bluuebunny/crossref_metadata_2025 huggingface dataset:
Thorough GABAergic innervation of the entire axon initial segment revealed by an optogenetic ‘laserspritzer’
Authors: Wang Xinjun , Hooks Bryan M. , Sun Qian‐Quan
Published: August 2014

Key points

The laserspritzer we developed activates axo‐axonic synapses (AASs) with spatial resolutions of less than 40 μm.

AASs innervate the entire length of the axon initial segment (AIS), as opposed to forming a highly concentrated cartridge.

AAS‐mediated synaptic potentials are inhibitory, with reversal potentials similar to those of basket synapses.

AAS inhibition impedes action potentials and epileptiform activity more robustly than perisomatic inhibitions.

AAS activation alone is sufficient to inhibit action potential generation and epileptiform activities in vitro.

GABAergic terminals of chandelier cells exclusively innervate the axon initial segment (AIS) of excitatory neurons. Although the anatomy of these synapses has been well‐studied in several brain areas, relatively little is known about their physiological properties. Using vesicular γ‐aminobutyric acid transporter–channelrhodopsin 2–enhanced yellow fluorescence protein (VGAT‐ChR2‐YFP)‐expressing mice and a novel fibreoptic ‘laserspritzer’ approach that we developed, we investigated the physiological properties of axo‐axonic synapses (AASs) in brain slices from the piriform cortex (PC) of mice. AASs were in close proximity to voltage‐gated Na+ (NaV) channels located at the AIS. AASs were selectively activated by a 5 μm laserspritzer placed in close proximity to the AIS. Under a minimal laser stimulation condition and using whole‐cell somatic voltage‐clamp recordings, the amplitudes and kinetics of IPSCs mediated by AASs were similar to those mediated by perisomatic inhibitions. Results were further validated with channelrhodopsin 2‐assisted circuit mapping (CRACM) of the entire inhibitory inputs map. For the first time, we revealed that the laserspritzer‐induced AAS‐IPSCs persisted in the presence of TTX and TEA but not 4‐AP. Next, using gramicidin‐based perforated patch recordings, we found that the GABA reversal potential (EGABA) was −73.6 ± 1.2 mV when induced at the AIS and −72.8 ± 1.1 mV when induced at the perisomatic site. Our anatomical and physiological results lead to the novel conclusions that: (1) AASs innervate the entire length of the AIS, as opposed to forming a highly concentrated cartridge, (2) AAS inhibition suppresses action potentials and epileptiform activity more robustly than perisomatic inhibitions, and (3) AAS activation alone can be sufficient to inhibit action potential generation and epileptiform activities in vitro.
Presynaptic actions of transcranial and local direct current stimulation in the red nucleus
Authors: Bączyk M. , Jankowska E.
Published: August 2014

Key points

Transcranial direct current stimulation (tDCS) may affect both postsynaptic neurons and presynaptic axons providing input to these neurons.

Here, we show that presynaptic subcortical actions of tDCS outlast the duration of its application by up to 1 h and may contribute to long‐lasting facilitation of activation of neurons in the red nucleus in experiments on deeply anaesthetized animals. This is demonstrated by increased axonal excitability of the interpositorubral neurons that, in turn, activate rubrospinal neurons.

We also show that effects of electric fields induced by tDCS may be reproduced by local cathodal polarization, by applying <1 μA direct current within the red nucleus. Such polarization replicates effects of anodal tDCS in the cat as well as cathodal tDCS in the rat and evokes similar long‐lasting facilitation.

The main aim of the present study was to examine to what extent long‐lasting subcortical actions of transcranial direct current stimulation (tDCS) may be related to its presynaptic actions. This was investigated in the red nucleus, where tDCS was recently demonstrated to facilitate transmission between interpositorubral and rubrospinal neurons. Changes in the excitability of preterminal axonal branches of interpositorubral neurons close to rubrospinal neurons were investigated during and after tDCS (0.2 mA) applied over the sensorimotor cortical area in deeply anaesthetized rats and cats. As a measure of the excitability, we used the probability of antidromic activation of individual interpositorubral neurons by electrical stimuli applied in the red nucleus. Our second aim was to compare effects of weak (≤1 μA) direct current applied within the red nucleus with effects of tDCS to allow the use of local depolarization in a further analysis of mechanisms of tDCS instead of widespread and more difficult to control depolarization evoked by distant electrodes. Local cathodal polarization was found to replicate all effects of cathodal tDCS hitherto demonstrated in the rat, including long‐lasting facilitation of trans‐synaptically evoked descending volleys and trisynaptically evoked EMG responses in neck muscles. It also replicated all effects of anodal tDCS in the cat. In both species, it increased the excitability of preterminal axonal branches of interpositorubral neurons up to 1 h post‐tDCS. Local anodal polarization evoked opposite effects. We thus show that presynaptic actions of polarizing direct current may contribute to both immediate and prolonged effects of tDCS.
Effects of hyperoxia and hypoxia on the physiological traits responsible for obstructive sleep apnoea
Authors: Edwards Bradley A. , Sands Scott A. , Owens Robert L. , White David P. , Genta Pedro R. , Butler James P. , Malhotra Atul , Wellman Andrew
Published: August 2014

Key points

Changes in the level of inspired oxygen have dramatic effects on the pathophysiology of obstructive sleep apnoea (OSA): hyperoxia reduces the severity of OSA in some but not all patients, whereas hypoxia transforms obstructive events into central events. Given that OSA is likely to result from the interaction of key pathophysiological traits, including a compromised pharyngeal anatomy, inadequate upper airway muscle function, a large ventilatory response to a disturbance in ventilation (high loop gain) and a low arousal threshold, we examined how changes in oxygen levels alter these traits.

Our study demonstrates that the beneficial effect of hyperoxia on OSA severity is solely based on its ability to attenuate loop gain, whereas hypoxia increases loop gain and the arousal threshold in addition to improving pharyngeal collapsibility.

Such effects help to explain why oxygen therapy may not work in every patient with OSA and explain the disappearance of OSA and the emergence of central events during hypoxic conditions.

Oxygen therapy is known to reduce loop gain (LG) in patients with obstructive sleep apnoea (OSA), yet its effects on the other traits responsible for OSA remain unknown. Therefore, we assessed how hyperoxia and hypoxia alter four physiological traits in OSA patients. Eleven OSA subjects underwent a night of polysomnography during which the physiological traits were measured using multiple 3‐min ‘drops’ from therapeutic continuous positive airway pressure (CPAP) levels. LG was defined as the ratio of the ventilatory overshoot to the preceding reduction in ventilation. Pharyngeal collapsibility was quantified as the ventilation at CPAP of 0 cmH2O. Upper airway responsiveness was defined as the ratio of the increase in ventilation to the increase in ventilatory drive across the drop. Arousal threshold was estimated as the level of ventilatory drive associated with arousal. On separate nights, subjects were submitted to hyperoxia (n = 9; FiO2 ∼0.5) or hypoxia (n = 10; FiO2 ∼0.15) and the four traits were reassessed. Hyperoxia lowered LG from a median of 3.4 [interquartile range (IQR): 2.6–4.1] to 2.1 (IQR: 1.3–2.5) (P < 0.01), but did not alter the remaining traits. By contrast, hypoxia increased LG [median: 3.3 (IQR: 2.3–4.0) vs. 6.4 (IQR: 4.5–9.7); P < 0.005]. Hypoxia additionally increased the arousal threshold (mean ± s.d. 10.9 ± 2.1 l min−1 vs. 13.3 ± 4.3 l min−1; P < 0.05) and improved pharyngeal collapsibility (mean ± s.d. 3.4 ± 1.4 l min−1 vs. 4.9 ± 1.3 l min−1; P < 0.05), but did not alter upper airway responsiveness (P = 0.7). This study demonstrates that the beneficial effect of hyperoxia on the severity of OSA is primarily based on its ability to reduce LG. The effects of hypoxia described above may explain the disappearance of OSA and the emergence of central sleep apnoea in conditions such as high altitude.
Hypercapnia attenuates ventilator‐induced lung injury via a disintegrin and metalloprotease‐17
Authors: Otulakowski Gail , Engelberts Doreen , Gusarova Galina A. , Bhattacharya Jahar , Post Martin , Kavanagh Brian P.
Published: August 2014

Key points

Hypercapnia is common in mechanically ventilated patients with lung injury; while CO2 can ameliorate experimental lung injury, it can also cause harm.

Because hypercapnia can protect against ventilator‐induced lung injury (VILI), understanding its impact on key signalling pathways may provide insight into the mechanisms of VILI.

We show that hypercapnia blocks stretch‐mediated activation of p44/42 mitogen‐activated protein kinase (MAPK) signalling in alveolar epithelial cells; this occurs through inhibition of sheddase (i.e. the metalloprotease, ADAM17), which releases ligands that bind to the epidermal growth factor receptor.

In vivo pharmacological blockade of ADAM17 reduces downstream MAPK activation and attenuates VILI in a two‐hit mouse model.

Thus, hypercapnia uncovered a novel ADAM17‐dependent mechanism of VILI, and this represents a potential therapeutic target.

Hypercapnic acidosis, common in mechanically ventilated patients, has been reported to exert both beneficial and harmful effects in models of lung injury. Understanding its effects at the molecular level may provide insight into mechanisms of injury and protection. The aim of this study was to establish the effects of hypercapnic acidosis on mitogen‐activated protein kinase (MAPK) activation, and determine the relevant signalling pathways. p44/42 MAPK activation in a murine model of ventilator‐induced lung injury (VILI) correlated with injury and was reduced in hypercapnia. When cultured rat alveolar epithelial cells were subjected to cyclic stretch, activation of p44/42 MAPK was dependent on epidermal growth factor receptor (EGFR) activity and on shedding of EGFR ligands; exposure to 12% CO2 without additional buffering blocked ligand shedding, as well as EGFR and p44/42 MAPK activation. The EGFR ligands are known substrates of the matrix metalloprotease ADAM17, suggesting stretch activates and hypercapnic acidosis blocks stretch‐mediated activation of ADAM17. This was corroborated in the isolated perfused mouse lung, where elevated CO2 also inhibited stretch‐activated shedding of the ADAM17 substrate TNFR1 from airway epithelial cells. Finally, in vivo confirmation was obtained in a two‐hit murine model of VILI where pharmacological inhibition of ADAM17 reduced both injury and p44/42 MAPK activation. Thus, ADAM17 is an important proximal mediator of VILI; its inhibition is one mechanism of hypercapnic protection and may be a target for clinical therapy.
Type Synthesis of Single-Loop Overconstrained 6R Spatial Mechanisms for Circular Translation
Authors: Kong Xianwen
Published: August 2014

To discover single-degree-of-freedom (DOF) single-loop overconstrained mechanisms is still an open problem. This paper deals with the type synthesis of single DOF single-loop overconstrained 6RMCTs (6R spatial mechanisms for circular translation). These mechanisms provide alternatives to planar parallelograms and are also associated with self-motion of several translational parallel mechanisms. 6RMCTs are to be obtained using a construction approach in combination with the approaches to the type synthesis of parallel mechanisms. By imposing certain conditions on the hybrid overconstrained 6R (plano-spherical, plano-Bennett, double-spherical, and spherico-Bennett) mechanisms, Bricard plane symmetric mechanism, and Bricard line symmetric mechanism, six special cases of 6RMCTs are obtained. By combining planar parallelograms with these special mechanisms, the general cases of 6RMCTs are then constructed. Finally, 4R2H, 2R4H, and 6H mechanisms for circular translation are obtained from the above 6RMCTs by replacing one or more pairs of R (revolute) joints with parallel axes each with a pair of H (helical) joints with parallel axes and the same pitch. This work contributes to the research on overconstrained six-bar mechanisms and further reveals that the research areas of parallel mechanisms and single-loop overconstrained mechanisms are closely related.
Analysis of a Fixed-Guided Compliant Beam With an Inflection Point Using the Pseudo-Rigid-Body Model Concept
Authors: Midha Ashok , Bapat Sushrut G. , Mavanthoor Adarsh , Chinta Vivekananda
Published: August 2014

This paper provides an efficient method of analysis for a fixed-guided compliant beam with an inflection point, subjected to beam end load or displacement boundary conditions, or a combination thereof. To enable this, such a beam is modeled as a pair of well-established pseudo-rigid-body models (PRBMs) for fixed-free compliant beam segments. The analysis procedure relies on the properties of inflection in developing the necessary set of parametric, static equilibrium and compatibility equations for solution. The paper further discusses the multiplicity of possible solutions, including displacement configurations, for any two specified beam end displacement boundary conditions, depending on the locations and types of the effecting loads on the beam to meet these boundary conditions. A unique solution may exist when a third beam end displacement boundary condition is specified; however, this selection is not unconditional. A concept of characteristic deflection domain is proposed to assist with the selection of the third boundary condition to yield a realistic solution. The analysis method is also used to synthesize a simple, fully compliant mechanism utilizing the fixed-guided compliant segments.
Head and Neck Response of a Finite Element Anthropomorphic Test Device and Human Body Model During a Simulated Rotary-Wing Aircraft Impact
Authors: White Nicholas A. , Danelson Kerry A. , Scott Gayzik F. , Stitzel Joel D.
Published: August 2014

A finite element (FE) simulation environment has been developed to investigate aviator head and neck response during a simulated rotary-wing aircraft impact using both an FE anthropomorphic test device (ATD) and an FE human body model. The head and neck response of the ATD simulation was successfully validated against an experimental sled test. The majority of the head and neck transducer time histories received a CORrelation and Analysis (CORA) rating of 0.7 or higher, indicating good overall correlation. The human body model simulation produced a more biofidelic head and neck response than the ATD experimental test and simulation, including change in neck curvature. While only the upper and lower neck loading can be measured in the ATD, the shear force, axial force, and bending moment were reported for each level of the cervical spine in the human body model using a novel technique involving cross sections. This loading distribution provides further insight into the biomechanical response of the neck during a rotary-wing aircraft impact.
Computational Modeling of Thrombotic Microparticle Deposition in Nonparallel Flow Regimes
Authors: Hall Connie L. , Calt Melissa
Published: August 2014

Thrombotic microparticles (MPs) released from cells and platelets in response to various stimuli are present in elevated numbers in various disease states that increase the risk for thrombotic events. In order to understand how particles of this size may localize in nonparallel flow regimes and increase thrombotic risk, a computational analysis of flow and MP deposition was performed for 3 deg of stenosis at moderate Reynolds number (20 < Re < 80) and for recirculation zones at low Reynolds (∼1) number. The results indicate that MP deposition results primarily from impaction and not by diffusive flux.
Modeling and Analysis of Drug-Eluting Stents With Biodegradable PLGA Coating: Consequences on Intravascular Drug Delivery
Authors: Zhu Xiaoxiang , Braatz Richard D.
Published: August 2014

Increasing interests have been raised toward the potential applications of biodegradable poly(lactic-co-glycolic acid) (PLGA) coatings for drug-eluting stents in order to improve the drug delivery and reduce adverse outcomes in stented arteries in patients. This article presents a mathematical model to describe the integrated processes of drug release in a stent with PLGA coating and subsequent drug delivery, distribution, and drug pharmacokinetics in the arterial wall. The integrated model takes into account the PLGA degradation and erosion, anisotropic drug diffusion in the arterial wall, and reversible drug binding. The model simulations first compare the drug delivery from a biodegradable PLGA coating with that from a biodurable coating, including the drug release profiles in the coating, average arterial drug levels, and arterial drug distribution. Using the model for the PLGA stent coating, the simulations further investigate drug internalization, interstitial fluid flow in the arterial wall, and stent embedment for their impact on drug delivery. Simulation results show that these three factors, while imposing little change in the drug release profiles, can greatly change the average drug concentrations in the arterial wall. In particular, each of the factors leads to significant and yet distinguished alterations in the arterial drug distribution that can potentially influence the treatment outcomes. The detailed integrated model provides insights into the design and evaluation of biodegradable PLGA-coated drug-eluting stents for improved intravascular drug delivery.
Microdosimetry of proton and carbon ions
Authors: Liamsuwan Thiansin , Hultqvist Martha , Lindborg Lennart , Uehara Shuzo , Nikjoo Hooshang
Published: August 2014

Purpose:

To investigate microdosimetry properties of 160 MeV/u protons and 290 MeV/u12C ion beams in small volumes of diameters 10–100 nm.

Methods:

Energy distributions of primary particles and nuclear fragments in the beams were calculated from simulations with the general purpose code SHIELD‐HIT, while energy depositions by monoenergetic ions in nanometer volumes were obtained from the event‐by‐event Monte Carlo track structure ion code PITS99 coupled with the electron track structure code KURBUC.

Results:

The results are presented for frequencies of energy depositions in cylindrical targets of diameters 10–100 nm, dose distributionsyd(y) in lineal energy y, and dose‐mean lineal energies . For monoenergetic ions, the was found to increase with an increasing target size for high‐linear energy transfer (LET) ions, but decrease with an increasing target size for low‐LET ions. Compared to the depth dose profile of the ion beams, the maximum of the depth profile for the 160 MeV proton beam was located at ∼0.5 cm behind the Bragg peak maximum, while the peak of the 290 MeV/u 12C beam coincided well with the peak of the absorbed dose profile. Differences between the and dose‐averaged linear energy transfer (LETD) were large in the proton beam for both target volumes studied, and in the 12C beam for the 10 nm diameter cylindrical volumes. The determined for 100 nm diameter cylindrical volumes in the 12C beam was approximately equal to the LETD. The contributions from secondary particles to the of the beams are presented, including the contributions from secondary protons in the proton beam and from fragments with atomic number Z = 1–6 in the 12C beam.

Conclusions:

The present investigation provides an insight into differences in energy depositions in subcellular‐size volumes when irradiated by proton and carbon ion beams. The results are useful for characterizing ion beams of practical importance for biophysical modeling of radiation‐induced DNA damage response and repair in the depth profiles of protons and carbon ions used in radiotherapy.
Motion tracking in the liver: Validation of a method based on 4D ultrasound using a nonrigid registration technique
Authors: Vijayan Sinara , Klein Stefan , Hofstad Erlend Fagertun , Lindseth Frank , Ystgaard Brynjulf , Langø Thomas
Published: August 2014

Purpose:

Treatments like radiotherapy and focused ultrasound in the abdomen require accurate motion tracking, in order to optimize dosage delivery to the target and minimize damage to critical structures and healthy tissues around the target. 4D ultrasound is a promising modality for motion tracking during such treatments. In this study, the authors evaluate the accuracy of motion tracking in the liver based on deformable registration of 4D ultrasound images.

Methods:

The offline analysis was performed using a nonrigid registration algorithm that was specifically designed for motion estimation from dynamic imaging data. The method registers the entire 4D image data sequence in a groupwise optimization fashion, thus avoiding a bias toward a specifically chosen reference time point. Three healthy volunteers were scanned over several breathing cycles (12 s) from three different positions and angles on the abdomen; a total of nine 4D scans for the three volunteers. Well‐defined anatomic landmarks were manually annotated in all 96 time frames for assessment of the automatic algorithm. The error of the automatic motion estimation method was compared with interobserver variability. The authors also performed experiments to investigate the influence of parameters defining the deformation field flexibility and evaluated how well the method performed with a lower temporal resolution in order to establish the minimum frame rate required for accurate motion estimation.

Results:

The registration method estimated liver motion with an error of 1 mm (75% percentile over all datasets), which was lower than the interobserver variability of 1.4 mm. The results were only slightly dependent on the degrees of freedom of the deformation model. The registration error increased to 2.8 mm with an eight times lower temporal resolution.

Conclusions:

The authors conclude that the methodology was able to accurately track the motion of the liver in the 4D ultrasound data. The authors believe that the method has potential in interventions on moving abdominal organs such as MR or ultrasound guided focused ultrasound therapy and radiotherapy, pending the method is enabled to run in real‐time. The data and the annotations used for this study are made publicly available for those who would like to test other methods on 4D liver ultrasound data.
Postmortem validation of breast density using dual‐energy mammography
Authors: Molloi Sabee , Ducote Justin L. , Ding Huanjun , Feig Stephen A.
Published: August 2014

Purpose:

Mammographic density has been shown to be an indicator of breast cancer risk and also reduces the sensitivity of screening mammography. Currently, there is no accepted standard for measuring breast density. Dual energy mammography has been proposed as a technique for accurate measurement of breast density. The purpose of this study is to validate its accuracy in postmortem breasts and compare it with other existing techniques.

Methods:

Forty postmortem breasts were imaged using a dual energy mammography system. Glandular and adipose equivalent phantoms of uniform thickness were used to calibrate a dual energy basis decomposition algorithm. Dual energy decomposition was applied after scatter correction to calculate breast density. Breast density was also estimated using radiologist reader assessment, standard histogram thresholding and a fuzzy C‐mean algorithm. Chemical analysis was used as the reference standard to assess the accuracy of different techniques to measure breast composition.

Results:

Breast density measurements using radiologist reader assessment, standard histogram thresholding, fuzzy C‐mean algorithm, and dual energy were in good agreement with the measured fibroglandular volume fraction using chemical analysis. The standard error estimates using radiologist reader assessment, standard histogram thresholding, fuzzy C‐mean, and dual energy were 9.9%, 8.6%, 7.2%, and 4.7%, respectively.

Conclusions:

The results indicate that dual energy mammography can be used to accurately measure breast density. The variability in breast density estimation using dual energy mammography was lower than reader assessment rankings, standard histogram thresholding, and fuzzy C‐mean algorithm. Improved quantification of breast density is expected to further enhance its utility as a risk factor for breast cancer.
Reconstructing transient acoustic radiation from an arbitrary object with a uniform surface velocity distribution
Authors: Wu Sean F.
Published: August 2014

This paper presents the general formulations for reconstructing the transient acoustic field generated by an arbitrary object with a uniformly distributed surface velocity in free space. These formulations are derived from the Kirchhoff–Helmholtz integral theory that correlates the transient acoustic pressure at any field point to those on the source surface. For a class of acoustic radiation problems involving an arbitrarily oscillating object with a uniformly distributed surface velocity, for example, a loudspeaker membrane, the normal surface velocity is frequency dependent but is spatially invariant. Accordingly, the surface acoustic pressure is expressible as the product of the surface velocity and the quantity that can be solved explicitly by using the Kirchhoff–Helmholtz integral equation. This surface acoustic pressure can be correlated to the field acoustic pressure using the Kirchhoff–Helmholtz integral formulation. Consequently, it is possible to use nearfield acoustic holography to reconstruct acoustic quantities in entire three-dimensional space based on a single set of acoustic pressure measurements taken in the near field of the target object. Examples of applying these formulations to reconstructing the transient acoustic pressure fields produced by various arbitrary objects are demonstrated.
Clotrimazole as a Potent Agent for Treating the Oomycete Fish Pathogen Saprolegnia parasitica through Inhibition of Sterol 14α-Demethylase (CYP51)
Authors: Warrilow Andrew G. S. , Hull Claire M. , Rolley Nicola J. , Parker Josie E. , Nes W. David , Smith Stephen N. , Kelly Diane E. , Kelly Steven L.
Published: August 2014

A candidate CYP51 gene encoding sterol 14α-demethylase from the fish oomycete pathogen
Saprolegnia parasitica
(SpCYP51) was identified based on conserved CYP51 residues among CYPs in the genome. It was heterologously expressed in
Escherichia coli
, purified, and characterized. Lanosterol, eburicol, and obtusifoliol bound to purified SpCYP51 with similar binding affinities (

K
s

, 3 to 5 μM). Eight pharmaceutical and six agricultural azole antifungal agents bound tightly to SpCYP51, with posaconazole displaying the highest apparent affinity (

K
d

, ≤3 nM) and prothioconazole-desthio the lowest (
K
d
, ∼51 nM). The efficaciousness of azole antifungals as SpCYP51 inhibitors was confirmed by 50% inhibitory concentrations (IC
50
s) of 0.17 to 2.27 μM using CYP51 reconstitution assays. However, most azole antifungal agents were less effective at inhibiting
S. parasitica
,
Saprolegnia diclina
, and
Saprolegnia ferax
growth. Epoxiconazole, fluconazole, itraconazole, and posaconazole failed to inhibit
Saprolegnia
growth (MIC
100
, >256 μg ml
−1
). The remaining azoles inhibited
Saprolegnia
growth only at elevated concentrations (MIC
100
[the lowest antifungal concentration at which growth remained completely inhibited after 72 h at 20°C], 16 to 64 μg ml
−1
) with the exception of clotrimazole, which was as potent as malachite green (MIC
100
, ∼1 μg ml
−1
). Sterol profiles of azole-treated
Saprolegnia
species confirmed that endogenous CYP51 enzymes were being inhibited with the accumulation of lanosterol in the sterol fraction. The effectiveness of clotrimazole against SpCYP51 activity (IC
50
, ∼1 μM) and the concentration inhibiting the growth of
Saprolegnia
species
in vitro
(MIC
100
, ∼1 to 2 μg ml
−1
) suggest that clotrimazole could be used against
Saprolegnia
infections, including as a preventative measure by pretreatment of fish eggs, and for freshwater-farmed fish as well as in leisure activities.
Punicalagin Inhibits Salmonella Virulence Factors and Has Anti-Quorum-Sensing Potential
Authors: Li Guanghui , Yan Chunhong , Xu Yunfeng , Feng Yuqing , Wu Qian , Lv Xiaoying , Yang Baowei , Wang Xin , Xia Xiaodong
Published: August 2014

Punicalagin, an essential component of pomegranate rind, has been demonstrated to possess antimicrobial activity against several food-borne pathogens, but its activity on the virulence of pathogens and its anti-quorum-sensing (anti-QS) potential have been rarely reported. This study investigated the efficacy of subinhibitory concentrations of punicalagin on
Salmonella
virulence factors and QS systems. A broth microdilution method was used to determine the MICs of punicalagin for 10
Salmonella
strains. Motility assay and quantitative reverse transcription (RT)-PCR were performed to evaluate the effects of punicalagin on the virulence attributes and QS-related genes of
Salmonella
. The MICs of punicalagin for several
Salmonella
strains ranged from 250 to 1,000 μg/ml. Motility assays showed that punicalagin, at 1/16× MIC and 1/32× MIC, significantly decreased bacterial swimming and swarming motility, which corresponded to downregulation of the motility-related genes (
fliA
,
fliY
,
fljB
,
flhC
, and
fimD
) in RT-PCR assays. RT-PCR also revealed that punicalagin downregulated the expression of most of the selected genes involved in
Salmonella
virulence. Moreover, a QS inhibition assay indicated that punicalagin dose dependently inhibited the production of violacein by
Chromobacterium violaceum
and repressed the expression of QS-related genes (
sdiA
and
srgE
) in
Salmonella
. In addition, punicalagin significantly reduced
Salmonella
invasion of colonic cells (
P
< 0.01) with no impact on adhesion. These findings suggest that punicalagin has the potential to be developed as an alternative or supplemental agent for prevention of
Salmonella
infection.
Efficient PCR-Based Amplification of Diverse Alcohol Dehydrogenase Genes from Metagenomes for Improving Biocatalysis: Screening of Gene-Specific Amplicons from Metagenomes
Authors: Itoh Nobuya , Kariya Satomi , Kurokawa Junji
Published: August 2014

Screening of gene-specific amplicons from metagenomes (S-GAM) has tremendous biotechnological potential. We used this approach to isolate alcohol dehydrogenase (
adh
) genes from metagenomes based on the
Leifsonia
species
adh
gene (
lsadh
), the enzyme product of which can produce various chiral alcohols. A primer combination was synthesized by reference to homologs of
lsadh
, and PCR was used to amplify nearly full-length
adh
genes from metagenomic DNAs. All
adh
preparations were fused with
lsadh
at the terminal region and used to construct
Escherichia coli
plasmid libraries. Of the approximately 2,000 colonies obtained, 1,200 clones were identified as
adh
positive (∼60%). Finally, 40
adh
genes,
Hladh
-001 to
Hladh
-040 (for
h
omologous

L
eifsonia

adh

), were identified from 223 clones with high efficiency, which were randomly sequenced from the 1,200 clones. The
Hladh
genes obtained via this approach encoded a wide variety of amino acid sequences (8 to 99%). After screening, the enzymes obtained (HLADH-012 and HLADH-021) were confirmed to be superior to LSADH in some respects for the production of anti-Prelog chiral alcohols.
Genetic Diversity and Evolution of Bradyrhizobium Populations Nodulating Erythrophleum fordii, an Evergreen Tree Indigenous to the Southern Subtropical Region of China
Authors: Yao Yao , Wang Rui , Lu Jun Kun , Sui Xin Hua , Wang En Tao , Chen Wen Xin
Published: August 2014

The nodulation of
Erythrophleum fordii
has been recorded recently, but its microsymbionts have never been studied. To investigate the diversity and biogeography of rhizobia associated with this leguminous evergreen tree, root nodules were collected from the southern subtropical region of China. A total of 166 bacterial isolates were obtained from the nodules and characterized. In a PCR-based restriction fragment length polymorphism (RFLP) analysis of ribosomal intergenic sequences, the isolates were classified into 22 types within the genus
Bradyrhizobium
. Sequence analysis of 16S rRNA, ribosomal intergenic spacer (IGS), and the housekeeping genes
recA
and
glnII
classified the isolates into four groups: the
Bradyrhizobium elkanii
and
Bradyrhizobium pachyrhizi
groups, comprising the dominant symbionts,
Bradyrhizobium yuanmingense
, and an unclassified group comprising the minor symbionts. The
nodC
and
nifH
phylogenetic trees defined five or six lineages among the isolates, which was largely consistent with the definition of genomic species. The phylogenetic results and evolutionary analysis demonstrated that mutation and vertical transmission of genes were the principal processes for the divergent evolution of
Bradyrhizobium
species associated with
E. fordii
, while lateral transfer and recombination of housekeeping and symbiotic genes were rare. The distribution of the dominant rhizobial populations was affected by soil pH and effective phosphorus. This is the first report to characterize
E. fordii
rhizobia.
Engineering of Bacillus subtilis Strains To Allow Rapid Characterization of Heterologous Diguanylate Cyclases and Phosphodiesterases
Authors: Gao Xiaohui , Dong Xiao , Subramanian Sundharraman , Matthews Paige M. , Cooper Caleb A. , Kearns Daniel B. , Dann Charles E.
Published: August 2014

Microbial processes, including biofilm formation, motility, and virulence, are often regulated by changes in the available concentration of cyclic dimeric guanosine monophosphate (c-di-GMP). Generally, high c-di-GMP concentrations are correlated with decreased motility and increased biofilm formation and low c-di-GMP concentrations are correlated with an increase in motility and activation of virulence pathways. The study of c-di-GMP is complicated, however, by the fact that organisms often encode dozens of redundant enzymes that synthesize and hydrolyze c-di-GMP, diguanylate cyclases (DGCs), and c-di-GMP phosphodiesterases (PDEs); thus, determining the contribution of any one particular enzyme is challenging. In an effort to develop a facile system to study c-di-GMP metabolic enzymes, we have engineered a suite of
Bacillus subtilis
strains to assess the effect of individual heterologously expressed proteins on c-di-GMP levels. As a proof of principle, we characterized all 37 known genes encoding predicted DGCs and PDEs in
Clostridium difficile
using parallel readouts of swarming motility and fluorescence from green fluorescent protein (GFP) expressed under the control of a c-di-GMP-controlled riboswitch. We found that 27 of the 37 putative
C. difficile
630 c-di-GMP metabolic enzymes had either active cyclase or phosphodiesterase activity, with agreement between our motility phenotypes and fluorescence-based c-di-GMP reporter. Finally, we show that there appears to be a threshold level of c-di-GMP needed to inhibit motility in
Bacillus subtilis
.
Genomic Insights into the Evolutionary Origin of Xanthomonas axonopodis pv. citri and Its Ecological Relatives
Authors: Midha Samriti , Patil Prabhu B.
Published: August 2014

Xanthomonas axonopodis
pv. citri (Xac) is the causal agent of citrus bacterial canker (CBC) and is a serious problem worldwide. Like CBC, several important diseases in other fruits, such as mango, pomegranate, and grape, are also caused by
Xanthomonas
pathovars that display remarkable specificity toward their hosts. While citrus and mango diseases were documented more than 100 years ago, the pomegranate and grape diseases have been known only since the 1950s and 1970s, respectively. Interestingly, diseases caused by all these pathovars were noted first in India. Our genome-based phylogenetic studies suggest that these diverse pathogens belong to a single species and these pathovars may be just a group of rapidly evolving strains. Furthermore, the recently reported pathovars, such as those infecting grape and pomegranate, form independent clonal lineages, while the citrus and mango pathovars that have been known for a long time form one clonal lineage. Such an understanding of their phylogenomic relationship has further allowed us to understand major and unique variations in the lineages that give rise to these pathovars. Whole-genome sequencing studies including ecological relatives from their putative country of origin has allowed us to understand the evolutionary history of Xac and other pathovars that infect fruits.
Functional and Phylogenetic Divergence of Fungal Adenylate-Forming Reductases
Authors: Kalb Daniel , Lackner Gerald , Hoffmeister Dirk
Published: August 2014

A key step in fungal
l
-lysine biosynthesis is catalyzed by adenylate-forming
l
-α-aminoadipic acid reductases, organized in domains for adenylation, thiolation, and the reduction step. However, the genomes of numerous ascomycetes and basidiomycetes contain an unexpectedly large number of additional genes encoding similar but functionally distinct enzymes. Here, we describe the functional
in vitro
characterization of four reductases which were heterologously produced in
Escherichia coli
. The
Ceriporiopsis subvermispora
serine reductase Nps1 features a terminal ferredoxin-NADP
+
reductase (FNR) domain and thus belongs to a hitherto undescribed class of fungal multidomain enzymes. The second major class is characterized by the canonical terminal short-chain dehydrogenase/reductase domain and represented by
Ceriporiopsis subvermispora
Nps3 as the first biochemically characterized
l
-α-aminoadipic acid reductase of basidiomycete origin.
Aspergillus flavus
l
-tyrosine reductases LnaA and LnbA are members of a distinct phylogenetic clade. Phylogenetic analysis supports the view that fungal adenylate-forming reductases are more diverse than previously recognized and belong to four distinct classes.
Chemolithotrophic Primary Production in a Subglacial Ecosystem
Authors: Boyd Eric S. , Hamilton Trinity L. , Havig Jeff R. , Skidmore Mark L. , Shock Everett L.
Published: August 2014

Glacial comminution of bedrock generates fresh mineral surfaces capable of sustaining chemotrophic microbial communities under the dark conditions that pervade subglacial habitats. Geochemical and isotopic evidence suggests that pyrite oxidation is a dominant weathering process generating protons that drive mineral dissolution in many subglacial systems. Here, we provide evidence correlating pyrite oxidation with chemosynthetic primary productivity and carbonate dissolution in subglacial sediments sampled from Robertson Glacier (RG), Alberta, Canada. Quantification and sequencing of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) transcripts suggest that populations closely affiliated with
Sideroxydans lithotrophicus
, an iron sulfide-oxidizing autotrophic bacterium, are abundant constituents of microbial communities at RG. Microcosm experiments indicate sulfate production during biological assimilation of radiolabeled bicarbonate. Geochemical analyses of subglacial meltwater indicate that increases in sulfate levels are associated with increased calcite and dolomite dissolution. Collectively, these data suggest a role for biological pyrite oxidation in driving primary productivity and mineral dissolution in a subglacial environment and provide the first rate estimate for bicarbonate assimilation in these ecosystems. Evidence for lithotrophic primary production in this contemporary subglacial environment provides a plausible mechanism to explain how subglacial communities could be sustained in near-isolation from the atmosphere during glacial-interglacial cycles.
Improvement of 2,3-Butanediol Yield in Klebsiella pneumoniae by Deletion of the Pyruvate Formate-Lyase Gene
Authors: Jung Moo-Young , Mazumdar Suman , Shin Sang Heum , Yang Kap-Seok , Lee Jinwon , Oh Min-Kyu
Published: August 2014

Klebsiella pneumoniae
is considered a good host strain for the production of 2,3-butanediol, which is a promising platform chemical with various industrial applications. In this study, three genes, including those encoding glucosyltransferase (
wabG
), lactate dehydrogenase (
ldhA
), and pyruvate formate-lyase (
pflB
), were disrupted in
K. pneumoniae
to reduce both its pathogenic characteristics and the production of several by-products. In flask cultivation with minimal medium, the yield of 2,3-butanediol from rationally engineered
K. pneumoniae
(Δ
wabG
Δ
ldhA
Δ
pflB
) reached 0.461 g/g glucose, which was 92.2% of the theoretical maximum, with a significant reduction in by-product formation. However, the growth rate of the
pflB
mutant was slightly reduced compared to that of its parental strain. Comparison with similar mutants of
Escherichia coli
suggested that the growth defect of
pflB
-deficient
K. pneumoniae
was caused by redox imbalance rather than reduced level of intracellular acetyl coenzyme A (acetyl-CoA). From an analysis of the transcriptome, it was confirmed that the removal of
pflB
from
K. pneumoniae
significantly repressed the expression of genes involved in the formate hydrogen lyase (FHL) system.
The Gene Cluster for
para
-Nitrophenol Catabolism Is Responsible for 2-Chloro-4-Nitrophenol Degradation in Burkholderia sp. Strain SJ98
Authors: Min Jun , Zhang Jun-Jie , Zhou Ning-Yi
Published: August 2014

Burkholderia
sp. strain SJ98 (DSM 23195) utilizes 2-chloro-4-nitrophenol (2C4NP) or
para
-nitrophenol (PNP) as a sole source of carbon and energy. Here, by genetic and biochemical analyses, a 2C4NP catabolic pathway different from those of all other 2C4NP utilizers was identified with chloro-1,4-benzoquinone (CBQ) as an intermediate. Reverse transcription-PCR analysis showed that all of the
pnp
genes in the
pnpABA1CDEF
cluster were located in a single operon, which is significantly different from the genetic organization of all other previously reported PNP degradation gene clusters, in which the structural genes were located in three different operons. All of the Pnp proteins were purified to homogeneity as His-tagged proteins. PnpA, a PNP 4-monooxygenase, was found to be able to catalyze the monooxygenation of 2C4NP to CBQ. PnpB, a 1,4-benzoquinone reductase, has the ability to catalyze the reduction of CBQ to chlorohydroquinone. Moreover, PnpB is also able to enhance PnpA activity
in vitro
in the conversion of 2C4NP to CBQ. Genetic analyses indicated that
pnpA
plays an essential role in the degradation of both 2C4NP and PNP by gene knockout and complementation. In addition to being responsible for the lower pathway of PNP catabolism, PnpCD, PnpE, and PnpF were also found to be likely involved in that of 2C4NP catabolism. These results indicated that the catabolism of 2C4NP and that of PNP share the same gene cluster in strain SJ98. These findings fill a gap in our understanding of the microbial degradation of 2C4NP at the molecular and biochemical levels.
Microbial Ecology Dynamics Reveal a Succession in the Core Microbiota Involved in the Ripening of Pasta Filata Caciocavallo Pugliese Cheese
Authors: De Pasquale Ilaria , Di Cagno Raffaella , Buchin Solange , De Angelis Maria , Gobbetti Marco
Published: August 2014

Pyrosequencing of the 16S rRNA targeting RNA, community-level physiological profiles made with Biolog EcoPlates, proteolysis, and volatile component (VOC) analyses were mainly used to characterize the manufacture and ripening of the pasta filata cheese Caciocavallo Pugliese. Plate counts revealed that cheese manufacture affected the microbial ecology. The results agreed with those from culture-independent approaches. As shown by urea-PAGE, reverse-phase high pressure liquid chromatography (RP-HPLC), and free-amino-acid (FAA) analyses, the extent of secondary proteolysis mainly increased after 30 to 45 days of ripening. VOCs and volatile free fatty acids (VFFA) were identified by a purge-and-trap method (PT) and solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS), respectively. Except for aldehydes, the levels of most of VOCs and VFFA mainly increased from 30 to 45 days onwards. As shown through pyrosequencing analysis, raw cows' milk was contaminated by
Firmicutes
(53%),
Proteobacteria
(39%),
Bacteroidetes
(7.8%),
Actinobacteria
(0.06%), and
Fusobacteria
(0.03%), with heterogeneity at the genus level. The primary starter
Streptococcus thermophilus
dominated the curd population. Other genera occurred at low incidence or sporadically. The microbial dynamics reflected on the overall physiological diversity. At 30 days, a microbial succession was clearly highlighted. The relative abundance of
Streptococcus
sp. and especially
St. thermophilus
decreased, while that of
Lactobacillus casei
,
Lactobacillus
sp., and especially
Lactobacillus paracasei
increased consistently. Despite the lower relative abundance compared to
St. thermophilus
, mesophilic lactobacilli were the only organisms positively correlated with the concentration of FAAs, area of hydrophilic peptide peaks, and several VOCs (e.g., alcohols, ketones, esters and all furans). This study showed that a core microbiota was naturally selected during middle ripening, which seemed to be the main factor responsible for cheese ripening.
ApnI, a Transmembrane Protein Responsible for Subtilomycin Immunity, Unveils a Novel Model for Lantibiotic Immunity
Authors: Deng Yun , Li Cong-Zhi , Zhu Yi-Guang , Wang Peng-Xia , Qi Qing-Dong , Fu Jing-Jing , Peng Dong-Hai , Ruan Li-Fang , Sun Ming
Published: August 2014

Subtilomycin was detected from the plant endophytic strain
Bacillus subtilis
BSn5 and was first reported from
B. subtilis
strain MMA7. In this study, a gene cluster that has been proposed to be related to subtilomycin biosynthesis was isolated from the BSn5 genome and was experimentally validated by gene inactivation and heterologous expression. Comparison of the subtilomycin gene cluster with other verified related lantibiotic gene clusters revealed a particular organization of the genes
apnI
and
apnT
downstream of
apnAPBC
, which may be involved in subtilomycin immunity. Through analysis of expression of the
apnI
and/or
apnT
genes in the subtilomycin-sensitive strain CU1065 and inactivation of
apnI
and
apnT
in the producer strain BSn5, we showed that the single gene
apnI
, encoding a putative transmembrane protein, was responsible for subtilomycin immunity. To our knowledge, evidence for lantibiotic immunity that is solely dependent on a transmembrane protein is quite rare. Further bioinformatic analysis revealed the abundant presence of ApnI-like proteins that may be responsible for lantibiotic immunity in
Bacillus
and
Paenibacillus
. We cloned the
paeI
gene, encoding one such ApnI-like protein, into CU1065 and showed that it confers resistance to paenibacillin. However, no cross-resistance was detected between ApnI and PaeI, even though subtilomycin and paenibacillin share similar structures, suggesting that the protection provided by ApnI/ApnI-like proteins involves a specific-sequence recognition mechanism. Peptide release/binding assays indicated that the recombinant
B. subtilis
expressing
apnI
interacted with subtilomycin. Thus, ApnI represents a novel model for lantibiotic immunity that appears to be common.