bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanomyogram amplitude correlates with human gastrocnemius medialis muscle and tendon stiffness both before and after acute passive stretching
Authors: Longo Stefano , Cè Emiliano , Rampichini Susanna , Devoto Michela , Limonta Eloisa , Esposito Fabio
Published: June 2014

New Findings

What is the central question of this study?

The aim of this study was to determine the possible correlations between the mechanomyogram signal amplitude and the stiffness at the muscle–tendon unit, muscle and tendon levels, which were only hypothesised in previous investigations.

What is the main finding and its importance?

The main findings of this study are that the amplitude of the mechanomyogram was correlated inversely, albeit with small r2 values, with muscle–tendon unit, muscle and tendon stiffness both before and after application of stretching.

The study aimed to assess the level of correlation between muscle–tendon unit (MTU) stiffness and mechanomyogram (MMG) signal amplitude of the human gastrocnemius medialis muscle, both before and after acute passive stretching. The passive torque (Tpass), electrically evoked peak torque (pT) and myotendinous junction displacement were determined at different angles of dorsiflexion (0, 10 and 20 deg), while maximum voluntary isometric torque (Tmax) was assessed only at 0 deg. Measurements were repeated after a bout of passive stretching. From the MMG signal, the root mean square (RMS) and peak to peak (p‐p) were calculated. The MTU, muscle and tendon stiffness were determined by ultrasound and Tpass measurements. Before stretching, correlations between MMG RMS and MTU, muscle and tendon stiffness were found (R2 = 0.22–0.46). After stretching, Tpass, Tmax, pT and MTU, muscle and tendon stiffness decreased by 25 ± 7, 16 ± 2, 9 ± 2, 22 ± 7, 23 ± 8 and 28 ± 5%, respectively (P < 0.05). During voluntary and electrically evoked contractions, MMG p‐p decreased by 9 ± 2 and 5 ± 1%, while MMG RMS increased by 48 ± 7 and 50 ± 8%, respectively (P < 0.05). Correlations between MMG RMS and MTU, muscle and tendon stiffness were still present after stretching (R2 = 0.44–0.60). In conclusion, correlations between MMG RMS and stiffness exist both before and after stretching, suggesting that a slacker MTU leads to larger muscle fibre oscillations. However, care must be taken in using MMG amplitude as an indirect index to estimate stiffness owing to the relatively small R2 values of the investigated correlations.
Rapidly alternating photoperiods disrupt central and peripheral rhythmicity and decrease plasma glucose, but do not affect glucose tolerance or insulin secretion in sheep
Authors: Varcoe Tamara J. , Gatford Kathryn L. , Voultsios Athena , Salkeld Mark D. , Boden Michael J. , Rattanatray Leewen , Kennaway David J.
Published: June 2014

New Findings

What is the central question of this study?

Disrupting circadian rhythms in rodents perturbs glucose metabolism and increases adiposity. In this study, we asked whether circadian rhythm disruption, induced by exposure of sheep to rapidly alternating photoperiods (RAPs), also disrupts metabolic homeostasis in a large diurnal animal model.

What is the main finding and its importance?

Exposure to RAPs disrupted central (melatonin and core body temperature) and peripheral rhythmicity (skeletal muscle clock gene expression). This led to reduced nocturnal plasma glucose concentrations, but did not affect glucose tolerance and glucose‐stimulated insulin secretion. These results suggest that RAP‐induced circadian rhythm disruption has minimal effect on glucose homeostasis in the sheep.

Disrupting circadian rhythms in rodents perturbs glucose metabolism and increases adiposity. To determine whether these effects occur in a large diurnal animal, we assessed the impact of circadian rhythm disruption upon metabolic function in sheep. Adult ewes (n = 7) underwent 3 weeks of a control 12 h light–12 h dark photoperiod, followed by 4 weeks of rapidly alternating photoperiods (RAPs) whereby the time of light exposure was reversed twice each week. Measures of central (melatonin secretion and core body temperature) and peripheral rhythmicity (clock and metabolic gene expression in skeletal muscle) were obtained over 24 h in both conditions. Metabolic homeostasis was assessed by glucose tolerance tests and 24 h glucose and insulin profiles. Melatonin and core body temperature rhythms resynchronized within 2 days of the last photoperiod shift. High‐amplitude Bmal1, Clock, Nr1d1, Cry2 and Per3 mRNA rhythms were apparent in skeletal muscle, which were phase advanced by up to 3.5 h at 2 days after the last phase shift, whereas Per1 expression was downregulated at this time. Pparα, Pgc1α and Nampt mRNA were constitutively expressed in both conditions. Nocturnal glucose concentrations were reduced following chronic phase shifts (zeitgeber time 0, −5.5%; zeitgeber time 12, −2.9%; and zeitgeber time 16, −5.7%), whereas plasma insulin, glucose tolerance and glucose‐stimulated insulin secretion were not altered. These results demonstrate that clock gene expression within ovine skeletal muscle oscillates over 24 h and responds to changing photoperiods. However, metabolic genes which link circadian and metabolic clocks in rodents were arrhythmic in sheep. Differences may be due to the ruminant versus monogastric digestive organization in each species. Together, these results demonstrate that despite disruptions to central and peripheral rhythmicity following exposure to rapidly alternating photoperiods, there was minimal impact on glucose homeostasis in the sheep.
Effects of detraining on the temporal expression of positive and negative angioregulatory proteins in skeletal muscle of mice
Authors: Olenich Sara A. , Audet Gerald N. , Roberts Kathleen A. , Olfert I. Mark
Published: June 2014

Key points

Skeletal muscle capillary regression is associated with elevated thrombospondin‐1 and vascular endothelial growth factor protein expression with detraining.

Vascular endothelial growth factor and nucleolin responses to acute exercise are blunted in the triceps surae muscles following exercise training. In the plantaris and soleus muscles, this blunted response persists up to 28 days after cessation of training.

Effects of detraining on skeletal muscle microvascular density appears to be similar among skeletal muscle of varying oxidative potential and is poorly associated with expression of matrix metalloproteinases‐2 and ‐9 and endostatin.

Temporal expression of positive and negative angiogenic factors in response to detraining is poorly understood. We report the protein expression of anti‐angiogenic peptides (thrombospondin‐1, TSP‐1; and endostatin) as well as pro‐angiogenic factors (vascular endothelial growth factor, VEGF; matrix metalloproteinases‐2 and ‐9), and nucleolin (a nuclear protein involved with synthesis and maturation of ribosomes) in response to detraining in triceps surae muscles of C57BL/6 mice. Male mice were allowed to exercise voluntarily for 21 days, and then basal and acute response to exercise were evaluated at 1, 7, 14 and 28 days detraining (D1, D7, D14, D28, respectively, n = 12/group). As seen in the D1 mice, training resulted in the increased muscle capillary‐to‐fibre ratio (C/F), increased maximal running time and elevated basal expression of VEGF and matrix metalloproteinase‐9 (P < 0.05). After 7 days of detraining (D7), C/F levels were similar to control levels, but both basal VEGF and TSP‐1 were elevated (P < 0.05). At D14 and D28, TSP‐1 protein was not different compared to baseline levels; however, VEGF was elevated in gastrocnemius (GA), but not the soleus (SOL) or plantaris (PLT) muscles, of D14 mice. Endostatin tended to decrease in D14 and D28 compared to controls. Timing of nucleolin protein expression differed between muscle groups, with increases at D1, D7 and D14 in the PLT, SOL and GA muscles, respectively. The response of VEGF and nucleolin to acute exercise was blunted with training, and remained blunted in the PLT and SOL even after 28 days of detraining, at a time point long after muscle capillarization was observed to be similar to pre‐training levels. These data suggest that TSP‐1 may be a mediator of capillary regression with detraining, even in the face of elevated VEGF, suggesting that pro‐angiogenic regulators may not be able to prevent the regression of skeletal muscle capillaries under physiological conditions. The responses of matrix metalloproteinases, endostatin and nucleolin poorly correlated with detraining‐induced capillary regression.
Asymmetric temporal interactions of sound‐evoked excitatory and inhibitory inputs in the mouse auditory midbrain
Authors: Ono Munenori , Oliver Douglas L.
Published: June 2014

Key points

The temporal coding of sound is critical for the auditory system.

Little is known about how sound evokes the timing of synaptic currents at higher levels of the auditory system and how the integration of these currents results in neural firing.

In the inferior colliculus, excitatory and inhibitory synaptic currents evoked by long‐duration sounds have different temporal properties with excitatory currents more variable than inhibitory currents.

Within a neuron, the sequence of synaptic events suggests the fastest sound‐evoked excitatory postsynaptic current (EPSCs) usually followed the inhibitory postsynaptic currents (IPSCs), and the slowest EPSCs preceded by IPSCs.

Knowledge about the timing of neural activity in the midbrain is important for understanding how temporally complex sounds such as speech are processed and communicated to higher centres of the system.

In the auditory midbrain, synaptic mechanisms responsible for the precise temporal coding of inputs in the brainstem are absent. Instead, in the inferior colliculus (IC), the diverse temporal firing patterns must be coded by other synaptic mechanisms, about which little is known. Here, we demonstrate the temporal characteristics of sound‐evoked excitatory and inhibitory postsynaptic currents (seEPSCs and seIPSCs, respectively) in vivo in response to long‐duration tones. The seEPSCs and seIPSCs differ in the variability of their temporal properties. The seEPSCs have either early or late current peaks, and the early‐peaked currents may be either transient or sustained varieties. The seIPSCs have only early‐peaked sustained responses but often have offset responses. When measured in a single neuron, the seIPSC peaks usually follow early, transient seEPSCs, but the seIPSCs precede latest‐peaking seEPSCs. A model of the firing produced by the integration of asymmetric seEPSCs and seIPSCs showed that the temporal pattern of the early‐peaked sustained neurons was easily modified by changing the parameters of the seIPSC. These results suggest that the considerable variability in the peak time and duration of the seEPSCs shapes the overall time course of firing and often precedes or follows the less variable seIPSC. Despite this, the inhibitory currents are potent in modifying the firing patterns, and the inhibitory response to sound offset appears to be one area where the integration of excitatory and inhibitory synaptic currents is lacking. Thus, the integration of sound‐evoked activity in the IC often employs the asymmetric temporal interaction of excitatory and inhibitory synaptic currents to shape the firing pattern of the neuron.
Mineralocorticoid and AT1 receptors in the paraventricular nucleus contribute to sympathetic hyperactivity and cardiac dysfunction in rats post myocardial infarct
Authors: Huang Bing S. , Chen Aidong , Ahmad Monir , Wang Hong‐Wei , Leenen Frans H. H.
Published: June 2014

Key points

Central mineralocorticoid receptor (MR) and angiotensin II type 1 receptor (AT1R) activation play a critical role in sympathetic hyperactivity and progressive left ventricle (LV) remodelling and dysfunction after a myocardial infarction (MI).

Intra‐paraventricular nucleus (PVN) infusion of adeno‐associated virus (AAV) carrying small interfering RNA (siRNA) against MR (AAV‐MR‐siRNA) markedly decreases both MR and AT1R expression in the PVN post MI, whereas AAV‐AT1aR‐siRNA only decreases AT1R expression. Both AAVs largely prevent sympathetic hyperactivity and inhibit part of LV remodelling and dysfunction post MI.

These findings indicate that enhanced MR–AT1R signalling in the PVN is critical for sympathetic hyperactivity post MI, and contributes to part of LV dysfunction post MI.

Intracerebroventricular infusion of a mineralocorticoid receptor (MR) or angiotensin II type 1 receptor (AT1R) blocker in rats attenuates sympathetic hyperactivity and progressive left ventricular (LV) dysfunction post myocardial infarction (MI). The present study examined whether knockdown of MRs or AT1Rs specifically in the paraventricular nucleus (PVN) contributes to these effects, and compared cardiac effects with those of systemic treatment with the β1‐adrenergic receptor blocker metoprolol. The PVN of rats was infused with adeno‐associated virus carrying small interfering RNA against either MR (AAV‐MR‐siRNA) or AT1R (AAV‐AT1R‐siRNA), or as control scrambled siRNA. At 4 weeks post MI, AT1R but not MR expression was increased in the PVN, excitatory renal sympathetic nerve activity and pressor responses to air stress were enhanced, and arterial baroreflex function was impaired; LV end‐diastolic pressure (LVEDP) was increased and LV peak systolic pressure (LVPSP), ejection fraction (EF) and dP/dtmax decreased. AAV‐MR‐siRNA and AAV‐AT1R‐siRNA both normalized AT1R expression in the PVN, similarly ameliorated sympathetic and pressor responses to air stress, largely prevented baroreflex desensitization, and improved LVEDP, EF and dP/dtmax as well as cardiac interstitial (but not perivascular) fibrosis. In a second set of rats, metoprolol at 70 or 250 mg kg−1 day−1 in the drinking water for 4 weeks post MI did not improve LV function except for a decrease in LVEDP at the lower dose. These results suggest that in rats MR‐dependent upregulation of AT1Rs in the PVN contributes to sympathetic hyperactivity, and LV dysfunction and remodelling post MI. In rats, normalizing MR–AT1R signalling in the PVN is a more effective strategy to improve LV dysfunction post MI than systemic β1 blockade.
Validation of a Newly Developed n-Heptane Reduced Chemistry and Its Application to Simulations of Ignition Quality Tester, Diesel, and HCCI Combustion
Authors: Tsai Hsin-Luen , Chen J.-Y. , Chin Gregory T.
Published: June 2014

A skeletal mechanism (144 species) and a corresponding reduced mechanism (62 species) were developed on the basis of the most recent detailed n-heptane mechanism by Lawrence Livermore National Laboratories (LLNL, version 3.1, 2012) (Mehl et al., 2011, “Kinetic Modeling of Gasoline Surrogate Components and Mixtures Under Engine Conditions,” Proc. Combust. Inst., 33, pp. 193–200), in order to assess the mechanism's performance under various practical combustion conditions. These simplified mechanisms were constructed and validated under shock tube conditions. Three-dimensional computational fluid dynamics (3D CFD) simulations with both simplified mechanisms were conducted for the following modeling applications: ignition quality tester (IQT), diesel engine, and homogeneous charge compression ignition (HCCI) engine. In comparison with experimental data, the simulation results were found satisfactory under the diesel condition but inaccurate for both the IQT and HCCI conditions. For HCCI, the intake temperature used in the simulation had to be increased 30 K in order to be consistent with the engine data provided by Guo et al. (2010, “An Experimental and Modeling Study of HCCI Combustion Using n-Heptane,” ASME J. Eng. Gas Turbines Power, 132(2), 022801). Exploration of possible causes is conducted leading to the conclusion that refinement in the mechanism is needed for accurate prediction of combustion under IQT and HCCI conditions.
Elucidation of the Molecular Basis for Arabinoxylan-Debranching Activity of a Thermostable Family GH62 α-l-Arabinofuranosidase from Streptomyces thermoviolaceus
Authors: Wang Weijun , Mai-Gisondi Galina , Stogios Peter J. , Kaur Amrit , Xu Xiaohui , Cui Hong , Turunen Ossi , Savchenko Alexei , Master Emma R.
Published: June 2014

Xylan-debranching enzymes facilitate the complete hydrolysis of xylan and can be used to alter xylan chemistry. Here, the family GH62 α-l-arabinofuranosidase fromStreptomyces thermoviolaceus(SthAbf62A) was shown to have a half-life of 60 min at 60°C and the ability to cleave α-1,3l-arabinofuranose (l-Araf) from singly substituted xylopyranosyl (Xylp) backbone residues in wheat arabinoxylan; low levels of activity on arabinan as well as 4-nitrophenyl α-l-arabinofuranoside were also detected. After selective removal of α-1,3l-Arafsubstituents from disubstituted Xylpresidues present in wheat arabinoxylan, SthAbf62A could also cleave the remaining α-1,2l-Arafsubstituents, confirming the ability of SthAbf62A to remove α-l-Arafresidues that are (1→2) and (1→3) linked to monosubstituted β-d-Xylpsugars. Three-dimensional structures of SthAbf62A and its complex with xylotetraose andl-arabinose confirmed a five-bladed β-propeller fold and revealed a molecular Velcro in blade V between the β1 and β21 strands, a disulfide bond between Cys27 and Cys297, and a calcium ion coordinated in the central channel of the fold. The enzyme-arabinose complex structure further revealed a narrow and seemingly rigidl-arabinose binding pocket situated at the center of one side of the β propeller, which stabilized the arabinofuranosyl substituent through several hydrogen-bonding and hydrophobic interactions. The predicted catalytic amino acids were oriented toward this binding pocket, and the catalytic essentiality of Asp53 and Glu213 was confirmed by site-specific mutagenesis. Complex structures with xylotetraose revealed a shallow cleft for xylan backbone binding that is open at both ends and comprises multiple binding subsites above and flanking thel-arabinose binding pocket.
Effect of Microstructure on Population Growth Parameters of Escherichia coli in Gelatin-Dextran Systems
Authors: Boons Kathleen , Noriega Estefanía , Van den Broeck Rob , David Charlotte C. , Hofkens Johan , Van Impe Jan F.
Published: June 2014

Current literature acknowledges the effect of food structure on bacterial dynamics. Most studies introduce this “structure” factor using a single gelling agent, resulting in a homogeneous environment, whereas in practice most food products are heterogeneous. Therefore, this study focuses on heterogeneous protein-polysaccharide mixtures, based on gelatin and dextran. These mixtures show phase separation, leading to a range of heterogeneous microstructures by adjusting relative concentrations of both gelling agents. Based on confocal microscope observations, the growth of
Escherichia coli
in gelatin-dextran systems was observed to occur in the dextran phase. To find a relation between microscopic and population behavior, growth experiments were performed in binary and singular gelatin-dextran systems and culture broth at 23.5°C, with or without adding 2.9% (wt/vol) NaCl. The Baranyi and Roberts growth model was fitted to the experimental data and parameter estimates were statistically compared. For salted binary mixtures, a decrease in the population maximum cell density was observed with increasing gelatin concentration. In this series, for one type of microstructure, i.e., a gelatin matrix phase with a disperse dextran phase, the maximum cell density decreased with decreasing percentage of dextran phase. However, this relation no longer held when other types of microstructure were observed. Compared to singular systems, adding a second gelling agent in the presence of NaCl had an effect on population lag phases and maximum cell densities. For unsalted media, the growth parameters of singular and binary mixtures were comparable. Introducing this information into mathematical models leads to more reliable growth predictions and enhanced food safety.
Analysis of Strains Lacking Known Osmolyte Accumulation Mechanisms Reveals Contributions of Osmolytes and Transporters to Protection against Abiotic Stress
Authors: Murdock Lindsay , Burke Tangi , Coumoundouros Chelsea , Culham Doreen E. , Deutch Charles E. , Ellinger James , Kerr Craig H. , Plater Samantha M. , To Eric , Wright Geordie , Wood Janet M.
Published: June 2014

Osmolyte accumulation and release can protect cells from abiotic stresses. In
Escherichia coli
, known mechanisms mediate osmotic stress-induced accumulation of K
+
glutamate, trehalose, or zwitterions like glycine betaine. Previous observations suggested that additional osmolyte accumulation mechanisms (OAMs) exist and their impacts may be abiotic stress specific. Derivatives of the uropathogenic strain CFT073 and the laboratory strain MG1655 lacking known OAMs were created. CFT073 grew without osmoprotectants in minimal medium with up to 0.9 M NaCl. CFT073 and its OAM-deficient derivative grew equally well in high- and low-osmolality urine pools. Urine-grown bacteria did not accumulate large amounts of known or novel osmolytes. Thus, CFT073 showed unusual osmotolerance and did not require osmolyte accumulation to grow in urine. Yeast extract and brain heart infusion stimulated growth of the OAM-deficient MG1655 derivative at high salinity. Neither known nor putative osmoprotectants did so. Glutamate and glutamine accumulated after growth with either organic mixture, and no novel osmolytes were detected. MG1655 derivatives retaining individual OAMs were created. Their abilities to mediate osmoprotection were compared at 15°C, 37°C without or with urea, and 42°C. Stress protection was not OAM specific, and variations in osmoprotectant effectiveness were similar under all conditions. Glycine betaine and dimethylsulfoniopropionate (DMSP) were the most effective. Trimethylamine-
N
-oxide (TMAO) was a weak osmoprotectant and a particularly effective urea protectant. The effectiveness of glycine betaine, TMAO, and proline as osmoprotectants correlated with their preferential exclusion from protein surfaces, not with their propensity to prevent protein denaturation. Thus, their effectiveness as stress protectants correlated with their ability to rehydrate the cytoplasm.
Norovirus Transmission between Hands, Gloves, Utensils, and Fresh Produce during Simulated Food Handling
Authors: Rönnqvist M. , Aho E. , Mikkelä A. , Ranta J. , Tuominen P. , Rättö M. , Maunula L.
Published: June 2014

Human noroviruses (HuNoVs), a leading cause of food-borne gastroenteritis worldwide, are easily transferred via ready-to-eat (RTE) foods, often prepared by infected food handlers. In this study, the transmission of HuNoV and murine norovirus (MuNoV) from virus-contaminated hands to latex gloves during gloving, as well as from virus-contaminated donor surfaces to recipient surfaces after simulated preparation of cucumber sandwiches, was inspected. Virus transfer was investigated by swabbing with polyester swabs, followed by nucleic acid extraction from the swabs with a commercial kit and quantitative reverse transcription-PCR. During gloving, transfer of MuNoV dried on the hand was observed 10/12 times. HuNoV, dried on latex gloves, was disseminated to clean pairs of gloves 10/12 times, whereas HuNoV without drying was disseminated 11/12 times. In the sandwich-preparing simulation, both viruses were transferred repeatedly to the first recipient surface (left hand, cucumber, and knife) during the preparation. Both MuNoV and HuNoV were transferred more efficiently from latex gloves to cucumbers (1.2% ± 0.6% and 1.5% ± 1.9%) than vice versa (0.7% ± 0.5% and 0.5% ± 0.4%). We estimated that transfer of at least one infective HuNoV from contaminated hands to the sandwich prepared was likely to occur if the hands of the food handler contained 3 log
10
or more HuNoVs before gloving. Virus-contaminated gloves were estimated to transfer HuNoV to the food servings more efficiently than a single contaminated cucumber during handling. Our results indicate that virus-free food ingredients and good hand hygiene are needed to prevent HuNoV contamination of RTE foods.
Wastewater Irrigation Increases the Abundance of Potentially Harmful Gammaproteobacteria in Soils in Mezquital Valley, Mexico
Authors: Broszat Melanie , Nacke Heiko , Blasi Ronja , Siebe Christina , Huebner Johannes , Daniel Rolf , Grohmann Elisabeth
Published: June 2014

Wastewater contains large amounts of pharmaceuticals, pathogens, and antimicrobial resistance determinants. Only a little is known about the dissemination of resistance determinants and changes in soil microbial communities affected by wastewater irrigation. Community DNAs from Mezquital Valley soils under irrigation with untreated wastewater for 0 to 100 years were analyzed by quantitative real-time PCR for the presence of
sul
genes, encoding resistance to sulfonamides. Amplicon sequencing of bacterial 16S rRNA genes from community DNAs from soils irrigated for 0, 8, 10, 85, and 100 years was performed and revealed a 14% increase of the relative abundance of
Proteobacteria
in rainy season soils and a 26.7% increase in dry season soils for soils irrigated for 100 years with wastewater. In particular,
Gammaproteobacteria
, including potential pathogens, such as
Pseudomonas
,
Stenotrophomonas
, and
Acinetobacter
spp., were found in wastewater-irrigated fields. 16S rRNA gene sequencing of 96 isolates from soils irrigated with wastewater for 100 years (48 from dry and 48 from rainy season soils) revealed that 46% were affiliated with the
Gammaproteobacteria
(mainly potentially pathogenic
Stenotrophomonas
strains) and 50% with the
Bacilli
, whereas all 96 isolates from rain-fed soils (48 from dry and 48 from rainy season soils) were affiliated with the
Bacilli
. Up to six types of antibiotic resistance were found in isolates from wastewater-irrigated soils; sulfamethoxazole resistance was the most abundant (33.3% of the isolates), followed by oxacillin resistance (21.9% of the isolates). In summary, we detected an increase of potentially harmful bacteria and a larger incidence of resistance determinants in wastewater-irrigated soils, which might result in health risks for farm workers and consumers of wastewater-irrigated crops.
LacR Is a Repressor of
lacABCD
and LacT Is an Activator of
lacTFEG
, Constituting the
lac
Gene Cluster in Streptococcus pneumoniae
Authors: Afzal Muhammad , Shafeeq Sulman , Kuipers Oscar P.
Published: June 2014

Comparison of the transcriptome of
Streptococcus pneumoniae
strain D39 grown in the presence of either lactose or galactose with that of the strain grown in the presence of glucose revealed the elevated expression of various genes and operons, including the
lac
gene cluster, which is organized into two operons, i.e.,
lac
operon I (
lacABCD
) and
lac
operon II (
lacTFEG
). Deletion of the DeoR family transcriptional regulator
lacR
that is present downstream of the
lac
gene cluster revealed elevated expression of
lac
operon I even in the absence of lactose. This suggests a function of LacR as a transcriptional repressor of
lac
operon I, which encodes enzymes involved in the phosphorylated tagatose pathway in the absence of lactose or galactose. Deletion of
lacR
did not affect the expression of
lac
operon II, which encodes a lactose-specific phosphotransferase. This finding was further confirmed by β-galactosidase assays with P
lacA-lacZ
and P
lacT-lacZ
in the presence of either lactose or glucose as the sole carbon source in the medium. This suggests the involvement of another transcriptional regulator in the regulation of
lac
operon II, which is the BglG-family transcriptional antiterminator LacT. We demonstrate the role of LacT as a transcriptional activator of
lac
operon II in the presence of lactose and CcpA-independent regulation of the
lac
gene cluster in
S. pneumoniae
.
Genetic Characterization of Plasmid-Associated Triphenylmethane Reductase in Listeria monocytogenes
Authors: Dutta Vikrant , Elhanafi Driss , Osborne Jason , Martinez Mira Rakic , Kathariou Sophia
Published: June 2014

The enzyme triphenylmethane reductase (TMR) reduces toxic triphenylmethane dyes into colorless, nontoxic derivatives, and TMR-producing microorganisms have been proposed as bioremediation tools. Analysis of the genome of
Listeria monocytogenes
H7858 (1998-1999 hot dog outbreak) revealed that the plasmid (pLM80) of this strain harboring a gene cassette (
bcrABC
) conferring resistance to benzalkonium chloride (BC) and other quaternary ammonium disinfectants also harbored a gene (
tmr
) highly homologous to TMR-encoding genes from diverse Gram-negative bacteria. The pLM80-associated
tmr
was located two genes downstream of
bcrABC
as part of a putative IS
1216
composite transposon. To confirm the role of
tmr
in triphenylmethane dye detoxification, we introduced various
tmr
-harboring fragments of pLM80 in a pLM80-cured derivative of strain H7550, from the same outbreak as H7858, and assessed the resistance of the constructs to the triphenylmethane dyes crystal violet (CV) and malachite green. Transcriptional and subcloning data suggest that the regulation of TMR is complex. Constructs harboring fragments spanning
bcrABC
and
tmr
were CV resistant, and in such constructs
tmr
transcription was induced by sublethal levels of either BC or CV. However, constructs harboring only
tmr
and its upstream intergenic region could also confer resistance to CV, albeit at lower levels. Screening a panel of BC-resistant
L. monocytogenes
strains revealed that all those harboring
bcrABC
and adjacent pLM80 sequences, including
tmr
, were resistant to CV and decolorized this dye. The findings suggest a potential role of TMR as a previously unknown adaptive attribute for environmental persistence of
L. monocytogenes
.
TgaA, a VirB1-Like Component Belonging to a Putative Type IV Secretion System of Bifidobacterium bifidum MIMBb75
Authors: Guglielmetti Simone , Balzaretti Silvia , Taverniti Valentina , Miriani Matteo , Milani Christian , Scarafoni Alessio , Corona Silvia , Ciranna Alessandro , Arioli Stefania , Santala Ville , Iametti Stefania , Bonomi Francesco , Ventura Marco , Mora Diego , Karp Matti
Published: June 2014

Bifidobacterium bifidum
MIMBb75 is a human intestinal isolate demonstrated to be interactive with the host and efficacious as a probiotic. However, the molecular biology of this microorganism is yet largely unknown. For this reason, we undertook whole-genome sequencing of
B. bifidum
MIMBb75 to identify potential genetic factors that would explain the metabolic and probiotic attributes of this bacterium. Comparative genomic analysis revealed a 45-kb chromosomal region that comprises 19 putative genes coding for a potential type IV secretion system (T4SS). Thus, we undertook the initial characterization of this genetic region by studying the putative
virB1
-like gene, named
tgaA
. Gene
tgaA
encodes a peptidoglycan lytic enzyme containing two active domains: lytic murein transglycosylase (LT, cd00254.3) and cysteine- and histidine-dependent amidohydrolase/peptidase (CHAP, pfam05257.4). By means of several
in vitro
assays, we experimentally confirmed that protein TgaA, consistent with its computationally assigned role, has peptidoglycan lytic activity, which is principally associated to the LT domain. Furthermore, immunofluorescence and immunogold labeling showed that the protein TgaA is abundantly expressed on the cell surface of
B. bifidum
MIMBb75. According to the literature, the T4SSs, which have not been characterized before in bifidobacteria, can have important implications for bacterial cell-to-cell communication as well as cross talk with host cells, justifying the interest for further studies aimed at the investigation of this genetic region.
Inhibition of Biofilm Formation by T7 Bacteriophages Producing Quorum-Quenching Enzymes
Authors: Pei Ruoting , Lamas-Samanamud Gisella R.
Published: June 2014

Bacterial growth in biofilms is the major cause of recalcitrant biofouling in industrial processes and of persistent infections in clinical settings. The use of bacteriophage treatment to lyse bacteria in biofilms has attracted growing interest. In particular, many natural or engineered phages produce depolymerases to degrade polysaccharides in the biofilm matrix and allow access to host bacteria. However, the phage-produced depolymerases are highly specific for only the host-derived polysaccharides and may have limited effects on natural multispecies biofilms. In this study, an engineered T7 bacteriophage was constructed to encode a lactonase enzyme with broad-range activity for quenching of quorum sensing, a form of bacterial cell-cell communication via small chemical molecules (acyl homoserine lactones [AHLs]) that is necessary for biofilm formation. Our results demonstrated that the engineered T7 phage expressed the AiiA lactonase to effectively degrade AHLs from many bacteria. Addition of the engineered T7 phage to mixed-species biofilms containing
Pseudomonas aeruginosa
and
Escherichia coli
resulted in inhibition of biofilm formation. Such quorum-quenching phages that can lyse host bacteria and express quorum-quenching enzymes to affect diverse bacteria in biofilm communities may become novel antifouling and antibiofilm agents in industrial and clinical settings.
Iron Triggers λSo Prophage Induction and Release of Extracellular DNA in Shewanella oneidensis MR-1 Biofilms
Authors: Binnenkade Lucas , Teichmann Laura , Thormann Kai M.
Published: June 2014

Prophages are ubiquitous elements within bacterial chromosomes and affect host physiology and ecology in multiple ways. We have previously demonstrated that phage-induced lysis is required for extracellular DNA (eDNA) release and normal biofilm formation in
Shewanella oneidensis
MR-1. Here, we investigated the regulatory mechanisms of prophage λSo spatiotemporal induction in biofilms. To this end, we used a functional fluorescence fusion to monitor λSo activation in various mutant backgrounds and in response to different physiological conditions. λSo induction occurred mainly in a subpopulation of filamentous cells in a strictly RecA-dependent manner, implicating oxidative stress-induced DNA damage as the major trigger. Accordingly, mutants affected in the oxidative stress response (Δ
oxyR
) or iron homeostasis (Δ
fur
) displayed drastically increased levels of phage induction and abnormal biofilm formation, while planktonic cells were not or only marginally affected. To further investigate the role of oxidative stress, we performed a mutant screen and identified two independent amino acid substitutions in OxyR (T104N and L197P) that suppress induction of λSo by hydrogen peroxide (H
2
O
2
). However, λSo induction was not suppressed in biofilms formed by both mutants, suggesting a minor role of intracellular H
2
O
2
in this process. In contrast, addition of iron to biofilms strongly enhanced λSo induction and eDNA release, while both processes were significantly suppressed at low iron levels, strongly indicating that iron is the limiting factor. We conclude that uptake of iron during biofilm formation triggers λSo-mediated lysis of a subpopulation of cells, likely by an increase in iron-mediated DNA damage sensed by RecA.
The Functional Structure of Central Carbon Metabolism in Pseudomonas putida KT2440
Authors: Sudarsan Suresh , Dethlefsen Sarah , Blank Lars M. , Siemann-Herzberg Martin , Schmid Andreas
Published: June 2014

What defines central carbon metabolism? The classic textbook scheme of central metabolism includes the Embden-Meyerhof-Parnas (EMP) pathway of glycolysis, the pentose phosphate pathway, and the citric acid cycle. The prevalence of this definition of central metabolism is, however, equivocal without experimental validation. We address this issue using a general experimental approach that combines the monitoring of transcriptional and metabolic flux changes between steady states on alternative carbon sources. This approach is investigated by using the model bacterium
Pseudomonas putida
with glucose, fructose, and benzoate as carbon sources. The catabolic reactions involved in the initial uptake and metabolism of these substrates are expected to show a correlated change in gene expressions and metabolic fluxes. However, there was no correlation for the reactions linking the 12 biomass precursor molecules, indicating a regulation mechanism other than mRNA synthesis for central metabolism. This result substantiates evidence for a (re)definition of central carbon metabolism including all reactions that are bound to tight regulation and transcriptional invariance. Contrary to expectations, the canonical Entner-Doudoroff and EMP pathways
sensu stricto
are not a part of central carbon metabolism in
P. putida
, as they are not regulated differently from the aromatic degradation pathway. The regulatory analyses presented here provide leads on a qualitative basis to address the use of alternative carbon sources by deregulation and overexpression at the transcriptional level, while rate improvements in central carbon metabolism require careful adjustment of metabolite concentrations, as regulation resides to a large extent in posttranslational and/or metabolic regulation.
Interplay between Candida albicans and the Antimicrobial Peptide Armory
Authors: Swidergall Marc , Ernst Joachim F.
Published: June 2014

Antimicrobial peptides (AMPs) are key elements of innate immunity, which can directly kill multiple bacterial, viral, and fungal pathogens. The medically important fungus
Candida albicans
colonizes different host niches as part of the normal human microbiota. Proliferation of
C. albicans
is regulated through a complex balance of host immune defense mechanisms and fungal responses. Expression of AMPs against pathogenic fungi is differentially regulated and initiated by interactions of a variety of fungal pathogen-associated molecular patterns (PAMPs) with pattern recognition receptors (PRRs) on human cells. Inflammatory signaling and other environmental stimuli are also essential to control fungal proliferation and to prevent parasitism. To persist in the host,
C. albicans
has developed a three-phase AMP evasion strategy, including secretion of peptide effectors, AMP efflux pumps, and regulation of signaling pathways. These mechanisms prevent
C. albicans
from the antifungal activity of the major AMP classes, including cathelicidins, histatins, and defensins leading to a basal resistance. This minireview summarizes human AMP attack and
C. albicans
resistance mechanisms and current developments in the use of AMPs as antifungal agents.
VeA Is Associated with the Response to Oxidative Stress in the Aflatoxin Producer Aspergillus flavus
Authors: Baidya Sachin , Duran Rocio M. , Lohmar Jessica M. , Harris-Coward Pamela Y. , Cary Jeffrey W. , Hong Sung-Yong , Roze Ludmila V. , Linz John E. , Calvo Ana M.
Published: June 2014

Survival of fungal species depends on the ability of these organisms to respond to environmental stresses. Osmotic stress or high levels of reactive oxygen species (ROS) can cause stress in fungi resulting in growth inhibition. Both eukaryotic and prokaryotic cells have developed numerous mechanisms to counteract and survive the stress in the presence of ROS. In many fungi, the HOG signaling pathway is crucial for the oxidative stress response as well as for osmotic stress response. This study revealed that while the osmotic stress response is only slightly affected by the master regulator
veA
, this gene, also known to control morphological development and secondary metabolism in numerous fungal species, has a profound effect on the oxidative stress response in the aflatoxin-producing fungus
Aspergillus flavus
. We found that the expression of
A. flavus
homolog genes involved in the HOG signaling pathway is regulated by
veA
. Deletion of
veA
resulted in a reduction in transcription levels of oxidative stress response genes after exposure to hydrogen peroxide. Furthermore, analyses of the effect of VeA on the promoters of
cat1
and
trxB
indicate that the presence of VeA alters DNA-protein complex formation. This is particularly notable in the
cat1
promoter, where the absence of VeA results in abnormally stronger complex formation with reduced
cat1
expression and more sensitivity to ROS in a
veA
deletion mutant, suggesting that VeA might prevent binding of negative transcription regulators to the
cat1
promoter. Our study also revealed that
veA
positively influences the expression of the transcription factor gene
atfB
and that normal formation of DNA-protein complexes in the
cat1
promoter is dependent on AtfB.
Aspergillus parasiticus SU-1 Genome Sequence, Predicted Chromosome Structure, and Comparative Gene Expression under Aflatoxin-Inducing Conditions: Evidence that Differential Expression Contributes to Species Phenotype
Authors: Linz John E. , Wee Josephine , Roze Ludmila V.
Published: June 2014

The filamentous fungi
Aspergillus parasiticus
and
Aspergillus flavus
produce the carcinogenic secondary metabolite aflatoxin on susceptible crops. These species differ in the quantity of aflatoxins B
1
, B
2
, G
1
, and G
2
produced in culture, in the ability to produce the mycotoxin cyclopiazonic acid, and in morphology of mycelia and conidiospores. To understand the genetic basis for differences in biochemistry and morphology, we conducted next-generation sequence (NGS) analysis of the
A. parasiticus
strain SU-1 genome and comparative gene expression (RNA sequence analysis [RNA Seq]) analysis of
A. parasiticus
SU-1 and
A. flavus
strain NRRL 3357 (3357) grown under aflatoxin-inducing and -noninducing culture conditions. Although
A. parasiticus
SU-1 and
A. flavus
3357 are highly similar in genome structure and gene organization, we observed differences in the presence of specific mycotoxin gene clusters and differential expression of specific mycotoxin genes and gene clusters that help explain differences in the type and quantity of mycotoxins synthesized. Using computer-aided analysis of secondary metabolite clusters (antiSMASH), we demonstrated that
A. parasiticus
SU-1 and
A. flavus
3357 may carry up to 93 secondary metabolite gene clusters, and surprisingly, up to 10% of the genome appears to be dedicated to secondary metabolite synthesis. The data also suggest that fungus-specific zinc binuclear cluster (C6) transcription factors play an important role in regulation of secondary metabolite cluster expression. Finally, we identified uniquely expressed genes in
A. parasiticus
SU-1 that encode C6 transcription factors and genes involved in secondary metabolism and stress response/cellular defense. Future work will focus on these differentially expressed
A. parasiticus
SU-1 loci to reveal their role in determining distinct species characteristics.
Application of Coamplification at Lower Denaturation Temperature-PCR Sequencing for Early Detection of Antiviral Drug Resistance Mutations of Hepatitis B Virus
Authors: Wong Danny Ka-Ho , Tsoi Ottilia , Huang Fung-Yu , Seto Wai-Kay , Fung James , Lai Ching-Lung , Yuen Man-Fung
Published: June 2014

Nucleoside/nucleotide analogue for the treatment of chronic hepatitis B virus (HBV) infection is hampered by the emergence of drug resistance mutations. Conventional PCR sequencing cannot detect minor variants of <20%. We developed a modifiedco-amplification atlowerdenaturation temperature-PCR (COLD-PCR) method for the detection of HBV minority drug resistance mutations. The critical denaturation temperature for COLD-PCR was determined to be 78°C. Sensitivity of COLD-PCR sequencing was determined using serially diluted plasmids containing mixed proportions of HBV reverse transcriptase (rt) wild-type and mutant sequences. Conventional PCR sequencing detected mutations only if they existed in ≥25%, whereas COLD-PCR sequencing detected mutations when they existed in 5 to 10% of the viral population. The performance of COLD-PCR was compared to conventional PCR sequencing and a line probe assay (LiPA) using 215 samples obtained from 136 lamivudine- or telbivudine-treated patients with virological breakthrough. Among these 215 samples, drug resistance mutations were detected in 155 (72%), 148 (69%), and 113 samples (53%) by LiPA, COLD-PCR, and conventional PCR sequencing, respectively. Nineteen (9%) samples had mutations detectable by COLD-PCR but not LiPA, while 26 (12%) samples had mutations detectable by LiPA but not COLD-PCR, indicating both methods were comparable (P= 0.371). COLD-PCR was more sensitive than conventional PCR sequencing. Thirty-five (16%) samples had mutations detectable by COLD-PCR but not conventional PCR sequencing, while none had mutations detected by conventional PCR sequencing but not COLD-PCR (P< 0.0001). COLD-PCR sequencing is a simple method which is comparable to LiPA and superior to conventional PCR sequencing in detecting minor lamivudine/telbivudine resistance mutations.
Use of Anidulafungin as a Surrogate Marker To Predict Susceptibility and Resistance to Caspofungin among 4,290 Clinical Isolates of Candida by Using CLSI Methods and Interpretive Criteria
Authors: Pfaller Michael A. , Diekema Daniel J. , Jones Ronald N. , Castanheira Mariana
Published: June 2014

This study addressed the application of anidulafungin as a surrogate marker to predict the susceptibility ofCandidato caspofungin due to unacceptably high interlaboratory variation of caspofungin MIC values. CLSI reference broth microdilution methods and species-specific interpretive criteria were used to test 4,290 strains ofCandida(eight species), including 71 strains with documentedfksmutations. Caspofungin MIC values were compared with those of anidulafungin to determine the percentage of categorical agreement (CA) and very major (VME), major (ME), and minor error rates, as well as the ability to detectfksmutants. For all 4,290 isolates the CA was 97.1% (0.2% VME and ME, 2.5% minor errors) using anidulafungin as the surrogate. Among the 62 isolates ofCandida albicans(4 isolates),C. tropicalis(5 isolates),C. krusei(4 isolates),C. kefyr(2 isolates), andC. glabrata(47 isolates) that were nonsusceptible (NS; either intermediate [I] or resistant [R]) to both caspofungin and anidulafungin, 52 (83.8%) contained a mutation infks1orfks2. Eight mutants ofC. glabrata, two ofC. albicans, and one each ofC. tropicalisandC. kruseiwere classified as susceptible (S) to both antifungal agents. The remaining 7 mutants (2C. albicansand 5C. glabrata) were susceptible to one of the agents and either intermediate or resistant to the other. Using the epidemiological cutoff value (ECV) of 0.12 μg/ml for both caspofungin and anidulafungin to differentiate wild-type (WT) from non-WT strains ofC. glabrata, 42 of the 55 (76.4%)C. glabratamutants were non-WT and 8 of the 55 (14.5%) were WT for both agents (90.9% concordance). Anidulafungin can accurately serve as a surrogate marker to predict S and R ofCandidato caspofungin.
Extended Characterization of Corynebacterium pyruviciproducens Based on Clinical Strains from Canada and Switzerland
Authors: Goldenberger D. , Hinić V. , Turan S. , Schultheiss E. , Pacheco A. L. , Frei R. , Bernard K.
Published: June 2014

The species
Corynebacterium pyruviciproducens
was described in 2010 based on the features of a single strain. In this report, we describe the chemotaxonomic and phenotypic characteristics of 11
C. pyruviciproducens
clinical strains isolated in Switzerland and Canada in comparison to the strain 06-17730
T
. Heterogeneities within the type strain were found in the 16S rRNA gene and in biochemical markers. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identification of this species could not be achieved since currently this bacterial species is not included in the corresponding database. Reliable identification is obtained only with sequence-based identification tools. Results of antimicrobial susceptibility testing of this species with an extended panel of antimicrobials are presented here for the first time.