Revisiting the Preparation and Catalytic Performance of a Phosphine-Modified Co(II) Hydroformylation Precatalyst.

In light of recent conflicting reports regarding the hydroformylation catalytic activity derived from cationic Co(II) precatalysts of the form [Co(acac)(bis(phosphine))]BF4, the synthetic procedures and characterization of [Co(acac)(dppBz)]BF4, 1, are evaluated. Leveraging calibrated ESI-TOF MS methodologies, substantial quantities of Co(acac)2(dppBz), 2, were observed within samples of 1. The source of the impurity, 2, is determined to derive from incomplete protonolysis of the Co(acac)2 precursor and ligand scrambling occurring during the synthesis of 1. Revised synthetic procedures using lower temperature conditions and longer reaction times afford analytically pure samples of 1 based on ESI-TOF MS and NMR spectroscopic analysis. Complex 1 is demonstrated to act as a hydroformylation precatalyst for the conversion of 1-hexene to 1-heptanal under relatively mild conditions at 51.7 bar and 140 °C. The presence of impurity 2 is shown to dramatically decrease the catalytic performance derived from 1.

Underwater spectral reflectance measurements: the reflectance standard submersion factor and its impact on derived target reflectance.

For Earth observation remote sensing, high quality reflectance spectra are necessary for model input, algorithm development, and validation of derived products. In the aquatic environment, a common approach for making spectral reflectance measurements involves using a calibrated reflectance standard such as a Spectralon plaque underwater. The manufacturer provides a National Institute of Standards and Technology traceable reflectance curve with each standard, measured in air. Here, we demonstrate how the reflectance factor changes when submerged in water based on the standard albedo and viewing geometry. Target reflectances calculated incorrectly with the air calibrated values are 10%-60% lower than those estimated with submerged plaque calibrations. We provide guidelines for proper use and calibration of standards underwater.

Chloride facilitates Mn(III) formation during photoassembly of the Photosystem II oxygen-evolving complex.

The Mn4Ca oxygen-evolving complex (OEC) in Photosystem II (PSII) is assembled in situ from free Mn2+, Ca2+, and water. In an early light-driven step, Mn2+ in a protein high-affinity site is oxidized to Mn3+. Using dual-mode electron paramagnetic resonance spectroscopy, we observed that Mn3+ accumulation increases as chloride concentration increases in spinach PSII membranes depleted of all extrinsic subunits. At physiologically relevant pH values, this effect requires the presence of calcium. When combined with pH studies, we conclude that the first Mn2+ oxidation event in OEC assembly requires a deprotonation that is facilitated by chloride.

Mycobacterium tuberculosis Transfer RNA Induces IL-12p70 via Synergistic Activation of Pattern Recognition Receptors within a Cell Network.

Upon recognition of a microbial pathogen, the innate and adaptive immune systems are linked to generate a cell-mediated immune response against the foreign invader. The culture filtrate of Mycobacterium tuberculosis contains ligands, such as M. tuberculosis tRNA, that activate the innate immune response and secreted Ags recognized by T cells to drive adaptive immune responses. In this study, bioinformatics analysis of gene-expression profiles derived from human PBMCs treated with distinct microbial ligands identified a mycobacterial tRNA-induced innate immune network resulting in the robust production of IL-12p70, a cytokine required to instruct an adaptive Th1 response for host defense against intracellular bacteria. As validated by functional studies, this pathway contained a feed-forward loop, whereby the early production of IL-18, type I IFNs, and IL-12p70 primed NK cells to respond to IL-18 and produce IFN-γ, enhancing further production of IL-12p70. Mechanistically, tRNA activates TLR3 and TLR8, and this synergistic induction of IL-12p70 was recapitulated by the addition of a specific TLR8 agonist with a TLR3 ligand to PBMCs. These data indicate that M. tuberculosis tRNA activates a gene network involving the integration of multiple innate signals, including types I and II IFNs, as well as distinct cell types to induce IL-12p70.

Oxidation of phosphorothioate DNA modifications leads to lethal genomic instability.

Genomic modification by sulfur in the form of phosphorothioate (PT) is widespread among prokaryotes, including human pathogens. Apart from its physiological functions, PT sulfur has redox and nucleophilic properties that suggest effects on bacterial fitness in stressful environments. Here we show that PTs are dynamic and labile DNA modifications that cause genomic instability during oxidative stress. In experiments involving isotopic labeling coupled with mass spectrometry, we observed sulfur replacement in PTs at a rate of ∼2% h-1 in unstressed Escherichia coli and Salmonella enterica. Whereas PT levels were unaffected by exposure to hydrogen peroxide (H2O2) or hypochlorous acid (HOCl), PT turnover increased to 3.8-10% h-1 after HOCl treatment and was unchanged by H2O2, consistent with the repair of HOCl-induced sulfur damage. PT-dependent sensitivity to HOCl extended to cytotoxicity and DNA strand breaks, which occurred at HOCl doses that were orders of magnitude lower than the corresponding doses of H2O2. The genotoxicity of HOCl in PT-containing bacteria suggests reduced fitness in competition with HOCl-producing organisms and during infections in humans.

Self-referenced single-shot THz detection.

We demonstrate a self-referencing method to reduce noise in a single-shot terahertz detection scheme. By splitting a single terahertz pulse and using a reflective echelon, both the signal and reference terahertz time-domain waveforms were measured using one laser pulse. Simultaneous acquisition of these waveforms significantly reduces noise originating from shot-to-shot fluctuations. We show that correlation function based referencing, which is not limited to polarization dependent measurements, can achieve a noise floor that is comparable to state-of-the-art polarization-gated balanced detection. Lastly, we extract the DC conductivity of a 30 nm free-standing gold film using a single THz pulse. The measured value of σ0 = 1.3 ± 0.4 × 107 S m-1 is in good agreement with the value measured by four-point probe, indicating the viability of this method for measuring dynamical changes and small signals.

Conformational changes in a Photosystem II hydrogen bond network stabilize the oxygen-evolving complex.

The Mn4CaO5 oxygen-evolving complex (OEC) in Photosystem II (PSII) is assembled in situ and catalyzes water oxidation. After OEC assembly, the PsbO extrinsic subunit docks to the lumenal face of PSII and both stabilizes the OEC and facilitates efficient proton transfer to the lumen. D1 residue R334 is part of a hydrogen bond network involved in proton release during catalysis and interacts directly with PsbO. D1-R334 has recently been observed in different conformations in apo- and holo-OEC PSII structures. We generated a D1-R334G point mutant in Synechocystis sp. PCC 6803 to better understand this residue's function. D1-R334G PSII is active under continuous light, but the OEC is unstable in darkness. Isolated D1-R334G core complexes have little bound PsbO and less manganese as the wild type control. The S2 intermediate is stabilized in D1-R334G indicating that the local environment around the OEC has been altered. These results suggest that the hydrogen bond network that includes D1-R334 exists in a different functional conformation during PSII biogenesis in the absence of PsbO.

Assembly and Repair of Photosystem II in Chlamydomonas reinhardtii.

Oxygenic photosynthetic organisms use Photosystem II (PSII) to oxidize water and reduce plastoquinone. Here, we review the mechanisms by which PSII is assembled and turned over in the model green alga Chlamydomonas reinhardtii. This species has been used to make key discoveries in PSII research due to its metabolic flexibility and amenability to genetic approaches. PSII subunits originate from both nuclear and chloroplastic gene products in Chlamydomonas. Nuclear-encoded PSII subunits are transported into the chloroplast and chloroplast-encoded PSII subunits are translated by a coordinated mechanism. Active PSII dimers are built from discrete reaction center complexes in a process facilitated by assembly factors. The phosphorylation of core subunits affects supercomplex formation and localization within the thylakoid network. Proteolysis primarily targets the D1 subunit, which when replaced, allows PSII to be reactivated and completes a repair cycle. While PSII has been extensively studied using Chlamydomonas as a model species, important questions remain about its assembly and repair which are presented here.

Benthic effects on the polarization of light in shallow waters.

Measurements of the upwelling polarized radiance in relatively shallow waters of varying depths and benthic conditions are compared to simulations, revealing the depolarizing nature of the seafloor. The simulations, executed with the software package RayXP, are solutions to the vector radiative transfer equation, which depends on the incident light field and three types of parameters: inherent optical properties, the scattering matrix, and the benthic reflectance. These were measured directly or calculated from measurements with additional assumptions. Specifically, the Lambertian model used to simulate benthic reflectances is something of a simplification of reality, but the bottoms used in this study are found to be crucial for accurate simulations of polarization. Comparisons of simulations with and without bottom contributions show that only the former corroborate measurements of the Stokes components and the degree of linear polarization (DoLP) collected by the polarimeter developed at the City College of New York. Because this polarimeter is multiangular and hyperspectral, errors can be computed point-wise over a large range of scattering angles and wavelengths. Trends also become apparent. DoLP is highly sensitive to the benthic reflectance and to the incident wavelength, peaking in the red band, but the angle of linear polarization is almost spectrally constant and independent of the bottom. These results can thus facilitate the detection of benthic materials as well as future studies of camouflage by benthic biota; to hide underwater successfully, animals must reflect light just as depolarized as that reflected by benthic materials.

A system for exposing molecules and cells to biologically relevant and accurately controlled steady-state concentrations of nitric oxide and oxygen.

Nitric oxide (NO) plays key roles in cell signaling and physiology, with diverse functions mediated by NO concentrations varying over three orders-of-magnitude. In spite of this critical concentration dependence, current approaches to NO delivery in vitro result in biologically irrelevant and poorly controlled levels, with hyperoxic conditions imposed by ambient air. To solve these problems, we developed a system for controlled delivery of NO and O(2) over large concentration ranges to mimic biological conditions. Here we describe the fabrication, operation and calibration of the delivery system. We then describe applications for delivery of NO and O(2) into cell culture media, with a comparison of experimental results and predictions from mass transfer models that predict the steady-state levels of various NO-derived reactive species. We also determined that components of culture media do not affect the steady-state levels of NO or O(2) in the device. This system provides critical control of NO delivery for in vitro models of NO biology and chemistry.

An automated liquid jet for fluorescence dosimetry and microsecond radiolytic labeling of proteins.

X-ray radiolytic labeling uses broadband X-rays for in situ hydroxyl radical labeling to map protein interactions and conformation. High flux density beams are essential to overcome radical scavengers. However, conventional sample delivery environments, such as capillary flow, limit the use of a fully unattenuated focused broadband beam. An alternative is to use a liquid jet, and we have previously demonstrated that use of this form of sample delivery can increase labeling by tenfold at an unfocused X-ray source. Here we report the first use of a liquid jet for automated inline quantitative fluorescence dosage characterization and sample exposure at a high flux density microfocused synchrotron beamline. Our approach enables exposure times in single-digit microseconds while retaining a high level of side-chain labeling. This development significantly boosts the method's overall effectiveness and efficiency, generates high-quality data, and opens up the arena for high throughput and ultrafast time-resolved in situ hydroxyl radical labeling.

microRNA modulation of circadian-clock period and entrainment.

microRNAs (miRNAs) are a class of small, noncoding RNAs that regulate the stability or translation of mRNA transcripts. Although recent work has implicated miRNAs in development and in disease, the expression and function of miRNAs in the adult mammalian nervous system have not been extensively characterized. Here, we examine the role of two brain-specific miRNAs, miR-219 and miR-132, in modulating the circadian clock located in the suprachiasmatic nucleus. miR-219 is a target of the CLOCK and BMAL1 complex, exhibits robust circadian rhythms of expression, and the in vivo knockdown of miR-219 lengthens the circadian period. miR-132 is induced by photic entrainment cues via a MAPK/CREB-dependent mechanism, modulates clock-gene expression, and attenuates the entraining effects of light. Collectively, these data reveal miRNAs as clock- and light-regulated genes and provide a mechanistic examination of their roles as effectors of pacemaker activity and entrainment.

Molting strategies of Arctic seals drive annual patterns in metabolism.

Arctic seals, including spotted (Phoca largha), ringed (Pusa hispida) and bearded (Erignathus barbatus) seals, are directly affected by sea ice loss. These species use sea ice as a haul-out substrate for various critical functions, including their annual molt. Continued environmental warming will inevitably alter the routine behavior and overall energy budgets of Arctic seals, but it is difficult to quantify these impacts as their metabolic requirements are not well known-due in part to the difficulty of studying wild individuals. Thus, data pertaining to species-specific energy demands are urgently needed to better understand the physiological consequences of rapid environmental change. We used open-flow respirometry over a four-year period to track fine-scale, longitudinal changes in the resting metabolic rate (RMR) of four spotted seals, three ringed seals and one bearded seal trained to participate in research. Simultaneously, we collected complementary physiological and environmental data. Species-specific metabolic demands followed expected patterns based on body size, with the largest species, the bearded seal, exhibiting the highest absolute RMR (0.48 ± 0.04 L O2 min-1) and the lowest mass-specific RMR (4.10 ± 0.47 ml O2 min-1 kg-1), followed by spotted (absolute: 0.33 ± 0.07 L O2 min-1; mass-specific: 6.13 ± 0.73 ml O2 min-1 kg-1) and ringed (absolute: 0.20 ± 0.04 L O2 min-1; mass-specific: 7.01 ± 1.38 ml O2 min-1 kg-1) seals. Further, we observed clear and consistent annual patterns in RMR that related to the distinct molting strategies of each species. For species that molted over relatively short intervals-spotted (33 ± 4 days) and ringed (28 ± 6 days) seals-metabolic demands increased markedly in association with molt. In contrast, the bearded seal exhibited a prolonged molting strategy (119 ± 2 days), which appeared to limit the overall cost of molting as indicated by a relatively stable annual RMR. These findings highlight energetic trade-offs associated with different molting strategies and provide quantitative data that can be used to assess species-specific vulnerabilities to changing conditions.

Reciprocal regulation of TORC signaling and tRNA modifications by Elongator enforces nutrient-dependent cell fate.

Nutrient availability has a profound impact on cell fate. Upon nitrogen starvation, wild-type fission yeast cells uncouple cell growth from cell division to generate small, round-shaped cells that are competent for sexual differentiation. The TORC1 (TOR complex 1) and TORC2 complexes exert opposite controls on cell growth and cell differentiation, but little is known about how their activity is coordinated. We show that transfer RNA (tRNA) modifications by Elongator are critical for this regulation by promoting the translation of both key components of TORC2 and repressors of TORC1. We further identified the TORC2 pathway as an activator of Elongator by down-regulating a Gsk3 (glycogen synthase kinase 3)-dependent inhibitory phosphorylation of Elongator. Therefore, a feedback control is operating between TOR complex (TORC) signaling and tRNA modification by Elongator to enforce the advancement of mitosis that precedes cell differentiation.

Photocatalytic effects of titanium dioxide nanoparticles on aquatic organisms-Current knowledge and suggestions for future research.

Nanoparticles are entering natural systems through product usage, industrial waste and post-consumer material degradation. As the production of nanoparticles is expected to increase in the next decade, so too are predicted environmental loads. Engineered metal-oxide nanomaterials, such as titanium dioxide, are known for their photocatalytic capabilities. When these nanoparticles are exposed to ultraviolet radiation in the environment, however, they can produce radicals that are harmful to aquatic organisms. There have been a number of studies that have reported the toxicity of titanium dioxide nanoparticles in the absence of light. An increasing number of studies are assessing the interactive effects of nanoparticles and ultraviolet light. However, most of these studies neglect environmentally-relevant experimental conditions. For example, researchers are using nanoparticle concentrations and light intensities that are too high for natural systems, and are ignoring water constituents that can alter the light field. The purpose of this review is to summarize the current knowledge of the photocatalytic effects of TiO2 nanoparticles on aquatic organisms, discuss the limitations of these studies, and outline environmentally-relevant factors that need to be considered in future experiments.

Use of Hyperspectral Imagery to Assess Cryptic Color Matching in Sargassum Associated Crabs.

Mats of the pelagic macroalgae Sargassum represent a complex environment for the study of marine camouflage at the air-sea interface. Endemic organisms have convergently evolved similar colors and patterns, but quantitative assessments of camouflage strategies are lacking. Here, spectral camouflage of two crab species (Portunus sayi and Planes minutus) was assessed using hyperspectral imagery (HSI). Crabs matched Sargassum reflectance across blue and green wavelengths (400-550 nm) and diverged at longer wavelengths. Maximum discrepancy was observed in the far-red (i.e., 675 nm) where Chlorophyll a absorption occurred in Sargassum and not the crabs. In a quantum catch color model, both crabs showed effective color matching against blue/green sensitive dichromat fish, but were still discernible to tetrachromat bird predators that have visual sensitivity to far red wavelengths. The two species showed opposing trends in background matching with relation to body size. Variation in model parameters revealed that discrimination of crab and background was impacted by distance from the predator, and the ratio of cone cell types for bird predators. This is one of the first studies to detail background color matching in this unique, challenging ecosystem at the air-sea interface.

Open-ocean fish reveal an omnidirectional solution to camouflage in polarized environments.

Despite appearing featureless to our eyes, the open ocean is a highly variable environment for polarization-sensitive viewers. Dynamic visual backgrounds coupled with predator encounters from all possible directions make this habitat one of the most challenging for camouflage. We tested open-ocean crypsis in nature by collecting more than 1500 videopolarimetry measurements from live fish from distinct habitats under a variety of viewing conditions. Open-ocean fish species exhibited camouflage that was superior to that of both nearshore fish and mirrorlike surfaces, with significantly higher crypsis at angles associated with predator detection and pursuit. Histological measurements revealed that specific arrangements of reflective guanine platelets in the fish's skin produce angle-dependent polarization modifications for polarocrypsis in the open ocean, suggesting a mechanism for natural selection to shape reflectance properties in this complex environment.

Quantitative analysis of ribonucleoside modifications in tRNA by HPLC-coupled mass spectrometry.

Post-transcriptional modification of RNA is an important determinant of RNA quality control, translational efficiency, RNA-protein interactions and stress response. This is illustrated by the observation of toxicant-specific changes in the spectrum of tRNA modifications in a stress-response mechanism involving selective translation of codon-biased mRNA for crucial proteins. To facilitate systems-level studies of RNA modifications, we developed a liquid chromatography-mass spectrometry (LC-MS) technique for the quantitative analysis of modified ribonucleosides in tRNA. The protocol includes tRNA purification by HPLC, enzymatic hydrolysis, reversed-phase HPLC resolution of the ribonucleosides, and identification and quantification of individual ribonucleosides by LC-MS via dynamic multiple reaction monitoring (DMRM). In this approach, the relative proportions of modified ribonucleosides are quantified in several micrograms of tRNA in a 15-min LC-MS run. This protocol can be modified to analyze other types of RNA by modifying the steps for RNA purification as appropriate. By comparison, traditional methods for detecting modified ribonucleosides are labor- and time-intensive, they require larger RNA quantities, they are modification-specific or require radioactive labeling.

Safety, immunogenicity, and efficacy of an alphavirus replicon-based swine influenza virus hemagglutinin vaccine.

A single-cycle, propagation-defective replicon particle (RP) vaccine expressing a swine influenza virus hemagglutinin (HA) gene was constructed and evaluated in several different animal studies. Studies done in both the intended host (pigs) and non-host (mice) species demonstrated that the RP vaccine is not shed or spread by vaccinated animals to comingled cohorts, nor does it revert to virulence following vaccination. In addition, vaccinated pigs develop both specific humoral and IFN-γ immune responses, and young pigs are protected against homologous influenza virus challenge.