bluuebunny/crossref_metadata_2025 huggingface dataset:
Investigating student understanding of operational-amplifier circuits
Authors: Papanikolaou Christos P. , Tombras George S. , Van De Bogart Kevin L. , Stetzer MacKenzie R.
Published: November 2015

The research reported in this article represents a systematic, multi-year investigation of student understanding of the behavior of basic operational-amplifier (op-amp) circuits. The participants in this study were undergraduates enrolled in upper-division physics courses on analog electronics at three different institutions, as well as undergraduates in introductory and upper-division electrical engineering courses at one of the institutions. The findings indicate that many students complete these courses without developing a functional understanding of the behavior of op-amp circuits. This article describes the most prevalent conceptual and reasoning difficulties identified (typically after lecture and hands-on laboratory experience) as well as several implications for electronics instruction that have emerged from this investigation.
The surprising influence of longitudinal motion in vibrating strings: Comment on “Video-based spatial portraits of a nonlinear vibrating string” [Am. J. Phys. 80(10), 862–869 (2012)]
Authors: Rowland David R.
Published: November 2015

An error in the quoted nonlinear coefficient that is commonly found in the literature is identified. The subtle origin of this error is identified as the neglect of longitudinal displacements of points in the string, which leads to a nonlinear coefficient that is a factor of 3/2 too large. A correct derivation is outlined and numerical simulations verify the correction.
Structural-functional connectivity deficits of neocortical circuits in the
Fmr1

−/y

mouse model of autism
Authors: Haberl Matthias G. , Zerbi Valerio , Veltien Andor , Ginger Melanie , Heerschap Arend , Frick Andreas
Published: November 2015

Structural and functional connectivity phenotype in the neocortex of
Fmr1

−/y

mice supports a prominent hypothesis of autism.
Coherent driving and freezing of bosonic matter wave in an optical Lieb lattice
Authors: Taie Shintaro , Ozawa Hideki , Ichinose Tomohiro , Nishio Takuei , Nakajima Shuta , Takahashi Yoshiro
Published: November 2015

Matter-wave dynamics reveals a flat energy band engineered in a novel optical lattice.
Mechanism of strength reduction along the graphenization pathway
Authors: Gamboa Antonio , Farbos Baptiste , Aurel Philippe , Vignoles Gérard L. , Leyssale Jean-Marc
Published: November 2015

Stable single-bond cracks retard failure in low-temperature polycrystalline graphenes with respect to heat-treated samples.
Early genome duplications in conifers and other seed plants
Authors: Li Zheng , Baniaga Anthony E. , Sessa Emily B. , Scascitelli Moira , Graham Sean W. , Rieseberg Loren H. , Barker Michael S.
Published: November 2015

A new phylogenomic approach reveals that conifer genomes are duplicated despite rare polyploidy among extant species.
Electronic plants
Authors: Stavrinidou Eleni , Gabrielsson Roger , Gomez Eliot , Crispin Xavier , Nilsson Ove , Simon Daniel T. , Berggren Magnus
Published: November 2015

Integrated organic electronic analog and digital circuits can be formed within plant parts and live plants.
Dibenzylideneacetones Are Potent Trypanocidal Compounds That Affect the Trypanosoma cruzi Redox System
Authors: Lazarin-Bidóia Danielle , Desoti Vânia Cristina , Martins Solange Cardoso , Ribeiro Fabianne Martins , Ud Din Zia , Rodrigues-Filho Edson , Ueda-Nakamura Tânia , Nakamura Celso Vataru , de Oliveira Silva Sueli
Published: November 2015

Despite ongoing efforts, the available treatments for Chagas' disease are still unsatisfactory, especially in the chronic phase of the disease. Our previous study reported the strong trypanocidal activity of the dibenzylideneacetones A3K2A1 and A3K2A3 against
Trypanosoma cruzi
(Z. Ud Din, T. P. Fill, F. F. de Assis, D. Lazarin-Bidóia, V. Kaplum, F. P. Garcia, C. V. Nakamura, K. T. de Oliveira, and E. Rodrigues-Filho, Bioorg Med Chem 22:1121–1127, 2014,
http://dx.doi.org/10.1016/j.bmc.2013.12.020
). In the present study, we investigated the mechanisms of action of these compounds that are involved in parasite death. We showed that A3K2A1 and A3K2A3 induced oxidative stress in the three parasitic forms, especially trypomastigotes, reflected by an increase in oxidant species production and depletion of the endogenous antioxidant system. This oxidative imbalance culminated in damage in essential cell structures of
T. cruzi
, reflected by lipid peroxidation and DNA fragmentation. Consequently, A3K2A1 and A3K2A3 induced vital alterations in
T. cruzi
, leading to parasite death through the three pathways, apoptosis, autophagy, and necrosis.
Tree-Based Models for Predicting Mortality in Gram-Negative Bacteremia: Avoid Putting the CART before the Horse
Authors: Rhodes Nathaniel J. , O'Donnell J. Nicholas , Lizza Bryan D. , McLaughlin Milena M. , Esterly John S. , Scheetz Marc H.
Published: November 2015

Increasingly, infectious disease studies employ tree-based approaches, e.g., classification and regression tree modeling, to identify clinical thresholds. We present tree-based-model-derived thresholds along with their measures of uncertainty. We explored individual and pooled clinical cohorts of bacteremic patients to identify modified acute physiology and chronic health evaluation (II) (m-APACHE-II) score mortality thresholds using a tree-based approach. Predictive performance measures for each candidate threshold were calculated. Candidate thresholds were examined according to binary logistic regression probabilities of the primary outcome, correct classification predictive matrices, and receiver operating characteristic curves. Three individual cohorts comprising a total of 235 patients were studied. Within the pooled cohort, the mean (± standard deviation) m-APACHE-II score was 13.6 ± 5.3, with an in-hospital mortality of 16.6%. The probability of death was greater at higher m-APACHE II scores in only one of three cohorts (odds ratio for cohort 1 [OR
1
] = 1.15, 95% confidence interval [CI] = 0.99 to 1.34; OR
2
= 1.04, 95% CI = 0.94 to 1.16; OR
3
= 1.18, 95% CI = 1.02 to 1.38) and was greater at higher scores within the pooled cohort (OR
4
= 1.11, 95% CI = 1.04 to 1.19). In contrast, tree-based models overcame power constraints and identified m-APACHE-II thresholds for mortality in two of three cohorts (
P
= 0.02, 0.1, and 0.008) and the pooled cohort (
P
= 0.001). Predictive performance at each threshold was highly variable among cohorts. The selection of any one predictive threshold value resulted in fixed sensitivity and specificity. Tree-based models increased power and identified threshold values from continuous predictor variables; however, sample size and data distributions influenced the identified thresholds. The provision of predictive matrices or graphical displays of predicted probabilities within infectious disease studies can improve the interpretation of tree-based model-derived thresholds.
Neomycin Sulfate Improves the Antimicrobial Activity of Mupirocin-Based Antibacterial Ointments
Authors: Blanchard Catlyn , Brooks Lauren , Beckley Andrew , Colquhoun Jennifer , Dewhurst Stephen , Dunman Paul M.
Published: November 2015

In the midst of the current antimicrobial pipeline void, alternative approaches are needed to reduce the incidence of infection and decrease reliance on last-resort antibiotics for the therapeutic intervention of bacterial pathogens. In that regard, mupirocin ointment-based decolonization and wound maintenance practices have proven effective in reducing
Staphylococcus aureus
transmission and mitigating invasive disease. However, the emergence of mupirocin-resistant strains has compromised the agent's efficacy, necessitating new strategies for the prevention of staphylococcal infections. Herein, we set out to improve the performance of mupirocin-based ointments. A screen of a Food and Drug Administration (FDA)-approved drug library revealed that the antibiotic neomycin sulfate potentiates the antimicrobial activity of mupirocin, whereas other library antibiotics did not. Preliminary mechanism of action studies indicate that neomycin's potentiating activity may be mediated by inhibition of the organism's RNase P function, an enzyme that is believed to participate in the tRNA processing pathway immediately upstream of the primary target of mupirocin. The improved antimicrobial activity of neomycin and mupirocin was maintained in ointment formulations and reduced
S. aureus
bacterial burden in murine models of nasal colonization and wound site infections. Combination therapy improved upon the effects of either agent alone and was effective in the treatment of contemporary methicillin-susceptible, methicillin-resistant, and high-level mupirocin-resistant
S. aureus
strains. From these perspectives, combination mupirocin-and-neomycin ointments appear to be superior to that of mupirocin alone and warrant further development.
Broadly Neutralizing Anti-HIV Antibodies Prevent HIV Infection of Mucosal Tissue
Ex Vivo
Authors: Scott Yanille M. , Park Seo Young , Dezzutti Charlene S.
Published: November 2015

Broadly neutralizing monoclonal antibodies (nAbs) specific for HIV are being investigated for use in HIV prevention. Due to their ability to inhibit HIV attachment to and entry into target cells, nAbs may be suitable for use as topical HIV microbicides. As such, they would present an alternative intervention for individuals who may not benefit from using antiretroviral-based products for HIV prevention. We theorize that nAbs can inhibit viral transmission through mucosal tissue, thus reducing the incidence of HIV infection. The efficacy of the PG9, PG16, VRC01, and 4E10 antibodies was evaluated in an
ex vivo
human model of mucosal HIV transmission. nAbs reduced HIV transmission, causing 1.5- to 2-log
10
reductions in HIV replication in ectocervical tissues and ≈3-log
10
reductions in HIV replication in colonic tissues over 21 days. These antibodies demonstrated greater potency in colonic tissues, with a 50-fold higher dose being required to reduce transmission in ectocervical tissues. Importantly, nAbs retained their potency and reduced viral transmission in the presence of whole semen. No changes in tissue viability or immune activation were observed in colonic or ectocervical tissue after nAb exposure. Our data suggest that topically applied nAbs are safe and effective against HIV infection of mucosal tissue and support further development of nAbs as a topical microbicide that could be used for anal as well as vaginal protection.
Determination of Evolutionary Relationships of Outbreak-Associated Listeria monocytogenes Strains of Serotypes 1/2a and 1/2b by Whole-Genome Sequencing
Authors: Bergholz Teresa M. , den Bakker Henk C. , Katz Lee S. , Silk Benjamin J. , Jackson Kelly A. , Kucerova Zuzana , Joseph Lavin A. , Turnsek Maryann , Gladney Lori M. , Halpin Jessica L. , Xavier Karen , Gossack Joseph , Ward Todd J. , Frace Michael , Tarr Cheryl L.
Published: November 2015

We used whole-genome sequencing to determine evolutionary relationships among 20 outbreak-associated clinical isolates of
Listeria monocytogenes
serotypes 1/2a and 1/2b. Isolates from 6 of 11 outbreaks fell outside the clonal groups or “epidemic clones” that have been previously associated with outbreaks, suggesting that epidemic potential may be widespread in
L. monocytogenes
and is not limited to the recognized epidemic clones. Pairwise comparisons between epidemiologically related isolates within clonal complexes showed that genome-level variation differed by 2 orders of magnitude between different comparisons, and the distribution of point mutations (core versus accessory genome) also varied. In addition, genetic divergence between one closely related pair of isolates from a single outbreak was driven primarily by changes in phage regions. The evolutionary analysis showed that the changes could be attributed to horizontal gene transfer; members of the diverse bacterial community found in the production facility could have served as the source of novel genetic material at some point in the production chain. The results raise the question of how to best utilize information contained within the accessory genome in outbreak investigations. The full magnitude and complexity of genetic changes revealed by genome sequencing could not be discerned from traditional subtyping methods, and the results demonstrate the challenges of interpreting genetic variation among isolates recovered from a single outbreak. Epidemiological information remains critical for proper interpretation of nucleotide and structural diversity among isolates recovered during outbreaks and will remain so until we understand more about how various population histories influence genetic variation.
Tuning Chocolate Flavor through Development of Thermotolerant Saccharomyces cerevisiae Starter Cultures with Increased Acetate Ester Production
Authors: Meersman Esther , Steensels Jan , Struyf Nore , Paulus Tinneke , Saels Veerle , Mathawan Melissa , Allegaert Leen , Vrancken Gino , Verstrepen Kevin J.
Published: November 2015

Microbial starter cultures have extensively been used to enhance the consistency and efficiency of industrial fermentations. Despite the advantages of such controlled fermentations, the fermentation involved in the production of chocolate is still a spontaneous process that relies on the natural microbiota at cocoa farms. However, recent studies indicate that certain thermotolerantSaccharomyces cerevisiaecultures can be used as starter cultures for cocoa pulp fermentation. In this study, we investigate the potential of specifically developed starter cultures to modulate chocolate aroma. Specifically, we developed several newS. cerevisiaehybrids that combine thermotolerance and efficient cocoa pulp fermentation with a high production of volatile flavor-active esters. In addition, we investigated the potential of two strains of two non-Saccharomycesspecies that produce very large amounts of fruity esters (Pichia kluyveriandCyberlindnera fabianii) to modulate chocolate aroma. Gas chromatography-mass spectrometry (GC-MS) analysis of the cocoa liquor revealed an increased concentration of various flavor-active esters and a decrease in spoilage-related off-flavors in batches inoculated withS. cerevisiaestarter cultures and, to a lesser extent, in batches inoculated withP. kluyveriandCyb. fabianii. Additionally, GC-MS analysis of chocolate samples revealed that while most short-chain esters evaporated during conching, longer and more-fat-soluble ethyl and acetate esters, such as ethyl octanoate, phenylethyl acetate, ethyl phenylacetate, ethyl decanoate, and ethyl dodecanoate, remained almost unaffected. Sensory analysis by an expert panel confirmed significant differences in the aromas of chocolates produced with different starter cultures. Together, these results show that the selection of different yeast cultures opens novel avenues for modulating chocolate flavor.
Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16
Authors: Wenning Leonie , Stöveken Nadine , Wübbeler Jan Hendrik , Steinbüchel Alexander
Published: November 2015

Cysteine dioxygenases (Cdos), which catalyze the sulfoxidation of cysteine to cysteine sulfinic acid (CSA), have been extensively studied in eukaryotes because of their roles in several diseases. In contrast, only a few prokaryotic enzymes of this type have been investigated. In
Ralstonia eutropha
H16, two Cdo homologues (CdoA and CdoB) have been identified previously.
In vivo
studies showed that
Escherichia coli
cells expressing CdoA could convert 3-mercaptopropionate (3MP) to 3-sulfinopropionate (3SP), whereas no 3SP could be detected in cells expressing CdoB. The objective of this study was to confirm these findings and to study both enzymes in detail by performing an
in vitro
characterization. The proteins were heterologously expressed and purified to apparent homogeneity by immobilized metal chelate affinity chromatography (IMAC). Subsequent analysis of the enzyme activities revealed striking differences with regard to their substrate ranges and their specificities for the transition metal cofactor, e.g., CdoA catalyzed the sulfoxidation of 3MP to a 3-fold-greater extent than the sulfoxidation of cysteine, whereas CdoB converted only cysteine. Moreover, the dependency of the activities of the Cdos from
R. eutropha
H16 on the metal cofactor in the active center could be demonstrated. The importance of CdoA for the metabolism of the sulfur compounds 3,3′-thiodipropionic acid (TDP) and 3,3′-dithiodipropionic acid (DTDP) by further converting their degradation product, 3MP, was confirmed. Since 3MP can also function as a precursor for polythioester (PTE) synthesis in
R. eutropha
H16, deletion of
cdoA
might enable increased synthesis of PTEs.
From Rare to Dominant: a Fine-Tuned Soil Bacterial Bloom during Petroleum Hydrocarbon Bioremediation
Authors: Fuentes Sebastián , Barra Bárbara , Caporaso J. Gregory , Seeger Michael
Published: November 2015

Hydrocarbons are worldwide-distributed pollutants that disturb various ecosystems. The aim of this study was to characterize the short-lapse dynamics of soil microbial communities in response to hydrocarbon pollution and different bioremediation treatments. Replicate diesel-spiked soil microcosms were inoculated with either a defined bacterial consortium or a hydrocarbonoclastic bacterial enrichment and incubated for 12 weeks. The microbial community dynamics was followed weekly in microcosms using Illumina 16S rRNA gene sequencing. Both the bacterial consortium and enrichment enhanced hydrocarbon degradation in diesel-polluted soils. A pronounced and rapid bloom of a native gammaproteobacterium was observed in all diesel-polluted soils. A unique operational taxonomic unit (OTU) related to the
Alkanindiges
genus represented ∼0.1% of the sequences in the original community but surprisingly reached >60% after 6 weeks. Despite this
Alkanindiges
-related bloom, inoculated strains were maintained in the community and may explain the differences in hydrocarbon degradation. This study shows the detailed dynamics of a soil bacterial bloom in response to hydrocarbon pollution, resembling microbial blooms observed in marine environments. Rare community members presumably act as a reservoir of ecological functions in high-diversity environments, such as soils. This rare-to-dominant bacterial shift illustrates the potential role of a rare biosphere facing drastic environmental disturbances. Additionally, it supports the concept of “conditionally rare taxa,” in which rareness is a temporary state conditioned by environmental constraints.
Influence of
oxyR
on Growth, Biofilm Formation, and Mobility of Vibrio parahaemolyticus
Authors: Chung Chun-Hui , Fen Shin-yuan , Yu Shu-Chuan , Wong Hin-chung
Published: November 2015

Vibrio parahaemolyticus
is a common marine food-borne enteropathogen. In this study, we examined the antioxidative activity, growth, biofilm formation, and cell mobility of an
oxyR
deletion mutant and its genetically complementary strain of
V. parahaemolyticus
.
oxyR
is the regulator of catalase and
ahpC
genes. Protection against extrinsic H
2
O
2
and against the organic peroxides cumene hydroperoxide and
tert
-butyl hydroperoxide was weaker in the deletion mutant than in its parent strain. Expression of the major functional antioxidative genes,
ahpC1
and VPA1418, was markedly decreased in the
oxyR
mutant. Growth of this mutant on agar medium was significantly inhibited by autoclaved 0.25% glucose and by 0.25% dipotassium hydrogen phosphate, 0.5% monosaccharides (glucose, galactose, xylose, and arabinose), or 114.8 mM phosphates. The inhibition of the growth of this
oxyR
mutant by extrinsic peroxides, autoclaved sugars, and phosphates was eliminated by the complementary
oxyR
gene or by the addition of catalase to the autoclaved medium, while no inhibition of growth was observed when filter-sterilized sugars were used. The formation of biofilm and swimming mobility were significantly inhibited in the
oxyR
mutant relative to that in the wild-type strain. This investigation demonstrates the antioxidative function of
oxyR
in
V. parahaemolyticus
and its possible roles in biofilm formation, cell mobility, and the protection of growth in heated rich medium.
Purification and Host Specificity of Predatory Halobacteriovorax Isolates from Seawater
Authors: Richards Gary P. , Fay Johnna P. , Uknalis Joseph , Olanya O. Modesto , Watson Michael A.
Published: November 2015

Halobacteriovorax
(formerly
Bacteriovorax
) is a small predatory bacterium found in the marine environment and modulates bacterial pathogens in shellfish. Four strains of
Halobacteriovorax
originally isolated in
Vibrio parahaemolyticus
O3:K6 host cells were separated from their prey by an enrichment-filtration-dilution technique for specificity testing in other bacteria. This technique was essential, since 0.45-μm filtration alone was unable to remove infectious
Vibrio
minicells, as determined by scanning electron microscopy and cultural methods. Purified
Halobacteriovorax
strains were screened for predation against other
V. parahaemolyticus
strains and against
Vibrio vulnificus
,
Vibrio alginolyticus
,
Escherichia coli
O157:H7, and
Salmonella enterica
serovar Typhimurium DT104, all potential threats to seafood safety. They showed high host specificity and were predatory only against strains of
V. parahaemolyticus
. In addition, strains of
Halobacteriovorax
that were predatory for
E. coli
O157:H7 and
S
. Typhimurium DT104 were isolated from a tidal river at 5 ppt salinity. In a modified plaque assay agar, they killed their respective prey over a broad range of salinities (5 to 30 ppt). Plaques became smaller as the salinity levels rose, suggesting that the lower salinities were optimal for the predators' replication. These species also showed broader host specificity, infectious against each other's original hosts as well as against
V. parahaemolyticus
strains. In summary, this study characterized strains of
Halobacteriovorax
which may be considered for use in the development of broad-based biocontrol technologies to enhance the safety of commercially marketed shellfish and other foods.
Expanding the Limits of Thermoacidophily in the Archaeon Sulfolobus solfataricus by Adaptive Evolution
Authors: McCarthy Samuel , Johnson Tyler , Pavlik Benjamin J. , Payne Sophie , Schackwitz Wendy , Martin Joel , Lipzen Anna , Keffeler Erica , Blum Paul
Published: November 2015

Extremely thermoacidophilic
Crenarchaeota
belonging to the order
Sulfolobales
flourish in hot acidic habitats that are strongly oxidizing. The pH extremes of these habitats, however, often exceed the acid tolerance of type species and strains. Here, adaptive laboratory evolution was used over a 3-year period to test whether such organisms harbor additional thermoacidophilic capacity. Three distinct cell lines derived from a single type species were subjected to high-temperature serial passage while culture acidity was gradually increased. A 178-fold increase in thermoacidophily was achieved after 29 increments of shifted culture pH resulting in growth at pH 0.8 and 80°C. These strains were named super-acid-resistant
Crenarchaeota
(SARC). Mathematical modeling using growth parameters predicted the limits of acid resistance, while genome resequencing and transcriptome resequencing were conducted for insight into mechanisms responsible for the evolved trait. Among the mutations that were detected, a set of eight nonsynonymous changes may explain the heritability of increased acid resistance despite an unexpected lack of transposition. Four multigene components of the SARC transcriptome implicated oxidative stress as a primary challenge accompanying growth at acid extremes. These components included accelerated membrane biogenesis, induction of the
mer
operon, and an increased capacity for the generation of energy and reductant.
Use of Substrate-Induced Gene Expression in Metagenomic Analysis of an Aromatic Hydrocarbon-Contaminated Soil
Authors: Meier Matthew J. , Paterson E. Suzanne , Lambert Iain B.
Published: November 2015

Metagenomics allows the study of genes related to xenobiotic degradation in a culture-independent manner, but many of these studies are limited by the lack of genomic context for metagenomic sequences. This study combined a phenotypic screen known as substrate-induced gene expression (SIGEX) with whole-metagenome shotgun sequencing. SIGEX is a high-throughput promoter-trap method that relies on transcriptional activation of a green fluorescent protein (GFP) reporter gene in response to an inducing compound and subsequent fluorescence-activated cell sorting to isolate individual inducible clones from a metagenomic DNA library. We describe a SIGEX procedure with improved library construction from fragmented metagenomic DNA and improved flow cytometry sorting procedures. We used SIGEX to interrogate an aromatic hydrocarbon (AH)-contaminated soil metagenome. The recovered clones contained sequences with various degrees of similarity to genes (or partial genes) involved in aromatic metabolism, for example,
nahG
(salicylate oxygenase) family genes and their respective upstream
nahR
regulators. To obtain a broader context for the recovered fragments, clones were mapped to contigs derived from
de novo
assembly of shotgun-sequenced metagenomic DNA which, in most cases, contained complete operons involved in aromatic metabolism, providing greater insight into the origin of the metagenomic fragments. A comparable set of contigs was generated using a significantly less computationally intensive procedure in which assembly of shotgun-sequenced metagenomic DNA was directed by the SIGEX-recovered sequences. This methodology may have broad applicability in identifying biologically relevant subsets of metagenomes (including both novel and known sequences) that can be targeted computationally by
in silico
assembly and prediction tools.
The Exiguobacteriumsibiricum 255-15 GtfC Enzyme Represents a Novel Glycoside Hydrolase 70 Subfamily of 4,6-α-Glucanotransferase Enzymes
Authors: Gangoiti Joana , Pijning Tjaard , Dijkhuizen Lubbert
Published: November 2015

The glycoside hydrolase 70 (GH70) family originally was established for glucansucrase enzymes found solely in lactic acid bacteria synthesizing α-glucan polysaccharides from sucrose (e.g., GtfA). In recent years, we have characterized GtfB and related
Lactobacillus
enzymes as 4,6-α-glucanotransferase enzymes. These GtfB-type enzymes constitute the first GH70 subfamily of enzymes that are unable to act on sucrose as a substrate but are active with maltodextrins and starch, cleave α1→4 linkages, and synthesize linear α1→6-glucan chains. The GtfB disproportionating type of activity results in the conversion of malto-oligosaccharides into isomalto/malto-polysaccharides with a relatively high percentage of α1→6 linkages. This paper reports the identification of the members of a second GH70 subfamily (designated GtfC enzymes) and the characterization of the
Exiguobacterium
sibiricum
255-15 GtfC enzyme, which is also inactive with sucrose and displays 4,6-α-glucanotransferase activity with malto-oligosaccharides. GtfC differs from GtfB in synthesizing isomalto/malto-oligosaccharides. Biochemically, the GtfB- and GtfC-type enzymes are related, but phylogenetically, they clearly constitute different GH70 subfamilies, displaying only 30% sequence identity. Whereas the GtfB-type enzyme largely has the same domain order as glucansucrases (with α-amylase domains A, B, and C plus domains IV and V), this GtfC-type enzyme differs in the order of these domains and completely lacks domain V. In GtfC, the sequence of conserved regions I to IV of clan GH-H is identical to that in GH13 (I-II-III-IV) but different from that in GH70 (II-III-IV-I because of a circular permutation of the (β/α)
8
barrel. The GtfC 4,6-α-glucanotransferase enzymes thus represent structurally and functionally very interesting evolutionary intermediates between α-amylase and glucansucrase enzymes.
Land Spreading of Wastewaters from the Fruit-Packaging Industry and Potential Effects on Soil Microbes: Effects of the Antioxidant Ethoxyquin and Its Metabolites on Ammonia Oxidizers
Authors: Papadopoulou Evangelia S. , Tsachidou Bella , Sułowicz Sławomir , Menkissoglu-Spiroudi Urania , Karpouzas Dimitrios G.
Published: November 2015

Thiabendazole (TBZ), imazalil (IMZ),
ortho
-phenylphenol (OPP), diphenylamine (DPA), and ethoxyquin (EQ) are used in fruit-packaging plants (FPP) with the stipulation that wastewaters produced by their application would be depurated on site. However, no such treatment systems are currently in place, leading FPP to dispose of their effluents in agricultural land. We investigated the dissipation of those pesticides and their impact on soil microbes known to have a key role on ecosystem functioning. OPP and DPA showed limited persistence (50% dissipation time [DT
50
], 0.6 and 1.3 days) compared to TBZ and IMZ (DT
50
, 47.0 and 150.8 days). EQ was rapidly transformed to the short-lived quinone imine (QI) (major metabolite) and the more persistent 2,4-dimethyl-6-ethoxyquinoline (EQNL) (minor metabolite). EQ and OPP exerted significant inhibition of potential nitrification, with the effect of the former being more persistent. This was not reflected in the abundance (determined by quantitative PCR [qPCR]) of the
amoA
gene of ammonia-oxidizing bacteria (AOB) and archaea (AOA). Considering the above discrepancy and the metabolic pattern of EQ, we further investigated the hypothesis that its metabolites and not only EQ were toxic to ammonia oxidizers. Potential nitrification,
amoA
gene abundance, and
amoA
gene transcripts of AOB and AOA showed that QI was probably responsible for the inhibition of nitrification. Our findings have serious ecological and practical implications for soil productivity and N conservation in agriculturally impacted ecosystems and stress the need to include metabolites and RNA-based methods when the soil microbial toxicity of pesticides is assessed.
Genomics of the Genus Bifidobacterium Reveals Species-Specific Adaptation to the Glycan-Rich Gut Environment
Authors: Milani Christian , Turroni Francesca , Duranti Sabrina , Lugli Gabriele Andrea , Mancabelli Leonardo , Ferrario Chiara , van Sinderen Douwe , Ventura Marco
Published: November 2015

Bifidobacteria represent one of the dominant microbial groups that occur in the gut of various animals, being particularly prevalent during the suckling period of humans and other mammals. Their ability to compete with other gut bacteria is largely attributed to their saccharolytic features. Comparative and functional genomic as well as transcriptomic analyses have revealed the genetic background that underpins the overall saccharolytic phenotype for each of the 47 bifidobacterial (sub)species representing the genus
Bifidobacterium
, while also generating insightful information regarding carbohydrate resource sharing and cross-feeding among bifidobacteria. The abundance of bifidobacterial saccharolytic features in human microbiomes supports the notion that metabolic accessibility to dietary and/or host-derived glycans is a potent evolutionary force that has shaped the bifidobacterial genome.
Tyrosine Phosphorylation and Dephosphorylation in Burkholderia cenocepacia Affect Biofilm Formation, Growth under Nutritional Deprivation, and Pathogenicity
Authors: Andrade Angel , Tavares-Carreón Faviola , Khodai-Kalaki Maryam , Valvano Miguel A.
Published: November 2015

Burkholderia cenocepacia
, a member of the
B. cepacia
complex (Bcc), is an opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis. Tyrosine phosphorylation has emerged as an important posttranslational modification modulating the physiology and pathogenicity of Bcc bacteria. Here, we investigated the predicted bacterial tyrosine kinases BCAM1331 and BceF and the low-molecular-weight protein tyrosine phosphatases BCAM0208, BceD, and BCAL2200 of
B. cenocepacia
K56-2. We show that BCAM1331, BceF, BCAM0208, and BceD contribute to biofilm formation, while BCAL2200 is required for growth under nutrient-limited conditions. Multiple deletions of either tyrosine kinase or low-molecular-weight protein tyrosine phosphatase genes resulted in the attenuation of
B. cenocepacia
intramacrophage survival and reduced pathogenicity in the
Galleria mellonella
larval infection model. Experimental evidence indicates that BCAM1331 displays reduced tyrosine autophosphorylation activity compared to that of BceF. With the artificial substrate
p
-nitrophenyl phosphate, the phosphatase activities of the three low-molecular-weight protein tyrosine phosphatases demonstrated similar kinetic parameters. However, only BCAM0208 and BceD could dephosphorylate BceF. Further, BCAL2200 became tyrosine phosphorylated
in vivo
and catalyzed its autodephosphorylation. Together, our data suggest that despite having similar biochemical activities, low-molecular-weight protein tyrosine phosphatases and tyrosine kinases have both overlapping and specific roles in the physiology of
B. cenocepacia
.
Specific CD4+T-Cell Reactivity and Cytokine Release in Different Clinical Presentations of Leptospirosis
Authors: Volz Magdalena Sarah , Moos Verena , Allers Kristina , Luge Enno , Mayer-Scholl Anne , Nöckler Karsten , Loddenkemper Christoph , Jansen Andreas , Schneider Thomas
Published: November 2015

Clinical manifestations of leptospirosis are highly variable: from asymptomatic to severe and potentially fatal. The outcome of the disease is usually determined in the immunological phase, beginning in the second week of symptoms. The underlying mechanisms, predictive factors, and individual immune responses that contribute to clinical variations are not well understood. The aim of this study was to determine the specifics of CD4+T-cell reactivity and cytokine release after stimulation with leptospiral antigens in patients with leptospirosis of different disease severities (patients with mild and severe symptoms) and in control subjects (with and without proven exposure toLeptospira). Whole-blood specimens were stimulated withLeptospiraantigensin vitro. Subsequently, intracellular staining of cytokines was performed, and flow cytometry was used to assess the expression of CD40 ligand (CD40L) and the production of gamma interferon (IFN-γ), interleukin-10 (IL-10), IL-2, and tumor necrosis factor alpha (TNF-α) by CD4+T cells. The production of inflammatory cytokines such as TNF-α by CD4+T cells after stimulation with leptospiral antigens was highest in patients with severe disease. In contrast, the ratio of IL-10 production to TNF-α production was higher in exposed subjects than in patients with mild and severe disease. Levels of proinflammatory cytokines such as TNF-α may be useful markers of the severity of the immunological phase of leptospirosis. IL-10 production by T cells after antigen-specific stimulation may indicate a more successful downregulation of the inflammatory response and may contribute to an asymptomatic course of the disease.
Functional Analysis of Bacteriophage Immunity through a Type I-E CRISPR-Cas System in Vibrio cholerae and Its Application in Bacteriophage Genome Engineering
Authors: Box Allison M. , McGuffie Matthew J. , O'Hara Brendan J. , Seed Kimberley D.
Published: November 2015

The classical and El Tor biotypes of
Vibrio cholerae
serogroup O1, the etiological agent of cholera, are responsible for the sixth and seventh (current) pandemics, respectively. A genomic island (GI), GI-24, previously identified in a classical biotype strain of
V. cholerae
, is predicted to encode clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins (Cas proteins); however, experimental evidence in support of CRISPR activity in
V. cholerae
has not been documented. Here, we show that CRISPR-Cas is ubiquitous in strains of the classical biotype but excluded from strains of the El Tor biotype. We also provide
in silico
evidence to suggest that CRISPR-Cas actively contributes to phage resistance in classical strains. We demonstrate that transfer of GI-24 to
V. cholerae
El Tor via natural transformation enables CRISPR-Cas-mediated resistance to bacteriophage CP-T1 under laboratory conditions. To elucidate the sequence requirements of this type I-E CRISPR-Cas system, we engineered a plasmid-based system allowing the directed targeting of a region of interest. Through screening for phage mutants that escape CRISPR-Cas-mediated resistance, we show that CRISPR targets must be accompanied by a 3′ TT protospacer-adjacent motif (PAM) for efficient interference. Finally, we demonstrate that efficient editing of
V. cholerae
lytic phage genomes can be performed by simultaneously introducing an editing template that allows homologous recombination and escape from CRISPR-Cas targeting.

IMPORTANCE
Cholera, caused by the facultative pathogen
Vibrio cholerae
, remains a serious public health threat. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) provide prokaryotes with sequence-specific protection from invading nucleic acids, including bacteriophages. In this work, we show that one genomic feature differentiating sixth pandemic (classical biotype) strains from seventh pandemic (El Tor biotype) strains is the presence of a CRISPR-Cas system in the classical biotype. We demonstrate that the CRISPR-Cas system from a classical biotype strain can be transferred to a
V. cholerae
El Tor biotype strain and that it is functional in providing resistance to phage infection. Finally, we show that this CRISPR-Cas system can be used as an efficient tool for the editing of
V. cholerae
lytic phage genomes.
Regulation of Type IV Pili Contributes to Surface Behaviors of Historical and Epidemic Strains of Clostridium difficile
Authors: Purcell Erin B. , McKee Robert W. , Bordeleau Eric , Burrus Vincent , Tamayo Rita
Published: November 2015

The intestinal pathogen
Clostridium difficile
is an urgent public health threat that causes antibiotic-associated diarrhea and is a leading cause of fatal nosocomial infections in the United States.
C. difficile
rates of recurrence and mortality have increased in recent years due to the emergence of so-called “hypervirulent” epidemic strains. A great deal of the basic biology of
C. difficile
has not been characterized. Recent findings that flagellar motility, toxin synthesis, and type IV pilus (TFP) formation are regulated by cyclic diguanylate (c-di-GMP) reveal the importance of this second messenger for
C. difficile
gene regulation. However, the function(s) of TFP in
C. difficile
remains largely unknown. Here, we examine TFP-dependent phenotypes and the role of c-di-GMP in controlling TFP production in the historical 630 and epidemic R20291 strains of
C. difficile
. We demonstrate that TFP contribute to
C. difficile
biofilm formation in both strains, but with a more prominent role in R20291. Moreover, we report that R20291 is capable of TFP-dependent surface motility, which has not previously been described in
C. difficile
. The expression and regulation of the
pilA1
pilin gene differs between R20291 and 630, which may underlie the observed differences in TFP-mediated phenotypes. The differences in
pilA1
expression are attributable to greater promoter-driven transcription in R20291. In addition, R20291, but not 630, upregulates c-di-GMP levels during surface-associated growth, suggesting that the bacterium senses its substratum. The differential regulation of surface behaviors in historical and epidemic
C. difficile
strains may contribute to the different infection outcomes presented by these strains.

IMPORTANCE
How
Clostridium difficile
establishes and maintains colonization of the host bowel is poorly understood. Surface behaviors of
C. difficile
are likely relevant during infection, representing possible interactions between the bacterium and the intestinal environment. Pili mediate bacterial interactions with various surfaces and contribute to the virulence of many pathogens. We report that type IV pili (TFP) contribute to biofilm formation by
C. difficile
. TFP are also required for surface motility, which has not previously been demonstrated for
C. difficile
. Furthermore, an epidemic-associated
C. difficile
strain showed higher pilin gene expression and greater dependence on TFP for biofilm production and surface motility. Differences in TFP regulation and their effects on surface behaviors may contribute to increased virulence in recent epidemic strains.
Is there an association between tendinopathy and diabetes mellitus? A systematic review with meta-analysis
Authors: Ranger Tom A , Wong Andrea M Y , Cook Jill L , Gaida Jamie E
Published: November 2015

Background

Musculoskeletal symptoms limit adherence to exercise interventions for individuals with type 2 diabetes. People with diabetes may be susceptible to tendinopathy due to chronically elevated blood glucose levels. Therefore, we aimed to investigate this potential association by systematically reviewing and meta-analysing case–control, cross-sectional, and studies that considered both of these conditions.

Methods

Nine medical databases and hand searching methods were used without year limits to identify all relevant English language articles that considered diabetes and tendinopathy. Two authors applied exclusion criteria and one author extracted data with verification by a second author. Meta-analysis was conducted using a random effects model. Results were expressed as odds ratio (OR), mean difference or standardised mean difference with a confidence intervals (95% CI). Heterogeneity was assessed by I2.

Findings

31 studies were included in the final analysis of which 26 recruited people with diabetes and five recruited people with tendinopathy. Tendinopathy was more prevalent in people with diabetes (17 studies, OR 3·67, 95% CI 2·71 to 4·97), diabetes was more prevalent in people with tendinopathy (5 studies, OR 1·28, 95% CI 1·10 to 1·49), people with diabetes and tendinopathy had a longer duration of diabetes than people with diabetes only (6 studies, mean difference 5·26 years, 95% CI 4·15 to 6·36) and people with diabetes had thicker tendons than controls (9 studies, standardised mean difference 0·79 95% CI 0·47 to 1·12).

Interpretation

These findings provide strong evidence that diabetes is associated with higher risk of tendinopathy. This is clinically relevant as tendinopathy may affect adherence to exercise interventions for diabetes.
Epidemiology of haemodialysis catheter complications: a survey of 865 dialysis patients from 14 haemodialysis centres in Henan province in China
Authors: Wang Kai , Wang Pei , Liang Xianhui , Lu Xiaoqing , Liu Zhangsuo
Published: November 2015

Objectives

To investigate the incidence rates and risk factors for catheter-related complications in different districts and populations in Henan Province in China.

Design

Cross-sectional.

Setting

Fourteen hospitals in Henan Province.

Participants

865 patients with renal dysfunction undergoing dialysis using catheters between October 2013 and October 2014.

Main outcome measures

The main outcome measures were complications, risk factors and patient characteristics. Catheter-related complications included catheter-related infection (catheter exit-site infection, catheter tunnel infection and catheter-related bloodstream infection), catheter dysfunction (thrombosis, catheter malposition or kinking, and fibrin shell formation) and central vein stenosis.

Results

The overall incidence rate was 7.74/1000 catheter-days, affecting 38.61% of all patients, for catheter infections, 10.58/1000 catheter-days, affecting 56.65% of all patients, for catheter dysfunction, and 0.68/1000 catheter-days, affecting 8.79% of all patients, for central vein stenosis. Multivariate analysis showed that increased age, diabetes, primary educational level or below, rural residence, lack of a nephropathy visit before dialysis and pre-established permanent vascular access, not taking oral drugs to prevent catheter thrombus, lower serum albumin levels and higher ferritin levels were independently associated with catheter infections. Rural residence, not taking oral drugs to prevent thrombus, lack of an imaging examination after catheter insertion, non-tunnel catheter type, lack of medical insurance, lack of nephropathy visit before dialysis and pre-established permanent vascular access, left-sided catheter position, access via the femoral vein and lower haemoglobin level were independently associated with catheter dysfunction. Diabetes, lack of nephropathy visit before dialysis and pre-established permanent vascular access, lack of oral drugs to prevent catheter thrombus, left-sided catheter location and higher number of catheter insertions, were independently associated with central vein stenosis.

Conclusions

The rate of catheter-related complications was high in patients with end-stage renal disease in Henan Province. Our finding suggest that strategies should be implemented to decrease complication rates.
Disparities in road crash mortality among pedestrians using wheelchairs in the USA: results of a capture–recapture analysis
Authors: Kraemer John D , Benton Connor S
Published: November 2015

Objective

This study aims to quantify and describe the burden of fatal pedestrian crashes among persons using wheelchairs in the USA from 2006 to 2012.

Design

The occurrence of fatal pedestrian crashes among pedestrians using wheelchairs was assessed using two-source capture-recapture. Descriptive analysis of fatal crashes was conducted using customary approaches.

Setting

Two registries were constructed, both of which likely undercounted fatalities among pedestrians who use wheelchairs. The first used data from the Fatality Analysis Reporting System, and the second used a LexisNexis news search.

Outcome measures

Mortality rate (per 100 000 person-years) and crash-level, driver-level and pedestrian-level characteristics of fatal crashes.

Results

This study found that, from 2006 to 2012, the mortality rate for pedestrians using wheelchairs was 2.07/100 000 person-years (95% CI 1.60 to 2.54), which was 36% higher than the overall population pedestrian mortality rate (p=0.02). Men's risk was over fivefold higher than women's risk (p<0.001). Compared to the overall population, persons aged 50–64 using wheelchairs had a 38% increased risk (p=0.04), and men who use wheelchairs aged 50–64 had a 75% increased risk over men of the same age in the overall population (p=0.006). Almost half (47.6%; 95% CI 42.8 to 52.5) of fatal crashes occurred in intersections and 38.7% (95% CI 32.0 to 45.0) of intersection crashes occurred at locations without traffic control devices. Among intersection crashes, 47.5% (95% CI 40.6 to 54.5) involved wheelchair users in a crosswalk; no crosswalk was available for 18.3% (95% CI 13.5 to 24.4). Driver failure to yield right-of-way was noted in 21.4% (95% CI 17.7 to 25.7) of crashes, and no crash avoidance manoeuvers were detected in 76.4% (95% CI 71.0 to 81.2).

Conclusions

Persons who use wheelchairs experience substantial pedestrian mortality disparities calling for behavioural and built environment interventions.