bluuebunny/crossref_metadata_2025_split huggingface dataset:
Directed Evolution of Brain-Derived Neurotrophic Factor for Improved Folding and Expression in Saccharomyces cerevisiae
Authors: Burns Michael L. , Malott Thomas M. , Metcalf Kevin J. , Hackel Benjamin J. , Chan Jonah R. , Shusta Eric V. 
Published: July 2014

ABSTRACT

Brain-derived neurotrophic factor (BDNF) plays an important role in nervous system function and has therapeutic potential. Microbial production of BDNF has resulted in a low-fidelity protein product, often in the form of large, insoluble aggregates incapable of binding to cognate TrkB or p75 receptors. In this study, employing
Saccharomyces cerevisiae
display and secretion systems, it was found that BDNF was poorly expressed and partially inactive on the yeast surface and that BDNF was secreted at low levels in the form of disulfide-bonded aggregates. Thus, for the purpose of increasing the compatibility of yeast as an expression host for BDNF, directed-evolution approaches were employed to improve BDNF folding and expression levels. Yeast surface display was combined with two rounds of directed evolution employing random mutagenesis and shuffling to identify BDNF mutants that had 5-fold improvements in expression, 4-fold increases in specific TrkB binding activity, and restored p75 binding activity, both as displayed proteins and as secreted proteins. Secreted BDNF mutants were found largely in the form of soluble homodimers that could stimulate TrkB phosphorylation in transfected PC12 cells. Site-directed mutagenesis studies indicated that a particularly important mutational class involved the introduction of cysteines proximal to the native cysteines that participate in the BDNF cysteine knot architecture. Taken together, these findings show that yeast is now a viable alternative for both the production and the engineering of BDNF.
Transcriptome-Based Analysis of the Pantoea stewartii Quorum-Sensing Regulon and Identification of EsaR Direct Targets
Authors: Ramachandran Revathy , Burke Alison Kernell , Cormier Guy , Jensen Roderick V. , Stevens Ann M. 
Published: July 2014

ABSTRACT

Pantoea
stewartii
subsp.
stewartii
is a proteobacterium that causes Stewart's wilt disease in corn plants. The bacteria form a biofilm in the xylem of infected plants and produce capsule that blocks water transport, eventually causing wilt. At low cell densities, the quorum-sensing (QS) regulatory protein EsaR is known to directly repress expression of
esaR
itself as well as the genes for the capsular synthesis operon transcription regulator,
rcsA
, and a 2,5-diketogluconate reductase,
dkgA
. It simultaneously directly activates expression of genes for a putative small RNA,
esaS
, the glycerol utilization operon,
glpFKX
, and another transcriptional regulator,
lrhA
. At high bacterial cell densities, all of this regulation is relieved when EsaR binds an acylated homoserine lactone signal, which is synthesized constitutively over growth. QS-dependent gene expression is critical for the establishment of disease in the plant. However, the identity of the full set of genes controlled by EsaR/QS is unknown. A proteomic approach previously identified around 30 proteins in the QS regulon. In this study, a whole-transcriptome, next-generation sequencing analysis of rRNA-depleted RNA from QS-proficient and -deficient
P. stewartii
strains was performed to identify additional targets of EsaR. EsaR-dependent transcriptional regulation of a subset of differentially expressed genes was confirmed by quantitative reverse transcription-PCR (qRT-PCR). Electrophoretic mobility shift assays demonstrated that EsaR directly bound 10 newly identified target promoters. Overall, the QS regulon of
P. stewartii
orchestrates three major physiological responses: capsule and cell envelope biosynthesis, surface motility and adhesion, and stress response.
Comprehensive Comparison of Cultivable Norovirus Surrogates in Response to Different Inactivation and Disinfection Treatments
Authors: Cromeans Theresa , Park Geun Woo , Costantini Veronica , Lee David , Wang Qiuhong , Farkas Tibor , Lee Alvin , Vinjé Jan 
Published: July 2014

ABSTRACT

Human norovirus is the leading cause of epidemic and sporadic acute gastroenteritis. Since no cell culture method for human norovirus exists, cultivable surrogate viruses (CSV), including feline calicivirus (FCV), murine norovirus (MNV), porcine enteric calicivirus (PEC), and Tulane virus (TuV), have been used to study responses to inactivation and disinfection methods. We compared the levels of reduction in infectivities of CSV and Aichi virus (AiV) after exposure to extreme pHs, 56°C heating, alcohols, chlorine on surfaces, and high hydrostatic pressure (HHP), using the same matrix and identical test parameters for all viruses, as well as the reduction of human norovirus RNA levels under these conditions. At pH 2, FCV was inactivated by 6 log
10
units, whereas MNV, TuV, and AiV were resistant. All CSV were completely inactivated at 56°C within 20 min. MNV was inactivated 5 log
10
units by alcohols, in contrast to 2 and 3 log
10
units for FCV and PEC, respectively. TuV and AiV were relatively insensitive to alcohols. FCV was reduced 5 log
10
units by 1,000 ppm chlorine, in contrast to 1 log
10
unit for the other CSV. All CSV except FCV, when dried on stainless steel surfaces, were insensitive to 200 ppm chlorine. HHP completely inactivated FCV, MNV, and PEC at ≥300 MPa, and TuV at 600 MPa, while AiV was completely resistant to HHP up to 800 MPa. By reverse transcription-quantitative PCR (RT-qPCR), genogroup I (GI) noroviruses were more sensitive than GII noroviruses to alcohols, chlorine, and HHP. Although inactivation profiles were variable for each treatment, TuV and MNV were the most resistant CSV overall and therefore are the best candidates for studying the public health outcomes of norovirus infections.
Influence of Populus Genotype on Gene Expression by the Wood Decay Fungus Phanerochaete chrysosporium
Authors: Gaskell Jill , Marty Amber , Mozuch Michael , Kersten Philip J. , Splinter BonDurant Sandra , Sabat Grzegorz , Azarpira Ali , Ralph John , Skyba Oleksandr , Mansfield Shawn D. , Blanchette Robert A. , Cullen Dan 
Published: July 2014

ABSTRACTWe examined gene expression patterns in the lignin-degrading fungusPhanerochaete chrysosporiumwhen it colonizes hybrid poplar (Populus alba×tremula) and syringyl (S)-rich transgenic derivatives. A combination of microarrays and liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowed detection of a total of 9,959 transcripts and 793 proteins. Comparisons ofP. chrysosporiumtranscript abundance in medium containing poplar or glucose as a sole carbon source showed 113 regulated genes, 11 of which were significantly higher (>2-fold,P< 0.05) in transgenic line 64 relative to the parental line. Possibly related to the very large amounts of syringyl (S) units in this transgenic tree (94 mol% S), several oxidoreductases were among the upregulated genes. Peptides corresponding to a total of 18 oxidoreductases were identified in medium consisting of biomass from line 64 or 82 (85 mol% S) but not in the parental clone (65 mol% S). These results demonstrate thatP. chrysosporiumgene expression patterns are substantially influenced by lignin composition.
Factors Limiting the Spread of the Protective Symbiont Hamiltonella defensa in Aphis craccivora Aphids
Authors: Dykstra Hannah R. , Weldon Stephanie R. , Martinez Adam J. , White Jennifer A. , Hopper Keith R. , Heimpel George E. , Asplen Mark K. , Oliver Kerry M. 
Published: July 2014

ABSTRACT

Many insects are associated with heritable symbionts that mediate ecological interactions, including host protection against natural enemies. The cowpea aphid,
Aphis craccivora
, is a polyphagous pest that harbors
Hamiltonella defensa
, which defends against parasitic wasps. Despite this protective benefit, this symbiont occurs only at intermediate frequencies in field populations. To identify factors constraining
H. defensa
invasion in
Ap. craccivora
, we estimated symbiont transmission rates, performed fitness assays, and measured infection dynamics in population cages to evaluate effects of infection. Similar to results with the pea aphid,
Acyrthosiphon pisum
, we found no consistent costs to infection using component fitness assays, but we did identify clear costs to infection in population cages when no enemies were present. Maternal transmission rates of
H. defensa
in
Ap. craccivora
were high (ca. 99%) but not perfect. Transmission failures and infection costs likely limit the spread of protective
H. defensa
in
Ap. craccivora
. We also characterized several parameters of
H. defensa
infection potentially relevant to the protective phenotype. We confirmed the presence of
H. defensa
in aphid hemolymph, where it potentially interacts with endoparasites, and performed real-time quantitative PCR (qPCR) to estimate symbiont and phage abundance during aphid development. We also examined strain variation of
H. defensa
and its bacteriophage at multiple loci, and despite our lines being collected in different regions of North America, they were infected with a nearly identical strains of
H. defensa
and APSE4 phage. The limited strain diversity observed for these defensive elements may result in relatively static protection profile for this defensive symbiosis.
Hydrogen Photoproduction by Immobilized N
2
-Fixing Cyanobacteria: Understanding the Role of the Uptake Hydrogenase in the Long-Term Process
Authors: Kosourov Sergey , Leino Hannu , Murukesan Gayathri , Lynch Fiona , Sivonen Kaarina , Tsygankov Anatoly A. , Aro Eva-Mari , Allahverdiyeva Yagut 
Published: July 2014

ABSTRACT

We have investigated two approaches to enhance and extend H
2
photoproduction yields in heterocystous, N
2
-fixing cyanobacteria entrapped in thin alginate films. In the first approach, periodic CO
2
supplementation was provided to alginate-entrapped, N-deprived cells. N deprivation led to the inhibition of photosynthetic activity in vegetative cells and the attenuation of H
2
production over time. Our results demonstrated that alginate-entrapped Δ
hupL
cells were considerably more sensitive to high light intensity, N deficiency, and imbalances in C/N ratios than wild-type cells. In the second approach,
Anabaena
strain PCC 7120, its Δ
hupL
mutant, and
Calothrix
strain 336/3 films were supplemented with N
2
by periodic treatments of air, or air plus CO
2
. These treatments restored the photosynthetic activity of the cells and led to a high level of H
2
production in
Calothrix
336/3 and Δ
hupL
cells (except for the treatment air plus CO
2
) but not in the
Anabaena
PCC 7120 strain (for which H
2
yields did not change after air treatments). The highest H
2
yield was obtained by the air treatment of Δ
hupL
cells. Notably, the supplementation of CO
2
under an air atmosphere led to prominent symptoms of N deficiency in the Δ
hupL
strain but not in the wild-type strain. We propose that uptake hydrogenase activity in heterocystous cyanobacteria not only supports nitrogenase activity by removing excess O
2
from heterocysts but also indirectly protects the photosynthetic apparatus of vegetative cells from photoinhibition, especially under stressful conditions that cause an imbalance in the C/N ratio in cells.
Exopolysaccharides Synthesized by Lactobacillus reuteri Protect against Enterotoxigenic Escherichia coli in Piglets
Authors: Chen Xiao Yan , Woodward Adrienne , Zijlstra Ruurd T. , Gänzle Michael G. 
Published: July 2014

ABSTRACT

Enterotoxigenic
Escherichia coli
(ETEC) is a major cause of diarrhea in piglets; ETEC cells colonize the intestinal mucosa with adhesins and deliver toxins that cause fluid loss. This study determined the antiadhesive properties of bacterial exopolysaccharides (reuteran and levan) and related glycans (dextran and inulin) in a small intestinal segment perfusion (SISP) model. The SISP model used 10 jejunal segments from 5-week-old piglets. Five segments were infected with ETEC expressing K88 fimbriae (ETEC K88), while five segments were treated with saline. Every two segments (ETEC and non-ETEC infected) were infused with 65 ml of 10 g liter
−1
of glycans or saline (control) for 8 h. High-resolution melting-curve (HRM) quantitative PCR (qPCR) indicated that
E. coli
is the dominant bacterium in infected segments, while other bacteria were predominant in noninfected segments. Infection by ETEC K88 was also verified by qPCR; gene copy numbers of K88 fimbriae and the heat-labile toxin (LT) in mucosal scrapings and outflow fluid of infected segments were significantly higher than those in noninfected segments. Genes coding for K88 fimbriae and LT were also detected in noninfected segments. LT amplicons from infected and noninfected segments were 99% identical over 481 bp, demonstrating the presence of autochthonous ETEC K88. All glycans reduced fluid loss caused by ETEC K88 infection. Reuteran tended (
P
= 0.06) to decrease ETEC K88 levels in mucosal scraping sample, as judged by qPCR. Fluorescent
in situ
hybridization analysis demonstrated that reuteran significantly (
P
= 0.012) decreased levels of adherent ETEC K88. Overall, reuteran may prevent piglet diarrhea by reducing adhesion of ETEC K88.
Effect of Lineage-Specific Metabolic Traits of Lactobacillus reuteri on Sourdough Microbial Ecology
Authors: Lin Xiaoxi B. , Gänzle Michael G. 
Published: July 2014

ABSTRACT

This study determined the effects of specific metabolic traits of
Lactobacillus reuteri
on its competitiveness in sourdoughs. The competitiveness of lactobacilli in sourdough generally depends on their growth rate; acid resistance additionally contributes to competitiveness in sourdoughs with long fermentation times. Glycerol metabolism via glycerol dehydratase (
gupCDE
) accelerates growth by the regeneration of reduced cofactors; glutamate metabolism via glutamate decarboxylase (
gadB
) increases acid resistance by generating a proton motive force. Glycerol and glutamate metabolisms are lineage-specific traits in
L. reuteri
; therefore, this study employed glycerol dehydratase-positive sourdough isolates of human-adapted
L. reuteri
lineage I, glutamate decarboxylase-positive strains of rodent-adapted
L. reuteri
lineage II, as well as mutants with deletions in
gadB
or
gupCDE
. The competitivenesses of the strains were quantified by inoculation of wheat and sorghum sourdoughs with defined strains, followed by propagation of doughs with a 10% inoculum and 12-h or 72-h fermentation cycles. Lineage I
L. reuteri
strains dominated sourdoughs propagated with 12-h fermentation cycles; lineage II
L. reuteri
strains dominated sourdoughs propagated with 72-h fermentation cycles.
L. reuteri
100-23Δ
gadB
was outcompeted by its wild-type strain in sourdoughs fermented with 72-h fermentation cycles;
L. reuteri
FUA3400Δ
gupCDE
was outcompeted by its wild-type strain in sourdoughs fermented with both 12-h and 72-h fermentation cycles. Competition experiments with isogenic pairs of strains resulted in a constant rate of strain displacement of the less competitive mutant strain. In conclusion, lineage-specific traits of
L. reuteri
determine the competitiveness of this species in sourdough fermentations.
The Underlying Structure of Adaptation under Strong Selection in 12 Experimental Yeast Populations
Authors: Kohn Linda M. , Anderson James B. 
Published: July 2014

ABSTRACT

The aims of this study were to determine (i) whether adaptation under strong selection occurred through mutations in a narrow target of one or a few nucleotide sites or a broad target of numerous sites and (ii) whether the programs of adaptation previously observed from three experimental populations were unique or shared among populations that underwent parallel evolution. We used archived population samples from a previous study, representing 500 generations of experimental evolution in 12 populations under strong selection, 6 populations in a high-salt environment and 6 populations in a low-glucose environment. Each set of six populations included four with sexual reproduction and two with exclusively asexual reproduction. Populations were sampled as resequenced genomes of 115 individuals and as bulk samples from which frequencies of mutant alleles were estimated. In a high-salt environment, a broad target of 11 mutations within the proton exporter,
PMA1
, was observed among the six populations, in addition to expansions of the
ENA
gene cluster. This pattern was shared among populations that underwent parallel evolution. In a low-glucose environment, two programs of adaptation were observed. The originally observed pattern of mutation in
MDS3
/
MKT1
in population M8 was a narrow target of a single nucleotide, unique to this population. Among the other five populations, the three mutations were shared in a broad target, sensing/signaling genes
RAS1
and
RAS2. RAS1
/
RAS2
mutations were not observed in the high-salt populations;
PMA1
mutations were observed only in a high-salt environment.
Toxoplasma gondii Ingests and Digests Host Cytosolic Proteins
Authors: Dou Zhicheng , McGovern Olivia L. , Di Cristina Manlio , Carruthers Vern B. 
Published: July 2014

ABSTRACT

The protozoan parasite
Toxoplasma gondii
resides within a nonfusogenic vacuole during intracellular replication. Although the limiting membrane of this vacuole provides a protective barrier to acidification and degradation by lysosomal hydrolases, it also physically segregates the parasite from the host cytosol. Accordingly, it has been suggested that
T. gondii
acquires material from the host via membrane channels or transporters. The ability of the parasite to internalize macromolecules via endocytosis during intracellular replication has not been tested. Here, we show that
Toxoplasma
ingests host cytosolic proteins and digests them using cathepsin L and other proteases within its endolysosomal system. Ingestion was reduced in mutant parasites lacking an intravacuolar network of tubular membranes, implicating this apparatus as a possible conduit for trafficking to the parasite. Genetic ablation of proteins involved in the pathway is associated with diminished parasite replication and virulence attenuation. We show that both virulent type I and avirulent type II strain parasites ingest and digest host-derived protein, indicating that the pathway is not restricted to highly virulent strains. The findings provide the first definitive evidence that
T. gondii
internalizes proteins from the host during intracellular residence and suggest that protein digestion within the endolysosomal system of the parasite contributes to toxoplasmosis.

IMPORTANCE
Toxoplasma gondii
causes significant disease in individuals with weak immune systems. Treatment options for this infection have drawbacks, creating a need to understand how this parasite survives within the cells it infects as a prelude to interrupting its survival strategies. This study reveals that
T. gondii
internalizes proteins from the cytoplasm of the cells it infects and degrades such proteins within a digestive compartment within the parasite. Disruption of proteins involved in the pathway reduced parasite replication and lessened disease severity. The identification of a novel parasite ingestion pathway opens opportunities to interfere with this process and improve the outcome of infection.
Apoptosis-Like Death, an Extreme SOS Response in Escherichia coli
Authors: Erental Ariel , Kalderon Ziva , Saada Ann , Smith Yoav , Engelberg-Kulka Hanna 
Published: July 2014

ABSTRACT

In bacteria, SOS is a global response to DNA damage, mediated by the
recA-lexA
genes, resulting in cell cycle arrest, DNA repair, and mutagenesis. Previously, we reported that
Escherichia coli
responds to DNA damage via another
recA-lexA
-mediated pathway resulting in programmed cell death (PCD). We called it apoptosis-like death (ALD) because it is characterized by membrane depolarization and DNA fragmentation, which are hallmarks of eukaryotic mitochondrial apoptosis. Here, we show that ALD is an extreme SOS response that occurs only under conditions of severe DNA damage. Furthermore, we found that ALD is characterized by additional hallmarks of eukaryotic mitochondrial apoptosis, including (i) rRNA degradation by the endoribonuclease YbeY, (ii) upregulation of a unique set of genes that we called
e
xtensive-
d
amage-
i
n
duced (
Edin
) genes, (iii) a decrease in the activities of complexes I and II of the electron transport chain, and (iv) the formation of high levels of OH˙ through the Fenton reaction, eventually resulting in cell death. Our genetic and molecular studies on ALD provide additional insight for the evolution of mitochondria and the apoptotic pathway in eukaryotes.

IMPORTANCE
The SOS response is the first described and the most studied bacterial response to DNA damage. It is mediated by a set of two genes,
recA-lexA
, and it results in DNA repair and thereby in the survival of the bacterial culture. We have shown that
Escherichia coli
responds to DNA damage by an additional
recA-lexA
-mediated pathway resulting in an apoptosis-like death (ALD). Apoptosis is a mode of cell death that has previously been reported only in eukaryotes. We found that
E. coli
ALD is characterized by several hallmarks of eukaryotic mitochondrial apoptosis. Altogether, our results revealed that
recA-lexA
is a DNA damage response coordinator that permits two opposite responses: life, mediated by the SOS, and death, mediated by the ALD. The choice seems to be a function of the degree of DNA damage in the cell.
Coverage of common cancer types in UK national newspapers: a content analysis
Authors: Konfortion Julie , Jack Ruth H , Davies Elizabeth A 
Published: July 2014

ObjectiveTo determine whether recent newspaper coverage of the four most common cancer types relates to their relative burden and national awareness months, and to identify the subject focus during high-coverage periods.DesignContent analysis using the Nexis newspaper article database.SettingUK 2011–2012.Outcome measuresAnnual number and ranking, monthly proportions and subject of articles on breast, lung, bowel and prostate cancers.Results9178 articles were identified during 2011 and 2012 featuring breast (4237), prostate (1757), lung (1746) and bowel (1438) cancer. Peaks in monthly proportions above the 99% upper confidence limit were identified for each. Breast cancer had the highest coverage of 12% and 17% during its awareness month. Smaller peaks (11%) were identified during Bowel Cancer Awareness month. Prostate cancer received high coverage in relation to the case of the man convicted of the Lockerbie bombing who had been diagnosed with the cancer, and lung cancer in relation to the deaths of celebrities. Breast cancer was covered most often overall and by newspaper category while the lower coverage of other cancer types did not consistently mirror the relative number of new cases each year. The peaks by newspaper category were similar to the overall coverage with few exceptions.ConclusionsUK newspaper coverage of common cancer types other than of the breast appears under-represented relative to their population burden. Coverage of breast cancer and bowel cancer appears to be influenced by their awareness months, while that of prostate cancer and lung cancer is influenced by other media stories. Health-promoting public bodies and campaigners could learn from the success of Breast Cancer Awareness Month and work more closely with journalists to ensure that the relevant messages reach wider audiences.