Characterization of a Novel Thermostable DNA Lyase Used To Prepare DNA for Next-Generation Sequencing.

Recently, we constructed a hybrid thymine DNA glycosylase (hyTDG) by linking a 29-amino acid sequence from the human thymine DNA glycosylase with the catalytic domain of DNA mismatch glycosylase (MIG) from M. thermoautotrophicum, increasing the overall activity of the glycosylase. Previously, it was shown that a tyrosine to lysine (Y126K) mutation in the catalytic site of MIG could convert the glycosylase activity to a lyase activity. We made the corresponding mutation to our hyTDG to create a hyTDG-lyase (Y163K). Here, we report that the hybrid mutant has robust lyase activity, has activity over a broad temperature range, and is active under multiple buffer conditions. The hyTDG-lyase cleaves an abasic site similar to endonuclease III (Endo III). In the presence of ß-mercaptoethanol (ß-ME), the abasic site unsaturated aldehyde forms a ß-ME adduct. The hyTDG-lyase maintains its preference for cleaving opposite G, as with the hyTDG glycosylase, and the hyTDG-lyase and hyTDG glycosylase can function in tandem to cleave T:G mismatches. The hyTDG-lyase described here should be a valuable tool in studies examining DNA damage and repair. Future studies will utilize these enzymes to quantify T:G mispairs in cells, tissues, and genomic DNA using next-generation sequencing.

Kinetics measurements of G-quadruplex binding and unfolding by helicases.

G-quadruplex structures (G4s) form readily in DNA and RNA and play diverse roles in gene expression and other processes, and their inappropriate formation and stabilization are linked to human diseases. G4s are inherently long-lived, such that their timely unfolding depends on a suite of DNA and RNA helicase proteins. Biochemical analysis of G4 binding and unfolding by individual helicase proteins is important for establishing their levels of activity, affinity, and specificity for G4s, including individual G4s of varying sequence and structure. Here we describe a set of simple, accessible methods in which electrophoretic mobility shift assays (EMSA) are used to measure the kinetics of G4 binding, dissociation, and unfolding by helicase proteins. We focus on practical considerations and the pitfalls that are most likely to arise when these methods are used to study the activities of helicases on G4s.

Comprehensive MR imaging QA of 0.35 T MR-Linac using a multi-purpose large FOV phantom: A single-institution experience.

PURPOSE: Magnetic resonance-guided radiotherapy (MRgRT) is desired for the treatment of diseases in the abdominothoracic region, which has a broad imaging area and continuous motion. To ensure accurate treatment delivery, an effective image quality assurance (QA) program, with a phantom that covers the field of view (FOV) similar to a human torso, is required. However, routine image QA for a large FOV is not readily available at many MRgRT centers. In this work, we present the clinical experience of the large FOV MRgRT Insight phantom for periodic daily and monthly comprehensive magnetic resonance imaging (MRI)-QA and its feasibility compared to the existing institutional routine MRI-QA procedures in 0.35 T MRgRT. METHODS: Three phantoms; ViewRay cylindrical water phantom, Fluke 76-907 uniformity and linearity phantom, and Modus QA large FOV MRgRT Insight phantom, were imaged on the 0.35 T MR-Linac. The measurements were made in MRI mode with the true fast imaging with steady-state free precession (TRUFI) sequence. The ViewRay cylindrical water phantom was imaged in a single-position setup whereas the Fluke phantom and Insight phantom were imaged in three different orientations: axial, sagittal, and coronal. Additionally, the phased array coil QA was performed using the horizontal base plate of the Insight phantom by placing the desired coil around the base section which was compared to an in-house built Polyurethane foam phantom for reference. RESULT: The Insight phantom captured image artifacts across the entire planar field of view, up to 400 mm, in a single image acquisition, which is beyond the FOV of the conventional phantoms. The geometric distortion test showed a similar distortion of 0.45 ± 0.01  and 0.41 ± 0.01 mm near the isocenter, that is, within 300 mm lengths for Fluke and Insight phantoms, respectively, but showed higher geometric distortion of 0.8 ± 0.4 mm in the peripheral region between 300 and 400 mm of the imaging slice for the Insight phantom. The Insight phantom with multiple image quality features and its accompanying software utilized the modulation transform function (MTF) to evaluate the image spatial resolution. The average MTF values were 0.35 ± 0.01, 0.35 ± 0.01, and 0.34 ± 0.03 for axial, coronal, and sagittal images, respectively. The plane alignment and spatial accuracy of the ViewRay water phantom were measured manually. The phased array coil test for both the Insight phantom and the Polyurethane foam phantoms ensured the proper functionality of each coil element. CONCLUSION: The multifunctional large FOV Insight phantom helps in tracking MR imaging quality of the system to a larger extent compared to the routine daily and monthly QA phantoms currently used in our institute. Also, the Insight phantom is found to be more feasible for routine QA with easy setup.

DNA Base Excision Repair Intermediates Influence Duplex-Quadruplex Equilibrium.

Although genomic DNA is predominantly duplex under physiological conditions, particular sequence motifs can favor the formation of alternative secondary structures, including the G-quadruplex. These structures can exist within gene promoters, telomeric DNA, and regions of the genome frequently found altered in human cancers. DNA is also subject to hydrolytic and oxidative damage, and its local structure can influence the type of damage and its magnitude. Although the repair of endogenous DNA damage by the base excision repair (BER) pathway has been extensively studied in duplex DNA, substantially less is known about repair in non-duplex DNA structures. Therefore, we wanted to better understand the effect of DNA damage and repair on quadruplex structure. We first examined the effect of placing pyrimidine damage products uracil, 5-hydroxymethyluracil, the chemotherapy agent 5-fluorouracil, and an abasic site into the loop region of a 22-base telomeric repeat sequence known to form a G-quadruplex. Quadruplex formation was unaffected by these analogs. However, the activity of the BER enzymes were negatively impacted. Uracil DNA glycosylase (UDG) and single-strand selective monofunctional uracil DNA glycosylase (SMUG1) were inhibited, and apurinic/apyrimidinic endonuclease 1 (APE1) activity was completely blocked. Interestingly, when we performed studies placing DNA repair intermediates into the strand opposite the quadruplex, we found that they destabilized the duplex and promoted quadruplex formation. We propose that while duplex is the preferred configuration, there is kinetic conversion between duplex and quadruplex. This is supported by our studies using a quadruplex stabilizing molecule, pyridostatin, that is able to promote quadruplex formation starting from duplex DNA. Our results suggest how DNA damage and repair intermediates can alter duplex-quadruplex equilibrium.

The DHX36-specific-motif (DSM) enhances specificity by accelerating recruitment of DNA G-quadruplex structures.

DHX36 is a eukaryotic DEAH/RHA family helicase that disrupts G-quadruplex structures (G4s) with high specificity, contributing to regulatory roles of G4s. Here we used a DHX36 truncation to examine the roles of the 13-amino acid DHX36-specific motif (DSM) in DNA G4 recognition and disruption. We found that the DSM promotes G4 recognition and specificity by increasing the G4 binding rate of DHX36 without affecting the dissociation rate. Further, for most of the G4s measured, the DSM has little or no effect on the G4 disruption step by DHX36, implying that contacts with the G4 are maintained through the transition state for G4 disruption. This result suggests that partial disruption of the G4 from the 3' end is sufficient to reach the overall transition state for G4 disruption, while the DSM remains unperturbed at the 5' end. Interestingly, the DSM does not contribute to G4 binding kinetics or thermodynamics at low temperature, indicating a highly modular function. Together, our results animate recent DHX36 crystal structures, suggesting a model in which the DSM recruits G4s in a modular and flexible manner by contacting the 5' face early in binding, prior to rate-limiting capture and disruption of the G4 by the helicase core.

Blockchain-Enabled Asynchronous Federated Learning in Edge Computing.

The fast proliferation of edge computing devices brings an increasing growth of data, which directly promotes machine learning (ML) technology development. However, privacy issues during data collection for ML tasks raise extensive concerns. To solve this issue, synchronous federated learning (FL) is proposed, which enables the central servers and end devices to maintain the same ML models by only exchanging model parameters. However, the diversity of computing power and data sizes leads to a significant difference in local training data consumption, and thereby causes the inefficiency of FL. Besides, the centralized processing of FL is vulnerable to single-point failure and poisoning attacks. Motivated by this, we propose an innovative method, federated learning with asynchronous convergence (FedAC) considering a staleness coefficient, while using a blockchain network instead of the classic central server to aggregate the global model. It avoids real-world issues such as interruption by abnormal local device training failure, dedicated attacks, etc. By comparing with the baseline models, we implement the proposed method on a real-world dataset, MNIST, and achieve accuracy rates of 98.96% and 95.84% in both horizontal and vertical FL modes, respectively. Extensive evaluation results show that FedAC outperforms most existing models.

Characterization of radiotherapy component impact on MR imaging quality for an MRgRT system.

Radiotherapy components of an magnetic resonnace-guided radiotherapy (MRgRT) system can alter the magnetic fields, causing spatial distortion and image deformation, altering imaging and radiation isocenter coincidence and the accuracy of dose calculations. This work presents a characterization of radiotherapy component impact on MR imaging quality in terms of imaging isocenter variation and spatial integrity changes on a 0.35T MRgRT system, pre- and postupgrade of the system. The impact of gantry position, MLC field size, and treatment table power state on imaging isocenter and spatial integrity were investigated. A spatial integrity phantom was used for all tests. Images were acquired for gantry angles 0-330° at 30° increments to assess the impact of gantry position. For MLC and table power state tests all images were acquired at the home gantry position (330°). MLC field sizes ranged from 1.66 to 27.4 cm edge length square fields. Imaging isocenter shift caused by gantry position was reduced from 1.7 mm at gantry 150° preupgrade to 0.9 mm at gantry 120° postupgrade. Maximum spatial integrity errors were 0.5 mm or less pre- and postupgrade for all gantry angles, MLC field sizes, and treatment table power states. However, when the treatment table was powered on, there was significant reduction in SNR. This study showed that gantry position can impact imaging isocenter, but spatial integrity errors were not dependent on gantry position, MLC field size, or treatment table power state. Significant isocenter variation, while reduced postupgrade, is cause for further investigation.

Early detection of depression using a conversational AI bot: A non-clinical trial.

BACKGROUND: Artificial intelligence (AI) has gained momentum in behavioural health interventions in recent years. However, a limited number of studies use or apply such methodologies in the early detection of depression. A large population needing psychological-intervention is left unidentified due to barriers such as cost, location, stigma and a global shortage of health workers. Therefore, it is essential to develop a mass screening integrative approach that can identify people with depression at its early stage to avoid a potential crisis. OBJECTIVES: This study aims to understand the feasibility and efficacy of using AI-enabled chatbots in the early detection of depression. METHODS: We use Dialogflow as a conversation interface to build a Depression Analysisn (DEPRA) chatbot. A structured and authoritative early detection depression interview guide, which contains 27 questions combining the structured interview guide for the Hamilton Depression Scale (SIGH-D) and the inventory of depressive symptomatology (IDS-C), underpins the design of the conversation flow. To attain better accuracy and a wide variety of responses, we train Dialogflow with the utterances collected from a focus group of 10 people. The occupation of the focus group members included academics and HDR candidates who are conscious, vigilant and have a clear understanding of the questions. In addition, DEPRA is integrated with a social media platform to provide practical access to all the participants. For the non-clinical trial, we recruited 50 participants aged between 18 and 80 from across Australia. To evaluate the practicability and performance of DEPRA, we also asked participants to submit a user satisfaction survey at the end of the conversation. RESULTS: A sample of 50 participants, with an average age of 34.7 years, completed this non-clinical trial. More than half of the participants (54%) are male and the major ethnicities are Asian (63%), Middle Eastern (25%), and others 12%. The first group comprises professional academic staff and HDR candidates, the second and third groups comprise relatives, friends, and volunteers who were recruited via social media promotions. DEPRA uses two scientific scoring systems, QIDS-SR and IDS-SR to verify the results of early depression detection. As the results indicate, both scoring systems return a similar outcome with slight variations for different depression levels. According to IDS-SR, 30% of participants were healthy, 14% mild, 22% moderate, 14% severe, and 20% very severe. QIDS-SR suggests 32% were healthy, 18% mild, 10% moderate, 18% severe, and 22% very severe. Furthermore, the overall satisfaction rate of using DEPRA was 79% indicating that the participants had a high rate of user satisfaction and engagement. CONCLUSION: DEPRA shows promises as a feasible option for developing a mass screening integrated approach for early detection of depression. Although the chatbot is not intended to replace the functionality of mental health professionals, it does show promise as a means of assisting with automation and concealed communication with verified scoring systems.

Characterizing MR Imaging isocenter variation in MRgRT.

We characterized MRI isocenter variation at various gantry positions in two 0.35 T MRgRT systems using two independent methods. First, image center-based quantification was employed on 3D volumetric and 2D cine images of a 24 cm diameter spherical phantom at various gantry positions in the MRI QA mode. The center of the phantom images was identified to quantify the variation of the imaging center at each gantry position. Second, image registration-based quantification was used in radiotherapy mode. 3D volumetric MRIs of a cylindrical phantom were acquired and corresponding image registration from MRI to planning CT was performed. The shifts of the couch were identified to quantify the variation of the imaging center. For verification of noticeable MRI isocenter variation, star-shot pattern measurements with five beams were delivered on the radio-chromic film inserted into the phantom after the couch was shifted. The center of the star-shot pattern was identified to quantify the variation of the imaging center. The proposed methods for measuring MRI isocenter variation were demonstrated with MR-LINAC and MR-60Co systems. Both of the MRgRT systems had field inhomogeneities <5 ppm over a 24 cm diameter spherical volume (DSV) and spatial integrity distortion: <1 mm within 100 mm radius and <2 mm within 175 mm radius. The MRI isocenter of the MR-LINAC system showed noticeable 3D variation (max magnitude: 1.8 mm) compared to that of MR-60Co system (max magnitude: 0.9 mm) relative to the reference gantry positions. In addition, 2D variations (max magnitude) of the MRI isocenter from sagittal cine images were 0.9 mm for the MR-LINAC system and 0.5 mm for the MR-60Co system. Two proposed methods quantified the MRI isocenter variation for various gantry positions in two 0.35 T MRgRT systems. The results of significant isocenter variation in the MR-LINAC system requires further investigation to determine the cause.

FMEA of manual and automated methods for commissioning a radiotherapy treatment planning system.

PURPOSE: To evaluate the level of risk involved in treatment planning system (TPS) commissioning using a manual test procedure, and to compare the associated process-based risk to that of an automated commissioning process (ACP) by performing an in-depth failure modes and effects analysis (FMEA). METHODS: The authors collaborated to determine the potential failure modes of the TPS commissioning process using (a) approaches involving manual data measurement, modeling, and validation tests and (b) an automated process utilizing application programming interface (API) scripting, preloaded, and premodeled standard radiation beam data, digital heterogeneous phantom, and an automated commissioning test suite (ACTS). The severity (S), occurrence (O), and detectability (D) were scored for each failure mode and the risk priority numbers (RPN) were derived based on TG-100 scale. Failure modes were then analyzed and ranked based on RPN. The total number of failure modes, RPN scores and the top 10 failure modes with highest risk were described and cross-compared between the two approaches. RPN reduction analysis is also presented and used as another quantifiable metric to evaluate the proposed approach. RESULTS: The FMEA of a MTP resulted in 47 failure modes with an RPNave of 161 and Save of 6.7. The highest risk process of "Measurement Equipment Selection" resulted in an RPNmax of 640. The FMEA of an ACP resulted in 36 failure modes with an RPNave of 73 and Save of 6.7. The highest risk process of "EPID Calibration" resulted in an RPNmax of 576. CONCLUSIONS: An FMEA of treatment planning commissioning tests using automation and standardization via API scripting, preloaded, and pre-modeled standard beam data, and digital phantoms suggests that errors and risks may be reduced through the use of an ACP.