorescent protein (EYFP) with PhaZd1 and PhaZd2 in which the active-site serines (S190 and Ser193) were replaced with alanine resulted in the colocalization of only PhaZd1 fusions with PHB granules. C-terminal fusions of inactive PhaZd2(S193A) with EYFP revealed the presence of spindle-like structures, and no colocalization with PHB granules was observed. Chromosomal deletion of
phaZd1
,
phaZd2
, or both depolymerase genes had no significant effect on PHB accumulation and mobilization during growth in nutrient broth (NB) or NB-gluconate medium. Moreover, neither proteome analysis of purified native PHB granules nor
lacZ
fusion studies gave any indication that PhaZd1 or PhaZd2 was detectably present in the PHB granule fraction or expressed at all during growth on NB-gluconate medium. In conclusion, PhaZd1 and PhaZd2 are two PHB depolymerases with a high capacity to degrade PHB when artificially expressed but are apparently not involved in PHB mobilization in the wild type. The true
in vivo
functions of PhaZd1 and PhaZd2 remain obscure.
Revision of N2O-Producing Pathways in the Ammonia-Oxidizing Bacterium Nitrosomonas europaea ATCC 19718
Authors: Kozlowski Jessica A. , Price Jennifer , Stein Lisa Y.
Published: June 2014

Nitrite reductase (NirK) and nitric oxide reductase (NorB) have long been thought to play an essential role in nitrous oxide (N2O) production by ammonia-oxidizing bacteria. However, essential gaps remain in our understanding of how and when NirK and NorB are active and functional, putting into question their precise roles in N2O production by ammonia oxidizers. The growth phenotypes of theNitrosomonas europaeaATCC 19718 wild-type and mutant strains deficient in expression of NirK, NorB, and both gene products were compared under atmospheric and reduced O2tensions. Anoxic resting-cell assays and instantaneous nitrite (NO2−) reduction experiments were done to assess the ability of the wild-type and mutantN. europaeastrains to produce N2O through the nitrifier denitrification pathway. Results confirmed the role of NirK for efficient substrate oxidation ofN. europaeaand showed that NorB is involved in N2O production during growth at both atmospheric and reduced O2tensions. Anoxic resting-cell assays and measurements of instantaneous NO2−reduction using hydrazine as an electron donor revealed that an alternate nitrite reductase to NirK is present and active. These experiments also clearly demonstrated that NorB was the sole nitric oxide reductase for nitrifier denitrification. The results of this study expand the enzymology for nitrogen metabolism and N2O production byN. europaeaand will be useful to interpret pathways in other ammonia oxidizers that lack NirK and/or NorB genes.
Use of Silica-Encapsulated Pseudomonas sp. Strain NCIB 9816-4 in Biodegradation of Novel Hydrocarbon Ring Structures Found in Hydraulic Fracturing Waters
Authors: Aukema Kelly G. , Kasinkas Lisa , Aksan Alptekin , Wackett Lawrence P.
Published: June 2014

The most problematic hydrocarbons in hydraulic fracturing (fracking) wastewaters consist of fused, isolated, bridged, and spiro ring systems, and ring systems have been poorly studied with respect to biodegradation, prompting the testing here of six major ring structural subclasses using a well-characterized bacterium and a silica encapsulation system previously shown to enhance biodegradation. The direct biological oxygenation of spiro ring compounds was demonstrated here. These and other hydrocarbon ring compounds have previously been shown to be present in flow-back waters and waters produced from hydraulic fracturing operations.
Pseudomonas
sp. strain NCIB 9816-4, containing naphthalene dioxygenase, was selected for its broad substrate specificity, and it was demonstrated here to oxidize fundamental ring structures that are common in shale-derived waters but not previously investigated with this or related enzymes.
Pseudomonas
sp. NCIB 9816-4 was tested here in the presence of a silica encasement, a protocol that has previously been shown to protect bacteria against the extremes of salinity present in fracking wastewaters. These studies demonstrate the degradation of highly hydrophobic compounds by a silica-encapsulated model bacterium, demonstrate what it may not degrade, and contribute to knowledge of the full range of hydrocarbon ring compounds that can be oxidized using
Pseudomonas
sp. NCIB 9816-4.
Biosynthesis of the β-Methylarginine Residue of Peptidyl Nucleoside Arginomycin in Streptomyces arginensis NRRL 15941
Authors: Feng Jun , Wu Jun , Gao Jie , Xia Zhigui , Deng Zixin , He Xinyi
Published: June 2014

The peptidyl nucleoside arginomycin is active against Gram-positive bacteria and fungi but displays much lower toxicity to mice than its analog blasticidin S. It features a rare amino acid, β-methylarginine, which is attached to the deoxyhexose moiety via a 4′-aminoacyl bond. We here report cloning of the complete biosynthetic gene cluster for arginomycin from
Streptomyces arginensis
NRRL 15941. Among the 14 putative essential open reading frames,
argM
, encoding an aspartate aminotransferase (AAT), and adjacent
argN
, encoding an
S
-adenosyl methionine (SAM)-dependent methyltransferase, are coupled to catalyze arginine and yield β-methylarginine in
Escherichia coli
. Purified ArgM can transfer the α-amino group of
l
-arginine to α-ketoglutaric acid to give glutamate and thereby converts
l
-arginine to 5-guanidino-2-oxopentanoic acid, which is methylated at the C-3 position by ArgN to form 5-guanidino-3-methyl-2-oxopentanoic acid. Iteratively, ArgM specifically catalyzes transamination from the donor
l
-aspartate to the resulting 5-guanidino-3-methyl-2-oxopentanoic acid, generating β-methylarginine. The complete and concise biosynthetic pathway for the rare and bioactive amino acid revealed by this study may pave the way for the production of β-methylarginine either by enzymatic conversion or by engineered living cells.
Elucidation of Insertion Elements Carried on Plasmids and
In Vitro
Construction of Shuttle Vectors from the Toxic Cyanobacterium Planktothrix
Authors: Christiansen Guntram , Goesmann Alexander , Kurmayer Rainer
Published: June 2014

Several gene clusters that are responsible for toxin synthesis in bloom-forming cyanobacteria have been found to be associated with transposable elements (TEs). In particular, insertion sequence (IS) elements were shown to play a role in the inactivation or recombination of the genes responsible for cyanotoxin synthesis. Plasmids have been considered important vectors of IS element distribution to the host. In this study, we aimed to elucidate the IS elements propagated on the plasmids and the chromosome of the toxic cyanobacterium
Planktothrix agardhii
NIVA-CYA126/8 by means of high-throughput sequencing. In total, five plasmids (pPA5.5, pPA14, pPA50, pPA79, and pPA115, of 5, 6, 50, 79, and 120 kbp, respectively) were elucidated, and two plasmids (pPA5.5, pPA115) were found to propagate full IS element copies. Large stretches of shared DNA information between plasmids were constituted of TEs. Two plasmids (pPA5.5, pPA14) were used as candidates to engineer shuttle vectors (named pPA5.5SV and pPA14SV, respectively)
in vitro
by PCR amplification and the subsequent transposition of the Tn
5 cat
transposon containing the R6Kγ origin of replication of
Escherichia coli
. While pPA5.5SV was found to be fully segregated, pPA14SV consistently co-occurred with its wild-type plasmid even under the highest selective pressure. Interestingly, the Tn
5 cat
transposon became transferred by homologous recombination into another plasmid, pPA50. The availability of shuttle vectors is considered to be of relevance in investigating genome plasticity as a consequence of homologous recombination events. Combining the potential of high-throughput sequencing and
in vitro
production of shuttle vectors makes it simple to produce species-specific shuttle vectors for many cultivable prokaryotes.
Identification and Characterization of a Halotolerant, Cold-Active Marine Endo-β-1,4-Glucanase by Using Functional Metagenomics of Seaweed-Associated Microbiota
Authors: Martin Marjolaine , Biver Sophie , Steels Sébastien , Barbeyron Tristan , Jam Murielle , Portetelle Daniel , Michel Gurvan , Vandenbol Micheline
Published: June 2014

A metagenomic library was constructed from microorganisms associated with the brown alga
Ascophyllum nodosum
. Functional screening of this library revealed 13 novel putative esterase loci and two glycoside hydrolase loci. Sequence and gene cluster analysis showed the wide diversity of the identified enzymes and gave an idea of the microbial populations present during the sample collection period. Lastly, an endo-β-1,4-glucanase having less than 50% identity to sequences of known cellulases was purified and partially characterized, showing activity at low temperature and after prolonged incubation in concentrated salt solutions.
Rubber Oxygenase and Latex Clearing Protein Cleave Rubber to Different Products and Use Different Cleavage Mechanisms
Authors: Birke Jakob , Jendrossek Dieter
Published: June 2014

Two types of enzyme for oxidative cleavage of poly(
cis
-1,4-isoprene) are known. One is rubber oxygenase (RoxA) that is secreted by
Xanthomonas
sp. strain 35Y and a few other Gram-negative rubber-degrading bacteria during growth on polyisoprene. RoxA was studied in the past, and the recently solved structure showed a structural relationship to bacterial cytochrome
c
peroxidases (J. Seidel et al., Proc. Natl. Acad. Sci. U. S. A. 110:13833–13838, 2013,
http://dx.doi.org/10.1073/pnas.1305560110
). The other enzyme is latex-clearing protein (Lcp) that is secreted by rubber-degrading actinomycetes, but Lcp has not yet been purified. Here, we expressed Lcp of
Streptomyces
sp. strain K30 in a Δ
roxA
background of
Xanthomonas
sp. strain 35Y and purified native (untagged) Lcp. The specific activities of Lcp and RoxA were 0.70 and 0.48 U/mg, respectively. Lcp differed from RoxA in the absence of heme groups and other characteristics. Notably, Lcp degraded polyisoprene via endo-type cleavage to tetra-C
20
and higher oligo-isoprenoids with aldehyde and keto end groups, whereas RoxA used an exo-type cleavage mechanism to give the main end product 12-oxo-4,8-dimethyltrideca-4,8-diene-1-al (ODTD). RoxA was able to cleave isolated Lcp-derived oligo-isoprenoid molecules to ODTD. Inhibitor studies, spectroscopic investigations and metal analysis gave no indication for the presence of iron, other metals, or cofactors in Lcp. Our results suggest that Lcp could be a member of the growing group of cofactor-independent oxygenases and differs in the cleavage mechanism from heme-dependent RoxA. In conclusion, RoxA and Lcp represent two different answers to the same biochemical problem, the cleavage of polyisoprene, a polymer that has carbon-carbon double bonds as the only functional groups for enzymatic attack.
VibA, a Homologue of a Transcription Factor for Fungal Heterokaryon Incompatibility, Is Involved in Antifungal Compound Production in the Plant-Symbiotic Fungus Epichloë festucae
Authors: Niones Jennifer T. , Takemoto Daigo
Published: June 2014

Symbiotic association of epichloae endophytes (
Epichloë/Neotyphodium
species) with cool-season grasses of the subfamily Pooideae confers bioprotective benefits to the host plants against abiotic and biotic stresses. While the production of fungal bioprotective metabolites is a well-studied mechanism of host protection from insect herbivory, little is known about the antibiosis mechanism against grass pathogens by the mutualistic endophyte. In this study, an
Epichloë festucae
mutant defective in antimicrobial substance production was isolated by a mutagenesis approach. In an isolated mutant that had lost antifungal activity, the exogenous DNA fragment was integrated into the promoter region of the
vibA
gene, encoding a homologue of the transcription factor VIB-1. VIB-1 in
Neurospora crassa
is a regulator of genes essential in vegetative incompatibility and promotion of cell death. Here we show that deletion of the
vibA
gene severely affected the antifungal activity of the mutant against the test pathogen
Drechslera erythrospila
. Further analyses showed that overexpressing
vibA
enhanced the antifungal activity of the wild-type isolate against test pathogens. Transformants overexpressing
vibA
showed an inhibitory activity on test pathogens that the wild-type isolate could not. Moreover, overexpressing
vibA
in a nonantifungal
E. festucae
wild-type Fl1 isolate enabled the transformant to inhibit the mycelial and spore germination of
D. erythrospila
. These results demonstrate that enhanced expression of
vibA
is sufficient for a nonantifungal isolate to obtain antifungal activity, implicating the critical role of VibA in antifungal compound production by epichloae endophytes.
PFP1
, a Gene Encoding an Epc-N Domain-Containing Protein, Is Essential for Pathogenicity of the Barley Pathogen Rhynchosporium commune
Authors: Siersleben Sylvia , Penselin Daniel , Wenzel Claudia , Albert Sylvie , Knogge Wolfgang
Published: June 2014

Scald caused by
Rhynchosporium commune
is an important foliar disease of barley. Insertion mutagenesis of
R. commune
generated a nonpathogenic fungal mutant which carries the inserted plasmid in the upstream region of a gene named
PFP1
. The characteristic feature of the gene product is an Epc-N domain. This motif is also found in homologous proteins shown to be components of histone acetyltransferase (HAT) complexes of fungi and animals. Therefore, PFP1 is suggested to be the subunit of a HAT complex in
R. commune
with an essential role in the epigenetic control of fungal pathogenicity. Targeted
PFP1
disruption also yielded nonpathogenic mutants which showed wild-type-like growth
ex planta
, except for the occurrence of hyphal swellings. Complementation of the deletion mutants with the wild-type gene reestablished pathogenicity and suppressed the hyphal swellings. However, despite wild-type-level
PFP1
expression, the complementation mutants did not reach wild-type-level virulence. This indicates that the function of the protein complex and, thus, fungal virulence are influenced by a position-affected long-range control of
PFP1
expression.
The Volatome of Aspergillus fumigatus
Authors: Heddergott C. , Calvo A. M. , Latgé J. P.
Published: June 2014

Early detection of invasive aspergillosis is absolutely required for efficient therapy of this fungal infection. The identification of fungal volatiles in patient breath can be an alternative for the detection of
Aspergillus fumigatus
that still remains problematic. In this work, we investigated the production of volatile organic compounds (VOCs) by
A. fumigatus
in vitro
, and we show that volatile production depends on the nutritional environment.
A. fumigatus
produces a multiplicity of VOCs, predominantly terpenes and related compounds. The production of sesquiterpenoid compounds was found to be strongly induced by increased iron concentrations and certain drugs, i.e., pravastatin. Terpenes that were always detectable in large amounts were α-pinene, camphene, and limonene, as well as sesquiterpenes, identified as α-bergamotene and β-
trans
-bergamotene. Other substance classes that were found to be present in the volatome, such as 1-octen-3-ol, 3-octanone, and pyrazines, were found only under specific growth conditions. Drugs that interfere with the terpene biosynthesis pathway influenced the composition of the fungal volatome, and most notably, a block of sesquiterpene biosynthesis by the bisphosphonate alendronate fundamentally changed the VOC composition. Using deletion mutants, we also show that a terpene cyclase and a putative kaurene synthase are essential for the synthesis of volatile terpenes by
A. fumigatus
. The present analysis of
in vitro
volatile production by
A. fumigatus
suggests that VOCs may be used in the diagnosis of infections caused by this fungus.
Interaction of Cryptococcus neoformans Extracellular Vesicles with the Cell Wall
Authors: Wolf Julie M. , Espadas-Moreno Javier , Luque-Garcia Jose L. , Casadevall Arturo
Published: June 2014

Cryptococcus neoformans
produces extracellular vesicles containing a variety of cargo, including virulence factors. To become extracellular, these vesicles not only must be released from the plasma membrane but also must pass through the dense matrix of the cell wall. The greatest unknown in the area of fungal vesicles is the mechanism by which these vesicles are released to the extracellular space given the presence of the fungal cell wall. Here we used electron microscopy techniques to image the interactions of vesicles with the cell wall. Our goal was to define the ultrastructural morphology of the process to gain insights into the mechanisms involved. We describe single and multiple vesicle-leaving events, which we hypothesized were due to plasma membrane and multivesicular body vesicle origins, respectively. We further utilized melanized cells to “trap” vesicles and visualize those passing through the cell wall. Vesicle size differed depending on whether vesicles left the cytoplasm in single versus multiple release events. Furthermore, we analyzed different vesicle populations for vesicle dimensions and protein composition. Proteomic analysis tripled the number of proteins known to be associated with vesicles. Despite separation of vesicles into batches differing in size, we did not identify major differences in protein composition. In summary, our results indicate that vesicles are generated by more than one mechanism, that vesicles exit the cell by traversing the cell wall, and that vesicle populations exist as a continuum with regard to size and protein composition.
Normal Telomere Length Maintenance in Saccharomyces cerevisiae Requires Nuclear Import of the Ever Shorter Telomeres 1 (Est1) Protein via the Importin Alpha Pathway
Authors: Hawkins Charlene , Friedman Katherine L.
Published: June 2014

The Est1 (ever shorter telomeres 1) protein is an essential component of yeast telomerase, a ribonucleoprotein complex that restores the repetitive sequences at chromosome ends (telomeres) that would otherwise be lost during DNA replication. Previous work has shown that the telomerase RNA component (
TLC1
) transits through the cytoplasm during telomerase biogenesis, but mechanisms of protein import have not been addressed. Here we identify three nuclear localization sequences (NLSs) in Est1p. Mutation of the most N-terminal NLS in the context of full-length Est1p reduces Est1p nuclear localization and causes telomere shortening—phenotypes that are rescued by fusion with the NLS from the simian virus 40 (SV40) large-T antigen. In contrast to that of the
TLC1
RNA, Est1p nuclear import is facilitated by Srp1p, the yeast homolog of importin α. The reduction in telomere length observed at the semipermissive temperature in a
srp1
mutant strain is rescued by increased Est1p expression, consistent with a defect in Est1p nuclear import. These studies suggest that at least two nuclear import pathways are required to achieve normal telomere length homeostasis in yeast.
Reality check: the cost–effectiveness of removing body checking from youth ice hockey
Authors: Lacny Sarah , Marshall Deborah A , Currie Gillian , Kulin Nathalie A , Meeuwisse Willem H , Kang Jian , Emery Carolyn A
Published: June 2014

Background/aim

The risk of injury among Pee Wee (ages 11–12 years) ice hockey players in leagues that allow body checking is threefold greater than in leagues that do not allow body checking. We estimated the cost–effectiveness of a no body checking policy versus a policy that allows body checking in Pee Wee ice hockey.

Methods

Cost–effectiveness analysis alongside a prospective cohort study during the 2007–2008 season, including players in Quebec (n=1046), where policy did not allow body checking, and in Alberta (n=1108), where body checking was allowed. Injury incidence rates (injuries/1000 player-hours) and incidence proportions (injuries/100 players), adjusted for cluster using Poisson regression, allowed for standardised comparisons and meaningful translation to community stakeholders. Based on Alberta fee schedules, direct healthcare costs (physician visits, imaging, procedures) were adjusted for cluster using bootstrapping. We examined uncertainty in our estimates using cost–effectiveness planes.

Results

Associated with significantly higher injury rates, healthcare costs where policy allowed body checking were over 2.5 times higher than where policy disallowed body checking ($C473/1000 player-hours (95% CI $C358 to $C603) vs $C184/1000 player-hours (95% CI $C120 to $C257)). The difference in costs between provinces was $C289/1000 player-hours (95% CI $C153 to $C432). Projecting results onto Alberta Pee Wee players registered in the 2011–2012 season, an estimated 1273 injuries and $C213 280 in healthcare costs would be avoided during just one season with the policy change.

Conclusion

Our study suggests that a policy disallowing body checking in Pee Wee ice hockey is cost-saving (associated with fewer injuries and lower costs) compared to a policy allowing body checking. As we did not account for long-term outcomes, our results underestimate the economic impact of these injuries.
Study protocol for the nutritional route in oesophageal resection trial: a single-arm feasibility trial (NUTRIENT trial)
Authors: Weijs Teus J , Nieuwenhuijzen Grard A P , Ruurda Jelle P , Kouwenhoven Ewout A , Rosman Camiel , Sosef Meindert , v Hillegersberg Richard , Luyer Misha D P
Published: June 2014

Introduction

The best route of feeding for patients undergoing an oesophagectomy is unclear. Concerns exist that early oral intake would increase the incidence and severity of pneumonia and anastomotic leakage. However, in studies including patients after many other types of gastrointestinal surgery and in animal experiments, early oral intake has been shown to be beneficial and enhance recovery. Therefore, we aim to determine the feasibility of early oral intake after oesophagectomy.

Methods and analysis

This study is a feasibility trial in which 50 consecutive patients will start oral intake directly following oesophagectomy. Primary outcomes will be the frequency and severity of anastomotic leakage and (aspiration) pneumonia. Clinical parameters will be registered prospectively and nutritional requirements and intake will be assessed by a dietician. Surgical complications will be registered.

Ethics and dissemination

Approval for this study has been obtained from the Medical Ethical Committee of the Catharina Hospital Eindhoven and the study has been registered at the Dutch Trial Register, NTR4136. Results will be published and presented at international congresses.

Discussion

We hypothesise that the oral route of feeding is safe and feasible following oesophagectomy, as has been shown previously for other types of gastrointestinal surgery. It is expected that early oral nutrition will result in enhanced recovery. Furthermore, complications related to artificial feeding, such as jejunostomy tube feeding, are believed to be reduced. However, (aspiration) pneumonia and anastomotic leakage are potential risks that are carefully monitored.

Trial registration number

NTR4136.
An instrument for the assessment of diarrhoeal severity based on a longitudinal community-based study
Authors: Lee Gwenyth , Peñataro Yori Pablo , Paredes Olortegui Maribel , Caulfield Laura E , Sack David A , Fischer-Walker Christa , Black Robert E , Kosek Margaret
Published: June 2014

Objective

Diarrhoea is a significant contributer to morbidity and is among the leading causes of death of children living in poverty. As such, the incidence, duration and severity of diarrhoeal episodes in the household are often key variables of interest in a variety of community-based studies. However, there currently exists no means of defining diarrhoeal severity that are (A) specifically designed and adapted for community-based studies, (B) associated with poorer child outcomes and (C) agreed on by the majority of researchers. Clinical severity scores do exist and are used in healthcare settings, but these tend to focus on relatively moderate-to-severe dehydrating and dysenteric disease, require trained observation of the child and, given the variability of access and utilisation of healthcare, fail to sufficiently describe the spectrum of disease in the community setting.

Design

Longitudinal cohort study.

Setting

Santa Clara de Nanay, a rural community in the Northern Peruvian Amazon.

Participants

442 infants and children 0–72 months of age.

Main outcome measures

Change in weight over 1-month intervals and change in length/height over 9-month intervals.

Results

Diarrhoeal episodes with symptoms of fever, anorexia, vomiting, greater number of liquid stools per day and greater number of total stools per day were associated with poorer weight gain compared with episodes without these symptoms. An instrument to measure the severity was constructed based on the duration of these symptoms over the course of a diarrhoeal episode.

Conclusions

In order to address limitations of existing diarrhoeal severity scores in the context of community-based studies, we propose an instrument comprised of diarrhoea-associated symptoms easily measured by community health workers and based on the association of these symptoms with poorer child growth. This instrument can be used to test the impact of interventions on the burden of diarrhoeal disease.
How weight change is modelled in population studies can affect research findings: empirical results from a large-scale cohort study
Authors: Paige E , Korda R J , Banks E , Rodgers B
Published: June 2014

Objectives

To investigate how results of the association between education and weight change vary when weight change is defined and modelled in different ways.

Design

Longitudinal cohort study.

Participants

60 404 men and women participating in the Social, Environmental and Economic Factors (SEEF) subcomponent of the 45 and Up Study—a population-based cohort study of people aged 45 years or older, residing in New South Wales, Australia.

Outcome measures

The main exposure was self-reported education, categorised into four groups. The outcome was annual weight change, based on change in self-reported weight between the 45 and Up Study baseline questionnaire and SEEF questionnaire (completed an average of 3.3 years later). Weight change was modelled in four different ways: absolute change (kg) modelled as (1) a continuous variable and (2) a categorical variable (loss, maintenance and gain), and relative (%) change modelled as (3) a continuous variable and (4) a categorical variable. Different cut-points for defining weight-change categories were also tested.

Results

When weight change was measured categorically, people with higher levels of education (compared with no school certificate) were less likely to lose or to gain weight. When weight change was measured as the average of a continuous measure, a null relationship between education and annual weight change was observed. No material differences in the education and weight-change relationship were found when comparing weight change defined as an absolute (kg) versus a relative (%) measure. Results of the logistic regression were sensitive to different cut-points for defining weight-change categories.

Conclusions

Using average weight change can obscure important directional relationship information and, where possible, categorical outcome measurements should be included in analyses.
Association of lifestyle-related factors with circadian onset patterns of acute myocardial infarction: a prospective observational study in Japan
Authors: Edahiro Ryuya , Sakata Yasuhiko , Nakatani Daisaku , Suna Shinichiro , Usami Masaya , Matsumoto Sen , Hara Masahiko , Kitamura Tetsuhisa , Sato Hiroshi , Yamashita Shizuya , Nanto Shinsuke , Hikoso Shungo , Sakata Yasushi , Hori Masatsugu , Hamasaki Toshimitsu , Komuro Issei ,   on behalf of the OACIS Investigators
Published: June 2014

Objective

The onset of acute myocardial infarction (AMI) shows characteristic circadian variations involving a definite morning peak and a less-defined night-time peak. However, the factors influencing the circadian patterns of AMI onset and their influence on morning and night-time peaks have not been fully elucidated.

Design, setting and participants

An analysis of patients registered between 1998 and 2008 in the Osaka Acute Coronary Insufficiency Study, which is a prospective, multicentre observational study of patients with AMI in the Osaka region of Japan. The present study included 7755 consecutive patients with a known time of AMI onset.

Main outcomes and measures

A mixture of two von Mises distributions was used to examine whether a circadian pattern of AMI had uniform, unimodal or bimodal distribution, and the likelihood ratio test was then used to select the best circadian pattern among them. The hierarchical likelihood ratio test was used to identify factors affecting the circadian patterns of AMI onset. The Kaplan-Meier method was used to estimate survival curves of 1-year mortality according to AMI onset time.

Results

The overall population had a bimodal circadian pattern of AMI onset characterised by a high and sharp morning peak and a lower and less-defined night-time peak (bimodal p<0.001). Although several lifestyle-related factors had a statistically significant association with the circadian patterns of AMI onset, serum triglyceride levels had the most prominent association with the circadian patterns of AMI onset. Patients with triglyceride ≥150 mg/dL on admission had only one morning peak in the circadian pattern of AMI onset during weekdays, with no peaks detected on weekends, whereas all other subgroups had two peaks throughout the week.

Conclusions

The circadian pattern of AMI onset was characterised by bimodality. Notably, several lifestyle-related factors, particularly serum triglyceride levels, had a strong relation with the circadian pattern of AMI onset.

Trial registration number

UMIN000004575.
Marital status and survival after oesophageal cancer surgery: a population-based nationwide cohort study in Sweden
Authors: Brusselaers Nele , Mattsson Fredrik , Johar Asif , Wikman Anna , Lagergren Pernilla , Lagergren Jesper , Ljung Rickard
Published: June 2014

Objectives

A beneficial effect of being married on survival has been shown for several cancer types, but is unclear for oesophageal cancer. The objective of this study was to clarify the potential influence of the marital status on the overall and disease-specific survival after curatively intended treatment of oesophageal cancer using a nationwide population-based design, taking into account the known major prognostic variables.

Design

Prospective, population-based cohort.

Setting

All Swedish hospitals performing surgery for oesophageal cancer during 2001–2005.

Participants

This study included 90% of all patients with oesophageal or junctional cancer who underwent surgical resection in Sweden in 2001–2005, with follow-up until death or the end of the study period (2012).

Primary and secondary outcome measures

Cox regression was used to estimate associations between the marital status and the 5-year overall and disease-specific mortality, expressed as HRs with 95% CIs, with adjustment for sex, age, tumour stage, histological type, complications, comorbidities and annual surgeon volume.

Results

Of all 606 included patients (80.4% men), 55.1% were married, 9.2% were remarried, 22.6% were previously married and 13% were never married. Compared with the married patients, the never married (HR 1.02, 95% CI 0.77 to 1.35), previously married (HR 0.90, 95% CI 0.71 to 1.15) and remarried patients (HR 0.79, 95% CI 0.55 to 1.13) had no increased overall 5-year mortality. The corresponding HRs for disease-specific survival, and after excluding the initial 90 days of surgery, were similar to the HRs for the overall survival.

Conclusions

This study showed no evidence of a better 5-year survival in married patients compared with non-married patients undergoing surgery for oesophageal cancer.
Mitigating errors caused by interruptions during medication verification and administration: interventions in a simulated ambulatory chemotherapy setting
Authors: Prakash Varuna , Koczmara Christine , Savage Pamela , Trip Katherine , Stewart Janice , McCurdie Tara , Cafazzo Joseph A , Trbovich Patricia
Published: June 2014

Background

Nurses are frequently interrupted during medication verification and administration; however, few interventions exist to mitigate resulting errors, and the impact of these interventions on medication safety is poorly understood.

Objective

The study objectives were to (A) assess the effects of interruptions on medication verification and administration errors, and (B) design and test the effectiveness of targeted interventions at reducing these errors.

Methods

The study focused on medication verification and administration in an ambulatory chemotherapy setting. A simulation laboratory experiment was conducted to determine interruption-related error rates during specific medication verification and administration tasks. Interventions to reduce these errors were developed through a participatory design process, and their error reduction effectiveness was assessed through a postintervention experiment.

Results

Significantly more nurses committed medication errors when interrupted than when uninterrupted. With use of interventions when interrupted, significantly fewer nurses made errors in verifying medication volumes contained in syringes (16/18; 89% preintervention error rate vs 11/19; 58% postintervention error rate; p=0.038; Fisher's exact test) and programmed in ambulatory pumps (17/18; 94% preintervention vs 11/19; 58% postintervention; p=0.012). The rate of error commission significantly decreased with use of interventions when interrupted during intravenous push (16/18; 89% preintervention vs 6/19; 32% postintervention; p=0.017) and pump programming (7/18; 39% preintervention vs 1/19; 5% postintervention; p=0.017). No statistically significant differences were observed for other medication verification tasks.

Conclusions

Interruptions can lead to medication verification and administration errors. Interventions were highly effective at reducing unanticipated errors of commission in medication administration tasks, but showed mixed effectiveness at reducing predictable errors of detection in medication verification tasks. These findings can be generalised and adapted to mitigate interruption-related errors in other settings where medication verification and administration are required.
A new clinical cut-off of cytokeratin 19 mRNA copy number in sentinel lymph node better identifies patients eligible for axillary lymph node dissection in breast cancer
Authors: Deambrogio Cristina , Castellano Isabella , Paganotti Alessia , Zorini Elisabetta Omodeo , Corsi Fabio , Bussone Riccardo , Franchini Roberto , Antona Jlenia , Miglio Umberto , Sapino Anna , Antonacci Concetta , Boldorini Renzo
Published: June 2014

Aims

Cytokeratin 19 (CK19) mRNA copy number predicts the probability of tumour load in axillary lymph nodes (ALN) and can help in decision-making regarding the axillary dissection. The purpose of this study was to define a new cut-off of CK19 mRNA copy number using the one-step nucleic acid amplification (OSNA) assay on metastatic sentinel lymph nodes (SLN) in order to identify cases at risk of having one or more positive ALN.

Methods

1296 SLN from 1080 patients were analysed with the OSNA assay. 194 patients with positive SLN underwent ALN dissection and the mean value of CK19 copy number (320 000) of their SLN was set as initial cut-off. Receiver operative characteristics curve identify a best cut-off of 7700 (sensitivity 78%, specificity 57%). A comparison between our and the traditional cut-off (5000) was performed.

Results

The cut-off of 7700 successfully identifies patients with positive ALN (p=0.001, false- negative cases: 17%). In the range between 5000 and 7700, one patient with positive ALN would not undergo axillary dissection, whereas eight patients with negative ALN would be correctly identified.

Conclusions

We suggest that the level of CK19 mRNA copy number could be the only parameter to consider in the intraoperative management of the axilla.
Drivers of inequality in disability-free expectancy at birth and age 85 across space and time in Great Britain
Authors: Wohland Pia , Rees Phil , Gillies Clare , Alvanides Seraphim , Matthews Fiona E , O'Neill Vikki , Jagger Carol
Published: June 2014

Background

Although mortality and health inequalities at birth have increased both geographically and in socioeconomic terms, little is known about inequalities at age 85, the fastest growing sector of the population in Great Britain (GB).

Aim

To determine whether trends and drivers of inequalities in life expectancy (LE) and disability-free life expectancy (DFLE) at age 85 between 1991 and 2001 are the same as those at birth.

Methods

DFLE at birth and age 85 for 1991 and 2001 by gender were calculated for each local authority in GB using the Sullivan method. Regression modelling was used to identify area characteristics (rurality, deprivation, social class composition, ethnicity, unemployment, retirement migration) that could explain inequalities in LE and DFLE.

Results

Similar to values at birth, LE and DFLE at age 85 both increased between 1991 and 2001 (though DFLE increased less than LE) and gaps across local areas widened (and more for DFLE than LE). The significantly greater increases in LE and DFLE at birth for less-deprived compared with more-deprived areas were still partly present at age 85. Considering all factors, inequalities in DFLE at birth were largely driven by social class composition and unemployment rate, but these associations appear to be less influential at age 85.

Conclusions

Inequalities between areas in LE and DFLE at birth and age 85 have increased over time though factors explaining inequalities at birth (mainly social class and unemployment rates) appear less important for inequalities at age 85.
An optimized method for estimating the tidal volume from intracardiac or body surface electrocardiographic signals: implications for estimating minute ventilation
Authors: Sayadi Omid , Weiss Eric H. , Merchant Faisal M. , Puppala Dheeraj , Armoundas Antonis A.
Published: June 2014

The ability to accurately monitor tidal volume (TV) from electrocardiographic (ECG) signals holds significant promise for improving diagnosis treatment across a variety of clinical settings. The objective of this study was to develop a novel method for estimating the TV from ECG signals. In 10 mechanically ventilated swine, we collected intracardiac electrograms from catheters in the coronary sinus (CS), left ventricle (LV), and right ventricle (RV), as well as body surface electrograms, while TV was varied between 0 and 750 ml at respiratory rates of 7–14 breaths/min. We devised an algorithm to determine the optimized respirophasic modulation of the amplitude of the ECG-derived respiratory signal. Instantaneous measurement of respiratory modulation showed an absolute error of 72.55, 147.46, 85.68, 116.62, and 50.89 ml for body surface, CS, LV, RV, and RV-CS leads, respectively. Minute TV estimation demonstrated a more accurate estimation with an absolute error of 69.56, 153.39, 79.33, 122.16, and 48.41 ml for body surface, CS, LV, RV, and RV-CS leads, respectively. The RV-CS and body surface leads provided the most accurate estimations that were within 7 and 10% of the true TV, respectively. Finally, the absolute error of the bipolar RV-CS lead was significantly lower than any other lead configuration ( P < 0.0001). In conclusion, we have demonstrated that ECG-derived respiratory modulation provides an accurate estimation of the TV using intracardiac or body surface signals, without the need for additional hardware.