Institute for Collaborative Biotechnologies

Abalone shell nacre proteins act as surfactants to promote ion attachment at calcite steps, causing acceleration of the molecular-scale kinetics of calcite crystal growth. The proteins modify the shape of growing calcite (see Figure) through step-specific interactions, even though the proteins are larger than the atomic-scale steps. Understanding of crystal-growth control by interactions with proteins may give better control of new crystalline materials.

We report a general combinatorial approach to identify optimal substrates of a given protease by using quantitative kinetic screening of cellular libraries of peptide substrates (CLiPS). A whole-cell protease activity assay was developed by displaying fluorescent reporter substrates on the surface of Escherichia coli as N-terminal fusions. This approach enabled generation of substrate libraries of arbitrary amino acid composition and length that are self-renewing. Substrate hydrolysis by a target protease was measured quantitatively via changes in whole-cell fluorescence by using FACS. FACS enabled efficient screening to identify optimal substrates for a given protease and characterize their cleavage kinetics. The utility of CLiPS was demonstrated by determining the substrate specificity of two unrelated proteases, caspase-3 and enteropeptidase (or enterokinase). CLiPS unambiguously identified the caspase-3 consensus cleavage sequence DXVDG. Enteropeptidase was unexpectedly promiscuous, but exhibited a preference for substrates with the motif (D/E)RM, which were cleaved substantially faster than the canonical DDDDK recognition sequence, widely used for protein purification. CLiPS provides a straightforward and versatile approach to determine protease specificity and discover optimal substrates on the basis of cleavage kinetics.

Bacterial cell-surface display systems coupled with quantitative screening methods offer the potential to expand protein engineering capabilities. To more fully exploit this potential, a unique bacterial surface display scaffold was engineered to display peptides more efficiently from the surface exposed C- and N-termini of a circularly permuted outer membrane protein. Using directed evolution, efficient membrane localization of a circularly permuted OmpX (CPX) display scaffold was rescued, thereby improving the presentation of diverse passenger peptides on the cell surface. Random and targeted mutagenesis directed towards linkers joining the native N- and C-termini of OmpX coupled with screening by FACS yielded an enhanced CPX (eCPX) variant which localized to the outer membrane as efficiently as the non-permuted parent. Interestingly, enhancing substitutions coincided with a C-terminal motif conserved in outer membrane proteins. Surface localization of various passenger peptides and mini-proteins was expedited using eCPX relative to that achieved with the parent scaffold. The new variant also permitted simultaneous display and labeling of distinct peptides on structurally adjacent C- and N-termini, thus enabling display level normalization during library screening and the display of bidentate or dimeric peptides. Consequently, the evolved scaffold, eCPX, expands the range of applications for bacterial display. Finally, this approach provides a route to improve the performance of cell-surface display vectors for protein engineering and design.

The recent discovery and characterization of silicatein, a mineral-synthesizing enzyme that assembles to form the filamentous organic core of the glassy skeletal elements (spicules) of a marine sponge, has led to the development of new low-temperature synthetic routes to metastable semiconducting metal oxides. These protein filaments were shown in vitro to catalyze the hydrolysis and structurally direct the polycondensation of metal oxides at neutral pH and low temperature. Based on the confirmation of the catalytic mechanism and the essential participation of specific serine and histidine residues (presenting a nucleophilic hydroxyl and a nucleophilicity-enhancing hydrogen-bonding imidazole nitrogen) in silicatein's catalytic active site, we therefore sought to develop a synthetic mimic that provides both catalysis and the surface determinants necessary to template and structurally direct heterogeneous nucleation through condensation. Using lithographically patterned poly(dimethylsiloxane) stamps, bifunctional self-assembled monolayer surfaces containing the essential catalytic and templating elements were fabricated by using alkane thiols microcontact-printed on gold substrates. The interface between chemically distinct self-assembled monolayer domains provided the necessary juxtaposition of nucleophilic (hydroxyl) and hydrogen-bonding (imidazole) agents to catalyze the hydrolysis of a gallium oxide precursor and template the condensed product to form gallium oxohydroxide (GaOOH) and the defect spinel, gamma-gallium oxide (gamma-Ga(2)O(3)). Using this approach, the production of patterned substrates for catalytic synthesis and templating of semiconductors for device applications can be envisioned.

We demonstrate a method for high-bandwidth, high-sensitivity particle sensing and cell counting in a microfluidic system. Our approach employs a tuned radiofrequency probe, which forms the radiofrequency analog of a Coulter counter. By measuring the reflected rf power, this approach provides an unprecedented detection rate, with a theoretical bandwidth in excess of 10 MHz. Particle detection was performed in a continuous flow mode in a microfluidic channel, using 15μm diameter polystyrene beads suspended in a sucrose-saline solution. We demonstrate 30 kHz counting rates and show high-resolution bead time-of-flight data, comprising the fastest electronic particle detection on-chip to date.

The mechanisms of formation of biogenic magnesium-rich calcite remain an enigma. Here we present ultrastructural and compositional details of ossicles from the seastar Pisaster giganteus (Echinodermata, Asteroidea). Powder X-ray diffraction, infrared spectroscopy and elemental analyses confirm that the ossicles are composed of magnesium-rich calcite, whilst also containing about 0.01 % (w/w) of soluble organic matrix (SOM) as an intracrystalline component. Amino acid analysis and N-terminal sequencing revealed that this mixture of intracrystalline macromolecules consists predominantly of glycine-rich polypeptides. In vitro calcium carbonate precipitation experiments indicate that the SOM accelerates the conversion of amorphous calcium carbonate (ACC) into its final crystalline product. From this observation and from the discovery of ACC in other closely related taxa, it is suggested that substitution of magnesium into the calcite lattice through a transient precursor phase may be a universal phenomenon prevalent across the phylum echinodermata.

Moral Foundations Theory (MFT) and the Model of Intuitive Morality and Exemplars (MIME) contend that moral judgments are built on a universal set of basic moral intuitions. A large body of research has supported many of MFT’s and the MIME’s central hypotheses. Yet, an important prerequisite of this research—the ability to extract latent moral content represented in media stimuli with a reliable procedure—has not been systematically studied. In this article, we subject different extraction procedures to rigorous tests, underscore challenges by identifying a range of reliabilities, develop new reliability test and coding procedures employing computational methods, and provide solutions that maximize the reliability and validity of moral intuition extraction. In six content analytical studies, including a large crowd-based study, we demonstrate that: (1) traditional content analytical approaches lead to rather low reliabilities; (2) variation in coding reliabilities can be predicted by both text features and characteristics of the human coders; and (3) reliability is largely unaffected by the detail of coder training. We show that a coding task with simplified training and a coding technique that treats moral foundations as fast, spontaneous intuitions leads to acceptable inter-rater agreement, and potentially to more valid moral intuition extractions. While this study was motivated by issues related to MFT and MIME research, the methods and findings in this study have implications for extracting latent content from text narratives that go beyond moral information. Accordingly, we provide a tool for researchers interested in applying this new approach in their own work.

The way nature evolves and sculpts materials using proteins inspires new approaches to materials engineering but is still not completely understood. Here, we present a cell-free synthetic biological platform to advance studies of biologically synthesized solid-state materials. This platform is capable of simultaneously exerting many of the hierarchical levels of control found in natural biomineralization, including genetic, chemical, spatial, structural, and morphological control, while supporting the evolutionary selection of new mineralizing proteins and the corresponding genetically encoded materials that they produce. DNA-directed protein expression and enzymatic mineralization occur on polystyrene microbeads in water-in-oil emulsions, yielding synthetic surrogates of biomineralizing cells that are then screened by flow sorting, with light-scattering signals used to sort the resulting mineralized composites differentially. We demonstrate the utility of this platform by evolutionarily selecting newly identified silicateins, biomineralizing enzymes previously identified from the silica skeleton of a marine sponge, for enzyme variants capable of synthesizing silicon dioxide (silica) or titanium dioxide (titania) composites. Mineral composites of intermediate strength are preferentially selected to remain intact for identification during cell sorting, and then to collapse postsorting to expose the encoding genes for enzymatic DNA amplification. Some of the newly selected silicatein variants catalyze the formation of crystalline silicates, whereas the parent silicateins lack this ability. The demonstrated bioengineered route to previously undescribed materials introduces in vitro enzyme selection as a viable strategy for mimicking genetic evolution of materials as it occurs in nature.

PREVIOUS STUDIES HAVE EXAMINED THE INFLUENCE OF MEDITATION ON THREE FUNCTIONALLY DIFFERENT COMPONENTS OF ATTENTION: executive control, alerting, and orienting. These studies have consistently found that meditation training improves both executive attention and alerting, but there has not been a consistent and clear effect of meditation training on orienting. In addition, while previous studies have shown that the functional coupling of the alerting and executive networks increases the processing of task irrelevant stimuli, it is unknown if participating in a meditation retreat can decouple these components of attention and lead to improved performance. The current study investigated the influence of a week-long intensive meditation retreat on three components of attention by randomly assigning participants to either pre- or postretreat testing groups. A modified attention network test (ANT) was used. Executive attention was measured as the difference in response time (RT) between congruent and incongruent task irrelevant flankers (conflict effect). Reflexive and volitional orienting were measured by manipulating cue validity and stimulus onset asynchrony (SOA). The coupling of executive attention and alerting was measured by examining flanker interference as a function of the SOA of an alerting cue. The meditation retreat improved task based indices of executive attention, but not reflexive or volitional orienting. There was clear behavioral evidence of coupling between executive attention and alerting in the preretreat group, as the conflict effect peaked when an alerting cue was presented 300 ms before the target. Importantly, there was no increase in the conflict effect for the postretreat group. This is consistent with the notion that the retreat decoupled the executive and alerting networks. These results suggest that previously reported improvements in the executive and alerting networks after meditation training might be mediated by the same underlying mechanism.

Surface-immobilized, densely packed gold nanoparticles in contact with aqueous silver ions and exposed to red light rapidly photoreduce silver ions in solution producing radially symmetric metal deposits with diameters many times larger than the diameter of the illuminating laser beam. The average particle sizes in the deposit increase with radial distance from the center of the deposit. This reduction-at-a-distance effect arises from surface-plasmon-mediated photoemission, with the photoemitted electrons conducting along percolating silver pathways, reducing silver ions along these conducting channels and especially at their periphery, thereby propagating the effect of the illuminating laser outward.

Highly efficient bacterial removal and disinfection of drinking water by recyclable magnetic barium phosphate nanoflakes with embedded iron oxide nanoparticles.

Graded-index, moth eye-inspired anti-reflective features were fabricated in ZnS and ZnSe via nanosphere lithography using a Langmuir–Blodgett dip-coating method with plasma-based mask reduction and pattern transfer. Arrays of hexagonally close-packed conical frusta (top diameter = 300 nm, pitch = 690 nm, height = 2800 nm) were realized by isotropic etching (size-reduction) of the colloidal mask with CF4/Ar, followed by pattern transfer into the substrate using CH4/H2 plasma etching. Substantial increases in broadband transmission were achieved across the 2–20 μm range, yielding 23% and 26% single-side transmission improvement, and 92% and 88% absolute double-side transmission for ZnS and ZnSe, respectively, in excellent agreement with finite difference time domain (FDTD) optical simulations. Experimental differences in direct versus total transmission, and the general fall off of transmission at short wavelengths, were attributed to diffuse forward scattering and diffractive effects, as predicted by far-field scattering patterns using FDTD. The fabrication method presented can be used to enhance efficiency for multiple IR applications by minimizing reflective losses, while offering the further advantages of scalability and low cost.

<bold>Purpose</bold>: With three-dimensional (3-D) images displayed as stacks of 2-D images, radiologists rely more heavily on vision away from their fixation point to visually process information, guide eye movements, and detect abnormalities. Thus the ability to detect targets away from the fixation point, commonly characterized as the useful field of view (UFOV), becomes critical for these 3-D imaging modalities. We investigate how the UFOV, defined as the eccentricity, in which detection performance degrades to a given probability, varies across imaging modalities and targets. <bold>Approach</bold>: We measure the detectability of different targets at various distances from gaze locations for single slices of liver computed tomography (CT), 2-D digital mammograms (DM), and single slices of digital breast tomosynthesis (DBT) cases. Observers with varying expertise were instructed to maintain their gaze at a point while a short display of the image was flashed and an eye tracker verified observer’s steady fixation. Display times were 200 and 1000 ms for CT images and 500 ms for DM and DBT images. <bold>Results</bold>: We find variations in the UFOV from 9 to 12 deg for liver CT to as small as 2.5 to 5 deg for calcification clusters in breast images (DM and DBT). We compare our results to those reported in the literature for lung nodules and discuss the differences across methods used to measure the UFOV, their dependence on case selection/task difficulty, viewing conditions, and observer expertise. We propose a complementary measure defined in terms of performance degradation relative to the peak foveal performance (relative-UFOV) to circumvent UFOV’s variations with case selection/task difficulty. <bold>Conclusion</bold>: Our results highlight the variations in the UFOV across imaging modalities, target types, observer expertise, and measurement methods and suggest an additional relative-UFOV measure to more thoroughly characterize the detection performance away from point of fixation.

Anthropomorphic model observers are mathe- matical algorithms which are applied to images with the ultimate goal of predicting human signal detection and classification accuracy across varieties of backgrounds, image acquisitions and display conditions. A limitation of current channelized model observers is their inability to handle irregularly-shaped signals, which are common in clinical images, without a high number of directional channels. Here, we derive a new linear model observer based on convolution channels which we refer to as the "Filtered Channel observer" (FCO), as an extension of the channelized Hotelling observer (CHO) and the nonprewhitening with an eye filter (NPWE) observer. In analogy to the CHO, this linear model observer can take the form of a single template with an external noise term. To compare with human observers, we tested signals with irregular and asymmetrical shapes spanning the size of lesions down to those of microcalfications in 4-AFC breast tomosynthesis detection tasks, with three different contrasts for each case. Whereas humans uniformly outperformed conventional CHOs, the FCO observer outperformed humans for every signal with only one exception. Additive internal noise in the models allowed us to degrade model performance and match human performance. We could not match all the human performances with a model with a single internal noise component for all signal shape, size and contrast conditions. This suggests that either the internal noise might vary across signals or that the model cannot entirely capture the human detection strategy. However, the FCO model offers an efficient way to apprehend human observer performance for a non-symmetric signal.

Understanding the actions performed by other people is a key aspect of social interaction, including in clinical settings where patients are learning from therapists and caregivers. While lesions of the left cerebral hemisphere induce praxic disorders, the hemispheric specialisation of intention understanding remains unclear. Do patients with a right hemispheric lesion understand the intentions of other people properly? The present study investigates how a split-brain patient understands the means (what) and intentions (why) of the actions of other people. Results show a significant left hemispheric dominance for understanding what is done, and a significant right hemispheric dominance for understanding why an action is carried out. This discovery might have important clinical implications in neurological patients, especially when those with right hemisphere lesions are faced with important decisions related to the interpretation of other's intentions.

Protease cleavage site recognition motifs can be identified using protease substrate discovery methodologies, but typically exhibit non-optimal specificity and activity. To enable evolutionary optimization of substrate cleavage kinetics, a two-color cellular library of peptide substrates (CLiPS) methodology was developed. Two-color CLiPS was applied to identify peptide substrates for the tobacco etch virus (TEV) protease from a random pentapeptide library, which were then optimized by screening of a focused, extended substrate library. Quantitative library screening yielded seven amino acid substrates exhibiting rapid hydrolysis by TEV protease and high sequence similarity to the native seven-amino-acid substrate, with a strong consensus of EXLYPhiQG. Comparison of hydrolysis rates for a family of closely related substrates indicates that the native seven-residue TEV substrate co-evolved with TEV protease to facilitate highly efficient hydrolysis. Consensus motifs revealed by screening enabled database identification of a family of related, putative viral protease substrates. More generally, our results suggest that substrate evolution using CLiPS may be useful for optimizing substrate selectivity and activity to enable the design of more effective protease activity probes, molecular imaging agents, and prodrugs.

A simple and scalable method, based on dip-coat colloidal lithography, mask reduction, and plasma-based pattern transfer, is presented to create graded-index, moth eye-inspired antireflective features on II–VI semiconductors. Hexagonal arrays of isolated conical frusta with tunable geometry (top diameter = 200–1300 nm, pitch = 310–2530 nm, and height = 790–7100 nm) were realized by isotropic etching of various size silica colloid masks before pattern transfer into the underlying substrate. Substantial increases in single-side direct and total infrared (IR) transmission across the 4–20 μm range (9%–15% for CdTe thin films and 18% for bulk CdTe) were achieved, in excellent agreement with transfer matrix calculations and finite difference time domain optical simulations. The fabrication method presented can be used to enhance efficiency in multiple IR application areas including photovoltaics, optical system components, detectors, and focal plane array imagers.

Sandwich assays are among the most powerful tools in molecular detection. These assays use “pairs” of affinity reagents so that the detection signal is generated only when both reagents bind simultaneously to different sites on the target molecule, enabling highly sensitive and specific measurements in complex samples. Thus, the capability to efficiently screen affinity reagent pairs at a high throughput is critical. In this work, we describe an experimental strategy for screening “aptamer pairs” at a throughput of 106 aptamer pairs per hour—which is many orders of magnitude higher than the current state of the art. The key step in our process is the conversion of solution-phase aptamers into “aptamer particles” such that we can directly measure the simultaneous binding of multiple aptamers to a target protein based on fluorescence signals and sort individual particles harboring aptamer pairs via the fluorescence-activated cell-sorter instrument. As proof of principle, we successfully isolated a high-quality DNA aptamer pair for plasminogen activator inhibitor 1 (PAI-1). Within only two rounds of screening, we discovered DNA aptamer pairs with low-nanomolar sensitivity in dilute serum and excellent specificity with minimal off-target binding even to closely related proteins such as PAI-2.

The suprachiasmatic nucleus (SCN) is the master clock of the brain. It is a network of neurons that behave like biological oscillators, capable of synchronizing and maintaining daily rhythms. The detailed structure of this network is still unknown, and the role that the connectivity pattern plays in the network's ability to generate robust oscillations has yet to be fully elucidated. In recent work, we used an information theory-based technique to infer the structure of the functional network for synchronization, from bioluminescence reporter data. Here, we propose a computational method to determine the directionality of the connections between the neurons. We find that most SCN neurons have a similar number of incoming connections, but the number of outgoing connections per neuron varies widely, with the most highly connected neurons residing preferentially in the core.

A mental representation of the location of an object can be constructed using sensory information selected from the environment and information stored internally. Human electrophysiological evidence indicates that behaviorally relevant locations, regardless of the source of sensory information, are represented in alpha-band oscillations suggesting a shared process. Here, we present evidence from human subjects of either sex for two distinct alpha-band-based processes that separately support the representation of location, exploiting sensory evidence sampled either externally or internally. <b>SIGNIFICANCE STATEMENT</b> Our sensory environment and our internal trains of thought are coded in patterns of brain activity and are used to guide coherent behavior. Oscillations in the alpha-frequency band are a predominant feature of human brain activity. This oscillation plays a central role in both selective attention and working memory, suggesting that these important cognitive functions are mediated by a unitary mechanism. We show that the alpha oscillation reflects two distinct processes, one that is supported by continuous sampling of the external sensory environment, and one that is based on sampling from internal representations coded in visual short-term memory. This represents a significant change in our understanding of the nature of alpha oscillations and their relationship to attention and memory.