Diego P. Fernandez

A review of exosome separation techniques and characterization of B16-F10 mouse melanoma exosomes with AF4-UV-MALS-DLS-TEM

AbstractExosomes participate in cancer metastasis, but studying them presents unique challenges as a result of their small size and purification difficulties. Asymmetrical field flow fractionation with in-line ultraviolet absorbance, dynamic light scattering, and multi-angle light scattering was applied to the size separation and characterization of non-labeled B16-F10 exosomes from an aggressive mouse melanoma cell culture line. Fractions were collected and further analyzed using batch mode dynamic light scattering, transmission electron microscopy and compared with known size standards. Fractogram peak positions and computed radii show good agreement between samples and across fractions. Ultraviolet absorbance fractograms in combination with transmission electron micrographs were able to resolve subtle heterogeneity of vesicle retention times between separate batches of B16-F10 exosomes collected several weeks apart. Further, asymmetrical field flow fractionation also effectively separated B16-F10 exosomes into vesicle subpopulations by size. Overall, the flow field flow fractionation instrument combined with multiple detectors was able to rapidly characterize and separate exosomes to a degree not previously demonstrated. These approaches have the potential to facilitate a greater understanding of exosome function by subtype, as well as ultimately allow for “label-free” isolation of large scale clinical exosomes for the purpose of developing future exosome-based diagnostics and therapeutics. FigureFlow path of exosome sample through the asymmetrical field flow fractionation instrument, detectors, and transmission electron microscope.

Effects of fungicides on decomposer communities and litter decomposition in vineyard streams

Large amounts of fungicides are applied globally and partly enter freshwater ecosystems. A few laboratory studies examined their effects on decomposer communities and the ecosystem process of litter decomposition (LD), whereas the field situation remains largely unknown. We conducted a field study with 17 stream sites in a German vineyard area where fungicides represent the dominant pest control agent. Passive samplers were used to monitor 15 fungicides and 4 insecticides in streams and their toxicity was described using the toxic unit approach, whereas sediment samples were taken to characterise total copper concentrations. Microbial and leaf-shredding invertebrate community composition and related LD rates were assessed at each site. The structure of microbial and shredder communities as well as fungal biomass changed along the fungicide toxicity gradient. The changes in microbial endpoints were associated with a reduction of microbial LD rate of up to 40% in polluted streams. By contrast, neither the invertebrate LD rate nor in-situ measured gammarid feeding rates correlated with fungicide toxicity, but both were negatively associated with sediment copper concentrations. A subsequent laboratory experiment employing field fungicide concentrations suggested that the microbial community changes are causal. Overall, our results suggest that fungicides can affect LD under field conditions.

Strontium isotopes delineate fine-scale natal origins and migration histories of Pacific salmon

Strontium isotopes simultaneously delineate fine-scale natal habitats and migrations of Pacific salmon harvested during a coastal commercial fishery. Highly migratory organisms present major challenges to conservation efforts. This is especially true for exploited anadromous fish species, which exhibit long-range dispersals from natal sites, complex population structures, and extensive mixing of distinct populations during exploitation. By tracing the migratory histories of individual Chinook salmon caught in fisheries using strontium isotopes, we determined the relative production of natal habitats at fine spatial scales and different life histories. Although strontium isotopes have been widely used in provenance research, we present a new robust framework to simultaneously assess natal sources and migrations of individuals within fishery harvests through time. Our results pave the way for investigating how fine-scale habitat production and life histories of salmon respond to perturbations—providing crucial insights for conservation.

Dust-mediated loading of trace and major elements to Wasatch Mountain snowpack

Depth-integrated snow columns were collected at 12 sites across the central Wasatch Mountains, Utah, during March and April 2010 to determine concentrations of trace elements, major anions and cations, and pH. Sample collection was conducted at or near maximum snow accumulation prior to the onset of melt, and included spring dust events driven by southerly pre-frontal winds. Snow samples were melted in the laboratory and subsampled for analyses on filtered (0.45 μm) and unfiltered fractions. All measured elements (Al, As, Ba, Ca, Co, Cr, Cu, Fe, Hg, K, Li, Mg, Mn, Na, Ni, Pb, Sb, Sr, Ti, Tl, U, V, and Zn) and major anions (Cl, NO(3), and SO(4)) displayed significant increases in concentration (for example, factor of 2 to 5 increases for As, Cr, Hg, and Pb) between the six sites sampled in March (prior to dust events) and the six sites sampled in April (after dust events). Acid neutralizing capacity and pH were also elevated in April relative to March snowpack. Comparison of elemental concentration in the particulate (>0.45 μm; difference between unfiltered and filtered concentration) and soluble (<0.45 μm; filtered concentration) fractions shows that the concentration increase between March and April snowpack for the trace elements is primarily a result of association with dust particles >0.45 μm. The results suggest that the majority of trace element loading to the Wasatch snowpack occurs via dust deposition. The major elements were primarily loaded in the <0.45 μm fraction, suggesting deposition of soluble dust particles. The overall findings of this paper are similar to other studies regarding the role of dust on nutrient and trace element accumulation in soils and lake sediments, but to our knowledge this is the first study that compares trace element chemistry of seasonal snowpack before and after dust deposition events.

Relationships of surface water, pore water, and sediment chemistry in wetlands adjacent to Great Salt Lake, Utah, and potential impacts on plant community health.

We collected surface water, pore water, and sediment samples at five impounded wetlands adjacent to Great Salt Lake, Utah, during 2010 and 2011 in order to characterize pond chemistry and to compare chemistry with plant community health metrics. We also collected pore water and sediment samples along multiple transects at two sheet flow wetlands during 2011 to investigate a potential link between wetland chemistry and encroachment of invasive emergent plant species. Samples were analyzed for a suite of trace and major elements, nutrients, and relevant field parameters. The extensive sampling campaign provides a broad assessment of Great Salt Lake wetlands, including a range of conditions from reference to highly degraded. We used nonmetric multidimensional scaling (NMS) to characterize the wetland sites based on the multiple parameters measured in surface water, pore water, and sediment. NMS results showed that the impounded wetlands fall along a gradient of high salinity/low trace element concentrations to low salinity/high trace element concentrations, whereas the sheet flow wetlands have both elevated salinity and high trace element concentrations, reflecting either different sources of element loading or different biogeochemical/hydrological processes operating within the wetlands. Other geochemical distinctions were found among the wetlands, including Fe-reducing conditions at two sites and sulfate-reducing conditions at the remaining sites. Plant community health metrics in the impounded wetlands showed negative correlations with specific metal concentrations in sediment (THg, Cu, Zn, Cd, Sb, Pb, Ag, Tl), and negative correlations with nutrient concentrations in surface water (nitrite, phosphate, nitrate). In the sheet flow wetlands, invasive plant species were inversely correlated with pore water salinity. These results indicate that sediment and pore water chemistry play an important role in wetland plant community health, and that monitoring and remediation efforts should consider pore water and sediment chemistry in addition to surface water chemistry.

Strontium isotopes in tap water from the coterminous USA

Strontium isotope analysis has proven useful in geo-location investigations of organic and inorganic materials and may complement the region-of-origin information provided by hydrogen and oxygen stable isotope analysis. In this study, we analyzed 99 drinking (tap) water samples collected from 95 municipal water systems across the USA to investigate the potential that 87 Sr/ 86 Sr can be used to provenance samples from managed hydrological systems. Results from a leaching and exchange experiment demonstrated that non-ideal storage conditions did not prohibit Sr isotope analysis of previously archived water samples stored in glass. Tap water samples were analyzed via multi-collector inductively coupled plasma mass spectrometry, which was preceded by a novel, automated, in-line Sr purification method. Measured tap water 87 Sr/ 86 Sr was compared to expected 87 Sr/ 86 Sr for collection location, which was predicted using four published isotope landscape (isoscape) models: age of bedrock (bedrock model), age plus major and minor lithology of bedrock (major bedrock model), weathering of Sr from rock (local water model), and surface fluxes within watershed (catchment model). The geologic history of the geographic regions represented by collected tap waters was diverse and we therefore expected significant covariation in measured and modeled 87 Sr/ 86 Sr values. Tap water exhibited large ranges in both Sr concentration (0-1.9 mg/L) and 87 Sr/ 86 Sr (0.7037-0.7320). Measured tap water 87 Sr/ 86 Sr ratios were significantly and positively correlated with predictions based on bedrock and catchment models. However, these bedrock and catchment models explained relatively little of the tap water Sr isotopic variation (;10% and 17%, respectively), suggesting that the factors affecting drinking water 87 Sr/ 86 Sr are complex and more numerous than the variables included in current water models. This could be due to the reliance of some municipal water systems on groundwater, rather than surficial water sources; the natural movement of water across distinct geologic gradients; and/or the managed transport of water from source to point-of-use. Although published isoscape models for predicting Sr isotopic variation within the continental USA are reasonable approaches for estimating surface water 87 Sr/ 86 Sr, additional efforts are needed to develop a prediction model specifically for tap water 87 Sr/ 86 Sr.

Particulate and Dissolved Trace Element Concentrations in Three Southern Ecuador Rivers Impacted by Artisanal Gold Mining

Water and sediment samples were collected along river transects at three artisanal gold mining areas in southern Ecuador: Nambija, Portovelo-Zaruma, and Ponce Enriquez. Samples were analyzed for a suite of major and trace elements, including filtered/unfiltered water samples and stream flow measurements to determine dissolved/particulate loads. Results show that the Q. Calixto, Calera, and Siete rivers (corresponding to Nambija, Portovelo-Zaruma, and Ponce Enriquez mining areas, respectively) have substantial trace element contamination due to mining inputs. Dissolved concentrations were elevated at Calera and Siete relative to Q. Calixto, possibly reflecting the input of soluble cyano-metal complexes in mining zones where cyanidation is used in ore processing. A negative correlation was found between MeHg:THg ratios and pH, indicating an inverse relationship of mercury methylation with cyanidation (since cyanidation increases water pH). This was the first comprehensive study to examine an extensive suite of trace elements in both water and sediment at the three main gold mining areas of southern Ecuador, including dissolved and particulate loads, and the first study to report MeHg concentrations in the mercury-contaminated rivers.

Isolation of strontium pools and isotope ratios in modern human hair

The elements of human hair record specific information about an individuals health, diet, and surrounding environment. Strontium isotope ratios of human hair have attracted interest as they potentially record an individuals environment. Yet, separating the external environmental signals from the internal dietary indicators has remained a challenge. Here, we examined the effects of five different hair-cleaning methodologies to determine the extent that internal and external strontium signals can be isolated from human hair. In the first study of its kind, we employed an in-line strontium purification methodology and a multi-collector inductively coupled plasma mass spectrometer to obtain high-precision strontium isotope ratio of human hair and of leachates of the different washing treatments. We found that the different applications of an individual treatment removed a consistent amount of strontium from hair and that replicate analyses showed each treatment altered the strontium isotope ratios of hair consistently. A mass-balance approach was applied to demonstrate that strontium was quantitatively removed and was accounted for in either the treated hair or the leachate. We observed that strontium isotope ratio varied as a function of treatment aggressiveness so as to suggest that there was a fine-scale structuring of strontium within hair (transverse cross-sectional variations); these variations existed as differences in strontium concentrations and isotope ratios. As a result, the Sr isotope ratio of hair and hair leachates treated with the most aggressive cleaning methods reflected the isotope ratios of the interior and total exterior strontium signatures, respectively. The results of this study indicate that external environmental strontium signals can be distinguished from the internal signals and therefore permit the application of strontium isotope ratios of modern human hair for geospatial applications.

Dendritic network models: Improving isoscapes and quantifying influence of landscape and in‐stream processes on strontium isotopes in rivers

A critical challenge for the Earth sciences is to trace the transport and flux of matter within and among aquatic, terrestrial, and atmospheric systems. Robust descriptions of isotopic patterns across space and time, called “isoscapes,” form the basis of a rapidly growing and wide-ranging body of research aimed at quantifying connectivity within and among Earths systems. However, isoscapes of rivers have been limited by conventional Euclidean approaches in geostatistics and the lack of a quantitative framework to apportion the influence of processes driven by landscape features versus in-stream phenomena. Here we demonstrate how dendritic network models substantially improve the accuracy of isoscapes of strontium isotopes and partition the influence of hydrologic transport versus local geologic features on strontium isotope ratios in a large Alaska river. This work illustrates the analytical power of dendritic network models for the field of isotope biogeochemistry, particularly for provenance studies of modern and ancient animals.

Biased cyclical electrical field flow fractionation for separation of sub 50 nm particles.

Cyclical electrical field flow fractionation (CyElFFF) is a technique for characterizing and separating nanoparticles based on their size and charge using cyclical electric fields. The high diffusion rate of nanoparticles has prevented CyElFFF from being applicable to particles smaller than 100 nm. In this work, the diffusion challenges associated with nanoparticles was resolved using biased cyclical electric fields. This new method, biased cyclical electrical field flow fractionation (BCyElFFF), achieves baseline separation of 15 and 40 nm gold nanoparticles. Theoretical considerations show that the optimal resolution is achieved when the applied bias yields electrical transport that counteracts the diffusive transport of nanoparticles. BCyElFFF greatly extends separation capabilities of the cyclical electrical field flow fractionation to sub 50 nm nanoparticles and provides a powerful alternative to other separation and characterization techniques capable of separating nanoparticles smaller than 50 nm.