A rapid method for counting nucleated erythrocytes on stained blood smears by digital image analysis

Measures of parasitemia by intraerythrocytic hematozoan parasites are normally expressed as the number of infected erythrocytes per n erythrocytes and are notoriously tedious and time consuming to measure. We describe a protocol for generating rapid counts of nucleated erythrocytes from digital micrographs of thin blood smears that can be used to estimate intensity of hematozoan infections in nonmammalian vertebrate hosts. This method takes advantage of the bold contrast and relatively uniform size and morphology of erythrocyte nuclei on Giemsa-stained blood smears and uses ImageJ, a java-based image analysis program developed at the U.S. National Institutes of Health and available on the internet, to recognize and count these nuclei. This technique makes feasible rapid and accurate counts of total erythrocytes in large numbers of microscope fields, which can be used in the calculation of peripheral parasitemias in low-intensity infections.     

Modelling the impacts of two exotic invasive species on a native butterfly: top-down vs. bottom-up effects

1. Exotic invasive species can influence population dynamics of native species through top-down or bottom-up forces. The present study examined separate and interactive effects of multiple exotic species invasions on the native mustard white butterfly, Pieris napi oleracea Harris (Lepidoptera: Pieridae), using a stochastic simulation model. 2. P. n. oleracea populations in North America have decreased regionally since the 1860s. Competition with an exotic congener (P. rapae L.), loss of native host plants and parasitism by the introduced broconid wasp (Cotesia glomerata L.), have been suggested to be independently responsible for its decline. The present study examined these hypotheses, as well as an alternative, invasion by an exotic crucifer, garlic mustard (Alliaria petiolata[Bieb.] Cavara & Grande). 3. A stochastic simulation model of P. n. oleracea population dynamics revealed that decreasing the number of host plants available for oviposition and larval development (i.e. habitat loss), sharply reduced the probability of populations persistence and decreased population size for those that persisted. 4. Simulated invasion by garlic mustard also substantially decreased both probability of persistence (= 0 at approximately 50% cover) and mean population size. Persistence probability never reached zero under any C. glomerata scenarios, even when larval mortality in the second generation due to parasitism was 100%. The impact of garlic mustard was intensified by the addition of C. glomerata parasitism. 5. Results suggest that bottom-up forces, loss of host plants through forest understorey loss and/or garlic mustard invasion are the most important forces driving P. n. oleracea population decline. Parasitism by C. glomerata may interact to reduce P. n. oleracea populations more rapidly, but appears insufficient alone to cause local extinction.     

AHR2 mutant reveals functional diversity of aryl hydrocarbon receptors in zebrafish

The aryl hydrocarbon receptor (AHR) is well known for mediating the toxic effects of TCDD and has been a subject of intense research for over 30 years. Current investigations continue to uncover its endogenous and regulatory roles in a wide variety of cellular and molecular signaling processes. A zebrafish line with a mutation in ahr2 (ahr2(hu3335)), encoding the AHR paralogue responsible for mediating TCDD toxicity in zebrafish, was developed via Targeting Induced Local Lesions IN Genomes (TILLING) and predicted to express a non-functional AHR2 protein. We characterized AHR activity in the mutant line using TCDD and leflunomide as toxicological probes to investigate function, ligand binding and CYP1A induction patterns of paralogues AHR2, AHR1A and AHR1B. By evaluating TCDD-induced developmental toxicity, mRNA expression changes and CYP1A protein in the AHR2 mutant line, we determined that ahr2(hu3335) zebrafish are functionally null. In silico modeling predicted differential binding of TCDD and leflunomide to the AHR paralogues. AHR1A is considered a non-functional pseudogene as it does not bind TCCD or mediate in vivo TCDD toxicity. Homology modeling, however, predicted a ligand binding conformation of AHR1A with leflunomide. AHR1A-dependent CYP1A immunohistochemical expression in the liver provided in vivo confirmation of the in silico docking studies. The ahr2(hu3335) functional knockout line expands the experimental power of zebrafish to unravel the role of the AHR during development, as well as highlights potential activity of the other AHR paralogues in ligand-specific toxicological responses.     

Grape seed proanthocyanidins inhibit the invasiveness of human HNSCC cells by targeting EGFR and reversing the epithelial-to-mesenchymal transition

Head and neck squamous cell carcinoma (HNSCC) is responsible for approximately 20,000 deaths per year in the United States. Most of the deaths are due to the metastases. To develop more effective strategies for the prevention of metastasis of HNSCC cells, we have determined the effect of grape seed proanthocyanidins (GSPs) on the invasive potential of HNSCC cell and the mechanisms underlying these effects using OSC19 cells as an in vitro model. Using cell invasion assays, we established that treatment of the OSC19 cells with GSPs resulted in a dose-dependent inhibition of cell invasion. EGFR is over-expressed in 90% of HNSCCs and the EGFR inhibitors, erlotinib and gefitinib, are being explored as therapies for this disease. We found that GSPs treatment reduced the levels of expression of EGFR in the OSC19 cells as well as reducing the activation of NF-κB/p65, a downstream target of EGFR, and the expression of NF-κB-responsive proteins. GSPs treatment also reduced the activity of ERK1/2, an upstream regulator of NF-κB and treatment of the cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, inhibited cell invasion. Overexpression of EGFR and high NF-κB activity play a key role in the epithelial-to-mesenchymal transition, which is of critical importance in the processes underlying metastasis, and we found treatment with GSPs enhanced the levels of epithelial (E-cadherin, cytokeratins and desmoglein-2) and reduced the levels of mesenchymal (vimentin, fibronectin, N-cadherin and Slug) biomarkers in the OSC19 cells. These results indicate that GSPs have the ability to inhibit HNSCC cell invasion, and do so by targeting the expression of EGFR and activation of NF-κB as well as inhibiting the epithelial-to-mesenchymal transition.     

Forest nitrogen sinks in large eastern U.S. watersheds: estimates from forest inventory and an ecosystem model

The eastern U.S. receives elevated rates of Ndeposition compared to preindustrial times, yetrelatively little of this N is exported indrainage waters. Net uptake of N into forestbiomass and soils could account for asubstantial portion of the difference between Ndeposition and solution exports. We quantifiedforest N sinks in biomass accumulation andharvest export for 16 large river basins in theeastern U.S. with two separate approaches: (1)using growth data from the USDA ForestService's Forest Inventory and Analysis (FIA)program, and (2) using a model of forestnitrogen cycling (PnET-CN) linked to FIAinformation on forest age-class structure. Themodel was also used to quantify N sinks in soiland dead wood, and nitrate losses below therooting zone. Both methods agreed that netgrowth rates were highest in the relativelyyoung forests on the Schuylkill watershed, andlowest in the cool forests of northern Maine. Across the 16 watersheds, wood export removedan average of 2.7 kg N ha^–1 yr^–1(range: 1–5 kg N ha^–1 yr^–1), andstanding stocks increased by 4.0 kg N ha^–1yr^–1 (–3 to 8 kg N ha^–1 yr^–1). Together, these sinks for N in woody biomassamounted to a mean of 6.7 kg N ha^–1yr^–1 (2–9 kg N ha^–1 yr^–1), or73% (15–115%) of atmospheric N deposition. Modeled rates of net N sinks in dead wood andsoil were small; soils were only a significantnet sink for N during simulations ofreforestation of degraded agricultural sites. Predicted losses of nitrate depended on thecombined effects of N deposition, and bothshort- and long-term effects of disturbance. Linking the model with forest inventoryinformation on age-class structure provided auseful step toward incorporating realisticpatterns of forest disturbance status acrossthe landscape.     

Anthropogenic nitrogen sources and relationships to riverine nitrogen export in the northeastern U.S.A.

Human activities have greatly altered the nitrogen (N) cycle, accelerating the rate of N fixation in landscapes and delivery of N to water bodies. To examine relationships between anthropogenic N inputs and riverine N export, we constructed budgets describing N inputs and losses for 16 catchments, which encompass a range of climatic variability and are major drainages to the coast of the North Atlantic Ocean along a latitudinal profile from Maine to Virginia. Using data from the early 1990's, we quantified inputs of N to each catchment from atmospheric deposition, application of nitrogenous fertilizers, biological nitrogen fixation, and import of N in agricultural products (food and feed). We compared these inputs with N losses from the system in riverine export.The importance of the relative sources varies widely by catchment and is related to land use. Net atmospheric deposition was the largest N source (>60%) to the forested basins of northern New England (e.g. Penobscot and Kennebec); net import of N in food was the largest source of N to the more populated regions of southern New England (e.g. Charles & Blackstone); and agricultural inputs were the dominant N sources in the Mid-Atlantic region (e.g. Schuylkill & Potomac). Over the combined area of the catchments, net atmospheric deposition was the largest single source input (31%), followed by net imports of N in food and feed (25%), fixation in agricultural lands (24%), fertilizer use (15%), and fixation in forests (5%). The combined effect of fertilizer use, fixation in crop lands, and animal feed imports makes agriculture the largest overall source of N. Riverine export of N is well correlated with N inputs, but it accounts for only a fraction (25%) of the total N inputs. This work provides an understanding of the sources of N in landscapes, and highlights how human activities impact N cycling in the northeast region.     

Assemblage structure and dynamics of terrestrial birds in the southwest Amazon: a camera‐trap case study

Habitat spatial distribution, seasonal variation, and activity patterns influence changes in vertebrate assemblages over time. Terrestrial birds play major roles in the dynamics of tropical forests, but there are few effective methods to study these species due to their cryptic coloration and elusive behavior. We used camera‐trap data collected during 16 mo (February 2017–June 2018) to describe the terrestrial avifauna in southeastern Peru, assess to what extent the composition of terrestrial avifauna changes among seasons and across two major habitats (terra firme and floodplain forests), and determine daily activity patterns of terrestrial birds. We used overlap analyses to examine temporal co‐occurrence between ecologically similar and sympatric species. Camera traps recorded 16 species, including eight species in the family Tinamidae. Capture rates were highest for Pale‐winged Trumpeters (Psophia leucoptera; Psophiidae) and Gray‐fronted Doves (Leptolila rufaxilla; Columbidae). Species composition did not differ between habitats or seasons, and capture rates between habitats only differed for White‐throated Tinamous (Tinamus guttatus). Overlaps of activity patterns were high between ecologically similar species and species found in terra firme habitats (White‐throated Tinamous and Cinereous Tinamous, Crypturellus cinereus) and in both habitat types (Pale‐winged Trumpeters and Gray‐fronted Doves). Low numbers of captures of possibly locally rare or less abundant species hindered a complete analysis of spatial and seasonal patterns of terrestrial bird assemblages. We suggest a greater sampling effort and greater spatial replication to better understand the spatial and seasonal dynamics of the terrestrial avifauna. Further studies that assess the mechanisms that allow the coexistence of sympatric tinamous would be valuable, both in our study area and elsewhere. The use of camera traps in long‐term monitoring projects proved to be an effective tool for monitoring terrestrial birds, identifying cryptic and often rare animals to species level, and providing valuable ecological information at species and community levels.     

Estimating and interpreting secondary attack risk: Binomial considered biased

The household secondary attack risk (SAR), often called the secondary attack rate or secondary infection risk, is the probability of infectious contact from an infectious household member A to a given household member B, where we define infectious contact to be a contact sufficient to infect B if he or she is susceptible. Estimation of the SAR is an important part of understanding and controlling the transmission of infectious diseases. In practice, it is most often estimated using binomial models such as logistic regression, which implicitly attribute all secondary infections in a household to the primary case. In the simplest case, the number of secondary infections in a household with m susceptibles and a single primary case is modeled as a binomial(m, p) random variable where p is the SAR. Although it has long been understood that transmission within households is not binomial, it is thought that multiple generations of transmission can be neglected safely when p is small. We use probability generating functions and simulations to show that this is a mistake. The proportion of susceptible household members infected can be substantially larger than the SAR even when p is small. As a result, binomial estimates of the SAR are biased upward and their confidence intervals have poor coverage probabilities even if adjusted for clustering. Accurate point and interval estimates of the SAR can be obtained using longitudinal chain binomial models or pairwise survival analysis, which account for multiple generations of transmission within households, the ongoing risk of infection from outside the household, and incomplete follow-up. We illustrate the practical implications of these results in an analysis of household surveillance data collected by the Los Angeles County Department of Public Health during the 2009 influenza A (H1N1) pandemic.     

THE EFFECT OF ULTRA-VIOLET,X-RAY,AND RADIUM RADIATION ON THE TOXICITY OF NORMAL BLOOD

While the number of experiments performed is perhaps somewhat limited the results obtained were quite definite and warrant the following conclusions. The toxicity of normal blood for living plant protoplasm as studied on the growth of Lupinus albus seedlings is definitely influenced by various radiations. Ultra-violet rays produce no effect on normal blood or may even render it slightly less toxic. Roentgen rays render normal blood more toxic. The toxicity is greater in the case of the blood plasma as compared with the blood cells and a more toxic effect is produced with the Coolidge tube as compared with the gas tube. Radium emanations in the few experiments performed produced changes very much the same as those given by the x-rays.