Reconstitution and resonance assignments of yeast OST subunit Ost4 and its critical mutant Ost4V23D in liposomes by solid-state NMR.

N-linked glycosylation is an essential and highly conserved co- and post-translational protein modification in all domains of life. In humans, genetic defects in N-linked glycosylation pathways result in metabolic diseases collectively called Congenital Disorders of Glycosylation. In this modification reaction, a mannose rich oligosaccharide is transferred from a lipid-linked donor substrate to a specific asparagine side-chain within the -N-X-T/S- sequence (where X ≠ Proline) of the nascent protein. Oligosaccharyltransferase (OST), a multi-subunit membrane embedded enzyme catalyzes this glycosylation reaction in eukaryotes. In yeast, Ost4 is the smallest of nine subunits and bridges the interaction of the catalytic subunit, Stt3, with Ost3 (or its homolog, Ost6). Mutations of any C-terminal hydrophobic residues in Ost4 to a charged residue destabilizes the enzyme and negatively impacts its function. Specifically, the V23D mutation results in a temperature-sensitive phenotype in yeast. Here, we report the reconstitution of both purified recombinant Ost4 and Ost4V23D each in a POPC/POPE lipid bilayer and their resonance assignments using heteronuclear 2D and 3D solid-state NMR with magic-angle spinning. The chemical shifts of Ost4 changed significantly upon the V23D mutation, suggesting a dramatic change in its chemical environment.

Deep Learning-Based TEM Image Analysis for Fully Automated Detection of Gold Nanoparticles Internalized Within Tumor Cell.

Transmission electron microscopy (TEM) imaging can be used for detection/localization of gold nanoparticles (GNPs) within tumor cells. However, quantitative analysis of GNP-containing cellular TEM images typically relies on conventional/thresholding-based methods, which are manual, time-consuming, and prone to human errors. In this study, therefore, deep learning (DL)-based methods were developed for fully automated detection of GNPs from cellular TEM images. Several models of "you only look once (YOLO)" v5 were implemented, with a few adjustments to enhance the model's performance by applying the transfer learning approach, adjusting the size of the input image, and choosing the best optimization algorithm. Seventy-eight original (12,040 augmented) TEM images of GNP-laden tumor cells were used for model implementation and validation. A maximum F1 score (harmonic mean of the precision and recall) of 0.982 was achieved by the best-trained models, while mean average precision was 0.989 and 0.843 at 0.50 and 0.50-0.95 intersection over union threshold, respectively. These results suggested the developed DL-based approach was capable of precisely estimating the number/position of internalized GNPs from cellular TEM images. A novel DL-based TEM image analysis tool from this study will benefit research/development efforts on GNP-based cancer therapeutics, for example, by enabling the modeling of GNP-laden tumor cells using nanometer-resolution TEM images.

High-sensitivity imaging and quantification of intratumoral distributions of gold nanoparticles using a benchtop x-ray fluorescence imaging system.

A high-sensitivity benchtop x-ray fluorescence (XRF) imaging system, based on a high-power x-ray source and silicon drift detector, has been developed. This system allows gold L-shell XRF-based quantitative imaging of gold nanoparticles (GNPs) at concentrations as low as 0.007 mg/cm3 (7 ppm) in biological tissues/water. Its capability for biomedical applications was demonstrated by imaging the GNP distribution within a small (∼12×11×2 mm3) ex vivo sample (extracted from a murine tumor after intravenous GNP administration). The results suggest direct translatability for routine preclinical ex vivo imaging tasks involving GNPs, as well as the possibility for in vivo imaging of small/superficial animal tumors.

Solution structure and DNA-binding properties of the winged helix domain of the meiotic recombination HOP2 protein.

The HOP2 protein is required for efficient double-strand break repair which ensures the proper synapsis of homologous chromosomes and normal meiotic progression. We previously showed that in vitro HOP2 shows two distinctive activities: when it is incorporated into a HOP2-MND1 heterodimer, it stimulates DMC1 and RAD51 recombination activities, and the purified HOP2 alone is proficient in promoting strand invasion. The structural and biochemical basis of HOP2 action in recombination are poorly understood; therefore, they are the focus of this work. Herein, we present the solution structure of the amino-terminal portion of mouse HOP2, which contains a typical winged helix DNA-binding domain. Together with NMR spectral changes in the presence of double-stranded DNA, protein docking on DNA, and mutation analysis to identify the amino acids involved in DNA coordination, our results on the three-dimensional structure of HOP2 provide key information on the fundamental structural and biochemical requirements directing the interaction of HOP2 with DNA. These results, in combination with mutational experiments showing the role of a coiled-coil structural feature involved in HOP2 self-association, allow us to explain important aspects of the function of HOP2 in recombination.

Membrane attachment and structure models of lipid storage droplet protein 1.

Neutral lipid triglycerides, a main reserve for fat and energy, are stored in organelles called lipid droplets. The storage and release of triglycerides are actively regulated by several proteins specific to the droplet surface, one of which in insects is PLIN1. PLIN1 plays a key role in the activation of triglyceride hydrolysis upon phosphorylation. However, the structure of PLIN1 and its relation to functions remain elusive due to its insolubility and crystallization difficulty. Here we report the first solid-state NMR study on the Drosophila melanogaster PLIN1 in combination with molecular dynamics simulation to show the structural basis for its lipid droplet attachment. NMR spin diffusion experiments were consistent with the predicted membrane attachment motif of PLIN1. The data indicated that PLIN1 has close contact with the terminal methyl groups of the phospholipid acyl chains. Structure models for the membrane attachment motif were generated based on hydrophobicity analysis and NMR membrane insertion depth information. Simulated NMR spectra from a trans-model agreed with experimental spectra. In this model, lipids from the bottom leaflet were very close to the surface in the region enclosed by membrane attachment motif. This may imply that in real lipid droplet, triglyceride molecules might be brought close to the surface by the same mechanism, ready to leave the droplet in the event of lipolysis. Juxtaposition of triglyceride lipase structure to the trans-model suggested a possible interaction of a conserved segment with the lipase by electrostatic interactions, opening the lipase lid to expose the catalytic center.

Prompt gamma ray detection and imaging for boron neutron capture therapy using CdTe detector and novel detector shield - Monte Carlo study.

BACKGROUND: For boron neutron capture therapy (BNCT), the improvements in patient dosimetry will require information about the spatial variation of 10 B concentration in the tumor and critical organs. A non-invasive approach, based on the detection of prompt gamma (PG) rays from the BNC reaction, may be well-suited to obtain such information. The detectability of the BNC PG rays has been shown experimentally utilizing energy-resolving cadmium telluride (CdTe) detectors. However, the feasibility of this approach under the clinically relevant conditions of BNCT is currently unknown. PURPOSE: The present work aimed to investigate the aforementioned feasibility by performing Monte Carlo (MC) simulations under the phantom irradiation geometry relevant to accelerator-based BNCT (a-BNCT). Especially, this investigation focused on demonstrating the enhanced detection of the BNC PG rays using a novel neutron shield for CdTe detectors. Upon demonstrating the efficacy of the proposed detector shield, the BNC PG ray-based quantitative imaging of clinically relevant concentrations of 10 B was also demonstrated. METHODS: The Geant4 MC simulation toolkit was used to model the phantom irradiation by an epithermal neutron beam as well as the detection of the BNC PG rays from the phantom by CdTe detectors with and without the proposed gadolinium (Gd)-based detector shield. It was also used to model the BNC PG ray-based quantitative imaging of 10 B concentrations under a-BNCT scenarios. Each model included a 20 cm-diameter/24 cm-height cylindrical PMMA phantom containing 10 B inserts at various concentrations. Arrays of CdTe crystals of 5 × 5 × 1 mm3 each (up to 120 in the case of a ring detector) were modeled for acquiring the BNC PG ray signals and quantitative imaging. RESULTS: According to the MC simulations, thermalized neutrons from the phantom were found to reach the CdTe detector and captured by Cd and Te, resulting in the gamma ray background noise that directly interfered with the BNC PG ray signal. The proposed Gd-based detector shield was found to be highly effective in shielding thermal neutrons from the phantom, thereby reducing the unwanted gamma ray background noise. Owing to this shield, the detection of as low as seven parts-per-million (ppm) of 10 B within the phantom of clinically relevant size was possible using 20 billion incident neutron histories. Furthermore, quantitative imaging of 10 B distributed at low concentration (down to 50 ppm) within the phantom was demonstrated using computed tomography (CT) simulations with 16 billion incident neutron histories per angular projection. The 10 B detection limit (7.5 ppm) was also estimated using the reconstructed CT image. Both 10 B detection limits determined from this investigation are deemed clinically relevant for BNCT. CONCLUSIONS: The proposed Gd-based detector shield played an essential role for achieving the currently reported 10 B detection limits. Overall, the present MC simulation work demonstrated highly sensitive BNC PG ray detection and imaging under a-BNCT scenarios using CdTe detectors coupled with a novel detector shield.

Sensitivity enhancement of an experimental benchtop x-ray fluorescence imaging system by deploying a single crystal cadmium telluride detector system optimized for high flux x-ray operations.

In this work, an energy-resolving thermoelectrically cooled single crystal cadmium telluride (CdTe) detector system upgraded with the latest firmware was optimized for high x-ray flux operations using high bias voltage and fast peaking time. This detector system was deployed into an experimental benchtop x-ray fluorescence (XRF) imaging/computed tomography (XFCT) system developed for quantitative imaging of metal nanoprobes such as gold nanoparticles (GNPs). Using the firmware-upgraded and existing/old CdTe detector systems, the Compton/XRF spectra from small (8 mm diameter) GNP-containing phantoms were acquired. The phantoms were irradiated with 1.8 mm Sn-filtered 125 kVp cone beam x-rays at 24 mA. The firmware-upgraded detector system produced relatively lower dead time under high x-ray flux, compared with the old detector system, and performed well with the spectral resolution of ~0.7 keV (in full width at half maximum) at 69 keV photon energy. Given the same 2 mm aperture detector collimator and irradiation time of 10 s, this detector system managed to score nearly 50% more gold XRF signals than the existing one at all GNP concentrations tested. This improvement resulted in the GNP detection limit of 0.02 wt. % which was lower than that (0.03 wt. %) achievable with the existing detector system. When combined with the detector collimator containing a larger (3 mm) aperture, the firmware-upgraded detector system produced drastically more gold XRF signal at a given GNP concentration (e.g., 9 times more for 1 wt. % GNP solution and irradiation time of 10 s), leading to further reduction in the GNP detection limit (i.e., 0.01 wt. %). The present investigation showed that the firmware upgraded CdTe detector system optimized for high x-ray flux operations allowed for better photon counting efficiency, thus leading to sensitivity enhancement of an experimental benchtop XRF/XFCT imaging system.

Use of the Fully Spectroscopic Pixelated Cadmium Telluride Detector for Benchtop X-Ray Fluorescence Computed Tomography.

In this work, we integrated a commercially-available fully-spectroscopic pixelated cadmium telluride (CdTe) detector system as a two-dimensional (2D) array detector into our existing benchtop cone-beam x-ray fluorescence computed tomography (XFCT) system. After integrating this detector, known as High-Energy X-ray Imaging Technology (HEXITEC), we performed quantitative imaging of gold nanoparticle (GNP) distribution in a small animal-sized phantom using our benchtop XFCT system. Owing to the upgraded detector component within our benchtop XFCT system, we were able to conduct this phantom imaging in an unprecedented manner by volumetric XFCT scans followed by XFCT image reconstruction in 3D. The current results showed that adoption of HEXITEC, in conjunction with a custom-made parallel-hole collimator, drastically reduced the XFCT scan time/dose. Compared with the previous work performed with our original benchtop XFCT system adopting a single crystal CdTe detector, the currently observed reduction was up to a factor of 5, while achieving comparable GNP detection limit under similar experimental conditions. Overall, we demonstrated, for the first time to the best our knowledge, the feasibility of benchtop XFCT imaging of small animal-sized objects containing biologically relevant GNP concentrations (on the order of 0.1 mg Au/cm3 or 100 parts-per-million/ppm), with the scan time (on the order of 1 minute)/x-ray dose (on the order of 10 cGy) that are likely meeting the minimum requirements for routine preclinical imaging applications.

Bias-voltage dependent operational characteristics of a fully spectroscopic pixelated cadmium telluride detector system within an experimental benchtop x-ray fluorescence imaging setup.

Commercially available fully spectroscopic pixelated cadmium telluride (CdTe) detector systems have been adopted lately for benchtop x-ray fluorescence (XRF) imaging/computed tomography (XFCT) of objects containing metal nanoprobes such as gold nanoparticles (GNPs). To date, however, some important characteristics of such detector systems under typical operating conditions of benchtop XRF/XFCT imaging systems are not well known. One important but poorly studied characteristic is the effect of detector bias-voltage on photon counting efficiency, energy resolution, and the resulting material detection limit. In this work, therefore, we investigated these characteristics for a commercial pixelated detector system adopting a 1-mm-thick CdTe sensor (0.25-mm pixel-pitch), known as HEXITEC, incorporated into an experimental benchtop cone-beam XFCT system with parallel-hole detector collimation. The detector system, operated at different bias-voltages, was used to acquire the gold XRF/Compton spectra from 1.0 wt% GNP-loaded phantom irradiated with 125 kVp x-rays filtered by 1.8-mm Tin. At each bias-voltage, the gold XRF signal, and the full-width-at-half-maximum at gold Kα2XRF peak (∼67 keV) provided photon counting efficiency and energy resolution, respectively. Under the current experimental conditions, the detector photon counting efficiency and energy resolution improved with increasing bias-voltage by ∼41 and ∼29% at -300V; ∼54 and ∼35% at -500V, respectively, when compared to those at -100V. Consequently, the GNP detection limit improved by ∼26% at -300V and ∼30% at -500V. Furthermore, the homogeneity of per-pixel energy resolution within the collimated detector area improved by ∼34% at -300V and ∼54% at -500V. These results suggested the gradual improvements in the detector performance with increasing bias-voltage up to -500V. However, at and beyond -550V, there were no discernible improvements in photon counting efficiency and energy resolution. Thus, the bias-voltage range of -500 to -550V was found optimal under the current experimental conditions that are considered typical of benchtop XRF/XFCT imaging tasks.

Characterization of a Pixelated Cadmium Telluride Detector System Using a Polychromatic X-Ray Source and Gold Nanoparticle-Loaded Phantoms for Benchtop X-Ray Fluorescence Imaging.

Pixelated semi-conductor detectors providing high energy resolution enable parallel acquisition of x-ray fluorescence (XRF) signals, potentially leading to performance enhancement of benchtop XRF imaging or computed tomography (XFCT) systems utilizing ordinary polychromatic x-ray sources. However, little is currently known about the characteristics of such detectors under typical operating conditions of benchtop XRF imaging/XFCT. In this work, a commercially available pixelated cadmium telluride (CdTe) detector system, HEXITEC (High Energy X-ray Imaging Technology), was characterized to address this issue. Specifically, HEXITEC was deployed into our benchtop cone-beam XFCT system, and used to detect gold Kα XRF photons from gold nanoparticle (GNP)-loaded phantoms. To facilitate the detection of XRF photons, various parallel-hole stainless steel collimators were fabricated and coupled with HEXITEC. A pixel-by-pixel spectrum merging algorithm was introduced to obtain well-defined XRF + scatter spectra with parallel-hole collimators. The effect of charge sharing addition (CSA) and discrimination (CSD) algorithms was also investigated for pixel-level CS correction. Finally, the detector energy resolution, in terms of the full-width at half-maximum (FWHM) values at two gold Kα XRF peaks (~68 keV), was also determined. Under the current experimental conditions, CSD provided the best energy resolution of HEXITEC (~1.05 keV FWHM), compared with CSA and no CS correction. This FWHM value was larger (by up to ~0.35 keV) than those reported previously for HEXITEC (at ~60 keV Am-241 peak) and single-crystal CdTe detectors (at two gold Kα XRF peaks). This investigation highlighted characteristics of HEXITEC as well as the necessity for application-specific detector characterization.

Monte Carlo study of x-ray detection configurations for benchtop x-ray fluorescence computed tomography of gold nanoparticle-loaded objects.

Over the last decade, the performance of benchtop x-ray fluorescence computed tomography (XFCT) systems has been significantly enhanced through hardware and software optimizations. Recent studies have indicated the need of energy-resolving pixelated/array detectors in the x-ray detection component to further improve the sensitivity and image resolution of benchtop XFCT systems while meeting the realistic constraints of dose and scan time. Thus, it is of immediate interest in the research community to conduct the following investigations: (a) delineation of strengths/weaknesses of detection configurations that incorporate pixelated/array detectors in combination with two most frequently used (parallel-hole and pinhole) collimators; (b) one-to-one comparison of their performance under identical imaging conditions of benchtop XFCT. In this study, we developed a Geant4-based Monte Carlo model to investigate the effects of the aforementioned detection configurations on the sensitivity and image resolution of a benchtop XFCT system. Using this model, we simulated the detection of x-ray fluorescence and scattered photons from gold nanoparticle-containing phantoms using energy-resolving pixelated detectors coupled with parallel-hole and pinhole collimators. Simulation results demonstrated that the detector consisting of large pixels (1 mm × 1 mm) combined with a parallel-hole collimator had better sensitivity (i.e. lower detection limit) than the detector made of smaller pixels (0.25 mm × 0.25 mm) coupled with a pinhole collimator. In comparison, although slightly less sensitive, the latter detector configuration achieved better image resolution than did the former. Thus, a detection configuration consisting of a pixelated detector with submillimeter pixels and a pinhole collimator is preferable when image resolution is critical for benchtop XFCT applications. On the other hand, the detector with larger pixels coupled with a parallel-hole collimator is better suited for benchtop XFCT applications in which higher sensitivity and shorter scan time are essential.

Wing 1 of protein HOP2 is as important as helix 3 in DNA binding by MD simulation.

The repair of programmed DNA double-strand breaks through recombination is required for proper association and disjunction of the meiotic homologous chromosomes. Meiosis-specific protein HOP2 plays essential roles in recombination by promoting recombinase activities. The N-terminal domain of HOP2 interacts with DNA through helix 3 (H3) and wing 1 (W1). Mutations in wing 1 (Y65A/K67A/Q68A) slightly weakened the binding but mutations in helices 2 and 3 (Q30A/K44A/K49A) nearly abolished the binding. To better understand such differential effects at atomic level, molecular dynamics simulations were employed. Despite losing some hydrogen bonds, the W1-mutant DNA complex was rescued by stronger hydrophobic interactions. For the wild type and W1-mutant, the protein was found to slide along the DNA grooves as the DNA rolls along its double-helix axis. This motion could be functionally important to facilitate the precise positioning of the single-stranded DNA with the homologous double-stranded DNA. The sliding motion was reduced in the W1-mutant. The H-mutant nearly lost all intermolecular interactions. Moreover, an additional mutation in wing 1 (Y65A/K67A/Q68A/K69A) also caused complete complex dissociation. Therefore, both wing 1 and helix 3 make important contribution to the DNA binding, which could be important to the strand invasion function of HOP2 homodimer and HOP2-MND1 heterodimer. Similar to cocking a medieval crossbow with the archer's foot placed in the stirrup, wing 1 may push the minor groove to cause distortion while helix 3 grabs the major groove.