Biology subjects

Shin, T.

Publications and source records attributed to Shin, T..

8 recordsLinked to original sources

bioRxiv2026

Holotomography reveals biophysical remodeling of mouse oocytes during post-ovulatory aging

Routine assessment of oocyte state in reproductive biology and clinical embryology still relies largely on two-dimensional transmitted-light morphology, leaving the underlying three-dimensional intracellular organization largely unmeasured. Here, we use holotomography to reconstruct volumetric refractive index (RI) distributions in unlabeled live mouse oocytes and combine this imaging with sparse-annotation, AI-assisted compartment segmentation and multidimensional feature extraction across geometry, RI statistics, dry-mass density, and intracellular texture. Applied to an in vitro model of post-ovulatory aging, holotomography-derived biophysical profiles separated fresh and aged oocytes more strongly than two-dimensional brightfield-derived features, and feature-family ablation indicated that non-morphological descriptors contributed substantially to this separation. At the population level, aged oocytes showed coordinated ooplasmic remodeling, including reduced volume, increased RI heterogeneity, and increased dry-mass density while total dry mass was broadly conserved. These findings demonstrate a label-free quantitative framework for detecting post-ovulatory aging-associated biophysical remodeling in intact mammalian oocytes. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=167 SRC="FIGDIR/small/733271v1_ufig1.gif" ALT="Figure 1"> View larger version (89K): org.highwire.dtl.DTLVardef@c7257aorg.highwire.dtl.DTLVardef@6d92f0org.highwire.dtl.DTLVardef@1c9f7eborg.highwire.dtl.DTLVardef@b36637_HPS_FORMAT_FIGEXP M_FIG C_FIG

Developmental Biology↗

bioRxiv2025

Cation-exchange synthesized Zn-doped Ag2S Nanostructures for Photothermal and Photodynamic Therapies across Breast Cancer Subtypes

Photothermal therapy (PTT) and photodynamic therapy (PDT) require nanostructures capable of efficiently converting red-light energy into both heat and reactive oxygen species (ROS). However, simultaneously achieving high photothermal conversion efficiency and strong ROS generation remains challenging. Here, we report zinc-doped Ag2S (ZSS) nanostructures, synthesized via controlled cation exchange, in which Zn incorporation modulates the electronic structure of Ag2S and improves charge separation, thereby enhancing red-light-activated PTT/PDT performance while preserving intrinsic biocompatibility. Among the series, ZSS(0.15) demonstrated optimized charge-carrier dynamics, a high photothermal conversion efficiency of 67.26%, and approximately four-fold higher singlet oxygen (1O2) generation relative to methylene blue under 660 nm irradiation. These physicochemical enhancements translated into potent therapeutic outcomes: in-vitro, ZSS(0.15) achieved an IC50 of 15 {micro}g/mL under irradiation, corresponding to approximately a 1.5-fold enhancement in cytotoxic potency compared to pristine Ag2S, and induced apoptosis via activation of the p53/Bax/Caspase pathway. In-vivo, ZSS(0.15) with laser irradiation achieved [~]97% tumor volume suppression without systemic toxicity. Extending evaluation across multiple breast cancer cell lines representing distinct molecular subtypes further confirmed broad-spectrum in-vitro efficacy. Altogether, these findings demonstrate that controlled Zn incorporation into Ag2S effectively enhances red-light-driven photothermal conversion and 1O2 generation, establishing a rational materials strategy for improving synergistic PTT/PDT performance. GRAPHICAL ABSTRACTA Zn-doped Ag2S nanoplatform (ZSS(0.15)) enables synergistic photothermal and photodynamic therapy under 660-nm laser irradiation, triggering p53-mediated mitochondrial apoptosis via ROS generation and caspase-3 activation, achieving [~]97% tumor regression in-vivo without detectable systemic toxicity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/684667v2_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@1d93519org.highwire.dtl.DTLVardef@12d18f4org.highwire.dtl.DTLVardef@1c14608org.highwire.dtl.DTLVardef@1dcd9f8_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Noninvasive time-lapse 3D subcellular analysis of embryo development for machine learning-enabled prediction of blastocyst formation

Accurate embryo quality assessment is central to improving outcomes in in vitro fertilization (IVF), yet current practice relies mainly on subjective two-dimensional (2D) morphology. Here we present a label-free framework for quantitative three-dimensional (3D) embryo phenotyping using low-coherence holotomography (HT). Time-lapse HT enabled volumetric imaging of mouse embryos from the 2-cell stage to the blastocyst without affecting developmental competence, capturing subcellular features at high resolution. Quantitative analysis revealed that matured embryos exhibited higher blastomere counts, greater spatial variability, and tighter nuclear packing, whereas arrested embryos showed enlarged blastomeres, elevated cytoplasmic heterogeneity, and fewer, larger nuclei. Machine learning models trained on these features achieved robust prediction of blastocyst formation (AUC up to 0.958). Together, these findings demonstrate that HT provides objective and interpretable 3D biomarkers that could augment and transform embryo selection in IVF.

developmental biology↗

bioRxiv2023

Impact of CRISPR/HDR-editing versus lentiviral transduction on long-term engraftment and clonal dynamics of HSPCs in rhesus macaques

For precise genome editing via CRISPR/homology-directed repair (HDR), effective and safe editing of long-term engrafting hematopoietic stem cells (LT-HSCs) requires both sufficient HDR efficiency and protection of LT-HSC function and number. The impact of HDR on true LT-HSCs clonal dynamics in a relevant large animal model has not previously been studied. To track the HDR-edited cells, autologous rhesus macaque (RM) CD34+ cells were electroporated with the gRNA/Cas9 ribonucleoprotein (RNP) and HDR cassette barcode library structure and reinfused into RMs following myeloablation. For competitive model animals, fractionated CD34+ cells were transduced with a barcoded GFP-expressing lentiviral vector (LV) and electroporated via HDR machinery, respectively. CD33 knockout (KO) neutrophils were prevalent early following engraftment and then rapidly decreased, resulting in less than 1% total editing efficiency. Interestingly, in competitive animals, a higher concentration of i53 mRNA result in a less steep reduction in CD33 KO cells, presented a modest decrease in HDR rate (0.1-0.2%) and total indels (1.5-6.5%). In contrast, the drop off of LV-transduced GFP+ cells stabilized at 20% after 2 months. We next retrieved embedded barcodes and revealed that various clones contributed to early hematopoietic reconstitution, then after dominant clones appeared at steady state throughout the animals. In conclusion, CRISPR/HDR edited cells disappeared rapidly after the autologous transplantation in RM despite substantial gene editing outcome, whereas LV-transduced cells were relatively well maintained. Clonality of HDR-edited cells drastically shrank at early stage and then relied on several dominant clones, which can be mildly mitigated by the introduction of i53 mRNA.

cell biology↗

bioRxiv2023

Somatic Mosaicism in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Reveals Widespread Degeneration from Focal Mutations

Although mutations in many genes cause familial amyotrophic lateral sclerosis and frontotemporal dementia, most cases are sporadic (sALS and sFTD) with unclear etiology. We tested whether somatic mutations contribute to sALS and sFTD by deep targeted sequencing of 88 neurodegeneration-related genes in postmortem brain and spinal cord samples from 399 sporadic cases and 144 controls. Predicted deleterious somatic variants in ALS/FTD genes were observed in 2.1% of sporadic cases lacking deleterious germline variants. These variants occurred at very low allele fractions (typically <2%) and were often focal and enriched in disease-affected regions. Analysis of bulk RNA-seq data from an additional cohort identified deleterious somatic variants in DYNC1H1 and LMNA, genes associated with pediatric motor neuron degeneration. Targeted long-read sequencing further identified one sFTD case with de novo somatic C9orf72 repeat expansions. Together, these findings suggest that rare, focal somatic variants can contribute to sALS and sFTD and drive widespread neurodegeneration.

genetics↗

bioRxiv2023

HiExM: high-throughput expansion microscopy enables scalable super-resolution imaging

Expansion microscopy (ExM) enables nanoscale imaging using a standard confocal microscope through the physical, isotropic expansion of fixed immunolabeled specimens. ExM is widely employed to image proteins, nucleic acids, and lipid membranes in single cells; however, current methods limit the number of samples that can be processed simultaneously. We developed High-throughput Expansion Microscopy (HiExM), a robust platform that enables expansion microscopy of cells cultured in a standard 96-well plate. Our method enables [~]4.2x expansion of cells within individual wells, across multiple wells, and between plates. We also demonstrate that HiExM can be combined with high-throughput confocal imaging platforms to greatly improve the ease and scalability of image acquisition. As an example, we analyzed the effects of doxorubicin, a known cardiotoxic agent, on human cardiomyocytes (CMs) as measured by Hoechst signal across the nucleus. We show a dose dependent effect on nuclear DNA that is not observed in unexpanded CMs, suggesting that HiExM improves the detection of cellular phenotypes in response to drug treatment. Our method broadens the application of ExM as a tool for scalable super-resolution imaging in biological research applications. Significance StatementExpansion microscopy (ExM) is an accessible and widely used technique for super-resolution imaging of fixed biological specimens. For many ExM users, slide-based sample preparation and manual imaging limit the number of experimental conditions and samples that can be processed in parallel. Here, we develop a simple and inexpensive device that enables ExM within the wells of a standard 96-well cell culture plate. We show that samples prepared with our workflow can be imaged with a high-throughput autonomous confocal microscope, allowing for scalable super-resolution image acquisition, greatly increasing data output. Our device retains the accessibility of ExM while extending its application to research questions that require the analysis of many conditions, treatments, and time points.

bioengineering↗

bioRxiv2022

Ongoing Production of Tissue-Resident Macrophages from Hematopoietic Stem Cells in Healthy Adult Macaques

Macrophages are critical orchestrators of tissue immunity, from the initiation and resolution of antimicrobial immune responses to the subsequent repair of damaged tissue. Murine studies have demonstrated that tissue-resident macrophages are comprised of a mixture of yolk sac-derived cells that populate the tissue before birth and hematopoietic-derived replacements that are recruited in adult tissues both at steady-state and in increased numbers in response to tissue damage or infection. While resident macrophages in some murine tissues are readily turned over and replaced, other tissues primarily retain their original, yolk sac-derived complement of macrophages. How this translates to species that are constantly under immunologic challenge, such as humans, is unknown. To understand the ontogeny and longevity of tissue-resident macrophages in nonhuman primates (NHPs), we employ a model of autologous HSPC transplantation with HSPCs genetically modified to express individual bar-coded markers allowing for subsequent analysis of clonal differentiation of leukocyte subsets. We study the contribution of HSPC to tissue-macrophages and their clonotypic profiles relative to leukocyte subsets across tissues and peripheral blood. We also use in vivo bromodeoxyuracil infusions to monitor tissue macrophage turnover in NHPs at steady state. We find that, in all anatomic sites we studied, HSPC contribute to tissue-resident macrophage populations. Their clonotypic profile is dynamic and overlaps significantly with the clonal hierarchy of contemporaneous monocytes in peripheral blood. Moreover, we find evidence of tissue-macrophage turnover at steady state in otherwise unmanipulated NHPs. These data demonstrate the life span of tissue-resident macrophages can be limited and they can be replenished from HSPCs. Thus, in primates not all yolk-sac derived tissue-macrophages survive for the duration of the hosts life.

immunology↗

bioRxiv2021

Whole-genome analysis of de novo and polymorphic retrotransposon insertions in Autism Spectrum Disorder

Retrotransposons are dynamic forces in evolutionary genomics and have been implicated as causes of Mendelian disease and hereditary cancer, but their role in Autism Spectrum Disorder (ASD) has never been systematically defined. Here, we report 86,154 polymorphic retrotransposon insertions including >60% not previously reported and 158 de novo retrotransposition events identified in whole genome sequencing (WGS) data of 2,288 families with ASD from the Simons Simplex Collection (SSC). As expected, the overall burden of de novo events was similar between ASD individuals and unaffected siblings, with 1 de novo insertion per 29, 104, and 192 births for Alu, L1, and SVA respectively, and 1 de novo insertion per 20 births total, while the location of transposon insertions differed between ASD and unaffected individuals. ASD cases showed more de novo L1 insertions than expected in ASD genes, and we also found de novo intronic retrotransposition events in known syndromic ASD genes in affected individuals but not in controls. Additionally, we observed exonic insertions in genes with a high probability of being loss-of-function intolerant, including a likely causative exonic insertion in CSDE1, only in ASD individuals. Although de novo retrotransposition occurs less frequently than single nucleotide and copy number variants, these findings suggest a modest, but important, impact of intronic and exonic retrotransposition mutations in ASD and highlight the utility of developing specific bioinformatic tools for high-throughput detection of transposable element insertions.

genetics↗