Discriminating Residue Substitutions in a Single Protein Molecule Using a Sub-nanopore

It is now possible to create, in a thin inorganic membrane, a single, sub-nanometer-diameter pore (<i>i.e.</i>, a sub-nanopore) about the size of an amino acid residue. To explore the prospects for sequencing protein with it, measurements of the force and current were performed as two denatured histones, which differed by four amino acid residue substitutions, were impelled systematically through the sub-nanopore one at a time using an atomic force microscope. The force measurements revealed that once the denatured protein, stabilized by sodium dodecyl sulfate (SDS), translocated through the sub-nanopore, a disproportionately large force was required to pull it back. This was interpreted to mean that the SDS was cleaved from the protein during the translocation. The force measurements also exposed a dichotomy in the translocation kinetics: either the molecule slid nearly frictionlessly through the pore or it slipped-and-stuck. When it slid frictionlessly, regardless of whether the molecule was pulled N-terminus or C-terminus first through the pore, regular patterns were observed intermittently in the force and blockade current fluctuations that corresponded to the distance between stretched residues. Furthermore, the amplitude of the fluctuations in the current blockade were correlated with the occluded volume associated with the amino acid residues in the pore. Finally, a comparison of the patterns in the current fluctuations associated with the two practically identical histones supported the conclusion that a sub-nanopore was sensitive enough to discriminate amino acid substitutions in the sequence of <i>a single protein molecule</i> by measuring volumes of 0.1 nm³ per read

Discriminating Residue Substitutions in a Single Protein Molecule Using a Sub-nanopore

It is now possible to create, in a thin inorganic membrane, a single, sub-nanometer-diameter pore (<i>i.e.</i>, a sub-nanopore) about the size of an amino acid residue. To explore the prospects for sequencing protein with it, measurements of the force and current were performed as two denatured histones, which differed by four amino acid residue substitutions, were impelled systematically through the sub-nanopore one at a time using an atomic force microscope. The force measurements revealed that once the denatured protein, stabilized by sodium dodecyl sulfate (SDS), translocated through the sub-nanopore, a disproportionately large force was required to pull it back. This was interpreted to mean that the SDS was cleaved from the protein during the translocation. The force measurements also exposed a dichotomy in the translocation kinetics: either the molecule slid nearly frictionlessly through the pore or it slipped-and-stuck. When it slid frictionlessly, regardless of whether the molecule was pulled N-terminus or C-terminus first through the pore, regular patterns were observed intermittently in the force and blockade current fluctuations that corresponded to the distance between stretched residues. Furthermore, the amplitude of the fluctuations in the current blockade were correlated with the occluded volume associated with the amino acid residues in the pore. Finally, a comparison of the patterns in the current fluctuations associated with the two practically identical histones supported the conclusion that a sub-nanopore was sensitive enough to discriminate amino acid substitutions in the sequence of <i>a single protein molecule</i> by measuring volumes of 0.1 nm³ per read

Photoluminescence Blinking from Single CdSeS/ZnS Quantum Dots in a Conducting Polymer Matrix

Quantum dot nanocrystals (NQDs) present within organic conducting (polymer) host environments form hybrid organic–inorganic materials that are applied in a range of technologies such as light emitting diodes or solar cells. Understanding hole-transport and exciton dynamics in these hybrid materials is central to device performance and efficiency. Integral to hole-transport is the understanding of multiexciton processes such as charged excitons as well as neighbor–neighbor NQD interactions (on the nano and micrometer length scales). Studied here are the photoluminescence dynamics of single alloyed NQDs in conducting (or insulating) polymer environments. We find that conducting polymers (through hole transport) affect the presence and dynamics of charged excitons relative to insulating environments. The presence of such charged excitons induces a change in blinking dynamics with a corresponding increase in photoluminescence correlation between neighboring NQDs found using spatiotemporal statistical analysis. Understanding such phenomena advances the understanding of photoluminescence processes central to device design

Single-molecule protein identification by sub-nanopore sensors - Fig 2

<p>(a) An example of a pore current trace acquired from a denatured H3.3 histone translocating through sub-nanopore with a nominal diameter of 0.5-nm. (b) The bottom trace is a magnified view of a 600 ms region of a top trace, showing a current blockade associated with the translocation of a single protein molecule. In the figure, higher values correspond to larger blockade currents. Blockades, associated with the translocation of single proteins were identified as regions with fluctuations five standard deviations above the noise level and with duration > 1 ms.); and then the raw current <i>I</i> was converted into <i>fractional blockade current</i>.</p

TEM micrograph of sub-nanopore is shown with a nominal diameter of 0.5 nm sputtered through silicon nitride membrane about 10-nm thick.

<p>The shot noise is associated with electron transmission through the pore. (center) Multi-slice simulations of the TEM image are consistent with the experimental imaging conditions. The simulations correspond to a bi-conical pore with a 0.5 x 0.4 <i>nm</i>² cross-section and a 15 cone angle at defocus of -40 nm. (right) Space-filled model of the same pore is shown where the <i>Si</i> atoms are represented by spheres with a 0.235 nm diameter and <i>N</i> atoms by spheres with a 0.13 nm diameter. The scale bars are 1 nm.</p

Datasets summary.

<p>Datasets summary.</p

Live Bacterial Physiology Visualized with 5 nm Resolution Using Scanning Transmission Electron Microscopy

It is now possible to visualize at nanometer resolution the infection of a living biological cell with virus without compromising cell viability using scanning transmission electron microscopy (STEM). To provide contrast while preserving viability, <i>Escherichia coli</i> and P1 bacteriophages were first positively stained with a very low concentration of uranyl acetate in minimal phosphate medium and then imaged with low-dose STEM in a microfluidic liquid flow cell. Under these conditions, it was established that the median lethal dose of electrons required to kill half the tested population was LD₅₀ = 30 e^–/nm², which coincides with the disruption of a wet biological membrane, according to prior reports. Consistent with the lateral resolution and high-contrast signal-to-noise ratio (SNR) inferred from Monte Carlo simulations, images of the <i>E. coli</i> membrane, flagella, and the bacteriophages were acquired with 5 nm resolution, but the cumulative dose exceeded LD₅₀. On the other hand, with a cumulative dose below LD₅₀ (and lower SNR), it was still possible to visualize the infection of <i>E. coli</i> by P1, showing the insertion of viral DNA within 3 s, with 5 nm resolution

An example of H3.3 nanospectra identification (for a cluster of size five) against all human proteins of length 100–160 AAs.

<p>The total database size is 14 293, which covers approximately 20% of the human proteome taken from the UniProt database. The correct protein (H3F3A) is shown in bold. Proteins from the H3 family exhibit the highest <i>R</i>² scores among other proteins from the database.</p

Method for Dynamically Detecting Secretions from Single Cells Using a Nanopore

Secreted proteins mediate cell-to-cell communications. Thus, eavesdropping on the secretome could reveal the cellular phenotype, but it is challenging to detect the proteins because they are secreted only in minute amounts and then diluted in blood plasma or contaminated by cell culture medium or the lysate. In this pilot study, it is demonstrated that secretions from single cancer cells can be detected and dynamically analyzed through measurements of blockades in the electrolytic current due to single molecules translocating through a nanopore in a thin inorganic membrane. It is established that the distribution of blockades can be used to differentiate three different cancer cell lines (U937, MDA-MB-231, and MCF-7) in real time and quickly (<20 s). Importantly, the distinctive blockades associated with the chemokine CCL5, a prognostic factor for disease progression in breast cancer, along with other low-mass biomarkers of breast cancer (PI3, TIMP1, and MMP1) were identified in the context of the secretome of these three cell types, tracked with time, and used to provide information on the cellular phenotype

Data_Sheet_1_Identifying clinical phenotypes of frontotemporal dementia in post-9/11 era veterans using natural language processing.pdf

IntroductionFrontotemporal dementia (FTD) encompasses a clinically and pathologically diverse group of neurodegenerative disorders, yet little work has quantified the unique phenotypic clinical presentations of FTD among post-9/11 era veterans. To identify phenotypes of FTD using natural language processing (NLP) aided medical chart reviews of post-9/11 era U.S. military Veterans diagnosed with FTD in Veterans Health Administration care.MethodsA medical record chart review of clinician/provider notes was conducted using a Natural Language Processing (NLP) tool, which extracted features related to cognitive dysfunction. NLP features were further organized into seven Research Domain Criteria Initiative (RDoC) domains, which were clustered to identify distinct phenotypes.ResultsVeterans with FTD were more likely to have notes that reflected the RDoC domains, with cognitive and positive valence domains showing the greatest difference across groups. Clustering of domains identified three symptom phenotypes agnostic to time of an individual having FTD, categorized as Low (16.4%), Moderate (69.2%), and High (14.5%) distress. Comparison across distress groups showed significant differences in physical and psychological characteristics, particularly prior history of head injury, insomnia, cardiac issues, anxiety, and alcohol misuse. The clustering result within the FTD group demonstrated a phenotype variant that exhibited a combination of language and behavioral symptoms. This phenotype presented with manifestations indicative of both language-related impairments and behavioral changes, showcasing the coexistence of features from both domains within the same individual.DiscussionThis study suggests FTD also presents across a continuum of severity and symptom distress, both within and across variants. The intensity of distress evident in clinical notes tends to cluster with more co-occurring conditions. This examination of phenotypic heterogeneity in clinical notes indicates that sensitivity to FTD diagnosis may be correlated to overall symptom distress, and future work incorporating NLP and phenotyping may help promote strategies for early detection of FTD.</p