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Integration of Droplet Microfluidic Tools for Single-cell Functional Metagenomics:An Engineering Head Start

Genomics, Proteomics & Bioinformatics 2021年第3期19卷 504-518页

作者： David Conchouso Amani Al-Ma’abadi Hayedeh Behzad Mohammed Alarawi Masahito Hosokawa Yohei Nishikawa Haruko Takeyama Katsuhiko Mineta Takashi Gojobori Department of Industrial and Mechanical Engineering Universidad de las Américas PueblaPuebla 72810Mexico Computational Bioscience Research Center King Abdullah University of Science and TechnologyThuwal 23955-6900Saudi Arabia Biological and Environmental Sciences and Engineering Division King Abdullah University of Science and TechnologyThuwal 23955-6900Saudi Arabia Research Organization for Nano&Life Innovation Waseda UniversityTokyo 162-0041Japan Department of Life Science and Medical Bioscience Waseda UniversityTokyo 162-8480Japan Institute for Advanced Research of Biosystem Dynamics Waseda Research Institute for Science and EngineeringWaseda UniversityTokyo 169-8555Japan Computational Bio Big-Data Open Innovation Laboratory AIST-Waseda UniversityTokyo 169–0072Japan Computer Electricaland Mathematical Sciences and Engineering DivisionKing Abdullah University of Science and TechnologyThuwal 23955-6900Saudi Arabia

Droplet microfluidic techniques have shown promising outcome to study single cells at high throughput.However,their adoption in laboratories studying“-omics”sciences is still irrelevant due to the complex and multidisciplinary nature of the field.To facilitate their use,here we provide engineering details and organized protocols for integrating three droplet-based microfluidic technologies into the metagenomic pipeline to enable functional screening of bioproducts at high throughput.First,a device encapsulating single cells in droplets at a rate of~250 Hz is described considering droplet size and cell growth.Then,we expand on previously reported fluorescence-activated droplet sorting systems to integrate the use of 4 independent fluorescence-exciting lasers(i.e.,405,488,561,and 637 nm)in a single platform to make it compatible with different fluorescence-emitting biosensors.For this sorter,both hardware and software are provided and optimized for effortlessly sorting droplets at 60 Hz.Then,a passive droplet merger is also integrated into our pipeline to enable adding new reagents to already-made droplets at a rate of 200 Hz.Finally,we provide an optimized recipe for manufacturing these chips using silicon dry-etching tools.Because of the overall integration and the technical details presented here,our approach allows biologists to quickly use microfluidic technologies and achieve both single-cell resolution and high-throughput capability(>50,000 cells/day)for mining and bioprospecting metagenomic data.

关键词： Droplet microfluidics Single cell Metagenomics Droplet sorter Biotechnology

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Cortical transcriptome analysis after spinal cord injury reveals the regenerative mechanism of central nervous system in CRMP2 knock-in mice

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Neural Regeneration Research 2021年第7期16卷 1258-1265页

作者： Ayaka Sugeno Wenhui Piao Miki Yamazaki Kiyofumi Takahashi Koji Arikawa Hiroko Matsunaga Masahito Hosokawa Daisuke Tominaga Yoshio Goshima Haruko Takeyama Toshio Ohshima Laboratory for Molecular Brain Science Department of Life Science and Medical BioscienceGraduate School of Advanced Science and EngineeringWaseda UniversityTokyoJapan Biomolecular Engineering Laboratory Department of Life Science and Medical BioscienceGraduate School of Advanced Science and EngineeringWaseda UniversityTokyoJapan Computational Bio Big-Data Open Innovation Laboratory(CBBD-OIL) National Institute of Advanced Industrial Science and Technology(AIST)TokyoJapan Research Organization for Nano and Life Innovation Waseda UniversityTokyoJapan Institute for Advanced Research of Biosystem Dynamics Waseda Research Institute for Science and EngineeringGraduate School of Advanced Science and EngineeringWaseda UniversityTokyoJapan Cellular and Molecular Biotechnology Research Institute National Institute of Advanced Industrial Science and Technology(AIST)TsukubaJapan Department of Molecular Pharmacology and Neurobiology Graduate School of MedicineYokohama City UniversityYokohamaJapan

Recent studies have shown that mutation at Ser522 causes inhibition of collapsin response mediator protein 2(CRMP2) phosphorylation and induces axon elongation and partial recovery of the lost sensorimotor function after spinal cord injury(SCI).We aimed to reveal the intracellular mechanism in axotomized neurons in the CRMP2 knock-in(CRMP2KI) mouse model by performing transcriptome analysis in mouse sensorimotor cortex using micro-dissection punching system.Prior to that, we analyzed the structural pathophysiology in axotomized or neighboring neurons after SCI and found that somatic atrophy and dendritic spine reduction in sensorimotor cortex were suppressed in CRMP2KI mice.Further analysis of the transcriptome has aided in the identification of four hemoglobin genes Hba-a1, Hba-a2, Hbb-bs, and Hbb-bt that are significantly upregulated in wild-type mice with concomitant upregulation of genes involved in the oxidative phosphorylation and ribosomal pathways after SCI.However, we observed substantial upregulation in channel activity genes and downregulation of genes regulating vesicles, synaptic function, glial cell differentiation in CRMP2KI mice.Moreover, the transcriptome profile of CRMP2KI mice has been discussed wherein energy metabolism and neuronal pathways were found to be differentially regulated.Our results showed that CRMP2KI mice displayed improved SCI pathophysiology not only via microtubule stabilization in neurons, but also possibly via the whole metabolic system in the central nervous system, response changes in glial cells, and synapses.Taken together, we reveal new insights on SCI pathophysiology and the regenerative mechanism of central nervous system by the inhibition of CRMP2 phosphorylation at Ser522.All these experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee at Waseda University, Japan(2017-A027 approved on March 21, 2017;2018-A003 approved on March 25, 2018;2019-A026 approved on March 25, 2019).

关键词： CNS regeneration cortex CRMP2 hemoglobin metabolic pathway spinal cord injury spine transcriptome

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