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Human immunodeficiency virus type 2 (HIV-2) accumulates fewer mutations during replication than HIV type 1 (HIV-1). Advanced studies of HIV-2 mutagenesis, however, have historically been confounded by high background error rates in traditional next-generation sequencing techniques. In this study, we describe the adaptation of the previously described maximum-depth sequencing (MDS) technique to studies of both HIV-1 and HIV-2 for the ultra-accurate characterization of viral mutagenesis. We also present the development of a user-friendly Galaxy workflow for the bioinformatic analyses of sequencing data generated using the MDS technique, designed to improve replicability and accessibility to molecular virologists. This adapted MDS technique and analysis pipeline were validated by comparisons with previously published analyses of the frequency and spectra of mutations in HIV-1 and HIV-2 and is readily expandable to studies of viral mutation across the genomes of both viruses. Using this novel sequencing pipeline, we observed that the background error rate was reduced 100-fold over standard Illumina error rates, and 10-fold over traditional unique molecular identifier (UMI)-based sequencing. This technical advancement will allow for the exploration of novel and previously unrecognized sources of viral mutagenesis in both HIV-1 and HIV-2, which will expand our understanding of retroviral diversity and evolution.

Summary The SUSD4 (Sushi domain-containing protein 4) gene encodes a complement inhibitor that is frequently deleted in 1q41q42 microdeletion syndrome, a multisystem congenital disorder that includes neurodevelopmental abnormalities. To understand SUSD4's role in the mammalian nervous system, we analyzed Susd4 knockout (KO) mice. Susd4 KO mice exhibited significant defects in motor performance and significantly higher levels of anxiety-like behaviors. Susd4 KO brain had abnormal “hairy” basket cells surrounding Purkinje neurons within the cerebellum and significantly reduced dendritic spine density in hippocampal pyramidal neurons. Neurons and oligodendrocyte lineage cells of wild-type mice were found to express Susd4 mRNA. Protein expression of the complement component C1q was increased in the brains of Susd4 KO mice. Our data indicate that SUSD4 plays an important role in neuronal functions, possibly via the complement pathway, and that SUSD4 deletion may contribute to the nervous system abnormalities in patients with 1q41q42 deletions., Graphical Abstract, Highlights • Susd4 is expressed in neurons and oligodendrocyte lineage cells • Susd4 knockout mice have abnormal hippocampal and cerebellar neuronal morphologies • Susd4 knockout mice exhibit anxiety-like behaviors and impaired motor function • Susd4 knockout mice have elevated brain levels of the complement component C1q, Behavioral Neuroscience; Cellular Neuroscience; Components of the Immune System; Neuroscience

Human immunodeficiency virus (HIV) mutagenesis is driven by a variety of internal and external sources, including the host APOBEC3 (apolipoprotein B mRNA editing enzyme catalytic polypetide-like 3; A3) family of restriction factors, which catalyze G-to-A transition mutations during virus replication. HIV-2 replication is characterized by a relative lack of G-to-A mutations, suggesting infrequent restriction by A3 proteins. To date, the activity of the A3 repertoire against HIV-2 has remained largely uncharacterized, and the mutagenic activity of these proteins against HIV-2 remains to be elucidated. In this study, we provide the first comprehensive characterization of the restrictive capacity of A3 proteins against HIV-2 in cell culture using a dual fluorescent reporter HIV-2 vector virus. We found that A3F, A3G, and A3H restricted HIV-2 infectivity in the absence of Vif and were associated with significant increases in the frequency of viral mutants. These proteins increased the frequency of G-to-A mutations within the proviruses of infected cells as well. A3G and A3H also reduced HIV-2 infectivity via inhibition of reverse transcription and the accumulation of DNA products during replication. In contrast, A3D, did not exhibit any restrictive activity against HIV-2, even at higher expression levels. Taken together, these results provide evidence that A3F, A3G, and A3H, but not A3D, are capable of HIV-2 restriction. Differences in A3-mediated restriction of HIV-1 and HIV-2 may serve to provide new insights in the observed mutation profiles of these viruses.

A long-standing question of human immunodeficiency virus (HIV) genetic variation and evolution has been whether differences exist in mutation rate and/or mutation spectra among HIV types (i.e., HIV-1 versus HIV-2) and among HIV groups (i.e., HIV-1 groups M–P and HIV-2 groups A–H) and HIV-1 Group M subtypes (i.e., subtypes A–D, F–H, and J–K). To address this, we developed a new single-strand consensus sequencing assay for the determination of HIV mutation frequencies and spectra using the Illumina sequencing platform. This assay enables parallel and standardized comparison of HIV mutagenesis among various viral vectors with lower background error than traditional methods of Illumina library preparation. We found significant differences in viral mutagenesis between HIV types but intriguingly no significant differences among HIV-1 Group M subtypes. More specifically, HIV-1 exhibited higher transition frequencies than HIV-2, due mostly to single G-to-A mutations and (to a lesser extent) G-to-A hypermutation. These data suggest that HIV-2 RT exhibits higher fidelity during viral replication, and taken together, these findings demonstrate that HIV type but not subtype significantly affects viral mutation frequencies and spectra. These differences may inform antiviral and vaccine strategies.

Optical spectroscopic imaging of biological systems has important applications in medical diagnosis, biochemistry, and image-guided surgery. Vibrational spectroscopy, such as Raman scattering, provides high chemical selectivity but is limited by weak signals and a large fluorescence background. Fluorescence imaging is often used by introducing specific dyes in biological systems to label different system parts and to increase the image contrast. However, the extrinsic fluorescence of the staining molecules often masks the intrinsic vibrational signals of biomolecules, which could also be simultaneously detected using the same excitation laser source. Therefore, fluorescence staining is often accompanied by the loss of other important complimentary information. For example, the high laser power often used for the rapid, high-quality imaging could lead to photo-induced suppression or bleaching of the fluorescence and Raman signals resulting in sample photodamage. Therefore, simultaneous imaging and photodamage analysis need to be performed in a controlled bioimaging experiment. Here we perform simultaneous spectroscopic bioimaging and photostability analysis of rhodamine 6G (R6G) stained red blood cells (RBCs) using both fluorescence and resonance Raman imaging in a single 532 nm laser excitation experiment. We develop a corresponding data processing algorithm which allows separation of the two spectroscopic signals. We control the relative intensity of the R6G and RBC signals by varying the excitation laser power and simultaneously monitor the photostability of RBCs. We observe no significant photodamage of RBCs through the absence of changes in the relative Raman peak intensities. Conversely, the R6G molecules show bleaching with the suppression of both the fluorescence and resonance Raman signals. Our approach may be generalized to other types of stained cells with the appropriate selection of fluorescent dyes and excitation sources.

This article focuses on implementing multivariate analysis to evaluate biochar-derived carbonaceous nanomaterials (BCN) from three different feedstocks for the detection and differentiation of heavy metal ions in aqueous systems. The BCN were produced from dairy manure, rice straw and sorghum straw biochar using our NanoRefinery process. The NanoRefinery process transforms biochar into advanced nanomaterials using depolymerisation/chemical oxidation and purification of nanomaterials using solvent extraction. Dairy manure biochar-derived carbonaceous nanomaterials (DMB-CN), rice straw biochar-derived carbonaceous nanomaterials (RSB-CN) and sorghum straw biochar-derived carbonaceous nanomaterials (SSB-CN) were utilised as probes for the evaluation of their fluorescent properties and the detection of heavy metal ions. The BCN fluorescence quenching and fluorescence recovery was tested with lead (Pb2+), nickel (Ni2+), copper (Cu2+) and mercury (Hg2+). Principal component analysis (PCA) and discriminant analysis were used to differentiate among heavy metal ions in water samples. The BCN from different feedstocks had different characteristics and produced different interactions with heavy metal ions. DMB-CN had the highest quenching for Hg2+ and Ni2+ while SSB-CN and RSB-CN responded best to Cu2+ and Pb2+, respectively. The fluorescence quenching was modelled using linear and empirical functions. PCA and discriminant analysis used the quenching measurements to differentiate heavy metal ions in aqueous system. A key result was that the discriminant analysis had a 100% accuracy to detect Pb2+, 66% for Ni2+ and Cu2+, and 33% for Hg2+. This study has shown that biochar-derived carbonaceous nanomaterials could be used in heavy metal ions sensing applications. This is the first step in the development of a fast and accurate method for the detection of heavy metal ions in waters using environmentally friendly BCN.

This research seeks a coupled solution for managing the large amounts of biochar produced by microalgae biofuel production, and the necessity for novel, economic and accurate heavy metal sensing methods. Therefore, this study evaluated the transformation of microalgae biochar (MAB) into carbon dots (Cdots) and their subsequent application as heavy metal ion sensors in aqueous systems. The experimental phase included the transformation of MAB into microalgae biochar-derived carbon dots (MAB–Cdots), MAB–Cdot characterisation and the evaluation of the MAB–Cdots as transducers for the detection of four heavy metal ions (Pb2+, Cu2+, Cd2+, and Ni2+). MAB–Cdot fluorescence was stable over a wide range of pH and resistant to photo-bleaching, making them suitable as fluorescence probes. The MAB–Cdot fluorescence was quenched by all of the metal ions and displayed different quenching levels. Depending upon the ions involved, MAB–Cdots were used to detect the presence of heavy metal ions from concentrations of 0.012 μM up to 2 mM by measuring the reduction in fluorescence intensity. Neutral and slightly alkaline pHs were optimal for Cu2+ Ni2+ and Pb2+ heavy metal quenching. To quantify the concentration of the heavy metal ions, linear and logarithmic functions were used to model the MAB–Cdot fluorescence quenching. The sensing mechanism was determined to be reversible and purely collisional with some fluorophores less accessible than the others. This work demonstrated the ability to produce Cdots from microalgae biochar, examined their application as a transducer for detecting heavy metal ions in aqueous systems and paves the way for novel sensing systems using MAB-Cdots.

This article describes an efficient method, combining chemical oxidation and acetone extraction, to produce carbonaceous nanomaterials from dairy manure biochar. The optical and mechanical properties are similar to methods previously reported carbonaceous nanomaterials from biomass. Our novel process cuts the processing time in half and drastically reduces the energy input required. The acetone extraction produced 10 fractions with dairy manure biochar-derived carbonaceous nanomaterials (DMB–CNs). The fraction with the carbonaceous nanomaterials, DMB–CN-E1, with highest fluorescence was selected for in-depth characterisation and for initial testing across a range of applications. DMB–CN-E1 was characterised using atomic force microscope, electrophoresis, and spectrophotometric methods. DMB–CN-E1 exhibited a lateral dimension between 11 and 28 nm, a negative charge, and excitation/emission maxima at 337/410 nm, respectively. The bioimaging potential of DMB–CN-E1 evidenced different locations and different interactions with the cellular models evaluated. DMB–CN-E1 was quenched by several heavy metal ions showing a future application of these materials in heavy metal ion detection and/or removal. The demonstrated capabilities in bioimaging and environmental sensing create the opportunity for generating added-value nanomaterials (NanoRefinery) from dairy manure biochar gasification and, thus, increasing the economic viability of gasification plants.

Alterations in ocular blood flow have been implicated in mechanisms that lead to vision loss in patients with various ocular disorders such as diabetic retinopathy, glaucoma, and age-related macular degeneration. Assessment of retinal and choroidal blood flow is also a window to evaluate systemic diseases that affect microvasculature. Quantification and qualification of the blood flow in the retina and choroid help us understand pathophysiology, stratify disease risk, and monitor disease progression in these disorders. Multiple methods are used by researchers for assessment of blood flow, but a gold standard is lacking. We review commonly used methods, both invasive and noninvasive, for evaluation of blood flow, including intravital microscopy, laser Doppler velocimetry, laser Doppler flowmetry, laser interferometry, confocal scanning laser Doppler flowmetry, laser speckle flowgraphy, Doppler optical coherence tomography, blue-field entoptic simulation, retinal vessel caliber assessment, optical coherence tomography angiography, retinal function imaging, color Doppler imaging, and scanning laser ophthalmoscope angiogram. As technology evolves, better evaluation of blood flow in various ocular and systemic diseases will likely bring new perspectives into clinical practice and translate to better diagnosis and treatment.

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 cell-to-cell transmission is dependent on the release of infectious virus particles into the virological synapse. The HTLV-1 particle structure is still poorly understood, and previous studies analyzed viruses produced by transformed lymphocytic cell lines chronically infected with HTLV-1, particularly the MT-2 cell line, which harbors truncated proviruses and expresses aberrant forms of the Gag protein. In this study, we demonstrate that the chronically infected SP cell line harbors a relatively low number of proviruses, making it a more promising experimental system for the study of the HTLV-1 particle structure. We first identified the genomic sites of integration and characterized the genetic structure of the gag region in each provirus. We also determined that despite encoding a truncated Gag protein, only the full-length Gag protein was incorporated into virus particles. Cryo-transmission electron microscopy analyses of the purified virus particles revealed three classes of particles based upon capsid core morphology: complete cores, incomplete cores, and particles without distinct electron densities that would correlate with the capsid region of a core structure. Observed cores were generally polygonal, and virus particles were on average 115 nm in diameter. These data corroborate particle morphologies previously observed for MT-2 cells and provide evidence that the known poor infectivity of HTLV-1 particles may correlate with HTLV-1 particle populations containing few virus particles possessing a complete capsid core structure. IMPORTANCE Studies of retroviral particle core morphology have demonstrated a correlation between capsid core stability and the relative infectivity of the virus. In this study, we used cryo-transmission electron microscopy to demonstrate that HTLV-1 particles produced from a distinct chronically infected cell line are polymorphic in nature, with many particles lacking organized electron densities that would correlate with a complete core structure. These findings have important implications for infectious HTLV-1 spread, particularly in the context of cell-to-cell transmission, a critical step in HTLV-1 transmission and pathogenesis.

The recombinant protein Ultrabithorax (Ubx), a Drosophila melanogaster Hox transcription factor, self-assembles into biocompatible materials in vitro that are remarkably extensible and strong. Here, we demonstrate that the strength of Ubx materials is due to intermolecular dityrosine bonds. Ubx materials auto-fluoresce blue, a characteristic of dityrosine, and bind dityrosine-specific antibodies. Monitoring the fluorescence of reduced Ubx fibers upon oxygen exposure reveals biphasic bond formation kinetics. Two dityrosine bonds in Ubx were identified by site-directed mutagenesis followed by measurements of fiber fluorescent intensity. One bond is located between the N-terminus and the homeodomain (Y4/Y296 or Y12/Y293), and another bond is formed by Y167 and Y240. Fiber fluorescence closely correlates with fiber strength, demonstrating that these bonds are intermolecular. To our knowledge, this is the first identification of specific residues that participate in dityrosine bonds in protein-based materials. The percentage of Ubx molecules harboring both bonds can be decreased or increased by mutagenesis, providing an additional mechanism to control the mechanical properties of Ubx materials. Duplication of tyrosine-containing motifs in Ubx increases dityrosine content in Ubx fibers, suggesting these motifs could be inserted in other self-assembling proteins to strengthen the corresponding materials.

Quantum Dots (QDs) stabilized with dihydrolipoic acid (DHLA) were used as a template for layer-by-layer (LbL) modification to study the effect on the QD optical properties. We studied several different polyelectrolytes to determine that large quantities of monodisperse DHLA-QDs could only be obtained with the weak polyelectrolyte pair of poly(allylamine hydrochloride) (PAH) and poly(acrylic acid) (PAA). The key to this success was the development of a two-step method to split the LbL process into adsorption and centrifugation phases, which require different pH solutions for optimum success. Solution pH is highlighted as an important factor to achieve sufficient QD surface coverage and QD recovery during wash cycles. We optimized the process to scale up synthesis by introducing a solvent precipitation step before ultracentrifugation that, when coupled with the correct pH conditions, results in a mean QD recovery of 86-90% after three wash cycles. We found that adsorption of PAH had a negligible effect on the quantum yield and lifetime but an additional layer of PAA resulted in a substantial decrease in both quantum yield and lifetime that could not be recovered by the addition of more layers. The PAH coating provides a protective coating that extends DHLA-QDs stability, prevents photo-oxidation mediated aggregation, alleviates concerns over batch variability, and results in pH-dependent emission.

A half-scale three-bay by three-bay steel-concrete composite floor system, which represented gravity framing for a typical commercial building, was studied experimentally to evaluate its structural integrity under four separate column removal scenarios: a corner column, two edge columns, and an interior column. In each test, the load was incrementally applied in the bays that were tributary to the removed column using water in containers that were placed on top of the slab. The tests demonstrated that gravity systems for commercial buildings have a significant level of structural integrity—compared to the load redistribution capability expected for steel framing with simple shear connections—even without specific design against progressive collapse. In the corner and edge column removal scenarios, 2.9 kPa (60 psf) and 4.0 kPa (83 psf) were sustained, respectively, and these loads represent a range of 50–75% of the expected floor load. The interior column removal scenario had an unexpectedly low ca...

The pyrolytic decomposition of layered basic zinc acetate (LBZA) nanobelts (NBs) into nanocrystalline ZnO NBs is investigated using scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS) and photoluminescence (PL). We also report on the gas sensing response of the resulting ZnO nanomaterial to CO. The LBZA NBs are grown at 65 °C in an aqueous solution of zinc acetate dihydrate. AFM and SEM results show as-grown products possess the characteristic layered structure of the LBZA crystals. XRD and XPS results show that annealing as-grown products at 210 °C in air causes a transformation from zinc acetate to nanocrystalline ZnO NBs via thermal decomposition. The ZnO crystalline domain size increases with temperature from 9.2 nm at 200 °C to 94 nm at 1000 °C, as measured from XRD. SEM shows evidence of sintering at 600 °C. The thickness of the NBs, determined via AFM, ranges from 10 to 50 nm and remains approximately constant with annealing temperature. XPS confirmed the chemical transformation from zinc acetate to ZnO and showed a significant remaining zinc hydroxide component for the ZnO NBs consistent with published results. PL measurements at room temperature show a blue shift in peak emission as the nanobelts change from LBZA to ZnO at 200 °C. Above this transition temperature, the ZnO nanobelts possess strong band edge emission at 390 nm and little broad band emission in the visible region. The AFM and SEM images reveal that the crystallites within the nanobelts orientate in rows along the long axis during annealing. This structure provides a high surface area to volume ratio of aligned nanoparticles which is beneficial for gas sensing applications. Gas sensors fabricated from 400 °C annealed nanobelts showed a response of 1.62 when exposed to 200 ppm of CO in dry air at 400 °C, as defined by the ratio of resistance before and during exposure. This indicates that ZnO nanostructures obtained by thermal decomposition of LBZA NBs could provide a cost effective route to high sensitivity gas sensors.

To investigate the mechanism of interaction of gramicidin S-like antimicrobial peptides with biological membranes, a series of five decameric cyclic cationic beta-sheet-beta-turn peptides with all possible combinations of aromatic D-amino acids, Cyclo(Val-Lys-Leu-D-Ar1-Pro-Val-Lys-Leu-D-Ar2-Pro) (Ar identical with Phe, Tyr, Trp), were synthesized. Conformations of these cyclic peptides were comparable in aqueous solutions and lipid vesicles. Isothermal titration calorimetry measurements revealed entropy-driven binding of cyclic peptides to POPC and POPE/POPG lipid vesicles. Binding of peptides to both vesicle systems was endothermic-exceptions were peptides containing the Trp-Trp and Tyr-Trp pairs with exothermic binding to POPC vesicles. Application of one- and two-site binding (partitioning) models to binding isotherms of exothermic and endothermic binding processes, respectively, resulted in determination of peptide-lipid membrane binding constants (K(b)). The K(b1) and K(b2) values for endothermic two-step binding processes corresponded to high and low binding affinities (K(b1) >or= 100 K(b2)). Conformational change of cyclic peptides in transferring from buffer to lipid bilayer surfaces was estimated using fluorescence resonance energy transfer between the Tyr-Trp pair in one of the peptide constructs. The cyclic peptide conformation expands upon adsorption on lipid bilayer surface and interacts more deeply with the outer monolayer causing bilayer deformation, which may lead to formation of nonspecific transient peptide-lipid porelike zones causing membrane lysis.

Keys to successful treatment of disease include early diagnosis and timely treatment. It is hypothesized that early clotting events may contribute to a pro-thrombotic state that exacerbates atherothrombotic vascular disease. Brillouin spectroscopy involves inelastic coupling of light with phonons and enables viscoelastic characterization of samples at the microscale. In this work, we apply Brillouin spectroscopy to a model fibrinogen-thrombin clotting system with the goal of measuring clotting dynamics at the microscale and providing characterization that is not possible with standard rheometric techniques. Here, the clotting dynamics of the model clotting system are measured at various fibrinogen and thrombin concentrations.

Cross-system comparisons of drug delivery vectors are essential to ensure optimal design. An in-vitro experimental protocol is presented that separates the role of the delivery vector from that of its cargo in determining the cell response, thus allowing quantitative comparison of different systems. The technique is validated through benchmarking of the dose–response of human fibroblast cells exposed to the cationic molecule, polyethylene imine (PEI); delivered as a free molecule and as a cargo on the surface of CdSe nanoparticles and Silica microparticles. The exposure metrics are converted to a delivered dose with the transport properties of the different scale systems characterized by a delivery time, τ. The benchmarking highlights an agglomeration of the free PEI molecules into micron sized clusters and identifies the metric determining cell death as the total number of PEI molecules presented to cells, determined by the delivery vector dose and the surface density of the cargo.

The layer-by-layer modification of ≈5 nm mercaptocarboxylic acid stabilized gold nanoparticles was studied in an effort to illustrate effective means to overcome practical issues in handling and performing surface modification of such extremely small materials. To accomplish this, each layer deposition cycle was separated into a multi-step process wherein solution pH was controlled in two distinct phases of polyelectrolyte adsorption and centrifugation. Additionally, a solvent precipitation step was introduced to make processing more amenable by concentrating the sample and exchanging solution pH before ultracentrifugation. The pH-dependent assembly on gold nanoparticles was assessed after each layer deposition cycle by monitoring the plasmon peak absorbance location, surface charge, and the percentage of nanoparticles recovered. The selection of solution pH during the adsorption phase was found to be a critical parameter to enhance particle recovery and maximize surface charge when coating with weak polyelectrolytes. One bilayer was deposited with a high yield and the modified particles exhibited enhanced colloidal stability across a broad pH range and increased ionic strength. These findings support the adoption of this multi-step processing approach as an effective and generalizable approach to improve stability of high surface curvature particles.

The recombinant protein Ultrabithorax (Ubx), a Drosophila melanogaster Hox transcription factor, self-assembles into biocompatible materials in vitro that are remarkably extensible and strong. Here, we demonstrate that the strength of Ubx materials is due to intermolecular dityrosine bonds. Ubx materials auto-fluoresce blue, a characteristic of dityrosine, and bind dityrosine-specific antibodies. Monitoring the fluorescence of reduced Ubx fibers upon oxygen exposure reveals biphasic bond formation kinetics. Two dityrosine bonds in Ubx were identified by site-directed mutagenesis followed by measurements of fiber fluorescent intensity. One bond is located between the N-terminus and the homeodomain (Y4/Y296 or Y12/Y293), and another bond is formed by Y167 and Y240. Fiber fluorescence closely correlates with fiber strength, demonstrating that these bonds are intermolecular. To our knowledge, this is the first identification of specific residues that participate in dityrosine bonds in protein-based materials. The percentage of Ubx molecules harboring both bonds can be decreased or increased by mutagenesis, providing an additional mechanism to control the mechanical properties of Ubx materials. Duplication of tyrosine-containing motifs in Ubx increases dityrosine content in Ubx fibers, suggesting these motifs could be inserted in other self-assembling proteins to strengthen the corresponding materials.

The extensive use of nanomaterials in new products for the building envelope is expanding rapidly and continues to influence materials science and environmental health. However, there is still uncertainly around the safety of these novel materials to humans and the environment; therefore, more life cycle data is needed. One analytical technique to gather information critical to the development of safe nanomaterials is to characterize the physicochemical properties of the nano-enabled product along various stages of the product life cycle. This research investigates two different products fortified with nanoscale titania relevant to the building and construction industry: (1) a white paint and (2) a lacquer formulation. Three brands of commercially available titanium dioxide (TiO2) nanoparticles were incorporated into labformulated paint and lacquer mixtures. These fortified products were subjected to simulated degradation processes (a.k.a. “wear-and-tear” scenarios) to mimic the real world. Unincorporated TiO2, as well as TiO2 incorporated “wornand- torn” powders, were processed and analyzed for physical, chemical, and toxicological characteristics. Electron microscopy confirmed the presence of nanoscale TiO2 particles in the painted and lacquered matrices for all three nanoparticle-types. Cell viability and reactive oxygen species (ROS) generation was measured in immortalized human lung epithelial cells (A549). Results from bioassays showed that ROS generation between unincorporated TiO2 particles and TiO2 incorporated paint powders showed little difference, but, the TiO2 incorporated lacquered powders demonstrated significantly lower ROS generation and cell death. Thus, the polymer-based lacquer mitigated tissue damage. Our results show that using a life cycle approach may help provide data – and by extension, an understanding - of nanomaterial properties in the context of developed consumer products.

Erythrocyte abundance, mobility, and carrying capacity make them attractive as a platform for blood analyte sensing as well as for drug delivery. Sensor-loaded erythrocytes, dubbed erythrosensors, could be reinfused into the bloodstream, excited noninvasively through the skin, and used to provide measurement of analyte levels in the bloodstream. Several techniques to load erythrocytes, thus creating carrier erythrocytes, exist. However, their cellular characteristics remain largely unstudied. Changes in cellular characteristics lead to removal from the bloodstream. We hypothesize that erythrosensors need to maintain native erythrocytes’ (NEs) characteristics to serve as a long-term sensing platform. Here, we investigate two loading techniques and the properties of the resulting erythrosensors. For loading, hypotonic dilution requires a hypotonic solution while electroporation relies on electrical pulses to perforate the erythrocyte membrane. We analyze the resulting erythrosensor signal, size, morphology, and hemoglobin content. Although the resulting erythrosensors exhibit morphological changes, their size was comparable with NEs. The hypotonic dilution technique was found to load erythrosensors much more efficiently than electroporation, and the sensors were loaded throughout the volume of the erythrosensors. Finally, both techniques resulted in significant loss of hemoglobin. This study points to the need for continued development of loading techniques that better preserve NE characteristics.

Understanding the effect of variability in the interaction of individual cells with nanoparticles on the overall response of the cell population to a nanoagent is a fundamental challenge in bionanotechnology. Here, we show that the technique of time-resolved, high-throughput microscopy can be used in this endeavor. Mass measurement with single-cell resolution provides statistically robust assessments of cell heterogeneity, while the addition of a temporal element allows assessment of separate processes leading to deconvolution of the effects of particle supply and biological response. We provide a specific demonstration of the approach, in vitro, through time-resolved measurement of fibroblast cell (HFF-1) death caused by exposure to cationic nanoparticles. The results show that heterogeneity in cell area is the major source of variability with area-dependent nanoparticle capture rates determining the time of cell death and hence the form of the exposure–response characteristic. Moreover, due to the particulate nature of the nanoparticle suspension, there is a reduction in the particle concentration over the course of the experiment, eventually causing saturation in the level of measured biological outcome. A generalized mathematical description of the system is proposed, based on a simple model of particle depletion from a finite supply reservoir. This captures the essential aspects of the nanoparticle–cell interaction dynamics and accurately predicts the population exposure–response curves from individual cell heterogeneity distributions.

For diabetics, continuous glucose monitoring and the resulting tighter control of glucose levels ameliorate serious complications from hypoglycemia and hyperglycemia. Diabetics measure their blood glucose levels multiple times a day by finger pricks, or use implantable monitoring devices. Still, glucose and other analytes in the blood fluctuate throughout the day and the current monitoring methods are invasive, immunogenic, and/or present biodegradation problems. Using carrier erythrocytes loaded with a fluorescent sensor, we seek to develop a biodegradable, efficient, and potentially cost effective method to continuously sense blood analytes. We aim to reintroduce sensor-loaded erythrocytes to the bloodstream and conserve the erythrocytes lifetime of 120 days in the circulatory system. Here, we compare the efficiency of two loading techniques: hypotonic dilution and electroporation. Hypotonic dilution employs hypotonic buffer to create transient pores in the erythrocyte membrane, allowing dye entrance and a hypertonic buffer to restore tonicity. Electroporation relies on controlled electrical pulses that results in reversible pores formation to allow cargo entrance, follow by incubation at 37°C to reseal. As part of the cellular characterization of loaded erythrocytes, we focus on cell size, shape, and hemoglobin content. Cell recovery, loading efficiency and cargo release measurements render optimal loading conditions. The detected fluorescent signal from sensor-loaded erythrocytes can be translated into a direct measurement of analyte levels in the blood stream. The development of a suitable protocol to engineer carrier erythrocytes has profound and lasting implications in the erythrosensor’s lifespan and sensing capabilities.

Zum Thema Die nicht-invasive Beatmung (NIV) über eine Maske ermöglicht bei vielen Patienten eine effiziente Beatmung unter Vermeidung der Risiken und Nebenwirkungen der endotrachealen Intubation. Für die chronisch-obstruktive Lungenerkrankung (COPD) sind die Vorteile der Maskenbeatmung im Hinblick auf Beatmungsdauer, Dauer der Intensivbehandlung, Prognose und Behandlungskosten inzwischen relativ gut durch Studien belegt. Bei anderen Indikationen –“Nicht-COPD-Patienten”– erlauben die verfügbaren Daten noch kein gesichertes Urteil, rechtfertigen aber einen Behandlungsversuch auch außerhalb von Studien in klinischer Routine. Nachteilig sind der initial höhere personelle Betreuungsaufwand bei NIV sowie die stark von der Erfahrung abhängige Erfolgsrate. Die wichtigste Voraussetzung für eine erfolgreiche nicht-invasive Beatmung in der Intensivmedizin ist die Bereitschaft von Ärzten und Pflegepersonal, eine ungewohnte Beatmungsphilosophie zu akzeptieren und die relativ einfache Technik der Maskenbeatmung zu erlernen. Bei Beherrschung der Technik kann die NIV aber eine wertvolle Hilfe vom Notarztwagen bis zur Intensivstation sein.

Unfavourable cycling conditions may successively lead to a porous PbO 2 structure with poor mechanical and electrical connection between the particles of which the porous electrode is built. Increasing active material resistance of this skeleton will induce a more and more inhomogeneous current distribution over the active mass, with successive restriction of the discharge/recharge processes to the active material area near to the grid. This leads to excessive mass utilisation in this very region, which aggravates the situation from cycle to cycle. The results are active material softening and shedding. This change of PbO 2 structure can be reverted, when cycling is continued with modified regimes. By this and by other means, lost capacity may be recovered. This effect is called reversible capacity decay or reversible insufficient mass utilization (RIMU). The Aggregate-of-Spheres Model (AOS), based on the behaviour of sintered bodies, describes semi-quantitatively the phenomena at the connecting sites of the particles of the PbO 2 active material, explaining the experimental findings of reversible capacity decay. The main features of reversible capacity loss are discussed in terms of AOS explanations, in order to work out strategies and limits for the improvement of lead/acid battery performance.

We have developed a low-cost technique using a conventional microwave oven to grow layered basic zinc acetate (LBZA) nanosheets (NSs) from a zinc acetate, zinc nitrate and HMTA solution in only 2 min. The as-grown crystals and their pyrolytic decomposition into ZnO nanocrystalline NSs are characterized by scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDS), atomic force microscopy (AFM), X-ray diffraction (XRD) and photoluminescence (PL). SEM and AFM measurements show that the LBZA NSs have typical lateral dimensions of 1 to 5 μm and thickness of 20 to 100 nm. Annealing in air from 200°C to 1,000°C results in the formation of ZnO nanocrystalline NSs, with a nanocrystallite size ranging from 16 nm at 200°C to 104 nm at 1,000°C, as determined by SEM. SEM shows evidence of sintering at 600°C. PL shows that the shape of the visible band is greatly affected by the annealing temperature and that the exciton band to defect band intensity ratio is maximum at 400°C and decreases by a factor of 15 after annealing at 600°C. The shape and thickness of the ZnO nanocrystalline NSs are the same as LBZA NSs. This structure provides a high surface-to-volume ratio of interconnected nanoparticles that is favorable for applications requiring high specific area and low resistivity such as gas sensing and dye-sensitized solar cells (DSCs). We show that resistive gas sensors fabricated with the ZnO NSs showed a response of 1.12 and 1.65 to 12.5 ppm and 200 ppm of CO at 350°C in dry air, respectively, and that DSCs also fabricated from the material had an overall efficiency of 1.3%. PACS 81.07.-b; 62.23.Kn; 61.82.Fk

Quantum Dot (QD)/microsphere structures supporting Whispering Gallery Modes (WGMs) are attached to Atomic Force Microscope (AFM) cantilevers for characterization of the evanescent field around the QD/microsphere and utilization of the evanescent field for sensing at the apical surface of live cells. Following laser excitation, QD emission couples to WGMs that circumnavigate the microsphere via total internal reflection at the internal surfaces of the microsphere. The resulting evanescent field is characterized utilizing the high spatial control of an AFM in approaching a dye monolayer on a test surface. The measured evanescent field extends approximately 50 nm from the microsphere surface, matching theoretical predictions. This system was then used to sense the accumulation of integrin and formation of focal adhesions at the apical surface of cells.

Measurement of blood analytes provides crucial information about a patient’s health. Some such analytes, such as glucose in the case of diabetes, require long-term or near-continuous monitoring for proper disease management. However, current monitoring techniques are far from ideal: multiple-per-day finger stick tests are inconvenient and painful for the patient; implantable sensors have short functional life spans (i.e., 3-7 days). Due to analyte transporters on red blood cell (RBC) membranes that equilibrate intracellular and extracellular analyte levels, RBCs serve as an attractive alternative for encapsulating analyte sensors. Once reintroduced to the blood stream, the functionalized RBCs may continue to live for the remainder of their life span (120 days for humans). They are biodegradable and biocompatible, thereby eliminating the immune system response common for many implanted devices. The proposed sensing system utilizes the ability of the RBCs to swell in response to a decrease in the osmolarity of the extracellular solution. Just before lysis, they develop small pores on the scale of tens of nanometers. While at low temperature, analyte-sensitive dyes in the extracellular solution diffuse into the perforated RBCs and become entrapped upon restoration of temperature and osmolarity. Since the fluorescent signal from the entrapped dye reports on changes in the analyte level of the extracellular solution via the RBC transporters, interactions between the RBCs and the dye are critical to the efficacy of this technique. In this work, we study the use of a near infrared pH sensitive dye encapsulated within RBCs and assess the ability to measure dye fluorescence in vivo .

The influence of the addition of phosphoric acid to the electrolyte on the performance of gelled lead/acid electric-vehiicle batteries is investigated. This additive reduces the reversible capacity decay of the positive electrode significantly which is observed upon extended cycling when recharge of the battery is performed at low initial rate. This is important when low-rate on-board chargers are used. Pulsed discharge, typical for electric-vehicle application, induces reversible capacity decay more than constant-current discharge at a same depth-of-discharge, as well with as without the addition of phosphoric acid. By contrast, hindrance in presence of H3PO4 for both the recharge and the discharge reaction helps to homogenize the state of many individual cells during cycling in long battery strings. Reversible capacity loss, which occurs after extended cycling and when pulsed discharge is applied, can be recovered by a single discharge at very low rate with batteries with and without the addition of phosphoric acid. The discharge-rate dependency of the capacity is significantly reduced when phosphoric acid is added. The pulse discharge behaviour may be better, even if the nominal capacity is reduced. The experimental findings of the influence of phosphoric acid addition is discussed in terms of the aggregate-of-spheres model of reversible capacity decay.

We present a novel method for colloidal synthesis of one-dimensional ZnO nanopods by heterogeneous nucleation on zero-dimensional ZnO nanoparticle ‘seeds’. Ultra-small ZnO nanopods, multi-legged structures with sub-20 nm individual leg diameters, can be synthesized by hydrolysis of a Zn2+ precursor growth solution in presence of ∼4 nm ZnO seeds under hydrothermal conditions via microwave-assisted heating in as little as 20 min of reaction time. One-dimensional ZnO nanorods are initially generated in the reaction mixture by heterogeneous nucleation and growth along the [0001] direction of the ZnO crystal. Growth of one-dimensional nanorods subsequently yields to an ‘attachment’ and size-focusing phase where individual nanorods fuse together to form multi-legged nanopods having diameters ∼15 nm. ZnO nanopods exhibit broad orange-red defect-related photoluminescence in addition to a near-band edge emission at 373 nm when excited above the band-gap at 350 nm. The defect-related photoluminescence of the ZnO nanopods has been applied towards reversible optical humidity sensing at room temperature. The sensors demonstrated a linear response between 22% and 70% relative humidity with a 0.4% increase in optical intensity per % change in relative humidity. Due to their ultra-small dimensions, ZnO nanopods exhibit a large dynamic range and enhanced sensitivity to changes in ambient humidity, thus showcasing their ability as a platform for optical environmental sensing.

In this report, a technique for rapid synthesis of ZnO microstructures by microwave-assisted heating of precursors at hydrothermal conditions is demonstrated. Further, the reaction mechanism for the growth of ZnO microstructures is analyzed. An accelerated rate of reaction obtained using microwaves enables a dissolution-recrystallization mechanism for generation of one dimensional (1D) rod-like structures, thereby showing that time of reaction can be used to dictate ZnO microstructure morphology.

The pyrolytic decomposition of layered basic zinc acetate (LBZA) nanobelts (NBs) and nanosheets (NSs) into nanocrystalline ZnO NBs/NSs has been investigated using scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray diffraction (XRD) and Photoluminescence (PL). We also report on the gas sensing response of the resulting ZnO nanomaterials to CO. The LBZA NBs were grown at 65°C for 9h in an aqueous solution of zinc acetate dehydrate and the LBZA NSs were grown in 2min in a standard microwave oven. AFM, SEM and XRD showed as-grown products possess the characteristic layered structure of LBZA crystals. XRD results showed that annealing as-grown products a 400°C in air caused a transformation from zinc acetate to nanocrystalline ZnO via thermal decomposition and that the crystalline domain size increased with temperature. The thickness of the NBs and NSs, determined via AFM, ranged from 10 to 50 nm. PL shows a larger defect band for ZnO NBs, compared to NSs. Gas sensors fabricated from 400°C annealed NBs showed a response of 1.62 when exposed to 200ppm of CO in dry air at 400°C. The same response was observed at 325°C for the NSs, which also had better response at lower concentrations. Our results indicate that ZnO nanostructures obtained by thermal decomposition of LBZA NBs/NSs could provide a cost effective route to high sensitivity gas sensors.

Error-free demultiplexing of 40 Gbit/s channels out of a 160 Gbit/s optical time-division signal is demonstrated using four-wave mixing in a semiconductor optical amplifier.

When CdSe/ZnS-polyethyleneimine (PEI) quantum dots (QDs) are microencapsulated in polymeric microcapsules, human fibroblasts are protected from acute cytotoxic effects. Differences in cellular morphology, uptake, and viability were assessed after treatment with either microencapsulated or unencapsulated dots. Specifically, QDs contained in microcapsules terminated with polyethylene glycol (PEG) mitigate contact with and uptake by cells, thus providing a tool to retain particle luminescence for applications such as extracellular sensing and imaging. The microcapsule serves as the ‘‘first line of defense’’ for containing the QDs. This enables the individual QD coating to be designed primarily to enhance the function of the biosensor.

We investigate the use of energy delivery using microwave radiation for both synthesis of nanoparticles as well as a hybrid imaging technique known as thermoacoustic tomography (TAT). In each instance, the absorption of microwave radiation is converted into heat. In the case of nanoparticle synthesis, water is used as the solvent and heated to induce synthesis of the nanostructures. For this aqueous synthesis technique, we demonstrate the use of both pulsed and continuous wave (CW) microwave systems operating at 2.45 GHz. In this report, we concentrate on ZnO nanostructures including nanorods, nanowire arrays and nanobelts. These are compared with nanowire arrays and nanobelts grown by vapor transport through both electron microscopy and photo-excited luminescence. We also review the use of iron oxide (Fe3O4) nanoparticles as contrast agents in TAT as previously reported. Here, we measured the properties of the colloidal nanoparticles in the microwave regime and compared the absorption with the TAT signal produced by our thermoacoustic imaging system at 3 GHz. The nanoparticles directly absorb the microwave radiation and produce a thermo-acoustic signal. The results from nanoparticles are compared to the signal produced by deionized water. The results demonstrate that microwaves represent an efficient method for the delivery of energy for both synthesis and biomedical imaging.

Incorporation of nanoparticles during the hierarchical self-assembly of protein-based materials can impart function to the resulting composite materials. Herein we demonstrate that the structure and nanoparticle distribution of composite fibers are sensitive to the method of nanoparticle addition and the physicochemical properties of both the nanoparticle and the protein. Our model system consists of a recombinant enhanced green fluorescent protein-Ultrabithorax (EGFP-Ubx) fusion protein and luminescent CdSe-ZnS core-shell quantum dots (QDs), allowing us to optically assess the distribution of both the protein and nanoparticle components within the composite material. Although QDs favorably interact with EGFP-Ubx monomers, the relatively rough surface morphology of composite fibers suggests EGFP-Ubx-QD conjugates impact self-assembly. Indeed, QDs templated onto EGFP-Ubx film post-self-assembly can be subsequently drawn into smooth composite fibers. Additionally, the QD surface charge impacts QD distribution within the composite material, indicating that surface charge plays an important role in self-assembly. QDs with either positively or negatively charged coatings significantly enhance fiber extensibility. Conversely, QDs coated with hydrophobic moieties and suspended in toluene produce composite fibers with a heterogeneous distribution of QDs and severely altered fiber morphology, indicating that toluene severely disrupts Ubx self-assembly. Understanding factors that impact the protein-nanoparticle interaction enables manipulation of the structure and mechanical properties of composite materials. Since proteins interact with nanoparticle surface coatings, these results should be applicable to other types of nanoparticles with similar chemical groups on the surface.

Various organisms can sense light through a large family of signaling proteins known as photoreceptors. Upon absorption of a photon in the appropriate wavelength range, photoreceptors undergo structural changes in the chromophore, an organic pigment embedded in the photosensory module of the protein. Phytochromes are red-light photoreceptors originally discovered in plants and more recently in bacteria. They are unique in their ability to undergo reversible photoconversion between two photoisomerizable states, Pr (red light ∼ 700 nm) and Pfr (far-red light ∼ 750 nm). In the photosynthetic bacterium, Rhodopseudomonas palustris, a pair of bacteriophytochromes, RpBphP2 (P2) and RpBphP3 (P3) modulate synthesis of a light-harvesting complex. P2 and P3 have the same biliverdin chromophore (BV) and share 52% amino acid sequence identity, yet they have distinct photoconversion properties. P2, similar to classical bacteriophytochromes, alternates between Pr and Pfr states. P3 is unusual since it alternates between Pr and a unique Pnr (near-red light ∼ 650 nm) state. Our experimental goal is to identify amino acid residues that form specific interactions with BV during photoconversion and as such play key roles in forming distinct photoisomerizable states of P2 and P3. Through site-directed mutagenesis of conserved amino acids informed by structural and sequence analysis of bacteriophytochromes, we created a single-amino acid mutant variant of P3 T480P that undergoes classic reversible photoconversion between Pr and Pfr states. We also report on P2 and P3 mutants that show minimal absorption in the red region of visible light spectrum or are naturally more fluorescent than wild-type proteins. Currently, we are investigating chemical mechanisms that justify observed P2 and P3 mutant phenotypes.

Resonant coherent lattice vibrations in the vicinity of the indirect absorption edge in a ${\mathrm{BiI}}_{3}$ layered crystal are observed in a femtosecond pump-probe experiment. Coherent optical phonons that are impulsively excited by femtosecond pump pulses modulate the phase of probe pulses, causing oscillation of the probe spectrum in time. This oscillation, which has a period given by the period of the oscillation, continues for more than 100 cycles. We speculate that the coherent phonon-assisted indirect exciton transitions also contribute to the data.

Two-photon-excited fluorescence and second-harmonic generation are characterized as a function of laser pulse duration as short as sub-10-fs. A comparative study is performed where pulse duration is varied by introducing dispersion, as reported previously, and by tailoring pulse spectral width and minimizing its time-bandwidth product (transform-limited pulses). Experimental data and calculations show that characterizing a two-photon signal with the two schemes to vary pulse duration measures different phenomena. Two-photon signal characterization using dispersion-broadened pulses measures only the effect of chirp on the pulse two-photon-excitation spectrum and is independent of molecular response. Transform-limited pulses are used to measure the dependence of two-photon signal generation on pulse duration. Calculations show that deviation from the 1/T p relationship would be expected as the transform-limited pulse spectral width approaches the molecular two-photon absorption linewidth and exhibits asymptotic behavior for pulse spectral widths 10 times greater than the absorption linewidth. Experimental measurements are consistent with the predicted behavior. The impact of using ultrashort laser pulses on the performance characteristics of nonlinear optical microscopy is discussed.