Permanganate Oxidation of Organic Contaminants and Model Compounds.

Permanganate oxidation is an attractive environmental remediation strategy due to its low cost, ease of use, and wide range in reactivity. Here, permanganate reactivity trends are investigated for model organic compounds and organic contaminants. Second-order permanganate reaction rate constants were compiled for 215 compounds from 82 references (journal articles, conference proceedings, master's theses, and dissertations). Additionally, we validated some phenol rate constants and contribute a few additional phenol rate constants. Commonalities between contaminant oxidation products are also discussed, and we tentatively identify several model compound oxidation products. Aromatic rings, alcohols, and ether groups had low reaction rate constants with permanganate. Alkene reaction sites had the highest reaction rate constants, followed by phenols, anilines, and benzylic carbon-hydrogen bonds. Generally, permanganate reactivity follows electrophilic substitution trends at the reaction site where electron donating groups increase the rate of reaction, while electron withdrawing groups decrease the rate of reaction. Solution conditions, specifically, buffer type and concentration, may impact the rate of reaction, which could be due to either an ionic strength effect or the buffer ions acting as ligands. The impact of these solution conditions, unfortunately, precludes the development of a quantitative structure-activity relationship for permanganate reaction rate constants with the currently available data. We note that critical experimental details are often missing in the literature, which posed a challenge when comparing rate constants between studies. Future research directions on permanganate oxidation should seek to improve our understanding of buffer effects and to identify oxidation products for model compounds so that extrapolations can be made to more complex contaminant structures.

Molecular Docking as a Tool to Examine Organic Cation Sorption to Organic Matter.

Molecular docking simulations were performed to examine the structural effects of organic cations on their sorption to organic matter. A set of benzylamine compounds was used to assess the sorption trends arising from the systematic structural differences between ring or nitrogen substituents. Binding simulations were performed using AutoDock 4.2 with Schulten's proposed soil organic matter as a representative organic matter structure. The calculated binding energies for the sorbate compounds correlated strongly with the measured sorption energies for Pahokee peat, indicating that the simulated binding energies and their associated sorbate orientations were representative of the experimental conditions. Graphical docking orientations showed primary, secondary, and tertiary aminium compounds to form hydrogen-bond interactions with deprotonated carboxylic acid groups in a pocket of the organic matter structure. Quaternary ammonium compounds formed pi-pi or cation-pi interactions with the aromatic groups elsewhere in the same organic matter pocket. Ring substituents showed no clear trends in sorption energies with the substituent group type for primary aminium compounds. Rather, substituent groups altered the simulated van der Waals, electrostatic, hydrogen-bond, and desolvation energy contributions to the overall sorption energies, in part because of the variations in docking orientations between compounds. Increasing methyl substitution of the aminium nitrogen group was associated with an increase in van der Waals energy contributions and a decrease in electrostatic energy contributions to the overall compound sorption energies because of aminium charge delocalization into methyl substituents and steric hindrance from methyl substituents to form specific interactions. The findings illustrate how molecular docking can be used to explore the effects of organic cation structure on sorption interactions with organic matter.

Data-Based Chemical Class Regions for Van Krevelen Diagrams.

Ultrahigh resolution mass spectrometry (UHR-MS) is commonly used to characterize natural organic matter (NOM). The complexity of both NOM and the data set produced make data visualization challenging. Van Krevelen diagrams─plots of component hydrogen/carbon (H/C) against oxygen/carbon (O/C) elemental ratios─have become a popular way to visualize the chemical formulas identified by UHR-MS. Different regions on the van Krevelen diagram have been attributed to different chemical classes; however, the classifications vary between studies and the regions lack standard definitions. Here, chemical formulas were obtained from public databases to create H/C and O/C ranges for amino sugar, carbohydrate, lignin, lipid, peptide, and tannin chemical classes on van Krevelen diagrams. The recommended H/C and O/C ranges are presented in a table and can be adapted to any data analysis software programs. The regions recommended here agreed reasonably well with previous literature for amino sugar, carbohydrate, lignin, lipid, and peptide regions. However, the recommended tannin region appears at lower H/C ratio values and with a wider range of O/C ratio values compared to previous studies. The regions presented herein are strongly recommended for use as consistent reference points in future NOM characterization studies to aid in the discussion of data and to readily compare studies.

Solvent choice influences final toxicity results in Thamnocephalus platyurus bioassay when exposed to microcystin -LR.

Cyanobacterial blooms present a threat to many waterbodies around the world used for drinking water and recreational purposes. Toxicology tests, such as the Thamnotoxkit-F which uses the cladoceran T. platyurus, have been employed to assess the health hazards that these blooms may pose to the public. However, reported median lethal concentrations (LC50) of microcystin -LR to T. platyurus vary significantly from one study to the next. The variation in solvent type and concentrations used to dissolve microcystin -LR in preparation for toxicity experiments may be contributing to the variations in LC50 values found in the literature. The primary goal of this study was to determine what solvents and their corresponding concentrations can be used for microcystin -LR testing using T. platyurus without artifactually impacting LC50 values. All toxicity testing was completed using glassware as polystyrene containers have been shown to sorb microcystin. Microcystin -LR LC50 values for T. platyurus were determined using United States Environmental Protection Agency (US EPA) moderately hard standard freshwater as a control for comparison with systems that were prepared using dimethyl sulfoxide or methanol to dissolve microcystin -LR. Low levels of dimethyl sulfoxide (2%) or methanol (1%) did not impact LC50 values of microcystin -LR to T. platyurus compared to US EPA moderately hard standard freshwater diluted in microcystin -LR. However, higher levels of dimethyl sulfoxide (4%) and methanol (1.4% and 4%) did lower the LC50 for microcystin -LR to T. platyurus, consistent with the toxicity of these solvents to T. platyurus when dosed in the absence of microcystin -LR. Researchers need to report the type and concentrations of solvents used in toxicity tests using cyanotoxins in order to ensure that results can be intercompared appropriately. Furthermore, researchers need to use caution when using organic solvents such as dimethyl sulfoxide or methanol to ensure that these solvents are not causing significant mortality in toxicity testing.

Microcystin-LR degradation kinetics during chlorination: Role of water quality conditions.

Microcystin-LR (MCLR) produced during certain cyanobacteria blooms can contaminate drinking water sources and pose a threat to public health. Previous studies of MCLR degradation by free chlorine may have artifacts from using strong reducing agents to quench chlorination reactions, and they also have not explored the influence of water quality characteristics such as pH, alkalinity, temperature and dissolved organic matter (DOM). Using a novel quencher, 1,3,5-trimethoxybenzene (TMB), the apparent MCLR degradation rate constants were found to be higher than those obtained with thiosulfate (S2O32-), a traditionally used strong reducing quencher. Thiosulfate converted N-chlorinated MCLR degradation products back to the parent MCLR, thereby underestimating MCLR loss over time. The second-order rate constants for HOCl (kHOCl) and OCl- (kOCl-) during chlorination of MCLR were determined to be 72 ± 13 and 28 ± 1.8 M-1s-1, respectively, allowing for determination of the apparent MCLR rate constants (kapp,MCLR) for any known pH condition. The MCLR reaction with free chlorine was strongly affected by temperature and the presence of DOM, while changes in ionic strength and alkalinity had little effect. Free chlorine in the presence of DOM, originating from both terrestrial and microbial sources, exhibited two-stage decay. The initial chlorine demand in the first 15 s of reaction can be determined by the dissolved organic carbon (DOC) concentration (initial chlorine demand = 1.8 × DOC), and the second-order rate constants for the later slower decay correlated well with SUVA254 (kapp,DOM = 0.73 × SUVA254 - 0.41). The results yielded a practical model to predict the decay of MCLR during chlorination of waters with varied water quality characteristics.

Chemical Alterations of Dissolved Organic Matter by Permanganate Oxidation.

Dissolved organic matter (DOM) is ubiquitous in raw drinking water and can efficiently scavenge oxidants, such as permanganate. Here, changes to DOM induced by permanganate oxidation under typical drinking water treatment conditions (6 µM, 1 h) to bulk DOM properties, DOM functional groups, and DOM chemical formulae were examined for two DOM isolate types (terrestrial and microbial). Permanganate oxidation did not mineralize DOM, rather changes were compositional in nature. Optical properties suggest that permanganate oxidation decreased DOM aromaticity (decreased SUVA-254), decreased DOM electron-donating capacity, and decreased DOM average molecular weight (increased E2/E3 ratios). Fourier-transform-infrared spectroscopy second derivative analyses revealed that permanganate does not oxidize DOM alkene groups, suggesting permanganate access to functional groups may be important. Four ionization techniques were used with ultrahigh-resolution mass spectrometry: negative and positive ion mode electrospray ionization and negative and positive ion mode laser/desorption ionization. The results from all four techniques were combined to understand changes in DOM chemical formulae. It was concluded that nitrogen-containing aromatic compounds and alkylbenzenes were oxidized by permanganate to form nitrogen-containing aliphatic compounds and benzoic acid-containing compounds. This work highlights how multiple ionization techniques coupled with UHR-MS can enable a more detailed characterization of DOM.

Aluminosilicate Mineralogy and the Sorption of Organic Cations: Interplay between Electrostatic Barriers and Compound Structural Features.

Current predictive models of organic cation sorption assume that sorbates interact with all sites on aluminosilicate minerals in the same manner. To examine whether differences in aluminosilicate structure and the resultant changes in electrostatic potential influence the sorption of organic cations, seven smectites were chosen with different proportions of isomorphic substitutions (origin of clay charge) located in octahedral versus tetrahedral layers and with the presence or absence of aluminosilicate interlayers. Sorption coefficients for 14 benzylamine derivatives with systematic differences in compound structures were collected to understand the possible influence of aluminosilicate mineralogy. Benzylamine compounds with methyl group substitution on the charged amine or with electron-donating or -withdrawing ring substituents displayed decreases in cation exchange-normalized sorption coefficients (KCEC), by up to one order of magnitude, between hectorite (100% isomorphic substitution in the octahedral layer) and nontronite (100% isomorphic substitution in the tetrahedral layer). To understand this difference across aluminosilicates, stochastic molecular models of the various aluminosilicate minerals with interlayers were performed. These models showed that negative charge density associated with tetrahedral sites results in high positive electrostatic energy barriers within the interlayer, creating a penalty for compounds with positive charge spread over a larger compound surface area as occurs from primary to quaternary amines. Conversely, clays with charge originating from octahedral sites produce low electrostatic potential barriers within the interlayer, decreasing the penalty for quaternary amine sorption. Trends for nine cationic pharmaceutical compounds, which varied in size, group alkylation, and/or polar substituents, demonstrated similar decreases in KCEC values to aluminosilicate minerals with high electrostatic energy barriers. Overall, aluminosilicate mineralogy was found to exert a large influence (0.5-1 order of magnitude in sorption coefficients) on organic cation sorption. The application of atomistic electrostatic potential mapping of both sorbent and sorbate structures provided insights to explain trends in sorption coefficients that could not be described by the basic electrostatic potential theory or by assuming that sorbate structure moieties yielded additive sorption contributions.

Impact of bloom events on dissolved organic matter fluorophore signatures in Ohio waters.

Due to the increase in severe cyanobacterial blooms in drinking water sources and recreational waters across the globe, inexpensive and reliable methods of detecting oncoming blooms are needed. Cyanobacterial blooms can contribute substantially to the bulk chromophoric dissolved organic matter pool. Thus, the fluorescence signature of organic matter derived from these blooms may be an indicator of upcoming blooms. Water samples from five sites around Ohio were collected regularly between February and October 2017. A PARAFAC model was developed to determine if these protein-like fluorophores could be linked to bloom biomass and whether they were absent in dissolved organic matter from oligotrophic waters. One reference site at an oligotrophic reservoir was included to confirm the lack of protein-like fluorophores in the absence of a bloom event. We found that an increase in tryptophan-like and tyrosine-like fluorophores occurs before the increase in chlorophyll a levels, associated with bloom biomass, in some Ohio waters affected by cyanobacterial blooms; however, protein-like fluorophores are not correlated with levels of the cyanotoxin, microcystin. The excitation and emission wavelengths of these fluorophores (tryptophan-like: 239/341 nm, tyrosine-like: 248/306 nm) may be used to monitor impending blooms in waters heavily impacted by cyanobacteria but may not be applicable to waters receiving treated wastewater discharges.

Removal of cyanotoxins by potassium permanganate: Incorporating competition from natural water constituents.

In recent years, harmful algal blooms capable of producing toxins including microcystins, cylindrospermopsin, and saxitoxin have increased in occurrence and severity. These toxins can enter drinking water treatment plants and, if not effectively removed, pose a serious threat to human health. The work here investigated the efficacy of permanganate oxidation as a treatment strategy, with a focus on incorporating competition by cyanobacterial cells and dissolved organic matter (DOM). We report rate constants of 272 ±â€¯23 M-1 s-1 for the reaction between permanganate and microcystin-LR, 0.26 ±â€¯0.05 M-1 s-1 for the reaction between permanganate and cylindrospermopsin, and, using chemical analogs, estimate a maximum rate constant of 2.7 ±â€¯0.2 M-1 s-1 for the reaction between permanganate and saxitoxin. We conclude that permanganate only shows potential to remove microcystins. No pH (6-10) or alkalinity (0-50 mM) dependence was observed for the rate of reaction between microcystin-LR and permanganate; however, a temperature dependence was observed and can be characterized by an activation energy of 16 ±â€¯5 kJ mol-1. The competition posed by cyanobacterial cells was quantified by an apparent second order rate constant of 2.5 ±â€¯0.3 × 10-6 L µg chl-a-1 s-1. From this apparent second order rate constant, it was concluded that cyanobacterial cells are not efficient scavengers of permanganate within typical contact times but this second order rate constant can be used to accurately predict microcystin degradation in algal-impacted waters. The competition posed by DOM depended on both the amount of DOM present (as measured by TOC) and its electron donating capacity (as predicted by SUVA-254 or E2/E3 ratio). DOM was concluded to scavenge permanganate efficiently and we forward that this should be considered in permanganate dosing calculations.

Diethyl phenylene diamine (DPD) oxidation to measure low concentration permanganate in environmental systems.

Permanganate has been used traditionally in drinking water treatment for its oxidation properties and ease of use. The concentration of permanganate in treatment conditions is low and difficult to detect. A colorimetric method using diethylphenylene diamine (DPD) oxidation to measure low levels (i.e., less than 6 µM) of permanganate in water was developed and applied to quantify permanganate scavenging by dissolved organic matter (DOM). Manganese dioxide (MnO2) particles were shown to interfere with DPD oxidation; however, particles were removed effectively using 0.1 µm PVDF filters prior to reaction with DPD. DOM and complexed-Mn(III) were concluded to not interfere with the DPD reaction. The DPD method was validated by obtaining the second-order rate constant for permanganate reaction with phenol (1.7 ±â€¯0.2 M-1 s-1), and comparing to the rate constant obtained independently by monitoring phenol degradation (i.e., UPLC-UV) (1.6 ±â€¯0.2 M-1 s-1). Permanganate reaction with DOM isolate samples did not follow pseudo-first order kinetics. Faster reaction rates were observed with higher ionic strength (1 mM versus 5 mM carbonate). No change in reaction rates with pH was observed at lower ionic strength (1 mM); while at higher ionic strength, the reaction rate was faster at pH 7 than at pH 10. In contrast, linear kinetics were observed for permanganate reaction with DOM in filtered whole water samples. These samples showed similar trends with pH and ionic strength as for DOM isolates. The presented method is valid to quantify permanganate reaction rates with organic contaminants or with natural scavengers.

Predicting Organic Cation Sorption Coefficients: Accounting for Competition from Sorbed Inorganic Cations Using a Simple Probe Molecule.

With the increasing number of emerging contaminants that are cationic at environmentally relevant pH values, there is a need for robust predictive models of organic cation sorption coefficients (Kd). Current predictive models fail to account for the differences in the identity, abundance, and affinity of surface-associated inorganic exchange ions naturally present at negatively charged receptor sites on environmental solids. To better understand how organic cation sorption is influenced by surface-associated inorganic exchange ions, sorption coefficients of 10 organic cations (including eight pharmaceuticals and two simple probe organic amines) were determined for six homoionic forms of the aluminosilicate mineral, montmorillonite. Organic cation sorption coefficients exhibited consistent trends for all compounds across the various homoionic clays with sorption coefficients (Kd) decreasing as follows: KdNa+ > KdNH4+ ≥ KdK+ > KdCa2+ ≥ KdMg2+ > KdAl3+. This trend for competition between organic cations and exchangeable inorganic cations is consistent with the inorganic cation selectivity sequence, determined for exchange between inorganic ions. Such consistent trends in competition between organic and inorganic cations suggested that a simple probe cation, such as phenyltrimethylammonium or benzylamine, could capture soil-to-soil variations in native inorganic cation identity and abundance for the prediction of organic cation sorption to soils and soil minerals. Indeed, sorption of two pharmaceutical compounds to 30 soils was better described by phenyltrimethylammonium sorption than by measures of benzylamine sorption, effective cation exchange capacity alone, or a model from the literature (Droge, S., and Goss, K. Environ. Sci. Technol. 2013, 47, 14224). A hybrid approach integrating structural scaling factors derived from this literature model of organic cation sorption, along with phenyltrimethylammonium Kd values, allowed for estimation of Kd values for more structurally complex organic cations to homoionic montmorillonites and to heteroionic soils (mean absolute error of 0.27 log unit). Accordingly, we concluded that the use of phenyltrimethylammonium as a probe compound was a promising means to account for the identity, affinity, and abundance of natural exchange ions in the prediction of organic cation sorption coefficients for environmental solids.

Role of effluent organic matter in the photochemical degradation of compounds of wastewater origin.

The photoreactivity of treated wastewater effluent organic matter differs from that of natural organic matter, and the indirect phototransformation rates of micropollutants originating in wastewater are expected to depend on the fractional contribution of wastewater to total stream flow. Photodegradation rates of four common compounds of wastewater origin (sulfamethoxazole, sulfadimethoxine, cimetidine and caffeine) were measured in river water, treated municipal wastewater effluent and mixtures of both to simulate various effluent-stream water mixing conditions that could occur in environmental systems. Compounds were chosen for their unique photodegradation pathways with the photochemically produced reactive intermediates, triplet-state excited organic matter (3OM*), singlet oxygen (1O2), and hydroxyl radicals (OH). For all compounds, higher rates of photodegradation were observed in effluent relative to upstream river water. Sulfamethoxazole degraded primarily via direct photolysis, with some contribution from OH and possibly from carbonate radicals and other unidentified reactive intermediates in effluent-containing samples. Sulfadimethoxine also degraded mainly by direct photolysis, and natural organic matter appeared to inhibit this process to a greater extent than predicted by light screening. In the presence of effluent organic matter, sulfadimethoxine showed additional reactions with OH and 1O2. In all water samples, cimetidine degraded by reaction with 1O2 (>95%) and caffeine by reaction with OH (>95%). In river water mixtures, photodegradation rate constants for all compounds increased with increasing fractions of effluent. A conservative mixing model was able to predict reaction rate constants in the case of hydroxyl radical reactions, but it overestimated rate constants in the case of 3OM* and 1O2 pathways. Finally, compound degradation rate constants normalized to the rate of light absorption by water correlated with E2/E3 ratios (sample absorbance at 254 nm divided by sample absorbance at 365 nm), suggesting that organic matter optical properties may hold promise to predict indirect compound photodegradation rates for various effluent mixing ratios.

Column Chromatography To Obtain Organic Cation Sorption Isotherms.

Column chromatography was evaluated as a method to obtain organic cation sorption isotherms for environmental solids while using the peak skewness to identify the linear range of the sorption isotherm. Custom packed HPLC columns and standard batch sorption techniques were used to intercompare sorption isotherms and solid-water sorption coefficients (Kd) for four organic cations (benzylamine, 2,4-dichlorobenzylamine, phenyltrimethylammonium, oxytetracycline) with two aluminosilicate clay minerals and one soil. A comparison of Freundlich isotherm parameters revealed isotherm linearity or nonlinearity was not significantly different between column chromatography and traditional batch experiments. Importantly, skewness (a metric of eluting peak symmetry) analysis of eluting peaks can establish isotherm linearity, thereby enabling a less labor intensive means to generate the extensive data sets of linear Kd values required for the development of predictive sorption models. Our findings clearly show that column chromatography can reproduce sorption measures from conventional batch experiments with the benefit of lower labor-intensity, faster analysis times, and allow for consistent sorption measures across laboratories with distinct chromatography instrumentation.

Triplet photochemistry of effluent and natural organic matter in whole water and isolates from effluent-receiving rivers.

Effluent organic matter (EfOM), contained in treated municipal wastewater, differs in composition from naturally occurring dissolved organic matter (DOM). The presence of EfOM may thus alter the photochemical production of reactive intermediates in rivers that receive measurable contributions of treated municipal wastewater. Quantum yield coefficients for excited triplet-state OM (3OM*) and apparent quantum yields for singlet oxygen (1O2) were measured for both whole water samples and OM isolated by solid phase extraction from whole water samples collected upstream and downstream of municipal wastewater treatment plant discharges in three rivers receiving differing effluent contributions: Hockanum R., CT (22% (v/v) effluent flow), E. Fork Little Miami R., OH (11%), and Pomperaug R., CT (6%). While only small differences in production of these reactive intermediates were observed between upstream and downstream whole water samples collected from the same river, yields of 3OM* and 1O2 varied by 30-50% between the rivers. Apparent quantum yields of 1O2 followed similar trends to those of 3OM*, consistent with 3OM* as a precursor to 1O2 formation. Higher 3OM* reactivity was observed for whole water samples than for OM isolates of the same water, suggesting differential recoveries of photoreactive moieties by solid phase extraction. 3OM* and 1O2 yields increased with increasing E2/E3 ratio (A254 nm divided by A365 nm) and decreased with increasing electron donating capacities of the samples, thus exhibiting trends also observed for reference humic and fulvic acid isolates. Mixing experiments with EfOM and DOM isolates showed evidence of quenching of triplet DOM by EfOM when measured yields were compared to theoretical yields. Together, the results suggest that effluent contributions of up to 25% (v/v) to river systems have a negligible influence on photochemical production of 3OM* and 1O2 apparently because of quenching of triplet DOM by EfOM. Furthermore, the results highlight the importance of whole water studies for quantifying in situ photoreactivity, particularly for 3OM*.

Rapid isolation and purification of mitochondria for transplantation by tissue dissociation and differential filtration.

Previously described mitochondrial isolation methods using differential centrifugation and/or Ficoll gradient centrifugation require 60 to 100 min to complete. We describe a method for the rapid isolation of mitochondria from mammalian biopsies using a commercial tissue dissociator and differential filtration. In this protocol, manual homogenization is replaced with the tissue dissociator's standardized homogenization cycle. This allows for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization. Following tissue dissociation, the homogenate is filtered through nylon mesh filters, which eliminate repetitive centrifugation steps. As a result, mitochondrial isolation can be performed in less than 30 min. This isolation protocol yields approximately 2 x 10(10) viable and respiration competent mitochondria from 0.18 ± 0.04 g (wet weight) tissue sample.

Superparamagnetic iron oxide nanoparticles function as a long-term, multi-modal imaging label for non-invasive tracking of implanted progenitor cells.

The purpose of this study was to determine the ability of superparamagnetic iron oxide (SPIO) nanoparticles to function as a long-term tracking label for multi-modal imaging of implanted engineered tissues containing muscle-derived progenitor cells using magnetic resonance imaging (MRI) and X-ray micro-computed tomography (µCT). SPIO-labeled primary myoblasts were embedded in fibrin sealant and imaged to obtain intensity data by MRI or radio-opacity information by µCT. Each imaging modality displayed a detection gradient that matched increasing SPIO concentrations. Labeled cells were then incorporated in fibrin sealant, injected into the atrioventricular groove of rat hearts, and imaged in vivo and ex vivo for up to 1 year. Transplanted cells were identified in intact animals and isolated hearts using both imaging modalities. MRI was better able to detect minuscule amounts of SPIO nanoparticles, while µCT more precisely identified the location of heavily-labeled cells. Histological analyses confirmed that iron oxide particles were confined to viable, skeletal muscle-derived cells in the implant at the expected location based on MRI and µCT. These analyses showed no evidence of phagocytosis of labeled cells by macrophages or release of nanoparticles from transplanted cells. In conclusion, we established that SPIO nanoparticles function as a sensitive and specific long-term label for MRI and µCT, respectively. Our findings will enable investigators interested in regenerative therapies to non-invasively and serially acquire complementary, high-resolution images of transplanted cells for one year using a single label.

The day/night proteome in the murine heart.

Circadian rhythms are essential to cardiovascular health and disease. Temporal coordination of cardiac structure and function has focused primarily at the physiological and gene expression levels, but these analyses are invariably incomplete, not the least because proteins underlie many biological processes. The purpose of this study was to reveal the diurnal cardiac proteome and important contributions to cardiac function. The 24-h day-night murine cardiac proteome was assessed by two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography-mass spectrometry. Daily variation was considerable, as ∼7.8% (90/1,147) of spots exhibited statistical changes at paired times across the 24-h light- (L) dark (D) cycle. JTK_CYCLE was used to investigate underlying diurnal rhythms in corresponding mRNA. We next revealed that disruption of the L:D cycle altered protein profiles and diurnal variation in cardiac function in Langendorff-perfused hearts, relative to the L:D cycle. To investigate the role of the circadian clock mechanism, we used cardiomyocyte clock mutant (CCM) mice. CCM myofilaments exhibited a loss of time-of-day-dependent maximal calcium-dependent ATP consumption, and altered phosphorylation rhythms. Moreover, the cardiac proteome was significantly altered in CCM hearts, especially enzymes regulating vital metabolic pathways. Lastly, we used a model of pressure overload cardiac hypertrophy to demonstrate the temporal proteome during heart disease. Our studies demonstrate that time of day plays a direct role in cardiac protein abundance and indicate a novel mechanistic contribution of circadian biology to cardiovascular structure and function.

Seasonal and spatial variations in microbial activity at various phylogenetic resolutions at a groundwater - surface water interface.

We investigated the seasonal and spatial variation in activity and density of the metabolically active in situ microbial community (AIMC) at a landfill leachate-impacted groundwater - surface water interface (GSI). A series of AIMC traps were designed and implemented for AIMC sampling and microbial activity and density examinations. Measurements were made not only at the level of bacterial domain but also at the levels of alphaproteobacterial Rhizobiales order and gammaproteobacterial Pseudomonas genus, both of which included a large number of iron-oxidizing bacteria as revealed from previous analysis. Consistently higher microbial activities with less variation in depth were measured in the AIMC traps than in the ambient sediments. Flood disturbance appeared to control AIMC activity distributions at the gradually elevated GSI. The highest AIMC activities were generally obtained from locations closest to the free surface water boundary except during the dry season when microbial activities were similar across the entire GSI. A clone library of AIMC 16S rRNA genes was constructed, and it confirmed the predominant role of the targeted alphaproteobacterial group in AIMC activity and composition. This taxon constituted 2%-14% of all bacteria with similar activity distribution profiles. The Pseudomonas group occupied only 0.1‰-0.5‰ of the total bacterial density, but its activity was 27 times higher than the bacterial average. Of the 16S rRNA sequences in the AIMC clone library, 7.5% were phylogenetically related to putative IOB, supporting the occurrence and persistence of active microbial iron oxidation across the studied iron-rich GSI ecosystem.

Seasonal arsenic accumulation in stream sediments at a groundwater discharge zone.

Seasonal changes in arsenic and iron accumulation rates were examined in the sediments of a brook that receives groundwater discharges of arsenic and reduced iron. Clean glass bead columns were deployed in sediments for known periods over the annual hydrologic cycle to monitor changes in arsenic and iron concentrations in bead coatings. The highest accumulation rates occurred during the dry summer period (July-October) when groundwater discharges were likely greatest at the sample locations. The intermediate flow period (October-March), with higher surface water levels, was associated with losses of arsenic and iron from bead column coatings at depths below 2-6 cm. Batch incubations indicated iron releases from solids to be induced by biological reduction of iron (oxy)hydroxide solids. Congruent arsenic releases during incubation were limited by the high arsenic sorption capacity (0.536 mg(As)/mg(Fe)) of unreacted iron oxide solids. The flooded spring (March-June) with high surface water flows showed the lowest arsenic and iron accumulation rates in the sediments. Comparisons of accumulation rates across a shoreline transect were consistent with greater rates at regions exposed above surface water levels for longer times and greater losses at locations submerged below surface water. Iron (oxy)hydroxide solids in the shallowest sediments likely serve as a passive barrier to sorb arsenic released to pore water at depth by biological iron reduction.

An improved method for the preparation of type I collagen from skin.

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. Clinically, this protein is widely used for cosmetic surgery, dermal injections, bone grafting, and reconstructive surgery. The sources of COL1 for these procedures are commonly nonhuman, which increases the potential for inflammation and rejection as well as xenobiotic disease transmission. In view of this, a method to efficiently and quickly purify COL1 from limited quantities of autologously-derived tissues would circumvent many of these issues; however, standard isolation protocols are lengthy and often require large quantities of collagenous tissues. Here, we demonstrate an efficient COL1 extraction method that reduces the time needed to isolate and purify this protein from about 10 days to less than 3 hr. We chose the dermis as our tissue source because of its availability during many surgical procedures. This method uses traditional extraction buffers combined with forceful agitation and centrifugal filtration to obtain highly-pure, soluble COL1 from small amounts of corium. Briefly, dermal biopsies are washed thoroughly in ice-cold dH2O after removing fat, connective tissue, and hair. The skin samples are stripped of noncollagenous proteins and polysaccharides using 0.5 M sodium acetate and a high speed bench-top homogenizer. Collagen from residual solids is subsequently extracted with a 0.075 M sodium citrate buffer using the homogenizer. These extracts are purified using 100,000 MW cut-off centrifugal filters that yield COL1 preparations of comparable or superior quality to commercial products or those obtained using traditional procedures. We anticipate this method will facilitate the utilization of autologously-derived COL1 for a multitude of research and clinical applications.