Selective and Efficient Biomacromolecular Extraction of Rare-Earth Elements using Lanmodulin.

Lanmodulin (LanM) is a recently discovered protein that undergoes a large conformational change in response to rare-earth elements (REEs). Here, we use multiple physicochemical methods to demonstrate that LanM is the most selective macromolecule for REEs characterized to date and even outperforms many synthetic chelators. Moreover, LanM exhibits metal-binding properties and structural stability unseen in most other metalloproteins. LanM retains REE binding down to pH ≈ 2.5, and LanM-REE complexes withstand high temperature (up to 95 °C), repeated acid treatments, and up to molar amounts of competing non-REE metal ions (including Mg, Ca, Zn, and Cu), allowing the protein's use in harsh chemical processes. LanM's unrivaled properties were applied to metal extraction from two distinct REE-containing industrial feedstocks covering a broad range of REE and non-REE concentrations, namely, precombustion coal and electronic waste leachates. After only a single all-aqueous step, quantitative and selective recovery of the REEs from all non-REEs initially present (Li, Na, Mg, Ca, Sr, Al, Si, Mn, Fe, Co, Ni, Cu, Zn, and U) was achieved, demonstrating the universal selectivity of LanM for REEs against non-REEs and its potential application even for industrial low-grade sources, which are currently underutilized. Our work indicates that biosourced macromolecules such as LanM may offer a new paradigm for extractive metallurgy and other applications involving f-elements.

Pupillometry measures of autonomic nervous system regulation with advancing age in a healthy pediatric cohort.

PURPOSE: To determine if variables of the pupillary light response mature with age and sex in a healthy pediatric cohort and the utility of pupillometry in assessment among pediatric participants. METHODS: After 1 min in a dark room to establish baseline, pupillometry was performed on 323 healthy, pediatric participants (646 eyes; 2-21 years; 175 females). Variables included initial pupil diameter, pupil diameter after light stimulus, percent pupillary constriction, latency to onset of constriction, average constriction velocity, maximum constriction velocity, average dilation velocity, and time from light stimulus to 75% of the initial pupil diameter. Data analyses employed ANOVAs and non-linear regressions. RESULTS: Analyses of age group differences revealed that participants 12-21 years old had a larger initial pupil diameter and pupil diameter after light stimulus, with males aged 12-18 years demonstrating a larger pupil diameter than all younger participants (ps < 0.05). Participants 12-18 years old had a slower maximum constriction velocity than participants 6-11 years old, with no sex differences (ps < 0.05). Furthermore, males aged 12-18 years old had a smaller percent constriction than males 6-11 years old (ps < 0.05). Regressions revealed that percent constriction and dilation velocity seemed to mature linearly, initial pupil diameter and ending pupil diameter matured quadratically, and the constriction velocity terms matured cubically. CONCLUSIONS: Results revealed maturation of the pupillary light response by age and sex in healthy pediatric participants. Given the value of the pupillary light response as a biomarker, the results provide normative benchmarks for comparison in health and disease, including opiate-exposed and concussion patients.

Novel methods of imaging and analysis for the thermoregulatory sweat test.

The thermoregulatory sweat test (TST) can be central to the identification and management of disorders affecting sudomotor function and small sensory and autonomic nerve fibers, but the cumbersome nature of the standard testing protocol has prevented its widespread adoption. A high-resolution, quantitative, clean and simple assay of sweating could significantly improve identification and management of these disorders. Images from 89 clinical TSTs were analyzed retrospectively using two novel techniques. First, using the standard indicator powder, skin surface sweat distributions were determined algorithmically for each patient. Second, a fundamentally novel method using thermal imaging of forced evaporative cooling was evaluated through comparison with the standard technique. Correlation and receiver operating characteristic analyses were used to determine the degree of match between these methods, and the potential limits of thermal imaging were examined through cumulative analysis of all studied patients. Algorithmic encoding of sweating and nonsweating regions produces a more objective analysis for clinical decision-making. Additionally, results from the forced cooling method correspond well with those from indicator powder imaging, with a correlation across spatial regions of -0.78 (confidence interval: -0.84 to -0.71). The method works similarly across body regions, and frame-by-frame analysis suggests the ability to identify sweating regions within ~1 s of imaging. Although algorithmic encoding can enhance the standard sweat testing protocol, thermal imaging with forced evaporative cooling can dramatically improve the TST by making it less time consuming and more patient friendly than the current approach. NEW & NOTEWORTHY The thermoregulatory sweat test (TST) can be central to the identification and management of several common neurological disorders, but the cumbersome nature of the standard testing protocol has prevented its widespread adoption. In this study, images from 89 clinical TSTs were analyzed retrospectively using two novel techniques. Our results suggest that these improved methods could make sweat testing more reliable and acceptable for screening and management of a range of neurological disorders.

Compatibility of High-Moisture Storage for Biochemical Conversion of Corn Stover: Storage Performance at Laboratory and Field Scales.

Wet anaerobic storage of corn stover can provide a year-round supply of feedstock to biorefineries meanwhile serving an active management approach to reduce the risks associated with fire loss and microbial degradation. Wet logistics systems employ particle size reduction early in the supply chain through field-chopping which removes the dependency on drying corn stover prior to baling, expands the harvest window, and diminishes the biorefinery size reduction requirements. Over two harvest years, in-field forage chopping was capable of reducing over 60% of the corn stover to a particle size of 6 mm or less. Aerobic and anaerobic storage methods were evaluated for wet corn stover in 100 L laboratory reactors. Of the methods evaluated, traditional ensiling resulted in <6% total solid dry matter loss (DML), about five times less than the aerobic storage process and slightly less than half that of the anaerobic modified-Ritter pile method. To further demonstrate the effectiveness of the anaerobic storage, a field demonstration was completed with 272 dry tonnes of corn stover; DML averaged <5% after 6 months. Assessment of sugar release as a result of dilute acid or dilute alkaline pretreatment and subsequent enzymatic hydrolysis suggested that when anaerobic conditions were maintained in storage, sugar release was either similar to or greater than as-harvested material depending on the pretreatment chemistry used. This study demonstrates that wet logistics systems offer practical benefits for commercial corn stover supply, including particle size reduction during harvest, stability in storage, and compatibility with biochemical conversion of carbohydrates for biofuel production. Evaluation of the operational efficiencies and costs is suggested to quantify the potential benefits of a fully-wet biomass supply system to a commercial biorefinery.

Recovery of Rare Earth Elements from Low-Grade Feedstock Leachates Using Engineered Bacteria.

The use of biomass for adsorption of rare earth elements (REEs) has been the subject of many recent investigations. However, REE adsorption by bioengineered systems has been scarcely documented, and rarely tested with complex natural feedstocks. Herein, we engineered E. coli cells for enhanced cell surface-mediated extraction of REEs by functionalizing the OmpA protein with 16 copies of a lanthanide binding tag (LBT). Through biosorption experiments conducted with leachates from metal-mine tailings and rare earth deposits, we show that functionalization of the cell surface with LBT yielded several notable advantages over the nonengineered control. First, the efficiency of REE adsorption from all leachates was enhanced as indicated by a 2-10-fold increase in distribution coefficients for individual REEs. Second, the relative affinity of the cell surface for REEs was increased over all non-REEs except Cu. Third, LBT-display systematically enhanced the affinity of the cell surface for REEs as a function of decreasing atomic radius, providing a means to separate high value heavy REEs from more common light REEs. Together, our results demonstrate that REE biosorption of high efficiency and selectivity from low-grade feedstocks can be achieved by engineering the native bacterial surface.

Planarian regeneration in space: Persistent anatomical, behavioral, and bacteriological changes induced by space travel.

Regeneration is regulated not only by chemical signals but also by physical processes, such as bioelectric gradients. How these may change in the absence of the normal gravitational and geomagnetic fields is largely unknown. Planarian flatworms were moved to the International Space Station for 5 weeks, immediately after removing their heads and tails. A control group in spring water remained on Earth. No manipulation of the planaria occurred while they were in orbit, and space-exposed worms were returned to our laboratory for analysis. One animal out of 15 regenerated into a double-headed phenotype-normally an extremely rare event. Remarkably, amputating this double-headed worm again, in plain water, resulted again in the double-headed phenotype. Moreover, even when tested 20 months after return to Earth, the space-exposed worms displayed significant quantitative differences in behavior and microbiome composition. These observations may have implications for human and animal space travelers, but could also elucidate how microgravity and hypomagnetic environments could be used to trigger desired morphological, neurological, physiological, and bacteriomic changes for various regenerative and bioengineering applications.

Nodal expression in triple-negative breast cancer: Cellular effects of its inhibition following doxorubicin treatment.

Triple-negative breast cancer (TNBC) represents an aggressive cancer subtype characterized by the lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). The independence of TNBC from these growth promoting factors eliminates the efficacy of therapies which specifically target them, and limits TNBC patients to traditional systemic neo/adjuvant chemotherapy. To better understand the growth advantage of TNBC - in the absence of ER, PR and HER2, we focused on the embryonic morphogen Nodal (associated with the cancer stem cell phenotype), which is re-expressed in aggressive breast cancers. Most notably, our previous data demonstrated that inhibition of Nodal signaling in breast cancer cells reduces their tumorigenic capacity. Furthermore, inhibiting Nodal in other cancers has resulted in improved effects of chemotherapy, although the mechanisms for this remain unknown. Thus, we hypothesized that targeting Nodal in TNBC cells in combination with conventional chemotherapy may improve efficacy and represent a potential new strategy. Our preliminary data demonstrate that Nodal is highly expressed in TNBC when compared to invasive hormone receptor positive samples. Treatment of Nodal expressing TNBC cell lines with a neutralizing anti-Nodal antibody reduces the viability of cells that had previously survived treatment with the anthracycline doxorubicin. We show that inhibiting Nodal may alter response mechanisms employed by cancer cells undergoing DNA damage. These data suggest that development of therapies which target Nodal in TNBC may lead to additional treatment options in conjunction with chemotherapy regimens - by altering signaling pathways critical to cellular survival.

Bioadsorption of Rare Earth Elements through Cell Surface Display of Lanthanide Binding Tags.

With the increasing demand for rare earth elements (REEs) in many emerging clean energy technologies, there is an urgent need for the development of new approaches for efficient REE extraction and recovery. As a step toward this goal, we genetically engineered the aerobic bacterium Caulobacter crescentus for REE adsorption through high-density cell surface display of lanthanide binding tags (LBTs) on its S-layer. The LBT-displayed strains exhibited enhanced adsorption of REEs compared to cells lacking LBT, high specificity for REEs, and an adsorption preference for REEs with small atomic radii. Adsorbed Tb(3+) could be effectively recovered using citrate, consistent with thermodynamic speciation calculations that predicted strong complexation of Tb(3+) by citrate. No reduction in Tb(3+) adsorption capacity was observed following citrate elution, enabling consecutive adsorption/desorption cycles. The LBT-displayed strain was effective for extracting REEs from the acid leachate of core samples collected at a prospective rare earth mine. Our collective results demonstrate a rapid, efficient, and reversible process for REE adsorption with potential industrial application for REE enrichment and separation.

Degradation of phenolic compounds by the lignocellulose deconstructing thermoacidophilic bacterium Alicyclobacillus Acidocaldarius.

Alicyclobacillus acidocaldarius, a thermoacidophilic bacterium, has a repertoire of thermo- and acid-stable enzymes that deconstruct lignocellulosic compounds. The work presented here describes the ability of A. acidocaldarius to reduce the concentration of the phenolic compounds: phenol, ferulic acid, ρ-coumaric acid and sinapinic acid during growth conditions. The extent and rate of the removal of these compounds were significantly increased by the presence of micro-molar copper concentrations, suggesting activity by copper oxidases that have been identified in the genome of A. acidocaldarius. Substrate removal kinetics was first order for phenol, ferulic acid, ρ-coumaric acid and sinapinic acid in the presence of 50 µM copper sulfate. In addition, laccase enzyme assays of cellular protein fractions suggested significant activity on a lignin analog between the temperatures of 45 and 90 °C. This work shows the potential for A. acidocaldarius to degrade phenolic compounds, demonstrating potential relevance to biofuel production and other industrial processes.

Effects of a novel Nodal-targeting monoclonal antibody in melanoma.

Nodal is highly expressed in various human malignancies, thus supporting the rationale for exploring Nodal as a therapeutic target. Here, we describe the effects of a novel monoclonal antibody (mAb), 3D1, raised against human Nodal. In vitro treatment of C8161 human melanoma cells with 3D1 mAb shows reductions in anchorage-independent growth and vasculogenic network formation. 3D1 treated cells also show decreases of Nodal and downstream signaling molecules, P-Smad2 and P-ERK and of P-H3 and CyclinB1, with an increase in p27. Similar effects were previously reported in human breast cancer cells where Nodal expression was generally down-regulated; following 3D1 mAb treatment, both Nodal and P-H3 levels are reduced. Noteworthy is the reduced growth of human melanoma xenografts in Nude mice treated with 3D1 mAb, where immunostaining of representative tumor sections show diminished P-Smad2 expression. Similar effects both in vitro and in vivo were observed in 3D1 treated A375SM melanoma cells harboring the active BRAF(V600E) mutation compared to treatments with IgG control or a BRAF inhibitor, dabrafenib. Finally, we describe a 3D1-based ELISA for the detection of Nodal in serum samples from cancer patients. These data suggest the potential of 3D1 mAb for selecting and targeting Nodal expressing cancers.

Evaluating the potential of native ureolytic microbes to remediate a 90Sr contaminated environment.

This study was a preliminary evaluation of ureolytically driven calcite precipitation and strontium coprecipitation for remediating (90)Sr contamination at the Hanford 100-N Area in Washington; in particular the approach is suitable for treating sorbed (90)Sr that could otherwise be a long-term source for groundwater contamination. Geochemical conditions at the site are compatible with long-term calcite stability, and therefore groundwater and sediment samples were examined to assess the ureolytic capabilities of the native microbiota. Quantitative assays detected up to 2 × 10(4) putative ureC gene copies mL(-1) in water and up to 9 × 10(5) copies g(-1) in sediment. The ureC assays and laboratory-based estimates of ureolytic activity indicated that the distribution of in situ ureolytic potential was very heterogeneous with depth and also that the ureolytic activity was predominantly associated with attached organisms. A mixed kinetic-equilibrium model was developed for the 100-N site to simulate urea treatment and predict strontium removal. Together, the microbial characterization data and modeling suggest that the site has the requisite biogeochemical characteristics for application of the calcite precipitation remediation approach for (90)Sr.

Responses of ammonia-oxidizing bacterial and archaeal populations to organic nitrogen amendments in low-nutrient groundwater.

To evaluate the potential for organic nitrogen addition to stimulate the in situ growth of ammonia oxidizers during a field scale bioremediation trial, samples collected from the Eastern Snake River Plain Aquifer in Idaho before, during, and after the addition of molasses and urea were subjected to PCR analysis of ammonia monooxygenase subunit A (amoA) genes. Ammonia-oxidizing bacteria (AOB) and archaea (AOA) were present in all of the samples tested, with AOA amoA genes outnumbering AOB amoA genes in all of the samples. Following urea addition, nitrate levels rose and bacterial amoA copy numbers increased dramatically, suggesting that urea hydrolysis stimulated nitrification. Bacterial amoA diversity was limited to two Nitrosomonas phylotypes, whereas archaeal amoA analyses revealed 20 distinct operational taxonomic units, including several that were markedly different from all previously reported sequences. Results from this study demonstrate the likelihood of stimulating ammonia-oxidizing communities during field scale manipulation of groundwater conditions to promote urea hydrolysis.

Effects of 14 days of microgravity on fast hindlimb and diaphragm muscles of the rat.

The purpose of the present study was to determine the effects of 14 days of microgravity on specific rat fast-twitch muscles, and to compare these data with previous data from rat fast-twitch muscles exposed to microgravity for 10 days (Kraemer et al. 2000). Hindlimb muscles containing predominately fast fibers [extensor digitorum longus (EDL), superficial "white" (GSW) and deep "red" (GDR) gastrocnemius] and the diaphragm (DIA) were removed from flight and ground-based control animals and analyzed for: muscle mass, fiber type distribution, cross-sectional area, and myosin heavy chain (MHC) isoform content. Gravitational unloading for 14 days caused significant decreases in muscle mass (8-9%) and cross-sectional area of almost all fiber types (10-35%) from both EDL and gastrocnemius muscles. However, microgravity had little effect on fiber type composition in these muscles with significant changes occurring only in the EDL type IID fiber population (9.5% decrease). Similarly, relative MHC isoform content was only slightly altered by exposure to microgravity (increased content of MHCIIa in flight EDL). No changes in area, fiber type percentages, or MHC isoform content were detected in the DIA following the 14-day spaceflight. Similar to data gathered following a 10-day spaceflight (Kraemer et al. 2000), the 14-day flight did not appear to cause significant slow-to-fast (I --> IIA) or fast-to-faster (IIA --> IID --> IIB) transformations in hindlimb muscles containing predominantly fast-twitch fibers. However, the longer period of gravitational unloading did result in additional loss in muscle fiber cross-sectional area with involvement of more major fiber types.

Gravitropic moss cells default to spiral growth on the clinostat and in microgravity during spaceflight.

In addition to shoots and roots, the gravity (g)-vector orients the growth of specialized cells such as the apical cell of dark-grown moss protonemata. Each apical cell of the moss Ceratodon purpureus senses the g-vector and adjusts polar growth accordingly producing entire cultures of upright protonemata (negative gravitropism). The effect of withdrawing a constant gravity stimulus on moss growth was studied on two NASA Space Shuttle (STS) missions as well as during clinostat rotation on earth. Cultures grown in microgravity (spaceflight) on the STS-87 mission exhibited two successive phases of non-random growth and patterning, a radial outgrowth followed by the formation of net clockwise spiral growth. Also, cultures pre-aligned by unilateral light developed clockwise hooks during the subsequent dark period. The second spaceflight experiment flew on STS-107 which disintegrated during its descent on 1 February 2003. However, most of the moss experimental hardware was recovered on the ground, and most cultures, which had been chemically fixed during spaceflight, were retrieved. Almost all intact STS-107 cultures displayed strong spiral growth. Non-random culture growth including clockwise spiral growth was also observed after clinostat rotation. Together these data demonstrate the existence of default non-random growth patterns that develop at a population level in microgravity, a response that must normally be overridden and masked by a constant g-vector on earth.

Use of 3-hydroxyphenylacetylene for activity-dependent, fluorescent labeling of bacteria that degrade toluene via 3-methylcatechol.

3-hydroxyphenylacetylene (3-HPA) served as a novel, activity-dependent, fluorogenic and chromogenic probe for bacterial enzymes known to degrade toluene via meta ring fission of the intermediate, 3-methylcatechol. By this direct physiological analysis, cells grown with an aromatic substrate to induce the synthesis of toluene-degrading enzymes were fluorescently labeled.

Microbial communities from methane hydrate-bearing deep marine sediments in a forearc basin.

Microbial communities in cores obtained from methane hydrate-bearing deep marine sediments (down to more than 300 m below the seafloor) in the forearc basin of the Nankai Trough near Japan were characterized with cultivation-dependent and -independent techniques. Acridine orange direct count data indicated that cell numbers generally decreased with sediment depth. Lipid biomarker analyses indicated the presence of viable biomass at concentrations greater than previously reported for terrestrial subsurface environments at similar depths. Archaeal lipids were more abundant than bacterial lipids. Methane was produced from both acetate and hydrogen in enrichments inoculated with sediment from all depths evaluated, at both 10 and 35 degrees C. Characterization of 16S rRNA genes amplified from the sediments indicated that archaeal clones could be discretely grouped within the Euryarchaeota and Crenarchaeota domains. The bacterial clones exhibited greater overall diversity than the archaeal clones, with sequences related to the Bacteroidetes, Planctomycetes, Actinobacteria, Proteobacteria, and green nonsulfur groups. The majority of the bacterial clones were either members of a novel lineage or most closely related to uncultured clones. The results of these analyses suggest that the microbial community in this environment is distinct from those in previously characterized methane hydrate-bearing sediments.

Cellular localization of D-lactate dehydrogenase and NADH oxidase from Archaeoglobus fulgidus.

Members of the genus Archaeoglobus are hyperthermophilic sulfate reducers with an optimal growth temperature of 83 degrees C. Archaeoglobus fulgidus can utilize simple compounds including D-lactate, L-lactate and pyruvate as the sole substrate for carbon and electrons for dissimilatory sulfate reduction. Previously we showed that this organism makes a D-lactate dehydrogenase (Dld) that requires FAD and Zn2+ for activity. To determine the cellular location and topology of Dld and to identify proteins that interact with Dld, an antibody directed against Dld was prepared. Immunocytochemical studies using gold particle-coated secondary antibodies show that more than 85% of Dld is associated with the membrane. A truncated form of Dld was detected in immunoblots of whole cells treated with protease, showing that Dld is an integral membrane protein and that a significant portion of Dld, including part of the FAD-binding pocket, is outside the membrane facing the S-layer. The gene encoding Dld is part of an operon that includes noxA2, which encodes one of several NADH oxidases in A. fulgidus. Previous studies have shown that NoxA2 remains bound to Dld during purification. Thin sections of A. fulgidus probed simultaneously with antibodies against Dld and NoxA2 show that both proteins co-localized to the same sites in the membrane. Although these data show a tight interaction between NoxA2 and Dld, the role of NoxA2 in electron transport reactions is unknown. Rather, NoxA2 may protect proteins involved in electron transfer by reducing O2 to H2O2 or H2O.