Polluted wetlands contain multidrug-resistance plasmids encoding CTX-M-type extended-spectrum ß-lactamases.

While most detailed analyses of antibiotic resistance plasmids focus on those found in clinical isolates, less is known about the vast environmental reservoir of mobile genetic elements and the resistance and virulence factors they encode. We selectively isolated three strains of cefotaxime-resistant Escherichia coli from a wastewater-impacted coastal wetland. The cefotaxime-resistant phenotype was transmissible to a lab strain of E. coli after one hour, with frequencies as high as 10-3 transconjugants per recipient. Two of the plasmids also transferred cefotaxime resistance to Pseudomonas putida, but these were unable to back-transfer this resistance from P. putida to E. coli. In addition to the cephalosporins, E. coli transconjugants inherited resistance to at least seven distinct classes of antibiotics. Complete nucleotide sequences revealed large IncF-type plasmids with globally distributed replicon sequence types F31:A4:B1 and F18:B1:C4 carrying diverse antibiotic resistance and virulence genes. The plasmids encoded extended-spectrum ß-lactamases blaCTX-M-15 or blaCTX-M-55, each associated with the insertion sequence ISEc9, although in different local arrangements. Despite similar resistance profiles, the plasmids shared only one resistance gene in common, the aminoglycoside acetyltransferase aac(3)-IIe. Plasmid accessory cargo also included virulence factors involved in iron acquisition and defense against host immunity. Despite their sequence similarities, several large-scale recombination events were detected, including rearrangements and inversions. In conclusion, selection with a single antibiotic, cefotaxime, yielded conjugative plasmids conferring multiple resistance and virulence factors. Clearly, efforts to limit the spread of antibiotic resistance and virulence among bacteria must include a greater understanding of mobile elements in the natural and human-impacted environments.

A novel method of screening combinations of angiostatics identifies bevacizumab and temsirolimus as synergistic inhibitors of glioma-induced angiogenesis.

Tumor angiogenesis is critical for the growth and progression of cancer. As such, angiostasis is a treatment modality for cancer with potential utility for multiple types of cancer and fewer side effects. However, clinical success of angiostatic monotherapies has been moderate, at best, causing angiostatic treatments to lose their early luster. Previous studies demonstrated compensatory mechanisms that drive tumor vascularization despite the use of angiostatic monotherapies, as well as the potential for combination angiostatic therapies to overcome these compensatory mechanisms. We screened clinically approved angiostatics to identify specific combinations that confer potent inhibition of tumor-induced angiogenesis. We used a novel modification of the ex ovo chick chorioallantoic membrane (CAM) model that combined confocal and automated analyses to quantify tumor angiogenesis induced by glioblastoma tumor onplants. This model is advantageous due to its low cost and moderate throughput capabilities, while maintaining complex in vivo cellular interactions that are difficult to replicate in vitro. After screening multiple combinations, we determined that glioblastoma-induced angiogenesis was significantly reduced using a combination of bevacizumab (Avastin®) and temsirolimus (Torisel®) at doses below those where neither monotherapy demonstrated activity. These preliminary results were verified extensively, with this combination therapy effective even at concentrations further reduced 10-fold with a CI value of 2.42E-5, demonstrating high levels of synergy. Thus, combining bevacizumab and temsirolimus has great potential to increase the efficacy of angiostatic therapy and lower required dosing for improved clinical success and reduced side effects in glioblastoma patients.

Circadian activity patterns of mammalian predators and prey in Costa Rica.

Temporal niche shifts can shape predator-prey interactions by enabling predator avoidance, enhancing feeding success, and reducing competition among predators. Using a community-based conservation approach, we investigated temporal niche partitioning of mammalian predators and prey across 12 long-term camera trap surveys in the Pacific slope and Talamanca Cordillera of Costa Rica. Temporal overlap and segregation were investigated between predator-prey and predator-predator pairs using overlap analysis, circular statistics, and relative abundance after accounting for differences in habitat, season, and human impact among sites. We made the assumption that predators select abundant prey and adjust their activity to maximize their temporal overlap, thus we predicted that abundant prey with high overlap would be preferred prey species for that predator. We also predicted that similar-sized pairs of predator species with the greatest potential for competitive interactions would have the highest temporal segregation. Our results supported the existence of temporal niche separation among the eight species of predators-the smaller Leopardus felids (ocelot, margay, oncilla) were primarily nocturnal, the largest felids (jaguar and puma) and coyote were cathemeral, and the smaller jaguarundi and tayra were mostly diurnal. Most prey species (67%) were primarily nocturnal versus diurnal or cathemeral (33%). Hierarchical clustering identified relationships among species with the most similar activity patterns. We discuss the primary prey and competitor species predicted for each of the eight predators. Contrary to our prediction, the activity pattern of similar-sized intraguild competitors overlapped more than dissimilar-sized competitors, suggesting that similar-sized predators are hunting the same prey at the same time. From this we conclude that prey availability is more important than competition in determining circadian activity patterns of Neotropical predators. Our results indicate the presence of a delicate balance of tropical food webs that may be disrupted by overhunting, leading to a depauperate community consisting of ubiquitous generalists and endangered specialists. With Central America a hotspot for hunting-induced "empty forests," community-based conservation approaches may offer the best road to reduce illegal hunting and maintain the biodiversity and community structure of tropical forest systems.

Los cambios temporales de nicho pueden amoldar las interacciones predador-presa al permitir a la presa evadir los predadores, mejorar la eficacia de la alimentación y reducir la competición entre predadores. Mediante muestreos con cámaras trampa en 12 sitios de la pendiente del Pacífico y la Cordillera de Talamanca de Costa Rica, investigamos la división temporal de los nichos de mamíferos predadores y presas usando un enfoque de conservación basado en la comunidad. Investigamos la superposición y segregación temporal entre pares predador-presa y predador-predador utilizando análisis de superposición, estadísticas circulares y abundancia relativa, teniendo en cuenta diferencias de hábitat, estacionales y de impacto humano entre los sitios. Asumimos que los predadores seleccionan presas abundantes y ajustan su actividad para maximizar su superposición temporal con éstas; por lo tanto, predijimos que una presa abundante con alta superposición sería una especie de presa preferida para ese predador. También predijimos que pares de especies de predadores de tamaño similar con el mayor potencial para interacciones competitivas tendrían la mayor segregación temporal. Nuestros resultados respaldaron la existencia de una separación temporal de nicho entre las ocho especies de predadores­determinamos que los felinos más pequeños del género Leopardus (manigordo, caucel, tigrillo) son principalmente nocturnos, los felinos más grandes (jaguar y puma) y el coyote son catemerales, y los jaguarundi y tayra más pequeños son principalmente diurnos. La mayoría de las especies de presas (67%) son principalmente nocturnas versus diurnas o catemerales (33%). Mediante análisis de agrupación jerárquica, identificamos relaciones entre especies con los patrones de actividad más similares. Para cada uno de los predadores discutimos las presas primarias predichas y las especies competidoras. Contrario a nuestra predicción, el patrón de actividad de los predadores intragremios de tamaño similar se superpone más que el de los competidores de distinto tamaño, lo que indica que los predadores de tamaño similar están cazando la misma presa y están activos al mismo tiempo. De esto concluimos que la disponibilidad de presas es más importante que la competición en cuanto a determinar los patrones de actividad circadiana de los predadores neotropicales. Nuestros resultados revelan que el delicado equilibrio de las redes tróficas tropicales puede ser interferido por la caza excesiva, lo que lleva a una comunidad depauperada compuesta por generalistas ubicuos y especialistas en peligro de extinción. Con América Central como un centro de "bosques vacíos" inducidos por la caza, los enfoques de conservación basados en la comunidad pueden ofrecer el mejor camino para reducir la caza ilegal, y mantener la biodiversidad y estructura ecológica comunitaria de los sistemas forestales tropicales.

Supra-Versus Submaximal Cycle Ergometer Verification of VO2max in Males and Females.

This study was designed to determine the optimal intensity for verification phase testing (VP) in healthy, young adults. Thirty one young, active participants (16 females) completed a cycle ergometer graded exercise test (GXT) VO2max test and 4 VP tests at 80, 90, 100, and 105% of the maximum wattage achieved during the GXT. GXT and VP VO2max values showed a significant test x sex interaction (p = 0.02). The males elicited significantly higher VO2max values during the GXT, 80%, and 90% when compared to the 105%, (105 vs. GXT: p = 0.05; 105% vs. 80%: p < 0.01; 105% vs. 90%: p = 0.02). There were no significant differences in VO2max across the tests in the females (p > 0.05); 80% of the males achieved their highest VP VO2max during a submaximal VP test compared to only 37.5% of the females. A secondary study conducted showed excellent reliability (ICCs > 0.90) and low variation (CVs < 3%) for the 90% VP. Our findings show that a submaximal verification phase intensity is ideal for young healthy males to elicit the highest VO2max during cycle ergometer testing. For females, a range of intensities (80-105%) produce similar VO2max values. However, the 80% VP yields an unnecessarily high time to exhaustion.

Cryptic Species Account for the Seemingly Idiosyncratic Secondary Metabolism of Sarcophyton glaucum Specimens Collected in Palau.

Sarcophyton glaucum is one of the most abundant and chemically studied soft corals with over 100 natural products reported in the literature, primarily cembrane diterpenoids. Yet, wide variation in the chemistry observed from S. glaucum over the past 50 years has led to its reputation as a capricious producer of bioactive metabolites. Recent molecular phylogenetic analysis revealed that S. glaucum is not a single species but a complex of at least seven genetically distinct species not distinguishable using traditional taxonomic criteria. We hypothesized that perceived intraspecific chemical variation observed in S. glaucum was actually due to differences between cryptic species (interspecific variation). To test this hypothesis, we collected Sarcophyton samples in Palau, performed molecular phylogenetic analysis, and prepared chemical profiles of sample extracts using gas chromatography-flame ionization detection. Both unsupervised (principal component analysis) and supervised (linear discriminant analysis) statistical analyses of these profiles revealed a strong relationship between cryptic species membership and chemical profiles. Liquid chromatography with tandem mass spectrometry-based analysis using feature-based molecular networking permitted identification of the chemical drivers of this difference between clades, including cembranoid diterpenes (2R,11R,12R)-isosarcophytoxide (5), (2S,11R,12R)-isosarcophytoxide (6), and isosarcophine (7). Our results suggest that early chemical studies of Sarcophyton may have unknowingly conflated different cryptic species of S. glaucum, leading to apparently idiosyncratic chemical variation.

Characterization of Four Multidrug Resistance Plasmids Captured from the Sediments of an Urban Coastal Wetland.

Self-transmissible and mobilizable plasmids contribute to the emergence and spread of multidrug-resistant bacteria by enabling the horizontal transfer of acquired antibiotic resistance. The objective of this study was to capture and characterize self-transmissible and mobilizable resistance plasmids from a coastal wetland impacted by urban stormwater runoff and human wastewater during the rainy season. Four plasmids were captured, two self-transmissible and two mobilizable, using both mating and enrichment approaches. Plasmid genomes, sequenced with either Illumina or PacBio platforms, revealed representatives of incompatibility groups IncP-6, IncR, IncN3, and IncF. The plasmids ranged in size from 36 to 144 kb and encoded known resistance genes for most of the major classes of antibiotics used to treat Gram-negative infections (tetracyclines, sulfonamides, ß-lactams, fluoroquinolones, aminoglycosides, and amphenicols). The mobilizable IncP-6 plasmid pLNU-11 was discovered in a strain of Citrobacter freundii enriched from the wetland sediments with tetracycline and nalidixic acid, and encodes a novel AmpC-like ß-lactamase (blaWDC-1), which shares less than 62% amino acid sequence identity with the PDC class of ß-lactamases found in Pseudomonas aeruginosa. Although the IncR plasmid pTRE-1611 was captured by mating wetland bacteria with P. putida KT2440 as recipient, it was found to be mobilizable rather than self-transmissible. Two self-transmissible multidrug-resistance plasmids were also captured: the small (48 kb) IncN3 plasmid pTRE-131 was captured by mating wetland bacteria with Escherichia coli HY842 where it is seemed to be maintained at nearly 240 copies per cell, while the large (144 kb) IncF plasmid pTRE-2011, which was isolated from a cefotaxime-resistant environmental strain of E. coli ST744, exists at just a single copy per cell. Furthermore, pTRE-2011 bears the globally epidemic blaCTX-M-55 extended-spectrum ß-lactamase downstream of ISEcp1. Our results indicate that urban coastal wetlands are reservoirs of diverse self-transmissible and mobilizable plasmids of relevance to human health.

High Diversity of CTX-M Extended-Spectrum ß-Lactamases in Municipal Wastewater and Urban Wetlands.

The CTX-M-type extended-spectrum ß-lactamases (ESBLs) present a serious public health threat as they have become nearly ubiquitous among clinical gram-negative pathogens, particularly the enterobacteria. To aid in the understanding and eventual control of the spread of such resistance genes, we sought to determine the diversity of CTX-M ESBLs not among clinical isolates, but in the environment, where weaker and more diverse selective pressures may allow greater enzyme diversification. This was done by examining the CTX-M diversity in municipal wastewater and urban coastal wetlands in southern California, United States, by Sanger sequencing of polymerase chain reaction amplicons. Of the five known CTX-M phylogroups (1, 2, 8, 9, and 25), only genes from groups 1 and 2 were detected in both wastewater treatment plants (WWTPs), and group 1 genes were also detected in one of the two wetlands after a winter rain. The highest relative abundance of blaCTX-M group 1 genes was in the sludge of one WWTP (2.1 × 10(-4) blaCTX-M copies/16S rRNA gene copy). Gene libraries revealed surprisingly high nucleotide sequence diversity, with 157 new variants not found in GenBank, representing 99 novel amino acid sequences. Our results indicate that the resistomes of WWTPs and urban wetlands contain diverse blaCTX-M ESBLs, which may constitute a mobile reservoir of clinically relevant resistance genes.

THE HOPF BIFURCATION WITH BOUNDED NOISE.

We study Hopf-Andronov bifurcations in a class of random differential equations (RDEs) with bounded noise. We observe that when an ordinary differential equation that undergoes a Hopf bifurcation is subjected to bounded noise then the bifurcation that occurs involves a discontinuous change in the Minimal Forward Invariant set.