Biology subjects

Ohkubo, T.

Publications and source records attributed to Ohkubo, T..

5 recordsLinked to original sources

bioRxiv2023

Somatic Mosaicism in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Reveals Widespread Degeneration from Focal Mutations

Although mutations in many genes cause familial amyotrophic lateral sclerosis and frontotemporal dementia, most cases are sporadic (sALS and sFTD) with unclear etiology. We tested whether somatic mutations contribute to sALS and sFTD by deep targeted sequencing of 88 neurodegeneration-related genes in postmortem brain and spinal cord samples from 399 sporadic cases and 144 controls. Predicted deleterious somatic variants in ALS/FTD genes were observed in 2.1% of sporadic cases lacking deleterious germline variants. These variants occurred at very low allele fractions (typically <2%) and were often focal and enriched in disease-affected regions. Analysis of bulk RNA-seq data from an additional cohort identified deleterious somatic variants in DYNC1H1 and LMNA, genes associated with pediatric motor neuron degeneration. Targeted long-read sequencing further identified one sFTD case with de novo somatic C9orf72 repeat expansions. Together, these findings suggest that rare, focal somatic variants can contribute to sALS and sFTD and drive widespread neurodegeneration.

genetics↗

bioRxiv2023

In vivo visualization of intracellular pH gradient in brain using PET imaging

Intracellular pH (pHi) is a valuable index for predicting hypoxic brain damage. However, no positron emission tomography (PET) probe is currently available for monitoring pHi in vivo. In this study, we developed a new approach for visualizing monoacylglycerol lipase (MAGL) activity in the brain. This approach used PET with a new probe [11C]HC-A, an azetidine carbamate inhibitor, whose uptake and residence depended on the pHi gradient was evaluated with in silico, in vitro, and in vivo assessments. Molecular dynamics simulations predicted that complex (complex-A) between HC-A and MAGL would be difficult to hydrolyze under acidic conditions. In vitro assessment using rat brain homogenate showed that [11C]HC-A reacted with MAGL to yield [11C]complex-A, which was rapidly hydrolyzed to liberate 11CO2. The 11CO2 liberation rate was slower at lower pH. In PET with [11C]HC-A using ischemic rats, the radioactivity clearance rate, which reflects the production rate of 11CO2 in the brain, was lower in a remarkably hypoxic area than in the contralateral region. In conclusion, we successfully visualized the pHi gradient in the brain using PET imaging.

neuroscience↗

bioRxiv2023

A hybrid in silico/in-cell controller for microbial bioprocesses with process-model mismatch

The optimization of bioprocess inputs using mathematical models is widely practiced. However, the mismatch between model prediction and the actual process [called process-model mismatch (PMM)] is problematic; when a large PMM exists, the process inputs optimized using the mathematical model in advance are no longer optimal for the actual process. In this study, we propose a hybrid control system that combines model-based optimization (in silico feedforward controller) and feedback controllers using synthetic genetic circuits integrated into cells (in-cell feedback controller) - which we named the hybrid in silico/in-cell controller (HISICC) - as a solution to this PMM issue. As a proof of concept for HISICC, we constructed a mathematical model of an engineered Escherichia coli strain for the isopropanol production process that was previously developed. This strain contains an in-cell feedback controller, and its combination with an in silico controller can be regarded as an example of HISICC. We demonstrated the robustness of HISICC against PMM by comparing the strain with another strain with no in-cell feedback controller in simulations assuming PMM of various magnitudes.

synthetic biology↗

bioRxiv2022

On-line reoptimization of mammalian fed-batch culture using a nonlinear model predictive controller

Fed-batch culture enables high productivity by maintaining low substrate concentrations in the early stage of the culture to suppress the accumulation of by-products that are harmful to cell growth. Therefore, they are widely used in the production of biopharmaceuticals by mammalian cells. However, there exists a trade-off in the design of the fed-batch process: early feeding results in the accumulation of harmful by-products, whereas late feeding results in a shortage of substrates needed for cell growth and synthesis of the desired product. To manage this trade-off and maximize the product yield, model-based optimization of the feeding trajectory has been reported in several studies. A significant drawback of this off-line optimization approach is the mismatch between the predictions made using the model and the actual process states, called the process-model mismatch (PMM). If the PMM is large, the off-line optimized feeding trajectory is no longer optimal for the actual process, resulting in lower product yields. Mammalian cell culture models typically contain dozens of unknown parameters that must be estimated prior to optimization. Sufficient parameter estimation is often unachievable owing to the nonlinear nature of these models. We believe that reoptimizing the feeding trajectory in real time using a nonlinear model predictive controller (NLMPC) is an effective solution to this PMM. Although NLMPC is a model-based feedback controller widely utilised in mammalian fed-batch culture, only a few studies have applied it to on-line reoptimization, and it remains unclear whether NLMPC with a standard kinetic model can effectively compensate for a large PMM. In this study, we demonstrated the reoptimization of the feeding trajectory with a NLMPC using two previously reported standard monoclonal antibody (mAb) production models. In both models, NLMPC successfully suppressed the reduction in mAb yield caused by the intentional introduction of PMM.

bioengineering↗

bioRxiv2018

Development of a microfluidic cell culture and monitoring system for intracellular signaling studies

We describe a microfluidic cell culture and monitoring system that temporally controls molecule concentrations around cells cultured in a small space. The simple system consists of three syringe pumps and a microfluidic device with two inlet ports and two outlet ports. Each syringe pump discharges or draws culture medium, solutions containing signal molecules, or cell suspensions through a port in a programmed flow rate sequence. Signal molecule solutions of differing concentration are merged in a microchannel, mixed immediately, and transported into the cell culture chamber. Regulating the flow rate ratio of syringe pumps over time enables dynamic control of the concentration of signal molecules in the cell culture chamber. The system provides various time-dependent waveforms of concentration over cultured cells, including pulse, rectangular, and triangular. The practical performance of the system for concentration control was evaluated using fluorescent dye imaging. The system was also used with CHO-K1 cells to measure intracellular Ca2+ concentrations, which vary with extracellular ATP levels. When a rectangular pulse of ATP was applied to the cells, Ca2+ levels increased quickly. By contrast, several Ca2+ peaks were observed in response to stepwise increases in ATP concentration. Single-cell Ca2+ responses to ATP pulse stimulation were analyzed by quantitative fluorescence imaging. Hierarchical clustering and quantitative analysis of single-cell data revealed the diversity of Ca2+ responses to ATP pulse stimulation. These results demonstrate that the microfluidic cell culture system is useful for studying a variety of cellular responses, including cell signaling.

bioengineering↗