Showing papers by "Hiroki R. Ueda published in 2019"

Advanced CUBIC tissue clearing for whole-organ cell profiling.

Abstract: Tissue-clearing techniques are powerful tools for biological research and pathological diagnosis. Here, we describe advanced clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) procedures that can be applied to biomedical research. This protocol enables preparation of high-transparency organs that retain fluorescent protein signals within 7-21 d by immersion in CUBIC reagents. A transparent mouse organ can then be imaged by a high-speed imaging system (>0.5 TB/h/color). In addition, to improve the understanding and simplify handling of the data, the positions of all detected cells in an organ (3-12 GB) can be extracted from a large image dataset (2.5-14 TB) within 3-12 h. As an example of how the protocol can be used, we counted the number of cells in an adult whole mouse brain and other distinct anatomical regions and determined the number of cells transduced with mCherry following whole-brain infection with adeno-associated virus (AAV)-PHP.eB. The improved throughput offered by this protocol allows analysis of numerous samples (e.g., >100 mouse brains per study), providing a platform for next-generation biomedical research.

SMILES Transformer: Pre-trained Molecular Fingerprint for Low Data Drug Discovery

Abstract: In drug-discovery-related tasks such as virtual screening, machine learning is emerging as a promising way to predict molecular properties. Conventionally, molecular fingerprints (numerical representations of molecules) are calculated through rule-based algorithms that map molecules to a sparse discrete space. However, these algorithms perform poorly for shallow prediction models or small datasets. To address this issue, we present SMILES Transformer. Inspired by Transformer and pre-trained language models from natural language processing, SMILES Transformer learns molecular fingerprints through unsupervised pre-training of the sequence-to-sequence language model using a huge corpus of SMILES, a text representation system for molecules. We performed benchmarks on 10 datasets against existing fingerprints and graph-based methods and demonstrated the superiority of the proposed algorithms in small-data settings where pre-training facilitated good generalization. Moreover, we define a novel metric to concurrently measure model accuracy and data efficiency.

Reduced Neoantigen Expression Revealed by Longitudinal Multiomics as a Possible Immune Evasion Mechanism in Glioma.

Abstract: Immune-based therapies have shown limited efficacy in glioma thus far. This might be at least in part due to insufficient numbers of neoantigens, thought to be targets of immune attack. In addition, we hypothesized that dynamic genetic and epigenetic tumor evolution in gliomas might also affect the mutation/neoantigen landscape and contribute to treatment resistance through immune evasion. Here, we investigated changes in the neoantigen landscape and immunologic features during glioma progression using exome and RNA-seq of paired primary and recurrent tumor samples obtained from 25 WHO grade II-IV glioma patients (glioblastoma, IDH-wild-type, n = 8; grade II-III astrocytoma, IDH-mutant, n = 9; and grade II-III oligodendroglioma, IDH-mutant, 1p/19q-codeleted, n = 8). The number of missense mutations, predicted neoantigens, or expressed neoantigens was not significantly different between primary and recurrent tumors. However, we found that in individual patients the ratio of expressed neoantigens to predicted neoantigens, designated the "neoantigen expression ratio," decreased significantly at recurrence (P = 0.003). This phenomenon was particularly pronounced for "high-affinity," "clonal," and "passenger gene-derived" neoantigens. Gene expression and IHC analyses suggested that the decreased neoantigen expression ratio was associated with intact antigen presentation machinery, increased tumor-infiltrating immune cells, and ongoing immune responses. Our findings imply that decreased expression of highly immunogenic neoantigens, possibly due to persistent immune selection pressure, might be one of the immune evasion mechanisms along with tumor clonal evolution in some gliomas.

Comprehensive assay for the molecular profiling of cancer by target enrichment from formalin-fixed paraffin-embedded specimens

Abstract: Tumor molecular profiling is becoming a standard of care for patients with cancer, but the optimal platform for cancer sequencing remains undetermined. We established a comprehensive assay, the Todai OncoPanel (TOP), which consists of DNA and RNA hybridization capture-based next-generation sequencing panels. A novel method for target enrichment, named the junction capture method, was developed for the RNA panel to accurately and cost-effectively detect 365 fusion genes as well as aberrantly spliced transcripts. The TOP RNA panel can also measure the expression profiles of an additional 109 genes. The TOP DNA panel was developed to detect single nucleotide variants and insertions/deletions for 464 genes, to calculate tumor mutation burden and microsatellite instability status, and to infer chromosomal copy number. Clinically relevant somatic mutations were identified in 32.2% (59/183) of patients by prospective TOP testing, signifying the clinical utility of TOP for providing personalized medicine to cancer patients.

Insights into magnetoelectric coupling mechanism of the room-temperature multiferroic S r 3 C o 2 F e 24 O 41 from domain observation

Abstract: The mechanism of a magnetoelectric coupling in a room-temperature multiferroic, $\mathrm{S}{\mathrm{r}}_{3}\mathrm{C}{\mathrm{o}}_{2}\mathrm{F}{\mathrm{e}}_{24}{\mathrm{O}}_{41}$, with the Z-type hexaferrite structure, is examined by three approaches: observations of domain structures and their field responses, measurements of magnetic-field effect on electric polarization, i.e., magnetoelectric effect, and phenomenological discussions on the interplay among coexisting order parameters. With use of a resonant soft x-ray microdiffraction technique, we visualized magnetic-field responses of two types of magnetic domains ascribed to ferrimagnetic and spiral components inherent in a transverse conical magnetic structure of the hexaferrite. A simultaneous inversion of these magnetic domains by a magnetic-field reversal was observed, meaning that the process of a magnetization reversal corresponds to a 180\ifmmode^\circ\else\textdegree\fi{} rotation of the cone axis. The reversal process of the magnetic structure, together with experimental results of the magnetoelectric effect, leads us to the conclusion that the magnetoelectricity in the Z-type hexaferrite originates mainly from the spin-dependent metal-ligand orbital hybridization, with minor contribution from the asymmetric spin-exchange interaction. Furthermore, such a mechanism is discussed by the symmetry analysis based on the Landau theory and is well described in terms of couplings among the coexisting order parameters included in the free energy. Thus, observations on field responses of multiple domains in multiferroics provide insights into underlying microscopic magnetoelectric coupling mechanisms.

A period without PER: understanding 24-hour rhythms without classic transcription and translation feedback loops.

Abstract: Since Ronald Konopka and Seymour Benzer's discovery of the gene Period in the 1970s, the circadian rhythm field has diligently investigated regulatory mechanisms and intracellular transcriptional and translation feedback loops involving Period, and these investigations culminated in a 2017 Nobel Prize in Physiology or Medicine for Michael W. Young, Michael Rosbash, and Jeffrey C. Hall. Although research on 24-hour behavior rhythms started with Period, a series of discoveries in the past decade have shown us that post-transcriptional regulation and protein modification, such as phosphorylation and oxidation, are alternatives ways to building a ticking clock.

Genes and Ion Channels in the Circadian and Homeostatic Regulation of Sleep

Abstract: The timing and duration of sleep is an evolutionarily conserved process. Sleep timing is regulated by the circadian clock, and genetic and physiological studies in humans and animal models reveal that sleep timing is regulated by molecular networks of clock genes and cellular circuits of clock neurons. Although sleep duration seems to be regulated by networks of neural circuits, recent reverse and forward genetics have revealed several genes that regulate sleep duration and homeostasis, which suggests that sleep duration is also regulated by both cellular circuits and molecular networks. In this chapter, we discuss how timing of sleep is regulated by circadian clocks and how homeostatic regulation of sleep amount occurs.

A Microfluidic Platform Based on Robust Gas and Liquid Exchange for Long-term Culturing of Explanted Tissues.

Abstract: Microfluidic devices are important platforms to culture and observe biological tissues. Compared with conventional setups, microfluidic devices have advantages in perfusion, including an enhanced delivery of nutrients and gases to tissues. However, explanted tissues can maintain their functions for only hours to days in microfluidic devices, although their observations are desired for weeks. The suprachiasmatic nucleus (SCN) is a brain region composed of heterogeneous cells to control the biological clock system through synchronizing individual cells in this region. The synchronized and complicated cell-cell interactions of SCN cells are difficult to reproduce from seeded cells. Thus, the viability of explanted SCN contributes to the study of SCN functions. In this paper, we propose a new perfusion platform combining a PDMS microfluidic device with a porous membrane to culture an explanted SCN for 25 days. We expect that this platform will provide a universal interface for microfluidic manipulation of tissue explants.