Biology subjects

Lee, S. K.

Publications and source records attributed to Lee, S. K..

At least 19 recordsLinked to original sources

bioRxiv2026

Scalable and rare-variant aware genome inference across the 1kGP cohort

Pangenome graphs built from haplotype-resolved de novo assemblies enable accurate analysis of genetic variation. The short-read-based tool PanGenie efficiently genotypes variants discovered in a pangenome across large cohorts and outper-forms linear reference-based methods for structural variants (SVs). However, it cannot detect novel variants absent from the graph, missing many rare SVs (allele frequency < 1%) and was limited to graphs with 254 haplotypes. First, we introduce a haplotype sampling step that reduces the number of haplotypes using sample-specific k-mers before genotyping, decreasing runtime twelvefold and memory usage 1.4-fold at 30x coverage. Second, we present a polishing work-flow that corrects residual errors in haplotypes inferred from PanGenie genotypes and incorporates rare and private mutations. We genotype 3,202 samples from the 1000 Genomes Project and use low-coverage ONT data (967 samples) for polishing. We achieve a median QV of 46 and provide the 1,934 polished haplotype sequences as a community resource.

bioinformatics↗

bioRxiv2026

De novo design of a safe and potent respiratory syncytial virus immuno-focusing antigen

Respiratory syncytial virus (RSV) remains the leading cause of severe respiratory infections in infants, the elderly, and the immunocompromised. Although stabilized full-length pre-fusion (pre-F) protein vaccines are promising, enhanced respiratory disease (ERD) remains a critical safety concern. Here, we used artificial intelligence to design a de novo immuno-focused antigen that structurally preserves the RSV F head region containing critical neutralising epitopes-- site O, II and V-while replacing the non-neutralising stem with a computationally designed scaffold to minimise immunopathological risk. The lead candidate, aRF6, elicited robust protective immunity against RSV in mice and similar immunogenicity in non-human primates without detectable toxicity. Importantly, in stringent ERD-promoting models, aRF6 induced minimal pulmonary pathology and markedly attenuated Th2-biased cytokine responses, outperforming formalin-inactivated RSV and full-length-stabilized pre-F. The results of cryoelectron microscopy confirmed that the aRF6 structure precisely matched the computational predictions. These results demonstrated that computationally designed de novo immuno-focused antigens can yield safe and effective RSV vaccines, thereby providing a rational framework for next-generation vaccine development.

microbiology↗

bioRxiv2026

4-Hexylresorcinol Enhances Skeletal Muscle Glucose Handling through the AMPK-GLUT4 axis in Diabetic Rats

BackgroundSkeletal muscle insulin resistance in diabetes is characterized by impaired GLUT4 availability and dysregulated AMP-activated protein kinase (AMPK) signaling. We investigated whether 4-hexylresorcinol (4HR) modulates glucose metabolism and AMPK-acetyl-CoA carboxylase (ACC)-GLUT4 signaling in vitro and in streptozotocin (STZ)-induced diabetic rats. MethodsDifferentiated C2C12 myotubes were treated with 4HR (0.1-10 {micro}M) and assessed for glucose uptake, GLUT4 translocation, protein stability, proteasome involvement, and AMPK/ACC phosphorylation. In vivo, streptozotocin (STZ)-induced diabetic rats received subcutaneous 4HR (10 mg{middle dot}kgL{superscript 1}, 5 times/week for 7 weeks). Glycemic control, insulin secretion, muscle glycogen content, and serum protein profiles were evaluated. Results4HR markedly increased glucose uptake (>5-fold vs. control, p<0.05), accompanied by increased phosphorylation of AMPK (Thr172) and ACC (Ser79), and redistribution of GLUT4 toward the cell periphery. Cycloheximide chase and proteasome inhibition experiments indicated partial stabilization of GLUT4. In diabetic rats, 4HR reduced fasting glycemia, improved glucose tolerance, elevated circulating insulin, and restored glycogen-rich fibers in skeletal muscle. Serum profiling revealed a metabolic and anti-inflammatory shift, with increased AMPK, p-ACC, GLUT1, LGR4, LDHA, and PGC-1, alongside suppression of NF-{kappa}B and TNF-. Conclusion4HR improves skeletal muscle glucose metabolism in STZ-induced diabetes, associated with activation of AMPK-ACC signaling, preservation of GLUT4 expression, and restoration of glycogen content. These findings highlight 4HR as a promising candidate for further investigation in diabetic muscle dysfunction and metabolic disease.

molecular biology↗

bioRxiv2025

Cas9-enriched nanopore sequencing enables comprehensive and multiplexed detection of repeat expansions

Short tandem repeat (STR) expansion is a major genetic mechanism underlying numerous neurogenetic disorders. However, traditional PCR amplification and short-read next-generation sequencing (NGS)-based methods often fail to detect complex and large expansions including methylation information. Here, we modified an amplification-free nanopore Cas9-targeted sequencing (nCATS) platform and developed a dedicated analysis algorithm, STRiker, for the simultaneous assessment of all currently defined STR loci (56 sites) using a single test with genomic DNA from patient-derived blood cells. We ultimately identified pathogenic repeat expansions in 12 of 37 patients (32.4%) with cerebellar ataxia who remained genetically undiagnosed despite extensive prior genetic testing, in FGF14 (n=4), ATXN8OS, NOP56, RFC1 (n=2 each), and PRNP and NOTCH2NLC (n=1 each). Additionally, family-based cascade screening revealed six relatives with repeat expansions in five families. These results demonstrated a broader diversity of pathogenic repeat structures, particularly in FGF14, and illustrated that CpG methylation can mitigate the pathogenic effects of repeat expansions. This approach offers a powerful strategy for improving the diagnosis of STR-related neurogenetic diseases, for cerebellar ataxia and other STR-related diseases in the future. One Sentence SummaryImproved nanopore Cas9-targeted sequencing with STRiker enhances diagnosis of STR- related neurogenetic diseases and offers broader molecular insights.

bioengineering↗

bioRxiv2025

Machine learning-driven decoding of maternal immune signatures in repeated pregnancy loss

BackgroundRepeated pregnancy loss (RPL) is a multifactorial condition in which the underlying immunological mechanisms remain incompletely understood. Although immune tolerance at the maternal-fetal interface is critical for successful pregnancy, the immune disruptions that contribute to RPL, independent of fetal aneuploidy, remain poorly characterized. MethodsWe performed single-cell RNA sequencing of decidual tissues from RPL patients and gestational age-matched controls. We employed single-cell transcriptomics coupled with genotype-based origin analysis to dissect immune dysregulation in RPL. To identify cell type-specific RPL signatures, we mutually applied a supervised machine learning model and foundation model-based analysis, followed by pathway enrichment and network analysis. In addition, we prioritized drug repurposing candidates based on the drug response data, which reversed the RPL-associated transcriptional profiles. ResultsIn normal pregnancy, fetal immune cells were nearly absent but increased in RPL, whereas fetal trophoblasts were reduced, suggesting hindered placental development despite the absence of chromosomal abnormalities in fetal cells. Interestingly, by showing a strong common rejection module score, RPL immune cells resemble the transcriptional signatures of acute transplant rejection, indicating the contribution of the maternal immune system. Our machine learning and transformer-based models mutually identified T-cell-derived transcriptomic signatures that distinguished RPL immune cells. By examining biological confounding factors, including fetal-origin signatures, we prioritized CXCR4 and JUN in maternal T cells as RPL-associated signatures. For clinical application, we explored reversed transcriptional signatures from drug response data, and three compounds were highlighted as candidates for drug repurposing. ConclusionsTogether, our approach identifies maternal immune signatures such as those of CXCR4 and JUN and links them to potential drug repurposing candidates, thereby providing both mechanistic insights and therapeutic opportunities for RPL.

bioinformatics↗

bioRxiv2025

Genomic factors limiting the diversity of Saccharomycotina plant pathogens

The Saccharomycotina fungi have evolved to inhabit a vast diversity of habitats over their 400-million-year evolution. There are, however, only a few known fungal pathogens of plants in this subphylum, primarily belonging to the genera Eremothecium and Geotrichum. We compared the genomes of 12 plant-pathogenic Saccharomycotina strains to 360 plant-associated strains to identify features unique to the phytopathogens. Characterization of the oxylipin synthesis genes, a compound believed to be involved in Eremothecium pathogenicity, did not reveal any differences in gene presence within or between the plant-pathogenic and plant-associated strains. A reverse-ecological approach, however, revealed that plant pathogens lack several metabolic enzymes known to assist other phytopathogens in overcoming plant defenses. This includes L-rhamnose metabolism, formamidase and nitrilase genes. This result suggests that the Saccharomycotina plant pathogens are limited to infecting ripening fruits as they are without the necessary enzymes to degrade common phytohormones and secondary metabolites produced by plants.

genomics↗

bioRxiv2025

Perilysosomal Ca2+ overload impairs autophagic degradation in β-cell lipotoxicity

Saturated fatty acids impose lipotoxic stress on pancreatic {beta}-cells, leading to {beta}-cell failure and diabetes. In this study, we investigate the critical role of organellar Ca2+ disturbance on defective autophagy and {beta}-cell lipotoxicity. Palmitate, a saturated fatty acid, induced perilysosomal Ca2+ elevation, sustained mTORC1 activation on the lysosomal membrane, suppression of the lysosomal transient receptor potential mucolipin 1 (TRPML1) channel, and accumulation of undigested autophagosomes in {beta}-cells. These Ca2+ aberrations with autophagy defects by palmitate were prevented by a mTORC1 inhibitor or a mitochondrial superoxide scavenger. To alleviate perilysosomal Ca2+ overload, strategies such as lowering extracellular Ca2+, employing voltage-gated Ca2+ channel blocker or ATP-sensitive K+ channel opener effectively abrogated mTORC1 activation and preserved autophagy. Furthermore, redirecting perilysosomal Ca2+ into the endoplasmic reticulum (ER) with an ER Ca2+ ATPase activator, restores TRPML1 activity, promotes autophagic flux, and improves survival of {beta}-cells exposed to palmitate-induced lipotoxicity. Our findings suggest oxidative stress-Ca2+ overload-mTORC1 pathway involves in TRPML1 suppression and defective autophagy during {beta}-cell lipotoxicity. Restoring perilysosomal Ca2+ homeostasis emerges as a promising therapeutic strategy for metabolic diseases.

physiology↗

bioRxiv2024

Cyclic Electromagnetic DNA Simulation (CEDS) can enhance the Functions of Anticancer miR-26a-5p and miR-126-5p in RAW 264.7 cells

Since cyclic electromagnetic DNA simulation (CEDS) was found to increase the hybridization potential of double-stranded DNA (dsDNA) and affect the functions of plasmid DNA (1), in this study, Dodecagonal CEDS was applied to RAW 264.7 cells to target anticancer miRNAs, miR-26a-5p and miR-126-5p, at 20-25 Gauss for 20 min during cell culture, followed by cytological observation, quantitative polymerase chain reaction (qPCR), and immunoprecipitation-based high-performance liquid chromatography (IP-HPLC). Both of miR-26a-5p*-CEDS and miR-126-5p*-CEDS induced significant decrease in cell number, and strong immunoreaction of c-caspase 3 in the remaining cells compared to oncogenic miR-21-5p*-CEDS and untreated control. In qPCR results, miR-26a-5p*-CEDS and miR-126-5p*-CEDS markedly increased the expression of target primary and mature miRNAs. IP-HPLC analysis showed that both miR-26a-5p*-CEDS and miR-126-5p*-CEDS induced potent anticancer effect on RAW 264.7 cells by suppressing RAS signaling, proliferation-growth-cytodifferentiation signaling axis, and angiogenesis-survival-oncogenesis-chronic inflammation axis compared to the untreated control and the positive control performed with non-specific poly-A 12A*-CEDS. MiR-26a-5p*-CEDS increased miRNA biogenesis and ER stress signaling more than miR-126-5p*-CEDS, while miR-126-5p*-CEDS increased NFkB signaling-immunity-apoptosis signaling axis more than miR-26a-5p*-CEDS. Therefore, it is proposed that the sequential treatment of miR-26a-5p*-CEDS and miR-126-5p*-CEDS may give a synergistic anticancer effect on RAW 264.7 cells. It is postulated that CEDS using a miRNA sequence can stimulate hybridization between miRNA and target mRNA and also increase the expression of target miRNA, and subsequently decrease the expression of miRNA target proteins, resulting in the alteration of protein signaling pathways in cells.

molecular biology↗

bioRxiv2024

Cyclic Electromagnetic DNA Simulation (CEDS) targeting Telomere Repeat Sequence can enhance Anticancer Effect, while CEDS targeting Canonical E-box Sequence induce Oncogenic Effect in Cells

Cyclic electromagnetic DNA simulation (CEDS) was used in the previous study (1) to stimulate the base pair polarities of oligo-dsDNAs, plasmid DNAs, and miRNAs. This study applied dodecagonal CEDS to target telomere repeat sequence (TTAGGG*-CEDS), canonical E-box sequence (CACGTG*-CEDS) in genomic DNA of RAW 264.7 cells and KB cells. During cell culture, CEDS was performed at 20-25 Gauss for 20 min, and followed by cytological observation and immunoprecipitation high-performance liquid chromatography (IP-HPLC) using 350 antisera. KB cells treated with TTAGGG*-CEDS showed a significant decrease in cell density and metachromasia in toluidine blue staining compared to untreated control, while only a slight decrease in cell density and metachromasia was observed by telomere mutation sequence, TTTGGG*-CEDS. Conversely, CACGTG*-CEDS induced almost no change in cell density, cell number, metachromasia in toluidine blue staining compared to random sequence*-CEDS (2(ACGT)*-CEDS) and untreated control. IP-HPLC results showed that TTAGGG*-CEDS induced a potent anticancer effect by downregulating RAS, oncogenesis, and telomere signaling, and activating NFkB signaling-p53-and FAS-mediated apoptosis-innate and cellular immunity signaling axis compared to untreated control and positive control performed with nonspecific poly-A 12A*-CEDS. In contrast, CACGTG*-CEDS exhibited a significant oncogenic effect by enhancing of RAS and NFkB signaling and resulting oncogenesis-glycolysis-chronic inflammation signaling axis. Therefore, it is suggested that CEDS is able to target DNA motif sequences in genomic DNA, and that TTAGGG*-CEDS and CACGTG*-CEDS are favorable candidates to regulate important genes involved in oncogenesis, inflammation, apoptosis, etc.

molecular biology↗

bioRxiv2024

Cyclic Electromagnetic DNA Simulation (CEDS) influences the functions of Long Oligo-dsDNAs and Plasmid DNAs

Hydrogen bonding magnetic resonance-based cyclic electromagnetic DNA simulation (CEDS) was proposed to play a role in regulating the function of target short oligo-dsDNA in the previous study, as the decagonal CEDS was found to induce the hybridization and conformational changes of target short oligo-dsDNA in a sequence-specific manner. This study applied dodecagonal CEDS on 24 bps oligo-dsDNAs and plasmid DNAs. CEDS can affect 24 bps oligo-dsDNAs intercalated by ethidium bromide (EtBr) or condensed by spermidine by stimulating them to partially remove EtBr or spermidine, respectively, depending on CEDS time. When the multiple cloning site of pBluescript was digested with eight restriction endonucleases (RE) under CEDS separately, all RE digests were enhanced depending on CEDS time compared to the negative and positive controls both in EtBr intercalated electrophoresis and HPLC analysis. In vitro RNA transcription from human VEGFA, vWF or elafin cDNA subcloned into pBluescript DNA was increased by CEDS using each promoter sequence depending on CEDS time compared to negative and positive controls. In addition, the productions of green fluorescent protein (GFP) and {beta}-galactosidase were enhanced by CEDS using each promoter sequence from pE-GFP-1 and pBluescript, respectively. In the survival assay using E. coli transfected with pBluescript containing ampicillin resistance gene, the number of surviving E. coli was increased by CEDS using a T3 promoter sequence compared to the control during two days of culture, while it was decreased by CEDS using a T7 promoter, non-specific 12A, 6(TA), or mutation-2 (GG18,19CC) T3 promoter sequence. Therefore, it is suggested that dodecagonal CEDS can target 24 bps oligo-dsDNAs and promoter sequences of plasmid DNAs in a sequence-specific manner and influence their functions.

molecular biology↗

bioRxiv2024

Hydrogen Bonding Magnetic Resonance (HBMR)-based Cyclic Electromagnetic DNA Simulation (CEDS) can affect DNA Hybridization and Conformation

The proton magnetic resonance is commonly used in MRI machines with a strong magnetic field of over 1 T, while this study hypothesized that a weak magnetic field below 0.01 T can induce the proton magnetic resonance in hydrogen bonds of double-stranded DNA (dsDNA). This study found the hydrogen bonding magnetic resonance (HBMR) in dsDNA can modulate the conformations and functions of dsDNAs (1). Decagonal or dodecagonal cyclic electromagnetic DNA simulation (CEDS) was designed to target three-dimensional structures of randomly oriented dsDNAs with about 25% efficiency. This study found that the most effective magnetic exposure times for inducing an electric potential in A-T and G-C base pairs were 280 and 480 msec, respectively. Decagonal CEDS using a target sequence at 20-25 Gauss for 30 min was able to induce sequence-specific hybridization of target short oligo-dsDNAs in 0.005M NaCl solution and their unique conformation in 0.1M NaCl solution. There was a tendency that Pyu oligo-dsDNAs showed more hybridization and unique conformational changes by CEDS using a target sequence than Puy oligo-dsDNAs. This CEDS effect on Pyu ds6(2C2A) increased with CEDS time up to 90 min and gradually decreased to about half (51.8%) of the increase at 240 min resting time. CEDS influenced the oligo-dsDNAs to increase their infrared (IR) absorbance at approximately 3700-2800 cm-1 band compared to the positive and negative controls, which was more dominant in Pyu oligo-dsDNAs than in Puy oligo-dsDNAs. Therefore, it is postulated that HBMR-based CEDS using a weak magnetic field can increase the hybridization potential of oligo-dsDNAs and subsequently lead to unique DNA conformation required for the initiation of various DNA functions. Therefore, it is suggested that decagonal CEDS play a role in regulating the function of target short oligo-dsDNA.

molecular biology↗

bioRxiv2024

Cyclic Electromagnetic DNA Simulation (CEDS) can regulate the Functions of Oncogenesis-related miRNAs and DNA Motifs: Protein Signaling Pathways in RAW 264.7 Cells detected by IP-HPLC Analysis

Based on the hydrogen bonding magnetic resonance (HBMR) in double-stranded DNAs (dsDNAs) exhibiting unique base pair polarities, Cyclic Electromagnetic DNA Simulation (CEDS)1 was designed to target nine miRNAs and four protein binding site sequences, which are known to have an anti-oncogenic or oncogenic role in cells. RAW 264.7 cells were cultured and treated with CEDS at 20-25 Gauss in a 5% CO2 and 37 incubator for 20 min. Besides the cytological observation, the expressions of murine miRNAs and proteins were assessed by quantitative polymerase chain reaction (qPCR) and immunoprecipitation high-performance liquid chromatography (IP-HPLC) using 350 antisera, respectively. The results demonstrated that CEDS was found to increase the number of the corresponding mature and primary miRNAs and influenced the protein signaling in cells. For example, miR-34a-5p*-CEDS was able to downregulate 59 out of 43 target proteins (72.9%), and attenuated RAS-NFkB signaling axis, which subsequently led to the inactivation of proliferation-related signaling axis, protection-ER stress-survival signaling axis, telomere-glycolysis-fibrosis signaling axis, and FAS- and PARP-mediated apoptosis signaling axis. Conversely, miR-34a-5p*-CEDS activated the p53/Rb/E2F1-SHH/PTCH/GLI-Notch/Jagged proliferation signaling axis, epigenetic modification-RAS-p53 associated apoptosis signaling axis, and oncogenesis-senescence-innate immunity-chronic inflammation signaling axis. Among nine CEDS targeting mature miRNAs, miR-150-5p*-CEDSs exhibited a potent anti-oncogenic effect on RAW 264.7 cells, while miR-34a-5p*-, miR-181a-5p*-, miR-216a-5p*-, miR-365a-3p*-, and miR-655-3p*-CEDSs exhibited a dual effect, inducing both anti-oncogenic and oncogenic effects. Conversely, miR-155-5p*-CEDS appears to induce marked oncogenic effect, rather than anti-oncogenic effect. Among four CEDS targeting protein binding site sequences, p53 binding site sequence AGACATGCCT*-CEDS induced an oncogenic effect by upregulating proliferation, telomere, and oncogenesis signaling, while concomitantly inducing an anti-oncogenic effect by downregulating RAS and NFkB signaling and upregulating p53- and PARP-mediated apoptosis. Canonical E2Fs binding site sequence TTTC(C/G)CGC*-CEDS facilitated an oncogenic effect by enhancing RAS and NFkB signaling, resulting in the upregulation of proliferation, telomere, and senescence signaling. Consequently, it was found that CEDS using a miRNA or protein binding site sequence impacted the protein signaling in RAW 264.7 cells in a sequence-specific manner. It is therefore proposed that CEDS can target miRNAs and DNA motifs, influencing their hybridization and conformation, and subsequently regulate the protein signaling in the cells.

molecular biology↗

bioRxiv2024

DNA Base Pair Polarities of Pyrimidine(s)-Purine(s) (Pyu) dsDNA induce stronger Hybridization and unique DNA Conformation compared to Purine(s)-Pyrimidine(s) (Puy) dsDNA

The present study introduces a concept of DNA base pair polarity in double-stranded DNA (dsDNA), which has led the serial investigation of hydrogen bonding magnetic resonance-based gene regulation. DNA base pair polarity is characterized by hydrogen bonding between electrostatically positive purine and negative pyrimidine, exerting the stabilization and organization of dsDNA. Comparison of DNA hybridization potential between Pyu (pyrimidine(s)-purine(s)) and Puy (purine(s)-pyrimidine(s)) oligo-dsDNAs in EtBr intercalated electrophoresis showed that Pyu ds(nTnA) (n=3, 4,5,6,7,8,10,12) and ds(nCnA) (n=7,8,10,12) exhibit stronger hybridization than Puy ds(nAnT) and ds(nAnC), respectively. In the HPLC for DNA conformation change in 0.1M NaCl solution, Pyu oligo-dsDNA tended to have a larger peak area than Puy oligo-dsDNA, indicating that the former had a more extended and organized DNA conformation than the latter. FT-IR results showed that Pyu ds3T3A, ds3C3G, dsCTCGAG, and dsTCTAGA showed higher IR absorbance at 3400-3200 cm-1 than Puy ds3A3T, ds3G3C, dsGAGCTC, and dsAGATCT, respectively, indicating Pyu oligo-dsDNAs have increased N-H stretching in hybridization than Puy oligo-dsDNAs. In vitro RNA transcription from template plasmid DNAs inserted with six segments of Pyu ds6(TCTGA) and positive ds14(GA) produced more RNA than those with seven segments of palindromic Pyu ds(CATG) and negative ds14(CT), respectively, indicating the former plays a stimulating role for RNA transcription while the latter plays an inhibitory role. When comparing PCR primers between Pyu ds12/n(nCnA) and Puy ds12/n(nAnC) (n=1,2,3,4,6,12), Pyu primers can easily hybridize on template DNA and subsequently produce abundant DNA compared to Puy primers. The data suggest that Pyu oligo-dsDNAs hybridize strongly and form distinctive conformation, thereby stimulating RNA transcription more than Puy oligo-dsDNAs. Since Pyu oligo-dsDNA appears to preserve DNA signal and conformation relevant to DNA function more efficiently than Puy dsDNA, it is proposed that the entire DNA code can be divided into Pyu dsDNAs with unique DNA base pair polarities.

molecular biology↗

bioRxiv2024

Genetic tuning of intrinsically photosensitive retinal ganglion cell subtype identity to drive visual behavior

The melanopsin-expressing, intrinsically photosensitive retinal ganglion cells (ipRGCs) comprise a subset of the [~]40 retinal ganglion cell types in the mouse retina and drive a diverse array of light-evoked behaviors from circadian photoentrainment to pupil constriction to contrast sensitivity for visual perception. Central to the ability of ipRGCs to control this diverse array of behaviors is the distinct complement of morphophysiological features and gene expression patterns found in the M1-M6 ipRGC subtypes. However, the genetic regulatory programs that give rise to subtypes of ipRGCs are unknown. Here, we identify the transcription factor Brn3b (Pou4f2) as a key genetic regulator that shapes the unique functions of ipRGC subtypes and their diverse downstream visual behaviors.

neuroscience↗

bioRxiv2023

Focal acetylcholinergic modulation of the human midcingulo-insular network during attention: Meta-analytic neuroimaging and behavioral evidence

The basal forebrain cholinergic neurons provide acetylcholine to the cortex via large projections. Recent molecular imaging work in humans indicates that the cortical cholinergic innervation is not uniformly distributed, but rather may disproportionately innervate cortical areas relevant to supervisory attention. In this study, we therefore reexamined the spatial relationship between acetylcholinergic modulation and attention in the human cortex using meta-analytic strategies targeting both pharmacological and nonpharmacological neuroimaging studies. We found that pharmaco-modulation of acetylcholine evoked both increased activity in the anterior cingulate and decreased activity in the opercular and insular cortex. In large independent meta-analyses of non-pharmacological neuroimaging research, we demonstrate that during attentional engagement these cortical areas exhibit (1) task-related co-activation with the basal forebrain, (2) task-related co-activation with one another, and (3) spatial overlap with dense cholinergic innervations originating from the BF, as estimated by multimodal PET and MR imaging. Finally, we provide meta-analytic evidence that pharmaco-modulation of acetylcholine also induces a speeding of responses to targets with no apparent tradeoff in accuracy. In sum, we demonstrate in humans that acetylcholinergic modulation of midcingulo-insular hubs of the ventral attention/salience network via basal forebrain afferents may coordinate selection of task relevant information, thereby facilitating cognition and behavior.

neuroscience↗

bioRxiv2022

GATK-gCNV: A Rare Copy Number Variant Discovery Algorithm and Its Application to Exome Sequencing in the UK Biobank

Copy number variants (CNVs) are major contributors to genetic diversity and disease. To date, exome sequencing (ES) has been generated for millions of individuals in international biobanks, human disease studies, and clinical diagnostic screening. While standardized methods exist for detecting short variants (single nucleotide and insertion/deletion variants) using tools such as the Genome Analysis ToolKit (GATK), technical challenges have confounded similarly uniform large-scale CNV analyses from ES data. Given the profound impact of rare and de novo coding CNVs on genome organization and human disease, the lack of widely-adopted and robustly benchmarked rare CNV discovery tools has presented a barrier to routine exome-wide assessment of this critical class of variation. Here, we introduce GATK-gCNV, a flexible algorithm to discover rare CNVs from genome sequencing read-depth information, which we distribute as an open-source tool packaged in GATK. GATK-gCNV uses a probabilistic model and inference framework that accounts for technical biases while simultaneously predicting CNVs, which enables self-consistency between technical read-depth normalization and variant calling. We benchmarked GATK-gCNV in 7,962 exomes from individuals in quartet families with matched genome sequencing and microarray data. These analyses demonstrated 97% recall of rare ([≤]1% site frequency) coding CNVs detected by microarrays and 95% recall of rare coding CNVs discovered by genome sequencing at a resolution of more than two exons. We applied GATK-gCNV to generate a reference catalog of rare coding CNVs in 197,306 individuals with ES from the UK Biobank. We observed strong correlations between CNV rates per gene and measures of mutational constraint, as well as rare CNV associations with multiple traits. In summary, GATK-gCNV is a tunable approach for sensitive and specific CNV discovery in ES, which can easily be applied across trait association and clinical screening.

genomics↗

bioRxiv2022

Cost-efficient whole genome-sequencing using novel mostly natural sequencing-by-synthesis chemistry and open fluidics platform

We introduce a massively parallel novel sequencing platform that combines an open flow cell design on a circular wafer with a large surface area and mostly natural nucleotides that allow optical end-point detection without reversible terminators. This platform enables sequencing billions of reads with longer read length ([~]300bp) and fast runs times (<20hrs) with high base accuracy (Q30 > 85%), at a low cost of $1/Gb. We establish system performance by whole-genome sequencing of the Genome-In-A-Bottle reference samples HG001-7, demonstrating high accuracy for SNPs (99.6%) and Indels in homopolymers up to length 10 (96.4%) across the vast majority (>98%) of the defined high-confidence regions of these samples. We demonstrate scalability of the whole-genome sequencing workflow by sequencing an additional 224 selected samples from the 1000 Genomes project achieving high concordance with reference data.

genomics↗

bioRxiv2021

Pentoxifylline-induced Protein Expression Change in RAW 264.7 Cells as Determined by Immunoprecipitation-based High Performance Liquid Chromatography

Although pentoxifylline (PTX) was identified as a competitive non-selective phosphodiesterase inhibitor, its pharmacological effect has not been clearly elucidated. The present study explored the effect of low dose 10 g/mL PTX (therapeutic dose) compared to high dose 300 g/mL PTX (experimental dose) in RAW 264.7 cells through immunoprecipitation-based high performance liquid chromatography (IP-HPLC), immunohistochemistry, and western blot. 10 g/mL PTX increased the expression of proliferation (Ki-67, PCNA, cyclin D2, cdc25A), epigenetic modification (KDM4D, PCAF), protein translation (DOHH, DHPS, eIF5A1), RAS signaling (KRAS, pAKT1/2/3, PI3K), NFkB signaling (NFkB, GADD45, p38), protection (HSP70, SOD1, GSTO1/2), neuromuscular differentiation (NSE{gamma}, myosin-1a, desmin), osteoblastic differentiation (BMP2, RUNX2, osterix), acute inflammation (TNF, IL-1, CXCR4), innate immunity ({beta}-defensin 1, lactoferrin, TLR-3, -4), cell-mediated immunity (CD4, CD8, CD80), while decreased the expression of ER stress (eIF2, eIF2AK3, ATF6), fibrosis (FGF2, CTGF, collagen 3A1), and chronic inflammation (CD68, MMP-2, -3, COX2) versus the untreated controls. The activation of proliferation by 10 g/mL PTX was also supported by the increase of cMyc-MAX heterodimer and {beta}-catenin-TCF1 complex in double IP-HPLC. 10 g/mL PTX enhanced FAS-mediated apoptosis but diminished p53-mediated apoptosis, and downregulated many angiogenesis proteins (angiogenin, VEGF-A, and FLT4), but upregulated HIF1, VEGFR2, and CMG2 reactively. Whereas, 300 g/mL PTX consistently decreased proliferation, epigenetic modification, RAS and NFkB signaling, neuromuscular and osteoblastic differentiation, but increased apoptosis, ER stress, and fibrosis compared to 10 g/mL PTX. These data suggest PTX has different biological effect on RWA 264.7 cells depending on the concentration of 10 g/mL and 300 g/mL PTX. The low dose 10 g/mL PTX enhanced RAS/NFkB signaling, proliferation, differentiation, and inflammation, particularly, it stimulated neuromuscular and osteoblastic differentiation, innate immunity, and cell-mediated immunity, but attenuated ER stress, fibrosis, angiogenesis, and chronic inflammation, while the high dose 300 g/mL PTX was found to alleviate the 10 g/mL PTX-induced biological effects, resulted in the suppression of RAS/NFkB signaling, proliferation, neuromuscular and osteoblastic differentiation, and inflammation.

pharmacology and toxicology↗