Ligand-induced EGF receptor oligomerization is kinase-dependent and enhances internalization

The current activation model of the EGF receptor (EGFR) predicts that binding of EGF results in dimerization and oligomerization of the EGFR, leading to the allosteric activation of the intracellular tyrosine kinase. Little is known about the regulatory mechanism of receptor oligomerization. In this study, we have employed FRET between identical fluorophores (homo-FRET) to monitor the dimerization and oligomerization state of the EGFR before and after receptor activation. Our data show that, in the absence of ligand, ～40% of the EGFR molecules were present as inactive dimers or predimers. The monomer/predimer ratio was not affected by deletion of the intracellular domain. Ligand binding induced the formation of receptor oligomers, which were found in both the plasma membrane and intracellular structures. Ligand-induced oligomerization required tyrosine kinase activity and nine different tyrosine kinase substrate residues. This indicates that the binding of signaling molecules to activated EGFRs results in EGFR oligomerization. Induction of EGFR predimers or pre-oligomers using the EGFR fused to the FK506-binding protein did not affect signaling but was found to enhance EGF-induced receptor internalization. Our data show that EGFR oligomerization is the result of EGFR signaling and enhances EGFR internalization.     

Presence of Bacteroidales as a Predictor of Pathogens in Surface Waters of the Central California Coast

The value of Bacteroidales genetic markers and fecal indicator bacteria (FIB) to predict the occurrence of waterborne pathogens was evaluated in ambient waters along the central California coast. Bacteroidales host-specific quantitative PCR (qPCR) was used to quantify fecal bacteria in water and provide insights into contributing host fecal sources. Over 140 surface water samples from 10 major rivers and estuaries within the Monterey Bay region were tested over 14 months with four Bacteroidales-specific assays (universal, human, dog, and cow), three FIB (total coliforms, fecal coliforms, and enterococci), two protozoal pathogens (Cryptosporidium and Giardia spp.), and four bacterial pathogens (Campylobacter spp., Escherichia coli O157:H7, Salmonella spp., and Vibrio spp.). Indicator and pathogen distribution was widespread, and detection was not highly seasonal. Vibrio cholerae was detected most frequently, followed by Giardia, Cryptosporidium, Salmonella, and Campylobacter spp. Bayesian conditional probability analysis was used to characterize the Bacteroidales performance assays, and the ratios of concentrations determined using host-specific and universal assays were used to show that fecal contamination from human sources was more common than livestock or dog sources in coastal study sites. Correlations were seen between some, but not all, indicator-pathogen combinations. The ability to predict pathogen occurrence in relation to indicator threshold cutoff levels was evaluated using a weighted measure that showed the universal Bacteroidales genetic marker to have a comparable or higher mean predictive potential than standard FIB. This predictive ability, in addition to the Bacteroidales assays providing information on contributing host fecal sources, supports using Bacteroidales assays in water quality monitoring programs.Coastal waters worldwide have been influenced by human activities for centuries, as they are adjacent to densely populated areas, provide a means of transportation, and receive substantial recreational use. Consequently, impairments in nearshore water quality can result from enrichment of the coastal marine ecosystem with pollutants and nutrients that are transported down watersheds from land to sea. This poses health risks to humans and animals. Microbial pollution is caused by fecal contamination from a variety of sources, including humans, livestock, pets, and wildlife, and fecal pathogen pollution has been associated with numerous outbreaks of waterborne disease (14, 15, 27, 41, 49, 55).Fecal indicator bacteria (FIB) that normally reside in the gastrointestinal tracts of humans and animals are used throughout the world to assess the microbiological quality of drinking and recreational waters. In the United States, FIB are used to define bacterial water quality standards aimed at reducing health risks in recreational waters, as required by the Beaches Environmental Assessment and Coastal Health Act (5), which amended the Clean Water Act (11). Groups of standard FIB monitored in water include total coliforms (TC), fecal coliforms (FC), Escherichia coli bacteria, and enterococci. These bacterial groups have been considered indicators of health risks in epidemiologic and quantitative microbial risk assessment (QMRA) studies (38, 42, 59, 66).To date, many monitoring programs have focused only on FIB measurements and do not test for pathogens. However, substantial evidence has been collected that challenges the usefulness of FIB data alone. A few limitations of using standard FIB to represent pathogens in water include the fact that FIB have been shown to multiply in the environment, that they are not host specific, and that the absence of FIB is not necessarily evidence of pathogen absence (21, 50, 51, 56). Consequently, alternative indicators of fecal pollution that address the weaknesses of standard FIB are needed. Ideally, these indicators would decay at rates similar to those of pathogens, be present at high concentrations in fecal sources, and be present at low concentrations in unpolluted environments. Proposed alternative indicators include (i) anaerobic bacteria such as bifidobacteria (46), Clostridium perfringens (22), and Bacteroidales (20); (ii) viruses such as F-specific RNA (F-RNA)-specific coliphages (39), phages infecting Bacteroides fragilis (30), and host-specific viruses (25); and (iii) chemical compounds such as fecal sterols (29). An added benefit of using alternative indicators is that, in some cases, host sources of fecal contamination can be identified.Over a decade ago, PCR-based assays were developed to detect Bacteroides in an effort to monitor human fecal pollution in the environment (36, 37). This approach was adopted by others and further advanced to identify host-specific Bacteroidales 16S rRNA gene markers for different fecal sources. This has resulted in PCR and quantitative PCR (qPCR) assays for the detection of human, dog, pig, and cow Bacteroidales markers (6, 7, 16, 34, 57) as well as assays for the detection of general Bacteroidales markers (7, 34). The analysis of Bacteroidales markers has been incorporated in microbial source tracking (MST) studies, particularly in the United States, Japan, and Europe (24, 45, 52-54, 64).The objective of this study was to compare the abilities of Bacteroidales markers and FIB to predict the occurrence of waterborne pathogens in riverine and estuarine waters in California and to use several statistical approaches to better characterize the strengths and limitations of the assays. We hypothesized that Bacteroidales and FIB would correlate with bacterial and protozoal pathogen detection in surface waters. To test this hypothesis, four Bacteroidales-specific assays (universal, human, dog, and cow), three types of FIB (total coliforms, fecal coliforms, and enterococci), two protozoal pathogens (Cryptosporidium and Giardia spp.), and four bacterial pathogens (Campylobacter spp., E. coli O157, Salmonella spp., and Vibrio spp.) were monitored monthly for 14 months in 10 streams, rivers, and estuaries feeding into the Monterey Bay region of California.     

Dietary lipid composition affects the gene expression of gill Na+/K+-ATPase α1b but not the α1a isoform in juvenile fall chinook salmon (Oncorhynchus tshawytscha)

We assessed the effects of dietary fatty acid composition on sodium–potassium ATPase (Na⁺/K⁺-ATPase) activity and isoform expression in the gills of juvenile fall chinook salmon, Oncorhynchus tshawytscha by supplementing diets with either anchovy oil (AO) or AO blended with canola oil (CO) so that CO comprised 0% (0CO), 11% (11CO), 22% (22CO), 33% (33CO), 43% (43CO), or 54% (54CO) of the measured dietary lipid content. The effects of diet were assessed in freshwater (FW) following 104 days of diet manipulation, in response to 24-h seawater (SW) transfer at this time, and following an additional 35 days of SW acclimation. Gill Na⁺/K⁺-ATPase activity was not significantly affected by diet at any sampling time, and there were no consistent effects of diet on the expression of the Na⁺/K⁺-ATPase α1a isoform. As dietary CO increased, Na⁺/K⁺-ATPase α1b mRNA decreased in fish held in FW, with the 43CO and 54CO diet groups having significantly lower levels than fish fed the 0CO and 11CO diets. Twenty-four-hour SW challenge did not affect the expression of the Na⁺/K⁺-ATPase α1a isoform in any diet group, but this isoform was down-regulated in all diet groups following 35 days of SW acclimation. Na⁺/K⁺-ATPase α1b expression levels increased in response to 24-h SW transfer and SW acclimation only in fish fed the 54CO diet. The effects of the two extreme diets (0CO and 54CO) were also assessed at various time points during 104 days of rearing in FW. Na⁺/K⁺-ATPase α1b mRNA levels were greater in fish fed diet 0CO versus those fed diet 54CO at all times during the FW culture period. These data demonstrate that dietary fatty acid composition can influence the gill Na⁺/K⁺-ATPase isoform physiology of juvenile fall-run chinook salmon prior to SW transfer.     

In utero cortisol and testosterone exposure and fear reactivity in infancy

Fetal programming is emerging as a major conceptual model for understanding developmental origins of health and disease, including behavioral outcomes. As part of a larger study of prenatal stress and child development, we examined the association between prenatal hormone exposure and fear reactivity, a temperament dimension that is a predictor of long-term behavioral adjustment. Amniotic fluid was collected from a sample of women undergoing clinically indicated amniocentesis for later analysis of cortisol and testosterone. Children with normal birth outcomes were recalled for follow-up assessment at 17 months, at which time we administered an observational assessment of temperament (lab-TAB; n = 108). Information on pregnancy and obstetric outcome was included as covariates. Results indicated that there was a significant association between prenatal testosterone and observed fear reactivity in boys (r(53) = 0.34, p = 0.01); no significant effect was found in girls (r(54) = − 0.07, ns); the effect remained when obstetric, psychosocial, and parental anxiety were controlled for. There was not a significant association between fetal cortisol exposure and fear reactivity. The prediction from in utero testosterone exposure to fear reactivity in boys extends prior research on prenatal testosterone and may represent an association with a general predisposition to greater arousal and reactivity.     

Large-scale field application of RNAi technology reducing Israeli acute paralysis virus disease in honey bees (Apis mellifera, Hymenoptera: Apidae)

The importance of honey bees to the world economy far surpasses their contribution in terms of honey production; they are responsible for up to 30% of the world's food production through pollination of crops. Since fall 2006, honey bees in the U.S. have faced a serious population decline, due in part to a phenomenon called Colony Collapse Disorder (CCD), which is a disease syndrome that is likely caused by several factors. Data from an initial study in which investigators compared pathogens in honey bees affected by CCD suggested a putative role for Israeli Acute Paralysis Virus, IAPV. This is a single stranded RNA virus with no DNA stage placed taxonomically within the family Dicistroviridae. Although subsequent studies have failed to find IAPV in all CCD diagnosed colonies, IAPV has been shown to cause honey bee mortality. RNA interference technology (RNAi) has been used successfully to silence endogenous insect (including honey bee) genes both by injection and feeding. Moreover, RNAi was shown to prevent bees from succumbing to infection from IAPV under laboratory conditions. In the current study IAPV specific homologous dsRNA was used in the field, under natural beekeeping conditions in order to prevent mortality and improve the overall health of bees infected with IAPV. This controlled study included a total of 160 honey bee hives in two discrete climates, seasons and geographical locations (Florida and Pennsylvania). To our knowledge, this is the first successful large-scale real world use of RNAi for disease control.     

Quantitative determination of oxytocin receptor antagonist atosiban in rat plasma by liquid chromatography–tandem mass spectrometry

A kinetic study of atosiban was conducted following repeated intravenous administration in Wistar rats. Sample analysis was performed using liquid chromatography–tandem mass spectrometry (LC–MS/MS) following full validation of an in-house method. Eptifibatide, a cyclic peptide, was used as an internal standard (IS). The analyte and internal standard were extracted using solid phase extraction (SPE) method. Chromatographic separation was carried out using an ACE C18 5 μm 50 mm × 4.6 mm column with gradient elution. Mass spectrometric detection was performed using TSQ Quantum ultra AM. The lower limit of quantification was 0.01 μg/ml when 100 μl rat plasma was used. Plasma concentrations of atosiban were measured at 0 (pre-dose), 2, 15, 30, 45, 60, 120 min at the dosage levels of 0.125 mg/kg (low dose), 0.250 mg/kg (mid dose), and 0.500 mg/kg (high dose), respectively. Atosiban plasma concentration measured at Day 1 showed mean peak atosiban concentration (C_max) 0.40, 0.57, 1.95 μg/ml for low, mid and high dose treated animals and mean peak concentration on Day 28 was 0.41, 0.88, 1.31 μg/ml on Day 28 for low, mid and high dose treated animals.     

Methods in studying ECM degradation

Almost all tissues in our body contain specific cells associated with the tissue itself, and an extracellular matrix (ECM) that consists of a variety of proteins of which the bulk is formed by different types of collagens, glycoproteins and proteoglycans. The ECM plays a pivotal role in numerous processes not only related to the mechanical properties of a tissue, but also in modulating cellular activity. For a proper functioning of a tissue remodeling of the ECM is essential. Some connective tissues are characterized by a very rapid turnover (e.g. periodontal ligament) whereas others hardly show signs of turnover (e.g. cartilage). In all situations degradation of the ECM constituents occur. Under certain conditions, especially during a pathological situation, a high level of degradation may take place. In other situations matrix synthesis and deposition outstrips breakdown, leading to a fibrosis. In order to obtain information on the level of degradation of the different ECM components, various methods have been employed. A number of these methods will be discussed in this article.     

Ecological Thresholds in the Savanna Landscape: Developing a Protocol for Monitoring the Change in Composition and Utilisation of Large Trees

Background

Acquiring greater understanding of the factors causing changes in vegetation structure - particularly with the potential to cause regime shifts - is important in adaptively managed conservation areas. Large trees (≥5 m in height) play an important ecosystem function, and are associated with a stable ecological state in the African savanna. There is concern that large tree densities are declining in a number of protected areas, including the Kruger National Park, South Africa. In this paper the results of a field study designed to monitor change in a savanna system are presented and discussed.

Methodology/Principal Findings

Developing the first phase of a monitoring protocol to measure the change in tree species composition, density and size distribution, whilst also identifying factors driving change. A central issue is the discrete spatial distribution of large trees in the landscape, making point sampling approaches relatively ineffective. Accordingly, fourteen 10 m wide transects were aligned perpendicular to large rivers (3.0–6.6 km in length) and eight transects were located at fixed-point photographic locations (1.0–1.6 km in length). Using accumulation curves, we established that the majority of tree species were sampled within 3 km. Furthermore, the key ecological drivers (e.g. fire, herbivory, drought and disease) which influence large tree use and impact were also recorded within 3 km.

Conclusions/Significance

The technique presented provides an effective method for monitoring changes in large tree abundance, size distribution and use by the main ecological drivers across the savanna landscape. However, the monitoring of rare tree species would require individual marking approaches due to their low densities and specific habitat requirements. Repeat sampling intervals would vary depending on the factor of concern and proposed management mitigation. Once a monitoring protocol has been identified and evaluated, the next stage is to integrate that protocol into a decision-making system, which highlights potential leading indicators of change. Frequent monitoring would be required to establish the rate and direction of change. This approach may be useful in generating monitoring protocols for other dynamic systems.     

Ascertaining the Effect of Reductive Dehalogenation on Chlorinated Hydrocarbon Plume Lengths in Groundwater: Analyses of Multisite Data

Chlorinated hydrocarbon groundwater plume data from a multisite study were evaluated by a variety of statistical techniques (correlation, analysis of covariance, principal components) to quantify the effects of biotransformations (reductive dehalogenation) on plume length. After accounting for the effects of groundwater velocity, source strength, and biases in the data collection process, chlorinated hydrocarbon plume lengths at sites where reductive dehalogenation was occurring were found to be significantly shorter on average, by roughly a factor of two, than those where it was not. Moreover, principal component analyses indicated significant differences in the behavior of chlorinated hydrocarbon plumes between sites with and without evidence of reductive dehalogenation, respectively. The advantage in examining plume behavior from this population-oriented perspective is that overall trends in plume behavior can be evaluated despite site-specific influences such as heterogeneities and unique release histories. Ultimately, it is these average trends that would be of the most interest to policymakers because they represent the ranges of conditions that will be encountered. This is especially important in the case of chlorinated hydrocarbons because they will biotransform at rates significant for appreciable natural attenuation only in certain instances.