Cancer genomes accumulate somatic mutations over time, influenced by both intrinsic and extrinsic mutational processes. In metastatic cancer, disseminated tumor cells may acquire additional mutations at metastatic sites, shaped by extrinsic factors distinct from those at the primary tumor. As a result, cancer genomes at metastatic sites may bear mutational signatures originating from both primary and metastatic environments. However, the patterns and relative contributions of mutational signatures specific to metastatic sites remain poorly understood. To investigate this, we analyzed mutational signatures from seven metastatic cancer patients. We observed distinct mutational patterns between early and late mutation profiles within individual patients, where the early and late categories were based on their relative timing during tumor evolution. Early mutations were often dominated by a single mutational signature that accounted for more than half of the total signature burden. These dominant signatures tended to be shared among tumors of the same cancer type, suggesting that early mutations in metastatic cancers may be shaped by a single, highly active mutational process at the primary tumor site. In contrast, late mutations were often more poorly decomposed into distinct mutational signatures, reflecting more complex and diverse compositions. Overall, early mutations tended to preserve clearer signals of their origin.

Single-cell RNA sequencing (scRNA-seq) simultaneously provides gene-expression profiles and genetic variants from individual cells, creating an opportunity to relate cellular phenotypes to their somatic evolutionary histories. However, delineation of genetic type (GTs) from scRNA-seq remains difficult because most variant positions are unobserved in individual cells and the observed base calls contain substantial false-positive and false-negative errors. We evaluated some existing phylogenetic and imputation methods using one simulated dataset and two empirical tumor datasets. We found that extreme sparsity prevented reliable recovery of known or independently inferred GTs when multiple GTs were present. This led us to adapt the STICI transformer architecture to train a separate model de novo on each sparse cell-variant (CV) matrix. These data-specific models predicted millions of missing bases, greatly reducing matrix sparsity. Phylogenetic analyses of the imputed CV matrices showed substantially improved recovery of GTs in both simulated and empirical datasets. In the empirical dataset, transformer-based analysis also suggested finer-scale genetic structure within some previously reported GTs that was not apparent with the existing methods. These results demonstrate that highly sparse scRNA-seq datasets contain substantially more recoverable lineage information than previously appreciated and that de novo transformer modeling provides an effective approach for recovering much of this hidden information. Nevertheless, sequencing errors persisted, limiting reconstruction of cellular lineage structure and leaving significant room for methodological improvement before expression phenotypes can be examined reliably in the context of their cellular evolutionary relationships.

RNA sequencing (RNA-Seq) is a high-throughput sequencing approach that enables comprehensive quantification of transcriptomes at a genome-wide scale. As a result, RNA-Seq has become a routine component of molecular biology research, and more researchers are now expected to analyze RNA-Seq data as part of their projects. However, unlike the largely experimental nature of benchwork, RNA-Seq analysis demands proficiency with computational and statistical approaches to manage technical issues and large data sizes. Although numerous manuals and reviews on RNA-Seq data analysis are available, many are either highly specialized, fragmented, or overly superficial, leaving beginners to use tools without understanding the underlying principles. To address this gap, we provide a decision-oriented guide tailored for molecular biologists encountering RNA-Seq analysis for the first time. This review is designed for readers to enable to decide which tools and statistical approaches to use based on their data, goals, and constraints. We aim to equip beginners with the knowledge required to perform RNA-Seq analysis rigorously and with confidence.

Water is one of the most common molecules in the universe. Water is polarized, but it has many states besides the normal tetrahedron depicted in standard biology texts. Water is also the most ubiquitous molecule on Earth, the universal solvent. It is the internal and external habitat of cells. Ecologically, water is contiguous with life and the chemistry it nourishes. Water merges with everything from DNA to itself in the vast ocean; it is a constant molecule, and it does not change—or does it? Water, is the planet’s unwavering, flowing, but fixed liquid substrate, and it has an elemental and evolutionary story to tell. Water can independently regulate solute transport, entangling with cell proteins to create the aqueous conditions that support life metabolisms and the evolution of other molecules. Water dynamics are rarely mentioned in standard biology discussions, even though biomolecules are strongly influenced by the hydration shells around them. For water to affiliate with all things living requires specialized entry and exiting of water, achievable by a ubiquitous channel protein called an aquaporin. In this article, we will explore water’s often neglected complex relationship with all things biological from an aquaporin perspective. The aquaporin family of proteins is ancient and spans the tree of life in archaea, bacteria, protozoa, fungi, plants, animals, and viruses. From DNA to osmoregulation, aquaporins literally channel the water molecule through geological time. We will also explore the bigger picture of the aquaporin as a teaching tool for evolution. Through the genomic medicine paradigm, we examine diseases that manifest from defective aquaporins. From a visual and arts perspective, we reframe biological processes in the light of the most abundant but nominally understood molecule on Earth: water.

Water is one of the most common molecules in the universe. Water is polarized, but it has many states besides the normal tetrahedron depicted in standard biology texts. Water is also the most ubiquitous molecule on Earth, the universal solvent. It is the internal and external habitat of cells. Ecologically, water is contiguous with life and the chemistry it nourishes. Water merges with everything from DNA to itself in the vast ocean; it is a constant molecule, and it does not change-or does it? Water, is the planet's unwavering, flowing, but fixed liquid substrate, and it has an elemental and evolutionary story to tell. Water can independently regulate solute transport, entangling with cell proteins to create the aqueous conditions that support life metabolisms and the evolution of other molecules. Water dynamics are rarely mentioned in standard biology discussions, even though biomolecules are strongly influenced by the hydration shells around them. For water to affiliate with all things living requires specialized entry and exiting of water, achievable by a ubiquitous channel protein called an aquaporin. In this article, we will explore water's often neglected complex relationship with all things biological from an aquaporin perspective. The aquaporin family of proteins is ancient and spans the tree of life in archaea, bacteria, protozoa, fungi, plants, animals, and viruses. From DNA to osmoregulation, aquaporins literally channel the water molecule through geological time. We will also explore the bigger picture of the aquaporin as a teaching tool for evolution. Through the genomic medicine paradigm, we examine diseases that manifest from defective aquaporins. From a visual and arts perspective, we reframe biological processes in the light of the most abundant but nominally understood molecule on Earth: water.

DNA sequencing technologies are widely used to study tumor evolution within a cancer patient. However, analyses require various computational methods, including those to infer clone sequences (genotypes of cancer cell populations), clone frequencies within each tumor sample, clone phylogeny, mutational tree, dynamics of mutational signatures, and metastatic cell migration events. Therefore, we developed GenoPath, a streamlined pipeline of existing tools to perform tumor evolution analysis. We also developed and added tools to visualize results to assist interpretation and derive biological insights. We have illustrated GenoPath's utility through a case study of tumor evolution using metastatic prostate cancer data. By reducing computational barriers, GenoPath broadens access to tumor evolution analysis. The software is available at https://github.com/SayakaMiura/GP.

The phylogeny of a person’s hematopoietic stem cells (HSCs) can be used to quantify physiological aging of blood using a phyloAge model based on diversity decay metrics. However, this procedure currently requires accurate HSC genome sequences, which are expensive and time-consuming to obtain. We show that metrics of diversity decay can be derived from the somatic variant frequency spectrum (VFS) using more affordable, routine bulk sequencing, because HSCs evolve without recombination at a clock-like rate. We found that VFS-based models produce phyloAge estimates similar to those derived from HSC genome phylogenies. Customized for protein-coding variation and sequencing read depth, VFS-based HSC phyloAge estimates were, on average, 168 years more than chronological ages in 157 patients with acute myeloid leukemia, consistent with excess HSC aging observed in cancer patients using single cell genome phylogenies. We also tested the hypothesis that variants in cancer driver genes may confer longevity, as they occur in a significant fraction of long-lived individuals. Indeed, HSC phyloAge estimates were significantly lower, consistent with reduced hematologic cancer risk among extremely old individuals. Thus, the new metrics and models broaden the utility of the phyloAge approach, making it feasible and efficient for clinical and research applications.

Transdermal drug delivery minimizes pain and provides a controlled, stable release of drugs, but its effectiveness is limited by the skin’s natural barriers. Microneedles overcome this problem, enabling minimally invasive drug delivery. Microneedle patches (MNPs) with 80 µm-tall needles composed of hyaluronic acid (HA) were developed and evaluated for their formability, structural integrity, dissolution rate, skin penetration ability, and drug transmission capacity. The influence of the molecular weight of HA on these properties was also investigated. MNPs made from low-molecular-weight HA (30 kDa–50 kDa) demonstrated 12.5 times superior drug permeability in ex vivo human skin compared to needleless patches (NLPs). Furthermore, in the same test, low-molecular-weight HA MNPs had 1.7 times higher drug permeability than high-molecular-weight HA MNPs, suggesting superior transdermal administration. The molecular weight of HA significantly influenced its solubility and permeability, highlighting the potential effectiveness of MNPs as drug delivery systems. Puncture tests demonstrated a penetration depth of 50–60 µm, indicating minimal nerve irritation in the dermis and effective drug delivery to the superficial dermal layer. These results present a manufacturing technique for MNPs incorporating model drug compounds and highlight their potential as a novel and minimally invasive drug delivery method for the biomedical applications of soft gels.

Nanoimprint lithography, a technique within microfabrication, continues to progress due to its ability to pattern large areas with high resolution, efficiency, and cost-effectiveness. Among its variants, UV nanoimprint lithography offers rapid curing through UV light and exceeds thermal nanoimprint lithography in terms of the throughput. However, UV nanoimprint lithography often traps air during the imprinting process and molds made from non-gas-permeable materials such as quartz and metal can result in molding defects. In this study, a new TiO2-SiO2 radical-based gas-permeable mold surface material was developed using the sol-gel method to enhance ultraviolet-based nanoimprint lithography for precise processing. Compared to existing material, this surface material exhibited superior performance in terms of both gas permeability and mechanical properties, with oxygen gas permeability 1.2 times higher, carbon dioxide gas permeability 1.3 times higher, and flexural strength 1.1 times higher than those of existing material. Based on these performance enhancements, the microfabrication demonstrated superior transfer accuracy compared to master molds with specifications of (a) pitch: 20 mu m, height: 17.1 mu m, bottom diameter: 7.14 mu m, and (b) pitch: 50 mu m, height: 17.0 mu m, bottom diameter: 7.14 mu m, achieving (a) height 99.9%, bottom diameter 99.0%, and (b) height 99.8%, bottom diameter 94.1%. Additionally, these gas-permeable molds facilitated high-precision fine processing on the surface of lactic acid-glycolic acid copolymers, reaching (a) bottom diameter of 95.3% and (b) 93.7%, respectively. The findings from this study will advance precision processing technology, improve the accuracy of fine processing in machine tools, and enhance production efficiency. This is particularly anticipated to aid the development of advanced production systems in sectors such as medical devices, semiconductor manufacturing, optical components, and microfluidic devices, thereby promoting future industrial growth.

Polylactic acid (PLA), a biodegradable material derived from renewable sources, has been challenging to fabricate through injection molding, owing to its narrow crystallization range and poor heat resistance. Conventional injection molding also encounters difficulties with gas venting in the cavity, which impedes microscale processing. This study successfully performed micro-injection molding of standard PLA under typical conditions. Using an amine-containing gas-permeable hybrid molds, PLA microstructures measuring 1.2 mu m in height and 2.7 mu m in base diameter were successfully molded, demonstrating high moldability. This study established the viability of micro-injection molding for PLA and may provide an important foundation for the future development of micro-surface fabricated devices made of PLA in the biomedical field, such as blood coagulation prevention medical devices based on microfabrication

We attempted to perform surface microfabrication of the bioabsorbable material lactic acid–glycolic acid copolymer (LG-80) using a micro-imprint lithography technique with a gas-permeable porous mold at less than 5 °C. As a result, high-resolution surface micromachining with a height of 1.26 μm and a pitch of 2.97 μm was achieved using a convex sapphire mold with a height of 1.3 μm and a pitch of 3 μm. After processing, the LG-80 exhibited high water repellency, and FT-IR analysis of the surface showed no significant change in its chemical structure, confirming that the surface microfabrication was successful, while retaining the properties of the material. This demonstrated new possibilities for surface microfabrication technology for bioabsorbable materials, which are expected to be applied in the medical and life science fields in products such as surgical implants, tissue regeneration materials, and cell culture scaffold materials. In particular, the use of micro-imprint lithography enables low-cost and high-precision processing, which will be a major step toward the practical application of bioabsorbable materials.

In this study, water-developable photoresist materials capable of fine patterning were developed by imparting photosensitive groups to a molecule made from hemicellulose arabinoxylan extracted from corn bran, which were fabricated by UV exposure at 365 nm and 45 J/cm(2). The surface topography of the photoresist pattern developed with water was observed to be approximately 0.46 mu m. The etching resistance was improved in the photoresist material made from hemicellulose arabinoxylan compared with other water-soluble polymers. The development of water-developable photoresist materials derived from hemicellulose is expected to reduce environmental impact. In addition to the electronics field, where photoresist materials have been used in the past, applications in the medical field and biosystems are also expected since the material is derived from biomass.

Microneedles are of great interest in diverse fields, including cosmetics, drug delivery systems, chromatography, and biological sensing for disease diagnosis. Self-dissolving ultrafine microneedles of pure sodium hyaluronate hydrogels were fabricated using a UV-curing TiO2-SiO2 gas-permeable mold polymerized by sol-gel hydrolysis reactions in nanoimprint lithography processes under refrigeration at 5 °C, where thermal decomposition of microneedle components can be avoided. The moldability, strength, and dissolution behavior of sodium hyaluronate hydrogels with different molecular weights were compared to evaluate the suitability of ultrafine microneedles with a bottom diameter of 40 μm and a height of 80 μm. The appropriate molecular weight range and formulation of pure sodium hyaluronate hydrogels were found to control the dissolution behavior of self-dissolving ultrafine microneedles while maintaining the moldability and strength of the microneedles. This fabrication technology of ultrafine microneedles expands their possibilities as a next-generation technique for bioactive gels for controlling the blood levels of drugs and avoiding pain during administration.

Abstract In cancer, somatic mutations occur continuously, causing cell populations to evolve. These somatic mutations result in the evolution of cellular gene expression patterns that can also change due to epigenetic modifications and environmental changes. By exploring the concordance of gene expression changes with molecular evolutionary trajectories of cells, we can examine the role of somatic variation on the evolution of gene expression patterns. In these endeavors, reliable inference of the evolutionary relationship of single cells is a key step. However, single-cell sequences contain many errors and missing bases, which necessitate advancing standard molecular phylogenetics approaches for applications in analyzing these datasets. We first show our new computational approach (TopHap+) that integratively applies standard phylogenetic optimality principles and patterns of co-occurrence of sequence variations to produce more expansive and accurate cellular phylogenies from single-cell sequence datasets. We then present Multi-Omics Concordance Analysis (MOCA) software to jointly analyze gene expressions and genetic variations from single-cell RNA sequencing profiles. MOCA outputs cells and genes showing convergent and divergent gene expression patterns in functional genomics. Citation Format: Sayaka Miura, Sudhir Kumar. Reconstruction of cellular phylogenies and analysis of gene expression variation in single cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Cancer Evolution and Data Science: The Next Frontier; 2023 Dec 3-6; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_2):Abstract nr B007.

In tissue engineering and regenerative medicine, scaffold micropatterning plays an essential role in reproducing the microscopic cellular environment and cell-cell interactions. This study provides a novel molding process for surface microfabricaion of 100% pure fish-derived collagen without the use of photoinitiators, which can be cytotoxic, by low-temperature molding at 5℃ using nanoimprint lithography. TiO2-SiO2 gas-permeable porous mold was used to fabricate collagen micropatterns, which can improve gas entrapment during molding, one of the challenges in nanoimprint lithography. The excellent gas permeability of TiO2-SiO2 gas-permeable porous mold enabled fine patterning with a height of 80 μm and a bottom diameter of 40 μm without molding defects for a collagen solution containing 40 wt% water. FT-IR spectral measurements revealed that low-temperature drying at 5℃ during microfabrication to the collagen surface had almost no effect on the collagen components. This molding process, which does not require chemical modification of collagen and does not cause protein denaturation even at molding temperatures of 5℃, has the potential to be widely used as a next-generation medical application technology in the fields of tissue engineering and regenerative medicine.

In this study, UV-curing gas-permeable mold material was developed that can improve gas entrapment during fabricating, which is a challenge in nanoimprint lithography (NIL). An advanced nanofabrication technology that hybridizes the high-resolution of NIL with the mass productivity of injection molding has enabled nanofabrication of polypropylene with a height of 300 nm and a base diameter of 240 nm. This technology enables nanofabrication of plastics with short cycle times, and is expected to create functional surfaces such as antibacterial through nanofabrication.

Conventional photoresist materials are generally coated and developed using organic solvents, but the water-soluble photoresist material in this study can be coated and developed using water. Patterning of water-soluble photoresist material on PMMA, which was contaminated or damaged by the organic coating solvent and developer in the conventional photoresist material, resulted in the fabrication of 6 µm holes and 3 µm lines. The water-based coating and development process is expected to contribute to biomaterial applications and environmental impact.

In tissue engineering and regenerative medicine, scaffold micropatterning plays an essential role in reproducing the microscopic cellular environment and cell-cell interactions. This study provides a novel molding process for surface micro fabricaion of 100% pure fish derived collagen without the use of photo initiators, which can be cytotoxic, by low temperature molding at 5 degrees C using nanoimprint lithography. TiO2-SiO2 gas-permeable porous mold was used to fabricate collagen micropatterns, which can improve gas entrapment during molding, one of the challenges in nanoimprint lithography. The excellent gas permeability of TiO2-SiO2 gas-permeable porous mold enabled fine patterning with a height of 80 mu m and a bottom diameter of 40 mu m without molding defects for a collagen solution containing 40 wt% water. FT-IR spectral measurements revealed that low-temperature drying at 5 degrees C during microfabrication to the collagen surface had almost no effect on the collagen components. This molding process, which does not require chemical modification of collagen and does not cause protein denaturation even at molding temperatures of 5 degrees C , has the potential to be widely used as a next-generation medical application technology in the fields of tissue engineering and regenerative medicine.

Cationic gas-permeable molds fabricated via sol–gel polymerization undergo cationic polymerization using epoxide, resulting in gas permeability owing to their cross-linked structures. By applying this cationic gas-permeable mold to nano-injection molding, which is used for the mass production of resins, nano-protrusion structures with a height of approximately 300 nm and a pitch of approximately 400 nm were produced. The molding defects caused by gas entrapment in the air and cavities when using conventional gas-impermeable metal molds were improved, and the cationic gas-permeable mold could be continuously fabricated for 3000 shots under non-vacuum conditions. The results of the mechanical evaluations showed improved thermal stability and Martens hardness, which is expected to lead to the advanced production of resin nano-structures. Furthermore, the surface roughness of the nano-protrusion structures fabricated using injection molding improved the water contact angle by approximately 46°, contributing to the development of various hydrophobic materials in the future.