bluuebunny/crossref_metadata_2025_split huggingface dataset:
In Vivo
Analysis of NH
4
+
Transport and Central Nitrogen Metabolism in Saccharomyces cerevisiae during Aerobic Nitrogen-Limited Growth
Authors: Cueto-Rojas H. F. , Maleki Seifar R. , ten Pierick A. , van Helmond W. , Pieterse M. M. , Heijnen J. J. , Wahl S. A. 
Published: September 2016

ABSTRACT

Ammonium is the most common N source for yeast fermentations. Although its transport and assimilation mechanisms are well documented, there have been only a few attempts to measure the
in vivo
intracellular concentration of ammonium and assess its impact on gene expression. Using an isotope dilution mass spectrometry (IDMS)-based method, we were able to measure the intracellular ammonium concentration in N-limited aerobic chemostat cultivations using three different N sources (ammonium, urea, and glutamate) at the same growth rate (0.05 h
−1
). The experimental results suggest that, at this growth rate, a similar concentration of intracellular (IC) ammonium, about 3.6 mmol NH
4
+
/liter
IC
, is required to supply the reactions in the central N metabolism, independent of the N source. Based on the experimental results and different assumptions, the vacuolar and cytosolic ammonium concentrations were estimated. Furthermore, we identified a futile cycle caused by NH
3
leakage into the extracellular space, which can cost up to 30% of the ATP production of the cell under N-limited conditions, and a futile redox cycle between Gdh1 and Gdh2 reactions. Finally, using shotgun proteomics with protein expression determined relative to a labeled reference, differences between the various environmental conditions were identified and correlated with previously identified N compound-sensing mechanisms.

IMPORTANCE
In our work, we studied central N metabolism using quantitative approaches. First, intracellular ammonium was measured under different N sources. The results suggest that
Saccharomyces cerevisiae
cells maintain a constant NH
4
+
concentration (around 3 mmol NH
4
+
/liter
IC
), independent of the applied nitrogen source. We hypothesize that this amount of intracellular ammonium is required to obtain sufficient thermodynamic driving force. Furthermore, our calculations based on thermodynamic analysis of the transport mechanisms of ammonium suggest that ammonium is not equally distributed, indicating a high degree of compartmentalization in the vacuole. Additionally, metabolomic analysis results were used to calculate the thermodynamic driving forces in the central N metabolism reactions, revealing that the main reactions in the central N metabolism are far from equilibrium. Using proteomics approaches, we were able to identify major changes, not only in N metabolism, but also in C metabolism and regulation.
Peeking under the Iron Curtain: Development of a Microcosm for Imaging the Colonization of Steel Surfaces by Mariprofundus sp. Strain DIS-1, an Oxygen-Tolerant Fe-Oxidizing Bacterium
Authors: Mumford Adam C. , Adaktylou Irini J. , Emerson David 
Published: September 2016

ABSTRACT

Microbially influenced corrosion (MIC) is a major cause of damage to steel infrastructure in the marine environment. Despite their ability to grow directly on Fe(II) released from steel, comparatively little is known about the role played by neutrophilic iron-oxidizing bacteria (FeOB). Recent work has shown that FeOB grow readily on mild steel (1018 MS) incubated
in situ
or as a substrate for pure cultures
in vitro
; however, details of how they colonize steel surfaces are unknown yet are important for understanding their effects. In this study, we combine a novel continuously upwelling microcosm with confocal laser scanning microscopy (CLSM) to determine the degree of colonization of 1018 MS by the marine FeOB strain DIS-1. 1018 MS coupons were incubated with sterile seawater (pH 8) inoculated with strain DIS-1. Incubations were performed both under oxic conditions and in an anoxic-to-oxic gradient. Following incubations of 1 to 10 days, the slides were removed from the microcosms and stained to visualize both cells and stalk structures. Stained coupons were visualized by CLSM after being mounted in a custom frame to preserve the three-dimensional structure of the biofilm. The incubation of 1018 MS coupons with strain DIS-1 under oxic conditions resulted in initial attachment of cells within 2 days and nearly total coverage of the coupon with an ochre film within 5 days. CLSM imaging revealed a nonadherent biofilm composed primarily of the Fe-oxide stalks characteristic of strain DIS-1. When incubated with elevated concentrations of Fe(II), DIS-1 colonization of 1018 MS was inhibited.

IMPORTANCE
These experiments describe the growth of a marine FeOB in a continuous culture system and represent direct visualizations of steel colonization by FeOB. We anticipate that these experiments will lay the groundwork for studying the mechanisms by which FeOB colonize steel and help to elucidate the role played by marine FeOB in MIC. These observations of the interaction between an FeOB, strain DIS-1, and steel suggest that this experimental system will provide a useful model for studying the interactions between microbes and solid substrates.
Metabolism of Fructophilic Lactic Acid Bacteria Isolated from the Apis mellifera L. Bee Gut: Phenolic Acids as External Electron Acceptors
Authors: Filannino Pasquale , Di Cagno Raffaella , Addante Rocco , Pontonio Erica , Gobbetti Marco 
Published: September 2016

ABSTRACT

Fructophilic lactic acid bacteria (FLAB) are strongly associated with the gastrointestinal tracts (GITs) of
Apis mellifera
L. worker bees due to the consumption of fructose as a major carbohydrate. Seventy-seven presumptive lactic acid bacteria (LAB) were isolated from GITs of healthy
A. mellifera
L. adults, which were collected from 5 different geographical locations of the Apulia region of Italy. Almost all of the isolates showed fructophilic tendencies: these isolates were identified as
Lactobacillus kunkeei
(69%) or
Fructobacillus fructosus
(31%). A high-throughput phenotypic microarray targeting 190 carbon sources was used to determine that 83 compounds were differentially consumed. Phenotyping grouped the strains into two clusters, reflecting growth performance. The utilization of phenolic acids, such as
p
-coumaric, caffeic, syringic, or gallic acids, as electron acceptors was investigated in fructose-based medium. Almost all FLAB strains showed tolerance to high phenolic acid concentrations.
p
-Coumaric acid and caffeic acid were consumed by all FLAB strains through reductases or decarboxylases. Syringic and gallic acids were partially metabolized. The data collected suggest that FLAB require external electron acceptors to regenerate NADH. The use of phenolic acids as external electron acceptors by the 4 FLAB showing the highest phenolic acid reductase activity was investigated in glucose-based medium supplemented with
p
-coumaric acid. Metabolic responses observed through a phenotypic microarray suggested that FLAB may use
p
-coumaric acid as an external electron acceptor, enhancing glucose dissimilation but less efficiently than other external acceptors such as fructose or pyruvic acid.

IMPORTANCE
Fructophilic lactic acid bacteria (FLAB) remain to be fully explored. This study intends to link unique biochemical features of FLAB with their habitat. The quite unique FLAB phenome within the group lactic acid bacteria (LAB) may have practical relevance in food fermentations. The FLAB phenome may have implications for the levels of hexose metabolism products in fermented foods, as well as food probiotication. Due to the harsh conditions of honeybees' GITs, these bacteria had to develop specific physiological and biochemical characteristics, such as tolerance to phenolic acids. The screening of FLAB strains based on metabolic pathways involving phenolic acids may allow the selection of starter cultures with both technological and functional beneficial attributes. Bioconversion of phenolic compounds may contribute to the aroma attributes and biofunctionality of fermented foods. Thus, the selection of FLAB strains as starter cultures with specific enzymatic activities involving phenolic acids may have a promising role in food fermentations.
Microbial Herd Protection Mediated by Antagonistic Interaction in Polymicrobial Communities
Authors: Wong Megan J. Q. , Liang Xiaoye , Smart Matt , Tang Le , Moore Richard , Ingalls Brian , Dong Tao G. 
Published: September 2016

ABSTRACT

In host and natural environments, microbes often exist in complex multispecies communities. The molecular mechanisms through which such communities develop and persist, despite significant antagonistic interactions between species, are not well understood. The type VI secretion system (T6SS) is a lethal weapon commonly employed by Gram-negative bacteria to inhibit neighboring species through the delivery of toxic effectors. It is well established that intraspecies protection is conferred by immunity proteins that neutralize effector toxicities. In contrast, the mechanisms for interspecies protection are not clear. Here we use two T6SS-active antagonistic bacterial species,
Aeromonas hydrophila
and
Vibrio cholerae
, to demonstrate that interspecies protection is dependent on effectors.
A. hydrophila
and
V. cholerae
do not share conserved immunity genes but could coexist equally in a mixture. However, mutants lacking the T6SS or effectors were effectively eliminated by the competing wild-type strain. Time-lapse microscopic analyses showed that mutually lethal interactions drive the segregation of mixed species into distinct single-species clusters by eliminating interspersed single cells. Cluster formation provides herd protection by abolishing lethal interactions inside each cluster and restricting the interactions to the boundary. Using an agent-based modeling approach, we simulated the antagonistic interactions of two hypothetical species. The resulting simulations recapitulated our experimental observations. These results provide mechanistic insights regarding the general role of microbial weapons in determining the structures of complex multispecies communities.

IMPORTANCE
Investigating the warfare of microbes allows us to better understand the ecological relationships in complex microbial communities such as the human microbiota. Here we use the T6SS, a deadly bacterial weapon, as a model to demonstrate the importance of lethal interactions in determining community structures and the exchange of genetic materials. This simplified model elucidates a mechanism of microbial herd protection by which competing antagonistic species can coexist in the same niche, despite their diverse mutually destructive activities. Our results also suggest that antagonistic interactions impose strong selection that could promote multicellular organism-like social behaviors and contribute to the transition to multicellularity during evolution.
Generation of a Stable Plasmid for
In Vitro
and
In Vivo
Studies of Staphylococcus Species
Authors: Krute Christina N. , Krausz Kelsey L. , Markiewicz Mary A. , Joyner Jason A. , Pokhrel Srijana , Hall Pamela R. , Bose Jeffrey L. 
Published: September 2016

ABSTRACT

A major shortcoming to plasmid-based genetic tools is the necessity of using antibiotics to ensure plasmid maintenance. While selectable markers are very powerful, their use is not always practical, such as during
in vivo
models of bacterial infection. During previous studies, it was noted that the uncharacterized LAC-p01 plasmid in
Staphylococcus aureus
USA300 isolates was stable in the absence of a known selection and therefore could serve as a platform for new genetic tools for
Staphylococcus
species. LAC-p01 was genetically manipulated into an
Escherichia coli
-
S. aureus
shuttle vector that remained stable for at least 100 generations without antibiotic selection. The double- and single-stranded (
dso
and
sso
) origins were identified and found to be essential for plasmid replication and maintenance, respectively. In contrast, deletion analyses revealed that none of the four LAC-p01 predicted open reading frames were necessary for stability. Subsequent to this, the shuttle vector was used as a platform to generate two plasmids. The first plasmid, pKK22, contains all genes native to the plasmid for use in
S. aureus
USA300 strains, while the second, pKK30, lacks the four predicted open reading frames for use in non-USA300 isolates. pKK30 was also determined to be stable in
Staphylococcus epidermidis
. Moreover, pKK22 was maintained for 7 days postinoculation during a murine model of
S. aureus
systemic infection and successfully complemented an
hla
mutant in a dermonecrosis model. These plasmids that eliminate the need for antibiotics during both
in vitro
and
in vivo
experiments are powerful new tools for studies of
Staphylococcus
.

IMPORTANCE
Plasmid stability has been problematic in bacterial studies, and historically antibiotics have been used to ensure plasmid maintenance. This has been a major limitation during
in vivo
studies, where providing antibiotics for plasmid maintenance is difficult and has confounding effects. Here, we have utilized the naturally occurring plasmid LAC-p01 from an
S. aureus
USA300 strain to construct stable plasmids that obviate antibiotic usage. These newly modified plasmids retain stability over a multitude of generations
in vitro
and
in vivo
without antibiotic selection. With these plasmids, studies requiring genetic complementation, protein expression, or genetic reporter systems would not only overcome the burden of antibiotic usage but also eliminate the side effects of these antibiotics. Thus, our plasmids can be used as a powerful genetic tool for studies of
Staphylococcus
species.
The Redox Cofactor F
420
Protects Mycobacteria from Diverse Antimicrobial Compounds and Mediates a Reductive Detoxification System
Authors: Jirapanjawat Thanavit , Ney Blair , Taylor Matthew C. , Warden Andrew C. , Afroze Shahana , Russell Robyn J. , Lee Brendon M. , Jackson Colin J. , Oakeshott John G. , Pandey Gunjan , Greening Chris 
Published: September 2016

ABSTRACT

A defining feature of mycobacterial redox metabolism is the use of an unusual deazaflavin cofactor, F
420
. This cofactor enhances the persistence of environmental and pathogenic mycobacteria, including after antimicrobial treatment, although the molecular basis for this remains to be understood. In this work, we explored our hypothesis that F
420
enhances persistence by serving as a cofactor in antimicrobial-detoxifying enzymes. To test this, we performed a series of phenotypic, biochemical, and analytical chemistry studies in relation to the model soil bacterium
Mycobacterium smegmatis
. Mutant strains unable to synthesize or reduce F
420
were found to be more susceptible to a wide range of antibiotic and xenobiotic compounds. Compounds from three classes of antimicrobial compounds traditionally resisted by mycobacteria inhibited the growth of F
420
mutant strains at subnanomolar concentrations, namely, furanocoumarins (e.g., methoxsalen), arylmethanes (e.g., malachite green), and quinone analogues (e.g., menadione). We demonstrated that promiscuous F
420
H
2
-dependent reductases directly reduce these compounds by a mechanism consistent with hydride transfer. Moreover,
M. smegmatis
strains unable to make F
420
H
2
lost the capacity to reduce and detoxify representatives of the furanocoumarin and arylmethane compound classes in whole-cell assays. In contrast, mutant strains were only slightly more susceptible to clinical antimycobacterials, and this appeared to be due to indirect effects of F
420
loss of function (e.g., redox imbalance) rather than loss of a detoxification system. Together, these data show that F
420
enhances antimicrobial resistance in mycobacteria and suggest that one function of the F
420
H
2
-dependent reductases is to broaden the range of natural products that mycobacteria and possibly other environmental actinobacteria can reductively detoxify.

IMPORTANCE
This study reveals that a unique microbial cofactor, F
420
, is critical for antimicrobial resistance in the environmental actinobacterium
Mycobacterium smegmatis
. We show that a superfamily of redox enzymes, the F
420
H
2
-dependent reductases, can reduce diverse antimicrobials
in vitro
and
in vivo
.
M. smegmatis
strains unable to make or reduce F
420
become sensitive to inhibition by these antimicrobial compounds. This suggests that mycobacteria have harnessed the unique properties of F
420
to reduce structurally diverse antimicrobials as part of the antibiotic arms race. The F
420
H
2
-dependent reductases that facilitate this process represent a new class of antimicrobial-detoxifying enzymes with potential applications in bioremediation and biocatalysis.
Developmental trajectories in children with prolonged NICU stays
Authors: Subedi Dibya , DeBoer Mark D , Scharf Rebecca J 
Published: September 2016

ObjectiveTo evaluate the relationship between prolonged neonatal intensive care unit (NICU) stay after birth and childhood neurodevelopmental measures from age 9 months to kindergarten.DesignLongitudinal birth cohort study.Setting and patientsThis study examined a nationally representative sample of 10 700 participants from the Early Childhood Longitudinal Sample—Birth Cohort and selected those who had a NICU stay (n=2100). These children were followed from birth to kindergarten.PredictorsDays in the NICU.Main outcome measuresChildhood neurodevelopmental and early academic scores.ResultsIncreasing length of stay in the NICU had a significant negative relationship with the 9-month and 24-month Bayley mental and motor scores. Each additional week in the NICU increased the odds of scoring in the lowest 10% on the Bayley 9-month mental (OR 1.08, 95% CI 1.034 to 1.122) and motor (OR 1.11, CI 1.065 to 1.165) assessments and 24-month mental (OR 1.09, CI 1.041 to 1.144) and motor assessments (OR 1.07 CI 1.017 to 1.123). Gestational age was not significantly related with these measures in our model. Increasing socioeconomic status had a significant positive relationship with preschool and kindergarten reading and math scores and a lower odds of scoring in the lowest 10% in these measures.ConclusionIncreasing length of NICU stay was predictive of decreased child development measures in early childhood (9 and 24 months), while socioeconomic status was a better predictor at later assessments (preschool and kindergarten entries). Gestational category did not account for these differences. These data may have implications for counselling parents regarding potential neurodevelopmental consequences following NICU stay.
Gastric microbiota in the functional dyspepsia patients treated with probiotic yogurt
Authors: Nakae Hirohiko , Tsuda Ayumi , Matsuoka Takashi , Mine Tetsuya , Koga Yasuhiro 
Published: September 2016

ObjectiveTo investigate the structure of the gastric microbiota in functional dyspepsia (FD) and its role in the pathophysiology.DesignWe compared the basic physiological properties of the gastric fluid (GF) and the structure of the microbiota in the GF of 44 healthy control (HC) participants and 44 patients with FD. We then treated the patients with FD with a yogurt containing a probiotic strain ofLactobacillus gasseriOLL2716 (LG21 yogurt) and investigated the effects on the bacteriological parameters and symptoms to examine the relationship between them.ResultsThe volume of GF recovered from the stomach after overnight fasting was greater in the patients with FD than in the HCs, and decreased in the patients with FD whose symptoms were improved by the LG21 yogurt treatment. An analysis using a terminal restriction fragment polymorphism method demonstrated that the overall structure of the bacterial community and the abundance of genusPrevotellain the GF of the patients in the FD group were significantly different from those in the HC group. In the patients with FD, this bacteriological change was restored by treatment with LG21 yogurt. A significant inverse correlation was found between the abundance ofPrevotellaand the severity of postprandial distress-like symptoms in patients with FD who received LG21 yogurt.ConclusionsSignificant dysbiosis was found in the GF microbiota of patients with FD and considered to be involved in the pathogenesis. The abundance of genusPrevotellain the GF may be used as a biomarker of the efficacy of the treatment of FD.Trial registration numberUMINCTR000022026.
Barriers to making recommendations about medical tests: a qualitative study of European guideline developers
Authors: Gopalakrishna Gowri , Leeflang Mariska M G , Davenport Clare , Sanabria Andrea Juliana , Alonso-Coello Pablo , McCaffery Kirsten , Bossuyt Patrick , Langendam Miranda W 
Published: September 2016

ObjectivesDevelopment of medical test guidelines differs from intervention guideline development. These differences can pose unique challenges in building evidence-based recommendations to guide clinical practice. The aim of our study was to better understand these challenges, explore reasons behind them and identify possible solutions.Setting and participantsIn this qualitative study, we conducted in-depth interviews between February 2012 and April 2013 of a convenience sample of 17 European guideline developers experienced in medical test guideline development.Outcomes measuredWe used framework analysis with deductive and inductive approaches to generate the themes from the interviews. We kept interpretation grounded in the data.ResultsGuideline developers acknowledged that inclusion of patient important outcomes in their guideline development was necessary but lacking. This and other challenges raised fell into 3 broad and overlapping domains: methodological issues, resource limitations and a lack of awareness on the need for evidence that links testing to patient outcomes. Education was mentioned as a key solution to increase awareness and address the resources limitations mentioned.ConclusionsChallenges guideline developers face were interlinked across the domains of methodological issues, resource limitations and a lack of awareness. Solutions that addressed these challenges in parallel are needed. Raising awareness, education and training of relevant stakeholders such as medical doctors, funders and regulators to look beyond test accuracy is key to having a long-term resolution to the issues faced in medical test guideline development.