bluuebunny/crossref_metadata_2025 huggingface dataset:
TLR3 engagement induces IRF‐3‐dependent apoptosis in androgen‐sensitive prostate cancer cells and inhibits tumour growth in vivo
Authors: Gambara Guido , Desideri Marianna , Stoppacciaro Antonella , Padula Fabrizio , De Cesaris Paola , Starace Donatella , Tubaro Andrea , del Bufalo Donatella , Filippini Antonio , Ziparo Elio , Riccioli Anna
Published: December 2014

Toll‐like receptors (TLRs) are a family of highly conserved transmembrane proteins expressed in epithelial and immune cells that recognize pathogen associated molecular patterns. Besides their role in immune response against infections, numerous studies have shown an important role of different TLRs in cancer, indicating these receptors as potential targets for cancer therapy. We previously demonstrated that the activation of TLR3 by the synthetic double‐stranded RNA analogue poly I:C induces apoptosis of androgen‐sensitive prostate cancer (PCa) LNCaP cells and, much less efficiently, of the more aggressive PC3 cell line. Therefore, in this study we selected LNCaP cells to investigate the mechanism of TLR3‐mediated apoptosis and the in vivo efficacy of poly I:C‐based therapy. We show that interferon regulatory factor‐3 (IRF‐3) signalling plays an essential role in TLR3‐mediated apoptosis in LNCaP cells through the activation of the intrinsic and extrinsic apoptotic pathways. Interestingly, hardly any apoptosis was induced by poly I:C in normal prostate epithelial cells RWPE‐1. We also demonstrate for the first time the direct anticancer effect of poly I:C as a single therapeutic agent in a well‐established human androgen‐sensitive PCa xenograft model, by showing that tumour growth is highly impaired in poly I:C‐treated immunodeficient mice. Immunohistochemical analysis of PCa xenografts highlights the antitumour role of poly I:C in vivo both on cancer cells and, indirectly, on endothelial cells. Notably, we show the presence of TLR3 and IRF‐3 in both human normal and PCa clinical samples, potentially envisaging poly I:C‐based therapy for PCa.
Grain‐oriented segmentation of images of porous structures using ray casting and curvature energy minimization
Authors: LEE H.‐G. , CHOI M.‐K. , LEE S.‐C.
Published: November 2014

We segment an image of a porous structure by successively identifying individual grains, using a process that requires no manual initialization. Adaptive thresholding is used to extract an incomplete edge map from the image. Then, seed points are created on a rectangular grid. Rays are cast from each point to identify the local grain. The grain with the best shape is selected by energy minimization, and the grain is used to update the edge map. This is repeated until all the grains have been recognized. Tests on scanning electron microscope images of titanium oxide and aluminium oxide show that their process achieves better results than five other contour detection techniques.
Hepatitis C virus long‐term persistence in peripheral blood mononuclear cells in patients with haemophilia. Detection of occult genotype 1
Authors: Parodi C. , García G. , Monzani M. C. , Culasso A. , Aloisi N. , Corti M. , Campos R. , de E de Bracco M. M. , Baré P.
Published: November 2014

Peripheral blood mononuclear cells (PBMC) from chronic hepatitis C virus‐infected persons can harbour viral variants that are not detected in plasma samples. We explored the presence and persistence of HCV genotypes in plasma and PBMC cultures from 25 HCV‐monoinfected and 25 HIV/HCV‐coinfected patients with haemophilia. Cell cultures were performed at different time points between 1993 and 2010‐2011, and the HCV genome was examined in culture supernatants. Sequential plasma samples were studied during the same time period. Analysing sequential plasma samples, 21% of patients had mixed‐genotype infections, while 50% had mixed infections determined from PBMC culture supernatants. HIV coinfection was significantly associated with the presence of mixed infections (OR = 4.57, P = 0.02; 95% CI = 1.38–15.1). In our previous study, genotype 1 was found in 72% of 288 patients of this cohort. Similar results were obtained with the sequential plasma samples included in this study, 69% had genotype 1. However, when taking into account plasma samples and the results from PBMC supernatants, genotype 1 was identified in 94% of the population. The PBMC‐associated variants persisted for 10 years in some subjects, emphasizing their role as long‐term reservoirs. The presence of genotype 1 in PBMC may be associated with therapeutic failure and should not be disregarded when treating haemophilic patients who have been infected by contaminated factor concentrates. The clinical implications of persistent lymphotropic HCV variants deserve further examination among multiple exposed groups of HCV‐infected patients.
Influence of habitat and host morphology on macrofaunal assemblages associated with the sponge Halichondria melanadocia in an estuarine system of the southern Gulf of Mexico
Authors: Ávila Enrique , Ortega‐Bastida Alma Lilia
Published: November 2014

Sponges are inhabited by a wide variety of organisms and have been regarded as one of the richest biotopes in tropical seas. The aim of this study was to assess the influence of the host morphology and selected environmental conditions on macrofaunal assemblages associated with the sponge Halichondria melanadocia in an estuarine system of the southern Gulf of Mexico. This sponge exhibits different growth forms when it inhabits mangrove prop roots of Rhizophora mangle (thickly encrusting form) and adjacent seagrass beds (massive, amorphous or ramose form). From a total of 50 sponge specimens collected in each habitat, a total of eight taxa (of epi‐ and endobionts) was found associated with this sponge, with polychaetes, echinoderms and crustaceans the most abundant groups. In both morphotypes (thickly‐encrusting and massive‐ramose forms), taxon richness was positively related to sponge volume and oscular diameter. The overall mean abundance of associated fauna was also positively related to sponge volume in both morphotypes and with the oscular diameter (in the seagrass morphotype only). These findings suggest that H. melanadocia constitutes an important microhabitat for a wide variety of fauna, independent of its morphology and habitat type. However, when comparing the two morphotypes, the mangrove individuals, despite having smaller sizes, smaller oscular diameter and less structural complexity, displayed an overall mean abundance of fauna 17 times higher [0.36 ± 0.18 individuals (ind.)·ml·sponge−1] than that recorded in the seagrass individuals (0.021 ± 0.01 ind.·ml·sponge−1). There were also marked differences in the proportions of the major taxonomic groups; in fact, some of them were found exclusively in one morphotype. The variability recorded in the composition and abundance of associated fauna between the morphotypes seems to be influenced by differences in sponge morphology, environmental conditions (e.g. sedimentation rate and light intensity), substrate orientation and the fauna inhabiting the surrounding area.
Fine‐scale phylogeography reveals cryptic biodiversity in Pederson's cleaner shrimp,Ancylomenes pedersoni(Crustacea: Caridea: Palaemonidae), along the Florida Reef Tract
Authors: Titus Benjamin M. , Daly Marymegan
Published: November 2014

Populations of continuously distributed marine organisms that disperseviapelagic larvae are often assumed to exist in a state of genetic panmixia because of their potential ability for long‐distance dispersal. However, obligate symbionts may have more restricted gene flow due to recruitment limitations, making host specificity an important potential driver of biodiversity. To explore the tension between broad dispersal ability and limited recruitment potential, we used sequences of mtDNA cytochromecoxidase subunit I (COI) to assess the fine‐scale phylogeography of the cleaner shrimp,Ancylomenes pedersoni, an obligate symbiont of sea anemones, along 300 km of the Florida Reef Tract (FRT). The results indicate high genetic diversity withinA. pedersonipopulations along the FRT. Pair‐wise ϕSTvalues indicate significant genetic structuring between northern (Fort Lauderdale and Upper Keys) and southwestern (Lower Keys) populations, with significant isolation by distance. Two divergent COI haplotype lineages were detected through statistical parsimony analysis: one evenly distributed across the entire FRT (Clade 1) and one found primarily in two sample localities in the Lower Keys (Clade 2). Phylogenetic analyses using 16S‐rDNAindicates that this genetic diversity is of paraphyletic origin, and possibly the result of multiple colonization events. These results reveal a complex demographic and evolutionary history forA. pedersonipopulations and provide the first evidence of highly divergent intra‐specific lineages independently colonizing the FRT. Because the FRT is a highly impacted coral reef system, understanding phylogeographic patterns along it has value beyond documentation of the factors that generate genetic diversity in tropical reef systems: these data are critical for creating scientifically based management strategies.
Whale killers: Prevalence and ecological implications of killer whale predation on humpback whale calves off Western Australia
Authors: Pitman Robert L. , Totterdell John A. , Fearnbach Holly , Ballance Lisa T. , Durban John W. , Kemps Hans
Published: November 2014

Reports of killer whales (Orcinus orca) preying on large whales have been relatively rare, and the ecological significance of these attacks is controversial. Here we report on numerous observations of killer whales preying on neonate humpback whales (Megaptera novaeangliae) off Western Australia (WA) based on reports we compiled and our own observations. Attacking killer whales included at least 19 individuals from three stable social groupings in a highly connected local population; 22 separate attacks with known outcomes resulted in at least 14 (64%) kills of humpback calves. We satellite‐tagged an adult female killer whale and followed her group on the water for 20.3 h over six separate days. During that time, they attacked eight humpback calves, and from the seven known outcomes, at least three calves (43%) were killed. Overall, our observations suggest that humpback calves are a predictable, plentiful, and readily taken prey source for killer whales and scavenging sharks offWAfor at least 5 mo/yr. Humpback “escorts” vigorously assisted mothers in protecting their calves from attacking killer whales (and a white shark,Carcharodon carcharias). This expands the purported role of escorts in humpback whale social interactions, although it is not clear how this behavior is adaptive for the escorts.
Immature euphausiids do not appear to be prey for humpback whales (Megaptera novaeangliae) during spring and summer in Southeast Alaska
Authors: Szabo Andrew
Published: November 2014

Humpbacks whales (Megaptera novaeangliae) have shown a remarkable recovery in the North Pacific, raising concerns regarding their impact on marine communities. In Southeast Alaska, humpbacks feed heavily on euphausiids; however, it remains unclear whether they target immature individuals despite evidence that they do so elsewhere. I evaluate the hypothesis that humpbacks target immature euphausiids in late spring‐summer in Southeast Alaska. Plankton samples were collected at random sites (n = 44) and near whales (n = 53) between 8 June and 9 September 2008 in Frederick Sound and Stephens Passage. The proportion of samples containing immature euphausiids, and immature euphausiid abundance within those samples, were compared between the two sample types. Similar analyses were conducted for adult euphausiids (prey) and calanoid copepods (nonprey) for comparison. I found no statistical difference between the whale and random samples with respect to the occurrence or numerical density of immature euphausiids, which is consistent with the hypothesis that whales did not target them in 2008. Smaller size, insufficient numerical densities and lower energy density of immature euphausiids are suggested as possible reasons. These findings can assist in resolving regional humpback abundance and distribution patterns, and can contribute to an understanding of the trophic interactions characterizing the local ecosystem.
Activated microglia in ischemic stroke penumbra upregulate MCP‐1 and CCR2 expression in response to lysophosphatidylcholine derived from adjacent neurons and astrocytes
Authors: Inose Yuri , Kato Yoichiro , Kitagawa Kazuo , Uchiyama Shinichiro , Shibata Noriyuki
Published: December 2014

In acute stage of ischemic stroke, the surrounding zone of fresh infarcts is termed penumbra, where microglia are activated in response to damaged cell‐derived proinflammatory mediators. Rescuing penumbra by regulating inflammatory activity would minimize infarct volume, which positively correlates with functional outcome. To elucidate mechanisms by which inflammation occurs in penumbra, we performed immunohistochemical investigations using autopsied human brains affected by acute, subacute and chronic stages of cerebral infarction as well as cell culture experiments using a murine microglia‐derived cell line (BV‐2). In penumbra of fresh infarcts, immunoreactivity for secretory phospholipase A2 group X (sPLA2‐X), which is responsible for the production and release of the proinflammatory mediator lysophosphatidylcholine (LPC), was intensely detected in neurons and astrocytes. Furthermore, immunoreactivities for the LPC receptors G protein‐coupled receptor 132 (G2A) and P2X purinoreceptor 7 (P2X7R), as well as the CC chemokine monocyte chemoattractant protein‐1 (MCP‐1) and its receptor CCR2, were detectable in activated microglia. Prior to cell culture experiments, it was confirmed that BV‐2 cells were immunoreactive for ionized Ca2+‐binding adaptor molecule 1 (Iba1), G2A, P2X7R, MCP‐1 and CCR2. Reverse transcription‐quantitative polymerase chain reaction analysis revealed that MCP‐1 and CCR2 mRNA expression levels were significantly increased by LPC stimulation. The LPC‐driven increase in MCP‐1 transcripts was lowered by blockade of G2A or P2X7R or by inhibition of Rho‐associated protein kinase (ROCK) or inhibitor of κBα kinase. The LPC‐driven increase in CCR2 transcripts was lowered by blockade of G2A or P2X7R or by inhibition of ROCK, phosphatidylinositide 3‐kinanse, extracellular signal‐regulated kinase kinase, or p38 mitogen‐activated protein kinase. The present results provide in vivo and in vitro evidence that in acute stage of ischemic stroke, the sPLA2‐X enzyme product LPC is released from neurons and astrocytes and stimulates penumbra microglia via G2A and P2X7R, thereby exerting the MCP‐1/CCR2‐mediated neurotoxicity through distinct cell‐signaling pathways.
Tree carbon allocation dynamics determined using a carbon mass balance approach
Authors: Klein Tamir , Hoch Günter
Published: December 2014

Tree internal carbon (C) fluxes between compound and compartment pools are difficult to measure directly. Here we used a C mass balance approach to decipher these fluxes and provide a full description of tree C allocation dynamics.

We collected independent measurements of tree C sinks, source and pools in Pinus halepensis in a semi‐arid forest, and converted all fluxes to g C per tree d−1. Using this data set, a process flowchart was created to describe and quantify the tree C allocation on diurnal to annual time‐scales.

The annual C source of 24.5 kg C per tree yr−1 was balanced by C sinks of 23.5 kg C per tree yr−1, which partitioned into 70%, 17% and 13% between respiration, growth, and litter (plus export to soil), respectively. Large imbalances (up to 57 g C per tree d−1) were observed as C excess during the wet season, and as C deficit during the dry season. Concurrent changes in C reserves (starch) were sufficient to buffer these transient C imbalances.

The C pool dynamics calculated using the flowchart were in general agreement with the observed pool sizes, providing confidence regarding our estimations of the timing, magnitude, and direction of the internal C fluxes.
Ecological mechanisms for the coevolution of mating systems and defence
Authors: Campbell Stuart A.
Published: December 2014

The diversity of flowering plants is evident in two seemingly unrelated aspects of life history: sexual reproduction, exemplified by the stunning variation in flower form and function, and defence, often in the form of an impressive arsenal of secondary chemistry. Researchers are beginning to appreciate that plant defence and reproduction do not evolve independently, but, instead, may have reciprocal and interactive (coevolutionary) effects on each other. Understanding the mechanisms for mating–defence interactions promises to broaden our understanding of how ecological processes can generate these two rich sources of angiosperm diversity. Here, I review current research on the role of herbivory as a driver of mating system evolution, and the role of mating systems in the evolution of defence strategies. I outline different ecological mechanisms and processes that could generate these coevolutionary patterns, and summarize theoretical and empirical support for each. I provide a conceptual framework for linking plant defence with mating system theory to better integrate these two research fields.
Social cognitive theory and physical activity: a systematic review and meta‐analysis
Authors: Young M. D. , Plotnikoff R. C. , Collins C. E. , Callister R. , Morgan P. J.
Published: November 2014

This review investigated three research questions (i) What is the utility of social cognitive theory (SCT) to explain physical activity (PA)?; (ii) Is the effectiveness of SCT moderated by sample or methodological characteristics? and (iii) What is the frequency of significant associations between the core SCT constructs and PA? Ten electronic databases were searched with no date or sample restrictions. Forty‐four studies were retrieved containing 55 SCT models of PA. Methodological quality was assessed using a standardized tool. A random‐effects meta‐analysis revealed that SCT accounted for 31% of the variance in PA. However, methodological quality was mostly poor for these models. Methodological quality and sample age moderated the PA effect size, with increases in both associated with greater variance explained. Although self‐efficacy and goals were consistently associated with PA, outcome expectations and socio‐structural factors were not. This review determined that SCT is a useful framework to explain PA behaviour. Higher quality models explained more PA variance, but overall methodological quality was poor. As such, high‐quality studies examining the utility of SCT to explain PA are warranted.
The first wheezing episode: respiratory virus etiology, atopic characteristics, and illness severity
Authors: Turunen Riitta , Koistinen Annamari , Vuorinen Tytti , Arku Benedict , Söderlund‐Venermo Maria , Ruuskanen Olli , Jartti Tuomas
Published: December 2014

Background

Susceptibility to early rhinovirus‐induced wheezing has been recognized as an important risk factor for childhood asthma, but data on the first wheezing episode are limited. The aim of this selected population study was to investigate virus etiology, atopic characteristics, and illness severity, as well as their interrelation, among first‐time wheezing children.

Methods

We studied 111 first‐time wheezing children aged between 3 and 23 months (88/23 in‐/outpatients). The investigated factors included atopy, food, perennial and aeroallergen sensitization, eczema, atopic eczema, elevated blood eosinophil count, and parental allergic rhinitis, asthma, and smoking. Nasopharyngeal aspirates were analyzed for adenovirus, coronaviruses, enteroviruses, bocavirus‐1 (also serologically confirmed), influenza viruses, metapneumovirus, parainfluenza viruses, rhinovirus, and respiratory syncytial virus using PCR methods.

Results

The mean age of the study patients was 12 months (standard deviation 6.0). Atopic characteristics could be found in 56%, atopic eczema in 16%, and sensitization in 23% of the cases. In all samples (100%), ≥1 viruses were detected as follows: rhinovirus (76%), respiratory syncytial virus (29%), bocavirus (18%, acute infections), and other viruses <10% each. Virus coinfections occurred in 38% of the children. Rhinovirus infection was positively associated with age, blood eosinophil count, eczema, and duration of cough, as well as parental allergic rhinitis and smoking but negatively associated with virus coinfection (all p < 0.05).

Conclusions

A respiratory virus infection can be detected in all first‐time wheezing children. Rhinovirus dominated the findings and was linked to atopic characteristics, prolonged cough, and parental smoking.
Phenotypic and marker‐assisted pyramiding of genes for resistance to zucchini yellow mosaic virus in oilseed pumpkin (Cucurbita pepo)
Authors: Pachner Martin , Paris Harry S. , Winkler Johanna , Lelley Tamas
Published: December 2014

We report on the identification of phenotypic and molecular markers for genes introgressed into oilseed pumpkin Cucurbita pepo from C. moschata germplasm originating in Nigeria, Portugal and Puerto Rico, which provide resistance against zucchini yellow mosaic virus (ZYMV) and on pyramiding these genes for improved and long‐lasting field protection of oilseed pumpkins. One SCAR and two SSR markers have been found for three dominant resistance genes, Zym‐0, Zym‐1 and Zym‐2. Characteristic reactions to ZYMV inoculation of plants carrying the recessive genes for resistance zym‐4* and zym‐6 have been defined. Described are procedures and results of pyramiding various combinations of these genes in oilseed pumpkin using the three markers and the specific phenotypic reactions to infection of some of these genes. The putative combination of all six resistance genes in one genotype resulted in a resistance that appeared to be at least as strong as or even stronger than that of the resistance source germplasm in C. moschata.
Can Trade Agreements Curtail Trade Creation and Prevent Trade Diversion?
Authors: Cheong Juyoung , Kwak Do Won , Tang Kam Ki
Published: November 2014

This paper investigates how pre‐existing preferential trade agreements (PTAs) dilute the trade creation effect and shield the trade diversion effect of new PTAs. Countries having pre‐existing PTAs enjoy smaller gains in intra‐bloc trade because of the dilution effect and experience smaller losses or even gains in extra‐bloc trade because of the shielding effect. The findings support the proposition that PTAs could be used to fend off future trade diversion.
The Coaching Behavior Scale for Sport (CBS‐S): A psychometric evaluation of the Swedish version
Authors: Carlsson A. , Lundqvist C.
Published: December 2014

The present study validated a Swedish version of the 47‐item Coaching Behavior Scale for Sport (CBS‐S). Sample 1 consisted of 506 team sport athletes [262 men and 244 women; mean age: 22.20, standard deviation (SD) = 3.90] distributed across 41 coaches at the two highest national levels of various sports. Athletes completed the CBS‐S and established questionnaires of coaching behaviors (LSS), self‐confidence (CSAI‐2R), and coach–athlete relationship (CART‐Q). An additional sample of 39 basketball players (21 men and 18 women; mean age = 17.40, SD = 2.39) completed the CBS‐S twice, approximately 4 weeks apart. Confirmatory factor analysis showed an acceptable model fit for the seven‐factor version of the CBS‐S, although two items of the negative personal rapport subscale displayed insufficient factor loadings. Correlations between the subscales of the CBS‐S and established instruments were in accordance with theoretical expectations, supporting the concurrent validity. Cronbach's alpha (> 0.82) for all dimensions provided support for the reliability of the CBS‐S, and test‐retest correlations indicated moderate stability over time. Cultural differences in the assessment of coaching behaviors and the usability of the CBS‐S by coaches for self‐reflection and development are discussed.
Retrospective study of the prevalence of keratoconjunctivitis sicca in diabetic and nondiabetic dogs after phacoemulsification
Authors: Gemensky‐Metzler Anne J. , Sheahan Jennifer E. , Rajala‐Schultz Paivi J. , Wilkie David A. , Harrington Jay
Published: November 2014

Objective

To evaluate the prevalence of keratoconjunctivitis sicca (KCS) in diabetic and nondiabetic dogs following phacoemulsification.

Procedures

Medical records were reviewed for signalment, body weight, diabetes status, andSTTat the preoperative and all postoperative examinations. A clinical diagnosis ofKCSwas defined as aSTT<15 mm/min with compatible clinical signs. Schirmer tear test values and the prevalence ofKCSbased onSTT<15 mm/min were evaluated at 2–4, 5–8, 9–14, 15–20, and 41–52 weeks postoperatively. Size of dog (large, >10 kg; small, ≤10 kg) andSTTranges (<15 mm/min, 15–22 mm/min and >22 mm/min) were categorized for analysis of effect of body weight andSTTon prevalence ofKCS.

Results

One hundred and seventeen nondiabetic (198 eyes) and 118 diabetic dogs (228 eyes) were evaluated.KCSwas diagnosed in nearly twice as many diabetics as nondiabetics (27.4% vs. 15.4%) within 2 weeks postoperatively (P = 0.0088). Percentage of eyes withKCSamong all dogs decreased over time and did not differ significantly after the first postoperative visit. Diabetic small dogs were 1.7 times more likely to haveKCSthan nondiabetic small dogs (P = 0.0052). PreoperativeSTTwas significantly associated with likelihood of postoperativeKCSamong large (P < 0.0001), but not small dogs (P = 0.0781). Among large dogs, eyes withSTTs between 15 and 22 mm/min were approximately 3 times more likely to be diagnosed withKCSafter surgery than those withSTT>22 mm/min.

Conclusion

The greatest risk forKCSfor all dogs is during the first 2 weeks postoperatively. Populations at greatest risk for postoperativeKCSare small dogs, small diabetic dogs, and large dogs with preoperativeSTT≤22.
On the anticyclotomic Iwasawa main conjecture for modular forms
Authors: Chida Masataka , Hsieh Ming-Lun
Published: November 2014

We generalize the work of Bertolini and Darmon on the anticyclotomic main conjecture for elliptic curves to modular forms of higher weight.
Epitranscriptional regulation of cardiovascular development and disease
Authors: Dorn Gerald W. , Matkovich Scot J.
Published: November 2014

Development, homeostasis and responses to stress in the heart all depend on appropriate control of mRNA expression programmes, which may be enacted at the level of DNA sequence, DNA accessibility and RNA‐mediated control of mRNA output. Diverse mechanisms underlie promoter‐driven transcription of coding mRNAs and their translation into protein, and the ways in which sequence alteration of DNA can make an impact on these processes have been studied for some time. The field of epigenetics explores changes in DNA structure that influence its accessibility by transcriptional machinery, and we are continuing to develop our understanding of how these processes modify cardiac RNA production. In this topical review, we do not focus on how DNA sequence and methylation, and histone interactions, may alter its accessibility, but rather on newly described mechanisms by which some transcribed RNAs may alter initial transcription or downstream processing of other RNAs, involving both short non‐coding RNAs (microRNAs) and long non‐coding RNAs (lncRNAs). Here we present examples of how these two classes of non‐coding RNAs mediate widespread effects on cardiac transcription and protein output in processes for which we use the broad term ‘epitranscriptional regulation’ and that are complementary to the DNA methylation and histone modification events studied by classical epigenetics.
Molecular Mechanisms of Fluconazole Resistance in Candida parapsilosis Isolates from a U.S. Surveillance System
Authors: Grossman Nina T. , Pham Cau D. , Cleveland Angela A. , Lockhart Shawn R.
Published: December 2014

Candida parapsilosis
is the second or third most common cause of candidemia in many countries. The Infectious Diseases Society of America recommends fluconazole as the primary therapy for
C. parapsilosis
candidemia. Although the rate of fluconazole resistance among
C. parapsilosis
isolates is low in most U.S. institutions, the resistance rate can be as high as 7.5%. This study was designed to assess the mechanisms of fluconazole resistance in 706 incident bloodstream isolates from U.S. hospitals. We sequenced the
ERG11
and
MRR1
genes of 122
C. parapsilosis
isolates with resistant (30 isolates; 4.2%), susceptible dose-dependent (37 isolates; 5.2%), and susceptible (55 isolates) fluconazole MIC values and used real-time PCR of RNA from 17 isolates to investigate the regulation of
MDR1
. By comparing these isolates to fully fluconazole-susceptible isolates, we detected at least two mechanisms of fluconazole resistance: an amino acid substitution in the 14-α-demethylase gene
ERG11
and overexpression of the efflux pump
MDR1
, possibly due to point mutations in the
MRR1
transcription factor that regulates
MDR1
. The
ERG11
single nucleotide polymorphism (SNP) was found in 57% of the fluconazole-resistant isolates and in no susceptible isolates. The
MRR1
SNPs were more difficult to characterize, as not all resulted in overexpression of
MDR1
and not all
MDR1
overexpression was associated with an SNP in
MRR1
. Further work to characterize the
MRR1
SNPs and search for overexpression of other efflux pumps is needed.
Burkholderia cepacia Complex Phage-Antibiotic Synergy (PAS): Antibiotics Stimulate Lytic Phage Activity
Authors: Kamal Fatima , Dennis Jonathan J.
Published: December 2014

The
Burkholderia cepacia
complex (Bcc) is a group of at least 18 species of Gram-negative opportunistic pathogens that can cause chronic lung infection in cystic fibrosis (CF) patients. Bcc organisms possess high levels of innate antimicrobial resistance, and alternative therapeutic strategies are urgently needed. One proposed alternative treatment is phage therapy, the therapeutic application of bacterial viruses (or bacteriophages). Recently, some phages have been observed to form larger plaques in the presence of sublethal concentrations of certain antibiotics; this effect has been termed phage-antibiotic synergy (PAS). Those reports suggest that some antibiotics stimulate increased production of phages under certain conditions. The aim of this study is to examine PAS in phages that infect
Burkholderia cenocepacia
strains C6433 and K56-2. Bcc phages KS12 and KS14 were tested for PAS, using 6 antibiotics representing 4 different drug classes. Of the antibiotics tested, the most pronounced effects were observed for meropenem, ciprofloxacin, and tetracycline. When grown with subinhibitory concentrations of these three antibiotics, cells developed a chain-like arrangement, an elongated morphology, and a clustered arrangement, respectively. When treated with progressively higher antibiotic concentrations, both the sizes of plaques and phage titers increased, up to a maximum.
B. cenocepacia
K56-2-infected
Galleria mellonella
larvae treated with phage KS12 and low-dose meropenem demonstrated increased survival over controls treated with KS12 or antibiotic alone. These results suggest that antibiotics can be combined with phages to stimulate increased phage production and/or activity and thus improve the efficacy of bacterial killing.
Passage of Wolbachia pipientis through Mutant Drosophila melanogaster Induces Phenotypic and Genomic Changes
Authors: Newton Irene L. G. , Sheehan Kathy B.
Published: December 2014

Wolbachia pipientis
is a nearly ubiquitous, maternally transmitted bacterium that infects the germ line of insect hosts. Estimates are that
Wolbachia
infects 40 to 60% of insect species on the planet, making it one of the most prevalent infections on Earth. However, we know surprisingly little about the molecular mechanisms used by
Wolbachia
to infect its hosts. We passaged
Wolbachia
through normally restrictive
Drosophila melanogaster
hosts, bottlenecking
Wolbachia
through stochastic segregation while simultaneously selecting for mutants that could recolonize these previously restrictive hosts. Here, we show that
Wolbachia
alters its behavior when passaged through heterozygous mutant flies. After only three generations,
Wolbachia
was able to colonize the previously restrictive hosts at control titers. Additionally, the
Wolbachia
organisms passaged through heterozygous mutant
D. melanogaster
alter their pattern of tissue-specific Wsp protein production, suggesting a behavioral response to the host genotype. Using whole-genome resequencing, we identified the mutations accumulated by these lineages of
Wolbachia
and confirmed the existence and persistence of the mutations through clone library Sanger sequencing. Our results suggest that
Wolbachia
can quickly adapt to new host contexts, with genomic mutants arising after only two generations.
Use of an Endogenous Plasmid Locus for Stable in
trans
Complementation in Borrelia burgdorferi
Authors: Kasumba Irene N. , Bestor Aaron , Tilly Kit , Rosa Patricia A.
Published: December 2014

Targeted mutagenesis and complementation are important tools for studying genes of unknown function in the Lyme disease spirochete
Borrelia burgdorferi
. A standard method of complementation is reintroduction of a wild-type copy of the targeted gene on a shuttle vector. However, shuttle vectors are present at higher copy numbers than
B. burgdorferi
plasmids and are potentially unstable in the absence of selection, thereby complicating analyses in the mouse-tick infectious cycle.
B. burgdorferi
has over 20 plasmids, with some, such as linear plasmid 25 (lp25), carrying genes required by the spirochete
in vivo
but relatively unstable during
in vitro
cultivation. We propose that complementation on an endogenous plasmid such as lp25 would overcome the copy number and
in vivo
stability issues of shuttle vectors. In addition, insertion of a selectable marker on lp25 could ensure its stable maintenance by spirochetes in culture. Here, we describe the construction of a multipurpose allelic-exchange vector containing a multiple-cloning site and either of two selectable markers. This suicide vector directs insertion of the complementing gene into the
bbe02
locus, a site on lp25 that was previously shown to be nonessential during both
in vitro
and
in vivo
growth. We demonstrate the functional utility of this strategy by restoring infectivity to an
ospC
mutant through complementation at this site on lp25 and stable maintenance of the
ospC
gene throughout mouse infection. We conclude that this represents a convenient and widely applicable method for stable gene complementation in
B. burgdorferi
.
Draft Genome Sequences of Eight Enterohepatic
Helicobacter
Species Isolated from Both Laboratory and Wild Rodents
Authors: Sheh Alexander , Shen Zeli , Fox James G.
Published: November 2014

The draft genome sequences of eight enterohepatic
Helicobacter
species,
H. muridarum, H. trogontum, H. typhlonius
, and five unnamed helicobacters, are presented here. Using laboratory mice pervasively infected with helicobacters, we characterized the presence of known virulence factors.
Draft Genome Sequence of Fish Pathogenic Vibrio vulnificus Biotype 2
Authors: Koton Yael , Eghbaria Saleh , Gordon Michal , Chalifa-Caspi Vered , Bisharat Naiel
Published: November 2014

Vibrio vulnificus
is a marine pathogen capable of causing severe soft tissue infections and septicemia in humans.
V. vulnificus
biotype 2 is the etiological agent of fish vibriosis. We describe here the first draft genome sequence of
V. vulnificus
biotype 2, strain ES-7601, isolated from an infected eel in Japan.
Complete Genome Sequence of
Francisella endociliophora
Strain FSC1006, Isolated from a Laboratory Culture of the Marine Ciliate
Euplotes raikovi
Authors: Sjödin Andreas , Öhrman Caroline , Bäckman Stina , Lärkeryd Adrian , Granberg Malin , Lundmark Eva , Karlsson Edvin , Nilsson Elin , Vallesi Adriana , Tellgren-Roth Christian , Stenberg Per , Thelaus Johanna
Published: November 2014

A strain of
Francisella endociliophora
was isolated from a laboratory culture of the marine ciliate
Euplotes raikovi
. Here, we report the complete genome sequence of the bacterial strain FSC1006 (
Francisella
Strain Collection, Swedish Defence Research Agency, Umeå, Sweden).
Draft Genome Sequence of
Chryseobacterium
sp. Strain P1-3, a Keratinolytic Bacterium Isolated from Poultry Waste
Authors: Park Gun-Seok , Hong Sung-Jun , Lee Chang-Hyun , Khan Abdur Rahim , Ullah Ihsan , Jung Byung Kwon , Choi JungBae , Kwak Yunyoung , Back Chang-Gi , Jung Hee-Young , Shin Jae-Ho
Published: November 2014

Chryseobacterium
sp. strain P1-3, harboring keratin degrading activity, has recently been isolated from poultry waste. Here, we report the 4.6-Mbp draft genome sequence of the keratinolytic bacterium with a G+C content of 37.0% and 4,087 protein-coding genes.
Characteristics of Emerging Human-Pathogenic Escherichia coli O26:H11 Strains Isolated in France between 2010 and 2013 and Carrying the
stx
2d
Gene Only
Authors: Delannoy Sabine , Mariani-Kurkdjian Patricia , Bonacorsi Stephane , Liguori Sandrine , Fach Patrick
Published: November 2014

Strains of
Escherichia coli
O26:H11 that were positive for
stx
2
alone (
n
= 23), which were not epidemiologically related or part of an outbreak, were isolated from pediatric patients in France between 2010 and 2013. We were interested in comparing these strains with the new highly virulent
stx
2a
-positive
E. coli
O26 clone sequence type 29 (ST29) that has emerged recently in Europe, and we tested them by multilocus sequence typing (MLST),
stx
2
subtyping, clustered regularly interspaced short palindromic repeat (CRISPR) sequencing, and plasmid (
ehxA
,
katP
,
espP
, and
etpD
) and chromosomal (Z2098,
espK
, and
espV
) virulence gene profiling. We showed that 16 of the 23 strains appeared to correspond to this new clone, but the characteristics of 12 strains differed significantly from the previously described characteristics, with negative results for both plasmid and chromosomal genetic markers. These 12 strains exhibited a ST29 genotype and related CRISPR arrays (CRISPR2a alleles 67 or 71), suggesting that they evolved in a common environment. This finding was corroborated by the presence of
stx
2d
in 7 of the 12 ST29 strains. This is the first time that
E. coli
O26:H11 carrying
stx
2d
has been isolated from humans. This is additional evidence of the continuing evolution of virulent Shiga toxin-producing
E. coli
(STEC) O26 strains. A new O26:H11 CRISPR PCR assay, SP_O26_E, has been developed for detection of these 12 particular ST29 strains of
E. coli
O26:H11. This test is useful to better characterize the
stx
2
-positive O26:H11 clinical isolates, which are associated with severe clinical outcomes such as bloody diarrhea and hemolytic uremic syndrome.
Leukodepletion as a Point-of-Care Method for Monitoring HIV-1 Viral Load in Whole Blood
Authors: Titchmarsh Logan , Zeh Clement , Verpoort Thierry , Allain Jean-Pierre , Lee Helen
Published: November 2014

In order to limit the interference of HIV-1 cellular nucleic acids in estimating viral load (VL), the feasibility of leukodepletion of a small whole-blood (WB) volume to eliminate only leukocyte cell content was investigated, using a selection of filters. The efficacy of leukocyte filtration was evaluated by counting, CD45 quantitative PCR, and HIV-1 DNA quantification. Plasma HIV-1 was tested by real-time reverse transcription (RT)-PCR. A specific, miniaturized filter was developed and tested for leukocyte and plasma virus retention, WB sample dilution, and filtration parameters in HIV-1-spiked WB samples. This device proved effective to retain >99.9% of white blood cells in 100 μl of WB without affecting plasma VL. The Samba sample preparation chemistry was adapted to use a leukodepleted WB sample for VL monitoring using the point-of-care Samba-1 semiautomated system. The clinical performance of the assay was evaluated by testing 207 consecutive venous EDTA WB samples from HIV-1-infected patients attending a CD4 testing clinic. Most patients were on antiretroviral treatment (ART), but their VL status was unknown. Compared to the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test, the new Samba assay had a concordance of 96.5%. The use of the Samba system with a VL test for WB might contribute to HIV-1 ART management and reduce loss-to-follow-up rates in resource-limited settings.
Macropinocytosis Is the Entry Mechanism of Amphotropic Murine Leukemia Virus
Authors: Rasmussen Izabela , Vilhardt Frederik
Published: November 2014

The entry mechanism of murine amphotropic retrovirus (A-MLV) has not been unambiguously determined. We show here that A-MLV is internalized not by caveolae or other pinocytic mechanisms but by macropinocytosis. Thus, A-MLV infection of mouse embryonic fibroblasts deficient for caveolin or dynamin, and NIH 3T3 cells knocked down for caveolin expression, was unaffected. Conversely, A-MLV infection of NIH 3T3 and HeLa cells was sensitive to amiloride analogues and actin-depolymerizing drugs that interfere with macropinocytosis. Further manipulation of the actin cytoskeleton through conditional expression of dominant positive or negative mutants of Rac1, PAK1, and RhoG, to increase or decrease macropinocytosis, similarly correlated with an augmented or inhibited infection with A-MLV, respectively. The same experimental perturbations affected the infection of viruses that use clathrin-coated-pit endocytosis or other pathways for entry only mildly or not at all. These data agree with immunofluorescence studies and cryo-immunogold labeling for electron microscopy, which demonstrate the presence of A-MLV in protrusion-rich areas of the cell surface and in cortical fluid phase (dextran)-filled macropinosomes, which also account for up to a half of the cellular uptake of the cell surface-binding lectin concanavalin A. We conclude that A-MLV use macropinocytosis as the predominant entry portal into cells.

IMPORTANCE
Binding and entry of virus particles into mammalian cells are the first steps of infection. Understanding how pathogens and toxins exploit or divert endocytosis pathways has advanced our understanding of membrane trafficking pathways, which benefits development of new therapeutic schemes and methods of drug delivery. We show here that amphotropic murine leukemia virus (A-MLV) pseudotyped with the amphotropic envelope protein (which expands the host range to many mammalian cells) gains entry into host cells by macropinocytosis. Macropinosomes form as large, fluid-filled vacuoles (up to 10 μm) following the collapse of cell surface protrusions and membrane scission. We used drugs or the introduction of mutant proteins that affect the actin cytoskeleton and cell surface dynamics to show that macropinocytosis and A-MLV infection are correlated, and we provide both light- and electron-microscopic evidence to show the localization of A-MLV in macropinosomes. Finally, we specifically exclude some other potential entry portals, including caveolae, previously suggested to internalize A-MLV.
The Level of Viral Infection of Antigen-Presenting Cells Correlates with the Level of Development of Theiler's Murine Encephalomyelitis Virus-Induced Demyelinating Disease
Authors: Jin Young Hee , Kang Hyun Seok , Hou Wanqiu , Meng Liping , Kim Byung S.
Published: November 2014

Intracerebral infection with Theiler's murine encephalomyelitis virus (TMEV) induces immune-mediated demyelinating disease in susceptible SJL/J mice but not in resistant C57BL/6 mice. Previous studies have indicated that the major histocompatibility complex (MHC) genes play the most prominent role in the development of TMEV-induced demyelinating disease. In this study, we used C57BL/6.S (B6.S) congenic mice, which carryH-2sMHC genes instead ofH-2bMHC genes in conjunction with the C57BL/6 (B6) background genes. Our data show that virus-infected B6.S mice are free from disease and have significantly lower viral loads than susceptible SJL mice, particularly in the spinal cord. A strong protective Th1-type T helper response with virtually no pathogenic Th17 response was detected in B6.S mice, in contrast to the reduced Th1- and robust Th17-type responses in SJL mice. Notably, lower levels of viral infectivity in B6.S antigen-presenting cells (APCs) correlated with the disease resistance and T-cell-type response.In vitrostudies using APCs from B6.S and SJL mice show that TLR2, -3, -4, and -7, but not TLR9, signaling can replace viral infection and augment the effect of viral infection in the differentiation of the pathogenic Th17 cell type. Taken together, these results strongly suggest that the viral replication levels in APCs critically affect the induction of protective versus pathogenic Th cell types via the signaling of pattern recognition receptors for innate immune responses. Our current findings further imply that the levels of viral infectivity/replication and TLR-mediated signaling play critical roles in the pathogenesis of chronic viral diseases.

IMPORTANCEThis study indicates that innate immune cytokines produced in antigen-presenting cells stimulating the T cell immune responses during early viral infection play a critical role in determining the susceptibility of mice to the development of demyelinating disease. The level of innate immune cytokines reflects the level of initial viral infection in the antigen-presenting cells, and the level determines the development of T cell types, which are either protective or pathogenic. The level of initial viral infection to the cells is controlled by a gene or genes that are not associated with the major histocompatibility antigen complex genes. This finding has an important implication in controlling not only chronic viral infections but also infection-induced autoimmune-like diseases, which are closely associated with the pathogenic type of T cell responses.