Biology subjects

Ishida, Y.

Publications and source records attributed to Ishida, Y..

At least 19 recordsLinked to original sources

bioRxiv2026

A comprehensive atlas of somatic mutation rates and mutational signatures in normal human cells

Over the course of a lifetime, somatic mutations accrue in normal human cells, causing variation in cell phenotype and engendering somatic evolution with outcomes ranging from the adaptive immune system to cancer. To inform understanding of somatic evolution in the human body we report the mutation rates and mutational signatures of 53 normal cell types. Most show evidence of linear mutation accumulation over time with single base substitution mutation rates ranging from ~3.5/year/diploid genome in spermatogonia and sperm, to ~20/year in postmitotic neurons, ~50/year in mitotically active colorectal epithelial cells, ~60/year in kidney proximal tubule cells and hepatocytes, 100s/year in sun-exposed skin epidermal cells and 10-50/year in the remainder. Certain cell types, including skin epidermis, cardiac myocytes, bladder urothelium, kidney proximal tubule cells, and hepatocytes, show substantial variability in mutation burdens around the linear age trend, indicating the influence of additional factors which differ between individuals and modulate mutation accumulation, including exogenous mutagen exposures. At least 18 single-base substitution and nine small insertion and deletion mutational signatures are present, some in all cell types, some in a subset and others in a single cell type. Known exogenous mutagen exposures and endogenous mutational processes account for some mutational signatures, but the origins and mechanisms underlying many are uncertain. This comprehensive survey of mutagenesis provides a foundation for understanding somatic evolution of human cell populations in health and disease.

genomics↗

bioRxiv2026

A practical workflow for CAR screening in primary T cells using fitness-guided design and mRNA electroporation

Chimeric antigen receptor (CAR) T cell therapy has achieved remarkable therapeutic outcomes in hematological cancers. However, broader clinical use has uncovered substantial challenges arising from intrinsic properties of both T cells and tumor tissues. As the functional phenotype of CAR T cells is affected by the CAR molecular architecture, optimizing CAR constructs continues to be a critical and ongoing task. Here, we present a practical workflow for scalable screening of CAR variants in primary T cells using fitness-guided design and mRNA electroporation. Using a CD19-targeted second-generation CAR, we built a library of point mutants that focused mutagenesis on hinge and costimulatory domains. Amino acid substitutions were prioritized using the sequence-based zero-shot fitness predictor to enrich evolutionarily tolerated variants. From 340 designed variants, we electroporated mRNA encoding 85 constructs into primary human CD8+ T cells and quantified cytotoxicity against CD19-positive Nalm6 cells. Twenty-four variants reproducibly exceeded wild-type cytotoxicity across three runs, and three hits were selected for lentiviral validation. One of the selected variants showed significantly improved cytotoxicity despite lower expression frequency and exhibited higher CD62L within CAR-positive cells, suggesting enhanced intrinsic function with a less differentiated phenotype. This approach enables scalable, rapid discovery of improved CAR domain variants directly in primary T cells.

bioengineering↗

bioRxiv2026

Enterococcus faecalis is involved in the progression of the early stages of latent chronic pancreatitis surrounding pancreatic cancer tissue

(Objective) In our previous research, we identified latent chronic pancreatitis in the normal tissue surrounding pancreatic cancer. We also discovered the presence of Enterococcus faecalis (E. faecalis), a type of intestinal bacterium, in the pancreatic fluid and tissue of pancreatic cancer patients, suggesting it may be one of the factors contributing to the development of latent chronic pancreatitis. In this study, we performed pathological analyses to investigate its characteristics and investigate a possibility of E. faecalis infection. (Methods) Pathological analyses were performed, using 16 cases of pancreatic cancer and intraductal papillary mucinous neoplasia (IPMN) involving lesions in the pancreas tail. The involvement of E. faecalis was investigated with immunohistochemical analysis and serological methods. (Results) All cases exhibited inflammatory changes in pancreatic tissue without a clinical diagnosis of chronic pancreatitis, along with macrophage infiltration. These changes did not significantly differ according to preoperative treatment. DNA encoding E. faecalis 16s ribosomal RNA was detected in many cases, however, a positive immunostaining to E. faecalis was observed in only a few cases. Serum capsular polysaccharide (CPS) antibody levels exceeding the mean values were observed in patients with established chronic pancreatitis, while the level was not correlated with E. faecalis immunostaining. (Conclusion) These results suggest the E. faecalis infection is involved in the early stage of the progression of chronic latent pancreatitis and the diagnostic technology incorporating novel multi-biomarkers may be useful for identifying high-risk individuals for future pancreatic cancer development.

cancer biology↗

bioRxiv2026

Human 3D epithelioids enable continuous long-term clonal evolution studies across multiple epithelial tissues

Modeling human epithelia in vitro remains challenging because current systems do not fully preserve the combination of architecture, heterogeneity, clonal composition, and long-term dynamics shown in vivo. While 3D approaches such as organoids and organotypic cultures capture important aspects of lineage differentiation and niche signaling, they often lose stable organization over time, limiting studies of long-lasting processes such as clonal evolution and cell competition. Here, we present human epithelioids as continuous long-term, 3D epithelial cultures efficiently derived from eight adult human epithelia, including trachea, skin, buccal mucosa, esophagus, blader, urethra, submandibular gland and endometrium. Using immunostaining, electron microscopy, single-cell RNA sequencing, functional assays and somatic mutation analyses, we deeply characterized human epithelioids and confirmed that they recapitulate native architecture and cell diversity, sustain regenerative capacity, and preserve donor-specific mutational landscapes, establishing a robust and versatile platform for longitudinal interrogation of clonal evolution, tissue dynamics and responses to clinically relevant perturbations, including radiotherapy and chemotherapy, over extended timescales.

cancer biology↗

bioRxiv2026

Cytotoxicity-based High-throughput Screening System for CAR T Cell

Some chimeric antigen receptor (CAR) T cell therapies have shown strong clinical efficacy, yet systematic screening of new CAR designs remains constrained by labor-intensive, low-throughput evaluation methods. To address this limitation, we developed a cytotoxicity-centered, high-throughput screening platform that integrates single-cell pooled screening with fully automated arrayed screening enabling both large-scale library handling and quantitative functional resolution for systematic CAR design exploration. Using a mutation-based CAR design approach guided by protein fitness prediction, we generated a 4-1BB-based CAR library with approximately 10 theoretical variants while minimizing the prevalence of low-activity designs. In pooled screening, CAR T cells were evaluated at the single-cell level based on cytotoxicity and proliferation, enabling rapid enrichment of high-performing variants from a highly diverse library. Subsequent automated arrayed screening quantitatively measured cytotoxicity with high reproducibility, providing high-resolution functional data suitable for comparative ranking. Selected CAR variants demonstrated superior antitumor efficacy in a leukemia xenograft model compared with a template CAR. Furthermore, systematic analysis of mutation sites from an enhanced CAR variant identified essential mutation combinations underlying functional enhancement. Together, this study establishes a cytotoxicity-focused screening framework that provides a robust approach for optimizing CAR architectures and accelerating the development of CAR T-cell therapies.

synthetic biology↗

bioRxiv2025

Enhancing CAR-T cell activity prediction via fine-tuning protein language models with generated CAR sequences

Chimeric antigen receptor (CAR)-T cell therapy has shown remarkable success in treating hematological malignancies; however, several challenges remain, including limited efficacy against solid tumors, T cell exhaustion, and lack of T cell persistence, which have restricted its clinical efficacy across various indications. Sequence optimization of CAR constructs offers a promising strategy to enhance therapeutic efficacy of CAR-T cells. Recent advances in machine learning, especially protein language models (PLMs), enable prediction of mutational effects based on sequence representations. Nevertheless, applying PLMs to CARs is challenging due to the artificial nature of CARs and the absence of comprehensive CAR sequence databases. In this study, we developed a computational framework to predict CAR-T cell activity by fine-tuning ESM-2 with the CAR sequences generated using sequence augmentation. These CAR sequences were constructed by recombining homologous domains of CARs, enabling task-specific adaptation of the model. To evaluate prediction performance, we experimentally assessed the cytotoxicity of CAR-T cells expressing mutated CAR variants and compared these results with model predictions. Our results demonstrated that fine-tuned ESM-2 significantly improves prediction performance of CAR-T cell activity. Furthermore, we showed that training parameters--such as sequence diversity, number of training steps, and model size--substantially influence prediction performance. This work highlights the potential of combining sequence augmentation with fine-tuning PLMs to advance data-driven CAR-T cell design.

bioinformatics↗

bioRxiv2025

Modeling of hepatitis B virus infection spread in primary human hepatocytes

Chronic hepatitis B virus (HBV) infection poses a significant global health threat, causing severe liver diseases including cirrhosis and hepatocellular carcinoma. We characterized HBV DNA kinetics in primary human hepatocytes (PHH) over 32 days post-inoculation (pi) and used agent-based modeling (ABM) to gain insights into HBV lifecycle and spread. Parallel PHH cultures were mock-treated or HBV entry inhibitor Myr-preS1 (6.25 g/mL) was initiated 24h pi. In untreated PHH, 3 viral DNA kinetic patterns were identified: (1) an initial decline, followed by (2) rapid amplification, and (3) slower amplification/accumulation. In the presence of Myr-preS1, viral DNA and infected cell numbers in phase 3 were effectively blocked, with minimal to no increase. This suggests that phase 2 represents viral amplification in initially infected cells, while phase 3 corresponds to viral spread to naive cells. The ABM reproduced well the HBV kinetic patterns observed and predicted that the viral eclipse phase lasts between 18 and 38 hours. After the eclipse phase, the viral production rate increases over time, starting with a slow production cycle of 1 virion per day, which gradually accelerates to 1 virion per hour after 3 days. Approximately 4 days later, virion production reaches a steady state production rate of 4 virions/hour. The estimated median efficacy of Myr-preS1 in blocking HBV spread was 91% (range: 90-92%). The HBV kinetics and the predicted estimates of the HBV eclipse phase duration and HBV production cycles in PHH are similar of those predicted in uPA/SCID mice with human livers. ImportanceWhile primary human hepatocytes (PHH) are the most physiologically relevant culture system for studying HBV infection in vitro, comprehensive understanding of HBV infection kinetics and spread in PHH is lacking. In this study, we characterize HBV viral kinetics and employ agent-based modeling (ABM) to provide quantitative insights into the HBV production cycle and viral spread in PHH. The ABM provides an estimate of HBV eclipse phase duration, HBV production cycles and Myr-preS1 efficacy in blocking HBV spread in PHH. The results resemble those predicted in uPA/SCID mice with human livers, demonstrating that estimated HBV infection kinetic parameters in PHH in vitro mirrors that observed in in vivo HBV infection chimeric mouse model.

systems biology↗

bioRxiv2024

Zbtb38 inhibits apoptosis by transcriptionally regulating XIAP expression

The X-linked inhibitor of apoptosis protein (XIAP) is a key suppressor of apoptosis, crucial for cellular differentiation, embryogenesis, and cancer progression. However, its upstream regulatory mechanisms remain poorly understood. Here, we demonstrate that the zinc finger transcription factor Zbtb38, a negative regulator of apoptosis, modulates XIAP expression in both loss- and gain-of-function experiments, irrespective of p53 expression. Notably, XIAP overexpression rescues the apoptosis induced by Zbtb38 knockdown, indicating that Zbtb38-associated apoptosis is at least partially XIAP-dependent. Mechanistically, Zbtb38 binds to E-box motifs within upstream regulatory regions of XIAP and activates its transcription. During embryonic stem (ES) cell differentiation and embryogenesis, Zbtb38 depletion increases apoptosis and reduces XIAP and Bcl-2 expression, underscoring their functional relevance in these processes. Analysis of human tumor datasets reveals a strong positive correlation between ZBTB38 and XIAP expression, with elevated ZBTB38 levels associated with high-grade malignancies. Furthermore, Zbtb38 knockdown induces apoptosis in cancer cells with reduced XIAP expression, regardless of p53 expression. Collectively, these findings uncover a novel Zbtb38-XIAP axis that regulates apoptosis during cellular differentiation, development, and oncogenesis, and highlight its therapeutic potential in XIAP-driven and p53-deficient tumors.

cell biology↗

bioRxiv2024

ILC2s navigate tissue redistribution during infection using stage-specific S1P receptors

Lymphocytes can circulate as well as take residence within tissues. While the mechanisms by which circulating populations are recruited to infection sites have been extensively characterized, the molecular basis for the recirculation of tissue-resident cells is less understood. Here, we show that helminth infection- or IL-25-induced redistribution of intestinal group 2 innate lymphoid cells (ILC2s) requires access to the lymphatic vessel network. Although the secondary lymphoid structure is an essential signal hub for adaptive lymphocyte differentiation and dispatch, it is redundant for ILC2 migration and effector function. Upon IL-25 stimulation, a dramatic change in epigenetic landscape occurs in intestinal ILC2s, leading to the expression of sphingosine-1-phosphate receptors (S1PRs). Among the various S1PRs, we found that S1PR5 is critical for ILC2 exit from intestinal tissue to lymph. By contrast, S1PR1 plays a dominant role in ILC2 egress from mesenteric lymph nodes to blood circulation and then to distal tissues including the lung where the redistributed ILC2s contribute to tissue repair. The requirement of two S1PRs for ILC2 migration is largely due to the dynamic expression of the tissue-retention marker CD69, which mediates S1PR1 internalization. Thus, our study demonstrates a stage-specific requirement of different S1P receptors for ILC2 redistribution during infection. We therefore propose a fundamental paradigm that innate and adaptive lymphocytes utilize a shared vascular network frame and specialized navigation cues for migration.

immunology↗

bioRxiv2023

Bone marrow-derived CCR5-expressing macrophages promote venous thrombus resolution by up-regulating plasminogen activators and VEGF

BackgroundDeep vein thrombosis (DVT) is a common peripheral vascular disease arising from endothelial damage and can frequently result in pulmonary embolism. C-C chemokine receptor 5 (CCR5) is essentially involved in skin wound healing and pulmonary fibrosis, but the role of CCR5 still remains elusive. MethodsDeep vein thrombus was induced by the ligation of the inferior vena cava (IVC) in wild-type (WT) mice and Ccr5-/- mice, and thrombi were collected over time and various analyzes were performed. ResultsAfter ligation of the IVC in WT mice, a venous thrombus developed progressively until 5 days, and then resolved. Concomitantly, IVC ligation enhanced intrathrombotic gene and protein expression of Ccr5 and its ligand, Ccl5, and both were expressed mainly by intrathrombotic macrophages. The same treatment of Ccr5-/- mice resulted in significantly greater thrombus mass than WT mice. Moreover, the administration of a specific CCR5 inhibitor to WT mice recapitulated similar phenotypes as Ccr5-/- mice while that of CCL5 caused the opposite phenotypes. In Ccr5-/- mice, the production of intrathrombotic vascular endothelial growth factor (VEGF), tissue plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA) from macrophages was significantly reduced and intrathrombotic recanalization was also suppressed compared to WT mice. Moreover, CCL5 enhanced Vegf, Plat, and Plau gene expression in WT-derived peritoneal macrophages by activating the ERK MAPK signaling pathway in vitro. ConclusionsCCR5-deficient mice exhibited reduced expression of Vegf, Plat, and Plau, with concomitant attenuated neovascularization and reduced thrombus resolution. the Thus, the augmentation of the CCL5-CCR5 axis may be effective for DVT treatment by enhancing thrombolysis.

molecular biology↗

bioRxiv2023

Sensitization with an allogeneic MHC class I molecule induces anti-tumor immunity in the absence of PD-1 in mice

To investigate the effect of a major histocompatibility complex class I (MHC-I) overexpression to augment immune sensitivity against tumors, we have generated the murine colorectal carcinoma cell line MC38 (with the endogenous H-2b haplotype) overexpressing the allogeneic mouse MHC-I cell surface molecule H-2Kd (MC38 H-2Kd). The tumorigenicity of unmodified parental cells (MC38 PT) and MC38 H-2Kd was tested in vivo by subcutaneous injection into the flank of wild-type (WT) and programmed death-1 (PD-1) knockout (KO) mice in a C57BL/6 (H-2b) genetic background. MC38 PT cells readily formed tumors and grew progressively in both WT and PD-1 KO mice. The speed of MC38 PT tumor growth was slower in PD-1 KO mice than in WT mice. In contrast, MC38 H-2Kd cells showed full sensitivity to rejection by the immune system in both naive WT and PD-1 KO mice, indicated by spontaneous tumor regression. Next, we sought to determine the extent to which H-2Kd-overexpressing tumors could protect the mice against unmodified cancers. PD-1 KO mice were first sensitized with highly immunogenic MC38 H-2Kd cells and then challenged with weakly immunogenic MC38 PT cells. Intriguingly, all PD-1 KO mice gained immunity against the aggressive MC38 tumor and became tumor-free. Sensitizing PD-1 KO mice with growth-arrested (by the pre-treatment with mitomycin C, MMC) and the debris of MC38 H-2Kd tumors also provided full protection against the growth of secondary MC38 PT tumors. Most notably, sensitization with the debris of MC38 H-2Kd cells provided the long-term immunological memory against MC38 PT carcinoma cells. This finding implies that MC38 H-2Kd cells retain highly efficient and durable immunogenicity.

immunology↗

bioRxiv2023

Granulomatous inflammatory responses are elicited in the liver of PD-1 knockout mice by de novo genome mutagenesis

Aimsprogrammed death-1 (PD-1) is a negative regulator of immune responses. Upon deletion of PD-1 in mice, symptoms of autoimmunity developed only after they got old. In a model experiment in cancer immunotherapy, PD-1 was shown to prevent cytotoxic T lymphocytes from attacking cancer cells that expressed neoantigens derived from genome mutations. Furthermore, the larger number of genome mutations in cancer cells led to the more robust anti-tumor immune responses after the PD-1 blockade. In order to understand the common molecular mechanisms underlying these findings, we hypothesize that we might have acquired PD-1 during evolution in order to avoid/suppress autoimmune reactions against neoantigens derived from mutations in the genome of aged individuals. Main methods: to test the hypothesis, we introduced random mutations into the genome of young PD-1-/- and PD-1+/+ mice. We employed two different procedures of random mutagenesis: administration of a potent chemical mutagen N-ethyl-N-nitrosourea (ENU) into the peritoneal cavity of mice and deletion of MSH2, which is essential for the mismatch-repair activity in the nucleus and, therefore, for the suppression of accumulation of random mutations in the genome. Key findingswe observed granulomatous inflammatory changes in the liver of the ENU-treated PD-1 knockout (KO) mice, but not in the wild-type (WT) counterparts. Such lesions also developed in the PD-1/MSH2 double KO mice, but not in the MSH2 single KO mice. Significance: the results we obtained support our hypothesis: PD-1 probably functions to avoid/suppress inflammatory responses against neoantigens derived from genome mutations in aged individuals.

immunology↗

bioRxiv2023

Microbiome ssRNA as an environmental cue to activate TLR13-dependent tissue-protective programs in CD5Lhi hepatic macrophages

Hepatic macrophages maintain liver homeostasis, but little is known about the signals that activate the hepatoprotective programs within macrophages. Here, we show that toll-like receptor 13 (TLR13), a sensor of bacterial 23S ribosomal RNA (rRNA), senses microbiome RNAs to drive tissue-protective responses in CD5Lhi hepatic macrophages. Splenomegaly and hepatomegaly developed in the absence of the endosomal RNase, RNaseT2, via TLR13-dependent macrophage proliferation. Furthermore, TLR13 in hepatic Ly6Clo macrophages activated the transcription factors LXR and MafB, leading to expression of tissue-clearance molecules, such as CD5L, C1qb, and Axl. Consequently, Rnaset2-/- mice developed resistance to acute liver injury caused by challenges with acetaminophen and lipopolysaccharide + D-galactosamine. TLR13 responses in Rnaset2-/- mice were impaired by antibiotics, suggesting that TLR13 were activated by microbiome rRNAs, which was detected in the sera and hepatic macrophages. Repeated administration of wild-type mice with the TLR13 ligand, rather than other TLR ligands, selectively increased the number of Kupffer cells, which expressed immunoregulatory and tissue-clearance genes as hepatic macrophages in Rnaset2-/- mice did. Our results suggest that microbiome ssRNA serves as an environmental cue for initiating tissue-protective TLR13 responses in hepatic macrophages. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=192 SRC="FIGDIR/small/540294v1_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@69614eorg.highwire.dtl.DTLVardef@737c69org.highwire.dtl.DTLVardef@1ecd9eforg.highwire.dtl.DTLVardef@d7e10d_HPS_FORMAT_FIGEXP M_FIG In the absence of an endosomal RNase, RNase T2, microbiome RNAs circulating in the vasculature activate TLR13 in hepatic macrophages to drive hepatoprotective responses through expression of immunoregulatory and tissue-clearance molecules. Consequently, mice lacking RNase T2 are resistant against acute liver injuries caused by acetaminophen and LPS + D-galactosamine. C_FIG

immunology↗

bioRxiv2023

Coordinated multiple cellular processes in tongue development

Dysfunction of primary cilia leads to genetic disorder, ciliopathies, which shows various malformations in many vital organs such as brain. Multiple tongue deformities including cleft, hamartoma and ankyloglossia are also seen in ciliopathies, which yield difficulties in fundamental functions such as mastication and vocalization. Here, we found these tongue anomalies in mice with mutation of ciliary protein. Abnormal cranial neural crest-derived cells (CNCC) failed to evoke Hh signal for differentiation of mesoderm-derived cells into myoblasts, which resulted in abnormal differentiation of mesoderm-derived cells into adipocytes. The ectopic adipose subsequently arrested migration of other mesoderm-derived cells and CNCC. Some aberrant CNCC abnormally differentiated into osteoblasts due to the lack of Hh signal, which migrated into tongue to form ectopic bone. Ankyloglossia was caused by aberrant cell migration due to lack of non-canonical Wnt signaling. In addition to ciliopathies, these tongue anomalies are often observed as non-familial condition in human. We found that these tongue deformities could be reproduced in wild-type mice by simple mechanical manipulations in CNCC to disturb cellular processes which were disrupted in mutant mice. Thus, tongue development requires coordinated multiple cellular processes (cell-cell contact, migration and differentiation). Our results provide hints for possible future treatment in ciliopathies.

developmental biology↗

bioRxiv2022

NF-κB Decoy ODN-Loaded Poly lactic-co-glycolic Acid Nanospheres Inhibit Alveolar Ridge Resorption

Residual ridge resorption combined with dimensional loss resulting from tooth extraction has a prolonged correlation with early excessive inflammation. Nuclear factor-kappa B (NF-{kappa}B) decoy oligodeoxynucleotide (ODN) is a member of a group of double-stranded DNA capable of downregulating the expression of downstream genes of the NF-{kappa}B pathway. The healing action of its embellished effect combined with poly(lactic-co-glycolic acid) (PLGA) nanospheres on tooth extraction socket still remains unknown. Hence, the aim of this study was to investigate the therapeutic effect of NF-{kappa}B decoy ODN-loaded PLGA nanospheres (PLGA-NfD) transfected into extraction sockets in Wistar/ST rats. Micro-computed tomography and trabecular bone analysis following treatment with PLGA-NfD demonstrated inhibition of vertical alveolar bone loss with increased bone volume, smoother trabecular bone surface, thicker trabecular bone, larger trabecular number and separation, and fewer bone porosities. Histomorphometric and reverse transcription-quantitative polymerase chain reaction analysis revealed reduced tartrate-resistant acid phosphatase-expressing osteoclasts, interleukin-1{beta}, tumor necrosis factor-, receptor activator of NF-{kappa}B ligand, turnover rate and increased transforming growth factor-{beta}1 immunopositive reactions and relative gene expressions. These data demonstrate that local delivery of PLGA-NfD could be used as a substantial suppressor of inflammation during the healing process in a tooth extraction socket, with the potential of accelerated new bone formation.

immunology↗

bioRxiv2022

Non-invasive fecal DNA yields whole genome and metagenomic data for species conservation

Non-invasive biological samples benefit studies that investigate rare, elusive, endangered, and/or dangerous species. Integrating genomic techniques that use non-invasive biological samples with advances in computational approaches can benefit and inform wildlife conservation and management. Here we present a molecular pipeline that uses non-invasive fecal DNA samples to generate low- to medium-coverage genomes (e.g., >90% of the complete nuclear genome at 6X coverage) and metagenomic sequences, combining in a novel fashion widely available and accessible DNA collection cards with commonly used DNA extraction and library building approaches. DNA preservation cards are easy to transport and can be stored non-refrigerated, avoiding cumbersome and/or costly sample methods. The genomic library construction and shotgun sequencing approach did not require enrichment or targeted DNA amplification. The utility and potential of the data generated by this pipeline was demonstrated by the application of genome-scale analysis and metagenomics to zoo and free-ranging African savanna elephants (Loxodonta africana). Fecal samples collected from free-ranging individuals contained an average of 12.41% (5.54-21.65%) endogenous elephant DNA. Clustering of these elephants with others from the same geographic region was demonstrated by a principal component analysis of genetic variation using nuclear genome-wide SNPs. Metagenomic analyses generated compositional taxon classifications that included Loxodonta, green plants, fungi, arthropods, bacteria, viruses and archaea, showcasing the utility of our approach for addressing complementary questions based on host-associated DNA, e.g., pathogen and parasite identification. The molecular pipeline presented here extends applications beyond what has previously been shown for target-enriched datasets and contributes towards the expansion and application of genomic techniques to conservation science and practice.

genomics↗

bioRxiv2022

FT-GO: a multiplex fluorescent tyramide signal amplification system for histochemical analysis

Tyramide signal amplification (TSA) is a highly sensitive method for histochemical analysis. Previously, we reported a TSA system, BT-GO, for bright-filed imaging. Here, we develop FT-GO (Fluorochromized Tyramide-Glucose Oxidase) as a multiplex fluorescent TSA system. FT-GO involves peroxidase-catalyzed deposition of FT with hydrogen peroxide produced in enzymatic reaction between glucose and glucose oxidase. We showed that FT-GO enhanced immunofluorescence signals while maintaining low background signals. Compared with indirect immunofluorescence detections, FT-GO demonstrated a more widespread distribution of monoaminergic projection systems in mouse and marmoset brains. For multiplex labeling with FT-GO, we quenched Ab-conjugated peroxidase using sodium azide. We applied FT-GO to multiplex fluorescent in situ hybridization, and succeeded in labeling neocortical interneuron subtypes by coupling with immunofluorescence. FT-GO immunofluorescence further increased the detectability of an adeno-associated virus tracer. Given its simplicity and a staining with a high signal-to-noise ratio, FT-GO would provide a versatile platform for histochemical analysis.

neuroscience↗

bioRxiv2022

Circulating mast cell progenitors migrate to atopic dermatitis-like skin then adapt locally

BackgroundMast cells (MCs) are tissue-resident immune cells that are classified into two subsets in mice: connective tissue-type MCs (CTMCs) and mucosal type MCs (MMCs). Although both CTMCs and MMCs can be induced from bone marrow (BM)-derived hematopoietic stem cells (HSCs) in vitro, recent research on MC ontogeny has revealed that MMCs are maintained with a supply of BM-derived HSCs, while CTMCs are maintained locally by self-proliferation in steady state in vivo. However, how CTMCs, such as skin MCs, are maintained in an inflammatory state such as atopic dermatitis (AD) remains to be fully elucidated. Methods: MC903-induced AD model was used to identify BM-derived MCs in the skin. The infiltration and proliferation of MCs were evaluated by flow cytometry using CD45.1 BM-chimera mice and parabiosis. BM-derived MCs in AD-like skin were compared to resident MCs (rMCs) in gene expressions by RNA sequence analysis. The fate of BM-derived MCs in AD-like skin was investigated for expressions of CTMC markers and responses to compound 48/80. Results: In AD-like skin, significant increase of both rMCs and BM-derived MCs was observed. BM-derived MCs were derived from circulating MC progenitors (MCps) and were distinguished from rMCs by integrin{beta}7 expression, which was gradually downregulated in the skin. RNA sequence analysis showed that integrin{beta}7+ MCs in the skin shared characteristics of both MMC and CTMC. Integrin{beta}7+ MCs proliferated in situ and acquired the CTMC phenotypes in AD-like skin. Conclusions: Skin MCs are maintained in AD-like skin by both local proliferation of rMCs and infiltration/proliferation of BM-derived MCs, which differentiate toward CTMC in the skin.

immunology↗