bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of efficacy of prion reduction filters using blood from an endogenously infected 263K scrapie hamster model
Authors: McLeod Neil P. , Nugent Philip , Dixon Douglas , Dennis Mike , Cornwall Mark , Mallinson Gary , Watkins Nicholas , Thomas Stephen , Sutton J. Mark
Published: May 2015

BACKGROUND

The P‐Capt prion reduction filter (MacoPharma) removes prion infectivity in model systems. This independent evaluation assesses prion removal from endogenously infected animal blood, using CE‐marked P‐Capt filters, and replicates the proposed use of the filter within the UK Blood Services.

STUDY DESIGN AND METHODS

Two units of blood, generated from 263K scrapie–infected hamsters, were processed using leukoreduction filters (LXT‐quadruple, MacoPharma). Approximately 100 mL of the removed plasma was added back to the red blood cells (RBCs) and the blood was filtered through a P‐Capt filter. Samples of unfiltered whole blood, the prion filter input (RBCs plus plasma and SAGM [RBCPS]), and prion‐filtered leukoreduced blood (PFB) were injected intracranially into hamsters. Clinical symptoms were monitored for 500 ± 1 day, and brains were assessed for spongiosis and prion protein deposit.

RESULTS

In Filtration Run 1, none of the 50 challenged animals were diagnosed with scrapie after inoculation with the RBCPS fraction, while two of 190 hamsters injected with PFB were infected. In Filtration Run 2, one of 49 animals injected with RBCPS and two of 193 hamsters injected with PFB were infected. Run 1 reduced the infectious dose (ID) by 1.467 log (>1.187 log and <0.280 log for leukoreduction and prion filtration, respectively). Run 2 reduced prion infectivity by 1.424 log (1.127 and 0.297 log, respectively). Residual infectivity was estimated at 0.212 ± 0.149 IDs/mL (Run 1) and 0.208 ± 0.147 IDs/mL (Run 2).

CONCLUSION

Leukoreduction removed the majority of infectivity from 263K scrapie hamster blood. The P‐Capt filter removed a proportion of the remaining infectivity, but residual infectivity was observed in two independent processes.
Red blood cell storage duration is not associated with clinical outcomes for acute chest syndrome in children with sickle cell disease
Authors: Fields Melanie E. , Hulbert Monica L. , Chen Ling , Berlin Ari N. , Jackups Ron , Spinella Philip C.
Published: May 2015

BACKGROUND

Providers commonly transfuse sickle cell disease (SCD) patients with fresh red blood cells (RBCs) as treatment for acute chest syndrome (ACS). The objective of this study was to determine if there is an association between the storage duration of RBCs and length of hospitalization and oxygen requirement after transfusion in pediatric SCD patients with ACS.

STUDY DESIGN AND METHODS

This is a retrospective cohort study of pediatric SCD patients with ACS treated with a simple RBC transfusion over 8.5 years at a single institution. Multivariate generalized estimation equation analysis was used to identify associations between storage duration of RBCs and outcome measures.

RESULTS

A total of 234 ACS episodes in 131 subjects were included. The median storage duration of the oldest unit of transfused RBCs was 17 days (interquartile range, 11‐26). The majority of ACS episodes, 77.4%, were treated with 1 unit of transfused RBCs; 20.9% received 2 units; and 1.7% received 3 or more units of RBCs. There was no association between the storage duration of the oldest unit of transfused RBCs and either duration of hospitalization or supplemental oxygen requirement after transfusion in multivariate analyses.

CONCLUSION

This retrospective study is one of the first to investigate the role of the storage lesion in children with SCD and does not support the preferential transfusion of fresh RBCs for ACS. Ultimately, a randomized controlled trial is necessary to determine whether the storage age of RBCs affects outcomes for patients with SCD and ACS.
Romiplostim in the management of the thrombocytopenic surgical patient
Authors: Marshall Ariela L. , Goodarzi Katayoon , Kuter David J.
Published: May 2015

BACKGROUND

Thrombopoietin receptor agonists increase platelet (PLT) counts and are approved for the treatment of chronic immune thrombocytopenia (ITP). These agents may also be useful for the management of thrombocytopenia in patients requiring surgical procedures.

STUDY DESIGN AND METHODS

We conducted a retrospective review of patients with thrombocytopenia (baseline PLT count, <150 × 109/L) who received romiplostim before planned operative procedures. We characterized patient demographics, dosing and duration of romiplostim use, success in achieving PLT counts high enough for surgery, and clinical outcomes.

RESULTS

Eighteen patients underwent a total of 22 operative procedures, including three Jehovah's Witnesses who underwent five procedures. Etiologies of thrombocytopenia included mild ITP (not on romiplostim at baseline), liver disease, hematologic malignancy, and drug‐related thrombocytopenia. Median PLT count at romiplostim initiation was 47 × 109/L (range, 11 × 109‐120 × 109/L). All patients experienced a PLT count increase over a median of 4 weeks; median PLT count at surgery was 144 × 109/L (range, 28 × 109‐370 × 109/L). PLT counts increase to more than 150 × 109/L in four of five Jehovah's Witness patients by the time of surgery. There were no surgical delays or cancellations due to thrombocytopenia. Four bleeding events occurred; none were fatal and none occurred at a PLT count of fewer than 80 × 109/L. No definitive thromboembolic events occurred.

CONCLUSION

Romiplostim successfully increased preoperative PLT counts allowing operative interventions, was well tolerated, did not lead to any significant thromboembolic events, and avoided the need for transfusion. Romiplostim may be of clinical utility in the preoperative management of thrombocytopenic patients, especially those unable to receive or unresponsive to PLT transfusion.
How do we utilize a transfusion safety officer?
Authors: Dunbar Nancy M. , Szczepiorkowski Zbigniew M.
Published: May 2015

The hospital transfusion safety officer (TSO) serves an important role in improving transfusion safety outside of the laboratory through education, active surveillance of the transfusion process (patient identification, blood administration, appropriate ordering practices, transfusion reactions, incidents, and near misses), patient blood management (blood utilization review, minimization of perioperative blood loss, documentation review), quality improvement (transfusion guidelines development, transfusion committee or peer review participation, massive transfusion protocols), and research. We provide a description of how we utilize our hospital‐based TSO to improve transfusion safety and ensure compliance with regulatory requirements and maintenance of certification at our institution.
Expression of the cellular prion protein affects posttransfusion recovery and survival of red blood cells in mice
Authors: Glier Hana , Simak Jan , Panigaj Martin , Gelderman Monique P. , Vostal Jaroslav G. , Holada Karel
Published: May 2015

BACKGROUND

Cellular prion protein (PrPC) is expressed on various cell types including red blood cells (RBCs). The PrPC plays a key role in the pathogenesis of prion diseases, but its physiologic function remains unclear. PrPC is expressed on CD34+ hematopoietic stem cells and its expression is regulated during blood cell differentiation including the erythroid line.

STUDY DESIGN AND METHODS

We investigated the role of PrPC in RBC survival in circulation by transfusing a mix of biotin‐labeled RBCs from wild‐type (WT) and PrP knockout (KO) mice to groups of recipient mice (WT and KO). The proportion of biotinylated RBCs in peripheral blood was estimated by flow cytometry.

RESULTS

KO RBCs displayed a markedly higher first‐day posttransfusion recovery but had a decreased survival in circulation when compared to WT RBCs. Similar results were obtained in all groups of transfused mice, irrespective of RBCs biotinylation level. In addition, we confirmed this finding in an analogous study using Tga20 mice overexpressing PrPC and KO mice of a different genetic background.

CONCLUSION

Our results demonstrate that PrPC expression affects RBC recovery and survival in circulation.
Overcoming problems with the use of ratios as continuous characters for phylogenetic analyses
Authors: Mongiardino Koch Nicolás , Soto Ignacio M. , Ramírez Martín J.
Published: May 2015

The use of quantitative morphometric information for phylogenetic inference has been an intensely debated topic for most of the history of phylogenetic systematics. Despite several drawbacks, the most common strategy to include this sort of data into phylogenetic studies is the use of ratios, that is quotients between morphometric variables. Here, we discuss one particular problem associated with such methodology: the fact that the often arbitrary election of which variable serves as numerator and which as denominator affects the phylogenetic outcome of the analysis. We describe the cause for such an effect, and study its implications with the use of several published data matrices. Alternative coding schemes for ratio characters result in very different phylogenetic hypotheses, an effect that may even be strong enough to affect studies that combine continuous and discrete morphological information. Some of the resulting incongruence is produced by the differences in magnitude of the continuous characters involved, although different rescaling techniques are shown to decrease, but not eliminate, the confounding effect. To eliminate such problematic effect, ratios should be either log‐transformed before their use or replaced by more effective ways to capture morphometric information.
Patterns of reproductive‐mode evolution in Old World tree frogs (Anura, Rhacophoridae)
Authors: Meegaskumbura Madhava , Senevirathne Gayani , Biju S. D. , Garg Sonali , Meegaskumbura Suyama , Pethiyagoda Rohan , Hanken James , Schneider Christopher J.
Published: May 2015

The Old World tree frogs (Anura: Rhacophoridae), with 387 species, display a remarkable diversity of reproductive modes – aquatic breeding, terrestrial gel nesting, terrestrial foam nesting and terrestrial direct development. The evolution of these modes has until now remained poorly studied in the context of recent phylogenies for the clade. Here, we use newly obtained DNA sequences from three nuclear and two mitochondrial gene fragments, together with previously published sequence data, to generate a well‐resolved phylogeny from which we determine major patterns of reproductive‐mode evolution. We show that basal rhacophorids have fully aquatic eggs and larvae. Bayesian ancestral‐state reconstructions suggest that terrestrial gel‐encapsulated eggs, with early stages of larval development completed within the egg outside of water, are an intermediate stage in the evolution of terrestrial direct development and foam nesting. The ancestral forms of almost all currently recognized genera (except the fully aquatic basal forms) have a high likelihood of being terrestrial gel nesters. Direct development and foam nesting each appear to have evolved at least twice within Rhacophoridae, suggesting that reproductive modes are labile and may arise multiple times independently. Evolution from a fully aquatic reproductive mode to more terrestrial modes (direct development and foam nesting) occurs through intermediate gel nesting ancestral forms. This suggests that gel nesting is not only a possible transitional state for the evolution of terrestriality, but also that it is a versatile reproductive mode that may give rise to other terrestrial reproductive modes. Evolution of foam nesting may have enabled rhacophorids to lay a larger number of eggs in more open and drier habitats, where protection from desiccation is important. Terrestrial direct development allows frogs to lay eggs independent of bodies of water, in a diversity of humid habitats, and may represent a key innovation that facilitated the evolution of nearly half of all known rhacophorid species.
Seeing double: pseudocryptic diversity in the Doriopsilla albopunctata‐Doriopsilla gemela species complex of the north‐eastern Pacific
Authors: Hoover Craig , Lindsay Tabitha , Goddard Jeffrey H. R. , Valdés Ángel
Published: May 2015

Molecular analyses of samples of Doriopsilla albopunctata and its ‘twin’ species Doriopsilla gemela collected from their entire geographic ranges in the north‐eastern Pacific revealed an unexpected level of species diversity. Species delimitation analyses recovered five distinct species in the complex. When the specimens were re‐examined morphologically in the light of the molecular results, some external and internal differences between them became evident; thus, these species are considered pseudocryptic. The ranges of the three species previously named D. albopunctata overlap partially or entirely, and this along with anecdotal evidence of prezygotic isolation suggests sympatric speciation may be at play. On the contrary, the ranges of the two species previously named D. gemela do not overlap and their developmental modes differ. A review of the literature was conducted to clarify the taxonomy of the species complex, and we concluded that two available names (D. fulva and D. gemela) can be confidently assigned to two of the species. The original description of D. albopunctata is ambiguous, but the common usage of the name is fixed herein with the designation of a neotype for the most common intertidal southern California species. Finally, two new names are introduced for the remaining two species.
Particle formation and its control in dual frequency plasma etching reactors
Authors: Kim Munsu , Cheong Hee-Woon , Whang Ki-Woong
Published: May 2015

The behavior of a particle cloud in plasma etching reactors at the moment when radio frequency (RF) power changes, that is, turning off and transition steps, was observed using the laser-light-scattering method. Two types of reactors, dual-frequency capacitively coupled plasma (CCP) and the hybrid CCP/inductively coupled plasma (ICP), were set up for experiments. In the hybrid CCP/ICP reactor (hereafter ICP reactor), the position and shape of the cloud were strongly dependent on the RF frequency. The particle cloud becomes larger and approaches the electrode as the RF frequency increases. By turning the lower frequency power off later with a small delay time, the particle cloud is made to move away from the electrode. Maintaining lower frequency RF power only was also helpful to reduce the particle cloud size during this transition step. In the ICP reactor, a sufficient bias power is necessary to make a particle trap appear. A similar particle cloud to that in the CCP reactor was observed around the sheath region of the lower electrode. The authors can also use the low-frequency effect to move the particle cloud away from the substrate holder if two or more bias powers are applied to the substrate holder. The dependence of the particle behavior on the RF frequencies suggests that choosing the proper frequency at the right moment during RF power changes can reduce particle contamination effectively.
Northwestern Pacific typhoon intensity controlled by changes in ocean temperatures
Authors: Mei Wei , Xie Shang-Ping , Primeau François , McWilliams James C. , Pasquero Claudia
Published: May 2015

Ocean warming is a predicting factor for typhoon intensity.
Tailoring pathway modularity in the biosynthesis of erythromycin analogs heterologously engineered in
E. coli
Authors: Zhang Guojian , Li Yi , Fang Lei , Pfeifer Blaine A.
Published: May 2015

A redesigned production process enables expanded molecular variation and altered bioactivity for the antibiotic erythromycin.
Isotopically enhanced triple-quantum-dot qubit
Authors: Eng Kevin , Ladd Thaddeus D. , Smith Aaron , Borselli Matthew G. , Kiselev Andrey A. , Fong Bryan H. , Holabird Kevin S. , Hazard Thomas M. , Huang Biqin , Deelman Peter W. , Milosavljevic Ivan , Schmitz Adele E. , Ross Richard S. , Gyure Mark F. , Hunter Andrew T.
Published: May 2015

Three coupled quantum dots in isotopically purified silicon enable all-electrical qubit control with long coherence time.
CysK Plays a Role in Biofilm Formation and Colonization by Vibrio fischeri
Authors: Singh Priyanka , Brooks John F. , Ray Valerie A. , Mandel Mark J. , Visick Karen L.
Published: May 2015

A biofilm, or a matrix-embedded community of cells, promotes the ability of the bacterium
Vibrio fischeri
to colonize its symbiotic host, the Hawaiian squid
Euprymna scolopes
. Biofilm formation and colonization depend on
syp
, an 18-gene polysaccharide locus. To identify other genes necessary for biofilm formation, we screened for mutants that failed to form wrinkled colonies, a type of biofilm. We obtained several with defects in genes required for cysteine metabolism, including
cysH
,
cysJ
,
cysK
, and
cysN
. The
cysK
mutant exhibited the most severe wrinkling defect. It could be complemented with a wild-type copy of the
cysK
gene, which encodes
O
-acetylserine sulfhydrolase, or by supplementing the medium with additional cysteine. None of a number of other mutants defective for biosynthetic genes negatively impacted wrinkled colony formation, suggesting a specific role for CysK. CysK did not appear to control activation of Syp regulators or transcription of the
syp
locus, but it did influence production of the Syp polysaccharide. Under biofilm-inducing conditions, the
cysK
mutant retained the same ability as that of the parent strain to adhere to the agar surface. The
cysK
mutant also exhibited a defect in pellicle production that could be complemented by the
cysK
gene but not by cysteine, suggesting that, under these conditions, CysK is important for more than the production of cysteine. Finally, our data reveal a role for
cysK
in symbiotic colonization by
V. fischeri
. Although many questions remain, this work provides insights into additional factors required for biofilm formation and colonization by
V. fischeri
.
SnPKS19
Encodes the Polyketide Synthase for Alternariol Mycotoxin Biosynthesis in the Wheat Pathogen Parastagonospora nodorum
Authors: Chooi Yit-Heng , Muria-Gonzalez Mariano Jordi , Mead Oliver L. , Solomon Peter S.
Published: May 2015

Alternariol (AOH) is an important mycotoxin from the
Alternaria
fungi. AOH was detected for the first time in the wheat pathogen
Parastagonospora nodorum
in a recent study. Here, we exploited reverse genetics to demonstrate that SNOG_15829 (
SnPKS19
), a close homolog of
Penicillium aethiopicum
norlichexanthone (NLX) synthase gene
gsfA
, is required for AOH production. We further validate that
SnPKS19
is solely responsible for AOH production by heterologous expression in
Aspergillus nidulans
. The expression profile of
SnPKS19
based on previous
P. nodorum
microarray data correlated with the presence of AOH
in vitro
and its absence
in planta
. Subsequent characterization of the Δ
SnPKS19
mutants showed that
SnPKS19
and AOH are not involved in virulence and oxidative stress tolerance. Identification and characterization of the
P. nodorum
SnPKS19
cast light on a possible alternative AOH synthase gene in
Alternaria alternata
and allowed us to survey the distribution of AOH synthase genes in other fungal genomes. We further demonstrate that phylogenetic analysis could be used to differentiate between AOH synthases and the closely related NLX synthases. This study provides the basis for studying the genetic regulation of AOH production and for development of molecular diagnostic methods for detecting AOH-producing fungi in the future.
“Wigglesworthia morsitans” Folate (Vitamin B
9
) Biosynthesis Contributes to Tsetse Host Fitness
Authors: Snyder Anna K. , Rio Rita V. M.
Published: May 2015

Closely related ancient endosymbionts may retain minor genomic distinctions through evolutionary time, yet the biological relevance of these small pockets of unique loci remains unknown. The tsetse fly (Diptera: Glossinidae), the sole vector of lethal African trypanosomes (
Trypanosoma
spp.), maintains an ancient and obligate mutualism with species belonging to the gammaproteobacterium
Wigglesworthia
. Extensive concordant evolution with associated
Wigglesworthia
species has occurred through tsetse species radiation. Accordingly, the retention of unique symbiont loci between
Wigglesworthia
genomes may prove instrumental toward host species-specific biological traits. Genome distinctions between “
Wigglesworthia
morsitans
” (harbored within
Glossina morsitans
bacteriomes) and the basal species
Wigglesworthia glossinidia
(harbored within
Glossina brevipalpis
bacteriomes) include the retention of chorismate and downstream folate (vitamin B
9
) biosynthesis capabilities, contributing to distinct symbiont metabolomes. Here, we demonstrate that these
W. morsitans
pathways remain functionally intact, with folate likely being systemically disseminated through a synchronously expressed tsetse folate transporter within bacteriomes. The folate produced by
W. morsitans
is demonstrated to be pivotal for
G. morsitans
sexual maturation and reproduction. Modest differences between ancient symbiont genomes may still play key roles in the evolution of their host species, particularly if loci are involved in shaping host physiology and ecology. Enhanced knowledge of the
Wigglesworthia
-tsetse mutualism may also provide novel and specific avenues for vector control.
Resistance to Bacillus thuringiensis Toxin Cry2Ab in Trichoplusia ni Is Conferred by a Novel Genetic Mechanism
Authors: Song Xiaozhao , Kain Wendy , Cassidy Douglas , Wang Ping
Published: May 2015

The resistance to the
Bacillus thuringiensis
(Bt) toxin Cry2Ab in a greenhouse-originated
Trichoplusia ni
strain resistant to both Bt toxins Cry1Ac and Cry2Ab was characterized. Biological assays determined that the Cry2Ab resistance in the
T. ni
strain was a monogenic recessive trait independent of Cry1Ac resistance, and there existed no significant cross-resistance between Cry1Ac and Cry2Ab in
T. ni
. From the dual-toxin-resistant
T. ni
strain, a strain resistant to Cry2Ab only was isolated, and the Cry2Ab resistance trait was introgressed into a susceptible laboratory strain to facilitate comparative analysis of the Cry2Ab resistance with the susceptible
T. ni
strain. Results from biochemical analysis showed no significant difference between the Cry2Ab-resistant and -susceptible
T. ni
larvae in midgut proteases, including caseinolytic proteolytic activity and zymogram profile and serine protease activities, in midgut aminopeptidase and alkaline phosphatase activity, and in midgut esterases and hemolymph plasma melanization activity. For analysis of genetic linkage of Cry2Ab resistance with potential Cry toxin receptor genes, molecular markers for the midgut cadherin, alkaline phosphatase (ALP), and aminopeptidase N (APN) genes were identified between the original greenhouse-derived dual-toxin-resistant and the susceptible laboratory
T. ni
strains. Genetic linkage analysis showed that the Cry2Ab resistance in
T. ni
was not genetically associated with the midgut genes coding for the cadherin, ALP, and 6 APNs (APN1 to APN6) nor associated with the ABC transporter gene
ABCC2
. Therefore, the Cry2Ab resistance in
T. ni
is conferred by a novel but unknown genetic mechanism.
DNA Microarray-Based Identification of Genes Regulated by NtrC in Bradyrhizobium japonicum
Authors: Franck William L. , Qiu Jing , Lee Hae-In , Chang Woo-Suk , Stacey Gary
Published: May 2015

The
Bradyrhizobium japonicum
NtrBC two-component system is a critical regulator of cellular nitrogen metabolism, including the acquisition and catabolism of nitrogenous compounds. To better define the roles of this system, genome-wide transcriptional profiling was performed to identify the NtrC regulon during the response to nitrogen limitation. Upon cells perceiving low intracellular nitrogen, they stimulate the phosphorylation of NtrC, which induces genes responsible for alteration of the core glutamine synthetase/glutamate synthase nitrogen assimilation pathway, including the genes for the glutamine synthetases and PII proteins. In addition, genes responsible for the import and utilization of multiple nitrogen sources, specifically nitrate and nitrite, were upregulated by NtrC activation. Mutational analysis of a candidate nitrite reductase revealed a role for NtrC in regulating the assimilation of nitrite, since mutations in both
ntrC
and the gene encoding the candidate nitrite reductase abolished the ability to grow on nitrite as a sole nitrogen source.
Novel N4 Bacteriophages Prevail in the Cold Biosphere
Authors: Zhan Yuanchao , Buchan Alison , Chen Feng
Published: May 2015

Coliphage N4 is a lytic bacteriophage discovered nearly half a century ago, and it was considered to be a “genetic orphan” until very recently, when several additional N4-like phages were discovered to infect nonenteric bacterial hosts. Interest in this genus of phages is stimulated by their unique genetic features and propagation strategies. To better understand the ecology of N4-like phages, we investigated the diversity and geographic patterns of N4-like phages by examining 56 Chesapeake Bay viral communities, using a PCR-clone library approach targeting a diagnostic N4-like DNA polymerase gene. Many new lineages of N4-like phages were found in the bay, and their genotypes shift from the lower to the upper bay. Interestingly, signature sequences of N4-like phages were recovered only from winter month samples, when water temperatures were below 4°C. An analysis of existing metagenomic libraries from various aquatic environments supports the hypothesis that N4-like phages are most prolific in colder waters. In particular, a high number of N4-like phages were detected in Organic Lake, Antarctica, a cold and hypersaline system. The prevalence of N4-like phages in the cold biosphere suggests these viruses possess yet-to-be-determined mechanisms that facilitate lytic infections under cold conditions.
Phenotypic Heterogeneity, a Phenomenon That May Explain Why Quorum Sensing Does Not Always Result in Truly Homogenous Cell Behavior
Authors: Grote Jessica , Krysciak Dagmar , Streit Wolfgang R.
Published: May 2015

Phenotypic heterogeneity describes the occurrence of “nonconformist” cells within an isogenic population. The nonconformists show an expression profile partially different from that of the remainder of the population. Phenotypic heterogeneity affects many aspects of the different bacterial lifestyles, and it is assumed that it increases bacterial fitness and the chances for survival of the whole population or smaller subpopulations in unfavorable environments. Well-known examples for phenotypic heterogeneity have been associated with antibiotic resistance and frequently occurring persister cells. Other examples include heterogeneous behavior within biofilms, DNA uptake and bacterial competence, motility (i.e., the synthesis of additional flagella), onset of spore formation, lysis of phages within a small subpopulation, and others. Interestingly, phenotypic heterogeneity was recently also observed with respect to quorum-sensing (QS)-dependent processes, and the expression of autoinducer (AI) synthase genes and other QS-dependent genes was found to be highly heterogeneous at a single-cell level. This phenomenon was observed in several Gram-negative bacteria affiliated with the genera
Vibrio
,
Dinoroseobacter
,
Pseudomonas
,
Sinorhizobium
, and
Mesorhizobium
. A similar observation was made for the Gram-positive bacterium
Listeria monocytogenes
. Since AI molecules have historically been thought to be the keys to homogeneous behavior within isogenic populations, the observation of heterogeneous expression is quite intriguing and adds a new level of complexity to the QS-dependent regulatory networks. All together, the many examples of phenotypic heterogeneity imply that we may have to partially revise the concept of homogeneous and coordinated gene expression in isogenic bacterial populations.
Living in an Extremely Polluted Environment: Clues from the Genome of Melanin-Producing Aeromonas salmonicida subsp. pectinolytica 34mel
T
Authors: Pavan María Elisa , Pavan Esteban E. , López Nancy I. , Levin Laura , Pettinari M. Julia
Published: May 2015

Aeromonas salmonicida
subsp.
pectinolytica
34mel
T
can be considered an extremophile due to the characteristics of the heavily polluted river from which it was isolated. While four subspecies of
A. salmonicida
are known fish pathogens, 34mel
T
belongs to the only subspecies isolated solely from the environment. Genome analysis revealed a high metabolic versatility, the capability to cope with diverse stress agents, and the lack of several virulence factors found in pathogenic
Aeromonas
. The most relevant phenotypic characteristics of 34mel
T
are pectin degradation, a distinctive trait of
A. salmonicida
subsp.
pectinolytica
, and melanin production. Genes coding for three pectate lyases were detected in a cluster, unique to this microorganism, that contains all genes needed for pectin degradation. Melanin synthesis in 34mel
T
is hypothesized to occur through the homogentisate pathway, as no tyrosinases or laccases were detected and the homogentisate 1,2-dioxygenase gene is inactivated by a transposon insertion, leading to the accumulation of the melanin precursor homogentisate. Comparative genome analysis of other melanogenic
Aeromonas
strains revealed that this gene was inactivated by transposon insertions or point mutations, indicating that melanin biosynthesis in
Aeromonas
occurs through the homogentisate pathway. Horizontal gene transfer could have contributed to the adaptation of 34mel
T
to a highly polluted environment, as 13 genomic islands were identified in its genome, some of them containing genes coding for fitness-related traits. Heavy metal resistance genes were also found, along with others associated with oxidative and nitrosative stresses. These characteristics, together with melanin production and the ability to use different substrates, may explain the ability of this microorganism to live in an extremely polluted environment.
Comparison of Quantitative PCR and Droplet Digital PCR Multiplex Assays for Two Genera of Bloom-Forming Cyanobacteria, Cylindrospermopsis and Microcystis
Authors: Te Shu Harn , Chen Enid Yingru , Gin Karina Yew-Hoong
Published: May 2015

The increasing occurrence of harmful cyanobacterial blooms, often linked to deteriorated water quality and adverse public health effects, has become a worldwide concern in recent decades. The use of molecular techniques such as real-time quantitative PCR (qPCR) has become increasingly popular in the detection and monitoring of harmful cyanobacterial species. Multiplex qPCR assays that quantify several toxigenic cyanobacterial species have been established previously; however, there is no molecular assay that detects several bloom-forming species simultaneously.
Microcystis
and
Cylindrospermopsis
are the two most commonly found genera and are known to be able to produce microcystin and cylindrospermopsin hepatotoxins. In this study, we designed primers and probes which enable quantification of these genera based on the RNA polymerase C1 gene for
Cylindrospermopsis
species and the c-phycocyanin beta subunit-like gene for
Microcystis
species. Duplex assays were developed for two molecular techniques—qPCR and droplet digital PCR (ddPCR). After optimization, both qPCR and ddPCR assays have high linearity and quantitative correlations for standards. Comparisons of the two techniques showed that qPCR has higher sensitivity, a wider linear dynamic range, and shorter analysis time and that it was more cost-effective, making it a suitable method for initial screening. However, the ddPCR approach has lower variability and was able to handle the PCR inhibition and competitive effects found in duplex assays, thus providing more precise and accurate analysis for bloom samples.
Gene Replacement for the Generation of Designed Novel Avermectin Derivatives with Enhanced Acaricidal and Nematicidal Activities
Authors: Huang Jun , Chen An-Liang , Zhang Hui , Yu Zhen , Li Mei-Hong , Li Na , Lin Jia-Tan , Bai Hua , Wang Ji-Dong , Zheng Yu-Guo
Published: May 2015

Avermectin (AVM) and ivermectin (IVM) are potent pesticides and acaricides which have been widely used during the past 30 years. As insect resistance to AVM and IVM is greatly increasing, alternatives are urgently needed. Here, we report two novel AVM derivatives, tenvermectin A (TVM A) and TVM B, which are considered a potential new generation of agricultural and veterinary drugs. The molecules of the TVMs were designed based on structure and pharmacological property comparisons among AVM, IVM, and milbemycin (MBM). To produce TVMs, a genetically engineered strain, MHJ1011, was constructed from
Streptomyces avermitilis
G8-17, an AVM industrial strain. In MHJ1011, the native
aveA1
gene was seamlessly replaced with
milA1
from
Streptomyces hygroscopicus
. The total titer of the two TVMs produced by MHJ1011 reached 3,400 mg/liter. Insecticidal tests proved that TVM had enhanced activities against
Tetranychus cinnabarinus
and
Bursaphelenchus xylophilus
, as desired. This study provides a typical example of exploration for novel active compounds through a new method of polyketide synthase (PKS) reassembly for gene replacement. The results of the insecticidal tests may be of use in elucidating the structure-activity relationship of AVMs and MBMs.
Dual-Color Bioluminescence Imaging for Simultaneous Monitoring of the Intestinal Persistence of Lactobacillus plantarum and Lactococcus lactis in Living Mice
Authors: Daniel Catherine , Poiret Sabine , Dennin Véronique , Boutillier Denise , Lacorre Delphine Armelle , Foligné Benoit , Pot Bruno
Published: May 2015

Lactic acid bacteria are found in the gastrointestinal tract of mammals and have received tremendous attention due to their health-promoting properties. We report the development of two dual-color luciferase-producing
Lactobacillus
(
Lb
.)
plantarum
and
Lactococcus
(
Lc
.)
lactis
strains for noninvasive simultaneous tracking in the mouse gastrointestinal tract. We previously described the functional expression of the red luciferase mutant (CBRluc) from
Pyrophorus plagiophthalamus
in
Lb. plantarum
NCIMB8826 and
Lc. lactis
MG1363 (C. Daniel, S. Poiret, V. Dennin, D. Boutillier, and B. Pot, Appl Environ Microbiol
79:
1086–1094, 2013,
http://dx.doi.org/10.1128/AEM.03221-12
). In this study, we determined that CBRluc is a better-performing luciferase for
in vivo
localization of both lactic acid bacteria after oral administration than the green click beetle luciferase mutant construct developed in this study. We further established the possibility to simultaneously detect red- and green-emitting lactic acid bacteria by dual-wavelength bioluminescence imaging in combination with spectral unmixing. The difference in spectra of light emission by the red and green click beetle luciferase mutants and dual bioluminescence detection allowed
in vitro
and
in vivo
quantification of the red and green emitted signals; thus, it allowed us to monitor the dynamics and fate of the two bacterial populations simultaneously. Persistence and viability of both strains simultaneously administered to mice in different ratios was studied
in vivo
in anesthetized mice and
ex vivo
in mouse feces. The application of dual-luciferase-labeled bacteria has considerable potential to simultaneously study the interactions and potential competitions of different targeted bacteria and their hosts.
Tulane Virus as a Potential Surrogate To Mimic Norovirus Behavior in Oysters
Authors: Drouaz Najoua , Schaeffer Julien , Farkas Tibor , Le Pendu Jacques , Le Guyader Françoise S.
Published: May 2015

Oyster contamination by noroviruses is an important health and economic problem. The present study aimed to compare the behaviors of Norwalk virus (the prototype genogroup I norovirus) and two culturable viruses: Tulane virus and mengovirus. After bioaccumulation, tissue distributions were quite similar for Norwalk virus and Tulane virus, with the majority of viral particles detected in digestive tissues, while mengovirus was detected in large amounts in the gills and mantle as well as in digestive tissues. The levels of persistence of all three viruses over 8 days were comparable, but clear differences were observed over longer periods, with Norwalk and Tulane viruses displaying rather similar half-lives, unlike mengovirus, which was cleared more rapidly. These results indicate that Tulane virus may be a good surrogate for studying norovirus behavior in oysters, and they confirm the prolonged persistence of Norwalk virus in oyster tissues.
Genes Associated with Desiccation and Osmotic Stress in Listeria monocytogenes as Revealed by Insertional Mutagenesis
Authors: Hingston Patricia A. , Piercey Marta J. , Truelstrup Hansen Lisbeth
Published: May 2015

Listeria monocytogenes
is a foodborne pathogen whose survival in food processing environments may be associated with its tolerance to desiccation. To probe the molecular mechanisms used by this bacterium to adapt to desiccation stress, a transposon library of 11,700
L. monocytogenes
mutants was screened, using a microplate assay, for strains displaying increased or decreased desiccation survival (43% relative humidity, 15°C) in tryptic soy broth (TSB). The desiccation phenotypes of selected mutants were subsequently assessed on food-grade stainless steel (SS) coupons in TSB plus 1% glucose (TSB-glu). Single transposon insertions in mutants exhibiting a change in desiccation survival of >0.5 log CFU/cm
2
relative to that of the wild type were determined by sequencing arbitrary PCR products. Strain morphology, motility, and osmotic stress survival (in TSB-glu plus 20% NaCl) were also analyzed. The initial screen selected 129 desiccation-sensitive (DS) and 61 desiccation-tolerant (DT) mutants, out of which secondary screening on SS confirmed 15 DT and 15 DS mutants. Among the DT mutants, seven immotile and flagellum-less strains contained transposons in genes involved in flagellum biosynthesis (
fliP
,
flhB
,
flgD
,
flgL
) and motor control (
motB
,
fliM
,
fliY
), while others harbored transposons in genes involved in membrane lipid biosynthesis, energy production, potassium uptake, and virulence. The genes that were interrupted in the 15 DS mutants included those involved in energy production, membrane transport, protein metabolism, lipid biosynthesis, oxidative damage control, and putative virulence. Five DT and 14 DS mutants also demonstrated similar significantly (
P
< 0.05) different survival relative to that of the wild type when exposed to osmotic stress, demonstrating that some genes likely have similar roles in allowing the organism to survive the two water stresses.
Dynamics of the Streptococcus gordonii Transcriptome in Response to Medium, Salivary α-Amylase, and Starch
Authors: Haase Elaine M. , Feng Xianghui , Pan Jiachuan , Miecznikowski Jeffrey C. , Scannapieco Frank A.
Published: May 2015

Streptococcus gordonii
, a primary colonizer of the tooth surface, interacts with salivary α-amylase via amylase-binding protein A (AbpA). This enzyme hydrolyzes starch to glucose, maltose, and maltodextrins that can be utilized by various oral bacteria for nutrition. Microarray studies demonstrated that AbpA modulates gene expression in response to amylase, suggesting that the amylase-streptococcal interaction may function in ways other than nutrition. The goal of this study was to explore the role of AbpA in gene regulation through comparative transcriptional profiling of wild-type KS1 and AbpA
−
mutant KS1Ω
abpA
under various environmental conditions. A portion of the total RNA isolated from mid-log-phase cells grown in 5% CO
2
in (i) complex medium with or without amylase, (ii) defined medium (DM) containing 0.8% glucose with/without amylase, and (iii) DM containing 0.2% glucose and amylase with or without starch was reverse transcribed to cDNA and the rest used for RNA sequencing. Changes in the expression of selected genes were validated by quantitative reverse transcription-PCR. Maltodextrin-associated genes, fatty acid synthesis genes and competence genes were differentially expressed in a medium-dependent manner. Genes in another cluster containing a putative histidine kinase/response regulator, peptide methionine sulfoxide reductase, thioredoxin protein, lipoprotein, and cytochrome
c
-type protein were downregulated in KS1Ω
abpA
under all of the environmental conditions tested. Thus, AbpA appears to modulate genes associated with maltodextrin utilization/transport and fatty acid synthesis. Importantly, in all growth conditions AbpA was associated with increased expression of a potential two-component signaling system associated with genes involved in reducing oxidative stress, suggesting a role in signal transduction and stress tolerance.
Inactivation of Escherichia coli Cells in Aqueous Solution by Atmospheric-Pressure N
2
, He, Air, and O
2
Microplasmas
Authors: Zhou Renwu , Zhang Xianhui , Bi Zhenhua , Zong Zichao , Niu Jinhai , Song Ying , Liu Dongping , Yang Size
Published: May 2015

Atmospheric-pressure N
2
, He, air, and O
2
microplasma arrays have been used to inactivate
Escherichia coli
cells suspended in aqueous solution. Measurements show that the efficiency of inactivation of
E. coli
cells is strongly dependent on the feed gases used, the plasma treatment time, and the discharge power. Compared to atmospheric-pressure N
2
and He microplasma arrays, air and O
2
microplasma arrays may be utilized to more efficiently kill
E. coli
cells in aqueous solution. The efficiencies of inactivation of
E. coli
cells in water can be well described by using the chemical reaction rate model, where reactive oxygen species play a crucial role in the inactivation process. Analysis indicates that plasma-generated reactive species can react with
E. coli
cells in water by direct or indirect interactions.
Total Biosynthesis and Diverse Applications of the Nonribosomal Peptide-Polyketide Siderophore Yersiniabactin
Authors: Ahmadi Mahmoud Kamal , Fawaz Samar , Jones Charles H. , Zhang Guojian , Pfeifer Blaine A.
Published: May 2015

Yersiniabactin (Ybt) is a mixed nonribosomal peptide-polyketide natural product natively produced by the pathogenYersinia pestis. The compound enables iron scavenging capabilities upon host infection and is biosynthesized by a nonribosomal peptide synthetase featuring a polyketide synthase module. This pathway has been engineered for expression and biosynthesis usingEscherichia colias a heterologous host. In the current work, the biosynthetic process for Ybt formation was improved through the incorporation of a dedicated step to eliminate the need for exogenous salicylate provision. When this improvement was made, the compound was tested in parallel applications that highlight the metal-chelating nature of the compound. In the first application, Ybt was assessed as a rust remover, demonstrating a capacity of ∼40% compared to a commercial removal agent and ∼20% relative to total removal capacity. The second application tested Ybt in removing copper from a variety of nonbiological and biological solution mixtures. Success across a variety of media indicates potential utility in diverse scenarios that include environmental and biomedical settings.
Candida albicans Kinesin Kar3 Depends on a Cik1-Like Regulatory Partner Protein for Its Roles in Mating, Cell Morphogenesis, and Bipolar Spindle Formation
Authors: Frazer Corey , Joshi Monika , Delorme Caroline , Davis Darlene , Bennett Richard J. , Allingham John S.
Published: May 2015

Candida albicans
is a major fungal pathogen whose virulence is associated with its ability to transition from a budding yeast form to invasive hyphal filaments. The kinesin-14 family member
Ca
Kar3 is required for transition between these morphological states, as well as for mitotic progression and karyogamy. While kinesin-14 proteins are ubiquitous,
Ca
Kar3 homologs in hemiascomycete fungi are unique because they form heterodimers with noncatalytic kinesin-like proteins. Thus,
Ca
Kar3-based motors may represent a novel antifungal drug target. We have identified and examined the roles of a kinesin-like regulator of
Ca
Kar3. We show that
orf19.306
(dubbed
CaCIK1
) encodes a protein that forms a heterodimer with
Ca
Kar3, localizes
Ca
Kar3 to spindle pole bodies, and can bind microtubules and influence
Ca
Kar3 mechanochemistry despite lacking an ATPase activity of its own. Similar to
Ca
Kar3 depletion, loss of
Ca
Cik1 results in cell cycle arrest, filamentation defects, and an inability to undergo karyogamy. Furthermore, an examination of the spindle structure in cells lacking either of these proteins shows that a large proportion have a monopolar spindle or two dissociated half-spindles, a phenotype unique to the
C. albicans
kinesin-14 homolog. These findings provide new insights into mitotic spindle structure and kinesin motor function in
C. albicans
and identify a potentially vulnerable target for antifungal drug development.
Lipid Biosynthetic Genes Affect Candida albicans Extracellular Vesicle Morphology, Cargo, and Immunostimulatory Properties
Authors: Wolf Julie M. , Espadas Javier , Luque-Garcia Jose , Reynolds Todd , Casadevall Arturo
Published: May 2015

Microbial secretion is integral for regulating cell homeostasis as well as releasing virulence factors during infection. The genes encoding phosphatidylserine synthase (
CHO1
) and phosphatidylserine decarboxylase (
PSD1
and
PSD2
) are
Candida albicans
genes involved in phospholipid biosynthesis, and mutations in these genes affect mitochondrial function, cell wall thickness, and virulence in mice. We tested the roles of these genes in several agar-based secretion assays and observed that the
cho1
Δ/Δ and
psd1
Δ/Δ
psd2
Δ/Δ strains manifested less protease and phospholipase activity. Since extracellular vesicles (EVs) are surrounded by a lipid membrane, we investigated the effects of these mutations on EV structure, composition, and biological activity. The
cho1
Δ/Δ mutant releases EVs comparable in size to wild-type EVs, but EVs from the
psd1
Δ/Δ
psd2
Δ/Δ strain are much larger than those from the wild type, including a population of >100-nm EVs not observed in the EVs from the wild type. Proteomic analysis revealed that EVs from both mutants had a significantly different protein cargo than that of EVs from the wild type. EVs were tested for their ability to activate NF-κB in bone marrow-derived macrophage cells. While wild-type and
psd1
Δ/Δ
psd2
Δ/Δ mutant-derived EVs activated NF-κB, the
cho1
Δ/Δ mutant-derived EV did not. These studies indicate that the presence and absence of these
C. albicans
genes have qualitative and quantitative effects on EV size, composition, and immunostimulatory phenotypes that highlight a complex interplay between lipid metabolism and vesicle production.
Rgc2 Regulator of Glycerol Channel Fps1 Functions as a Homo- and Heterodimer with Rgc1
Authors: Lee Jongmin , Levin David E.
Published: May 2015

The plasma membrane aquaglyceroporin Fps1 is responsible for glycerol transport in yeast in response to changes in extracellular osmolarity. Fps1 functions as a homotetramer, and control of its channel activity in response to hyperosmotic shock involves a redundant pair of fungus-specific regulators, Rgc1 and Rgc2 (regulators of the glycerol channel), and the mitogen-activatd protein kinase (MAPK) Hog1 (high-osmolarity glycerol response). Rgc1 and Rgc2 maintain Fps1 in an open-channel state by binding to its C-terminal cytoplasmic domain. Phosphorylation of Rgc1 and Rgc2 by Hog1 induces their eviction from Fps1 and consequent channel closure. In the absence of Fps1 channel function, cells experience chronic cell wall stress, which may be exploited for antifungal drug development. We show here that Rgc1 and Rgc2 form homodimers and heterodimers with each other and that dimer formation of Rgc2 is mediated by its N-terminal domain. Mutations that prevent Rgc2 dimerization block its ability to open Fps1. Therefore, the Rgc-Rgc dimer interface might be an attractive drug target.