bluuebunny/crossref_metadata_2025 huggingface dataset:
Two Novel Type II Restriction-Modification Systems Occupying Genomically Equivalent Locations on the Chromosomes of Listeria monocytogenes Strains
Authors: Lee Sangmi , Ward T. J. , Siletzky R. M. , Kathariou S.
Published: February 2012

Listeria monocytogenes
is responsible for the potentially life-threatening food-borne disease listeriosis. One epidemic-associated clonal group of
L. monocytogenes
, epidemic clone I (ECI), harbors a Sau3AI-like restriction-modification (RM) system also present in the same genomic region in certain strains of other lineages. In this study, we identified and characterized two other, novel type II RM systems, LmoJ2 and LmoJ3, at this same locus. LmoJ2 and LmoJ3 appeared to recognize GCWGC (W = A or T) and GCNGC, respectively. Both RM systems consisted of genes with GC content below the genome average and were in the same genomic region in strains of different serotypes and lineages, suggesting site-specific horizontal gene transfer. Genomic DNA from the LmoJ2 and LmoJ3 strains grown at various temperatures (4 to 42°C) was resistant to digestion with restriction enzymes recognizing GCWGC or GCNGC, indicating that the methyltransferases were expressed under these conditions. Phages propagated in an LmoJ2-harboring strain exhibited moderately increased infectivity for this strain at 4 and 8°C but not at higher temperatures, while phages propagated in an LmoJ3 strain had dramatically increased infectivity for this strain at all temperatures. Among the sequenced
Listeria
phages, lytic phages possessed significantly fewer recognition sites for these RM systems than lysogenic phages, suggesting that in lytic phages sequence content evolved toward reduced susceptibility to such RM systems. The ability of LmoJ2 and LmoJ3 to protect against phages may affect the efficiency of phages as biocontrol agents for
L. monocytogenes
strains harboring these RM systems.
Anaerobic Methyl
tert
-Butyl Ether-Degrading Microorganisms Identified in Wastewater Treatment Plant Samples by Stable Isotope Probing
Authors: Sun Weimin , Sun Xiaoxu , Cupples Alison M.
Published: February 2012

Anaerobic methyl
tert
-butyl ether (MTBE) degradation potential was investigated in samples from a range of sources. From these 22 experimental variations, only one source (from wastewater treatment plant samples) exhibited MTBE degradation. These microcosms were methanogenic and were subjected to DNA-based stable isotope probing (SIP) targeted to both bacteria and archaea to identify the putative MTBE degraders. For this purpose, DNA was extracted at two time points, subjected to ultracentrifugation, fractioning, and terminal restriction fragment length polymorphism (TRFLP). In addition, bacterial and archaeal 16S rRNA gene clone libraries were constructed. The SIP experiments indicated bacteria in the phyla
Firmicutes
(family
Ruminococcaceae
) and
Alphaproteobacteria
(genus
Sphingopyxis
) were the dominant MTBE degraders. Previous studies have suggested a role for
Firmicutes
in anaerobic MTBE degradation; however, the putative MTBE-degrading microorganism in the current study is a novel MTBE-degrading phylotype within this phylum. Two archaeal phylotypes (genera
Methanosarcina
and
Methanocorpusculum
) were also enriched in the heavy fractions, and these organisms may be responsible for minor amounts of MTBE degradation or for the uptake of metabolites released from the primary MTBE degraders. Currently, limited information exists on the microorganisms able to degrade MTBE under anaerobic conditions. This work represents the first application of DNA-based SIP to identify anaerobic MTBE-degrading microorganisms in laboratory microcosms and therefore provides a valuable set of data to definitively link identity with anaerobic MTBE degradation.
Assessing the Contributions of the LiaS Histidine Kinase to the Innate Resistance of Listeria monocytogenes to Nisin, Cephalosporins, and Disinfectants
Authors: Collins Barry , Guinane Caitriona M. , Cotter Paul D. , Hill Colin , Ross R. Paul
Published: February 2012

The
Listeria monocytogenes
LiaSR two-component system (2CS) encoded by
lmo1021
and
lmo1022
plays an important role in resistance to the food preservative nisin. A nonpolar deletion in the histidine kinase-encoding component (Δ
liaS
) resulted in a 4-fold increase in nisin resistance. In contrast, the Δ
liaS
strain exhibited increased sensitivity to a number of cephalosporin antibiotics (and was also altered with respect to its response to a variety of other antimicrobials, including the active agents of a number of disinfectants). This pattern of increased nisin resistance and reduced cephalosporin resistance in
L. monocytogenes
has previously been associated with mutation of a second histidine kinase, LisK, which is a predicted regulator of
liaS
and a penicillin binding protein encoded by
lmo2229
. We noted that
lmo2229
transcription is increased in the Δ
liaS
mutant and in a Δ
liaS
Δ
lisK
double mutant and that disruption of
lmo2229
in the Δ
liaS
Δ
lisK
mutant resulted in a dramatic sensitization to nisin but had a relatively minor impact on cephalosporin resistance. We anticipate that further efforts to unravel the complex mechanisms by which LiaSR impacts on the antimicrobial resistance of
L. monocytogenes
could facilitate the development of strategies to increase the susceptibility of the pathogen to these agents.
Microbiota Dynamics and Diversity at Different Stages of Industrial Processing of Cocoa Beans into Cocoa Powder
Authors: Lima Lídia J. R. , van der Velpen Vera , Wolkers-Rooijackers Judith , Kamphuis Henri J. , Zwietering Marcel H. , Nout M. J. Rob
Published: February 2012

We sampled a cocoa powder production line to investigate the impact of processing on the microbial community size and diversity at different stages. Classical microbiological methods were combined with 16S rRNA gene PCR-denaturing gradient gel electrophoresis, coupled with clone library construction, to analyze the samples. Aerobic thermoresistant spores (ThrS) (100°C; 10 min) were also isolated and characterized (identity, genetic diversity, and spore heat resistance), in view of their relevance to the quality of downstream heat-treated cocoa-flavored drinks. In the nibs (broken, shelled cocoa beans), average levels of total aerobic microorganisms (TAM) (4.4 to 5.6 log CFU/g) and aerobic total spores (TS) (80°C; 10 min; 4.3 to 5.5 log CFU/g) were significantly reduced (
P
< 0.05) as a result of alkalizing, while fungi (4.2 to 4.4 log CFU/g) and
Enterobacteriaceae
(1.7 to 2.8 log CFU/g) were inactivated to levels below the detection limit, remaining undetectable throughout processing. Roasting further decreased the levels of TAM and TS, but they increased slightly during subsequent processing. Molecular characterization of bacterial communities based on enriched cocoa samples revealed a predominance of members of the
Bacillaceae
,
Pseudomonadaceae
, and
Enterococcaceae
. Eleven species of ThrS were found, but
Bacillus licheniformis
and the
Bacillus subtilis
complex were prominent and revealed great genetic heterogeneity. We concluded that the microbiota of cocoa powder resulted from microorganisms that could have been initially present in the nibs, as well as microorganisms that originated during processing.
B. subtilis
complex members, particularly
B. subtilis
subsp.
subtilis
, formed the most heat-resistant spores. Their occurrence in cocoa powder needs to be considered to ensure the stability of derived products, such as ultrahigh-temperature-treated chocolate drinks.
Ribosome Display of Combinatorial Antibody Libraries Derived from Mice Immunized with Heat-Killed Xylella fastidiosa and the Selection of MopB-Specific Single-Chain Antibodies
Authors: Azizi Armaghan , Arora Arinder , Markiv Anatoliy , Lampe David J. , Miller Thomas A. , Kang Angray S.
Published: February 2012

Pierce's disease is a devastating lethal disease of
Vitus vinifera
grapevines caused by the bacterium
Xylella fastidiosa
. There is no cure for Pierce's disease, and control is achieved predominantly by suppressing transmission of the glassy-winged sharpshooter insect vector. We present a simple robust approach for the generation of panels of recombinant single-chain antibodies against the surface-exposed elements of
X. fastidiosa
that may have potential use in diagnosis and/or disease transmission blocking studies.
In vitro
combinatorial antibody ribosome display libraries were assembled from immunoglobulin transcripts rescued from the spleens of mice immunized with heat-killed
X. fastidiosa
. The libraries were used in a single round of selection against an outer membrane protein, MopB, resulting in the isolation of a panel of recombinant antibodies. The potential use of selected anti-MopB antibodies was demonstrated by the successful application of the 4XfMopB3 antibody in an enzyme-linked immunosorbent assay (ELISA), a Western blot assay, and an immunofluorescence assay (IFA). These immortalized
in vitro
recombinant single-chain antibody libraries generated against heat-killed
X. fastidiosa
are a resource for the Pierce's disease research community that may be readily accessed for the isolation of antibodies against a plethora of
X. fastidiosa
surface-exposed antigenic molecules.
BrpA Is Involved in Regulation of Cell Envelope Stress Responses in Streptococcus mutans
Authors: Bitoun J. P. , Liao S. , Yao X. , Ahn S.-J. , Isoda R. , Nguyen A. H. , Brady L. J. , Burne R. A. , Abranches J. , Wen Z. T.
Published: February 2012

Previous studies have shown that BrpA plays a major role in acid and oxidative stress tolerance and biofilm formation by
Streptococcus mutans
. Mutant strains lacking BrpA also display increased autolysis and decreased viability, suggesting a role for BrpA in cell envelope integrity. In this study, we examined the impact of BrpA deficiency on cell envelope stresses induced by envelope-active antimicrobials. Compared to the wild-type strain UA159, the BrpA-deficient mutant (TW14D) was significantly more susceptible to antimicrobial agents, especially lipid II inhibitors. Several genes involved in peptidoglycan synthesis were identified by DNA microarray analysis as downregulated in TW14D. Luciferase reporter gene fusion assays also revealed that expression of
brpA
is regulated in response to environmental conditions and stresses induced by exposure to subinhibitory concentrations of cell envelope antimicrobials. In a
Galleria mellonella
(wax worm) model, BrpA deficiency was shown to diminish the virulence of
S. mutans
OMZ175, which, unlike
S. mutans
UA159, efficiently kills the worms. Collectively, these results suggest that BrpA plays a role in the regulation of cell envelope integrity and that deficiency of BrpA adversely affects the fitness and diminishes the virulence of OMZ175, a highly invasive strain of
S. mutans
.
Prevalence of Yersinia enterocolitica in Pigs Slaughtered in Chinese Abattoirs
Authors: Liang Junrong , Wang Xin , Xiao Yuchun , Cui Zhigang , Xia Shengli , Hao Qiong , Yang Jinchuan , Luo Longze , Wang Shukun , Li Kewei , Yang Haoshu , Gu Wenpeng , Xu Jianguo , Kan Biao , Jing Huaiqi
Published: February 2012

The distribution of
Yersinia enterocolitica
in slaughtered pigs in China was studied. A total of 8,773 samples were collected and examined from different pig abattoirs in 11 provinces from 2009 to 2011. Of these, 4,495 were oral-pharyngeal swab (tonsils) samples from pigs, 1,239 were from intestinal contents, and 3,039 were feces samples from abattoirs or local pigpens. The data showed that 1,132 strains were obtained, from which the isolation rate for
Yersinia enterocolitica
was 19.53% (878/4,495) from the tonsil samples, 7.51% (93/1,239) from intestinal contents, and 5.30% (161/3,039) from feces. Of the 850 pathogenic
Yersinia
strains, except for three of bioserotype 2/O:9 and three of bioserotype 4/O:3, most (844/850) were of bioserotype 3/O:3. Interestingly, pathogenic
Y. enterocolitica
accounted for the majority of the isolated strains from most provinces (85.17% to 100%), whereas from Heilongjiang, 96.52% (111/115) were classified as nonpathogenic biotype 1A with various serotypes, and only 3.48% of the strains (4/115) were pathogenic 3/O:3. All of the pathogenic strains were analyzed using pulsed-field gel electrophoresis (PFGE), and 49 patterns were obtained for the O:3 pathogenic strains; most of them were K6GN11C30021 (53.13%: 450/847) and K6GN11C30012 (21.37%: 181/847). Several strains from diarrhea patient samples revealed PFGE patterns identical to that from samples of local pigs, suggesting a possible link between porcine isolates and human infection. The results above suggested that
Yersinia enterocolitica
in slaughtered pigs from Chinese abattoirs was characterized by region-specific PFGE patterns and confirmed that strains isolated from pigs are closely related to those from human infections.
Rapid and Specific Detection of Escherichia coli Serogroups O26, O45, O103, O111, O121, O145, and O157 in Ground Beef, Beef Trim, and Produce by Loop-Mediated Isothermal Amplification
Authors: Wang Fei , Jiang Lin , Yang Qianru , Prinyawiwatkul Witoon , Ge Beilei
Published: February 2012

Escherichia coli
O157 and six additional serogroups of Shiga toxin-producing
E. coli
(STEC) (O26, O45, O103, O111, O121, and O145) account for the majority of STEC infections in the United States. In this study, O serogroup-specific genes (
wzx
or
wzy
) were used to design loop-mediated isothermal amplification (LAMP) assays for the rapid and specific detection of these leading STEC serogroups. The assays were evaluated in pure culture and spiked food samples (ground beef, beef trim, lettuce, and spinach) and compared with real-time quantitative PCR (qPCR). No false-positive or false-negative results were observed among 120 bacterial strains used to evaluate assay specificity. The limits of detection of various STEC strains belonging to these target serogroups were approximately 1 to 20 CFU/reaction mixture in pure culture and 10
3
to 10
4
CFU/g in spiked food samples, which were comparable to those of qPCR. Standard curves generated suggested good linear relationships between STEC cell numbers and LAMP turbidity signals. In various beef and produce samples spiked with two low levels (1 to 2 and 10 to 20 CFU/25 g) of respective STEC strains, the LAMP assays consistently achieved accurate detection after 6 to 8 h of enrichment. In conclusion, these newly developed LAMP assays may facilitate rapid and reliable detection of the seven major STEC serogroups in ground beef, beef trim, and produce during routine sample testing.
Regulation of Vacuolar H
+
-ATPase Activity by the Cdc42 Effector Ste20 in Saccharomyces cerevisiae
Authors: Lin Meng , Li Sheena Claire , Kane Patricia M. , Höfken Thomas
Published: February 2012

In the budding yeast
Saccharomyces cerevisiae
, the Cdc42 effector Ste20 plays a crucial role in the regulation of filamentous growth, a response to nutrient limitation. Using the split-ubiquitin technique, we found that Ste20 forms a complex with Vma13, an important regulatory subunit of vacuolar H
+
-ATPase (V-ATPase). This protein-protein interaction was confirmed by a pulldown assay and coimmunoprecipitation. We also demonstrate that Ste20 associates with vacuolar membranes and that Ste20 stimulates V-ATPase activity in isolated vacuolar membranes. This activation requires Ste20 kinase activity and does not depend on increased assembly of the V
1
and V
0
sectors of the V-ATPase, which is a major regulatory mechanism. Furthermore, loss of V-ATPase activity leads to a strong increase in invasive growth, possibly because these cells fail to store and mobilize nutrients efficiently in the vacuole in the absence of the vacuolar proton gradient. In contrast to the wild type, which grows in rather small, isolated colonies on solid medium during filamentation, hyperinvasive
vma
mutants form much bigger aggregates in which a large number of cells are tightly clustered together. Genetic data suggest that Ste20 and the protein kinase A catalytic subunit Tpk2 are both activated in the
vma13
Δ strain. We propose that during filamentous growth, Ste20 stimulates V-ATPase activity. This would sustain nutrient mobilization from vacuolar stores, which is beneficial for filamentous growth.
Genomic Rearrangements Leading to Overexpression of Aldo-Keto Reductase YafB of Escherichia coli Confer Resistance to Glyoxal
Authors: Kwon Minsuk , Lee Junghoon , Lee Changhan , Park Chankyu
Published: February 2012

Glyoxal is toxic and mutagenic
α
-oxoaldehyde generated
in vivo
as an oxidation by-product of sugar metabolism. We selected glyoxal-resistant mutants from an
Escherichia coli
strain lacking major glyoxal-detoxifying genes,
gloA
and
yqhD
, by growing cells in medium containing a lethal concentration of glyoxal. The mutants carried diverse genomic rearrangements, such as multibase deletions and recombination, in the upstream region of the
yafB
gene, encoding an aldo-keto reductase. Since these genomic lesions create transcriptional fusions of the
yafB
gene to the upstream
rrn
regulon or eliminate a negative regulatory site, the mutants generally enhanced an expression of the
yafB
gene. Glyoxal resistances of the mutants are correlated with the levels of
yafB
transcripts as well as the activities of aldo-keto reductase. An overproduction of YafB in the glyoxal-resistant mutant lacking the putative NsrR-binding site provides evidence that the
yafB
gene is negatively regulated by this protein. We also observed that the expression of
yafB
is enhanced with an increased concentration of glyoxal as well as a mutation in the
fnr
gene, encoding a putative regulator. The bindings of NsrR and Fnr to the
yafB
promoter were also demonstrated by gel mobility shift assays.
ExpR Is Not Required for Swarming but Promotes Sliding in Sinorhizobium meliloti
Authors: Nogales Joaquina , Bernabéu-Roda Lydia , Cuéllar Virginia , Soto María J.
Published: February 2012

Swarming is a mode of translocation dependent on flagellar activity that allows bacteria to move rapidly across surfaces. In several bacteria, swarming is a phenotype regulated by quorum sensing. It has been reported that the swarming ability of the soil bacterium
Sinorhizobium meliloti
Rm2011 requires a functional ExpR/Sin quorum-sensing system. However, our previous published results demonstrate that strains Rm1021 and Rm2011, both known to have a disrupted copy of
expR
, are able to swarm on semisolid minimal medium. In order to clarify these contradictory results, the role played by the LuxR-type regulator ExpR has been reexamined. Results obtained in this work revealed that
S. meliloti
can move over semisolid surfaces using at least two different types of motility. One type is flagellum-independent surface spreading or sliding, which is positively influenced by a functional
expR
gene mainly through the production of exopolysaccharide II (EPS II). To a lesser extent, EPS II-deficient strains can also slide on surfaces by a mechanism that is at least dependent on the siderophore rhizobactin 1021. The second type of surface translocation shown by
S. meliloti
is swarming, which is greatly dependent on flagella and rhizobactin 1021 but does not require ExpR. We have extended our study to demonstrate that the production of normal amounts of succinoglycan (EPS I) does not play a relevant role in surface translocation but that its overproduction facilitates both swarming and sliding motilities.
Novel, Highly Specific
N
-Demethylases Enable Bacteria To Live on Caffeine and Related Purine Alkaloids
Authors: Summers Ryan M. , Louie Tai Man , Yu Chi-Li , Gakhar Lokesh , Louie Kailin C. , Subramanian Mani
Published: February 2012

The molecular basis for the ability of bacteria to live on caffeine as a sole carbon and nitrogen source is unknown.
Pseudomonas putida
CBB5, which grows on several purine alkaloids, metabolizes caffeine and related methylxanthines via sequential
N
-demethylation to xanthine. Metabolism of caffeine by CBB5 was previously attributed to one broad-specificity methylxanthine
N
-demethylase composed of two subunits, NdmA and NdmB. Here, we report that NdmA and NdmB are actually two independent Rieske nonheme iron monooxygenases with
N
1
- and
N
3
-specific
N
-demethylation activity, respectively. Activity for both enzymes is dependent on electron transfer from NADH via a redox-center-dense Rieske reductase, NdmD. NdmD itself is a novel protein with one Rieske [2Fe-2S] cluster, one plant-type [2Fe-2S] cluster, and one flavin mononucleotide (FMN) per enzyme. All
ndm
genes are located in a 13.2-kb genomic DNA fragment which also contained a formaldehyde dehydrogenase.
ndmA
,
ndmB
, and
ndmD
were cloned as His
6
fusion genes, expressed in
Escherichia coli
, and purified using a Ni-NTA column. NdmA-His
6
plus His
6
-NdmD catalyzed
N
1
-demethylation of caffeine, theophylline, paraxanthine, and 1-methylxanthine to theobromine, 3-methylxanthine, 7-methylxanthine, and xanthine, respectively. NdmB-His
6
plus His
6
-NdmD catalyzed
N
3
-demethylation of theobromine, 3-methylxanthine, caffeine, and theophylline to 7-methylxanthine, xanthine, paraxanthine, and 1-methylxanthine, respectively. One formaldehyde was produced from each methyl group removed. Activity of an
N
7
-specific
N
-demethylase, NdmC, has been confirmed biochemically. This is the first report of bacterial
N
-demethylase genes that enable bacteria to live on caffeine. These genes represent a new class of Rieske oxygenases and have the potential to produce biofuels, animal feed, and pharmaceuticals from coffee and tea waste.
The Early Divisome Protein FtsA Interacts Directly through Its 1c Subdomain with the Cytoplasmic Domain of the Late Divisome Protein FtsN
Authors: Busiek Kimberly K. , Eraso Jesus M. , Wang Yipeng , Margolin William
Published: February 2012

InEscherichia coli, FtsN localizes late to the cell division machinery, only after a number of additional essential proteins are recruited to the early FtsZ-FtsA-ZipA complex. FtsN has a short, positively charged cytoplasmic domain (FtsNCyto), a single transmembrane domain (FtsNTM), and a periplasmic domain that is essential for FtsN function. Here we show that FtsA and FtsN interact directlyin vitro. FtsNCytois sufficient to bind to FtsA, but only when it is tethered to FtsNTMor to a leucine zipper. Mutation of a conserved patch of positive charges in FtsNCytoto negative charges abolishes the interaction with FtsA. We also show that subdomain 1c of FtsA is sufficient to mediate this interaction with FtsN. Finally, although FtsNCyto-TMis not essential for FtsN function, its overproduction causes a modest dominant-negative effect on cell division. These results suggest that basic residues within a dimerized FtsNCytoprotein interact directly with residues in subdomain 1c of FtsA. Since FtsA binds directly to FtsZ and FtsN interacts with enzymes involved in septum synthesis and splitting, this interaction between early and late divisome proteins may be one of several feedback controls for Z ring constriction.
The Branched-Chain Amino Acid Aminotransferase Encoded by
ilvE
Is Involved in Acid Tolerance in Streptococcus mutans
Authors: Santiago Brendaliz , MacGilvray Matthew , Faustoferri Roberta C. , Quivey Robert G.
Published: February 2012

The ability of
Streptococcus mutans
to produce and tolerate organic acids from carbohydrate metabolism represents a major virulence factor responsible for the formation of carious lesions. Pyruvate is a key metabolic intermediate that, when rerouted to other metabolic pathways such as amino acid biosynthesis, results in the alleviation of acid stress by reducing acid end products and aiding in maintenance of intracellular pH. Amino acid biosynthetic genes such as
ilvC
and
ilvE
were identified as being upregulated in a proteome analysis of
Streptococcus mutans
under acid stress conditions (A. C. Len, D. W. Harty, and N. A. Jacques, Microbiology
150:
1353–1366, 2004). In
Lactococcus lactis
and
Staphylococcus carnosus
, the
ilvE
gene product is involved with biosynthesis and degradation of branched-chain amino acids, as well as in the production of branched-chain fatty acids (B. Ganesan and B. C. Weimer, Appl. Environ. Microbiol.
70:
638–641, 2004; S. M. Madsen et al., Appl. Environ. Microbiol.
68:
4007–4014, 2002; and M. Yvon, S. Thirouin, L. Rijnen, D. Fromentier, and J. C. Gripon, Appl. Environ. Microbiol.
63:
414–419, 1997). Here we constructed and characterized an
ilvE
deletion mutant of
S. mutans
UA159. Growth experiments revealed that the
ilvE
mutant strain has a lag in growth when nutritionally limited for branched-chain amino acids. We further demonstrated that the loss of
ilvE
causes a decrease in acid tolerance. The
ilvE
strain exhibits a defect in F
1
-F
o
ATPase activity and has reduced catabolic activity for isoleucine and valine. Results from transcriptional studies showed that the
ilvE
promoter is upregulated during growth at low pH. Collectively, the results of this investigation show that amino acid metabolism is a component of the acid-adaptive repertoire of
S. mutans
.
Identification of Bacillus subtilis SipW as a Bifunctional Signal Peptidase That Controls Surface-Adhered Biofilm Formation
Authors: Terra Rebecca , Stanley-Wall Nicola R. , Cao Guoqiang , Lazazzera Beth A.
Published: February 2012

Biofilms of microbial cells encased in an exopolymeric matrix can form on solid surfaces, but how bacteria sense a solid surface and upregulate biofilm genes is largely unknown. We investigated the role of the
Bacillus subtilis
signal peptidase, SipW, which has a unique role in forming biofilms on a solid surface and is not required at an air-liquid interface. Surprisingly, we found that the signal peptidase activity of SipW was not required for solid-surface biofilms. Furthermore, a SipW mutant protein was constructed that lacks the ability to form a solid-surface biofilm but still retains signal peptidase activity. Through genetic and gene expression tests, the non-signal peptidase role of SipW was found to activate biofilm matrix genes specifically when cells were on a solid surface. These data provide the first evidence that a signal peptidase is bifunctional and that SipW has a regulatory role in addition to its role as a signal peptidase.
Evaluation of a novel nutrition education intervention for medical students from across England
Authors: Ray Sumantra , Udumyan Ruzan , Rajput-Ray Minha , Thompson Ben , Lodge Keri-Michele , Douglas Pauline , Sharma Poonam , Broughton Rachel , Smart Sandra , Wilson Rick , Gillam Stephen , van der Es Mike J , Fisher Ilana , Gandy Joan
Published: February 2012

Objectives

Problems such as hospital malnutrition (∼40% prevalence in the UK) may be managed better by improving the nutrition education of ‘tomorrow's doctors’. The Need for Nutrition Education Programme aimed to measure the effectiveness and acceptability of an educational intervention on nutrition for medical students in the clinical phase of their training.

Design

An educational needs analysis was followed by a consultative process to gain consensus on a suitable educational intervention. This was followed by two identical 2-day educational interventions with before and after analyses of Knowledge, Attitudes and Practices (KAP). The 2-day training incorporated six key learning outcomes.

Setting

Two constituent colleges of Cambridge University used to deliver the above educational interventions.

Participants

An intervention group of 100 clinical medical students from 15 medical schools across England were recruited to attend one of two identical intensive weekend workshops.

Primary and secondary outcome measures

The primary outcome measure consisted of change in KAP scores following intervention using a clinical nutrition questionnaire. Secondary outcome measures included change in KAP scores 3 months after the intervention as well as a student-led semiqualitative evaluation of the educational intervention.

Results

Statistically significant changes in KAP scores were seen immediately after the intervention, and this was sustained for 3 months. Mean differences and 95% CIs after intervention were Knowledge 0.86 (0.43 to 1.28); Attitude 1.68 (1.47 to 1.89); Practice 1.76 (1.11 to 2.40); KAP 4.28 (3.49 to 5.06). Ninety-seven per cent of the participants rated the overall intervention and its delivery as ‘very good to excellent’, reporting that they would recommend this educational intervention to colleagues.

Conclusion

Need for Nutrition Education Programme has highlighted the need for curricular innovation in the area of clinical health nutrition in medical schools. This project also demonstrates the effectiveness and acceptability of such a curriculum intervention for ‘tomorrow's doctors’. Doctors, dietitians and nutritionists worked well in an effective interdisciplinary partnership when teaching medical students, providing a good model for further work in a healthcare setting.
Benefits and risks of structuring and/or coding the presenting patient history in the electronic health record: systematic review
Authors: Fernando Bernard , Kalra Dipak , Morrison Zoe , Byrne Emma , Sheikh Aziz
Published: February 2012

Background

Patient histories in electronic health records currently exist mainly in free text format thereby limiting the possibility that decision support technology may contribute to the accuracy and timeliness of clinical diagnoses. Structuring and/or coding make patient histories potentially computable.

Methods

A systematic review was undertaken of the benefits and risks of structuring and/or coding patient history by searching nine international databases for published and unpublished studies over the period 1990–2010. The focus was on the current patient history, defined as information reported by a patient or the patient's caregiver about the patient's present health situation and health status. Findings were synthesised through a theoretically based textural analysis.

Findings

Of the 9207 potentially eligible papers identified, 10 studies satisfied the eligibility criteria. There was evidence of a modest number of benefits associated with structuring the current patient history, including obtaining more complete clinical histories, improved accuracy of patient self-documented histories, and better associated decision-making by professionals. However, no studies demonstrated any resulting improvements in patient care or outcomes. When more detailed records were obtained through the use of a structured format no attempt was made to confirm if this additional information was clinically useful. No studies investigated possible risks associated with structuring the patient history. No studies examined coding of the patient history.

Conclusions

There is an insufficient evidence base for sound policy making on the benefits and risks of structuring and/or coding patient history. The authors suggest this field of enquiry warrants further investigation given the interest in use of decision support technology to aid diagnoses.
Deliberate self-poisoning: characteristics of patients and impact on the emergency department of a large university hospital
Authors: Hendrix Lotte , Verelst Sandra , Desruelles Didier , Gillet Jean-Bernard
Published: February 2012

Study objective

The epidemiology, management and cost of emergency department (ED) visits for deliberate self-poisoning (DSP) are described.

Methods

In a retrospective study, the medical records of all DSP patients older than 16 years, who presented to the ED from 1 January 2009 to 31 December 2009, were reviewed.

Results

312 episodes of DSP were included, accounting for 0.6% of all ED visits. 190 patients were women, with a female to male ratio of 1.56:1. Mean patient age was 37 years. More than 60% (n=190) of DSP patients were <40 years of age. Most patients presented to the ED between 18:00 and 23:00. A single drug was ingested in 39% (n=121) of patients. Alcohol was co-ingested by 36% of patients who were mostly middle-aged men. Of the overdoses, 50.8% were due to benzodiazepines, 23.2% were due to antidepressants and 16.4% were due to antipsychotics. Two-thirds of patients were treated with oral activated charcoal and 89% were seen by a psychiatrist. Nearly 90% of patients were admitted to the ED observation ward, with a mean length of stay of 16.7 h. The estimated total cost was €;266 134.89, with an average of €;872.57 per patient.

Conclusion

Self-poisoning cases in Belgium are grossly similar to those in other Western countries. Supportive treatment alone should be considered in the majority of patients presenting with oral drug overdose. Overall, DSP leads to a significant financial burden on the community.
Pedestrians' perceptions of walkability and safety in relation to the built environment in Cali, Colombia, 2009–10
Authors: Villaveces Andrés , Nieto Luis Alfonso , Ortega Delia , Ríos José Fernando , Medina John Jairo , Gutiérrez María Isabel , Rodríguez Daniel
Published: February 2012

Objective

To assess pedestrians' perceptions of the walkability of the urban environment and pedestrian safety in Cali, Colombia.

Design

Standardised intercept interviews were conducted of 400 pedestrians walking in 20 randomly selected urban zones to ascertain frequency of walking, and perceptions of safety, the built environment and security. Four focus group meetings were held with community members and students addressing these issues in an open-ended forum. The study analysed quantitative data collected in street interviews and qualitative information from focus groups addressing respondents' views on problems for pedestrians, how the built environment affects walking and ideal walking conditions.

Results

Access to public transportation was deemed the most positive characteristic of the built environment and 61% of respondents liked walking in the street. When disaggregating street conditions, the majority of pedestrians were dissatisfied with their walking experiences. Pedestrians cited lack of respect for norms, personal safety and built environment characteristics as their main concerns. Among frequent walkers, residents tended to rate their neighbourhoods more negatively compared with non-residents. Through qualitative interviews it became apparent that narrow sidewalks that are frequently obstructed by motor vehicles are a key reason for pedestrian dissatisfaction. A perception of overall insecurity further determines how pedestrians assess or modulate their walking in this city.

Conclusions

Overall, walking in Cali was perceived negatively by pedestrians because of built environment characteristics and perceptions of insecurity. Qualitative information used to complement intercept surveys can provide a better way to identify pedestrian-specific transport-related problems.
Telecenter for secure, remote, collaborative child fatality review
Authors: Zonfrillo Mark R , Kumar Mahendra , Fortes Jose A , Winston Flaura K
Published: February 2012

Objectives

Child fatality review (CFR) is the systematic, interdisciplinary, multi-agency examination of paediatric deaths. While CFR findings may influence policies and reduce preventable fatalities, limited resources challenge accurate CFR data collection and prevention recommendations. Therefore, using technology to improve efficiency of reviews and access to remote participants could enhance the CFR experience. This project aimed to adapt a previously developed collaborative web-based appliance for remote, secure, collaborative review of crash investigations for use with CFR.

Methods

The user-centred design and evaluation process included: (1) key informant interviews and visits to CFR sessions to determine current practices, (2) an anonymous, qualitative, internet-based survey of 64 Pennsylvania CFR team leaders, (3) redesign and adaptation of the Telecenter appliance based on survey results and team feedback and (4) pilot-testing of the adapted Telecenter application at an actual local CFR.

Results

The qualitative informant interviews identified facilitators and barriers for adoption of the Telecenter. Facilitators included: team member training, improved communication and collaboration, more efficient reviews, and enhanced preventive efforts. Barriers identified included: concern for confidentiality and security, concern about accepting a novel CFR method, low interest in using technology, cost and maintenance. The survey of CFR team leaders identified themes for improving CFR team functioning including the need for evidence-based prevention resources, increased team training and efficient information gathering. The Telecenter was redesigned and adapted based on the information gathered and was successfully piloted for use with CFR.

Conclusions

Telecenter met the design goal to improve information dissemination and identification of fatality prevention strategies for CFR.
SERT and TPH-1 mRNA expression are reduced in irritable bowel syndrome patients regardless of visceral sensitivity state in large intestine
Authors: Kerckhoffs Angèle P. M. , ter Linde José J. M. , Akkermans Louis M. A. , Samsom Melvin
Published: February 2012

Colorectal visceral hypersensitivity has been demonstrated in a subset of irritable bowel syndrome (IBS) patients. Serine protease and serotonergic signaling modulate gastrointestinal visceral sensitivity. We evaluated whether altered mucosal serine protease and serotonergic pathway components are related to rectal visceral hypersensitivity in IBS patients. Colorectal mucosal biopsies of 23 IBS patients and 15 controls were collected. Gene transcripts of protease-activated receptor (PAR)-2, trypsinogen IV, tryptophan hydroxylase (TPH)-1, and serotonin reuptake transporter (SERT) were quantified using real-time polymerase chain reaction. Substance P and 5-HT contents were measured by ELISA. The number of enterochromaffin cells, mast cells, and intraepithelial lymphocytes was determined using immunohistochemistry. Rectal visceral sensitivity was determined in IBS patients using barostat programmed for phasic ascending distension. Rectal hypersensitivity (+) and (−) IBS patients showed lower TPH-1 and SERT mRNA levels in the rectum compared with controls ( P ≤ 0.05). Rectal hypersensitivity (+) IBS patients ( n = 12) showed lower TPH-1 mRNA level in the sigmoid compared with controls ( P = 0.015). No significant differences were observed in PAR-2 and trypsinogen IV expression between controls and IBS patients. Rectal substance P content was increased in IBS patients compared with controls ( P = 0.045). No significant differences were found in transcript levels, cell counts, and substance P and 5-HT contents between rectal hypersensitivity (+) and (−) IBS patients. In conclusion, regardless of visceral hypersensitivity state, several serotonergic signaling components are altered in IBS patients.
Upper esophageal sphincter impedance as a marker of sphincter opening diameter
Authors: Omari Taher I. , Ferris Lara , Dejaeger Eddy , Tack Jan , Vanbeckevoort Dirk , Rommel Nathalie
Published: February 2012

The measurement of the physical extent of opening of the upper esophageal sphincter (UES) during bolus swallowing has to date relied on videofluoroscopy. Theoretically luminal impedance measured during bolus flow should be influenced by luminal diameter. In this study, we measured the UES nadir impedance (lowest value of impedance) during bolus swallowing and assessed it as a potential correlate of UES diameter that can be determined nonradiologically. In 40 patients with dysphagia, bolus swallowing of liquids, semisolids, and solids was recorded with manometry, impedance, and videofluoroscopy. During swallows, the UES opening diameter (in the lateral fluoroscopic view) was measured and compared with automated impedance manometry (AIM)-derived swallow function variables and UES nadir impedance as well as high-resolution manometry-derived UES relaxation pressure variables. Of all measured variables, UES nadir impedance was the most strongly correlated with UES opening diameter. Narrower diameter correlated with higher impedance ( r = −0.478, P < 0.001). Patients with <10 mm, 10–14 mm (normal), and ≥15 mm UES diameter had average UES nadir impedances of 498 ± 39 Ohms, 369 ± 31 Ohms, and 293 ± 17 Ohms, respectively (ANOVA P = 0.005). A higher swallow risk index, indicative of poor pharyngeal swallow function, was associated with narrower UES diameter and higher UES nadir impedance during swallowing. In contrast, UES relaxation pressure variables were not significantly altered in relation to UES diameter. We concluded that the UES nadir impedance correlates with opening diameter of the UES during bolus flow. This variable, when combined with other pharyngeal AIM analysis variables, may allow characterization of the pathophysiology of swallowing dysfunction.
Pharmacological and genetic inhibition of calcineurin protects against carbachol-induced pathological zymogen activation and acinar cell injury
Authors: Muili Kamaldeen A. , Ahmad Mahwish , Orabi Abrahim I. , Mahmood Syeda M. , Shah Ahsan U. , Molkentin Jeffery D. , Husain Sohail Z.
Published: February 2012

Acute pancreatitis is a major health burden for which there are currently no targeted therapies. Premature activation of digestive proenzymes, or zymogens, within the pancreatic acinar cell is an early and critical event in this disease. A high-amplitude, sustained rise in acinar cell Ca2+is required for zymogen activation. We previously showed in a cholecystokinin-induced pancreatitis model that a potential target of this aberrant Ca2+signaling is the Ca2+-activated phosphatase calcineurin (Cn). However, in this study, we examined the role of Cn on both zymogen activation and injury, in the clinically relevant condition of neurogenic stimulation (by giving the acetylcholine analog carbachol) using three different Cn inhibitors or Cn-deficient acinar cells. In freshly isolated mouse acinar cells, pretreatment with FK506, calcineurin inhibitory peptide (CiP), or cyclosporine (CsA) blocked intra-acinar zymogen activation ( n = 3; P < 0.05). The Cn inhibitors also reduced leakage of lactate dehydrogenase (LDH) by 79%, 62%, and 63%, respectively ( n = 3; P < 0.05). Of the various Cn isoforms, the β-isoform of the catalytic A subunit (CnAβ) was strongly expressed in mouse acinar cells. For this reason, we obtained acinar cells from CnAβ-deficient mice (CnAβ−/−) and observed an 84% and 50% reduction in trypsin and chymotrypsin activation, respectively, compared with wild-type controls ( n = 3; P < 0.05). LDH release in the CnAβ-deficient cells was reduced by 50% ( n = 2; P < 0.05). The CnAβ-deficient cells were also protected against zymogen activation and cell injury induced by the cholecystokinin analog caerulein. Importantly, amylase secretion was generally not affected by either the Cn inhibitors or Cn deficiency. These data provide both pharmacological and genetic evidence that implicates Cn in intra-acinar zymogen activation and cell injury during pancreatitis.
Exercise training blunts oxidative stress in sickle cell trait carriers
Authors: Chirico Erica N. , Martin Cyril , Faës Camille , Féasson Léonard , Oyono-Enguéllé Samuel , Aufradet Emeline , Dubouchaud Hervé , Francina Alain , Canet-Soulas Emmanuelle , Thiriet Patrice , Messonnier Laurent , Pialoux Vincent
Published: February 2012

The aim of this study was to analyze the effects of exercise training on oxidative stress in sickle cell trait carriers. Plasma levels of oxidative stress [advanced oxidation protein products (AOPP), protein carbonyl, malondialdehyde (MDA), and nitrotyrosine], antioxidant markers [catalase, glutathione peroxidase (GPX), and superoxide dismutase (SOD)], and nitrite and nitrate (NOx) were assessed at baseline, immediately following a maximal exercise test (Tex), and during recovery (T1h, T2h, T24h) in trained (T: 8 h/wk minimum) and untrained (U: no regular physical activity) sickle cell trait (SCT) carriers or control (CON) subjects (T-SCT, n = 10; U-SCT, n = 8; T-CON, n = 11; and U-CON, n = 11; age: 23.5 ± 2.2 yr). The trained subjects had higher SOD activities (7.6 ± 5.4 vs. 5.2 ± 2.1 U/ml, P = 0.016) and lower levels of AOPP (142 ± 102 vs. 177 ± 102 μM, P = 0.028) and protein carbonyl (82.1 ± 26.0 vs. 107.3 ± 30.6 nm/ml, P = 0.010) than the untrained subjects in response to exercise. In response to exercise, U-SCT had a higher level of AOPP (224 ± 130 vs. 174 ± 121 μM, P = 0.012), nitrotyrosine (127 ± 29.1 vs.70.6 ± 46.6 nM, P = 0.003), and protein carbonyl (114 ± 34.0 vs. 86.9 ± 26.8 nm/ml, P = 0.006) compared with T-SCT. T-SCT had a higher SOD activity (8.50 ± 7.2 vs. 4.30 ± 2.5 U/ml, P = 0.002) and NOx (28.8 ± 11.4 vs. 14.6 ± 7.0 μmol·l−1·min−1, P = 0.003) in response to exercise than U-SCT. Our data indicate that the overall oxidative stress and nitric oxide response is improved in exercise-trained SCT carriers compared with their untrained counterparts. These results suggest that physical activity could be a viable method of controlling the oxidative stress. This could have a beneficial impact because of its involvement in endothelial dysfunction and subsequent vascular impairment in hemoglobin S carriers.
Modified iodine-paper technique for the standardized determination of sweat gland activation
Authors: Gagnon Daniel , Ganio Matthew S. , Lucas Rebekah A. I. , Pearson James , Crandall Craig G. , Kenny Glen P.
Published: February 2012

Quantifying sweat gland activation provides important information when explaining differences in sweat rate between populations and physiological conditions. However, no standard technique has been proposed to measure sweat gland activation, while the reliability of sweat gland activation measurements is unknown. We examined the interrater and internal reliability of the modified-iodine paper technique, as well as compared computer-aided analysis to manual counts of sweat gland activation. Iodine-impregnated paper was pressed against the skin of 35 participants in whom sweating was elicited by exercise in the heat or infusion of methylcholine. The number of active glands was subsequently determined by computer-aided analysis. In total, 382 measurements were used to evaluate: 1) agreement between computer analysis and manual counts; 2) the interrater reliability of computer analysis between independent investigators; and 3) the internal reliability of sweat gland activation measurements between duplicate samples. The number of glands identified with computer analysis did not differ from manual counts (68 ± 29 vs. 72 ± 24 glands/cm2; P = 0.27). These measures were highly correlated ( r = 0.77) with a mean bias ± limits of agreement of −4 ± 38 glands/cm2. When comparing computer analysis measures between investigators, values were highly correlated ( r = 0.95; P < 0.001) and the mean bias ± limits of agreement was 4 ± 18 glands/cm2. Finally, duplicate measures of sweat gland activation were highly correlated ( r = 0.88; P < 0.001) with a mean bias ± limits of agreement of 3 ± 29 glands/cm2. These results favor the use of the modified-iodine paper technique with computer-aided analysis as a standard technique to reliably evaluate the number of active sweat glands.
Apoptotic Cell Death and Efferocytosis in Atherosclerosis
Authors: Van Vré Emily A. , Ait-Oufella Hafid , Tedgui Alain , Mallat Ziad
Published: February 2012

Apoptotic cell death is an important feature of atherosclerotic plaques, and it seems to exert both beneficial and detrimental effects depending on the cell type and plaque stage. Because late apoptotic cells can launch proatherogenic inflammatory responses, adequate engulfment of apoptotic cells (efferocytosis) by macrophages is important to withstand atherosclerosis progression. Several efferocytosis systems, composed of different phagocytic receptors, apoptotic ligands, and bridging molecules, can be distinguished. Because phagocytes in atherosclerotic plaques are very much solicited, a fully operative efferocytosis system seems to be an absolute requisite. Indeed, recent studies demonstrate that deletion of just 1 of the efferocytosis pathways aggravates atherosclerosis. This review discusses the role of apoptosis in atherosclerosis and general mechanisms of efferocytosis, to end with indirect and direct indications of the significance of effective efferocytosis in atherosclerosis.