Classic indicators and diel dissolved oxygen versus trend analysis in assessing eutrophication of potable-water reservoirs.

Potable source-water reservoirs are the main water supplies in many urbanizing regions, yet their long-term responses to cultural eutrophication are poorly documented in comparison with natural lakes, creating major management uncertainties. Here, long-term discrete data (June 2006-June 2018) for classical eutrophication water quality indicators, continuous depth-profile data for dissolved oxygen (DO), and an enhanced hybrid statistical trend analysis model were used to evaluate the eutrophication status of a potable source-water reservoir. Based on classical indicators (nitrogen, N and phosphorus, P concentrations and ratios; phytoplankton biomass as chlorophyll a, chl a; and trophic state indices), the reservoir was eutrophic to hypereutrophic and stoichiometrically imbalanced. Anoxia/hypoxia occurred for 7-8 months annually systemwide, even throughout the water column for days to weeks in some years; and elevated total ammonia (up to ~900 µg tNH3 L-1 ) in surface waters from late summer/fall through late winter/early spring suggested substantial internal legacy nutrient loading. These surprising DO and tNH3 phenomena may characterize many reservoirs in urbanizing areas, and the associated cascade of negative impacts may increasingly affect them under global warming. Total organic carbon (TOC), seasonally influenced by phytoplankton biomass, commonly exceeded 6 mg L-1 , which is problematic for potable-water treatment, and significantly trended up over time. Wet-year inflow dilution influenced an apparent decreasing trend in nutrients within the hypereutrophic upper reservoir, which receives most tributary inputs. Nevertheless, significant reservoirwide trends (increasing total phosphorus [TP], phytoplankton chl a, TOC) and mid- and/or lower region trends (increasing total nitrogen [TN], tNH3 , decreasing TN:TP ratios) suggest that water quality degradation from eutrophication has worsened over time. These findings support broadly applicable recommendations to strengthen protection of potable source-water reservoirs in urbanizing watersheds: (1) protective numeric water quality criteria are needed for TOC as well as TN, TP, and chl a; (2) continuous diel data capture more realistic DO conditions than traditional sampling, and can provide important insights for water treatment managers; and (3) assessment of reservoir eutrophication status to track management progress over time should emphasize classic indicators equally as statistical trends, which are highly sensitive to short-term meteorological forcing.

Individual and environmental risk factors associated with fecal glucocorticoid metabolite concentrations in zoo-housed Asian and African elephants.

A recent large-scale welfare study in North America involving 106 Asian (Elephas maximus) and 131 African (Loxodonta africana) elephants at 64 accredited facilities identified links (i.e., risk factors) between zoo environmental factors and a number of welfare outcomes (stereotypic behavior, ovarian acyclicity, hyperprolactinemia, walking and recumbence, body condition, health status, serum cortisol). For this population of elephants, we used the same epidemiological methods to examine associations between those risk factors and two additional welfare outcomes, mean concentration and individual variability (CV) of fecal glucocorticoid metabolite concentrations (FGM) as indicators of stress. Results indicate that African elephants are more responsive to social stressors than Asians, and that poor joint health is a stress-related welfare problem for Asian, but not African elephants in the North American population. For both species, higher FGM concentrations were associated with zoos located at more northern latitudes, whereas lower FGM concentrations were associated with having free access to indoor/outdoor spaces, and spending more time in managed interactions with staff. Also important for captive management, elephants having diverse enrichment options and belonging to compatible social groups exhibited reduced intra-individual variability in FGM concentrations. Our findings show that aspects of the zoo environment can be potential sources of stress for captive elephants, and that there are management activities that may facilitate coping with zoo conditions. Given species differences in factors that affected FGM, targeted, species-specific management approaches likely are needed to ensure good welfare for all elephants.

Rapid and Sensitive Detection of Breast Cancer Cells in Patient Blood with Nuclease-Activated Probe Technology.

A challenge for circulating tumor cell (CTC)-based diagnostics is the development of simple and inexpensive methods that reliably detect the diverse cells that make up CTCs. CTC-derived nucleases are one category of proteins that could be exploited to meet this challenge. Advantages of nucleases as CTC biomarkers include: (1) their elevated expression in many cancer cells, including cells implicated in metastasis that have undergone epithelial-to-mesenchymal transition; and (2) their enzymatic activity, which can be exploited for signal amplification in detection methods. Here, we describe a diagnostic assay based on quenched fluorescent nucleic acid probes that detect breast cancer CTCs via their nuclease activity. This assay exhibited robust performance in distinguishing breast cancer patients from healthy controls, and it is rapid, inexpensive, and easy to implement in most clinical labs. Given its broad applicability, this technology has the potential to have a substantive impact on the diagnosis and treatment of many cancers.

RNA-Seq study reveals genetic responses of diverse wild soybean accessions to increased ozone levels.

BACKGROUND: Ozone is an air pollutant widely known to cause a decrease in productivity in many plant species, including soybean (Glycine max (L.) Merr). While the response of cultivated soybean to ozone has been studied, very little information is available regarding the ozone response of its wild relatives. RESULTS: Ozone-resistant wild soybean accessions were identified by measuring the response of a genetically diverse group of 66 wild soybean (Glycine soja Zucc. and Sieb.) accessions to elevated ozone levels. RNA-Seq analyses were performed on leaves of different ages from selected ozone-sensitive and ozone-resistant accessions that were subjected to treatment with an environmentally relevant level of ozone. Many more genes responded to elevated ozone in the two ozone-sensitive accessions than in the ozone-resistant accessions. Analyses of the ozone response genes indicated that leaves of different ages responded differently to ozone. Older leaves displayed a consistent reduction in expression of genes involved in photosynthesis in response to ozone, while changes in expression of defense genes dominated younger leaf tissue in response to ozone. As expected, there is a substantial difference between the response of ozone-sensitive and ozone-resistant accessions. Genes associated with photosystem 2 were substantially reduced in expression in response to ozone in the ozone-resistant accessions. A decrease in peptidase inhibitors was one of several responses specific to one of the ozone resistant accessions. CONCLUSION: The decrease in expression in genes associated with photosynthesis confirms that the photosynthetic apparatus may be an early casualty in response to moderate levels of ozone. A compromise of photosynthesis would substantially impact plant growth and seed production. However, the resistant accessions may preserve their photosynthetic apparatus in response to the ozone levels used in this study. Older leaf tissue of the ozone-resistant accessions showed a unique down-regulation of genes associated with endopeptidase inhibitor activity. This study demonstrates the existence of significant diversity in wild soybean for ozone response. Wild soybean accessions characterized in this study can be used by soybean breeders to enhance ozone tolerance of this important food crop.

CFTR gene transfer with AAV improves early cystic fibrosis pig phenotypes.

The physiological components that contribute to cystic fibrosis (CF) lung disease are steadily being elucidated. Gene therapy could potentially correct these defects. CFTR-null pigs provide a relevant model to test gene therapy vectors. Using an in vivo selection strategy that amplifies successful capsids by replicating their genomes with helper adenovirus coinfection, we selected an adeno-associated virus (AAV) with tropism for pig airway epithelia. The evolved capsid, termed AAV2H22, is based on AAV2 with 5 point mutations that result in a 240-fold increased infection efficiency. In contrast to AAV2, AAV2H22 binds specifically to pig airway epithelia and is less reliant on heparan sulfate for transduction. We administer AAV2H22-CFTR expressing the CF transmembrane conductance regulator (CFTR) cDNA to the airways of CF pigs. The transduced airways expressed CFTR on ciliated and nonciliated cells, induced anion transport, and improved the airway surface liquid pH and bacterial killing. Most gene therapy studies to date focus solely on Cl- transport as the primary metric of phenotypic correction. Here, we describe a gene therapy experiment where we not only correct defective anion transport, but also restore bacterial killing in CFTR-null pig airways.

Structural computational modeling of RNA aptamers.

RNA aptamers represent an emerging class of biologics that can be easily adapted for personalized and precision medicine. Several therapeutic aptamers with desirable binding and functional properties have been developed and evaluated in preclinical studies over the past 25years. However, for the majority of these aptamers, their clinical potential has yet to be realized. A significant hurdle to the clinical adoption of this novel class of biologicals is the limited information on their secondary and tertiary structure. Knowledge of the RNA's structure would greatly facilitate and expedite the post-selection optimization steps required for translation, including truncation (to reduce costs of manufacturing), chemical modification (to enhance stability and improve safety) and chemical conjugation (to improve drug properties for combinatorial therapy). Here we describe a structural computational modeling methodology that when coupled to a standard functional assay, can be used to determine key sequence and structural motifs of an RNA aptamer. We applied this methodology to enable the truncation of an aptamer to prostate specific membrane antigen (PSMA) with great potential for targeted therapy that had failed previous truncation attempts. This methodology can be easily applied to optimize other aptamers with therapeutic potential.

Smooth Muscle Cell-targeted RNA Aptamer Inhibits Neointimal Formation.

Inhibition of vascular smooth muscle cell (VSMC) proliferation by drug eluting stents has markedly reduced intimal hyperplasia and subsequent in-stent restenosis. However, the effects of antiproliferative drugs on endothelial cells (EC) contribute to delayed re-endothelialization and late stent thrombosis. Cell-targeted therapies to inhibit VSMC remodeling while maintaining EC health are necessary to allow vascular healing while preventing restenosis. We describe an RNA aptamer (Apt 14) that functions as a smart drug by preferentially targeting VSMCs as compared to ECs and other myocytes. Furthermore, Apt 14 inhibits phosphatidylinositol 3-kinase/protein kinase-B (PI3K/Akt) and VSMC migration in response to multiple agonists by a mechanism that involves inhibition of platelet-derived growth factor receptor (PDGFR)-ß phosphorylation. In a murine model of carotid injury, treatment of vessels with Apt 14 reduces neointimal formation to levels similar to those observed with paclitaxel. Importantly, we confirm that Apt 14 cross-reacts with rodent and human VSMCs, exhibits a half-life of ~300 hours in human serum, and does not elicit immune activation of human peripheral blood mononuclear cells. We describe a VSMC-targeted RNA aptamer that blocks cell migration and inhibits intimal formation. These findings provide the foundation for the translation of cell-targeted RNA therapeutics to vascular disease.

Oligonucleotide aptamers: A next-generation technology for the capture and detection of circulating tumor cells.

A critical challenge for treating cancer is the early identification of those patients who are at greatest risk of developing metastatic disease. The number of circulating tumor cells (CTCs) in cancer patients has recently been shown to be a valuable (and non-invasively accessible) diagnostic indicator of the state of metastatic disease. CTCs are rare cancer cells found in the blood circulation of cancer patients believed to provide a means of diagnosing the likelihood for metastatic spread and assessing response to therapy in advanced, as well as early stage disease settings. Numerous technical efforts have been made to reliably detect and quantify CTCs, but the development of a universal assay has proven quite difficult. Notable challenges for developing a broadly useful CTC-based diagnostic assay are the development of easy-to-operate methods that (1) are sufficiently sensitive to reliably detect the small number of CTCs that are present in the circulation and (2) can capture the molecular heterogeneity of tumor cells. In this review, we describe recent progress towards the application of synthetic oligonucleotide aptamers as promising, novel, robust tools for the isolation and detection of CTCs. Advantages and challenges of the aptamer approach are also discussed.

Method for Confirming Cytoplasmic Delivery of RNA Aptamers.

RNA aptamers are single-stranded RNA oligos that represent a powerful emerging technology with potential for treating numerous diseases. More recently, cell-targeted RNA aptamers have been developed for delivering RNA interference (RNAi) modulators (siRNAs and miRNAs) to specific diseased cells (e.g., cancer cells or HIV infected cells) in vitro and in vivo. However, despite initial promising reports, the broad application of this aptamer delivery technology awaits the development of methods that can verify and confirm delivery of aptamers to the cytoplasm of target cells where the RNAi machinery resides. We recently developed a functional assay (RIP assay) to confirm cellular uptake and subsequent cytoplasmic release of an RNA aptamer which binds to a cell surface receptor expressed on prostate cancer cells (PSMA). To assess cytoplasmic delivery, the aptamer was chemically conjugated to saporin, a ribosome inactivating protein toxin that is toxic to cells only when delivered to the cytoplasm (where it inhibits the ribosome) by a cell-targeting ligand (e.g., aptamer). Here, we describe the chemistry used to conjugate the aptamer to saporin and discuss a gel-based method to verify conjugation efficiency. We also detail an in vitro functional assay to confirm that the aptamer retains function following conjugation to saporin and describe a cellular assay to measure aptamer-mediated saporin-induced cytotoxicity.

A pilot study evaluating presurgery neuroanatomical biomarkers for postoperative cognitive decline after total knee arthroplasty in older adults.

BACKGROUND: Total knee arthroplasty improves quality of life but is associated with postoperative cognitive dysfunction in older adults. This prospective longitudinal pilot study with a parallel control group tested the hypotheses that (1) nondemented adults would exhibit primary memory and executive difficulties after total knee arthroplasty, and (2) reduced preoperative hippocampus/entorhinal volume would predict postoperative memory change, whereas preoperative leukoaraiosis and lacunae volumes would predict postoperative executive dysfunction. METHODS: Surgery (n = 40) and age-education-matched controls with osteoarthritis (n = 15) completed pre- and postoperative (3 weeks, 3 months, and 1 yr) memory and cognitive testing. Hypothesized brain regions of interest were measured in patients completing preoperative magnetic resonance scans (surgery, n = 31; control, n = 12). Analyses used reliable change methods to identify the frequency of cognitive change at each time point. RESULTS: The incidence of postoperative memory difficulties was shown with delay test indices (i.e., story memory test: 3 weeks = 17%, 3 months = 25%, 1 yr = 9%). Postoperative executive difficulty with measures of inhibitory function (i.e., Stroop Color Word: 3 weeks = 21%, 3 months = 22%, 1 yr = 9%). Hierarchical regression analysis assessing the predictive interaction of group (surgery, control) and preoperative neuroanatomical structures on decline showed that greater preoperative volumes of leukoaraiosis/lacunae were significantly contributed to postoperative executive (inhibitory) declines. CONCLUSIONS: This pilot study suggests that executive and memory declines occur in nondemented adults undergoing orthopedic surgery. Severity of preoperative cerebrovascular disease may be relevant for understanding executive decline, in particular.

CaMKII is essential for the proasthmatic effects of oxidation.

Increased reactive oxygen species (ROS) contribute to asthma, but little is known about the molecular mechanisms connecting increased ROS with characteristic features of asthma. We show that enhanced oxidative activation of the Ca(2+)/calmodulin-dependent protein kinase (ox-CaMKII) in bronchial epithelium positively correlates with asthma severity and that epithelial ox-CaMKII increases in response to inhaled allergens in patients. We used mouse models of allergic airway disease induced by ovalbumin (OVA) or Aspergillus fumigatus (Asp) and found that bronchial epithelial ox-CaMKII was required to increase a ROS- and picrotoxin-sensitive Cl(-) current (ICl) and MUC5AC expression, upstream events in asthma progression. Allergen challenge increased epithelial ROS by activating NADPH oxidases. Mice lacking functional NADPH oxidases due to knockout of p47 and mice with epithelial-targeted transgenic expression of a CaMKII inhibitory peptide or wild-type mice treated with inhaled KN-93, an experimental small-molecule CaMKII antagonist, were protected against increases in ICl, MUC5AC expression, and airway hyperreactivity to inhaled methacholine. Our findings support the view that CaMKII is a ROS-responsive, pluripotent proasthmatic signal and provide proof-of-concept evidence that CaMKII is a therapeutic target in asthma.

Hoechst increases adeno-associated virus-mediated transgene expression in airway epithelia by inducing the cytomegalovirus promoter.

BACKGROUND: In airway epithelia, the kinetics of recombinant adeno-associated virus (AAV) transgene expression is slow. This has negative practical implications for research, as well as for translation into therapy. The DNA minor groove-binding agent Hoechst-33342 has been shown to enhance AAV transgene expression. In the present study, we investigated the mechanism of Hoechst-related augmentation of AAV-mediated transgene expression. METHODS: We investigated the effect of Hoechst-33342 on HT1080, COS-7, mouse and human airway epithelia transduced with different AAV serotypes encoding enhanced green fluorescent protein (eGFP). We exposed cells to increasing concentrations of Hoechst-33342 at different time points. We evaluated the effect on second-strand DNA synthesis using AAV with a self-complementary genome. We also investigated the effect on expression from transfected plasmids with and without AAV2 inverted terminal repeats (ITRs). RESULTS: We found that Hoechst-33342 significantly accelerated AAV transgene expression for all serotypes tested. Hoechst-33342 only had an effect when the treatment was given during or after transduction, even 120 days post-transduction, suggesting an effect on transgene expression regulation. Hoechst-33342 increased transgene expression when cells were transduced with a self-complementary AAV with the cytomegalovirus promoter, although there was no effect on cells transduced with conventional single-stranded AAV encoding the Rous sarcoma virus promoter. Finally, Hoechst-33342 increases gene expression from transfected plasmids regardless of the presence of AAV2 ITRs. CONCLUSIONS: Hoechst dramatically augments and accelerates AAV-mediated transgene expression in airway epithelia without altering AAV-mediated gene transfer. Hoechst activation of the cytomegalovirus promoter is seen in plasmids, although it is drastically enhanced in the context of AAV.

Enhanced sialic acid-dependent endocytosis explains the increased efficiency of infection of airway epithelia by a novel adeno-associated virus.

We previously used directed evolution in human airway epithelia to create adeno-associated virus 2.5T (AAV2.5T), a highly infectious chimera of AAV2 and AAV5 with one point mutation (A581T). We hypothesized that the mechanism for its increased infection may be a higher binding affinity to the surface of airway epithelia than its parent AAV5. Here, we show that, like AAV5, AAV2.5T, uses 2,3N-linked sialic acid as its primary receptor; however, AAV2.5T binds to the apical surface of human airway epithelia at higher levels and has more receptors than AAV5. Furthermore, its binding affinity is similar to that of AAV5. An alternative hypothesis is that AAV2.5T interaction with 2,3N-linked sialic acid may instead be required for cellular internalization. Consistent with this, AAV2.5T binds but fails to be internalized by CHO cells that lack surface expression of sialic acid. Moreover, whereas AAV2.5T binds similarly to human (rich in 2,3N-linked sialic acid) and pig airway epithelia (2,6N-linked sialic acid), significantly more virus was internalized by human airway. Subsequent transduction correlated with the level of internalized rather than surface-bound virus. We also found that human airway epithelia internalized significantly more AAV2.5T than AAV5. These data suggest that AAV2.5T has evolved to utilize specific 2,3N-linked sialic acid residues on the surface of airway epithelia that mediate rapid internalization and subsequent infection. Thus, sialic acid serves as not just an attachment factor but is also required for AAV2.5T internalization, possibly representing an important rate-limiting step for other viruses that use sialic acids.

Directed evolution of adeno-associated virus to an infectious respiratory virus.

Respiratory viruses evolve to maintain infectivity levels that permit spread yet prevent host and virus extinction, resulting in surprisingly low infection rates. Respiratory viruses harnessed as gene therapy vectors have illustrated this limitation. We used directed evolution in an organotypic human airway model to generate a highly infectious adeno-associated virus. This virus mediated gene transfer more than 100-fold better than parental strains and corrected the cystic fibrosis epithelial Cl(-) transport defect. Thus, under appropriate selective pressures, viruses can evolve to be more infectious than observed in nature, a finding that holds significant implications for designing vectors for gene therapy and for understanding emerging pathogens.

Mercury(II) bioaccumulation and antioxidant physiology in four aquatic insects.

We examined Hg(II) bioaccumulation and compartmentalization patterns in conjunction with antioxidant responses in four aquatic insect species: two caddisflies (Chimarra sp. and Hydropsyche betteni) and two mayflies (Maccaffertium modestum and Isonychia sp). Total antioxidant capabilities differed among unexposed larvae, with both caddisfly species exhibiting elevated antioxidant activities relative to the mayflies. We were able to account for these differences by examining the constitutive activities of catalase (CAT), glutathione peroxidase (GPx), glutathione S-transferase (GST), and superoxide dismutase (SOD), in the four species. We also examined levels of reduced and oxidized glutathione and cysteine in the insects. Glutathione peroxidase and SOD were the most responsive to Hg exposure, with GPx catalytic activity increasing between 50 and 310%. Superoxide dismutase activity decreased between 35 and 50%. This SOD suppression was shown to be dose-dependent in both caddisflies, butthe strength of this suppression did not appear to be related to rates of uptake. Surprisingly, little Hg (<10%) was found in the heat-stable cytosolic protein subcellular compartment in each of the four species, suggesting that Hg was not well detoxified. By combining bioaccumulation studies with other physiological measures, we can begin to better understand the consequences of trace metal pollutants in nature.

The relation of stroke admissions to recent weather, airborne allergens, air pollution, seasons, upper respiratory infections, and asthma incidence, September 11, 2001, and day of the week.

BACKGROUND AND PURPOSE: Some previous research links stroke incidence to weather, some links strokes to air pollution, and some report seasonal effects. Alveolar inflammation was proposed as the mechanistic link. We present a unified model of time, weather, pollution, and upper respiratory infection (URI) incidence. METHODS: We combined existing databases: US Environmental Protection Agency pollution levels, National Weather Service data, counts of airborne allergens, and New York City Health and Hospitals Corporation counts of stroke, asthma, and URI patients. We used autoregressive integrated moving average modeling (a statistical time series modeling technique) with stroke admissions as the response variable and day of week, holidays, September 11th, and other counts and levels as explanatory variables. RESULTS: Using a broad definition of stroke, there were 5.1+/-2.3 stroke admissions per day: narrowly defined, 4.2+/-2.1 strokes per day. There are relatively fewer strokes on Sundays (0.50 strokes; P=0.0011), Saturdays (0.62; P<0.0001), Fridays (0.38; P=0.0009) and holidays (0.875; P=0.0016). We found relatively small, independent exacerbating effects of higher air temperature (P=0.0211), dry air (P=0.0187), URIs, (P<0.0001), grass pollen (P=0.0341), sulfur dioxide (SO2; P=0.0471), and suspended particles <10 microm in size (P=0.0404). These effects are modest: < or =0.6, 0.6, 2.4, 1, 0.9, and 0.7 strokes per day, respectively. We did not find statistically significant exacerbating effects of other variables. CONCLUSIONS: We found statistically significant, independent exacerbating effects of warmer, drier air, URIs, grass pollen, SO2, and particulate air pollution. The model supports the theory that links pulmonary inflammation to stroke.