bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolated Microbial Single Cells and Resulting Micropopulations Grow Faster in Controlled Environments
Authors: Dusny Christian , Fritzsch Frederik Sven Ole , Frick Oliver , Schmid Andreas
Published: July 2012

Singularized cells of
Pichia pastoris
,
Hansenula polymorpha
, and
Corynebacterium glutamicum
displayed specific growth rates under chemically and physically constant conditions that were consistently higher than those obtained in populations. This highlights the importance of single-cell analyses by uncoupling physiology and the extracellular environment, which is now possible using the Envirostat 2.0 concept.
Mechanisms by Which Ambient Humidity May Affect Viruses in Aerosols
Authors: Yang Wan , Marr Linsey C.
Published: July 2012

Many airborne viruses have been shown to be sensitive to ambient humidity, yet the mechanisms responsible for this phenomenon remain elusive. We review multiple hypotheses, including water activity, surface inactivation, and salt toxicity, that may account for the association between humidity and viability of viruses in aerosols. We assess the evidence and limitations for each hypothesis based on findings from virology, aerosol science, chemistry, and physics. In addition, we hypothesize that changes in pH within the aerosol that are induced by evaporation may trigger conformational changes of the surface glycoproteins of enveloped viruses and subsequently compromise their infectivity. This hypothesis may explain the differing responses of enveloped viruses to humidity. The precise mechanisms underlying the relationship remain largely unverified, and attaining a complete understanding of them will require an interdisciplinary approach.
Mycobacterial Shuttle Vectors Designed for High-Level Protein Expression in Infected Macrophages
Authors: Eitson Jennifer L. , Medeiros Jennifer J. , Hoover Ashley R. , Srivastava Shashikant , Roybal Kole T. , Aï¿½nsa Josï¿½ A. , Hansen Eric J. , Gumbo Tawanda , van Oers Nicolai S. C.
Published: July 2012

Mycobacterial shuttle vectors contain dual origins of replication for growth in both
Escherichia coli
and mycobacteria. One such vector, pSUM36, was re-engineered for high-level protein expression in diverse bacterial species. The modified vector (pSUM-
kan
-MCS2) enabled green fluorescent protein expression in
E. coli
,
Mycobacterium smegmatis
, and
M. avium
at levels up to 50-fold higher than that detected with the parental vector, which was originally developed with a
lacZ
α promoter. This high-level fluorescent protein expression allowed easy visualization of
M. smegmatis
and
M. avium
in infected macrophages. The
M. tuberculosis
gene
esat-6
was cloned in place of the green fluorescence protein gene (
gfp
) to determine the impact of ESAT-6 on the innate inflammatory response. The modified vector (pSUM-
kan
-MCS2) yielded high levels of ESAT-6 expression in
M. smegmatis
. The ability of ESAT-6 to suppress innate inflammatory pathways was assayed with a novel macrophage reporter cell line, designed with an interleukin-6 (IL-6) promoter-driven GFP cassette. This stable cell line fluoresces in response to diverse mycobacterial strains and stimuli, such as lipopolysaccharide.
M. smegmatis
clones expressing high levels of ESAT-6 failed to attenuate IL-6-driven GFP expression. Pure ESAT-6, produced in
E. coli
, was insufficient to suppress a strong inflammatory response elicited by
M. smegmatis
or lipopolysaccharide, with ESAT-6 itself directly activating the IL-6 pathway. In summary, a pSUM-protein expression vector and a mammalian IL-6 reporter cell line provide new tools for understanding the pathogenic mechanisms deployed by various mycobacterial species.
TetR-Based Gene Regulation Systems for Francisella tularensis
Authors: LoVullo Eric D. , Miller Cheryl N. , Pavelka Martin S. , Kawula Thomas H.
Published: July 2012

There are a number of genetic tools available for studying
Francisella tularensis
, the etiological agent of tularemia; however, there is no effective inducible or repressible gene expression system. Here, we describe inducible and repressible gene expression systems for
F. tularensis
based on the Tet repressor, TetR. For the inducible system, a
tet
operator sequence was cloned into a modified
F. tularensis groESL
promoter sequence and carried in a plasmid that constitutively expressed TetR. To monitor regulation the luminescence operon,
luxCDABE
, was cloned under the hybrid
Francisella
tetracycline-regulated promoter (
FTRp
), and transcription was initiated with addition of anhydrotetracycline (ATc), which binds TetR and alleviates TetR association with
tetO.
Expression levels measured by luminescence correlated with ATc inducer concentrations ranging from 20 to 250 ng ml
−1
. In the absence of ATc, luminescence was below the level of detection. The inducible system was also functional during the infection of J774A.1 macrophages, as determined by both luminescence and rescue of a mutant strain with an intracellular growth defect. The repressible system consists of
FTRp
regulated by a reverse TetR mutant (revTetR), TetR r1.7. Using this system with the
lux
reporter, the addition of ATc resulted in decreased luminescence, while in the absence of ATc the level of luminescence was not significantly different from that of a construct lacking TetR r1.7. Utilizing both systems, the essentiality of SecA, the protein translocase ATPase, was confirmed, establishing that they can effectively regulate gene expression. These two systems will be invaluable in exploring
F. tularensis
protein function.
Intact Polar and Core Glycerol Dibiphytanyl Glycerol Tetraether Lipids of Group I.1a and I.1b Thaumarchaeota in Soil
Authors: Sinninghe Damstï¿½ Jaap S. , Rijpstra W. Irene C. , Hopmans Ellen C. , Jung Man-Young , Kim Jong-Geol , Rhee Sung-Keun , Stieglmeier Michaela , Schleper Christa
Published: July 2012

Ecological studies of thaumarchaeota often apply glycerol dibiphytanyl glycerol tetraether (GDGT)-based intact membrane lipids. However, these components have only been characterized for thaumarchaeota from aquatic environments. Thaumarchaeota have been shown to play an important role in the nitrogen cycle in soil as ammonium oxidizers, and GDGTs are common lipids encountered in soil. We report the core and intact polar lipid (IPL) GDGTs produced by three newly available thaumarchaeota isolated from grassland soil in Austria (“
Nitrososphaera viennensis
,” group I.1b) and enriched from agricultural soils in South Korea (“
Candidatus
Nitrosoarchaeum koreensis” MY1, group I.1a; and “
Candidatus
Nitrososphaera” strain JG1, group I.1b). The soil thaumarchaeota all synthesize crenarchaeol as their major core GDGT, in agreement with the fact that crenarchaeol has also been detected in thaumarchaeota from aquatic environments. The crenarchaeol regioisomer apparently is produced in significant quantities only by soil thaumarchaeota of the I.1b subgroup. In addition, GDGTs with 0 to 4 cyclopentane moieties and GDGTs containing an additional hydroxyl group were detected. The IPL head groups of their membrane lipids comprised mainly monohexose, dihexose, trihexose, phosphohexose, and hexose-phosphohexose moieties. The hexose-phosphohexose head group bound to crenarchaeol occurred in all soil thaumarchaeota, and this IPL is at present the only lipid that is detected in all thaumarchaeota analyzed so far. This specificity and its lability indicate that it is the most suitable biomarker lipid to trace living thaumarchaeota. This study, in combination with previous studies, also suggests that hydroxylated GDGTs occur in the I.1a, but not in the I.1b, subgroup of the thaumarchaeota.
Involvement of the Major Capsid Protein and Two Early-Expressed Phage Genes in the Activity of the Lactococcal Abortive Infection Mechanism AbiT
Authors: Labrie Simon J. , Tremblay Denise M. , Moisan Maxim , Villion Manuela , Magadï¿½n Alfonso H. , Campanacci Valï¿½rie , Cambillau Christian , Moineau Sylvain
Published: July 2012

The dairy industry uses the mesophilic, Gram-positive, lactic acid bacterium (LAB)
Lactococcus lactis
to produce an array of fermented milk products. Milk fermentation processes are susceptible to contamination by virulent phages, but a plethora of phage control strategies are available. One of the most efficient is to use LAB strains carrying phage resistance systems such as abortive infection (Abi) mechanisms. Yet, the mode of action of most Abi systems remains poorly documented. Here, we shed further light on the antiviral activity of the lactococcal AbiT system. Twenty-eight AbiT-resistant phage mutants derived from the wild-type AbiT-sensitive lactococcal phages p2, bIL170, and P008 were isolated and characterized. Comparative genomic analyses identified three different genes that were mutated in these virulent AbiT-insensitive phage derivatives:
e14
(bIL170 [
e14
bIL170
]),
orf41
(P008 [
orf41
P008
]), and
orf6
(p2 [
orf6
p2
] and P008 [
orf6
P008
]). The genes
e14
bIL170
and
orf41
P008
are part of the early-expressed genomic region, but bioinformatic analyses did not identify their putative function.
orf6
is found in the phage morphogenesis module. Antibodies were raised against purified recombinant ORF6, and immunoelectron microscopy revealed that it is the major capsid protein (MCP). Coexpression in
L. lactis
of ORF6
p2
and ORF5
p2
, a protease, led to the formation of procapsids. To our knowledge, AbiT is the first Abi system involving distinct phage genes.
Chemotaxis of Marinobacter adhaerens and Its Impact on Attachment to the Diatom Thalassiosira weissflogii
Authors: Sonnenschein Eva C. , Syit Desalegne Abebew , Grossart Hans-Peter , Ullrich Matthias S.
Published: July 2012

Alga-bacterium interactions are crucial for aggregate formation and carbon cycling in aquatic systems. To understand the initiation of these interactions, we investigated bacterial chemotaxis within a bilateral model system.
Marinobacter adhaerens
HP15 has been demonstrated to attach to the diatom
Thalassiosira weissflogii
and induce transparent exopolymeric particle and aggregate formation.
M. adhaerens
possesses one polar flagellum and is highly motile. Bacterial cells were attracted to diatom cells, as demonstrated by addition of diatom cell homogenate or diatom culture supernatant to soft agar, suggesting that chemotaxis might be important for the interaction of
M. adhaerens
with diatoms. Three distinct chemotaxis-associated gene clusters were identified in the genome sequence of
M. adhaerens
, with the clusters showing significant sequence similarities to those of
Pseudomonas aeruginosa
PAO1. Mutations in the genes
cheA
,
cheB
,
chpA
, and
chpB
, which encode histidine kinases and methylesterases and which are putatively involved in either flagellum-associated chemotaxis or pilus-mediated twitching motility, were generated and mutants with the mutations were phenotypically analyzed. Δ
cheA
and Δ
cheB
mutants were found to be swimming deficient, and all four mutants were impaired in biofilm formation on abiotic surfaces. Comparison of the HP15 wild type and its chemotaxis mutants in cocultures with the diatom revealed that the fraction of bacteria attaching to the diatom decreased significantly for mutants in comparison to that for the wild type. Our results highlight the importance of
M. adhaerens
chemotaxis in initiation of its interaction with the diatom. In-depth knowledge of these basic processes in interspecies interactions is pivotal to obtain a systematic understanding of organic matter flux and nutrient cycling in marine ecosystems.
Heat Shock Protein 90 Is Required for Conidiation and Cell Wall Integrity in Aspergillus fumigatus
Authors: Lamoth Frédéric , Juvvadi Praveen R. , Fortwendel Jarrod R. , Steinbach William J.
Published: July 2012

Heat shock protein 90 (Hsp90) is a eukaryotic molecular chaperone. Its involvement in the resistance of
Candida albicans
to azole and echinocandin antifungals is well established. However, little is known about Hsp90's function in the filamentous fungal pathogen
Aspergillus fumigatus
. We investigated the role of Hsp90 in
A. fumigatus
by genetic repression and examined its cellular localization under various stress conditions. Failure to generate a deletion strain of
hsp90
suggested that it is essential. Genetic repression of Hsp90 was achieved by an inducible nitrogen-dependent promoter (
pniiA
-Hsp90) and led to decreased spore viability, decreased hyphal growth, and severe defects in germination and conidiation concomitant with the downregulation of the conidiation-specific transcription factors
brlA
,
wetA
, and
abaA
. Hsp90 repression potentiated the effect of cell wall inhibitors affecting the β-glucan structure of the cell wall (caspofungin, Congo red) and of the calcineurin inhibitor FK506, supporting a role in regulating cell wall integrity pathways. Moreover, compromising Hsp90 abolished the paradoxical effect of caspofungin. Pharmacological inhibition of Hsp90 by geldanamycin and its derivatives (17-AAG and 17-DMAG) resulted in similar effects. C-terminal green fluorescent protein (GFP) tagging of Hsp90 revealed mainly cytosolic distribution under standard growth conditions. However, treatment with caspofungin resulted in Hsp90 accumulation at the cell wall and at sites of septum formation, further highlighting its role in cell wall stress compensatory mechanisms. Targeting Hsp90 with fungal-specific inhibitors to cripple stress response compensatory pathways represents an attractive new antifungal strategy.
Hfq Influences Multiple Transport Systems and Virulence in the Plant Pathogen Agrobacterium tumefaciens
Authors: Wilms Ina , Möller Philip , Stock Anna-Maria , Gurski Rosemarie , Lai Erh-Min , Narberhaus Franz
Published: July 2012

The Hfq protein mediates gene regulation by small RNAs (sRNAs) in about 50% of all bacteria. Depending on the species, phenotypic defects of anhfqmutant range from mild to severe. Here, we document that the purified Hfq protein of the plant pathogen and natural genetic engineerAgrobacterium tumefaciensbinds to the previously described sRNA AbcR1 and its target mRNAatu2422, which codes for the substrate binding protein of an ABC transporter taking up proline and γ-aminobutyric acid (GABA). Several other ABC transporter components were overproduced in anhfqmutant compared to their levels in the parental strain, suggesting that Hfq plays a major role in controlling the uptake systems and metabolic versatility ofA. tumefaciens. Thehfqmutant showed delayed growth, altered cell morphology, and reduced motility. Although the DNA-transferring type IV secretion system was produced, tumor formation by the mutant strain was attenuated, demonstrating an important contribution of Hfq to plant transformation byA. tumefaciens.
Seventeen Sxy-Dependent Cyclic AMP Receptor Protein Site-Regulated Genes Are Needed for Natural Transformation in Haemophilus influenzae
Authors: Sinha Sunita , Mell Joshua C. , Redfield Rosemary J.
Published: July 2012

Natural competence is the ability of bacteria to actively take up extracellular DNA. This DNA can recombine with the host chromosome, transforming the host cell and altering its genotype. InHaemophilus influenzae, natural competence is induced by energy starvation and the depletion of nucleotide pools. This induces a 26-gene competence regulon (Sxy-dependent cyclic AMP receptor protein [CRP-S] regulon) whose expression is controlled by two regulators, CRP and Sxy. The role of most of the CRP-S genes in DNA uptake and transformation is not known. We have therefore created in-frame deletions of each CRP-S gene and studied their competence phenotypes. All but one gene (ssb) could be deleted. Although none of the remaining CRP-S genes were required for growth in rich medium or survival under starvation conditions, DNA uptake and transformation were abolished or reduced in most of the mutants. Seventeen genes were absolutely required for transformation, with 14 of these genes being specifically required for the assembly and function of the type IV pilus DNA uptake machinery. Only five genes were dispensable for both competence and transformation. This is the first competence regulon for which all genes have been mutationally characterized.
Cellulose Degradation by Sulfolobus solfataricus Requires a Cell-Anchored Endo-β-1-4-Glucanase
Authors: Girfoglio Michele , Rossi Mosé , Cannio Raffaele
Published: July 2012

A sequence encoding a putative extracellular endoglucanase (sso1354) was identified in the complete genome sequence ofSulfolobus solfataricus. The encoded protein shares signature motifs with members of glycoside hydrolases family 12. After an unsuccessful first attempt at cloning the full-length coding sequences inEscherichia coli, an active but unstable recombinant enzyme lacking a 27-residue N-terminal sequence was generated. This 27-amino-acid sequence shows significant similarity with corresponding regions in the sugar binding proteins AraS, GlcS, and TreS ofS. solfataricusthat are responsible for anchoring them to the plasma membrane. A strategy based on an effective vector/host genetic system forSulfolobusand on expression control by the promoter of theS. solfataricusgene which encodes the glucose binding protein allowed production of the enzyme in sufficient quantities for study. In fact, the enzyme expressed inS. solfataricuswas stable and highly thermoresistant and showed optimal activity at low pH and high temperature. The protein was detected mainly in the plasma membrane fraction, confirming the structural similarity to the sugar binding proteins. The results of the protein expression in the two different hosts showed that the SSO1354 enzyme is endowed with an endo-β-1-4-glucanase activity and specifically hydrolyzes cellulose. Moreover, it also shows significant but distinguishable specificity toward several other sugar polymers, such as lichenan, xylan, debranched arabinan, pachyman, and curdlan.
VirB-Mediated Positive Feedback Control of the Virulence Gene Regulatory Cascade of Shigella flexneri
Authors: Kane Kelly A. , Dorman Charles J.
Published: July 2012

Shigella flexneri
is a facultative intracellular pathogen that relies on a type III secretion system and its associated effector proteins to cause bacillary dysentery in humans. The genes that encode this virulence system are located on a 230-kbp plasmid and are transcribed in response to thermal, osmotic, and pH signals that are characteristic of the human lower gut. The virulence genes are organized within a regulatory cascade, and the nucleoid-associated protein H-NS represses each of the key promoters. Transcription derepression depends first on the VirF AraC-like transcription factor, a protein that antagonizes H-NS-mediated repression at the intermediate regulatory gene
virB
. The VirB protein in turn remodels the H-NS–DNA nucleoprotein complexes at the promoters of the genes encoding the type III secretion system and effector proteins, causing these genes to become derepressed. In this study, we show that the VirB protein also positively regulates the expression of its own gene (
virB
) via a
cis
-acting regulatory sequence. In addition, VirB positively regulates the gene coding for the VirF protein. This study reveals two hitherto uncharacterized feedback regulatory loops in the
S. flexneri
virulence cascade that provide a mechanism for the enhanced expression of the principal virulence regulatory genes.
Primer on Agar-Based Microbial Imaging Mass Spectrometry
Authors: Yang Jane Y. , Phelan Vanessa V. , Simkovsky Ryan , Watrous Jeramie D. , Trial Rachelle M. , Fleming Tinya C. , Wenter Roland , Moore Bradley S. , Golden Susan S. , Pogliano Kit , Dorrestein Pieter C.
Published: July 2012

Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) imaging mass spectrometry (IMS) applied directly to microbes on agar-based medium captures global information about microbial molecules, allowing for direct correlation of chemotypes to phenotypes. This tool was developed to investigate metabolic exchange factors of intraspecies, interspecies, and polymicrobial interactions. Based on our experience of the thousands of images we have generated in the laboratory, we present five steps of microbial IMS: culturing, matrix application, dehydration of the sample, data acquisition, and data analysis/interpretation. We also address the common challenges encountered during sample preparation, matrix selection and application, and sample adherence to the MALDI target plate. With the practical guidelines described herein, microbial IMS use can be extended to bio-based agricultural, biofuel, diagnostic, and therapeutic discovery applications.
Characterization of the Organic Hydroperoxide Resistance System of Brucella abortus 2308
Authors: Caswell Clayton C. , Baumgartner John E. , Martin Daniel W. , Roop R. Martin
Published: July 2012

The organic hydroperoxide resistance protein Ohr has been identified in numerous bacteria where it functions in the detoxification of organic hydroperoxides, and expression ofohris often regulated by a MarR-type regulator called OhrR. The genes annotated as BAB2_0350 and BAB2_0351 in theBrucella abortus2308 genome sequence are predicted to encode OhrR and Ohr orthologs, respectively. Using isogenicohrandohrRmutants andlacZpromoter fusions, it was determined that Ohr contributes to resistance to organic hydroperoxide, but not hydrogen peroxide, inB. abortus2308 and that OhrR represses the transcription of bothohrandohrRin this strain. Moreover, electrophoretic mobility shift assays and DNase I footprinting revealed that OhrR binds directly to a specific region in the intergenic region betweenohrandohrRthat shares extensive nucleotide sequence similarity with so-called “OhrR boxes” described in other bacteria. While Ohr plays a prominent role in protectingB. abortus2308 from organic hydroperoxide stress inin vitroassays, this protein is not required for the wild-type virulence of this strain in cultured murine macrophages or experimentally infected mice.
Cell Wall Amidase AmiC1 Is Required for Cellular Communication and Heterocyst Development in the Cyanobacterium Anabaena PCC 7120 but Not for Filament Integrity
Authors: Berendt Susanne , Lehner Josef , Zhang Yao Vincent , Rasse Tobias M. , Forchhammer Karl , Maldener Iris
Published: July 2012

Filamentous cyanobacteria of the order
Nostocales
display typical properties of multicellular organisms. In response to nitrogen starvation, some vegetative cells differentiate into heterocysts, where fixation of N
2
takes place. Heterocysts provide a micro-oxic compartment to protect nitrogenase from the oxygen produced by the vegetative cells. Differentiation involves fundamental remodeling of the Gram-negative cell wall by deposition of a thick envelope and by formation of a neck-like structure at the contact site to the vegetative cells. Cell wall-hydrolyzing enzymes, like cell wall amidases, are involved in peptidoglycan maturation and turnover in unicellular bacteria. Recently, we showed that mutation of the amidase homologue
amiC2
gene in
Nostoc punctiforme
ATCC 29133 distorts filament morphology and function. Here, we present the functional characterization of two
amiC
paralogues from
Anabaena
sp. strain PCC 7120. The
amiC1
(
alr0092
) mutant was not able to differentiate heterocysts or to grow diazotrophically, whereas the
amiC2
(
alr0093
) mutant did not show an altered phenotype under standard growth conditions. In agreement, fluorescence recovery after photobleaching (FRAP) studies showed a lack of cell-cell communication only in the AmiC1 mutant. Green fluorescent protein (GFP)-tagged AmiC1 was able to complement the mutant phenotype to wild-type properties. The protein localized in the septal regions of newly dividing cells and at the neck region of differentiating heterocysts. Upon nitrogen step-down, no mature heterocysts were developed in spite of ongoing heterocyst-specific gene expression. These results show the dependence of heterocyst development on amidase function and highlight a pivotal but so far underestimated cellular process, the remodeling of peptidoglycan, for the biology of filamentous cyanobacteria.
Rethinking the Evolution of Single-Stranded RNA (ssRNA) Bacteriophages Based on Genomic Sequences and Characterizations of Two R-Plasmid-Dependent ssRNA Phages, C-1 and Hgal1
Authors: Kannoly Sherin , Shao Yongping , Wang Ing-Nang
Published: July 2012

We have sequenced and characterized two R-plasmid-dependent single-stranded RNA bacteriophages (RPD ssRNA phages), C-1 and Hagl1. Phage C-1 requires a conjugative plasmid of the IncC group, while Hgal1 requires the IncH group. Both the adsorption rate constants and one-step growth curves are determined for both phages. We also empirically confirmed the lysis function of the predicted lysis genes. Genomic sequencing and phylogenetic analyses showed that both phages belong to theLevivirusgroup and are most closely related to another IncP-plasmid-dependent ssRNA phage, PRR1. Furthermore, our result strongly suggests that the stereotypical bauplans of genome organization found inLevivirusandAlloleviviruspredate phage specialization for conjugative plasmids, suggesting that the utilization of conjugative plasmids for cell attachment and entry comprises independent evolutionary events for these two main clades of ssRNA phages. Our result is also consistent with findings of a previous study, making theLevivirus-like genome organization ancestral and theAllolevivirus-like genome derived. To obtain a deeper insight into the evolution of ssRNA phages, more phages specializing for various conjugative plasmids and infecting different bacterial species would be needed.
The Small RNA RteR Inhibits Transfer of the Bacteroides Conjugative Transposon CTnDOT
Authors: Waters Jillian L. , Salyers Abigail A.
Published: July 2012

CTnDOT is a 65-kb conjugative transposon present in
Bacteroides
spp. that confers resistance to erythromycin [
erm
(F)] and tetracycline [
tet
(Q)]. An interesting feature of CTnDOT is that both excision from the chromosome and transfer of CTnDOT are stimulated by exposure to tetracycline. However, when no tetracycline is present, transfer of CTnDOT is not detectable. Previous studies suggested that a region containing a small RNA, RteR, appeared to mediate repression of CTnDOT transfer; however, virtually nothing was known about RteR. We have demonstrated that RteR is a 90-nucleotide transcript that is not further processed. RteR inhibits conjugative transfer of CTnDOT by targeting the transfer region, a 13-kb operon that encodes the
tra
genes required to assemble the mating apparatus. We report here that RteR interacts with the region downstream of
traA
. Levels of the downstream
tra
mRNA are dramatically reduced when RteR is present. Further, RteR does not appear to decrease the half-life of the
tra
mRNA transcript, suggesting that RteR does not bind to the transcript to initiate RNase-dependent decay, similar to other
trans
-acting small RNAs. We predict that RteR may act to enhance termination of the
tra
operon within
traB
, which could account for the decreased abundance of the
tra
transcript downstream of
traA
and explain why the
tra
mRNA has the same half-life whether or not RteR is present. RteR is the only small RNA that has been characterized so far within the
Bacteroidetes
phylum.
The Primosomal Protein DnaD Inhibits Cooperative DNA Binding by the Replication Initiator DnaA in Bacillus subtilis
Authors: Bonilla Carla Y. , Grossman Alan D.
Published: July 2012

DnaA is an AAA+ ATPase and the conserved replication initiator in bacteria. Bacteria control the timing of replication initiation by regulating the activity of DnaA. DnaA binds to multiple sites in the origin of replication (oriC) and is required for recruitment of proteins needed to load the replicative helicase. DnaA also binds to other chromosomal regions and functions as a transcription factor at some of these sites.Bacillus subtilisDnaD is needed during replication initiation for assembly of the replicative helicase atoriCand during replication restart at stalled replication forks. DnaD associates with DnaA atoriCand at other chromosomal regions bound by DnaA. Using purified proteins, we found that DnaD inhibited the ability of DnaA to bind cooperatively to DNA and caused a decrease in the apparent dissociation constant. These effects of DnaD were independent of the ability of DnaA to bind or hydrolyze ATP. Other proteins known to regulateB. subtilisDnaA also affect DNA binding, whereas much of the regulation ofEscherichia coliDnaA affects nucleotide hydrolysis or exchange. We found that the rate of nucleotide exchange forB. subtilisDnaA was high and not affected by DnaD. The rapid exchange is similar to that ofStaphylococcus aureusDnaA and in contrast to the low exchange rate ofEscherichia coliDnaA. We suggest that organisms in which DnaA has a high rate of nucleotide exchange predominantly regulate the DNA binding activity of DnaA and that those with low rates of exchange regulate hydrolysis and exchange.
Characterization of a New GlnR Binding Box in the Promoter of
amtB
in Streptomyces coelicolor Inferred a PhoP/GlnR Competitive Binding Mechanism for Transcriptional Regulation of
amtB
Authors: Wang Ying , Cen Xu-Feng , Zhao Guo-Ping , Wang Jin
Published: July 2012

The transcription of
amtB
in
Streptomyces coelicolor
has been proposed to be counter-regulated by GlnR (a global regulator for nitrogen metabolism) and PhoP (a global regulator for phosphate metabolism). However, the GlnR-protected region, which was deduced to be two 22-bp GlnR binding boxes (gTnAc-n6-GaAAc-n6-GtnAC-n6-GAAAc-n6, abbreviated as
a1-b1
and
a2-b2
), was separated from the PhoP-protected region in the promoter of
amtB
, leaving the mechanism for this regulation undefined. In this study, another 22-bp GlnR binding box, which consisted of
a3
-site-n6-
b3
-site (
a3-b3
) overlapping with the PhoP-binding sequences, was identified in the promoter region of
amtB
by a DNase I footprinting assay. An electrophoretic mobility shift assay (EMSA) using purified recombinant GlnR and the synthetic
amtB
promoter fragments with the three GlnR binding boxes individually mutated demonstrated that every box was involved in GlnR binding
in vitro
. Further
in vivo
assays using the
egfp
reporter gene fused to various kinds of mutated promoter regions of
amtB
demonstrated that all of the three GlnR binding boxes were required for GlnR-mediated activation of
amtB
transcription under the nitrogen-limited condition. The results of EMSA using the
amtB
promoter with mixtures of recombinant His-tagged GlnR and Trx-His-S-tagged PhoP inferred that PhoP might compete against GlnR from binding at the
a3-b3
site, attributable to the PhoP/GlnR counter-regulatory function subjected to further experimental proof.
Clinical assessment of the scapula: a review of the literature
Authors: Struyf Filip , Nijs Jo , Mottram Sarah , Roussel Nathalie A , Cools Ann M J , Meeusen Romain
Published: July 2012

Scientific evidence supporting a role for faulty scapular positioning in patients with various shoulder disorders is cumulating. Clinicians who manage patients with shoulder pain and athletes at risk of developing shoulder pain need to have the skills to assess static and dynamic scapular positioning and dynamic control. Several methods for the assessment of scapular positioning are described in scientific literature. However, the majority uses expensive and specialised equipment (laboratory methods), making their use in clinical practice nearly impossible. On the basis of biometric and kinematic studies, guidelines for interpreting the observation of static and dynamic scapular positioning pattern in patients with shoulder pain are provided. At this point, clinicians can use reliable clinical tests for the assessment of both static and dynamic scapular positioning in patients with shoulder pain. However, this review also provides clinicians several possible pitfalls when performing clinical scapular evaluation. On the basis of its clinical relevance, its proven reliability, its relation to body length and its applicability in a clinical setting, this review recommends to assess the scapula both static (visual observation and acromial distance or Baylor/double square method for shoulder protraction) and semidynamic (visual observation and inclinometry for scapular upward rotation). In addition, when the patient demonstrates with shoulder impingement symptoms, the scapular repositioning test and scapular assistant test are recommended for relating the patients’ symptoms to the position or movement of the scapula.
Impact of a hospital-wide hand hygiene initiative on healthcare-associated infections: results of an interrupted time series
Authors: Kirkland Kathryn B , Homa Karen A , Lasky Rosalind A , Ptak Judy A , Taylor Eileen A , Splaine Mark E
Published: July 2012

Background

Evidence that hand hygiene (HH) reduces healthcare-associated infections has been available for almost two centuries. Yet HH compliance among healthcare professionals continues to be low, and most efforts to improve it have failed.

Objective

To improve healthcare workers' HH, and reduce healthcare-associated infections.

Design

3-year interrupted time series with multiple sequential interventions and 1-year post-intervention follow-up.

Setting

Teaching hospital in rural New Hampshire.

Interventions

In five categories: (1) leadership/accountability; (2) measurement/feedback; (3) hand sanitiser availability; (4) education/training; and (5) marketing/communication.

Measurement

Monthly changes in observed HH compliance (%) and rates of healthcare-associated infection (includingStaphylococcus aureusinfections,Clostridium difficileinfections and bloodstream infections) per 1000 inpatient days. The subset ofS aureusinfections attributable to the operating room served as a tracer condition. We used statistical process control charts to identify significant changes.

Results

HH compliance increased significantly from 41% to 87% (p<0.01) during the initiative, and improved further to 91% (p<0.01) the following year. Nurses achieved higher HH compliance (93%) than physicians (78%). There was a significant, sustained decline in the healthcare-associated infection rate from 4.8 to 3.3 (p<0.01) per 1000 inpatient days. The rate ofS aureusinfections attributable to the operating room rose, while the rate of otherS aureusinfections fell.

Conclusions

Our initiative was associated with a large and significant hospital-wide improvement in HH which was sustained through the following year and a significant, sustained reduction in the incidence of healthcare-associated infection. The observed increased incidence of the tracer condition supports the assertion that HH improvement contributed to infection reduction. Persistent variation in HH performance among different groups requires further study.
Diagnostic errors in the intensive care unit: a systematic review of autopsy studies
Authors: Winters Bradford , Custer Jason , Galvagno Samuel M , Colantuoni Elizabeth , Kapoor Shruti G , Lee HeeWon , Goode Victoria , Robinson Karen , Nakhasi Atul , Pronovost Peter , Newman-Toker David
Published: July 2012

Context

Misdiagnoses may be an underappreciated cause of preventable morbidity and mortality in the intensive care unit (ICU). Their prevalence, nature, and impact remain largely unknown.

Objectives

To determine whether potentially fatal ICU misdiagnoses would be more common than in the general inpatient population (∼5%), and would involve more infections or vascular events.

Data sources

Systematic review of studies identified by electronic (MEDLINE, etc.) and manual searches (references in eligible articles) without language restriction (1966 through 2011).

Study selection and data abstraction

Observational studies examining autopsy-confirmed diagnostic errors in the adult ICU were included. Studies analysing misdiagnosis of one specific disease were excluded. Study results (autopsy rate, misdiagnosis prevalence, Goldman error class, diseases misdiagnosed) were abstracted and descriptive statistics calculated. We modelled the prevalence of Class I (potentially lethal) misdiagnoses as a non-linear function of the autopsy rate.

Results

Of 276 screened abstracts, 31 studies describing 5863 autopsies (median rate 43%) were analysed. The prevalence of misdiagnoses ranged from 5.5%–100% with 28% of autopsies reporting at least one misdiagnosis and 8% identifying a Class I diagnostic error. The projected prevalence of Class I misdiagnoses for a hypothetical autopsy rate of 100% was 6.3% (95% CI 4.0% to 7.5%). Vascular events and infections were the leading lethal misdiagnoses (41% each). The most common individual Class I misdiagnoses were PE, MI, pneumonia, and aspergillosis.

Conclusions

Our data suggest that as many as 40 500 adult patients in an ICU in USA may die with an ICU misdiagnoses annually. Despite this, diagnostic errors receive relatively little attention and research funding. Future studies should seek to prospectively measure the prevalence and impact of diagnostic errors and potential strategies to reduce them.
Removal of doctors from practice for professional misconduct in Australia and New Zealand
Authors: Elkin Katie , Spittal Matthew J , Elkin David , Studdert David M
Published: July 2012

Objective

To examine how disciplinary tribunals assess different forms of misconduct in deciding whether to remove doctors from practice for professional misconduct.

Design and setting

Multivariable regression analysis of 485 cases in which tribunals found doctors guilty of professional misconduct. The cases came from four Australian states (New South Wales, Victoria, Queensland and Western Australia) and New Zealand and were decided over a 10-year period (1 January 2000 – 30 September 2009).

Main outcome measures

Type of misconduct, the tribunal's explanation for why the misconduct occurred, and the disciplinary measure imposed.

Results

43% of the cases resulted in removal of the offending doctor from practice, 37% in restrictions on practice and 19% in non-restrictive sanctions. The odds of removal were very high in cases involving sexual relationships with patients (OR 22.59; 95% CI 10.18 to 50.14) and moderately high in cases involving inappropriate sexual conduct (not in the context of a relationship), commission of criminal offences, and forms of inappropriate conduct unrelated to patients. Cases in which the misconduct was judged to be due to willful wrongdoing (OR 17.14; 95% CI 8.62 to 34.09), incompetence (OR 6.02; 95% CI 2.87 to 12.63) and issues in the doctor's personal life (OR 4.17; 95% CI 2.07 to 8.41) also had higher odds removal from practice.

Conclusion

Tribunals in Australia and New Zealand tend to remove doctors from practice for behaviours indicative of character flaws and lack of insight, rather than behaviours exhibiting errors in care delivery, poor clinical judgement or lack of knowledge. The generalisability of these findings to regulatory regimes for health practitioners in other countries should be tested.
Improving outcome in severe trauma: what's new in ABC? Imaging, bleeding and brain injury
Authors: Harris Tim , Davenport Ross , Hurst Tom , Hunt Paul , Fotheringham Tim , Jones Jonathan
Published: July 2012

Appropriate imaging is critical in the initial assessment of patients with severe trauma. Plain radiographs remain integral to the primary survey. Focused ultrasonography is useful for identifying intraperitoneal fluid likely to represent haemorrhage in patients who are shocked and also has a role in identifying intrathoracic pathology. Modern scanners permit a greater role for CT, being more rapid and exposing the patient to less ionising radiation. ‘Whole body’ (head to pelvis) CT scanning has been shown to identify injuries missed by ‘traditional’ focused assessment and may be associated with an improved outcome. CT identifies more spinal injuries than plain radiographs, is the gold standard for diagnosing blunt aortic injury and facilitates non-operative management of solid organ injury and other bleeding. Coagulopathy occurs early in trauma as a direct result of injury and hypoperfusion. Damage control resuscitation with blood components is associated with an improved outcome in patients with trauma with massive haemorrhage. Packed cells and fresh frozen plasma should be used in a 1:1 to 1:2 ratio. Bedside measures of coagulopathy may prove useful. Adjuvant early treatment with tranexamic acid is of benefit in reducing blood loss and reducing mortality. Limited ‘damage control surgery’ with early optimisation of physiology augmented by interventional radiology to control haemorrhage is preferable to early definitive care. Limiting haemorrhage by correction of anticoagulation and minimising secondary brain injury through optimal supportive care is critical to improving outcome in neurotrauma.
Comparative outcomes of total hip and knee arthroplasty: a prospective cohort study
Authors: Hamilton David , Henderson G Robin , Gaston Paul , MacDonald Deborah , Howie Colin , Simpson A Hamish R W
Published: July 2012

Purpose

The comparative outcome of primary hip and knee arthroplasty is not well understood. This study aimed to investigate the outcome and satisfaction of these procedures and determine predictive models for 1 year patient outcome with a view to informing surgical management and patient expectations.

Study design

Prospective cohort study of all primary hip and knee arthroplasty procedures performed at the Royal Infirmary of Edinburgh between January 2006 and November 2008. General health (SF-12) and joint specific function (Oxford Score) was assessed pre-operatively and at 6 and 12 months post-operatively. Patient satisfaction was assessed at 12 months.

Results

1410 total hip arthroplasty (THA) and 1244 total knee arthroplasty (TKA) procedures were assessed. Oxford Score improved by 4.9 points more in THA patients than in TKA patients. SF-12 physical scores were on average 2.7 points greater in the THA patients at one year. Satisfaction was also greater (91%) following THA compared with TKA (81%). Regression modelling was not able to predict individual patient outcome; however, mean pre-operative Oxford Scores were found to be strong predictors of mean post-operative Oxford Scores for each procedure. Age, gender and pre-operative general health scores did not influence these models.

Conclusions

Both THA and TKA confer substantial improvement in patient outcome; however, greater joint specific, general health and satisfaction scores are reported following THA. This difference is physical in nature. Regression models are presented that can be applied to predict mean hip/knee arthroplasty outcome based on preoperative values.
Cyclophilin-A: a potential screening marker for vascular disease in type-2 diabetes
Authors: Ramachandran Surya , Kartha C.C.
Published: July 2012

The pathophysiology of vascular disease in diabetes involves abnormalities in endothelial cells, vascular smooth muscle cells, and monocytes. The metabolic abnormalities that characterize diabetes, such as hyperglycemia, increased free fatty acids, and insulin resistance, each provoke molecular mechanisms that contribute to vascular dysfunction. Several molecules have been identified as risk markers, and have been studied to prevent progression of disease and long-term complications. Markers such as C-reactive protein and monocyte chemoattractant protein-1 are used to assess risk for adverse cardiac events, but elevated levels are possible due to the presence of other risk factors as part of the natural physiological defense mechanism. In this review we discuss potential of cyclophilin-A, a secreted oxidative-stress-induced immunophilin with diverse functions. We present evidence for a significant role of cyclophilin-A in the pathogenesis of atherosclerosis in diabetes, and its potential as a marker for vascular disease in type-2 diabetes.
Receptors and ionic transporters in nuclear membranes: new targets for therapeutical pharmacological interventions
Authors: Bkaily Ghassan , Avedanian Levon , Al-Khoury Johny , Ahmarani Lena , Perreault Claudine , Jacques Danielle
Published: July 2012

Work from our group and other laboratories showed that the nucleus could be considered as a cell within a cell. This is based on growing evidence of the presence and role of nuclear membrane G-protein coupled receptors and ionic transporters in the nuclear membranes of many cell types, including vascular endothelial cells, endocardial endothelial cells, vascular smooth muscle cells, cardiomyocytes, and hepatocytes. The nuclear membrane receptors were found to modulate the functioning of ionic transporters at the nuclear level, and thus contribute to regulation of nuclear ionic homeostasis. Nuclear membranes of the mentioned types of cells possess the same ionic transporters; however, the type of receptors is cell-type dependent. Regulation of cytosolic and nuclear ionic homeostasis was found to be dependent upon a tight crosstalk between receptors and ionic transporters of the plasma membranes and those of the nuclear membrane. This crosstalk seems to be the basis for excitation–contraction coupling, excitation–secretion coupling, and excitation – gene expression coupling. Further advancement in this field will certainly shed light on the role of nuclear membrane receptors and transporters in health and disease. This will in turn enable the successful design of a new class of drugs that specifically target such highly vital nuclear receptors and ionic transporters.
KINETICS OF POLYMER EJECTION FROM CAPSID CONFINEMENT: SCALING CONSIDERATIONS AND COMPUTER EXPERIMENT
Authors: MILCHEV A. , BINDER K.
Published: July 2012

We investigate the ejection dynamics of a flexible polymer chain out of confined environment by means of scaling considerations and Monte Carlo simulations. Situations of this kind arise in different physical contexts, including a flexible synthetic polymer partially confined in a nanopore and a viral genome partially ejected from its capsid. In the case of cylindric confinement the entropic driving force which pulls the chain out of the pore is argued to be constant once a few persistent lengths are out of the pore. We demonstrate that in this case the ejection dynamics follows a [Formula: see text]-law with elapsed time t. The mean ejection time τ depends nonmonotonically on chain length N. However, if the geometric constraints comprise a wider capsid chamber attached to a narrow exit tube, the mechanism of ejection changes and involves the surmounting of an activation barrier. The driving force then varies in time. One finds good agreement of theory and computer simulation with recent experiments with DNA.
POLYMERIC NITROGEN CHAINS CONFINED IN CARBON NANOTUBE BUNDLE
Authors: ZHENG FAWEI , YANG YU , ZHANG PING
Published: July 2012

The hybrid structure of polymeric nitrogen chains and carbon nanotube bundle (CNTB) is studied using first-principle calculations. Our finite temperature molecular dynamics simulations show the hybrid system to be very stable at room temperature. The detailed electronic structure is also revealed in this study. According to the calculated electronic structure, the room temperature stabilization of the hybrid material has been demonstrated to originate from the electron transfer from carbon nanotubes (CNTS) to polymeric nitrogen chains. The present work provides a new way to synthesize nitrogen-based high-energy-density materials (HEDM).
COMPARATIVE STUDY OF THE PHENOMENA OF PRECIPITATION IN Al–Zn–Mg ALLOY BY THE DIFFERENTIAL SCANNING CALORIMETRIC AND THE DILATOMETRY AND INTERPRETATION OF DILATOMETRIC EFFECTS
Authors: HADJADJ L. , AMIRA R. , BOUCHEAR M.
Published: July 2012

Our aim is to contribute to comprehension of the phenomena of precipitation in the Al–Zn–Mg alloys. For this, we have made a comparative study of the transformations of phases using the differential scanning calorimetric and the dilatometry. This last technique is relatively new in the case of Al–Zn–Mg alloys. It consists of two opposite effects (contraction and expansion) observed on the dilatometric curves. These effects translate two opposite metallurgical phenomena which are generally the precipitation and the dissolution.