bluuebunny/crossref_metadata_2025_split huggingface dataset:
Survival of Airborne MS2 Bacteriophage Generated from Human Saliva, Artificial Saliva, and Cell Culture Medium
Authors: Zuo Zhili , Kuehn Thomas H. , Bekele Aschalew Z. , Mor Sunil K. , Verma Harsha , Goyal Sagar M. , Raynor Peter C. , Pui David Y. H. 
Published: February 2014

ABSTRACTLaboratory studies of virus aerosols have been criticized for generating airborne viruses from artificial nebulizer suspensions (e.g., cell culture media), which do not mimic the natural release of viruses (e.g., from human saliva). The objectives of this study were to determine the effect of human saliva on the infectivity and survival of airborne virus and to compare it with those of artificial saliva and cell culture medium. A stock of MS2 bacteriophage was diluted in one of three nebulizer suspensions, aerosolized, size selected (100 to 450 nm) using a differential mobility analyzer, and collected onto gelatin filters. Uranine was used as a particle tracer. The resulting particle size distribution was measured using a scanning mobility particle sizer. The amounts of infectious virus, total virus, and fluorescence in the collected samples were determined by infectivity assays, quantitative reverse transcription-PCR (RT-PCR), and spectrofluorometry, respectively. For all nebulizer suspensions, the virus content generally followed a particle volume distribution rather than a number distribution. The survival of airborne MS2 was independent of particle size but was strongly affected by the type of nebulizer suspension. Human saliva was found to be much less protective than cell culture medium (i.e., 3% tryptic soy broth) and artificial saliva. These results indicate the need for caution when extrapolating laboratory results, which often use artificial nebulizer suspensions. To better assess the risk of airborne transmission of viral diseases in real-life situations, the use of natural suspensions such as saliva or respiratory mucus is recommended.
Abundance of Broad Bacterial Taxa in the Sargasso Sea Explained by Environmental Conditions but Not Water Mass
Authors: Sjöstedt Johanna , Martiny Jennifer B. H. , Munk Peter , Riemann Lasse 
Published: February 2014

ABSTRACT

To explore the potential linkage between distribution of marine bacterioplankton groups, environmental conditions, and water mass, we investigated the factors determining the abundance of bacterial taxa across the hydrographically complex Subtropical Convergence Zone in the Sargasso Sea. Based on information from 16S rRNA gene clone libraries from various locations and two depths, abundances of the predominant taxa (eubacteria,
Archaea
,
Alphaproteobacteria
,
Gammaproteobacteria
,
Bacteroidetes
, and the
Roseobacter
, SAR11, and SAR86 clades) were quantified by real-time PCR. In addition, the abundances of
Synechococcus
,
Prochlorococcus
, and picoalgae were determined by flow cytometry. Linear multiple-regression models determining the relative effects of eight environmental variables and of water mass explained 35 to 86% of the variation in abundance of the quantified taxa, even though only one to three variables were significantly related to any particular taxon's abundance. Most of the variation in abundance was explained by depth and chlorophyll
a
. The predominant phototrophs,
Prochlorococcus
and picoalgae, were negatively correlated with phosphate, whereas eubacteria, heterotrophic bacteria, and SAR86 were negatively correlated with nitrite. Water mass showed limited importance for explaining the abundance of the taxonomical groups (significant only for
Roseobacter
, explaining 14% of the variation). The results suggest the potential for predicting the abundance of broad bacterioplankton groups throughout the Sargasso Sea using only a few environmental parameters.
The Lipopeptide Antibiotic Paenibacterin Binds to the Bacterial Outer Membrane and Exerts Bactericidal Activity through Cytoplasmic Membrane Damage
Authors: Huang En , Yousef Ahmed E. 
Published: February 2014

ABSTRACT

Paenibacterin is a broad-spectrum lipopeptide antimicrobial agent produced by
Paenibacillus thiaminolyticus
OSY-SE. The compound consists of a cyclic 13-residue peptide and an N-terminal C
15
fatty acyl chain. The mechanism of action of paenibacterin against
Escherichia coli
and
Staphylococcus aureus
was investigated in this study. The cationic lipopeptide paenibacterin showed a strong affinity for the negatively charged lipopolysaccharides (LPS) from the outer membrane of Gram-negative bacteria. Addition of LPS (100 μg/ml) completely eliminated the antimicrobial activity of paenibacterin against
E. coli
. The electrostatic interaction between paenibacterin and LPS may have displaced the divalent cations on the LPS network and thus facilitated the uptake of antibiotic into Gram-negative cells. Paenibacterin also damaged the bacterial cytoplasmic membrane, as evidenced by the depolarization of membrane potential and leakage of intracellular potassium ions from cells of
E. coli
and
S. aureus
. Therefore, the bactericidal activity of paenibacterin is attributed to disruption of the outer membrane of Gram-negative bacteria and damage of the cytoplasmic membrane of both Gram-negative and Gram-positive bacteria. Despite the evidence of membrane damage, this study does not rule out additional bactericidal mechanisms potentially exerted by paenibacterin.
Synthesis of Flavonoid
O
-Pentosides by Escherichia coli through Engineering of Nucleotide Sugar Pathways and Glycosyltransferase
Authors: Han So Hyun , Kim Bong Gyu , Yoon Jeong A. , Chong Youhoon , Ahn Joong-Hoon 
Published: February 2014

ABSTRACT

Plants produce two flavonoid
O
-pentoses, flavonoid
O
-xyloside and flavonoid
O
-arabinoside. However, analyzing their biological properties is difficult because flavonoids are not naturally produced in sufficient quantities. In this study,
Escherichia coli
was used to synthesize the plant-specific flavonoid
O
-pentosides quercetin 3-
O
-xyloside and quercetin 3-
O
-arabinoside. Two strategies were used. First,
E. coli
was engineered to express components of the biosynthetic pathways for UDP-xylose and UDP-arabinose. For UDP-xylose biosynthesis, two genes,
UXS
(UDP-xylose synthase) from
Arabidopsis thaliana
and
ugd
(UDP-glucose dehydrogenase) from
E. coli
, were overexpressed. In addition, the gene encoding ArnA (UDP-
l
-Ara4N formyltransferase/UDP-GlcA C-4″-decarboxylase), which competes with UXS for UDP-glucuronic acid, was deleted. For UDP-arabinose biosynthesis,
UXE
(UDP-xylose epimerase) was overexpressed. Next, we engineered UDP-dependent glycosyltransferases (UGTs) to ensure specificity for UDP-xylose and UDP-arabinose. The
E. coli
strains thus obtained synthesized approximately 160 mg/liter of quercetin 3-
O
-xyloside and quercetin 3-
O
-arabinoside.
Effect of C-Terminal Protein Tags on Pentitol and
l
-Arabinose Transport by Ambrosiozyma monospora Lat1 and Lat2 Transporters in Saccharomyces cerevisiae
Authors: Londesborough John , Richard Peter , Valkonen Mari , Viljanen Kaarina 
Published: February 2014

ABSTRACT

Functional expression in heterologous hosts is often less successful for integral membrane proteins than for soluble proteins. Here, two
Ambrosiozyma monospora
transporters were successfully expressed in
Saccharomyces cerevisiae
as tagged proteins. Growth of
A. monospora
on
l
-arabinose instead of glucose caused transport activities of
l
-arabinose,
l
-arabitol, and ribitol, measured using
l
-[1-
3
H]arabinose,
l
-[
14
C]arabitol, and [
14
C]ribitol of demonstrated purity.
A. monospora LAT1
and
LAT2
genes were cloned earlier by using their ability to improve the growth of genetically engineered
Saccharomyces cerevisiae
on
l
-arabinose. However, the
l
-arabinose and pentitol transport activities of
S. cerevisiae
carrying
LAT1
or
LAT2
are only slightly greater than those of control strains.
S. cerevisiae
carrying the
LAT1
or
LAT2
gene fused in frame to the genes for green fluorescent protein (GFP) or red fluorescent protein (mCherry) or adenylate kinase (AK) exhibited large (>3-fold for
LAT1
; >20-fold for
LAT2
) increases in transport activities. Lat1-mCherry transported
l
-arabinose with high affinity (

K
m

≈ 0.03 mM) and
l
-arabitol and ribitol with very low affinity (

K
m

≥ 75 mM). The Lat2-GFP, Lat2-mCherry, and Lat2-AK fusion proteins could not transport
l
-arabinose but were high-affinity pentitol transporters (

K
m

s ≈ 0.2 mM). The
l
-arabinose and pentitol transport activities of
A. monospora
could not be completely explained by any combination of the observed properties of tagged Lat1 and Lat2, suggesting either that tagging and expression in a foreign membrane alters the transport kinetics of Lat1 and/or Lat2 or that
A. monospora
contains at least one more
l
-arabinose transporter.
Genetic Validation of Aminoacyl-tRNA Synthetases as Drug Targets in Trypanosoma brucei
Authors: Kalidas Savitha , Cestari Igor , Monnerat Severine , Li Qiong , Regmi Sandesh , Hasle Nicholas , Labaied Mehdi , Parsons Marilyn , Stuart Kenneth , Phillips Margaret A. 
Published: February 2014

ABSTRACT

Human African trypanosomiasis (HAT) is an important public health threat in sub-Saharan Africa. Current drugs are unsatisfactory, and new drugs are being sought. Few validated enzyme targets are available to support drug discovery efforts, so our goal was to obtain essentiality data on genes with proven utility as drug targets. Aminoacyl-tRNA synthetases (aaRSs) are known drug targets for bacterial and fungal pathogens and are required for protein synthesis. Here we survey the essentiality of eight
Trypanosoma brucei
aaRSs by RNA interference (RNAi) gene expression knockdown, covering an enzyme from each major aaRS class: valyl-tRNA synthetase (ValRS) (class Ia), tryptophanyl-tRNA synthetase (TrpRS-1) (class Ib), arginyl-tRNA synthetase (ArgRS) (class Ic), glutamyl-tRNA synthetase (GluRS) (class 1c), threonyl-tRNA synthetase (ThrRS) (class IIa), asparaginyl-tRNA synthetase (AsnRS) (class IIb), and phenylalanyl-tRNA synthetase (α and β) (PheRS) (class IIc). Knockdown of mRNA encoding these enzymes in
T. brucei
mammalian stage parasites showed that all were essential for parasite growth and survival
in vitro
. The reduced expression resulted in growth, morphological, cell cycle, and DNA content abnormalities. ThrRS was characterized in greater detail, showing that the purified recombinant enzyme displayed ThrRS activity and that the protein localized to both the cytosol and mitochondrion. Borrelidin, a known inhibitor of ThrRS, was an inhibitor of
T. brucei
ThrRS and showed antitrypanosomal activity. The data show that aaRSs are essential for
T. brucei
survival and are likely to be excellent targets for drug discovery efforts.
The Entamoeba histolytica Dnmt2 Homolog (Ehmeth) Confers Resistance to Nitrosative Stress
Authors: Hertz Rivi , Tovy Ayala , Kirschenbaum Michael , Geffen Meirav , Nozaki Tomoyoshi , Adir Noam , Ankri Serge 
Published: February 2014

ABSTRACTNitric oxide (NO) has antimicrobial properties against many pathogens due to its reactivity as an S-nitrosylating agent. It inhibits many of the key enzymes that are involved in the metabolism and virulence of the parasiteEntamoeba histolyticathrough S-nitrosylation of essential cysteine residues. Very little information is available on the mechanism of resistance to NO by pathogens in general and by this parasite in particular. Here, we report that exposure of the parasites toS-nitrosoglutathione (GSNO), an NO donor molecule, strongly reduces their viability and protein synthesis. However, the deleterious effects of NO were significantly reduced in trophozoites overexpressing Ehmeth, the cytosine-5 methyltransferase of the Dnmt2 family. Since these trophozoites also exhibited high levels of tRNAAspmethylation, the high levels suggested that Ehmeth-mediated tRNAAspmethylation is part of the resistance mechanism to NO. We previously reported that enolase, another glycolytic enzyme, binds to Ehmeth and inhibits its activity. We observed that the amount of Ehmeth-enolase complex was significantly reduced in GSNO-treatedE. histolytica, which explains the aforementioned increase of tRNA methylation. Specifically, we demonstrated via site-directed mutagenesis that cysteine residues 228 and 229 of Ehmeth are susceptible to S-nitrosylation and are crucial for Ehmeth binding to enolase and for Ehmeth-mediated resistance to NO. These results indicate that Ehmeth has a central role in the response of the parasite to NO, and they contribute to the growing evidence that NO is a regulator of epigenetic mechanisms.
Trypanosome Alternative Oxidase Possesses both an N-Terminal and Internal Mitochondrial Targeting Signal
Authors: Hamilton VaNae , Singha Ujjal K. , Smith Joseph T. , Weems Ebony , Chaudhuri Minu 
Published: February 2014

ABSTRACT

Recognition of mitochondrial targeting signals (MTS) by receptor translocases of outer and inner membranes of mitochondria is one of the prerequisites for import of nucleus-encoded proteins into this organelle. The MTS for a majority of trypanosomatid mitochondrial proteins have not been well defined. Here we analyzed the targeting signal for trypanosome alternative oxidase (TAO), which functions as the sole terminal oxidase in the infective form of
Trypanosoma brucei
. Deleting the first 10 of 24 amino acids predicted to be the classical N-terminal MTS of TAO did not affect its import into mitochondria
in vitro
. Furthermore, ectopically expressed TAO was targeted to mitochondria in both forms of the parasite even after deletion of first 40 amino acid residues. However, deletion of more than 20 amino acid residues from the N terminus reduced the efficiency of import. These data suggest that besides an N-terminal MTS, TAO possesses an internal mitochondrial targeting signal. In addition, both the N-terminal MTS and the mature TAO protein were able to target a cytosolic protein, dihydrofolate reductase (DHFR), to a
T. brucei
mitochondrion. Further analysis identified a cryptic internal MTS of TAO, located within amino acid residues 115 to 146, which was fully capable of targeting DHFR to mitochondria. The internal signal was more efficient than the N-terminal MTS for import of this heterologous protein. Together, these results show that TAO possesses a cleavable N-terminal MTS as well as an internal MTS and that these signals act together for efficient import of TAO into mitochondria.
Dictyostelium discoideum Dgat2 Can Substitute for the Essential Function of Dgat1 in Triglyceride Production but Not in Ether Lipid Synthesis
Authors: Du Xiaoli , Herrfurth Cornelia , Gottlieb Thomas , Kawelke Steffen , Feussner Kristin , Rühling Harald , Feussner Ivo , Maniak Markus 
Published: February 2014

ABSTRACT

Triacylglycerol (TAG), the common energy storage molecule, is formed from diacylglycerol and a coenzyme A-activated fatty acid by the action of an acyl coenzyme A:diacylglycerol acyltransferase (DGAT). In order to conduct this step, most organisms rely on more than one enzyme. The two main candidates in
Dictyostelium discoideum
are Dgat1 and Dgat2. We show, by creating single and double knockout mutants, that the endoplasmic reticulum (ER)-localized Dgat1 enzyme provides the predominant activity, whereas the lipid droplet constituent Dgat2 contributes less activity. This situation may be opposite from what is seen in mammalian cells.
Dictyostelium
Dgat2 is specialized for the synthesis of TAG, as is the mammalian enzyme. In contrast, mammalian DGAT1 is more promiscuous regarding its substrates, producing diacylglycerol, retinyl esters, and waxes in addition to TAG. The
Dictyostelium
Dgat1, however, produces TAG, wax esters, and, most interestingly, also neutral ether lipids, which represent a significant constituent of lipid droplets. Ether lipids had also been found in mammalian lipid droplets, but the role of DGAT1 in their synthesis was unknown. The ability to form TAG through either Dgat1 or Dgat2 activity is essential for
Dictyostelium
to grow on bacteria, its natural food substrate.
The K
C
Channel in the
cbb
3
-Type Respiratory Oxygen Reductase from Rhodobacter capsulatus Is Required for Both Chemical and Pumped Protons
Authors: Yıldız Gülgez Gökçe , Gennis Robert B. , Daldal Fevzi , Öztürk Mehmet 
Published: February 2014

ABSTRACT

The heme-copper superfamily of proton-pumping respiratory oxygen reductases are classified into three families (A, B, and C families) based on structural and phylogenetic analyses. Most studies have focused on the A family, which includes the eukaryotic mitochondrial cytochrome
c
oxidase as well as many bacterial homologues. Members of the C family, also called the
cbb
3
-type oxygen reductases, are found only in prokaryotes and are of particular interest because of their presence in a number of human pathogens. All of the heme-copper oxygen reductases require proton-conducting channels to convey chemical protons to the active site for water formation and to convey pumped protons across the membrane. Previous work indicated that there is only one proton-conducting input channel (the K
C
channel) present in the
cbb
3
-type oxygen reductases, which, if correct, must be utilized by both chemical protons and pumped protons. In this work, the effects of mutations in the K
C
channel of the
cbb
3
-type oxygen reductase from
Rhodobacter capsulatus
were investigated by expressing the mutants in a strain lacking other respiratory oxygen reductases. Proton pumping was evaluated by using intact cells, and catalytic oxygen reductase activity was measured in isolated membranes. Two mutations, N346M and Y374F, severely reduced catalytic activity, presumably by blocking the chemical protons required at the active site. One mutation, T272A, resulted in a substantially lower proton-pumping stoichiometry but did not inhibit oxygen reductase activity. These are the first experimental data in support of the postulate that pumped protons are taken up from the bacterial cytoplasm through the K
C
channel.
The Flagellar Soluble Protein FliK Determines the Minimal Length of the Hook in Salmonella enterica Serovar Typhimurium
Authors: Uchida Kaoru , Aizawa Shin-Ichi 
Published: February 2014

ABSTRACT

The length of the flagellar hook is controlled by the soluble protein FliK. FliK is structurally divided into two halves with distinct functions; the N-terminal half determines hook length, while the C-terminal half switches the secretion substrate specificity, consequently terminating hook elongation. FliK properly achieves both functions only when it is secreted. In a previous paper, we showed that a temperature-sensitive
flgE
mutant of
Salmonella enterica
serovar Typhimurium, SJW2219, produced basal bodies with short hooks (average length, 25 nm) at 37°C. In this study, we show that the mutant cells grown at 37°C secrete FliK but not flagellin (FliC), indicating that FliK is abortively secreted into the medium when the hook is shorter than 30 nm. In contrast, FliK unfailingly switches the gate modes when the hook is longer than 30 nm. Taking the FliC, FliK, and FlgM secretion patterns into account, we conclude that FliK determines the minimal length of the hook. We will discuss how FliK detects the critical switching point of the secretion gate.
Analysis of the Loss in Heat and Acid Resistance during Germination of Spores of Bacillus Species
Authors: Luu Stephanie , Setlow Peter 
Published: February 2014

ABSTRACT

A major event in the nutrient germination of spores of
Bacillus
species is release of the spores' large depot of dipicolinic acid (DPA). This event is preceded by both commitment, in which spores continue through germination even if germinants are removed, and loss of spore heat resistance. The latter event is puzzling, since spore heat resistance is due largely to core water content, which does not change until DPA is released during germination. We now find that for spores of two
Bacillus
species, the early loss in heat resistance during germination is most likely due to release of committed spores' DPA at temperatures not lethal for dormant spores. Loss in spore acid resistance during germination also paralleled commitment and was also associated with the release of DPA from committed spores at acid concentrations not lethal for dormant spores. These observations plus previous findings that DPA release during germination is preceded by a significant release of spore core cations suggest that there is a significant change in spore inner membrane permeability at commitment. Presumably, this altered membrane cannot retain DPA during heat or acid treatments innocuous for dormant spores, resulting in DPA-less spores that are rapidly killed.
The LEAF questionnaire: a screening tool for the identification of female athletes at risk for the female athlete triad
Authors: Melin Anna , Tornberg Åsa B , Skouby Sven , Faber Jens , Ritz Christian , Sjödin Anders , Sundgot-Borgen Jorunn 
Published: February 2014

BackgroundLow energy availability (EA) in female athletes with or without an eating disorder (ED) increases the risk of oligomenorrhoea/functional hypothalamic amenorrhoea and impaired bone health, a syndrome called the female athlete triad (Triad). There are validated psychometric instruments developed to detect disordered eating behaviour (DE), but no validated screening tool to detect persistent low EA andTriadconditions, with or without DE/ED, is available.AimThe aim of this observational study was to develop and test a screening tool designed to identify female athletes at risk for theTriad.MethodsFemale athletes (n=84) with 18–39 years of age and training ≥5 times/week filled out the Low Energy Availability in Females Questionnaire (LEAF-Q), which comprised questions regarding injuries and gastrointestinal and reproductive function. Reliability and internal consistency were evaluated in a subsample of female dancers and endurance athletes (n=37). Discriminant as well as concurrent validity was evaluated by testing self-reported data against measured current EA, menstrual function and bone health in endurance athletes from sports such as long distance running and triathlon (n=45).ResultsThe 25-item LEAF-Q produced an acceptable sensitivity (78%) and specificity (90%) in order to correctly classify current EA and/or reproductive function and/or bone health.ConclusionsThe LEAF-Q is brief and easy to administer, and relevant as a complement to existing validated DE screening instruments, when screening female athletes at risk for theTriad, in order to enable early detection and intervention.