bluuebunny/crossref_metadata_2025 huggingface dataset:
Azole Affinity of Sterol 14α-Demethylase (CYP51) Enzymes from Candida albicans and Homo sapiens
Authors: Warrilow Andrew G. , Parker Josie E. , Kelly Diane E. , Kelly Steven L.
Published: December 2012

Candida albicansCYP51 (CaCYP51) (Erg11), full-lengthHomo sapiensCYP51 (HsCYP51), and truncated Δ60HsCYP51 were expressed inEscherichia coliand purified to homogeneity. CaCYP51 and both HsCYP51 enzymes bound lanosterol (Ks, 14 to 18 μM) and catalyzed the 14α-demethylation of lanosterol usingHomo sapienscytochrome P450 reductase and NADPH as redox partners. Both HsCYP51 enzymes bound clotrimazole, itraconazole, and ketoconazole tightly (dissociation constants [Kds], 42 to 131 nM) but bound fluconazole (Kd, ∼30,500 nM) and voriconazole (Kd, ∼2,300 nM) weakly, whereas CaCYP51 bound all five medical azole drugs tightly (Kds, 10 to 56 nM). Selectivity for CaCYP51 over HsCYP51 ranged from 2-fold (clotrimazole) to 540-fold (fluconazole) among the medical azoles. In contrast, selectivity for CaCYP51 over Δ60HsCYP51 with agricultural azoles ranged from 3-fold (tebuconazole) to 9-fold (propiconazole). Prothioconazole bound extremely weakly to CaCYP51 and Δ60HsCYP51, producing atypical type I UV-visible difference spectra (Kds, 6,100 and 910 nM, respectively), indicating that binding was not accomplished through direct coordination with the heme ferric ion. Prothioconazole-desthio (the intracellular derivative of prothioconazole) bound tightly to both CaCYP51 and Δ60HsCYP51 (Kd, ∼40 nM). These differences in binding affinities were reflected in the observed 50% inhibitory concentration (IC50) values, which were 9- to 2,000-fold higher for Δ60HsCYP51 than for CaCYP51, with the exception of tebuconazole, which strongly inhibited both CYP51 enzymes. In contrast, prothioconazole weakly inhibited CaCYP51 (IC50, ∼150 μM) and did not significantly inhibit Δ60HsCYP51.
Function of Cytochrome P450 Enzymes RosC and RosD in the Biosynthesis of Rosamicin Macrolide Antibiotic Produced by Micromonospora rosaria
Authors: Iizaka Yohei , Higashi Noriko , Ishida Masanari , Oiwa Reina , Ichikawa Yumi , Takeda Moeka , Anzai Yojiro , Kato Fumio
Published: December 2012

The cytochrome P450 enzyme-encoding genes
rosC
and
rosD
were cloned from the rosamicin biosynthetic gene cluster of
Micromonospora rosaria
IFO13697. The functions of RosC and RosD were demonstrated by gene disruption and complementation with
M. rosaria
and bioconversion of rosamicin biosynthetic intermediates with
Escherichia coli
expressing RosC and RosD. It is proposed that
M. rosaria
IFO13697 has two pathway branches that lead from the first desosaminyl rosamicin intermediate, 20-deoxo-20-dihydro-12,13-deepoxyrosamicin, to rosamicin.
Treatment with Telbivudine Positively Regulates Antiviral Immune Profiles in Chinese Patients with Chronic Hepatitis B
Authors: Ma Liang , Cai Yan-Jun , Yu Lei , Feng Jun-Yan , Wang Juan , Li Cong , Niu Jun-Qi , Jiang Yan-Fang
Published: December 2012

Chronic infection with hepatitis B virus (HBV) is associated with impairment of T and NK cell immunity. This study was aimed at investigating the impact of treatment with telbivudine (LDT) on T and NK cell immunity in patients with chronic hepatitis B (CHB). A total of 54 CHB patients and 30 healthy controls (HC) were recruited. Individual patients were treated orally with 600 mg LDT daily for 13 months. The serum HBV DNA loads, the levels of the HBV-related biomarkers alanine aminotransferase (ALT) and aspartate transaminase (AST), and the numbers of different subsets of peripheral T and NK cells in subjects were measured before and longitudinally after LDT treatment. Following treatment with LDT, the serum HBV DNA loads and the percentages of HBsAg- or HBeAg-seropositive cases were gradually reduced, accompanied by decreased levels of serum ALT and AST. In comparison with the HC, fewer CD3−CD56+and CD244+NK cells and CD3+CD8+T cells, lower frequencies of cytokine+CD4+T cells, and more CD3+CD4+, CD4+CD25+Foxp3+, CD4+CD25+CD127low, and CD8+PD-1+T cells were detected in CHB patients. Treatment with LDT increased the numbers of NK and CD8+cells and the frequencies of cytokine+CD4+T cells but reduced the numbers of CD4+CD25+Foxp3+, CD4+CD25+CD127low, and CD8+PD-1+T cells in CHB patients. The frequencies of cytokine+CD4+T cells were negatively associated with the levels of serum HBV DNA, ALT, and AST. Thus, treatment with LDT inhibits HBV replication, modulates T and NK cell immunity, and improves liver function in Chinese patients with CHB.
Combinations of Lambda Interferon with Direct-Acting Antiviral Agents Are Highly Efficient in Suppressing Hepatitis C Virus Replication
Authors: Friborg Jacques , Levine Steven , Chen Chaoqun , Sheaffer Amy K. , Chaniewski Susan , Voss Stacey , Lemm Julie A. , McPhee Fiona
Published: December 2012

The clinical efficacy of a pegylated form of human lambda 1 interferon (IFN-λ1; also referred to herein as lambda) has been demonstrated in patients chronically infected with hepatitis C virus (HCV) representing genotypes 1 through 4. In these proof-of-concept studies, lambda showed an improved safety profile compared to the pegylated form of alfa interferon (referred to herein as alfa). In the study described in this report, an assessment of the
in vitro
antiviral activity of type III IFNs toward different HCV replicons revealed that the unpegylated recombinant form of IFN-λ1 (rIFN-λ1) exerted the most robust effect, while rIFN-λ3 exhibited greater activity than rIFN-λ2. More importantly, cross-resistance to rIFN-λ1 was not observed in replicon cell lines known to have reduced susceptibility to investigational direct-acting antiviral (DAA) agents targeting the essential HCV nonstructural protein NS3, NS5A, or NS5B. When combined with either rIFN-α, the NS3 protease inhibitor (NS3 PI) asunaprevir (ASV), the NS5A replication complex inhibitor (NS5A RCI) daclatasvir (DCV), or the NS5B polymerase site I inhibitor (NS5B I) BMS-791325, rIFN-λ1 displayed a mixture of additive and synergistic effects. In three-drug combination studies, inclusion of lambda with ASV and DCV also yielded additive to synergistic effects. In line with these observations, it was demonstrated that a regimen that used a combination of rIFN-λ1 with one or two DAAs was superior to an IFN-free regimen in clearing HCV RNA in genotype 1a cell lines representing wild-type and NS3 protease inhibitor-resistant sequences. Overall, these data support further clinical development of lambda as part of alternative combination treatments with DAAs for patients chronically infected with HCV.
Azithromycin Kills Invasive Aggregatibacter actinomycetemcomitans in Gingival Epithelial Cells
Authors: Lai Pin-Chuang , Walters John D.
Published: December 2012

Aggregatibacter actinomycetemcomitans
invades periodontal pocket epithelium and is therefore difficult to eliminate by periodontal scaling and root planing. It is susceptible to azithromycin, which is taken up by many types of mammalian cells. This led us to hypothesize that azithromycin accumulation by gingival epithelium could enhance the killing of intraepithelial
A. actinomycetemcomitans
. [
3
H]azithromycin transport by Smulow-Glickman gingival epithelial cells and SCC-25 oral epithelial cells was characterized. To test our hypothesis, we infected cultured Smulow-Glickman cell monolayers with
A. actinomycetemcomitans
(Y4 or SUNY 465 strain) for 2 h, treated them with gentamicin to eliminate extracellular bacteria, and then incubated them with azithromycin for 1 to 4 h. Viable intracellular bacteria were released, plated, and enumerated. Azithromycin transport by both cell lines exhibited Michaelis-Menten kinetics and was competitively inhibited by
l-
carnitine and several other organic cations. Cell incubation in medium containing 5 μg/ml azithromycin yielded steady-state intracellular concentrations of 144 μg/ml in SCC-25 cells and 118 μg/ml in Smulow-Glickman cells. Azithromycin induced dose- and time-dependent intraepithelial killing of both
A. actinomycetemcomitans
strains. Treatment of infected Smulow-Glickman cells with 0.125 μg/ml azithromycin killed approximately 29% of the intraepithelial CFU of both strains within 4 h, while treatment with 8 μg/ml azithromycin killed ≥82% of the CFU of both strains (
P
< 0.05). Addition of carnitine inhibited the killing of intracellular bacteria by azithromycin (
P
< 0.05). Thus, human gingival epithelial cells actively accumulate azithromycin through a transport system that facilitates the killing of intraepithelial
A. actinomycetemcomitans
and is shared with organic cations.
Continuous Cellulosic Bioethanol Fermentation by Cyclic Fed-Batch Cocultivation
Authors: Jiang He-Long , He Qiang , He Zhili , Hemme Christopher L. , Wu Liyou , Zhou Jizhong
Published: December 2012

Cocultivation of cellulolytic and saccharolytic microbial populations is a promising strategy to improve bioethanol production from the fermentation of recalcitrant cellulosic materials. Earlier studies have demonstrated the effectiveness of cocultivation in enhancing ethanolic fermentation of cellulose in batch fermentation. To further enhance process efficiency, a semicontinuous cyclic fed-batch fermentor configuration was evaluated for its potential in enhancing the efficiency of cellulose fermentation using cocultivation. Cocultures of cellulolytic
Clostridium thermocellum
LQRI and saccharolytic
Thermoanaerobacter pseudethanolicus
strain X514 were tested in the semicontinuous fermentor as a model system. Initial cellulose concentration and pH were identified as the key process parameters controlling cellulose fermentation performance in the fixed-volume cyclic fed-batch coculture system. At an initial cellulose concentration of 40 g liter
−1
, the concentration of ethanol produced with pH control was 4.5-fold higher than that without pH control. It was also found that efficient cellulosic bioethanol production by cocultivation was sustained in the semicontinuous configuration, with bioethanol production reaching 474 mM in 96 h with an initial cellulose concentration of 80 g liter
−1
and pH controlled at 6.5 to 6.8. These results suggested the advantages of the cyclic fed-batch process for cellulosic bioethanol fermentation by the cocultures.
Rapid and Sensitive Method To Identify Mycobacterium avium subsp. paratuberculosis in Cow's Milk by DNA Methylase Genotyping
Authors: Mundo Silvia Leonor , Gilardoni Liliana Rosa , Hoffman Federico José , Lopez Osvaldo Jorge
Published: December 2012

Paratuberculosis is an infectious, chronic, and incurable disease that affects ruminants, caused byMycobacterium aviumsubsp.paratuberculosis. This bacterium is shed primarily through feces of infected cows but can be also excreted in colostrum and milk and might survive pasteurization. Since an association of genomic sequences ofM. aviumsubsp.paratuberculosisin patients with Crohn's disease has been described; it is of interest to rapidly detectM. aviumsubsp.paratuberculosisin milk for human consumption. IS900insertion is used as a target for PCR amplification to identify the presence ofM. aviumsubsp.paratuberculosisin biological samples. Two target sequences were selected: IS1(155 bp) and IS2(94 bp). These fragments have a 100% identity among allM. aviumsubsp.paratuberculosisstrains sequenced.M. aviumsubsp.paratuberculosiswas specifically concentrated from milk samples by immunomagnetic separation prior to performing PCR. The amplicons were characterized using DNA methylase Genotyping, i.e., the amplicons were methylated with 6-methyl-adenine and digested with restriction enzymes to confirm their identity. The methylated amplicons from 100 CFU ofM. aviumsubsp.paratuberculosiscan be visualized in a Western blot format using an anti-6-methyl-adenine monoclonal antibody. The use of DNA methyltransferase genotyping coupled to a scintillation proximity assay allows for the detection of up to 10 CFU ofM. aviumsubsp.paratuberculosisper ml of milk. This test is rapid and sensitive and allows for automation and thus multiple samples can be tested at the same time.
Heterologous Overexpression and Characterization of a Flavoprotein-Cytochrome
c
Complex Fructose Dehydrogenase of Gluconobacter japonicus NBRC3260
Authors: Kawai Shota , Goda-Tsutsumi Maiko , Yakushi Toshiharu , Kano Kenji , Matsushita Kazunobu
Published: December 2012

A heterotrimeric flavoprotein-cytochrome
c
complex fructose dehydrogenase (FDH) of
Gluconobacter japonicus
NBRC3260 catalyzes the oxidation of
d
-fructose to produce 5-keto-
d
-fructose and is used for diagnosis and basic research purposes as a direct electron transfer-type bioelectrocatalysis. The
fdhSCL
genes encoding the FDH complex of
G. japonicus
NBRC3260 were isolated by a PCR-based gene amplification method with degenerate primers designed from the amino-terminal amino acid sequence of the large subunit and sequenced. Three open reading frames for
fdhSCL
encoding the small, cytochrome
c
, and large subunits, respectively, were found and were presumably in a polycistronic transcriptional unit. Heterologous overexpression of
fdhSCL
was conducted using a broad-host-range plasmid vector, pBBR1MCS-4, carrying a DNA fragment containing the putative promoter region of the membrane-bound alcohol dehydrogenase gene of
Gluconobacter oxydans
and a
G. oxydans
strain as the expression host. We also constructed derivatives modified in the translational initiation codon to ATG from TTG, designated
TTG
FDH and
ATG
FDH. Membranes of the cells producing recombinant
TTG
FDH and
ATG
FDH showed approximately 20 times and 100 times higher specific activity than those of
G. japonicus
NBRC3260, respectively. The cells producing only FdhS and FdhL had no fructose-oxidizing activity, but showed significantly high
d
-fructose:ferricyanide oxidoreductase activity in the soluble fraction of cell extracts, whereas the cells producing the FDH complex showed activity in the membrane fraction. It is reasonable to conclude that the cytochrome
c
subunit is responsible not only for membrane anchoring but also for ubiquinone reduction.
Evolution of the Stx2-Encoding Prophage in Persistent Bovine Escherichia coli O157:H7 Strains
Authors: Park Dongjin , Stanton Eliot , Ciezki Kristin , Parrell Daniel , Bozile Matthew , Pike Daniel , Forst Steven A. , Jeong Kwang Cheol , Ivanek Renata , Döpfer Dörte , Kaspar Charles W.
Published: December 2012

Escherichia coli
O157:H7 is a human pathogen that resides asymptomatically in its bovine host. The level of Shiga toxin (Stx) produced is variable in bovine-derived strains in contrast to human isolates that mostly produce high levels of Stx. To understand the genetic basis for varied Stx production, chronological collections of bovine isolates from Wisconsin dairy farms, R and X, were analyzed for multilocus prophage polymorphisms,
stx
2
subtypes, and the levels of
stx
2
transcript and toxin. The
E. coli
O157:H7 that persisted on both farms were phylogenetically distinct and yet produced little to no Stx2 due to gene deletions in Stx2c-encoding prophage (farm R) or insertional inactivation of
stx
2a
by IS
1203v
(farm X). Loss of key regulatory and lysis genes in Stx2c-encoding prophage abolished
stx
2c
transcription and induction of the prophage and
stx
2a
::IS
1203v
in Stx2a-encoding prophage generated a truncated
stx
2a
mRNA without affecting phage production. Stx2-producing strains were transiently present (farm R) and became Stx2 negative on farm X (i.e.,
stx
2a
::IS
1203v
). To our knowledge, this is the first study that details the evolution of
E. coli
O157:H7 and its Stx2-encoding prophage in a chronological collection of natural isolates. The data suggest the bovine and farm environments can be niches where Stx2-negative
E. coli
O157:H7 emerge and persist, which explains the Stx variability in bovine isolates and may be part of an evolutionary step toward becoming bovine specialists.
Carbon Isotope Fractionation of 11 Acetogenic Strains Grown on H
2
and CO
2
Authors: Blaser Martin B. , Dreisbach Lisa K. , Conrad Ralf
Published: December 2012

Acetogenic bacteria are able to grow autotrophically on hydrogen and carbon dioxide by using the acetyl coenzyme A (acetyl-CoA) pathway. Acetate is the end product of this reaction. In contrast to the fermentative route of acetate production, which shows almost no fractionation of carbon isotopes, the acetyl-CoA pathway has been reported to exhibit a preference for light carbon. In
Acetobacterium woodii
the isotope fractionation factor (ε) for
13
C and
12
C has previously been reported to be ε = −58.6‰. To investigate whether such a strong fractionation is a general feature of acetogenic bacteria, we measured the stable carbon isotope fractionation factor of 10 acetogenic strains grown on H
2
and CO
2
. The average fractionation factor was ε
TIC
= −57.2‰ for utilization of total inorganic carbon and ε
acetate
= −54.6‰ for the production of acetate. The strongest fractionation was found for
Sporomusa sphaeroides
(ε
TIC
= −68.3‰), the lowest fractionation for
Morella thermoacetica
(ε
TIC
= −38.2‰). To investigate the reproducibility of our measurements, we determined the fractionation factor of 21 biological replicates of
Thermoanaerobacter kivui
. In general, our study confirmed the strong fractionation of stable carbon during chemolithotrophic acetate formation in acetogenic bacteria. However, the specific characteristics of the bacterial strain, as well as the cultural conditions, may have a moderate influence on the overall fractionation.
MiniUIB, a Novel Minitransposon-Based System for Stable Insertion of Foreign DNA into the Genomes of Gram-Negative and Gram-Positive Bacteria
Authors: Christie-Oleza Joseph Alexander , Brunet-Galmés Isabel , Lalucat Jorge , Nogales Balbina , Bosch Rafael
Published: December 2012

Transposition of the insertion sequence (IS) IS
Ppu12
is actively induced after conjugative interaction. The transposase of this IS can act in
trans
on structures flanked by inverted repeats similar to those of the transposon. Based on that fact, an IS
Ppu12
-based minitransposon, miniUIB, has been constructed in order to biotechnologically exploit the self-regulation of IS
Ppu12
and its increased activity after conjugative interaction. Mobilization of the miniUIB structure into the genome of
Pseudomonas stutzeri
AN10 after conjugative interaction was demonstrated. A single gene, i.e., the kanamycin resistance determinant, or large genetic structures of >12 kb, i.e.,
alkBFGHJKL
and
alkST
operons of
Pseudomonas putida
TF4-1L (GPo1), have been easily integrated in
P. stutzeri
AN10 by an RP4-based delivery system. Therefore, the integration of the
alk
determinants by use of the miniUIB system has extended the biodegradation capabilities of this strain. Plasmid pJOC100, containing the transposase and regulator genes of IS
Ppu12
adjacent to the miniUIB structure, was constructed in order to extend the host range of this biotechnologically useful genetic tool to other model and real-world bacteria. The effectiveness of the system for random mutagenesis in a phylogenetic wide range of bacteria and for the insertion of novel functions has been demonstrated, even in successive steps.
Prothioconazole and Prothioconazole-Desthio Activities against Candida albicans Sterol 14-α-Demethylase
Authors: Parker Josie E. , Warrilow Andrew G. S. , Cools Hans J. , Fraaije Bart A. , Lucas John A. , Rigdova Katarina , Griffiths William J. , Kelly Diane E. , Kelly Steven L.
Published: December 2012

Prothioconazole is a new triazolinthione fungicide used in agriculture. We have used
Candida albicans
CYP51 (CaCYP51) to investigate the
in vitro
activity of prothioconazole and to consider the use of such compounds in the medical arena. Treatment of
C. albicans
cells with prothioconazole, prothioconazole-desthio, and voriconazole resulted in CYP51 inhibition, as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol. We then compared the inhibitor binding properties of prothioconazole, prothioconazole-desthio, and voriconazole with CaCYP51. We observed that prothioconazole-desthio and voriconazole bind noncompetitively to CaCYP51 in the expected manner of azole antifungals (with type II inhibitors binding to heme as the sixth ligand), while prothioconazole binds competitively and does not exhibit classic inhibitor binding spectra. Inhibition of CaCYP51 activity in a cell-free assay demonstrated that prothioconazole-desthio is active, whereas prothioconazole does not inhibit CYP51 activity. Extracts from
C. albicans
grown in the presence of prothioconazole were found to contain prothioconazole-desthio. We conclude that the antifungal action of prothioconazole can be attributed to prothioconazole-desthio.
Phytophthora infestans Cholinephosphotransferase with Substrate Specificity for Very-Long-Chain Polyunsaturated Fatty Acids
Authors: Chen Yan , Chi Hsiang-yun , Meesapyodsuk Dauenpen , Qiu Xiao
Published: December 2012

The effective flux between phospholipids and neutral lipids is critical for a high level of biosynthesis and accumulation of very-long-chain polyunsaturated fatty acids (VLCPUFAs), such as arachidonic acid (ARA; 20:4n-6), eicosapentaenoic acid (EPA; 20:5n-3), and docosahexaenoic acid (DHA; 22:6n-3). Here we describe a cDNA (
PiCPT1
) from
Phytophthora infestans
, a VLCPUFA-producing oomycete, that may have a role in acyl trafficking between diacylglycerol (DAG) and phosphatidylcholine (PC) during the biosynthesis of VLCPUFAs. The cDNA encodes a polypeptide of 393 amino acids with a conserved CDP-alcohol phosphotransferase motif and approximately 27% amino acid identity to the
Saccharomyces cerevisiae
cholinephosphotransferase (ScCPT1).
In vitro
assays indicate that PiCPT1 has high cholinephosphotransferase (CPT) activity but no ethanolaminephosphotransferase (EPT) activity. Substrate specificity assays show that it prefers VLCPUFA-containing DAGs, such as ARA DAG and DHA DAG, as substrates. Real-time PCR analysis reveals that expression of
PiCPT1
was upregulated in
P. infestans
organisms fed with exogenous VLCPUFAs. These results lead us to conclude that PiCPT1 is a VLCPUFA-specific CPT which may play an important role in shuffling VLCPUFAs from DAG to PC in the biosynthesis of VLCPUFAs in
P. infestans
.
Molecular Analysis of theIn SituGrowth Rates of Subsurface Geobacter Species
Authors: Holmes Dawn E. , Giloteaux Ludovic , Barlett Melissa , Chavan Milind A. , Smith Jessica A. , Williams Kenneth H. , Wilkins Michael , Long Philip , Lovley Derek R.
Published: December 2012

Molecular tools that can provide an estimate of thein situgrowth rate ofGeobacterspecies could improve understanding of dissimilatory metal reduction in a diversity of environments. Whole-genome microarray analyses of a subsurface isolate ofGeobacter uraniireducens, grown under a variety of conditions, identified a number of genes that are differentially expressed at different specific growth rates. Expression of two genes encoding ribosomal proteins,rpsCandrplL, was further evaluated with quantitative reverse transcription-PCR (qRT-PCR) in cells with doubling times ranging from 6.56 h to 89.28 h. Transcript abundance ofrpsCcorrelated best (r2= 0.90) with specific growth rates. Therefore, expression patterns ofrpsCwere used to estimate specific growth rates ofGeobacterspecies during anin situuranium bioremediation field experiment in which acetate was added to the groundwater to promote dissimilatory metal reduction. Initially, increased availability of acetate in the groundwater resulted in higher expression ofGeobacter rpsC, and the increase in the number ofGeobactercells estimated with fluorescentin situhybridization compared well with specific growth rates estimated from levels ofin situ rpsCexpression. However, in later phases, cell number increases were substantially lower than predicted fromrpsCtranscript abundance. This change coincided with a bloom of protozoa and increased attachment ofGeobacterspecies to solid phases. These results suggest that monitoringrpsCexpression may better reflect the actual rate thatGeobacterspecies are metabolizing and growing duringin situuranium bioremediation than changes in cell abundance.
Engineering Synechococcus elongatus PCC 7942 for Continuous Growth under Diurnal Conditions
Authors: McEwen Jordan T. , Machado Iara M. P. , Connor Michael R. , Atsumi Shota
Published: December 2012

Synechococcus elongatus
strain PCC 7942 strictly depends upon the generation of photosynthetically derived energy for growth and is incapable of biomass increase in the absence of light energy. Obligate phototrophs' core metabolism is very similar to that of heterotrophic counterparts exhibiting diverse trophic behavior. Most characterized cyanobacterial species are obligate photoautotrophs under examined conditions. Here we determine that sugar transporter systems are the necessary genetic factors in order for a model cyanobacterium,
Synechococcus elongatus
PCC 7942, to grow continuously under diurnal (light/dark) conditions using saccharides such as glucose, xylose, and sucrose. While the universal causes of obligate photoautotrophy may be diverse, installing sugar transporters provides new insight into the mode of obligate photoautotrophy for cyanobacteria. Moreover, cyanobacterial chemical production has gained increased attention. However, this obligate phototroph is incapable of product formation in the absence of light. Thus, converting an obligate photoautotroph to a heterotroph is desirable for more efficient, economical, and controllable production systems.
Mycobacterium gilvum Illustrates Size-Correlated Relationships between Mycobacteria and Acanthamoeba polyphaga
Authors: Lamrabet Otmane , Drancourt Michel
Published: December 2012

Mycobacteria are isolated from soil and water environments, where free-living amoebae live. Free-living amoebae are bactericidal, yet some rapidly growing mycobacteria are amoeba-resistant organisms that survive in the amoebal trophozoites and cysts. Such a capacity has not been studied for the environmental rapidly growing organism
Mycobacterium gilvum
. We investigated the ability of
M. gilvum
to survive in the trophozoites of
Acanthamoeba polyphaga
strain Linc-AP1 by using optical and electron microscopy and culture-based microbial enumerations in the presence of negative controls. We observed that 29% of
A. polyphaga
cells were infected by
M. gilvum
mycobacteria by 6 h postinfection. Surviving
M. gilvum
mycobacteria did not multiply and did not kill the amoebal trophozoites during a 5-day coculture. Extensive electron microscopy observations indicated that
M. gilvum
measured 1.4 ± 0.5 μm and failed to find
M. gilvum
organisms in the amoebal cysts. Further experimental study of two other rapidly growing mycobacteria,
Mycobacterium rhodesiae
and
Mycobacterium thermoresistibile
, indicated that both measured <2 μm and exhibited the same amoeba-mycobacterium relationships as
M. gilvum
. In general, we observed that mycobacteria measuring <2 μm do not significantly grow within and do not kill amoebal trophozoites, in contrast to mycobacteria measuring >2 μm (
P
< 0.05). The mechanisms underlying such an observation remain to be determined.
Role of Nitrogen Limitation in Transformation of RDX (Hexahydro-1,3,5-Trinitro-1,3,5-Triazine) by Gordonia sp. Strain KTR9
Authors: Indest Karl J. , Hancock Dawn E. , Jung Carina M. , Eberly Jed O. , Mohn William W. , Eltis Lindsay D. , Crocker Fiona H.
Published: December 2012

The transcriptome of RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine)-degrading strain
Gordonia
sp. strain KTR9 and its
glnR
mutant were studied as a function of nitrogen availability to further investigate the observed ammonium-mediated inhibition of RDX degradation. The results indicate that nitrogen availability is a major determinant of RDX degradation and
xplA
gene expression in KTR9.
Sequencing and Functional Annotation of Avian Pathogenic Escherichia coli Serogroup O78 Strains Reveal the Evolution of E. coli Lineages Pathogenic for Poultry via Distinct Mechanisms
Authors: Dziva Francis , Hauser Heidi , Connor Thomas R. , van Diemen Pauline M. , Prescott Graham , Langridge Gemma C. , Eckert Sabine , Chaudhuri Roy R. , Ewers Christa , Mellata Melha , Mukhopadhyay Suman , Curtiss Roy , Dougan Gordon , Wieler Lothar H. , Thomson Nicholas R. , Pickard Derek J. , Stevens Mark P.
Published: December 2012

Avian pathogenic
Escherichia coli
(APEC) causes respiratory and systemic disease in poultry. Sequencing of a multilocus sequence type 95 (ST95) serogroup O1 strain previously indicated that APEC resembles
E. coli
causing extraintestinal human diseases. We sequenced the genomes of two strains of another dominant APEC lineage (ST23 serogroup O78 strains χ7122 and IMT2125) and compared them to each other and to the reannotated APEC O1 sequence. For comparison, we also sequenced a human enterotoxigenic
E. coli
(ETEC) strain of the same ST23 serogroup O78 lineage. Phylogenetic analysis indicated that the APEC O78 strains were more closely related to human ST23 ETEC than to APEC O1, indicating that separation of pathotypes on the basis of their extraintestinal or diarrheagenic nature is not supported by their phylogeny. The accessory genome of APEC ST23 strains exhibited limited conservation of APEC O1 genomic islands and a distinct repertoire of virulence-associated loci. In light of this diversity, we surveyed the phenotype of 2,185 signature-tagged transposon mutants of χ7122 following intra-air sac inoculation of turkeys. This procedure identified novel APEC ST23 genes that play strain- and tissue-specific roles during infection. For example, genes mediating group 4 capsule synthesis were required for the virulence of χ7122 and were conserved in IMT2125 but absent from APEC O1. Our data reveal the genetic diversity of
E. coli
strains adapted to cause the same avian disease and indicate that the core genome of the ST23 lineage serves as a chassis for the evolution of
E. coli
strains adapted to cause avian or human disease via acquisition of distinct virulence genes.
Disruption of Francisella tularensis Schu S4
iglI
,
iglJ
, and
pdpC
Genes Results in Attenuation for Growth in Human Macrophages and
In Vivo
Virulence in Mice and Reveals a Unique Phenotype for
pdpC
Authors: Long Matthew E. , Lindemann Stephen R. , Rasmussen Jed A. , Jones Bradley D. , Allen Lee-Ann H.
Published: December 2012

Francisella tularensis
is a facultative intracellular bacterial pathogen and the causative agent of tularemia. After infection of macrophages, the organism escapes from its phagosome and replicates to high density in the cytosol, but the bacterial factors required for these aspects of virulence are incompletely defined. Here, we describe the isolation and characterization of
Francisella tularensis
subsp.
tularensis
strain Schu S4 mutants that lack functional
iglI
,
iglJ
, or
pdpC
, three genes of the
Francisella
pathogenicity island. Our data demonstrate that these mutants were defective for replication in primary human monocyte-derived macrophages and murine J774 cells yet exhibited two distinct phenotypes. The
iglI
and
iglJ
mutants were similar to one another, exhibited profound defects in phagosome escape and intracellular growth, and appeared to be trapped in cathepsin D-positive phagolysosomes. Conversely, the
pdpC
mutant avoided trafficking to lysosomes, phagosome escape was diminished but not ablated, and these organisms replicated in a small subset of infected macrophages. The phenotype of each mutant strain was reversed by
trans
complementation.
In vivo
virulence was assessed by intranasal infection of BALB/c mice. The mutants appeared avirulent, as all mice survived infection with 10
8
CFU
iglJ-
or
pdpC
-deficient bacteria. Nevertheless, the
pdpC
mutant disseminated to the liver and spleen before being eliminated, whereas the
iglJ
mutant did not. Taken together, our data demonstrate that the pathogenicity island genes tested are essential for
F. tularensis
Schu S4 virulence and further suggest that
pdpC
may play a unique role in this process, as indicated by its distinct intermediate phenotype.
Helicobacter pylori Infection Impairs the Mucin Production Rate and Turnover in the Murine Gastric Mucosa
Authors: Navabi Nazanin , Johansson Malin E. V. , Raghavan Sukanya , Lindén Sara K.
Published: December 2012

To protect the surface of the stomach, the epithelial cells secrete a mucus layer, which is mainly comprised of the MUC5AC mucin. Further protection is provided by a thick glycocalyx on the apical surface of the epithelial cell, with the cell surface mucin MUC1 as a major component. Here, we investigate the production rate and turnover of newly synthesized mucin in mice and analyze the effects of early colonization and chronic infection with
H. pylori
. Metabolic incorporation of an azido GalNAc analog (GalNAz) was used as a nonradioactive method to perform pulse experiments in the whole animal. First, the subcellular movement of newly synthesized mucin and mucin turnover was determined in uninfected mice. Based on the time line for mucin transport and dissemination, 2, 6, and 12 h after GalNAz injection was selected to collect the stomachs from mice infected with
H. pylori
strain SS1 during early colonization (7 days) and chronic infection (90 days). The results demonstrated that the speed from the start of glycosylation to the final destination is faster for the membrane-bound mucin to reach the glycocalyx (2 h) than for the secretory mucins to become secreted into the mucus layer (5 h). Furthermore, infection with
H. pylori
reduces the rate of mucin turnover and decreases the levels of Muc1. Since
H. pylori
colonizes this mucus niche, the decreased turnover rate indicates that
H. pylori
creates a more stable and favorable environment for itself by impairing the defense mechanism for clearing the mucosal surface of pathogens by mucus flow.
Deletion of the Braun Lipoprotein-Encoding Gene and Altering the Function of Lipopolysaccharide Attenuate the Plague Bacterium
Authors: Sha Jian , Kirtley Michelle L. , van Lier Christina J. , Wang Shaofei , Erova Tatiana E. , Kozlova Elena V. , Cao Anthony , Cong Yingzi , Fitts Eric C. , Rosenzweig Jason A. , Chopra Ashok K.
Published: December 2012

Braun (murein) lipoprotein (Lpp) and lipopolysaccharide (LPS) are major components of the outer membranes ofEnterobacteriaceaefamily members that are capable of triggering inflammatory immune responses by activating Toll-like receptors 2 and 4, respectively. Expanding on earlier studies that demonstrated a role played by Lpp inYersinia pestisvirulence in mouse models of bubonic and pneumonic plague, we characterized anmsbBin-frame deletion mutant incapable of producing an acyltransferase that is responsible for the addition of lauric acid to the lipid A moiety of LPS, as well as a ΔlppΔmsbBdouble mutant of the highly virulentY. pestisCO92 strain. Although the ΔmsbBsingle mutant was minimally attenuated, the Δlppsingle mutant and the ΔlppΔmsbBdouble mutant were significantly more attenuated than the isogenic wild-type (WT) bacterium in bubonic and pneumonic animal models (mouse and rat) of plague. These data correlated with greatly reduced survivability of the aforementioned mutants in murine macrophages. Furthermore, the ΔlppΔmsbBdouble mutant was grossly compromised in its ability to disseminate to distal organs in mice and in evoking cytokines/chemokines in infected animal tissues. Importantly, mice that survived challenge with the ΔlppΔmsbBdouble mutant, but not the Δlppor ΔmsbBsingle mutant, in a pneumonic plague model were significantly protected against a subsequent lethal WT CO92 rechallenge. These data were substantiated by the fact that the ΔlppΔmsbBdouble mutant maintained an immunogenicity comparable to that of the WT strain and induced long-lasting T-cell responses against heat-killed WT CO92 antigens. Taken together, the data indicate that deletion of themsbBgene augmented the attenuation of the Δlppmutant by crippling the spread of the double mutant to the peripheral organs of animals and by inducing cytokine/chemokine responses. Thus, the ΔlppΔmsbBdouble mutant could provide a new live-attenuated background vaccine candidate strain, and this should be explored in the future.
Full-Length Plasmodium falciparum Circumsporozoite Protein Administered with Long-Chain Poly(I·C) or the Toll-Like Receptor 4 Agonist Glucopyranosyl Lipid Adjuvant-Stable Emulsion Elicits Potent Antibody and CD4+T Cell Immunity and Protection in Mice
Authors: Kastenmüller Kathrin , Espinosa Diego A. , Trager Lauren , Stoyanov Cristina , Salazar Andres M. , Pokalwar Santosh , Singh Sanjay , Dutta Sheetij , Ockenhouse Christian F. , Zavala Fidel , Seder Robert A.
Published: December 2012

ThePlasmodium falciparumcircumsporozoite (CS) protein (CSP) is a major vaccine target for preventing malaria infection. Thus, developing strong and durable antibody and T cell responses against CSP with novel immunogens and potent adjuvants may improve upon the success of current approaches. Here, we compare four distinct full-lengthP. falciparumCS proteins expressed inEscherichia coliorPichia pastorisfor their ability to induce immunity and protection in mice when administered with long-chain poly(I·C) [poly(I·C)LC] as an adjuvant. CS proteins expressed inE. coliinduced high-titer antibody responses against the NANP repeat region and potent CSP-specific CD4+T cell responses. Moreover,E. coli-derived CS proteins in combination with poly(I·C)LC induced potent multifunctional (interleukin 2-positive [IL-2+], tumor necrosis factor alpha-positive [TNF-α+], gamma interferon-positive [IFN-γ+]) CD4+effector T cell responses in blood, in spleen, and particularly in liver. Using transgenicPlasmodium bergheiexpressing the repeat region ofP. falciparumCSP [Pb-CS(Pf)], we showed that there was a 1- to 4-log decrease in malaria rRNA in the liver following a high-dose challenge and ∼50% sterilizing protection with a low-dose challenge compared to control levels. Protection was directly correlated with high-level antibody titers but not CD4+T cell responses. Finally, protective immunity was also induced using the Toll-like receptor 4 agonist glucopyranosyl lipid adjuvant-stable emulsion (GLA-SE) as the adjuvant, which also correlated with high antibody titers yet CD4+T cell immunity that was significantly less potent than that with poly(I·C)LC. Overall, these data suggest that full-length CS proteins and poly(I·C)LC or GLA-SE offer a simple vaccine formulation to be used alone or in combination with other vaccines for preventing malaria infection.
The Unique Structure of Haemophilus influenzae Protein E Reveals Multiple Binding Sites for Host Factors
Authors: Singh Birendra , Al-Jubair Tamim , Mörgelin Matthias , Thunnissen Marjolein M. , Riesbeck Kristian
Published: December 2012

Haemophilus influenzae
protein E (PE) is a multifunctional adhesin involved in direct interactions with lung epithelial cells and host proteins, including plasminogen and the extracellular matrix proteins vitronectin and laminin. We recently crystallized PE and successfully collected X-ray diffraction data at 1.8 Å. Here, we solved the structure of a recombinant version of PE and analyzed different functional regions. It is a dimer in solution and in the asymmetric unit of the crystals. The dimer has a structure that resembles a flattened β-barrel. It is, however, not a true β-barrel, as there are differences in both the hydrogen-bonding pattern and the shape. Each monomer consisted of a 6-stranded antiparallel β-sheet with a rigid α-helix at the C terminus tethered to the concave side of the sheet by a disulfide bridge. The laminin/plasminogen binding region (residues 41 to 68) is exposed, while the vitronectin binding region (residues 84 to 108) is partially accessible in the dimer. The dimerized PE explains the simultaneous interaction with laminin and vitronectin. In addition, we found this unique adhesin to be present in many bacterial genera of the family
Pasteurellaceae
and also orthologues in other, unrelated species (
Enterobacter cloacae
and
Listeria monocytogenes
). Peptides corresponding to the surface-exposed regions PE 24 to 37, PE 74 to 89, and PE 134 to 156 were immunogenic in the mouse. Importantly, these peptide-based antibodies also recognized PE at the bacterial surface. Taken together, our detailed structure of PE explains how this important virulence factor of
H. influenzae
simultaneously interacts with host vitronectin, laminin, or plasminogen, promoting bacterial pathogenesis.
Validity of theInternational Classification of Diseases 10th revisioncode for hospitalisation with hyponatraemia in elderly patients
Authors: Gandhi Sonja , Shariff Salimah Z , Fleet Jamie L , Weir Matthew A , Jain Arsh K , Garg Amit X
Published: December 2012

Objective

To evaluate the validity of theInternational Classification of Diseases, 10th Revision(ICD-10) diagnosis code for hyponatraemia (E87.1) in two settings: at presentation to the emergency department and at hospital admission.

Design

Population-based retrospective validation study.

Setting

Twelve hospitals in Southwestern Ontario, Canada, from 2003 to 2010.

Participants

Patients aged 66 years and older with serum sodium laboratory measurements at presentation to the emergency department (n=64 581) and at hospital admission (n=64 499).

Main outcome measures

Sensitivity, specificity, positive predictive value and negative predictive value comparing various ICD-10 diagnostic coding algorithms for hyponatraemia to serum sodium laboratory measurements (reference standard). Median serum sodium values comparing patients who were code positive and code negative for hyponatraemia.

Results

The sensitivity of hyponatraemia (defined by a serum sodium ≤132 mmol/l) for the best-performing ICD-10 coding algorithm was 7.5% at presentation to the emergency department (95% CI 7.0% to 8.2%) and 10.6% at hospital admission (95% CI 9.9% to 11.2%). Both specificities were greater than 99%. In the two settings, the positive predictive values were 96.4% (95% CI 94.6% to 97.6%) and 82.3% (95% CI 80.0% to 84.4%), while the negative predictive values were 89.2% (95% CI 89.0% to 89.5%) and 87.1% (95% CI 86.8% to 87.4%). In patients who were code positive for hyponatraemia, the median (IQR) serum sodium measurements were 123 (119–126) mmol/l and 125 (120–130) mmol/l in the two settings. In code negative patients, the measurements were 138 (136–140) mmol/l and 137 (135–139) mmol/l.

Conclusions

The ICD-10 diagnostic code for hyponatraemia differentiates between two groups of patients with distinct serum sodium measurements at both presentation to the emergency department and at hospital admission. However, these codes underestimate the true incidence of hyponatraemia due to low sensitivity.
Identifying clinical criteria to predict Type 1 diabetes, as defined by absolute insulin deficiency: a systematic review protocol
Authors: Shields Beverley M , Peters Jaime L , Cooper Chris , Powell Roy J , Knight Bridget A , Hyde Christopher , Hattersley Andrew T
Published: December 2012

Introduction

Management of a patient's diabetes is entirely dependent upon the type of diabetes they are deemed to have. Patients with Type 1 diabetes are insulin deficient so require multiple daily insulin injections, whereas patients with Type 2 diabetes still have some endogenous insulin production so insulin treatment is only required when diet and tablets do not establish good glycaemic control. Despite the importance of a correct diagnosis, classification of diabetes is based on aetiology and relies on clinical judgement. There are no clinical guidelines on how to determine whether a patient has Type 1 or Type 2 diabetes. We aim to systematically review the literature to derive evidence-based clinical criteria for the classification of the major subtypes of diabetes.

Methods and analysis

We will perform a systematic review of diagnostic accuracy studies to establish clinical criteria that predict the subsequent development of absolute insulin deficiency seen in Type 1 diabetes. Insulin deficiency will be determined by reference standard C-peptide concentrations. Synthesis of criteria identified will be undertaken using hierarchical summary receiver operating characteristic curves.

Ethics and dissemination

As this is a systematic review, there will be no ethical issues. We will disseminate results by writing up the final systematic review and synthesis for publication in a peer-reviewed journal and will present at national and international diabetes-related meetings.
Veterinarians' perceptions of behaviour support in small‐animal practice
Authors: Roshier A. L. , McBride E. A.
Published: December 2012

Veterinarians are professionals considered to be at the forefront of animal welfare, including behaviour medicine. However, concerns raised, both within the profession and without, highlight that the support offered is not optimal, due to deficiencies in veterinary training, which focuses on physical aspects and overlooks psychological aspects. This preliminary study explored the experiences and perceptions of six veterinarians (three male, three female, age range: 23–55 years) in two UK small‐animal practices. Seventeen annual booster consultations were videoed and conversations thematically analysed for welfare topics discussed. Both veterinarians and clients completed questionnaires to gather demographic information and perspectives. All veterinarians recognised behaviour as a component of their caseload, and acknowledged that clients expected them to provide behaviour support. Veterinarians varied in their experiences of and confidence in providing behaviour support. Five felt unable to meet client expectations; four did not feel their training had prepared them sufficiently. Only one provided dedicated behaviour consultations, the others referred cases. All provided suggestions for behaviour skills needed for new veterinary graduates. The study has afforded an insight into the experiences of a small opportunistic sample of veterinarians. The data indicated important limitations regarding time available in general consultations to discuss behaviour concerns, and practitioner knowledge and skill in detection, anamnesis, assessment and provision of appropriate behaviour information. Suggestions for veterinary training in behaviour are provided.
Canine behaviour problems: discussions between veterinarians and dog owners during annual booster consultations
Authors: Roshier A. L. , McBride E. A.
Published: December 2012

The veterinary profession recently acknowledged its responsibility to provide behaviour support, following criticism for focussing on the physiological aspects of welfare and overlooking the psychological. To further understand the practising of behavioural medicine, a ‘fly‐on‐the‐wall’ approach was used to investigate welfare discussions during dog booster vaccinations. Seventeen consultations involving six veterinarians in two UK small‐animal practices were videoed. Qualitative methods were used to analyse themes discussed and questionnaires completed to obtain participant information and perceptions. Five main topics of discussion were identified: navigation, medical, husbandry, behaviour and cost. Veterinarians led the discussion of all topics except behaviour which was instigated approximately equally by veterinarian and client. All clients reported one or more behaviours that were a concern to them, totalling 58 across the sample. Disconcertingly, only 10 were discussed during consultations and none fully explored nor managed beyond the consultation. Behaviour discussion varies between veterinarians; this may reflect their experience, confidence or clients' requests. Owners access welfare information from a variety of sources, not always from veterinarians. Where sources are not knowledgeable, both human and animal welfare can be seriously compromised. Veterinarians need to ensure that clients are enabled to discuss behaviour issues and are provided with appropriate support, be that in‐house or via referral.