Complete genome sequence of a Pseudomonas fluorescens bacteriophage UNO-G1W1 isolated from freshwater ice in Nebraska.

We provide the complete genome sequence for a novel Pseudomonas fluorescens bacteriophage named UNO-G1W1. This phage was isolated from a single ice cover sampling. The genome was sequenced on the Nanopore MinION, generated with the direct terminal repeat-phage-pipeline and polished with Illumina short reads. Sequence identity classifies the phage as an otagovirus.

Spatiotemporal dispersion compensation for a 200-THz noncollinear optical parametric amplifier.

A noncollinear optical parametric amplifier (NOPA) can produce few-cycle femtosecond laser pulses that are ideally suited for time-resolved optical spectroscopy measurements. However, the nonlinear-optical process giving rise to ultrabroadband pulses is susceptible to spatiotemporal dispersion problems. Here, we detail refinements, including chirped-pulse amplification (CPA) and pulse-front matching (PFM), that minimize spatiotemporal dispersion and thereby improve the properties of ultrabroadband pulses produced by a NOPA. The description includes a rationale behind the choices of optical and optomechanical components, as well as assessment protocols. We demonstrate these techniques using a 1 kHz, second-harmonic Ti:sapphire pump configuration, which produces ∼5-fs duration pulses that span from about 500 to 800 nm with a bandwidth of about 200 THz. To demonstrate the utility of the CPA-PFM-NOPA, we measure vibrational quantum beats in the transient-absorption spectrum of methylene blue, a dye molecule that serves as a reference standard.

Antimalarial Dibenzannulated Medium-Ring Keto Lactams.

We discovered dibenzannulated medium-ring keto lactams (11,12-dihydro-5H-dibenzo[b,g]azonine-6,13-diones) as a new antimalarial chemotype. Most of these had chromatographic LogD7.4 values ranging from <0 to 3 and good kinetic solubilities (12.5 to >100 µg/mL at pH 6.5). The more polar compounds in the series (LogD7.4 values of <2) had the best metabolic stability (CLint values of <50 µL/min/mg protein in human liver microsomes). Most of the compounds had relatively low cytotoxicity, with IC50 values >30 µM, and there was no correlation between antiplasmodial activity and cytotoxicity. The four most potent compounds had Plasmodium falciparum IC50 values of 4.2 to 9.4 nM and in vitro selectivity indices of 670 to >12,000. They were more than 4 orders-of-magnitude less potent against three other protozoal pathogens (Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania donovani) but did have relatively high potency against Toxoplasma gondii, with IC50 values ranging from 80 to 200 nM. These keto lactams are converted into their poorly soluble 4(1H)-quinolone transannular condensation products in vitro in culture medium and in vivo in mouse blood. The similar antiplasmodial potencies of three keto lactam-quinolone pairs suggest that the quinolones likely contribute to the antimalarial activity of the lactams.

Electronic Structure and Excited-State Dynamics of DNA-Templated Monomers and Aggregates of Asymmetric Polymethine Dyes.

Aggregates of conjugated organic molecules (i.e., dyes) may exhibit relatively large one- and two-exciton interaction energies, which has motivated theoretical studies on their potential use in quantum information science (QIS). In practice, one way of realizing large one- and two-exciton interaction energies is by maximizing the transition dipole moment (µ) and difference static dipole moment (Δd) of the constituent dyes. In this work, we characterized the electronic structure and excited-state dynamics of monomers and aggregates of four asymmetric polymethine dyes templated via DNA. Using steady-state and time-resolved absorption and fluorescence spectroscopy along with quantum-chemical calculations, we found the asymmetric polymethine dye monomers exhibited a large µ, an appreciable Δd, and a long excited-state lifetime (τp). We formed dimers of all four dyes and observed that one dye, Dy 754, displayed the strongest propensity for aggregation and exciton delocalization. Motivated by these results, we undertook a more comprehensive survey of Dy 754 dimer and tetramer aggregates using steady-state absorption and circular dichroism spectroscopy. Modeling these spectra revealed an appreciable excitonic hopping parameter (J). Lastly, we used femtosecond transient absorption spectroscopy to characterize τp of the dimer and tetramer, which we observed to be exceedingly short. This work revealed that asymmetric polymethine dyes exhibited µ, Δd, monomer τp, and J values promising for QIS; however, further work is needed to overcome excited-state quenching and achieve long aggregate τp.

Probing DNA structural heterogeneity by identifying conformational subensembles of a bicovalently bound cyanine dye.

DNA is a re-configurable, biological information-storage unit, and much remains to be learned about its heterogeneous structural dynamics. For example, while it is known that molecular dyes templated onto DNA exhibit increased photostability, the mechanism by which the structural dynamics of DNA affect the dye photophysics remains unknown. Here, we use femtosecond, two-dimensional electronic spectroscopy measurements of a cyanine dye, Cy5, to probe local conformations in samples of single-stranded DNA (ssDNA-Cy5), double-stranded DNA (dsDNA-Cy5), and Holliday junction DNA (HJ-DNA-Cy5). A line shape analysis of the 2D spectra reveals a strong excitation-emission correlation present in only the dsDNA-Cy5 complex, which is a signature of inhomogeneous broadening. Molecular dynamics simulations support the conclusion that this inhomogeneous broadening arises from a nearly degenerate conformer found only in the dsDNA-Cy5 complex. These insights will support future studies on DNA's structural heterogeneity.

Intramolecular Charge Transfer and Ultrafast Nonradiative Decay in DNA-Tethered Asymmetric Nitro- and Dimethylamino-Substituted Squaraines.

Molecular (dye) aggregates are a materials platform of interest in light harvesting, organic optoelectronics, and nanoscale computing, including quantum information science (QIS). Strong excitonic interactions between dyes are key to their use in QIS; critically, properties of the individual dyes govern the extent of these interactions. In this work, the electronic structure and excited-state dynamics of a series of indolenine-based squaraine dyes incorporating dimethylamino (electron donating) and/or nitro (electron withdrawing) substituents, so-called asymmetric dyes, were characterized. The dyes were covalently tethered to DNA Holliday junctions to suppress aggregation and permit characterization of their monomer photophysics. A combination of density functional theory and steady-state absorption spectroscopy shows that the difference static dipole moment (Δd) successively increases with the addition of these substituents while simultaneously maintaining a large transition dipole moment (µ). Steady-state fluorescence and time-resolved absorption and fluorescence spectroscopies uncover a significant nonradiative decay pathway in the asymmetrically substituted dyes that drastically reduces their excited-state lifetime (τ). This work indicates that Δd can indeed be increased by functionalizing dyes with electron donating and withdrawing substituents and that, in certain classes of dyes such as these asymmetric squaraines, strategies may be needed to ensure long τ, e.g., by rigidifying the π-conjugated network.

Queueing theory model of mTOR complexes' impact on Akt-mediated adipocytes response to insulin.

A queueing theory based model of mTOR complexes impact on Akt-mediated cell response to insulin is presented in this paper. The model includes several aspects including the effect of insulin on the transport of glucose from the blood into the adipocytes with the participation of GLUT4, and the role of the GAPDH enzyme as a regulator of mTORC1 activity. A genetic algorithm was used to optimize the model parameters. It can be observed that mTORC1 activity is related to the amount of GLUT4 involved in glucose transport. The results show the relationship between the amount of GAPDH in the cell and mTORC1 activity. Moreover, obtained results suggest that mTORC1 inhibitors may be an effective agent in the fight against type 2 diabetes. However, these results are based on theoretical knowledge and appropriate experimental tests should be performed before making firm conclusions.

Atomic Force Microscopy Cantilever-Based Nanoindentation: Mechanical Property Measurements at the Nanoscale in Air and Fluid.

An atomic force microscope (AFM) fundamentally measures the interaction between a nanoscale AFM probe tip and the sample surface. If the force applied by the probe tip and its contact area with the sample can be quantified, it is possible to determine the nanoscale mechanical properties (e.g., elastic or Young's modulus) of the surface being probed. A detailed procedure for performing quantitative AFM cantilever-based nanoindentation experiments is provided here, with representative examples of how the technique can be applied to determine the elastic moduli of a wide variety of sample types, ranging from kPa to GPa. These include live mesenchymal stem cells (MSCs) and nuclei in physiological buffer, resin-embedded dehydrated loblolly pine cross-sections, and Bakken shales of varying composition. Additionally, AFM cantilever-based nanoindentation is used to probe the rupture strength (i.e., breakthrough force) of phospholipid bilayers. Important practical considerations such as method choice and development, probe selection and calibration, region of interest identification, sample heterogeneity, feature size and aspect ratio, tip wear, surface roughness, and data analysis and measurement statistics are discussed to aid proper implementation of the technique. Finally, co-localization of AFM-derived nanomechanical maps with electron microscopy techniques that provide additional information regarding elemental composition is demonstrated.

Tunable Electronic Structure via DNA-Templated Heteroaggregates of Two Distinct Cyanine Dyes.

Molecular excitons are useful for applications in light harvesting, organic optoelectronics, and nanoscale computing. Electronic energy transfer (EET) is a process central to the function of devices based on molecular excitons. Achieving EET with a high quantum efficiency is a common obstacle to excitonic devices, often owing to the lack of donor and acceptor molecules that exhibit favorable spectral overlap. EET quantum efficiencies may be substantially improved through the use of heteroaggregates-aggregates of chemically distinct dyes-rather than individual dyes as energy relay units. However, controlling the assembly of heteroaggregates remains a significant challenge. Here, we use DNA Holliday junctions to assemble homo- and heterotetramer aggregates of the prototypical cyanine dyes Cy5 and Cy5.5. In addition to permitting control over the number of dyes within an aggregate, DNA-templated assembly confers control over aggregate composition, i.e., the ratio of constituent Cy5 and Cy5.5 dyes. By varying the ratio of Cy5 and Cy5.5, we show that the most intense absorption feature of the resulting tetramer can be shifted in energy over a range of almost 200 meV (1600 cm-1). All tetramers pack in the form of H-aggregates and exhibit quenched emission and drastically reduced excited-state lifetimes compared to the monomeric dyes. We apply a purely electronic exciton theory model to describe the observed progression of the absorption spectra. This model agrees with both the measured data and a more sophisticated vibronic model of the absorption and circular dichroism spectra, indicating that Cy5 and Cy5.5 heteroaggregates are largely described by molecular exciton theory. Finally, we extend the purely electronic exciton model to describe an idealized J-aggregate based on Förster resonance energy transfer (FRET) and discuss the potential advantages of such a device over traditional FRET relays.

Symmetry Breaking Charge Transfer in DNA-Templated Perylene Dimer Aggregates.

Molecular aggregates are of interest to a broad range of fields including light harvesting, organic optoelectronics, and nanoscale computing. In molecular aggregates, nonradiative decay pathways may emerge that were not present in the constituent molecules. Such nonradiative decay pathways may include singlet fission, excimer relaxation, and symmetry-breaking charge transfer. Singlet fission, sometimes referred to as excitation multiplication, is of great interest to the fields of energy conversion and quantum information. For example, endothermic singlet fission, which avoids energy loss, has been observed in covalently bound, linear perylene trimers and tetramers. In this work, the electronic structure and excited-state dynamics of dimers of a perylene derivative templated using DNA were investigated. Specifically, DNA Holliday junctions were used to template the aggregation of two perylene molecules covalently linked to a modified uracil nucleobase through an ethynyl group. The perylenes were templated in the form of monomer, transverse dimer, and adjacent dimer configurations. The electronic structure of the perylene monomers and dimers were characterized via steady-state absorption and fluorescence spectroscopy. Initial insights into their excited-state dynamics were gleaned from relative fluorescence intensity measurements, which indicated that a new nonradiative decay pathway emerges in the dimers. Femtosecond visible transient absorption spectroscopy was subsequently used to elucidate the excited-state dynamics. A new excited-state absorption feature grows in on the tens of picosecond timescale in the dimers, which is attributed to the formation of perylene anions and cations resulting from symmetry-breaking charge transfer. Given the close proximity required for symmetry-breaking charge transfer, the results shed promising light on the prospect of singlet fission in DNA-templated molecular aggregates.

Optimizing Magnetic Force Microscopy Resolution and Sensitivity to Visualize Nanoscale Magnetic Domains.

Magnetic force microscopy (MFM) enables mapping local magnetic fields across a sample surface with nanoscale resolution. To perform MFM, an atomic force microscopy (AFM) probe whose tip has been magnetized vertically (i.e., perpendicular to the probe cantilever) is oscillated at a fixed height above the sample surface. The resultant shifts in the oscillation phase or frequency, which are proportional to the magnitude and sign of the vertical magnetic force gradient at each pixel location, are then tracked and mapped. Although the spatial resolution and sensitivity of the technique increases with decreasing lift height above the surface, this seemingly straightforward path to improved MFM images is complicated by considerations such as minimizing topographical artifacts due to shorter range van der Waals forces, increasing the oscillation amplitude to further improve sensitivity, and the presence of surface contaminants (in particular water due to humidity under ambient conditions). In addition, due to the orientation of the probe's magnetic dipole moment, MFM is intrinsically more sensitive to samples with an out-of-plane magnetization vector. Here, high-resolution topographical and magnetic phase images of single and bicomponent nanomagnet artificial spin-ice (ASI) arrays obtained in an inert (argon) atmosphere glovebox with <0.1 ppm O2 and H2O are reported. Optimization of lift height and drive amplitude for high resolution and sensitivity while simultaneously avoiding the introduction of topographical artifacts is discussed, and detection of the stray magnetic fields emanating from either end of the nanoscale bar magnets (~250 nm long and <100 nm wide) aligned in the plane of the ASI sample surface is shown. Likewise, using the example of a Ni-Mn-Ga magnetic shape memory alloy (MSMA), MFM is demonstrated in an inert atmosphere with magnetic phase sensitivity capable of resolving a series of adjacent magnetic domains each ~200 nm wide.

Co-localizing Kelvin Probe Force Microscopy with Other Microscopies and Spectroscopies: Selected Applications in Corrosion Characterization of Alloys.

Kelvin probe force microscopy (KPFM), sometimes referred to as surface potential microscopy, is the nanoscale version of the venerable scanning Kelvin probe, both of which measure the Volta potential difference (VPD) between an oscillating probe tip and a sample surface by applying a nulling voltage equal in magnitude but opposite in sign to the tip-sample potential difference. By scanning a conductive KPFM probe over a sample surface, nanoscale variations in surface topography and potential can be mapped, identifying likely anodic and cathodic regions, as well as quantifying the inherent material driving force for galvanic corrosion. Subsequent co-localization of KPFM Volta potential maps with advanced scanning electron microscopy (SEM) techniques, including back scattered electron (BSE) images, energy dispersive spectroscopy (EDS) elemental composition maps, and electron backscattered diffraction (EBSD) inverse pole figures can provide further insight into structure-property-performance relationships. Here, the results of several studies co-localizing KPFM with SEM on a wide variety of alloys of technological interest are presented, demonstrating the utility of combining these techniques at the nanoscale to elucidate corrosion initiation and propagation. Important points to consider and potential pitfalls to avoid in such investigations are also highlighted: in particular, probe calibration and the potential confounding effects on the measured VPDs of the testing environment and sample surface, including ambient humidity (i.e., adsorbed water), surface reactions/oxidation, and polishing debris or other contaminants. Additionally, an example is provided of co-localizing a third technique, scanning confocal Raman microscopy, to demonstrate the general applicability and utility of the co-localization method to provide further structural insight beyond that afforded by electron microscopy-based techniques.

Characterizing Mode Anharmonicity and Huang-Rhys Factors Using Models of Femtosecond Coherence Spectra.

Femtosecond laser pulses readily produce coherent quantum beats in transient-absorption spectra. These oscillatory signals often arise from molecular vibrations and therefore may contain information about the excited-state potential energy surface near the Franck-Condon region. Here, by fitting the measured spectra of two laser dyes to microscopic models of femtosecond coherence spectra (FCS) arising from molecular vibrations, we classify coherent quantum-beat signals as fundamentals or overtones and quantify their Huang-Rhys factors and anharmonicity values. We discuss the extracted Huang-Rhys factors in the context of quantum-chemical computations. This work solidifies the use of FCS for analysis of coherent quantum beats arising from molecular vibrations, which will aid studies of molecular aggregates and photosynthetic proteins.

Mouse splenocyte enrichment strategies via negative selection for broadened single-cell transcriptomics.

Mammalian splenic tissue is rich in functional immune cells, primarily lymphocytes which can mask low-abundance populations in downstream analyses. This protocol enriches minority immune cell populations from mouse spleen via immunomagnetic negative depletion to generate an untouched enriched cell fraction. Enriched cells are then spiked with untouched splenocytes in a controlled repopulation, validated by flow cytometry and results in a single-cell transcriptomic clustering analysis with a broadened cellular landscape.

Electrochemically induced amorphous-to-rock-salt phase transformation in niobium oxide electrode for Li-ion batteries.

Intercalation-type metal oxides are promising negative electrode materials for safe rechargeable lithium-ion batteries due to the reduced risk of Li plating at low voltages. Nevertheless, their lower energy and power density along with cycling instability remain bottlenecks for their implementation, especially for fast-charging applications. Here, we report a nanostructured rock-salt Nb2O5 electrode formed through an amorphous-to-crystalline transformation during repeated electrochemical cycling with Li+. This electrode can reversibly cycle three lithiums per Nb2O5, corresponding to a capacity of 269 mAh g-1 at 20 mA g-1, and retains a capacity of 191 mAh g-1 at a high rate of 1 A g-1. It exhibits superb cycling stability with a capacity of 225 mAh g-1 at 200 mA g-1 for 400 cycles, and a Coulombic efficiency of 99.93%. We attribute the enhanced performance to the cubic rock-salt framework, which promotes low-energy migration paths. Our work suggests that inducing crystallization of amorphous nanomaterials through electrochemical cycling is a promising avenue for creating unconventional high-performance metal oxide electrode materials.

Tuning between Quenching and Energy Transfer in DNA-Templated Heterodimer Aggregates.

Molecular excitons, which propagate spatially via electronic energy transfer, are central to numerous applications including light harvesting, organic optoelectronics, and nanoscale computing; they may also benefit applications such as photothermal therapy and photoacoustic imaging through the local generation of heat via rapid excited-state quenching. Here we show how to tune between energy transfer and quenching for heterodimers of the same pair of cyanine dyes by altering their spatial configuration on a DNA template. We assemble "transverse" and "adjacent" heterodimers of Cy5 and Cy5.5 using DNA Holliday junctions. We find that the transverse heterodimers exhibit optical properties consistent with excitonically interacting dyes and fluorescence quenching, while the adjacent heterodimers exhibit optical properties consistent with nonexcitonically interacting dyes and disproportionately large Cy5.5 emission, suggestive of energy transfer between dyes. We use transient absorption spectroscopy to show that quenching in the transverse heterodimer occurs via rapid nonradiative decay to the ground state (∼31 ps) and that in the adjacent heterodimer rapid energy transfer from Cy5 to Cy5.5 (∼420 fs) is followed by Cy5.5 excited-state relaxation (∼700 ps). Accessing such drastically different photophysics, which may be tuned on demand for different target applications, highlights the utility of DNA as a template for dye aggregation.

Analogs of Marinopyrrole A Show Enhancement to Observed In Vitro Potency against Acute Toxoplasma gondii Infection.

The apicomplexan parasite Toxoplasma gondii is the causative agent of toxoplasmosis, a globally distributed infection with severe clinical consequences for immunocompromised individuals and developing fetuses. There are few available treatments, and these are associated with potentially severe adverse effects. Marinopyrrole A, a compound discovered in a marine Streptomyces species, has previously been found to exhibit potent antimicrobial activity, prompting our interest in exploring efficacy against Toxoplasma gondii. We found that marinopyrrole A was a highly potent anti-Toxoplasma molecule, with an in vitro 50% maximal inhibitory concentration (IC50) of 0.31 µM, corresponding to a higher potency than that of the current standard of care (pyrimethamine); however, addition of 20% serum led to abrogation of potency, and toxicity to human cell lines was observed. Yet, application of marinopyrrole A to an in vivo lethal acute infection model facilitated significantly enhanced survival at doses of 5, 10, and 20 mg/kg. We then tested a series of marinopyrrole A analogs (RL002, RL003, and RL125) and demonstrated significantly increased potency in vitro, with IC50 values ranging from 0.09 to 0.17 µM (3.6- to 6.8-fold increase relative to pyrimethamine). No detectable cytotoxicity was observed up to 50 µM in human foreskin fibroblasts, with cytotoxicity in HepG2 cells ranging from ∼28 to 50 µM, corresponding to >200-fold selectivity for parasites over host cells. All analogs additionally showed reduced sensitivity to serum. Further, RL003 potently inhibited in vitro-generated bradyzoites at 0.245 µM. Taken together, these data support further development of marinopyrrole A analogs as promising anti-Toxoplasma molecules to further combat this prevalent infection.

REVIEW OF DNA VACCINE APPROACHES AGAINST THE PARASITE TOXOPLASMA GONDII.

Toxoplasma gondii is an apicomplexan parasite that affects both humans and livestock. Transmitted to humans through ingestion, it is the second-leading cause of foodborne illness-related death. Currently, there exists no approved vaccine for humans or most livestock against the parasite. DNA vaccines, a type of subunit vaccine which uses segments of the pathogen's DNA to generate immunity, have shown varying degrees of experimental efficacy against infection caused by the parasite. This review compiles DNA vaccine efforts against Toxoplasma gondii, segmenting the analysis by parasite antigen, as well as a review of concomitant adjuvant usage. No single antigenic group was consistently more effective within in vivo trials relative to others.

Neurological and Neurobehavioral Disorders Associated with Toxoplasma gondii Infection in Humans.

The intracellular parasite Toxoplasma gondii is estimated to infect up to 30% of the world population, leading to lifelong chronic infection of the brain and muscle tissue. Although most latent T. gondii infections in humans have traditionally been considered asymptomatic, studies in rodents suggest phenotypic neurological changes are possible. Consequently, several studies have examined the link between T. gondii infection and diseases such as schizophrenia, epilepsy, depression, bipolar disorder, dysphoria, Alzheimer's disease, Parkinson's disease, and obsessive-compulsive disorder (OCD). To date, there is varying evidence of the relationship of T. gondii to these human neurological or neurobehavioral disorders. A thorough review of T. gondii literature was conducted to highlight and summarize current findings. We found that schizophrenia was most frequently linked to T. gondii infection, while sleep disruption showed no linkage to T. gondii infection, and other conditions having mixed support for a link to T. gondii. However, infection as a cause of human neurobehavioral disease has yet to be firmly established.

A Comparison of Solid Electrolyte Interphase Formation and Evolution on Highly Oriented Pyrolytic and Disordered Graphite Negative Electrodes in Lithium-Ion Batteries.

The presence and stability of solid electrolyte interphase (SEI) on graphitic electrodes is vital to the performance of lithium-ion batteries (LIBs). However, the formation and evolution of SEI remain the least understood area in LIBs due to its dynamic nature, complexity in chemical composition, heterogeneity in morphology, as well as lack of reliable in situ/operando techniques for accurate characterization. In addition, chemical composition and morphology of SEI are not only affected by the choice of electrolyte, but also by the nature of the electrode surface. While introduction of defects into graphitic electrodes has promoted their electrochemical properties, how such structural defects influence SEI formation and evolution remains an open question. Here, utilizing nondestructive operando electrochemical atomic force microscopy (EChem-AFM) the dynamic SEI formation and evolution on a pair of representative graphitic materials with and without defects, namely, highly oriented pyrolytic and disordered graphite electrodes, are systematically monitored and compared. Complementary to the characterization of SEI topographical and mechanical changes during electrochemical cycling by EChem-AFM, chemical analysis and theoretical calculations are conducted to provide mechanistic insights underlying SEI formation and evolution. The results provide guidance to engineer functional SEIs through design of carbon materials with defects for LIBs and beyond.