Hirofumi Kobayashi

Effects of reduced frequency breathing on arterial hypoxemia during exercise.

SummaryIt is uncertain that exercise with reduced frequency breathing (RFB) results in arterial hypoxemia. This study was designed to investigate whether RFB during exercise creates a true hypoxic condition in arterial blood by examining arterial oxygen saturation (SaO2) directly. Six subjects performed ten 30 s periods of exercise on a Monark bicycle ergometer at a work rate of 210 W alternating with 30 s rest intervals. The breath was controlled to use 1 s each for inspiration and expiration, and two trials with different breathing patterns were used; a continuous breathing (CB) trial and an RFB trial consisting of four seconds of breath-holding at functional residual capacity (FRC). Alveolar oxygen pressure during exercise showed a slight but significant (p<0.05) reduction with RFB as compared to CB. However, a marked increase in alveolar-arterial pressure difference for oxygen (A-aDO2) (p<0.05) with RFB over CB resulted in a marked (p<0.05) reduction in arterial oxygen pressure. Consequently, SaO2 fell as low as 88.8% on average. Additional examination of RFB with breath-holding at total lung capacity showed no increases in A-aDO2 in spite of the same amount of hypoventilation as compared with that at FRC. These results indicate that RFB during exercise can result in arterial hypoxemia if RFB is performed with breath-holding at FRC, this mechanism being closely related to the mechanical responses due to lung volume restriction.

High-throughput label-free image cytometry and image-based classification of live Euglena gracilis.

We demonstrate high-throughput label-free single-cell image cytometry and image-based classification of Euglena gracilis (a microalgal species) under different culture conditions. We perform it with our high-throughput optofluidic image cytometer composed of a time-stretch microscope with 780-nm resolution and 75-Hz line rate, and an inertial-focusing microfluidic device. By analyzing a large number of single-cell images from the image cytometer, we identify differences in morphological and intracellular phenotypes between E. gracilis cell groups and statistically classify them under various culture conditions including nitrogen deficiency for lipid induction. Our method holds promise for real-time evaluation of culture techniques for E. gracilis and possibly other microalgae in a non-invasive manner.

A unique form of light‐load training improves steadiness and performance on some functional tasks in older adults

Beginning Movement Load (BML) training is a unique form of light‐load training that comprises a lengthening–shortening sequence of muscle actions about multiple degrees of freedom. The purpose of the study was to determine the effectiveness of BML training at improving the performance of old adults on four functional tasks and to identify some of the neuromuscular adaptations that contributed to these gains. Healthy old adults (67.5 ± 5.23 years) were randomly assigned to either a BML training group (n = 17) or a control group (n = 7). The training group exercised with a 30% of the one repetition‐maximum (1‐RM) load and performed five to seven sets of 15 repetitions, three times per week for 8 weeks. BML training increased maximal voluntary contraction (MVC) force significantly for the knee extensors (31.6%), but not the elbow flexors (9.8%), and improved the steadiness of isometric contractions (10%, 30%, and 65% MVC forces). Training‐associated changes in times for ascending and descending stairs and one‐legged balance, but not the chair rise, were predicted by changes in selected combinations of MVC force and steadiness. The attributes of BML training that enabled it to elicit functionally meaningful adaptations in the neuromuscular system of older adults should be explored with more mechanistic studies.

High-throughput, label-free, single-cell, microalgal lipid screening by machine-learning-equipped optofluidic time-stretch quantitative phase microscopy

The development of reliable, sustainable, and economical sources of alternative fuels to petroleum is required to tackle the global energy crisis. One such alternative is microalgal biofuel, which is expected to play a key role in reducing the detrimental effects of global warming as microalgae absorb atmospheric CO2 via photosynthesis. Unfortunately, conventional analytical methods only provide population‐averaged lipid amounts and fail to characterize a diverse population of microalgal cells with single‐cell resolution in a non‐invasive and interference‐free manner. Here high‐throughput label‐free single‐cell screening of lipid‐producing microalgal cells with optofluidic time‐stretch quantitative phase microscopy was demonstrated. In particular, Euglena gracilis, an attractive microalgal species that produces wax esters (suitable for biodiesel and aviation fuel after refinement), within lipid droplets was investigated. The optofluidic time‐stretch quantitative phase microscope is based on an integration of a hydrodynamic‐focusing microfluidic chip, an optical time‐stretch quantitative phase microscope, and a digital image processor equipped with machine learning. As a result, it provides both the opacity and phase maps of every single cell at a high throughput of 10,000 cells/s, enabling accurate cell classification without the need for fluorescent staining. Specifically, the dataset was used to characterize heterogeneous populations of E. gracilis cells under two different culture conditions (nitrogen‐sufficient and nitrogen‐deficient) and achieve the cell classification with an error rate of only 2.15%. The method holds promise as an effective analytical tool for microalgae‐based biofuel production.

A Behavioral Mechanism of How Increases in Leg Strength Improve Old Adults’ Gait Speed

We examined a behavioral mechanism of how increases in leg strength improve healthy old adults’ gait speed. Leg press strength training improved maximal leg press load 40% (p = 0.001) and isometric strength in 5 group of leg muscles 32% (p = 0.001) in a randomly allocated intervention group of healthy old adults (age 74, n = 15) but not in no-exercise control group (age 74, n = 8). Gait speed increased similarly in the training (9.9%) and control (8.6%) groups (time main effect, p = 0.001). However, in the training group only, in line with the concept of biomechanical plasticity of aging gait, hip extensors and ankle plantarflexors became the only significant predictors of self-selected and maximal gait speed. The study provides the first behavioral evidence regarding a mechanism of how increases in leg strength improve healthy old adults’ gait speed.

Label-free detection of cellular drug responses by high-throughput bright-field imaging and machine learning

In the last decade, high-content screening based on multivariate single-cell imaging has been proven effective in drug discovery to evaluate drug-induced phenotypic variations. Unfortunately, this method inherently requires fluorescent labeling which has several drawbacks. Here we present a label-free method for evaluating cellular drug responses only by high-throughput bright-field imaging with the aid of machine learning algorithms. Specifically, we performed high-throughput bright-field imaging of numerous drug-treated and -untreated cells (N = ~240,000) by optofluidic time-stretch microscopy with high throughput up to 10,000 cells/s and applied machine learning to the cell images to identify their morphological variations which are too subtle for human eyes to detect. Consequently, we achieved a high accuracy of 92% in distinguishing drug-treated and -untreated cells without the need for labeling. Furthermore, we also demonstrated that dose-dependent, drug-induced morphological change from different experiments can be inferred from the classification accuracy of a single classification model. Our work lays the groundwork for label-free drug screening in pharmaceutical science and industry.

A replication-incompetent influenza virus bearing the HN glycoprotein of human parainfluenza virus as a bivalent vaccine

Influenza virus and human parainfluenza virus (HPIV) are major etiologic agents of acute respiratory illness in young children. Inactivated and live attenuated influenza vaccines are approved in several countries, yet no vaccine is licensed for HPIV. We previously showed that a replication-incompetent PB2-knockout (PB2-KO) virus that possesses a reporter gene in the coding region of the PB2 segment can serve as a platform for a bivalent vaccine. To develop a bivalent vaccine against influenza and parainfluenza virus, here, we generated a PB2-KO virus possessing the hemagglutinin-neuraminidase (HN) glycoprotein of HPIV type 3 (HPIV3), a major surface antigen of HPIV, in its PB2 segment. We confirmed that this virus replicated only in PB2-expressing cells and expressed HN. We then examined the efficacy of this virus as a bivalent vaccine in a hamster model. High levels of virus-specific IgG antibodies in sera and IgA, IgG, and IgM antibodies in bronchoalveolar lavage fluids against both influenza virus and HPIV3 were detected from hamsters immunized with this virus. The neutralizing capability of these serum antibodies was also confirmed. Moreover, the immunized hamsters were completely protected from virus challenge with influenza virus or HPIV3. These results indicate that PB2-KO virus expressing the HN of HPIV3 has the potential to be a novel bivalent vaccine against influenza and human parainfluenza viruses.

Preferred step frequency minimizes veering during natural human walking

In the absence of visual information, humans cannot maintain a straight walking path. We examined the hypothesis that step frequency during walking affects the magnitude of veering in healthy adults. Subject walked at a preferred (1.77 ± 0.18 Hz), low (0.8 × preferred, 1.41 ± 0.15 Hz), and high (1.2× preferred, 2.13 ± 0.20 Hz) step frequency with and without a blindfold. We compared the absolute differences between estimated and measured points of crossing a target line after 16 m of forward walking at the three step frequencies. There was no significant difference in veering when subjects walked at the different frequencies without a blindfold. However, the magnitude of veering was the smallest at the preferred (mean ± SE=91.6 ± 33.6 cm) compared with the low (204.3 ± 43.0 cm) and high (112.7 ± 34.0 cm) frequency gaits with a blindfold. Thus, walking at a preferred step frequency minimizes veering, which occurs in the absence of visual information. This phenomenon may be associated with the previously reported minimization of movement variability, energy cost, and attentional demand while walking at a preferred step frequency.

Optofluidic time-stretch quantitative phase microscopy

Innovations in optical microscopy have opened new windows onto scientific research, industrial quality control, and medical practice over the last few decades. One of such innovations is optofluidic time-stretch quantitative phase microscopy - an emerging method for high-throughput quantitative phase imaging that builds on the interference between temporally stretched signal and reference pulses by using dispersive properties of light in both spatial and temporal domains in an interferometric configuration on a microfluidic platform. It achieves the continuous acquisition of both intensity and phase images with a high throughput of more than 10,000 particles or cells per second by overcoming speed limitations that exist in conventional quantitative phase imaging methods. Applications enabled by such capabilities are versatile and include characterization of cancer cells and microalgal cultures. In this paper, we review the principles and applications of optofluidic time-stretch quantitative phase microscopy and discuss its future perspective.

Enhanced speed in fluorescence imaging using beat frequency multiplexing

Fluorescence imaging using radiofrequency-tagged emission (FIRE) is an emerging technique that enables higher imaging speed (namely, temporal resolution) in fluorescence microscopy compared to conventional fluorescence imaging techniques such as confocal microscopy and wide-field microscopy. It works based on the principle that it uses multiple intensity-modulated fields in an interferometric setup as excitation fields and applies frequency-division multiplexing to fluorescence signals. Unfortunately, despite its high potential, FIRE has limited imaging speed due to two practical limitations: signal bandwidth and signal detection efficiency. The signal bandwidth is limited by that of an acousto-optic deflector (AOD) employed in the setup, which is typically 100-200 MHz for the spectral range of fluorescence excitation (400-600 nm). The signal detection efficiency is limited by poor spatial mode-matching between two interfering fields to produce a modulated excitation field. Here we present a method to overcome these limitations and thus to achieve higher imaging speed than the prior version of FIRE. Our method achieves an increase in signal bandwidth by a factor of two and nearly optimal mode matching, which enables the imaging speed limited by the lifetime of the target fluorophore rather than the imaging system itself. The higher bandwidth and better signal detection efficiency work synergistically because higher bandwidth requires higher signal levels to avoid the contribution of shot noise and amplifier noise to the fluorescence signal. Due to its unprecedentedly high-speed performance, our method has a wide variety of applications in cancer detection, drug discovery, and regenerative medicine.