The common p.Ile291Val variant of ERLIN1 enhances TM6SF2 function and is associated with protection against MASLD.

BACKGROUND: The ERLIN1 p.Ile291Val single-nucleotide polymorphism (rs2862954) is associated with protection from steatotic liver disease (SLD), but effects of this variant on metabolic phenotypes remain uncertain. METHODS: Metabolic phenotypes and outcomes associated with ERLIN1 p.Ile291Val were analyzed by using a genome-first approach in the UK Biobank (UKB), Penn Medicine BioBank (PMBB), and All of Us cohort. FINDINGS: ERLIN1 p.Ile291Val carriers exhibited significantly lower serum levels of alanine aminotransferase and aspartate aminotransferase as well as higher levels of triglycerides, low-density lipoprotein cholesterol, Apolipoprotein B, high-density lipoprotein cholesterol, and Apolipoprotein A1 in UKB, and these values were affected by ERLIN1 p.Ile291Val in an allele-dose-dependent manner. Homozygous ERLIN1 p.Ile291Val carriers had a significantly reduced risk of developing metabolic dysfunction-associated SLD (MASLD, adjusted odds ratio [aOR] = 0.92, 95% confidence interval [CI], 0.88-0.96). The protective effect of this variant was enhanced in patients with alcoholic liver disease. Our results were replicated in PMBB and the All of Us cohort. Strikingly, the protective effects of ERLIN1 p.Ile291Val were not apparent in individuals carrying the TM6SF2 p.Glu167Lys variant associated with increased risk of SLD. We analyzed the effects of predicted loss-of-function ERLIN1 variants and found that they had opposite effects, namely reduced plasma lipids, suggesting that ERLIN1 p.Ile291Val may be a gain-of-function variant. CONCLUSION: Our study contributes to a better understanding of ERLIN1 by investigating a coding variant that has emerged as a potential gain-of-function mutation with protective effects against MASLD development.

Domain Switching Characteristics in Ga-Doped HfO2 Ferroelectric Thin Films with Low Coercive Field.

The gallium-doped hafnium oxide (Ga-HfO2) films with different Ga doping concentrations were prepared by adjusting the HfO2/Ga2O3 atomic layer deposition cycle ratio for high-speed and low-voltage operation in HfO2-based ferroelectric memory. The Ga-HfO2 ferroelectric films reveal a finely modulated coercive field (Ec) from 1.1 (HfO2/Ga2O3 = 32:1) to an exceptionally low 0.6 MV/cm (HfO2/Ga2O3 = 11:1). This modulation arises from the competition between domain nucleation and propagation speed during polarization switching, influenced by the intrinsic domain density and phase dispersion in the film with specific Ga doping concentrations. Higher Ec samples exhibit a nucleation-dominant switching mechanism, while lower Ec samples undergo a transition from a nucleation-dominant to a propagation-dominant reversal mechanism as the electric field increases. This work introduces Ga as a viable dopant for low Ec and offers insights into material design strategies for HfO2-based ferroelectric memory applications.

The associations of food environment with gastrointestinal cancer outcomes in the United States.

BACKGROUND: Social conditions and dietary behaviors have been implicated in the rising burden of gastrointestinal cancers (GIC). The "food environment" reflects influences on a community level relative to food availability, nutritional assistance, and social determinants of health. Using the US Department of Agriculture-Food Environment Atlas (FEA), we sought to characterize the association of food environment on GIC presenting stage and long-term survival. METHODS: Patients diagnosed with GIC between 2013 and 2017 were identified using the SEER database. FEA-scores were based on 282 county-level food security variables, store-restaurant availability, SNAP/WIC enrollment, pricing/taxes, and producer vicinity adjusted-for factors of socioeconomic status, race-ethnicity, transportation access, and comorbidities. Relative FEA rankings across US counties were averaged into a composite score and assigned to patients by county-of-residence. The association of FEA, cancer stage, and survival were analyzed using multiple logistic regression and cox-proportional hazard models relative to White/non-White race/ethnicity. RESULTS: Among 287,148 patients, the most common GIC-sites were colon (n = 97,942, 34%), pancreas (n = 49,785, 17.3%), liver (n = 31,098, 11.0%) and esophagus (n = 16,271, 5.7%). A worse food environment was independently associated with increased odds of late-stage diagnosis (esophageal odds ratio [OR]: 1.03, 95% confidence interval [CI]: 1.01-1.05; hepatic OR: 1.06, 95% CI: 1.03-1.08; pancreatic OR: 1.04, 95% CI: 1.01-1.06) among all patients; in contrast, food environment was associated with colorectal cancer stage among non-White patients only (OR: 1.04, 95% CI: 1.03-1.06). Worse food environment was associated with worse 3-year survival (colon OR: 1.03, 95% CI: 1.01-1.04; hepatic OR: 1.12, 95% CI: 1.08-1.17; gastric OR: 1.07, 95% CI: 1.01-1.13). Similar associations were noted relative to overall survival among the entire cohort (biliary tract hazard ratio [HR]: 1.03, 95% CI: 1.01-1.05; esophageal HR: 1.02, 95% CI: 1.01-1.04; hepatic HR: 1.07, 95% CI: 1.06-1.09; pancreatic HR: 1.04, 95% CI: 1.02-1.05; rectum HR: 1.03, 95% CI: 1.01-1.04; gastric HR: 1.05, 95% CI: 1.03-1.07), as well as among non-White patients (biliary HR: 1.04, 95% CI: 1.01-1.07; colon HR: 1.03, 95% CI: 1.01-1.05; esophageal HR: 1.05, 95% CI: 1.02-1.08; hepatic HR: 1.08, 95% CI: 1.06-1.10) (all p < 0.003). CONCLUSIONS: Food environment was independently associated with late-stage tumor presentation and worse 3-year and overall survival among GIC patients. Interventions to address inequities across communities relative to food environments are needed to alleviate disparities in cancer care.

To Combat Multiclass Imbalanced Problems by Aggregating Evolutionary Hierarchical Classifiers.

Real-world datasets are often imbalanced, posing frequent challenges to canonical machine learning algorithms that assume a balanced class distribution. Moreover, the imbalance problem becomes more complicated when the dataset is multiclass. Although many approaches have been presented for imbalanced learning (IL), research on the multiclass imbalanced problem is relatively limited and deficient. To alleviate these issues, we propose a forest of evolutionary hierarchical classifiers (FEHC) method for multiclass IL (MCIL). FEHC can be seen as a classifier fusion framework with a forest structure, and it aggregates several evolutionary hierarchical multiclassifiers (EHMCs) to reduce generalization error. Specifically, a multichromosome genetic algorithm (MCGA) is designed to simultaneously select (sub)optimal features, classifiers, and hierarchical structures when generating these EHMCs. The MCGA adopts a dynamic weighting module to learn difficult classes and promote the diversity of FEHC. We also present the "stratified underbagging" (SUB) strategy to address class imbalance and the "soft tree traversal" (STT) strategy to make FEHC converge faster and better. We thoroughly evaluate the proposed algorithm using 14 multiclass imbalanced datasets with various properties. Compared with popular and state-of-the-art approaches, FEHC obtains better performance under different evaluation metrics. Codes have been made publicly available on GitHub.https://github.com/CUHKSZ-NING/FEHCClassifier.

The World's consumption of free web-based Otolaryngology-Head and Neck Surgery educational resources: A global assessment of video usage trends.

Objectives: Online educational platforms with open access have seen a growing adoption in the field of medical education. However, the extent of their global usage is still unclear. To fill this knowledge gap, our objective is to examine the usage patterns of two renowned open-access resources in Otolaryngology. This includes identifying the most sought-after topics and understanding the demographics of their users. Methods: Retrospective study of web analytics data between 2016 and 2021 extracted from the Headmirror.com and Mayo Clinic Otolaryngology YouTube channel platforms analyzing demographic and education topic trends via descriptive, geospatial, time-series, t-tests, and ANOVA analyses. Results: Viewership spanned 124 countries in 7 different geographic regions, with 72 countries comprising low- to middle-income countries, mostly represented ages of 25-34 years old, came from high-income countries rather than low-income (p < .001), and used mobile phones followed by computers for device access. Video-educational material comprised of subspecialty topics on Rhinology and Sinus Surgery (25%) at the highest end and Facial Trauma (1%) at the lowest. Controlling for the age of the video content, the most-accessed videos comprised of subspecialty topics on Head and Neck Surgery at the highest end and Laryngology at the lowest with significant differentiation across topics of interest (p < .044). Conclusions: This assessment of web-analytics platforms from two widely used otolaryngology free, online-access materials showed increasing global usage trends with significant differentiating factors along viewership demographics, as well as sought-after subspecialty topics of interest. In turn, our results not only lay the groundwork for characterizing the global otolaryngology audience but also for future development of targeted educational materials and accessibility initiatives aimed at ameliorating global educational disparities in the field.

Assessing social vulnerabilities of salivary gland cancer care, prognosis, and treatment in the United States.

BACKGROUND: Salivary gland cancers (SGC)-social determinants of health (SDoH) investigations are limited by narrow scopes of SGC-types and SDoH. This Social Vulnerability Index (SVI)-study hypothesized that socioeconomic status (SES) most contributed to SDoH-associated SGC-disparities. METHODS: Retrospective cohort of 24 775 SGCs assessed SES, minority-language status (ML), household composition (HH), housing-transportation (HT), and composite-SDoH measured by the SVI via regressions with surveillance and survival length, late-staging presentation, and treatment (surgery, radio-, chemotherapy) receipt. RESULTS: Increasing social vulnerability showed decreases in surveillance/survival; increased odds of advanced-presenting-stage (OR: 1.12, 95% CI: 1.07, 1.17), chemotherapy receipt (OR: 1.13, 95% CI: 1.03, 1.23); decreased odds of primary surgery (0.89, 0.84, 0.94), radiotherapy (0.91, 0.85, 0.97, p = 0.003) for SGCs. Trends were differentially correlated with SES, ML, HH, and HT-vulnerabilities. CONCLUSIONS: Through quantifying SDoH-derived SGC-disparities, the SVI can guide targeted initiatives against SDoH that elicit the most detrimental associations for specific sociodemographics.

Microsatellite Instability Detection in Cancer: A Multiplex qPCR Approach that Obviates the Need for Matching Normal Samples.

BACKGROUND: Microsatellite instability (MSI) indicates DNA mismatch repair deficiency in certain types of cancer, such as colorectal cancer. The current gold standard technique, PCR-capillary electrophoresis (CE), requires matching normal samples and specialized instrumentation. We developed VarTrace, a rapid and low-cost quantitative PCR (qPCR) assay, to evaluate MSI using solely the tumor sample DNA, obviating the requirement for matching normal samples. METHODS: One hundred and one formalin-fixed paraffin-embedded (FFPE) tumor samples were tested using VarTrace and compared with the Promega OncoMate assay utilizing PCR-CE. Tumor percentage limit of detection was evaluated on contrived samples derived from clinical high MSI (MSI-H) samples. Analytical sensitivity, specificity, limit of detection, and input requirements were assessed using synthetic commercial reference standards. RESULTS: VarTrace successfully analyzed all 101 clinical FFPE samples, demonstrating 100% sensitivity and 98% specificity compared to OncoMate. It detected MSI-H with 97% accuracy down to 10% tumor. Analytical studies using synthetic samples showed a limit of detection of 5% variant allele frequency and a limit of input of 0.5 ng. CONCLUSIONS: This study validates VarTrace as a swift, accurate, and economical assay for MSI detection in samples with low tumor percentages without the need for matching normal DNA. VarTrace's capacity for highly sensitive MSI analysis holds potential for enhancing the efficiency of clinical work flows and broadening the availability of this test.

Activity-dependent compartmentalization of dendritic mitochondria morphology through local regulation of fusion-fission balance in neurons in vivo.

Neuronal mitochondria play important roles beyond ATP generation, including Ca2+ uptake, and therefore have instructive roles in synaptic function and neuronal response properties. Mitochondrial morphology differs significantly between the axon and dendrites of a given neuronal subtype, but in CA1 pyramidal neurons (PNs) of the hippocampus, mitochondria within the dendritic arbor also display a remarkable degree of subcellular, layer-specific compartmentalization. In the dendrites of these neurons, mitochondria morphology ranges from highly fused and elongated in the apical tuft, to more fragmented in the apical oblique and basal dendritic compartments, and thus occupy a smaller fraction of dendritic volume than in the apical tuft. However, the molecular mechanisms underlying this striking degree of subcellular compartmentalization of mitochondria morphology are unknown, precluding the assessment of its impact on neuronal function. Here, we demonstrate that this compartment-specific morphology of dendritic mitochondria requires activity-dependent, Ca2+ and Camkk2-dependent activation of AMPK and its ability to phosphorylate two direct effectors: the pro-fission Drp1 receptor Mff and the recently identified anti-fusion, Opa1-inhibiting protein, Mtfr1l. Our study uncovers a signaling pathway underlying the subcellular compartmentalization of mitochondrial morphology in dendrites of neurons in vivo through spatially precise and activity-dependent regulation of mitochondria fission/fusion balance.

Limb salvage reconstruction of the lower limb with complex ankle arthrodesis and magnetic internal lengthening nail.

PURPOSE: With advances in orthopedic implants, the use of intramedullary lengthening devices has gained increasing popularity as an alternative technique compared to lengthening with external fixators, with alleged comparable or better outcomes. The aim of this study is to report our single-center technique and outcomes of combined ankle arthrodesis and proximal tibial lengthening using external fixator with a motorized intramedullary nail, respectively. METHOD: Fourteen patients with post-traumatic advanced ankle arthritis underwent staged ankle arthrodesis with external fixator and proximal tibial lengthening using the PRECICE® ILN. Amount of shortening, length achieved, bone healing index, infection rate, ankle fusion rate, and ASAMI score were evaluated. RESULTS: The average age was 44 years old (range, 30-62). The mean follow up is 70 months (range, 43-121.4). The average amount of limb shortening for patients after ankle fusion was 36.7 mm (18-50) while lengthening was 35.9 mm (range, 18-50). Patients had the nail implanted for an average of 479 days (range, 248-730). Ankle fusions were healed in an average of 178.3 days. There were no surgical infections. All osteotomy-lengthening sites healed after an average 202 days (106-365). The mean bone healing index (BHI) was 56.0 days/cm (21.2-123.6) among the whole cohort. There were no cases of nonunion. ASAMI bone scores were excellent or good among all patients. CONCLUSION: Ankle arthrodesis with external fixation along with proximal tibial lengthening using motorized IMN yielded high rates of fusion and successful lengthening. This technique could be offered as a reasonable alternative to using external fixation for both purposes. LEVEL OF EVIDENCE: Level IV, Retrospective cohort study.

Redirecting raltitrexed from cancer cell thymidylate synthase to Mycobacterium tuberculosis phosphopantetheinyl transferase.

There is a compelling need to find drugs active against Mycobacterium tuberculosis (Mtb). 4'-Phosphopantetheinyl transferase (PptT) is an essential enzyme in Mtb that has attracted interest as a potential drug target. We optimized a PptT assay, used it to screen 422,740 compounds, and identified raltitrexed, an antineoplastic antimetabolite, as the most potent PptT inhibitor yet reported. While trying unsuccessfully to improve raltitrexed's ability to kill Mtb and remove its ability to kill human cells, we learned three lessons that may help others developing antibiotics. First, binding of raltitrexed substantially changed the configuration of the PptT active site, complicating molecular modeling of analogs based on the unliganded crystal structure or the structure of cocrystals with inhibitors of another class. Second, minor changes in the raltitrexed molecule changed its target in Mtb from PptT to dihydrofolate reductase (DHFR). Third, the structure-activity relationship for over 800 raltitrexed analogs only became interpretable when we quantified and characterized the compounds' intrabacterial accumulation and transformation.

High-performance ferroelectric field-effect transistors with ultra-thin indium tin oxide channels for flexible and transparent electronics.

With the development of wearable devices and hafnium-based ferroelectrics (FE), there is an increasing demand for high-performance flexible ferroelectric memories. However, developing ferroelectric memories that simultaneously exhibit good flexibility and significant performance has proven challenging. Here, we developed a high-performance flexible field-effect transistor (FeFET) device with a thermal budget of less than 400 °C by integrating Zr-doped HfO2 (HZO) and ultra-thin indium tin oxide (ITO). The proposed FeFET has a large memory window (MW) of 2.78 V, a high current on/off ratio (ION/IOFF) of over 108, and high endurance up to 2×107 cycles. In addition, the FeFETs under different bending conditions exhibit excellent neuromorphic properties. The device exhibits excellent bending reliability over 5×105 pulse cycles at a bending radius of 5 mm. The efficient integration of hafnium-based ferroelectric materials with promising ultrathin channel materials (ITO) offers unique opportunities to enable high-performance back-end-of-line (BEOL) compatible wearable FeFETs for edge intelligence applications.

Ionic Diffusive Nanomemristors with Dendritic Competition and Cooperation Functions for Ultralow Voltage Neuromorphic Computing.

Realization of dendric signal processing in the human brain is of great significance for spatiotemporal neuromorphic engineering. Here, we proposed an ionic dendrite device with multichannel communication, which could realize synaptic behaviors even under an ultralow action potential of 80 mV. The device not only could simulate one-to-one information transfer of axons but also achieve a many-to-one modulation mode of dendrites. By the adjustment of two presynapses, Pavlov's dog conditioning experiment was learned successfully. Furthermore, the device also could emulate the biological synaptic competition and synaptic cooperation phenomenon through the comodulation of three presynapses, which are crucial for artificial neural network (ANN) implementation. Finally, an ANN was further constructed to realize highly efficient and anti-interference recognition of fashion patterns. By introducing the cooperative device, synaptic weight updates could be improved for higher linearity and larger dynamic regulation range in neuromorphic computing, resulting in higher recognition accuracy and efficiency. Such an artificial dendric device has great application prospects in the processing of more complex information and the construction of an ANN system with more functions.

Enhanced Detection of Novel Low-Frequency Gene Fusions via High-Yield Ligation and Multiplexed Enrichment Sequencing.

Panel-based methods are commonly employed for the analysis of novel gene fusions in precision diagnostics and new drug development in cancer. However, these methods are constrained by limitations in ligation yield and the enrichment of novel gene fusions with low variant allele frequencies. In this study, we conducted a pioneering investigation into the stability of double-stranded adapter DNA, resulting in improved ligation yield and enhanced conversion efficiency. Additionally, we implemented blocker displacement amplification, achieving a remarkable 7-fold enrichment of novel gene fusions. Leveraging the pre-enrichment achieved with this approach, we successfully applied it to Nanopore sequencing, enabling ultra-fast analysis of novel gene fusions within one hour with high sensitivity. This method offers a robust and remarkably sensitive mean of analyzing novel gene fusions, promising the discovery of pivotal biomarkers that can significantly improve cancer diagnostics and the development of new therapeutic strategies.

Cortico-cortical evoked potentials of language tracts in minimally invasive glioma surgery guided by Penfield stimulation.

OBJECTIVE: We investigated the feasibility of recording cortico-cortical evoked potentials (CCEPs) in patients with low- and high-grade glioma. We compared CCEPs during awake and asleep surgery, as well as those stimulated from the functional Broca area and recorded from the functional Wernicke area (BtW), and vice versa (WtB). We also analyzed CCEP properties according to tumor location, histopathology, and aphasia. METHODS: We included 20 patients who underwent minimally invasive surgery in an asleep-awake-asleep setting. Strip electrode placement was guided by classical Penfield stimulation of positive language sites and fiber tracking of the arcuate fascicle. CCEPs were elicited with alternating monophasic single pulses of 1.1 Hz frequency and recorded as averaged signals. Intraoperatively, there was no post-processing of the signal. RESULTS: Ninety-seven CCEPs from 19 patients were analyzed. There was no significant difference in CCEP properties when comparing awake versus asleep, nor BtW versus WtB. CCEP amplitude and latency were affected by tumor location and histopathology. CCEP features after tumor resection correlated with short- and long-term postoperative aphasia. CONCLUSION: CCEP recordings are feasible during minimally invasive surgery. CCEPs might be surrogate markers for altered connectivity of the language tracts. SIGNIFICANCE: This study may guide the incorporation of CCEPs into intraoperative neurophysiological monitoring.

Focus Affinity Perception and Super-Resolution Embedding for Multifocus Image Fusion.

Despite the fact that there is a remarkable achievement on multifocus image fusion, most of the existing methods only generate a low-resolution image if the given source images suffer from low resolution. Obviously, a naive strategy is to independently conduct image fusion and image super-resolution. However, this two-step approach would inevitably introduce and enlarge artifacts in the final result if the result from the first step meets artifacts. To address this problem, in this article, we propose a novel method to simultaneously achieve image fusion and super-resolution in one framework, avoiding step-by-step processing of fusion and super-resolution. Since a small receptive field can discriminate the focusing characteristics of pixels in detailed regions, while a large receptive field is more robust to pixels in smooth regions, a subnetwork is first proposed to compute the affinity of features under different types of receptive fields, efficiently increasing the discriminability of focused pixels. Simultaneously, in order to prevent from distortion, a gradient embedding-based super-resolution subnetwork is also proposed, in which the features from the shallow layer, the deep layer, and the gradient map are jointly taken into account, allowing us to get an upsampled image with high resolution. Compared with the existing methods, which implemented fusion and super-resolution independently, our proposed method directly achieves these two tasks in a parallel way, avoiding artifacts caused by the inferior output of image fusion or super-resolution. Experiments conducted on the real-world dataset substantiate the superiority of our proposed method compared with state of the arts.

Assessment of Social Vulnerabilities of Care and Prognosis in Adult Ocular Melanomas in the US.

BACKGROUND: Prior works have studied the impact of social determinants on various cancers but there is limited analysis on eye-orbit cancers. Current literature tends to focus on socioeconomic status and race, with sparse analysis of interdisciplinary contributions. We examined social determinants as measured by the Centers for Disease Control and Prevention (CDC) Social Vulnerability Index (SVI), quantifying eye and orbit melanoma disparities across the United States. METHODS: A retrospective review of 15,157 patients diagnosed with eye-orbit cancers in the Surveillance, Epidemiology, and End Results (SEER) database from 1975 to 2017 was performed, extracting 6139 ocular melanomas. SVI scores were abstracted and matched to SEER patient data, with scores generated by weighted averages per population density of county's census tracts. Primary outcome was months survived, while secondary outcomes were advanced staging, high grading, and primary surgery receipt. RESULTS: With increased total SVI score, indicating more vulnerability, we observed significant decreases of 23.1% in months survival for melanoma histology (p < 0.001) and 19.6-39.7% by primary site. Increasing total SVI showed increased odds of higher grading (odds ratio [OR] 1.20, 95% confidence interval [CI] 1.02-1.43) and decreased odds of surgical intervention (OR 0.94, 95% CI 0.92-0.96). Of the four themes, higher magnitude contributions were observed with socioeconomic status (26.0%) and housing transportation (14.4%), while lesser magnitude contributions were observed with minority language status (13.5%) and household composition (9.0%). CONCLUSIONS: Increasing social vulnerability, as measured by the CDC SVI and its subscores, displayed significant detrimental trends in prognostic and treatment factors for adult eye-orbit melanoma. Subscores quantified which social determinants contributed most to disparities. This lays groundwork for providers to target the highest-impact social determinant for non-clinical factors in patient care.

Prediction of Visual Field Progression with Baseline and Longitudinal Structural Measurements Using Deep Learning.

PURPOSE: Identifying glaucoma patients at high risk of progression based on widely available structural data is an unmet task in clinical practice. We test the hypothesis that baseline or serial structural measures can predict visual field (VF) progression with deep learning (DL). DESIGN: Development of a DL algorithm to predict VF progression. METHODS: 3,079 eyes (1,765 patients) with various types of glaucoma and ≥5 VFs, and ≥3 years of follow-up from a tertiary academic center were included. Serial VF mean deviation (MD) rates of change were estimated with linear-regression. VF progression was defined as negative MD slope with p<0.05. A Siamese Neural Network with ResNet-152 backbone pre-trained on ImageNet was designed to predict VF progression using serial optic-disc photographs (ODP), and baseline retinal nerve fiber layer (RNFL) thickness. We tested the model on a separate dataset (427 eyes) with RNFL data from different OCT. The Main Outcome Measure was Area under ROC curve (AUC). RESULTS: Baseline average (SD) MD was 3.4 (4.9)dB. VF progression was detected in 900 eyes (29%). AUC (95% CI) for model incorporating baseline ODP and RNFL thickness was 0.813 (0.757-0.869). After adding the second and third ODPs, AUC increased to 0.860 and 0.894, respectively (p<0.027). This model also had highest AUC (0.911) for predicting fast progression (MD rate <1.0 dB/year). Model's performance was similar when applied to second dataset using RNFL data from another OCT device (AUC=0.893; 0.837-0.948). CONCLUSIONS: DL model predicted VF progression with clinically relevant accuracy using baseline RNFL thickness and serial ODPs and can be implemented as a clinical tool after further validation.

Extended Enrichment for Ultrasensitive Detection of Low-Frequency Mutations by Long Blocker Displacement Amplification.

Detecting low-frequency DNA mutations hotspots cluster is critical for cancer diagnosis but remains challenging. Quantitative PCR (qPCR) is constrained by sensitivity, and allele-specific PCR is restricted by throughput. Here we develop a long blocker displacement amplification (LBDA) coupled with qPCR for ultrasensitive and multiplexed variants detection. By designing long blocker oligos to perfectly match wildtype sequences while mispairing with mutants, long blockers enable 14-44 nt enrichment regions which is 2-fold longer than normal BDA in the experiments. For wild template with a specific nucleotide, LBDA can detect different mutation types down to 0.5 % variant allele frequency (VAF) in one reaction, with median enrichment fold of 1,000 on 21 mutant DNA templates compared to the wild type. We applied LBDA-qPCR to detect KRAS and NRAS mutation hotspots, utilizing a single plex assay capable of covering 81 mutations and tested in synthetic templates and colorectal cancer tissue samples. Moreover, the mutation types were verified through Sanger sequencing, demonstrating concordance with results obtained from next generation sequencing. Overall, LBDA-qPCR provides a simple yet ultrasensitive approach for multiplexed detection of low VAF mutations hotspots, presenting a powerful tool for cancer diagnosis and monitoring.