bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Electron Beam Sterilization on Mechanical Properties of a Porous Polymethylmethacrylate Space Maintenance Device
Authors: Tatara Alexander M. , Shah Sarita R. , Sotoudeh Mohammad , Henslee Allan M. , Wong Mark E. , Ratcliffe Anthony , Kurtis Kasper F. , Mikos Antonios G.
Published: February 2015

Sterilization is a vital component of the manufacturing process for any medical device. However, some sterilization techniques may alter device properties. While it is known that electron beam sterilization can change the mechanical properties of solid polymethylmethacrylate (PMMA) constructs, its effect on porous PMMA has not been explored. Therefore, porous PMMA space maintainer constructs designed for the treatment of craniofacial bone defects were sterilized at dosages of 30 kGy and 40 kGy. Electron beam sterilization was shown to increase the compressive properties of porous PMMA space maintainer devices.
Improving the Performance of a Biogas Powered Dual Fuel Diesel Engine Using Emulsified Rice Bran Biodiesel as Pilot Fuel Through Adjustment of Compression Ratio and Injection Timing
Authors: Bora Bhaskor J. , Saha Ujjwal K.
Published: February 2015

Emulsification is one of the proven techniques to control the pollutants of the diesel engines. The present work attempts to explore the effect of injection timing (IT) of pilot fuel and compression ratio (CR) for an emulsified rice bran biodiesel (RBB)–biogas powered dual fuel diesel engine. A two-phase stable water emulsion of rice bran methyl ester has been prepared by optimizing the factors such as water content (5% and 10%), surfactants (3%), and hydrophilic lipophilic balance (HLB) values (4.3, 5, and 6). The stability of the emulsions is determined on the basis of measurement of mean droplet diameter and stability test. For experimentation, a 3.5 kW single cylinder, direct injection (DI), water cooled, variable CR diesel engine is converted into a biogas run dual fuel diesel engine by connecting a venturi gas mixer at the inlet manifold. A set of combinations comprising CRs of 18, 17.5, and 17, and ITs of 23 deg, 26 deg, 29 deg, and 32 deg before top dead centers (BTDC) at different loading conditions are considered. The investigation demonstrates a maximum brake thermal efficiency (BTE) of 23.62% along with a liquid fuel replacement of 82.22% at pilot fuel IT of 29 deg BTDC and CR of 18. For the same combination, CO and HC emissions are found to be least in all the test cases.
Modeling of microwave diode on diamond like coating Si cathode
Authors: Goncharuk N. M. , Karushkin N. F.
Published: February 2015

Microwave diode structures were applied to the base of a silicon cathode with diamond like coating (DLC) to theoretically investigate resonant and nonresonant electron emission in the framework of small-signal theory. Negative conductance of the diode with resonant electron emission is caused at first by negative conductivity of the cathode due to resonant electron tunneling and at second an electron delay due to the finite time of an electron dwelling in a quantum well of DLC and its transit in the vacuum transit layer. Thus, its origin is the same as for a semiconductor resonant-tunneling diode and a negative conductivity takes place on a falling part of a resonant dependence of a direct emission current on an electric field. Owing to the sufficiently small electron mass of DLC, the duration of resonant electron emission for the studied diodes is considerably less than the optimal transit time, which is determined by the negative emission conductivity value. Therefore, resonant emission frequency of the diodes noticeably exceeds the optimal transit frequency, and, thus, the diode's frequency spectrum of negative conductance consists of a single band. Maximum negative conductance in the spectrum occurs at a few tenths or a few units of terahertz depending on the values of electric-field and parameters of the cathode coating and transit layer. These values determine the character of electron emission (resonant or nonresonant), negative conductivity, and delays in electron emission and transit. Negative conductance of the diodes with nonresonant emission results from combined delays of electron emission and transit. The delay in electron emission is caused a finite duration of electron tunneling under the vacuum potential barrier of electron affinity in DLC. The barrier is situated in the vacuum layer that is adjoined to the DLC and adjacent to the vacuum layer of electron transit. The delay in electron tunneling is comparable with transit delay for the studied diodes due to the great electron mass in vacuum that results in the multiband spectra of the diodes with nonresonant emission.
Mechanical strain mediated carrier scattering and its role in charge and thermal transport in freestanding nanocrystalline aluminum thin films
Authors: Kumar Sandeep , Garcia Davil , Jin Jiezhu , Haque Aman
Published: February 2015

In bulk metals, mechanical strain is known not to influence electrical and thermal transport. However, fundamentally different deformation mechanisms and strain localization at the grain boundaries may influence electron or phonon scattering in nanocrystalline materials. To investigate this hypothesis, the authors developed an experimental approach, where the authors performed thermal and electrical conductivity measurements on 100 nm thick freestanding nanocrystalline aluminum films with average grain size of 50 nm in situ inside a transmission electron microscope (TEM). The authors present experimental evidence of decrease in thermal conductivity and increase in electrical resistivity as a function of uniaxial tensile strain. In-situ TEM observations suggest that grain rotation induced by grain boundary diffusion, and not dislocation-based plasticity, is the dominant deformation mechanism in these thin films. The authors propose that diffusion causes rise in oxygen concentration resulting in increased defects at grain boundaries. Presence of oxygen only at the grain boundaries is confirmed by energy dispersive spectroscopy. Increased defect concentration by mechanical strain at grain boundary causes the change in thermal and charge transport.
FOXP1 potentiates Wnt/β-catenin signaling in diffuse large B cell lymphoma
Authors: Walker Matthew P. , Stopford Charles M. , Cederlund Maria , Fang Fang , Jahn Christopher , Rabinowitz Alex D. , Goldfarb Dennis , Graham David M. , Yan Feng , Deal Allison M. , Fedoriw Yuri , Richards Kristy L. , Davis Ian J. , Weidinger Gilbert , Damania Blossom , Major Michael B.
Published: February 2015

A common type of lymphoma overexpressing the transcription factor FOXP1 could be treated with Wnt/β-catenin inhibitors.
Better together
Authors: Nusinovich Yevgeniya
Published: February 2015

Colorectal cancers that display reduced sensitivity to EGFR inhibition and abundant IGF2 can be effectively treated by dual EGFR and IGF2 blockade.
Defining necessary circadian clock elements
Authors: Ray L. Bryan
Published: February 2015

Control of negative circadian elements in the simple fungus
Neurospora
goes beyond targeted proteolysis.
Differential CYP 2D6 Metabolism Alters Primaquine Pharmacokinetics
Authors: Potter Brittney M. J. , Xie Lisa H. , Vuong Chau , Zhang Jing , Zhang Ping , Duan Dehui , Luong Thu-Lan T. , Bandara Herath H. M. T. , Dhammika Nanayakkara N. P. , Tekwani Babu L. , Walker Larry A. , Nolan Christina K. , Sciotti Richard J. , Zottig Victor E. , Smith Philip L. , Paris Robert M. , Read Lisa T. , Li Qigui , Pybus Brandon S. , Sousa Jason C. , Reichard Gregory A. , Marcsisin Sean R.
Published: February 2015

Primaquine (PQ) metabolism by the cytochrome P450 (CYP) 2D family of enzymes is required for antimalarial activity in both humans (2D6) and mice (2D). Human CYP 2D6 is highly polymorphic, and decreased CYP 2D6 enzyme activity has been linked to decreased PQ antimalarial activity. Despite the importance of CYP 2D metabolism in PQ efficacy, the exact role that these enzymes play in PQ metabolism and pharmacokinetics has not been extensively studied
in vivo
. In this study, a series of PQ pharmacokinetic experiments were conducted in mice with differential CYP 2D metabolism characteristics, including wild-type (WT), CYP 2D knockout (KO), and humanized CYP 2D6 (KO/knock-in [KO/KI]) mice. Plasma and liver pharmacokinetic profiles from a single PQ dose (20 mg/kg of body weight) differed significantly among the strains for PQ and carboxy-PQ. Additionally, due to the suspected role of phenolic metabolites in PQ efficacy, these were probed using reference standards. Levels of phenolic metabolites were highest in mice capable of metabolizing CYP 2D6 substrates (WT and KO/KI 2D6 mice). PQ phenolic metabolites were present in different quantities in the two strains, illustrating species-specific differences in PQ metabolism between the human and mouse enzymes. Taking the data together, this report furthers understanding of PQ pharmacokinetics in the context of differential CYP 2D metabolism and has important implications for PQ administration in humans with different levels of CYP 2D6 enzyme activity.
Colostrum Hexasaccharide, a Novel Staphylococcus aureus Quorum-Sensing Inhibitor
Authors: Srivastava A. , Singh B. N. , Deepak D. , Rawat A. K. S. , Singh B. R.
Published: February 2015

The discovery of quorum-sensing (QS) systems regulating antibiotic resistance and virulence factors (VFs) has afforded a novel opportunity to prevent bacterial pathogenicity. Dietary molecules have been demonstrated to attenuate QS circuits of bacteria. But, to our knowledge, no study exploring the potential of colostrum hexasaccharide (CHS) in regulating QS systems has been published. In this study, we analyzed CHS for inhibiting QS signaling in
Staphylococcus aureus
. We isolated and characterized CHS from mare colostrum by high-performance thin-layer chromatography (HPTLC), reverse-phase high-performance liquid chromatography evaporative light-scattering detection (RP-HPLC-ELSD),
1
H and
13
C nuclear magnetic resonance (NMR), and electrospray ionization mass spectrometry (ESI-MS). Antibiofilm activity of CHS against
S. aureus
and its possible interference with bacterial QS systems were determined. The inhibition and eradication potentials of the biofilms were studied by microscopic analyses and quantified by 96-well-microtiter-plate assays. Also, the ability of CHS to interfere in bacterial QS by degrading acyl-homoserine lactones (AHLs), one of the most studied signal molecules for Gram-negative bacteria, was evaluated. The results revealed that CHS exhibited promising inhibitory activities against QS-regulated secretion of VFs, including spreading ability, hemolysis, protease, and lipase activities, when applied at a rate of 5 mg/ml. The results of biofilm experiments indicated that CHS is a strong inhibitor of biofilm formation and also has the ability to eradicate it. The potential of CHS to interfere with bacterial QS systems was also examined by degradation of AHLs. Furthermore, it was documented that CHS decreased antibiotic resistance in
S. aureus
. The results thus give a lead that mare colostrum can be a promising source for isolating a next-generation antibacterial.
Two Mechanisms of Killing of Pseudomonas aeruginosa by Tobramycin Assessed at Multiple Inocula via Mechanism-Based Modeling
Authors: Bulitta Jürgen B. , Ly Neang S. , Landersdorfer Cornelia B. , Wanigaratne Nicholin A. , Velkov Tony , Yadav Rajbharan , Oliver Antonio , Martin Lisandra , Shin Beom Soo , Forrest Alan , Tsuji Brian T.
Published: February 2015

Bacterial resistance is among the most serious threats to human health globally, and many bacterial isolates have emerged that are resistant to all antibiotics in monotherapy. Aminoglycosides are often used in combination therapies against severe infections by multidrug-resistant bacteria. However, models quantifying different antibacterial effects of aminoglycosides are lacking. While the mode of aminoglycoside action on protein synthesis has often been studied, their disruptive action on the outer membrane of Gram-negative bacteria remains poorly characterized. Here, we developed a novel quantitative model for these two mechanisms of aminoglycoside action, phenotypic tolerance at high bacterial densities, and adaptive bacterial resistance in response to an aminoglycoside (tobramycin) against three
Pseudomonas aeruginosa
strains. At low-intermediate tobramycin concentrations (<4 mg/liter), bacterial killing due to the effect on protein synthesis was most important, whereas disruption of the outer membrane was the predominant killing mechanism at higher tobramycin concentrations (≥8 mg/liter). The extent of killing was comparable across all inocula; however, the rate of bacterial killing and growth was substantially lower at the 10
8.9
CFU/ml inoculum than that at the lower inocula. At 1 to 4 mg/liter tobramycin for strain PAO1-RH, there was a 0.5- to 6-h lag time of killing that was modeled via the time to synthesize hypothetical lethal protein(s). Disruption of the outer bacterial membrane by tobramycin may be critical to enhance the target site penetration of antibiotics used in synergistic combinations with aminoglycosides and thereby combat multidrug-resistant bacteria. The two mechanisms of aminoglycoside action and the new quantitative model hold great promise to rationally design novel, synergistic aminoglycoside combination dosage regimens.
Sustained Release of a Novel Anti-Quorum-Sensing Agent against Oral Fungal Biofilms
Authors: Feldman Mark , Shenderovich Julia , Al-Quntar Abed Al Aziz , Friedman Michael , Steinberg Doron
Published: February 2015

Thiazolidinedione-8 (S-8) has recently been identified as a potential anti-quorum-sensing/antibiofilm agent against bacteria and fungi. Based on these results, we investigated the possibility of incorporating S-8 in a sustained-release membrane (SRM) to increase its pharmaceutical potential against
Candida albicans
biofilm. We demonstrated that SRM containing S-8 inhibits fungal biofilm formation in a time-dependent manner for 72 h, due to prolonged release of S-8. Moreover, the SRM effectively delivered the agent in its active form to locations outside the membrane reservoir. In addition, eradication of mature biofilm by the SRM containing S-8 was also significant. Of note, S-8-containing SRM affected the characteristics of mature
C. albicans
biofilm, such as thickness, exopolysaccharide (EPS) production, and morphogenesis of fungal cells. The concept of using an antibiofilm agent with no antifungal activity incorporated into a sustained-release delivery system is new in medicine and dentistry. This concept of an SRM containing a quorum-sensing quencher with an antibiofilm effect could pave the way for combating oral fungal infectious diseases.
Tuberculous Spondylitis in Russia and Prominent Role of Multidrug-Resistant Clone Mycobacterium tuberculosis Beijing B0/W148
Authors: Vyazovaya Anna , Mokrousov Igor , Solovieva Natalia , Mushkin Alexander , Manicheva Olga , Vishnevsky Boris , Zhuravlev Viacheslav , Narvskaya Olga
Published: February 2015

Extrapulmonary and, in particular, spinal tuberculosis (TB) constitutes a minor but significant part of the total TB incidence. In spite of this, almost no studies on the genetic diversity and drug resistance of
Mycobacterium tuberculosis
isolates from spinal TB patients have been published to date. Here, we report results of the first Russian and globally largest molecular study of
M. tuberculosis
isolates recovered from patients with tuberculous spondylitis (TBS). The majority of 107 isolates were assigned to the Beijing genotype (
n
= 80); the other main families were T (
n
= 11), Ural (
n
= 7), and LAM (
n
= 4). Multidrug resistance (MDR) was more frequently found among Beijing (90.5%) and, intriguingly, Ural (71.4%) isolates than other genotypes (5%;
P
< 0.001). The extremely drug-resistant (XDR) phenotype was exclusively found in the Beijing isolates (
n
= 7). A notable prevalence of the
rpoB531
and
katG315
mutations in Beijing strains that were similarly high in both TBS (this study) and published pulmonary TB (PTB) samples from Russia shows that TBS and PTB Beijing strains follow the same paradigm of acquisition of rifampin (RIF) and isoniazid (INH) resistance. The 24-locus mycobacterial interspersed repetitive unit–variable-number tandem-repeat (MIRU-VNTR) subtyping of 80 Beijing isolates further discriminated them into 24 types (Hunter Gaston index [HGI] = 0.83); types 100-32 and 94-32 represented the largest groups. A genotype of Russian successful clone B0/W148 was identified in 30 of 80 Beijing isolates. In conclusion, this study highlighted a crucial impact of the Beijing genotype and the especially prominent role of its MDR-associated successful clone B0/W148 cluster in the development of spinal MDR-TB in Russian patients.
Rapid Drug Tolerance and Dramatic Sterilizing Effect of Moxifloxacin Monotherapy in a Novel Hollow-Fiber Model of Intracellular Mycobacterium kansasii Disease
Authors: Srivastava Shashikant , Pasipanodya Jotam , Sherman Carleton M. , Meek Claudia , Leff Richard , Gumbo Tawanda
Published: February 2015

Mycobacterium kansasii
is the second most common mycobacterial cause of lung disease. Standard treatment consists of rifampin, isoniazid, and ethambutol for at least 12 months after negative sputum. Thus, shorter-duration therapies are needed. Moxifloxacin has good MICs for
M. kansasii
. However, good preclinical models to identify optimal doses currently are lacking. We developed a novel hollow fiber system model of intracellular
M. kansasii
infection. We indexed the efficacy of the standard combination regimen, which was a kill rate of −0.08 ± 0.05 log
10
CFU/ml/day (
r
2
= 0.99). We next performed moxifloxacin dose-effect and dose-scheduling studies at a half-life of 11.1 ± 6.47 h. Some systems also were treated with the efflux pump inhibitor reserpine. The highest moxifloxacin exposure, as well as lower exposures plus reserpine, sterilized the cultures by day 7. This suggests that efflux pump-mediated tolerance at low ratios of the area under the concentration-time curve from 0 to 24 h (AUC
0
–
24
) to MICs is an early bacterial defense mechanism but is overcome by higher exposures. The highest rate of moxifloxacin monotherapy sterilization was −0.82 ± 0.15 log
10
CFU/ml/day (
r
2
= 0.97). The moxifloxacin exposure associated with 80% of maximal kill (EC
80
) was an AUC
0–24
/MIC of 317 (the non-protein-bound moxifloxacin AUC
0–24
/MIC was 158.5). We performed Monte Carlo simulations of 10,000 patients in order to identify the moxifloxacin dose that would achieve or exceed the EC
80
. The simulations revealed an optimal moxifloxacin dose of 800 mg a day. The MIC susceptibility breakpoint at this dose was 0.25 mg/liter. Thus, moxifloxacin, at high enough doses, is suitable to study in patients for the potential to add rapid sterilization to the standard regimen.
Update on Linezolid
In Vitro
Activity through the Zyvox Annual Appraisal of Potency and Spectrum Program, 2013
Authors: Mendes Rodrigo E. , Hogan Patricia A. , Streit Jennifer M. , Jones Ronald N. , Flamm Robert K.
Published: February 2015

Linezolid showed MIC
50
s and MIC
90
s of 1 μg/ml (for both) against
Staphylococcus aureus
. Two
S. aureus
strains exhibited higher MICs (4 to 8 μg/ml) caused by
cfr
and/or target site mutations, including the first detection of
cfr
in Poland. Linezolid (MIC
50
and MIC
90
, 0.5 and 1 μg/ml) had potent MICs against coagulase-negative staphylococci (CoNS). Four CoNS had MICs of 16 to 128 μg/ml due to alterations in 23S rRNA and/or L3/L4. Linezolid inhibited all enterococci and streptococci at ≤2 μg/ml, except for one
Enterococcus faecium
strain (MIC, 8 μg/ml; G2576T [
Escherichia coli
numbering] mutation).
Efficacy of Postexposure Therapy against Glanders in Mice
Authors: Waag David M.
Published: February 2015

Burkholderia mallei
, the causative agent of glanders, is a CDC Tier 1 Select Agent for which there is no preventive vaccine and antibiotic therapy is difficult. In this study, we show that a combination of vaccination using killed cellular vaccine and therapy using moxifloxacin, azithromycin, or sulfamethoxazole-trimethoprim can protect BALB/c mice from lethal infection even when given 5 days after infectious challenge. Vaccination only, or antibiotic therapy only, was not efficacious. Although antibiotics evaluated experimentally can protect when given before or 1 day after challenge, this time course is not realistic in the cases of natural infection or biological attack, when the patient seeks treatment after symptoms develop or after a biological attack has been confirmed and the agent has been identified. Antibiotics can be efficacious after a prolonged interval between exposure and treatment, but only if the animals were previously vaccinated.
Decreasing Global Transcript Levels over Time Suggest that Phytoplasma Cells Enter Stationary Phase during Plant and Insect Colonization
Authors: Pacifico D. , Galetto L. , Rashidi M. , Abbà S. , Palmano S. , Firrao G. , Bosco D. , Marzachì C.
Published: January 2015

To highlight different transcriptional behaviors of the phytoplasma in the plant and animal host, expression of 14 genes of “CandidatusPhytoplasma asteris,” chrysanthemum yellows strain, was investigated at different times following the infection of a plant host (Arabidopsis thaliana) and two insect vector species (Macrosteles quadripunctulatusandEuscelidius variegatus). Target genes were selected among those encoding antigenic membrane proteins, membrane transporters, secreted proteins, and general enzymes. Transcripts were detected for all analyzed genes in the three hosts; in particular, those encoding the antigenic membrane protein Amp, elements of the mechanosensitive channel, and two of the four secreted proteins (SAP54 and TENGU) were highly accumulated, suggesting that they play important roles in phytoplasma physiology during the infection cycle. Most transcripts were present at higher abundance in the plant host than in the insect hosts. Generally, transcript levels of the selected genes decreased significantly during infection ofA. thalianaandM. quadripunctulatusbut were more constant inE. variegatus. Such decreases may be explained by the fact that only a fraction of the phytoplasma population was transcribing, while the remaining part was aging to a stationary phase. This strategy might improve long-term survival, thereby increasing the likelihood that the pathogen may be acquired by a vector and/or inoculated to a healthy plant.
Nitric Oxide Treatment for the Control of Reverse Osmosis Membrane Biofouling
Authors: Barnes Robert J. , Low Jiun Hui , Bandi Ratnaharika R. , Tay Martin , Chua Felicia , Aung Theingi , Fane Anthony G. , Kjelleberg Staffan , Rice Scott A.
Published: January 2015

Biofouling remains a key challenge for membrane-based water treatment systems. This study investigated the dispersal potential of the nitric oxide (NO) donor compound, PROLI NONOate, on single- and mixed-species biofilms formed by bacteria isolated from industrial membrane bioreactor and reverse osmosis (RO) membranes. The potential of PROLI NONOate to control RO membrane biofouling was also examined. Confocal microscopy revealed that PROLI NONOate exposure induced biofilm dispersal in all but two of the bacteria tested and successfully dispersed mixed-species biofilms. The addition of 40 μM PROLI NONOate at 24-h intervals to a laboratory-scale RO system led to a 92% reduction in the rate of biofouling (pressure rise over a given period) by a bacterial community cultured from an industrial RO membrane. Confocal microscopy and extracellular polymeric substances (EPS) extraction revealed that PROLI NONOate treatment led to a 48% reduction in polysaccharides, a 66% reduction in proteins, and a 29% reduction in microbial cells compared to the untreated control. A reduction in biofilm surface coverage (59% compared to 98%, treated compared to control) and average thickness (20 μm compared to 26 μm, treated compared to control) was also observed. The addition of PROLI NONOate led to a 22% increase in the time required for the RO module to reach its maximum transmembrane pressure (TMP), further indicating that NO treatment delayed fouling. Pyrosequencing analysis revealed that the NO treatment did not significantly alter the microbial community composition of the membrane biofilm. These results present strong evidence for the application of PROLI NONOate for prevention of RO biofouling.
Multifactorial Effects of Ambient Temperature, Precipitation, Farm Management, and Environmental Factors Determine the Level of Generic Escherichia coli Contamination on Preharvested Spinach
Authors: Park Sangshin , Navratil Sarah , Gregory Ashley , Bauer Arin , Srinath Indumathi , Szonyi Barbara , Nightingale Kendra , Anciso Juan , Jun Mikyoung , Han Daikwon , Lawhon Sara , Ivanek Renata
Published: January 2015

A repeated cross-sectional study was conducted to identify farm management, environment, weather, and landscape factors that predict the count of generic
Escherichia coli
on spinach at the preharvest level.
E. coli
was enumerated for 955 spinach samples collected on 12 farms in Texas and Colorado between 2010 and 2012. Farm management and environmental characteristics were surveyed using a questionnaire. Weather and landscape data were obtained from National Resources Information databases. A two-part mixed-effect negative binomial hurdle model, consisting of a logistic and zero-truncated negative binomial part with farm and date as random effects, was used to identify factors affecting
E. coli
counts on spinach. Results indicated that the odds of a contamination event (non-zero versus zero counts) vary by state (odds ratio [OR] = 108.1). Odds of contamination decreased with implementation of hygiene practices (OR = 0.06) and increased with an increasing average precipitation amount (mm) in the past 29 days (OR = 3.5) and the application of manure (OR = 52.2). On contaminated spinach,
E. coli
counts increased with the average precipitation amount over the past 29 days. The relationship between
E. coli
count and the average maximum daily temperature over the 9 days prior to sampling followed a quadratic function with the highest bacterial count at around 24°C. These findings indicate that the odds of a contamination event in spinach are determined by farm management, environment, and weather factors. However, once the contamination event has occurred, the count of
E. coli
on spinach is determined by weather only.
Microbial Safety and Sanitary Quality of Strawberry Primary Production in Belgium: Risk Factors for Salmonella and Shiga Toxin-Producing Escherichia coli Contamination
Authors: Delbeke Stefanie , Ceuppens Siele , Hessel Claudia Titze , Castro Irene , Jacxsens Liesbeth , De Zutter Lieven , Uyttendaele Mieke
Published: January 2015

Strawberries are an important fruit in Belgium in both production and consumption, but little information is available about the presence of
Salmonella
and Shiga toxin-producing
Escherichia coli
(STEC) in these berries, the risk factors in agricultural production, and possible specific mitigation options. In 2012, a survey was undertaken of three soil and three soilless cultivation systems in Belgium. No
Salmonella
spp. were isolated. No STEC was detected in the strawberry samples (0 of 72), but STEC was detected by PCR in 11 of 78 irrigation water and 2 of 24 substrate samples. Culture isolates were obtained for 2 of 11 PCR-positive irrigation water samples and 2 of 2 substrate samples. Multivariable logistic regression analysis revealed elevated generic
E. coli
numbers (the odds ratio [OR] for a 1 log increase being 4.6) as the most important risk factor for STEC, together with the berry-picking season (elevated risk in summer). The presence of generic
E. coli
in the irrigation water (≥1 CFU per 100 ml) was mainly influenced by the type of irrigation water (collected rainfall water stored in ponds was more often contaminated than groundwater pumped from boreholes [OR = 5.8]) and the lack of prior treatment (untreated water versus water subjected to sand filtration prior to use [OR = 19.2]). The follow-up study in 2013 at one of the producer locations indicated cattle to be the most likely source of STEC contamination of the irrigation water.
Novel Functions of (p)ppGpp and Cyclic di-GMP in Mycobacterial Physiology Revealed by Phenotype Microarray Analysis of Wild-Type and Isogenic Strains of Mycobacterium smegmatis
Authors: Gupta Kuldeepkumar Ramnaresh , Kasetty Sanjay , Chatterji Dipankar
Published: January 2015

The bacterial second messengers (p)ppGpp and bis-(3′-5′)-cyclic dimeric GMP (c-di-GMP) regulate important functions, such as transcription, virulence, biofilm formation, and quorum sensing. In mycobacteria, they regulate long-term survival during starvation, pathogenicity, and dormancy. Recently, a
Pseudomonas aeruginosa
strain lacking (p)ppGpp was shown to be sensitive to multiple classes of antibiotics and defective in biofilm formation. We were interested to find out whether
Mycobacterium smegmatis
strains lacking the gene for either (p)ppGpp synthesis (Δ
rel
Msm
) or c-di-GMP synthesis (Δ
dcpA
) would display similar phenotypes. We used phenotype microarray technology to compare the growth of the wild-type and the knockout strains in the presence of several antibiotics. Surprisingly, the Δ
rel
Msm
and Δ
dcpA
strains showed enhanced survival in the presence of many antibiotics, but they were defective in biofilm formation. These strains also displayed altered surface properties, like impaired sliding motility, rough colony morphology, and increased aggregation in liquid cultures. Biofilm formation and surface properties are associated with the presence of glycopeptidolipids (GPLs) in the cell walls of
M. smegmatis
. Thin-layer chromatography analysis of various cell wall fractions revealed that the levels of GPLs and polar lipids were reduced in the knockout strains. As a result, the cell walls of the knockout strains were significantly more hydrophobic than those of the wild type and the complemented strains. We hypothesize that reduced levels of GPLs and polar lipids may contribute to the antibiotic resistance shown by the knockout strains. Altogether, our data suggest that (p)ppGpp and c-di-GMP may be involved in the metabolism of glycopeptidolipids and polar lipids in
M. smegmatis
.
Evolutionary Expansion of the Amidohydrolase Superfamily in Bacteria in Response to the Synthetic Compounds Molinate and Diuron
Authors: Sugrue Elena , Fraser Nicholas J. , Hopkins Davis H. , Carr Paul D. , Khurana Jeevan L. , Oakeshott John G. , Scott Colin , Jackson Colin J.
Published: January 2015

The amidohydrolase superfamily has remarkable functional diversity, with considerable structural and functional annotation of known sequences. In microbes, the recent evolution of several members of this family to catalyze the breakdown of environmental xenobiotics is not well understood. An evolutionary transition from binuclear to mononuclear metal ion coordination at the active sites of these enzymes could produce large functional changes such as those observed in nature, but there are few clear examples available to support this hypothesis. To investigate the role of binuclear-mononuclear active-site transitions in the evolution of new function in this superfamily, we have characterized two recently evolved enzymes that catalyze the hydrolysis of the synthetic herbicides molinate (MolA) and phenylurea (PuhB). In this work, the crystal structures, mutagenesis, metal ion analysis, and enzyme kinetics of both MolA and PuhB establish that these enzymes utilize a mononuclear active site. However, bioinformatics and structural comparisons reveal that the closest putative ancestor of these enzymes had a binuclear active site, indicating that a binuclear-mononuclear transition has occurred. These proteins may represent examples of evolution modifying the characteristics of existing catalysts to satisfy new requirements, specifically, metal ion rearrangement leading to large leaps in activity that would not otherwise be possible.
Multigene Editing in the Escherichia coli Genome via the CRISPR-Cas9 System
Authors: Jiang Yu , Chen Biao , Duan Chunlan , Sun Bingbing , Yang Junjie , Yang Sheng
Published: January 2015

An efficient genome-scale editing tool is required for construction of industrially useful microbes. We describe a targeted, continual multigene editing strategy that was applied to the
Escherichia coli
genome by using the
Streptococcus pyogenes
type II CRISPR-Cas9 system to realize a variety of precise genome modifications, including gene deletion and insertion, with a highest efficiency of 100%, which was able to achieve simultaneous multigene editing of up to three targets. The system also demonstrated successful targeted chromosomal deletions in
Tatumella citrea
, another species of the
Enterobacteriaceae
, with highest efficiency of 100%.
The Putative Mevalonate Diphosphate Decarboxylase from Picrophilus torridus Is in Reality a Mevalonate-3-Kinase with High Potential for Bioproduction of Isobutene
Authors: Rossoni Luca , Hall Stephen J. , Eastham Graham , Licence Peter , Stephens Gill
Published: January 2015

Mevalonate diphosphate decarboxylase (MVD) is an ATP-dependent enzyme that catalyzes the phosphorylation/decarboxylation of (
R
)-mevalonate-5-diphosphate to isopentenyl pyrophosphate in the mevalonate (MVA) pathway. MVD is a key enzyme in engineered metabolic pathways for bioproduction of isobutene, since it catalyzes the conversion of 3-hydroxyisovalerate (3-HIV) to isobutene, an important platform chemical. The putative homologue from
Picrophilus torridus
has been identified as a highly efficient variant in a number of patents, but its detailed characterization has not been reported. In this study, we have successfully purified and characterized the putative MVD from
P. torridus.
We discovered that it is not a decarboxylase
per se
but an ATP-dependent enzyme, mevalonate-3-kinase (M3K), which catalyzes the phosphorylation of MVA to mevalonate-3-phosphate. The enzyme's potential in isobutene formation is due to the conversion of 3-HIV to an unstable 3-phosphate intermediate that undergoes consequent spontaneous decarboxylation to form isobutene. Isobutene production rates were as high as 507 pmol min
−1
g cells
−1
using
Escherichia coli
cells expressing the enzyme and 2,880 pmol min
−1
mg protein
−1
with the purified histidine-tagged enzyme, significantly higher than reported previously. M3K is a key enzyme of the novel MVA pathway discovered very recently in
Thermoplasma acidophilum
. We suggest that
P. torridus
metabolizes MVA by the same pathway.
Type III Secretion System and Virulence Markers Highlight Similarities and Differences between Human- and Plant-Associated Pseudomonads Related to Pseudomonas fluorescens and P. putida
Authors: Mazurier Sylvie , Merieau Annabelle , Bergeau Dorian , Decoin Victorien , Sperandio Daniel , Crépin Alexandre , Barbey Corinne , Jeannot Katy , Vicré-Gibouin Maïté , Plésiat Patrick , Lemanceau Philippe , Latour Xavier
Published: January 2015

Pseudomonas fluorescensis commonly considered a saprophytic rhizobacterium devoid of pathogenic potential. Nevertheless, the recurrent isolation of strains from clinical human cases could indicate the emergence of novel strains originating from the rhizosphere reservoir, which could be particularly resistant to the immune system and clinical treatment. The importance of type three secretion systems (T3SSs) in the relatedPseudomonas aeruginosanosocomial species and the occurrence of this secretion system in plant-associatedP. fluorescensraise the question of whether clinical isolates may also harbor T3SSs. In this study, isolates associated with clinical infections and identified in hospitals as belonging toP. fluorescenswere compared with fluorescent pseudomonads harboring T3SSs isolated from plants. Bacterial isolates were tested for (i) their genetic relationships based on their 16S rRNA phylogeny, (ii) the presence of T3SS genes by PCR, and (iii) their infectious potential on animals and plants under environmental or physiological temperature conditions. Two groups of bacteria were delineated among the clinical isolates. The first group encompassed thermotolerant (41°C) isolates from patients suffering from blood infections; these isolates were finally found to not belong toP. fluorescensbut were closely related and harbored highly conserved T3SS genes belonging to the Ysc-T3SS family, like the T3SSs fromP. aeruginosa. The second group encompassed isolates from patients suffering from cystic fibrosis; these isolates belonged toP. fluorescensand harbored T3SS genes belonging to the Hrp1-T3SS family found commonly in plant-associatedP. fluorescens.
The Flagellar Regulator TviA Reduces Pyroptosis by Salmonella enterica Serovar Typhi
Authors: Winter Sebastian E. , Winter Maria G. , Atluri Vidya , Poon Victor , Romão Everton L. , Tsolis Renée M. , Bäumler Andreas J.
Published: February 2015

To discern virulent from innocuous microbes, the innate immune system senses events associated with bacterial access to immunoprivileged sites such as the host cell cytosol. One such pathway is triggered by the cytosolic delivery of flagellin, the major subunit of the flagellum, by bacterial secretion systems. This leads to inflammasome activation and subsequent proinflammatory cell death (pyroptosis) of the infected phagocyte. In this study, we demonstrate that the causative agent of typhoid fever,
Salmonella enterica
serovar Typhi, can partially subvert this critical innate immune recognition event. The transcriptional regulator TviA, which is absent from
Salmonella
serovars associated with human gastroenteritis, repressed the expression of flagellin during infection of human macrophage-like (THP-1) cells. This mechanism allowed
S
. Typhi to dampen inflammasome activation, leading to reduced interleukin-1β (IL-1β) secretion and diminished cell death. Likewise, the introduction of the
tviA
gene in nontyphoidal
Salmonella enterica
serovar Typhimurium reduced flagellin-induced pyroptosis. These data suggest that gene regulation of virulence factors enables
S
. Typhi to evade innate immune recognition by concealing a pathogen-induced process from being sensed by the inflammasome.
A Novel Pyrroloquinoline Quinone-Dependent 2-Keto-
d
-Glucose Dehydrogenase from Pseudomonas aureofaciens
Authors: Umezawa Kiwamu , Takeda Kouta , Ishida Takuya , Sunagawa Naoki , Makabe Akiko , Isobe Kazuo , Koba Keisuke , Ohno Hiroyuki , Samejima Masahiro , Nakamura Nobuhumi , Igarashi Kiyohiko , Yoshida Makoto
Published: February 2015

A gene encoding an enzyme similar to a pyrroloquinoline quinone (PQQ)-dependent sugar dehydrogenase from filamentous fungi, which belongs to new auxiliary activities (AA) family 12 in the CAZy database, was cloned from
Pseudomonas aureofaciens
. The deduced amino acid sequence of the cloned enzyme showed only low homology to previously characterized PQQ-dependent enzymes, and multiple-sequence alignment analysis showed that the enzyme lacks one of the three conserved arginine residues that function as PQQ-binding residues in known PQQ-dependent enzymes. The recombinant enzyme was heterologously expressed in an
Escherichia coli
expression system for further characterization. The UV-visible (UV-Vis) absorption spectrum of the oxidized form of the holoenzyme, prepared by incubating the apoenzyme with PQQ and CaCl
2
, revealed a broad peak at approximately 350 nm, indicating that the enzyme binds PQQ. With the addition of 2-keto-
d
-glucose (2KG) to the holoenzyme solution, a sharp peak appeared at 331 nm, attributed to the reduction of PQQ bound to the enzyme, whereas no effect was observed upon 2KG addition to authentic PQQ. Enzymatic assay showed that the recombinant enzyme specifically reacted with 2KG in the presence of an appropriate electron acceptor, such as 2,6-dichlorophenol indophenol, when PQQ and CaCl
2
were added.
1
H nuclear magnetic resonance (
1
H-NMR) analysis of reaction products revealed 2-keto-
d
-gluconic acid (2KGA) as the main product, clearly indicating that the recombinant enzyme oxidizes the C-1 position of 2KG. Therefore, the enzyme was identified as a PQQ-dependent 2KG dehydrogenase (
Pa
2KGDH). Considering the high substrate specificity, the physiological function of
Pa
2KGDH may be for production of 2KGA.
SepD/SepL-Dependent Secretion Signals of the Type III Secretion System Translocator Proteins in Enteropathogenic Escherichia coli
Authors: Deng Wanyin , Yu Hong B. , Li Yuling , Finlay B. Brett
Published: February 2015

The type III protein secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE) is essential for the pathogenesis of attaching/effacing bacterial pathogens, including enteropathogenicEscherichia coli(EPEC), enterohemorrhagicE. coli(EHEC), andCitrobacter rodentium. These pathogens use the T3SS to sequentially secrete three categories of proteins: the T3SS needle and inner rod protein components; the EspA, EspB, and EspD translocators; and many LEE- and non-LEE-encoded effectors. SepD and SepL are essential for translocator secretion, and mutations in either lead to hypersecretion of effectors. However, how SepD and SepL control translocator secretion and secretion hierarchy between translocators and effectors is poorly understood. In this report, we show that the secreted T3SS components, the translocators, and both LEE- and non-LEE-encoded effectors all carry N-terminal type III secretion and translocation signals. These signals all behave like those of the effectors and are sufficient for mediating type III secretion and translocation by wild-type EPEC and hypersecretion by thesepDandsepLmutants. Our results extended previous observations and suggest that the secretion hierarchy of the different substrates is determined by a signal other than the N-terminal secretion signal. We identified a domain located immediately downstream of the N-terminal secretion signal in the translocator EspB that is required for SepD/SepL-dependent secretion. We further demonstrated that this EspB domain confers SepD/SepL- and CesAB-dependent secretion on the secretion signal of effector EspZ. Our results thus suggest that SepD and SepL control and regulate secretion hierarchy between translocators and effectors by recognizing translocator-specific export signals.

IMPORTANCEMany bacterial pathogens use a syringe-like protein secretion apparatus, termed the type III protein secretion system (T3SS), to secrete and inject numerous proteins directly into the host cells to cause disease. The secreted proteins perform different functions at various stages during infection and are classified into three substrate categories (T3SS components, translocators, and effectors). They all contain secretion signals at their N termini, but how their secretion hierarchy is determined is poorly understood. Here, we show that the N-terminal secretion signals from different substrate categories all behave the same and do not confer substrate specificity. We further characterize the secretion signals of the translocators and identify a translocator-specific signal, demonstrating that substrate-specific secretion signals are required in regulating T3SS substrate hierarchy.
Characterization of Novel Acyl Coenzyme A Dehydrogenases Involved in Bacterial Steroid Degradation
Authors: Ruprecht Amanda , Maddox Jaymie , Stirling Alexander J. , Visaggio Nicole , Seah Stephen Y. K.
Published: February 2015

The acyl coenzyme A (acyl-CoA) dehydrogenases (ACADs) FadE34 and CasC, encoded by the cholesterol and cholate gene clusters of
Mycobacterium tuberculosis
and
Rhodococcus jostii
RHA1, respectively, were successfully purified. Both enzymes differ from previously characterized ACADs in that they contain two fused acyl-CoA dehydrogenase domains in a single polypeptide. Site-specific mutagenesis showed that only the C-terminal ACAD domain contains the catalytic glutamate base required for enzyme activity, while the N-terminal ACAD domain contains an arginine required for ionic interactions with the pyrophosphate of the flavin adenine dinucleotide (FAD) cofactor. Therefore, the two ACAD domains must associate to form a single active site. FadE34 and CasC were not active toward the 3-carbon side chain steroid metabolite 3-oxo-23,24-bisnorchol-4-en-22-oyl-CoA (4BNC-CoA) but were active toward steroid CoA esters containing 5-carbon side chains. CasC has similar specificity constants for cholyl-CoA, deoxycholyl-CoA, and 3β-hydroxy-5-cholen-24-oyl-CoA, while FadE34 has a preference for the last compound, which has a ring structure similar to that of cholesterol metabolites. Knockout of the
casC
gene in
R. jostii
RHA1 resulted in a reduced growth on cholate as a sole carbon source and accumulation of a 5-carbon side chain cholate metabolite. FadE34 and CasC represent unique members of ACADs with primary structures and substrate specificities that are distinct from those of previously characterized ACADs.

IMPORTANCE
We report here the identification and characterization of acyl-CoA dehydrogenases (ACADs) involved in the metabolism of 5-carbon side chains of cholesterol and cholate. The two homologous enzymes FadE34 and CasC, from
M. tuberculosis
and
Rhodococcus jostii
RHA1, respectively, contain two ACAD domains per polypeptide, and we show that these two domains interact to form a single active site. FadE34 and CasC are therefore representatives of a new class of ACADs with unique primary and quaternary structures. The bacterial steroid degradation pathway is important for the removal of steroid waste in the environment and for survival of the pathogen
M. tuberculosis
within host macrophages. FadE34 is a potential target for development of new antibiotics against tuberculosis.
Stability of the Osmoregulated Promoter-DerivedproPmRNA Is Posttranscriptionally Regulated by RNase III in Escherichia coli
Authors: Lim Boram , Lee Kangseok
Published: February 2015

The enzymatic activity ofEscherichia coliendo-RNase III determines the stability of a subgroup of mRNA species, includingbdm,betT, andproU, whose protein products are associated with the cellular response to osmotic stress. Here, we report that the stability ofproPmRNA, which encodes a transporter of osmoprotectants, is controlled by RNase III in response to osmotic stress. We observed that steady-state levels ofproPmRNA and ProP protein are inversely correlated with cellular RNase III activity and, in turn, affect the proline uptake capacity of the cell.In vitroandin vivoanalyses ofproPmRNA revealed RNase III cleavage sites in a stem-loop within the 5′ untranslated region present only inproPmRNA species synthesized from the osmoregulated P1 promoter. Introduction of nucleotide substitutions in the cleavage site identified inhibited the ribonucleolytic activity of RNase III onproPmRNA, increasing the steady-state levels and half-life of the mRNA. In addition, decreased RNase III activity coincided with a significant increase in both the half-life and abundance ofproPmRNA under hyperosmotic stress conditions. Analysis of the RNA bound to RNase III viain vivocross-linking and immunoprecipitation indicated that this phenomenon is related to the decreased RNA binding capacity of RNase III. Our findings suggest the existence of an RNase III-mediated osmoregulatory network that rapidly balances the expression levels of factors associated with the cellular response to osmotic stress inE. coli.

IMPORTANCEOur results demonstrate that RNase III activity onproPmRNA degradation is downregulated inEscherichia colicells under osmotic stress. In addition, we show that the downregulation of RNase III activity is associated with decreased RNA binding capacity of RNase III under hyperosmotic conditions. In particular, our findings demonstrate a link between osmotic stress and RNase III activity, underscoring the growing importance of posttranscriptional regulation in modulating rapid physiological adjustment to environmental changes.