Biology subjects

Kitano, S.

Publications and source records attributed to Kitano, S..

9 recordsLinked to original sources

bioRxiv2026

Sequence-intrinsic barriers define a class of translation-restricted uORFs in the human genome

Ribosome profiling has revealed widespread translation of upstream open reading frames (uORFs) within the human 5' untranslated region, greatly expanding the apparent coding potential of the genome. However, despite pervasive translational activity, uORF-encoded proteins (uORFps) are only rarely detected by proteomic approaches, creating a major unresolved discrepancy between translation and protein accumulation. Here, we systematically quantified the intracellular accumulation of 111 evolutionarily conserved human uORFps using a unified reporter platform. Although we found that most conserved uORFps accumulated at extremely low levels despite robust transcript expression, protein length emerged as the strongest baseline predictor of this low accumulation. Intriguingly, a subset of conserved uORFs remained poorly accumulated even after experimental extension with a C-terminal EGFP tag, defining a distinct class of translation-restricted uORFs. Sequence and structural analysis of these translation-restricted uORFs revealed a cooperative enrichment of basic amino acids and stable local RNA secondary structures. Synonymous substitutions designed to disrupt these RNA structures substantially restored protein accumulation, demonstrating a major contribution of local transcript stratification to this translation-restricted phenotype. Using these identified rules, we developed a multivariable predictive model that classified 1,432 translation-restricted uORFs across the human transcriptome. Genes harboring these predicted translation-restricted uORFs exhibited significantly reduced downstream translation efficiency and elevated sequence conservation, demonstrating that these sequence-encoded barriers are under purifying selection to act as cis-regulatory elements scattered across the human transcriptome. Together, our findings identify a conserved class of human uORFs that encode intrinsic barriers to productive translation and provide a rigorous framework for understanding how noncanonical coding sequences shape the human proteome.

genetics↗

bioRxiv2026

Collagen Rope Trick: Cell-Laden Fibre Assembly at 1 Liquid Interfaces

Summary ParagraphTissues and organs in living organisms represent centimeter-scale hierarchical architectures comprising nano-to microscale, uniaxially aligned extracellular matrix (ECM) fibres with high mechanical strength, integrated with cellular components, as exemplified in tendon, skin, cartilage, bone, and blood vessels1. Here, we present a liquid-liquid interfacial spinning method to produce highly uniaxially aligned, centimeter-scale collagen fibres. The dried fibres exhibit exceptional mechanical properties, with fracture strength of 280 MPa, Youngs modulus of 6 GPa, and toughness of 17 MJ m-3, comparable to spider silk and tendon collagen, and exceeding supramolecular and double-network hydrogels1. Incorporating living cells into the collagen solution yielded centimeter-scale, cell-laden aligned fibres, with densely adherent, uniaxially aligned cells and over 80% viability. Myoblast-laden fibres recapitulate biological features of fibrotic muscle tissues, as observed in type II diabetes2. Interfacial collagen assembly further enables fabrication of dimension-controlled constructs, like 2D sheets, 0D capsules, and 1D tubes, thus providing modular building blocks for centimeter-scale 3D tissues and organ-like structures. This approach offers a versatile platform to engineer mechanically robust, cell-laden tissues with controlled hierarchical architecture.

bioengineering↗

bioRxiv2025

Meat the Fat: From High Lipid Content to Nutritional Enhancement to Reconstruct Wagyu Fat with Tunable Rheology and Organoleptic Fidelity for Cultivated Meat

Cultivated meat holds promise as a sustainable and ethical alternative to conventional meat, but reproducing the rich flavor, texture, and lipid composition of premium beef, such as Wagyu, remains a major hurdle. In this study, we redefine bovine adipogenesis by systematically optimizing edible biomaterials, fatty acid delivery systems, and culture conditions to engineer high-fat cultivated tissues with authentic sensory properties. Among various candidates, 1% alginate emerged as the optimal scaffold, providing mechanical softness compatible with adipogenic differentiation and mimicking the rheological properties of native fat. Supplementation with oleic acid complexed to edible albumins, especially whey protein (WP), significantly enhanced lipid accumulation and monounsaturated fatty acid content, particularly oleic acid, which reached up to 80.6% of total fatty acids. Engineered fat tissues displayed soft, spreadable textures and organoleptic characteristics consistent with high-quality beef fat. Fatty acid profiling revealed a bovine-like omega-6 signature, dominated by linoleic, arachidonic, and dihomo-{gamma}-linolenic acids, alongside detectable omega-3 subtypes including alpha-linolenic acid and trace levels of EPA and DHA. This composition not only validates the physiological relevance of the engineered fat, but also highlights its potential to deliver bioactive lipids rarely found in terrestrial animal products. Additionally, the WP-oleic acid system supported robust adipogenesis even under lower serum conditions. These findings establish a food-grade, tunable platform for producing physiologically and nutritionally enhanced fat tissues, advancing the realism, functionality, and health potential of next-generation cultivated meat.

bioengineering↗

bioRxiv2025

Cultivated Beef Meat Has the Potential to Maintain Original Characteristics of Beef Meat with Customizable Features

Cultivated meat (CM) is currently attracting much attention because of its promise to become a solution to issues of environmental sustainability, animal welfare, and decarbonization. While the fabrication process and materials are still the main focus, it is not yet clear what the characteristics of the constructed CMs are and how they take on the original characteristics of beef meat. In this study, the biological, physicochemical and sensory characteristics of three types of beef (Wagyu, Crossbreed and Holstein) meat were systematically analyzed and compared to muscle and fat fibers constructed by three-dimensional printing using satellite and adipose-derived stem cells isolated from these beef meats. The different characteristics of each beef meat were largely taken on by the CM fibers composed of bSC and bADSC of each meat. In addition, some differing properties from those of the respective beef meat were observed in CM fibers such as one of the omega-3 fatty acids, docosahexaenoic acid (DHA; Wagyu fat fibers showed the highest amount). Furthermore, we also found the important possibility to increase the composition of oleic acid to over 80% in monounsaturated fatty acid (Wagyu has around 50% oleic acid). This study revealed the importance of using cells isolated from each beef meat to provide CM that closely resemble the original texture and taste of each meat, and further the possibility of more carefully arranging their properties.

bioengineering↗

bioRxiv2023

Fluctuation of lysosomal protein degradation in neural stem cells of postnatal mouse brain

Lysosomes are intracellular organelles responsible for degrading diverse macromolecules delivered from several pathways, such as the endo-lysosomal and autophagic pathways. Recent reports have suggested that lysosomes are essential in regulating neural stem cells in developing, adult, and aged brains. However, the activity of these lysosomes has not yet been monitored in these brain tissues. Here, we report a new probe to measure lysosomal protein degradation in brain tissue by immunostaining. Our results demonstrate the fluctuation of lysosomal protein degradation in neural stem cells depending on age and brain disorder. Neural stem cells increase lysosomal activity during hippocampal development in the dentate gyrus, but aging and aging-related disease reduces their activity. In addition, physical exercise increases lysosomal activity in neural stem cells and astrocytes. We hypothesize three different stages of lysosomal activity: the increase in development, the stable state for the adult stage, and the reduction by damages with age or disease.

neuroscience↗

bioRxiv2022

Liver type 1 innate lymphoid cells lacking IL-7 receptor are a native killer cell subset fostered by parenchymal niches

Group 1 innate lymphoid cells (G1-ILCs), including circulating natural killer (NK) cells and tissue-resident type 1 ILCs (ILC1s), are innate immune sentinels critical for responses against infection and cancer. In contrast to relatively uniform NK cells through the body, diverse ILC1 subsets have been characterized across and within tissues in mice, but their developmental and functional heterogeneity remain unsolved. Here, using multimodal in vivo approaches including fate-mapping and targeting of the interleukin 15 (IL-15)-producing microenvironment, we demonstrate that liver parenchymal niches support the development of a cytotoxic ILC1 subset lacking IL-7 receptor (7R- ILC1s). During ontogeny, fetal liver (FL) G1-ILCs arise perivascularly and then differentiate into 7R- ILC1s within sinusoids. Hepatocyte-derived IL-15 supports parenchymal development of FL G1-ILCs to maintain adult pool of 7R- ILC1s. IL-7R+ (7R+) ILC1s in the liver, candidate precursors for 7R- ILC1s, are not essential for 7R- ILC1 development in physiological conditions. Functionally, 7R- ILC1s exhibit killing activity at steady state through granzyme B expression, which is underpinned by constitutive mTOR activity, unlike NK cells with exogenous stimulation-dependent cytotoxicity. Our study reveals the unique ontogeny and functions of liver-specific ILC1s, providing a detailed interpretation of ILC1 heterogeneity.

immunology↗

bioRxiv2020

Injectable prevascularized mature adipose tissues (iPAT) to achieve long-term survival in soft tissues regeneration

Soft tissue regeneration remains a challenge in reconstructive surgery. Current autologous fat implantations lead to high fat absorption ratios, while artificial implants can be associated with lymphoma occurrence. To overcome these limitations, our aim was to reproduce adipose tissue vasculature structure before implantation. Here, we developed injectable prevascularized adipose tissues (iPAT), using physiological collagen microfibers (CMF) mixed with human mature adipocytes, adipose-derived stem cells (ADSC) and human umbilical vein endothelial cells (HUVEC). Following murine subcutaneous implantation, higher cell survival (84{+/-}6% viability) and volume maintenance were shown after 3 months for the iPAT (up to twice heavier than the non-prevascularized balls). This higher survival can be explained by the greater amount of blood vessels (up to 1.6 folds increase), with balanced host anastomosis (51{+/-}1% of human/mouse lumens), also involving infiltration by the lymphatic and neural vasculature networks. These iPAT tissues allowed non-invasive soft tissue reconstruction for long-term outcomes, and the ability to cryopreserve them with maintained viability and functionality also enables a later reinjection usually required before reaching the final patient desired graft volume.

bioengineering↗

bioRxiv2018

Autonomous functionality of an upstream open reading frame in polycistronic mammalian mRNAs

Upstream open reading frames (uORFs) are established as cis-acting elements for eukaryotic translation of annotated ORFs (anORFs) located on the same mRNAs. Here, we identified a mammalian uORF with functions that are independent from anORF translation regulation. Bioinformatics screening using ribosome profiling data of human and mouse brains yielded 308 neurologically vital genes from which anORF and uORFs are polycistronically translated in both species. Among them, Arhgef9 contains a uORF named SPICA, which is highly conserved among vertebrates and stably translated only in specific brain regions of mice. Disruption of SPICA translation by ATG-to-TAG substitutions did not perturb translation or function of its anORF product, collybistin. SPICA-null mice displayed abnormal maternal reproductive performance and enhanced anxiety-like behavior, characteristic of ARHGEF9-associated neurological disorders. This study demonstrates that mammalian uORFs can be independent genetic units, revising the prevailing dogma of the monocistronic gene in mammals, and even eukaryotes.

molecular biology↗

bioRxiv2017

An engineered cellular Timer for analysing the time domain of cellular differentiation in vivo

Understanding the mechanisms of cellular differentiation is challenging because differentiation is initiated by signaling pathways that drive temporally dynamic processes, which are difficult to analyse in vivo. We establish a new Tool, Timer-of-cell-kinetics-and-activity (Tocky [toki], time in Japanese). Tocky uses the Fluorescent Timer protein, which spontaneously shifts its emission spectrum from blue-to-red, in combination with computer algorithms to reveal the dynamics of differentiation in vivo. Using a transcriptional target of T cell receptor (TCR)-signaling, we establish Nr4a3-Tocky to follow downstream effects of TCR signaling. Nr4a3-Tocky reveals the temporal sequence of events during regulatory T cell (Treg) differentiation and shows that persistent TCR signals occur during Treg generation. Remarkably, antigen-specific T cells at the site of autoimmune inflammation also show persistent TCR signaling. In addition, by generating Foxp3-Tocky, we reveal the in vivo dynamics of demethylation of the Foxp3 gene. Thus, Tocky is a Tool for cell biologists to address previously inaccessible questions by directly revealing dynamic processes in vivo.\n\nSummaryThe authors establish a new Tool, Timer-of-cell-kinetics-and-activity (Tocky) revealing the temporal dynamics of cellular activation and differentiation in vivo. The tool analyses the temporal sequence of molecular processes during cellular differentiation and identifies cells that receive persistent signals in vivo.

cell biology↗