Biology subjects

Ko, S.

Publications and source records attributed to Ko, S..

32 records · Page 2Linked to original sources

bioRxiv2023

GRable version 1.0: A software tool for site-specific glycoform analysis using the improved Glyco-RIDGE method with parallel clustering and MS2 information

High-throughput intact glycopeptide analysis is crucial for elucidating the physiological and pathological status of the glycans attached to each glycoprotein. Mass spectrometry-based glycoproteomic methods are challenging because of the diversity and heterogeneity of glycan structures. Therefore, we have developed an MS1-based site-specific glycoform analysis method named "Glycan heterogeneity-based Relational IDentification of Glycopeptide signals on Elution profile (Glyco-RIDGE)" for a more comprehensive analysis. This method detects glycopeptide signals as a cluster based on the mass and chromatographic properties of glycopeptides and then searches for each combination of core peptides and glycan compositions by matching their mass and retention time differences. Here we developed a novel browser-based software named GRable for semi-automated Glyco-RIDGE analysis with significant improvements in glycopeptide detection algorithms, including "parallel clustering." This unique function improved the comprehensiveness of glycopeptide detection and allowed the analysis to focus on specific glycan structures, such as pauci-mannose. The other notable improvement is evaluating the "confidence level" of the GRable results, especially using MS2 information. This function facilitated reduced misassignment of the core peptide and glycan composition and improved the interpretation of the results. Additional improved points are: "correction function" for accurate monoisotopic peak picking; one-to-one correspondence of clusters and core peptides even for multiply sialylated glycopeptides; and "inter-cluster analysis" function for understanding the reason for detected but unmatched clusters. The significance of these improvements was demonstrated using purified and crude glycoprotein samples, showing that GRable allowed site-specific glycoform analysis of intact sialylated glycoproteins on a large scale and in depth. Therefore, this software will help to analyze the status and changes in glycans to obtain biological and clinical insights into protein glycosylation by complementing the comprehensiveness of MS2-based glycoproteomics. GRable can run freely online using a web browser via the GlyCosmos Portal (https://glycosmos.org/grable). Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/564073v2_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@12ae798org.highwire.dtl.DTLVardef@1cac352org.highwire.dtl.DTLVardef@dd6b92org.highwire.dtl.DTLVardef@c16ddf_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2023

Neurogenesis-mediated circuit remodeling reduces engram reinstatement and promotes forgetting

Post-training increases in hippocampal neurogenesis are associated with forgetting of hippocampus-dependent memories in adult mice. This form of forgetting might be due to increased numbers of new neurons, remodeling of hippocampal circuitry or some combination of both. Here we tested the hypothesis that neurogenesis-mediated forgetting is caused by remodeling of hippocampal circuits by engineering mice in which adult-generated granule cells hypo- or hyper-integrate into hippocampal circuits. Using gene deletion, opto- and chemogenetic strategies, we find that hypo-integration of newborn neurons prevents post- training exercise-induced forgetting of contextual fear memories. Conversely, inducing hyper- integration of newborn neurons following contextual fear conditioning is sufficient to produce forgetting. Because these interventions did not affect survival of newborn neurons, these findings suggest that neurogenesis-mediated remodeling of hippocampal circuits represents a continuous and active form of interference that alters accessibility of engrams underlying hippocampal memories. Consistent with this, using engram-labeling approaches, we found that exercise-induced forgetting was associated with reduced engram reactivation.

neuroscience↗

bioRxiv2023

Estimation of Genetic Admixture Proportions via Haplotypes

Estimation of ethnic admixture is essential for creating personal genealogies, studying human history, and conducting genome-wide association studies (GWAS). Three methods exist for estimating admixture coefficients. The frequentist approach directly maximizes the binomial loglikelihood. The Bayesian approach adds a reasonable prior and samples the posterior distribution. Finally, the nonparametric approach decomposes the genotype matrix algebraically. Each approach scales successfully to data sets with a million individuals and a million single nucleotide polymorphisms (SNPs). Despite their variety, all current approaches assume independence between SNPs. To achieve independence requires performing LD (linkage disequilibrium) filtering before analysis. Unfortunately, this tactic loses valuable information and usually retains many SNPs still in LD. The present paper explores the option of explicitly incorporating haplotypes in ancestry estimation. Our program, HaploADMIXTURE, operates on adjacent SNP pairs and jointly estimates their haplotype frequencies along with admixture coefficients. This more complex strategy takes advantage of the rich information available in haplotypes and ultimately yields better admixture estimates and better clustering of real populations in curated data sets.

bioinformatics↗

bioRxiv2023

Consistent specificity and efficiency of tamoxifen-mediated cre induction across ages

Temporally controlling cre recombination through tamoxifen (Tam) induction has many advantages for biomedical research. Most studies report Tam induction at early post-natal/juvenile (<2 m.o.) mouse ages, but age-related neurodegeneration and aging studies can require cre induction in older mice (>12 m.o.). While anecdotally reported as problematic, there are no published comparisons of Tam mediated cre induction at early and late ages. Here, microglial-specific Cx3cr1creERT2 mice were crossed to a floxed NuTRAP reporter to compare cre induction at early (3-6 m.o.) and late (20 m.o.) ages. Specificity and efficiency of microglial labeling at 21-22 m.o. were identical in mice induced with Tam at 3-6 m.o. or 20 m.o. of age. Age-related microglial translatomic changes were also similar regardless of Tam induction age. Each cre and flox mouse line should be validated independently, however, these findings demonstrate that Tam-mediated cre induction can be performed even into older mouse ages.

genomics↗

bioRxiv2023

Assessing the Predictive Ability of Computational Epitope Prediction Methods on Fel d 1 Allergen

While computational epitope prediction methods have found broad application, their use, specifically in allergy-related contexts, remains relatively less explored. This study benchmarks several publicly available epitope prediction tools, focusing on the allergenic IgE and T-cell epitopes of Fel d 1, an extensively studied allergen. Using a variety of tools accessible via the Immune Epitope Database (IEDB) and other resources, we evaluate their ability to identify the known linear IgE and T-cell epitopes of Fel d 1. Our results show a limited effectiveness for B-cell epitope prediction methods, with most performing only marginally better than random selection. We also explored the general predictive abilities on other allergens, and the results were largely random. When predicting T-cell epitopes, ProPred successfully identified all known Fel d 1 T-cell epitopes, whereas the IEDB approach missed two known epitopes and demonstrated a tendency to over-predict. However, when applied to a larger test set, both methods performed only slightly better than random selection. Our findings show the limitations of current computational epitope prediction methods in accurately identifying allergenic epitopes, emphasizing the need for methodological advancements in allergen research.

bioinformatics↗

bioRxiv2023

A single-cell atlas of the aging murine ovary

Ovarian aging leads to diminished fertility, dysregulated endocrine signaling, and increased chronic disease burden. These effects begin to emerge long before follicular exhaustion. Around 35 years old, women experience a sharp decline in fertility, corresponding to declines in oocyte quality. Despite a growing body of work, the field lacks a comprehensive cellular map of the transcriptomic changes in the aging ovary to identify early drivers of ovarian decline. To fill this gap, we performed single-cell RNA sequencing on ovarian tissue from young (3-month-old) and reproductively aged (9-month-old) mice. Our analysis revealed a doubling of immune cells in the aged ovary, with lymphocyte proportions increasing the most, which was confirmed by flow cytometry. We also found an age-related downregulation of collagenase pathways in stromal fibroblasts, which corresponds to rises in ovarian fibrosis. Follicular cells displayed stress response, immunogenic, and fibrotic signaling pathway inductions with aging. This report raises provides critical insights into mechanisms responsible for ovarian aging phenotypes.

cell biology↗

bioRxiv2023

Mature microRNA-binding protein QKI promotes microRNA-mediated gene silencing

Although Argonaute (AGO) proteins have been the focus of microRNA (miRNA) studies, we observed AGO-free mature miRNAs directly interacting with RNA-binding proteins, implying the sophisticated nature of fine-tuning gene regulation by miRNAs. To investigate microRNA-binding proteins (miRBPs) globally, we analyzed PAR-CLIP data sets to identify RBP quaking (QKI) as a novel miRBP for let-7b. Potential existence of AGO-free miRNAs were further verified in genetically engineered AGO-depleted human and mouse cells. We have shown that QKI serves as an auxiliary factor empowering AGO2/let-7b-mediated gene silencing. Depletion of QKI decreases interaction of AGO2 with let-7b and target mRNA, consequently controlling target mRNA decay. QKI, however, also suppresses the dissociation of let-7b from AGO2, and slows assembly of AGO2/miRNA/target mRNA complexes at the single-molecule level. We also revealed that QKI suppresses cMYC expression at post-transcriptional level, and decreases proliferation and migration of HeLa cells, demonstrating that QKI is a tumor suppressor gene by in part augmenting let-7b activity. Our data show that QKI is a new type of RBP implicated in the versatile regulation of miRNA-mediated gene silencing.

biochemistry↗

bioRxiv2023

Microglial senescence contributes to female-biased neuroinflammation in the aging mouse hippocampus: implications for Alzheimer's disease

BackgroundMicroglia, the brains principal immune cells, have been implicated in the pathogenesis of Alzheimers disease (AD), a condition shown to affect more females than males. Although sex differences in microglial function and transcriptomic programming have been described across development and in disease models of AD, no studies have comprehensively identified the sex divergences that emerge in the aging mouse hippocampus. Further, existing models of AD generally develop pathology (amyloid plaques and tau tangles) early in life and fail to recapitulate the aged brain environment associated with late-onset AD. Here, we examined and compared transcriptomic and translatomic sex effects in young and old murine hippocampal microglia. MethodsHippocampal tissue from C57BL6/N and microglial NuTRAP mice of both sexes were collected at young (5-6 month-old [mo]) and old (22-25 mo) ages. Cell sorting and affinity purification techniques were used to isolate the microglial transcriptome and translatome for RNA-sequencing and differential expression analyses. Flow cytometry, qPCR, and imaging approaches were used to confirm the transcriptomic and translatomic findings. ResultsThere were marginal sex differences identified in the young hippocampal microglia, with most differentially expressed genes (DEGs) restricted to the sex chromosomes. Both sex chromosomally-and autosomally-encoded sex differences emerged with aging. These sex DEGs identified at old age were primarily female-biased and enriched in senescent and disease-associated microglial signatures. Normalized gene expression values can be accessed through a searchable web interface (https://neuroepigenomics.omrf.org/). Pathway analyses identified upstream regulators induced to a greater extent in females than in males, including inflammatory mediators IFNG, TNF, and IL1B, as well as AD-risk genes TREM2 and APP. ConclusionsThese data suggest that female microglia adopt disease-associated and senescent phenotypes in the aging mouse hippocampus, even in the absence of disease pathology, to a greater extent than males. This sexually divergent microglial phenotype may explain the difference in susceptibility and disease progression in the case of AD pathology. Future studies will need to explore sex differences in microglial heterogeneity in response to AD pathology and determine how sex-specific regulators (i.e., sex chromosomal or hormonal) elicit these sex effects.

neuroscience↗

bioRxiv2022

Branched germline cysts and female-specific cyst fragmentation facilitate oocyte determination in mice

During mouse gametogenesis, germ cells derived from the same progenitor are connected via intercellular bridges forming germline cysts, within which asymmetrical or symmetrical cell fate occurs in female and male germ cells respectively. Here, we have identified branched cyst structures in mice, and investigated their formation and function in oocyte determination. In fetal female cysts, 16.8% of the germ cells are connected by three or four bridges, namely branching germ cells. These germ cells are preferentially protected from cell death and cyst fragmentation, and to accumulate organelles and cytoplasm from sister germ cells to become primary oocytes. Changes in cyst structure and single-cell mRNA profiles suggested that cytoplasmic transport in germline cysts is conducted in a directional manner, in which cellular content is first transported locally between peripheral germ cells and further enriched in branching germ cells, a process causing selective germ cell loss in cysts. Cyst fragmentation occurs extensively in female cysts, but not in male cysts. Male cysts in fetal and adult testes have branched cyst structures, without differential cell fates between germ cells. During cyst formation, E-cadherin junctions between germ cells position intercellular bridges to form branched cysts. Disrupted junction formation in E-cadherin-depleted cysts led to an altered ratio in branched cysts. Germ cell-specific E-cadherin knockout resulted in reductions in primary oocyte number and oocyte size. These findings shed light on the mechanism of how the size of the ovarian reserve, the number of primary oocytes available to sustain adult ovarian function, is determined during ovary formation.

developmental biology↗

bioRxiv2022

Unsupervised Discovery of Ancestry Informative Markers and Genetic Admixture Proportions in Biobank-Scale Data Sets

Admixture estimation plays a crucial role in ancestry inference and genomewide association studies (GWAS). Computer programs such as ADMIXTURE and STRUCTURE are commonly employed to estimate the admixture proportions of sample individuals. However, these programs can be overwhelmed by the computational burdens imposed by the 105 to 106 samples and millions of markers commonly found in modern biobanks. An attractive strategy is to run these programs on a set of ancestry informative SNP markers (AIMs) that exhibit substantially different frequencies across populations. Unfortunately, existing methods for identifying AIMs require knowing ancestry labels for a subset of the sample. This supervised learning approach creates a chicken and the egg scenario. In this paper, we present an unsupervised, scalable framework that seamlessly carries out AIM selection and likelihood-based estimation of admixture proportions. Our simulated and real data examples show that this approach is scalable to modern biobank data sets. Our implementation of the method is called OpenADMIXTURE.

bioinformatics↗

bioRxiv2021

Restoring single-molecule localizations with wavefront sensing adaptive optics for deep-tissue super-resolution imaging

The specimen-induced aberration has been a major factor limiting the imaging depth of single-molecule localization microscopy (SMLM). Here, we report the application of label-free wavefront sensing adaptive optics to SMLM for deep-tissue super-resolution imaging. The proposed system measures complex tissue aberrations from intrinsic reflectance rather than fluorescence emission and physically corrects the wavefront distortion more than three-fold stronger than the previous limit. This enables us to resolve sub-diffraction morphologies of cilia and oligodendrocytes in whole intact zebrafish as well as dendritic spines in thick mouse brain tissues at the depth of up to 102 m with localization number enhancement by up to 37 times and localization precision comparable to aberration-free samples. The proposed approach can expand the application range of SMLM to intact animals that cause the loss of localization points owing to severe tissue aberrations.

cancer biology↗

bioRxiv2021

Multivariate Genomewide Association Analysis with IHT

1In genome-wide association studies (GWAS), analyzing multiple correlated traits is potentially superior to conducting multiple univariate analyses. Standard methods for multivariate GWAS operate marker-by-marker and are computationally intensive. We present a penalized regression algorithm for multivariate GWAS based on iterative hard thresholding (IHT) and implement it in a convenient Julia package MendelIHT.jl (https://github.com/OpenMendel/MendelIHT.jl). In simulation studies with up to 100 traits, IHT exhibits similar true positive rates, smaller false positive rates, and faster execution times than GEMMAs linear mixed models and mv-PLINKs canonical correlation analysis. On UK Biobank data, our IHT software completed a 3-trait joint analysis in 20 hours and an 18-trait joint analysis in 53 hours, requiring up to 80GB of computer memory. In short, our software enables geneticists to fit a single regression model that simultaneously considers the effect of all SNPs and dozens of traits.

genetics↗

bioRxiv2020

NOTCH-YAP1/TEAD-DNMT1 axis regulates hepatocytereprogramming into intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (ICC), a disease of poor prognosis, has increased in incidence. It is challenging to treat due to intra- and inter-tumoral heterogeneity, which in part is attributed to diverse cellular origin. Indeed, co-expression of AKT and NICD in hepatocytes (HCs) yielded ICC, with similarity to proliferative, Notch-activated, and stem cell-like subclasses of clinical ICC. NICD regulated SOX9 and YAP1 during ICC development. Yap1 deletion or TEAD inhibition impaired HC-to-biliary epithelial cell (BEC) reprogramming and ICC proliferation; Sox9 loss repressed tumor growth; and Yap1-Sox9 combined loss abolished ICC development in AKT-NICD model. DNMT1 was discovered as a novel downstream effector of YAP1-TEAD complex that directed HC-to-BEC/ICC fate-switch. DNMT1 loss prevented Notch-dependent HC-to-ICC development, and DNMT1 re-expression restored ICC development following TEAD repression. Coexpression of DNMT1 with AKT was sufficient to induce hepatic tumor development including ICC. Thus, we have identified a novel NOTCH-YAP1/TEAD-DNMT1 axis essential for HC-driven ICC development. SIGNIFICANCEWe evaluated the clinical relevance of hepatocyte-driven ICC model and revealed critical but distinct roles of YAP1 and SOX9 in AKT-NICD-driven hepatocyte-derived ICC. We also identified NOTCH-YAP1/TEAD-DNMT1 axis as a critical driver for hepatocyte-to-ICC reprogramming, which might have biological and therapeutic implications in ICC subsets.

cancer biology↗

bioRxiv2020

Compensatory hepatic adaptation accompanies permanent absence of intrahepatic biliary network due to YAP1 loss in liver progenitors

YAP1 regulates cell plasticity during liver injury, regeneration and cancer, but its role in liver development is unknown. YAP1 activity was detected in biliary cells and in cells at the hepato-biliary bifurcation in single-cell RNA-sequencing analysis of developing livers. Hepatoblast deletion of Yap1 led to no impairment in Notch-driven SOX9+ ductal plate formation, but prevented the formation of the abutting second layer of SOX9+ ductal cells, blocking the formation of a patent intrahepatic biliary tree. Intriguingly, the mice survived for 8 months with severe cholestatic injury and without any hepatocyte-to-biliary transdifferentiation. Ductular reaction in the perihilar region suggested extrahepatic biliary proliferation likely seeking the missing intrahepatic biliary network. Long-term survival of these mice occurred through hepatocyte adaptation via reduced metabolic and synthetic function including altered bile acid metabolism and transport. Overall, we show YAP1 as a key regulator of bile duct development while highlighting a profound adaptive capability of hepatocytes.

pathology↗