Isolation and characterization of rabbit limbal niche cells.

Limbal niche cells (LNCs) are one of the most important supporting cells for corneal epithelial stem cells (CES), however, research on LNCs has been mostly limited to humans and rats previously. To expand the research work into the rabbit animal model, one of the most often used animals in stem cell study, this study was carried out for the in vitro isolation and identification of rabbit LNCs. Rabbit LNCs were isolated by collagenase A digestion method and single cells were obtained, the cells were then seeded on 5% Matrigel-coated plastic surface and cultured in modified embryonic stem cell medium (MESCM). Three biological replicates of the isolating and characterization were recorded from New Zealand White rabbits aged from 2.5 months to 5 months. LNC markers (VIM/CD90/CD105/SCF/PDGFRß) were analyzed using tyramide signal amplification (TSA) staining, immunohistochemical staining (IHC), western blotting (WB), and real-time reverse transcription polymerase chain reaction (qPCR). TSA staining suggested that VIM was highly expressed in rabbit limbus stroma, which was confirmed by WB, and P63α was expressed in the basal limbus epithelium. Pan-CK and CK12 were highly expressed in the central corneal epithelium but lightly expressed in the limbal epithelium. The WB result indicated that PDGFRß and VIM expressions in rabbit-LNCs P4 were higher than in P1 and P7. In addition, rabbit corneal epithelium highly expressed Paired Box 6 (PAX6) and Epidermal growth factor-like domain 6(EGFL6). For the three repeat experiments, the cell expansion activity of rabbit-LNC was highest at P4. Rabbit-LNCs were passaged from P0 to P7, and the number of cell doublings (NCD) of P4 for the three repeat experiments was 2.816, 2.737, and 2.849. qPCR showed that high mRNA expression levels of VIM, CD90, CD105, SCF, and PDGFRß in rabbit-LNCs P4. In conclusion, rabbit-LNCs could be successfully isolated by the collagenase A digestion method as used in human tissue. There were similar characteristics between rabbit and human LNCs (VIM+/CD90+/CD105+/SCF+/PAX6+/PDGFRß+).

Personal exposure to airborne organic pollutants and lung function changes among healthy older adults.

Epidemiological evidence on the impact of airborne organic pollutants on lung function among the elderly is limited, and their underlying biological mechanisms remain largely unexplored. Herein, a longitudinal panel study was conducted in Jinan, Shandong Province, China, involving 76 healthy older adults monitored over a span of five months repetitively. We systematically evaluated personal exposure to a diverse range of airborne organic pollutants using a wearable passive sampler and their effects on lung function. Participants' pulmonary function indicators were assessed, complemented by comprehensive multi-omics analyses of blood and urine samples. Leveraging the power of interaction analysis, causal inference test (CIT), and integrative pathway analysis (IPA), we explored intricate relationships between specific organic pollutants, biomolecules, and lung function deterioration, elucidating the biological mechanisms underpinning the adverse impacts of these pollutants. We observed that bis (2-chloro-1-methylethyl) ether (BCIE) was significantly associated with negative changes in the forced vital capacity (FVC), with glycerolipids mitigating this adverse effect. Additionally, 31 canonical pathways [e.g., high mobility group box 1 (HMGB1) signaling, phosphatidylinositol 3-kinase (PI3K)/AKT pathway, epithelial mesenchymal transition, and heme and nicotinamide adenine dinucleotide (NAD) biosynthesis] were identified as potential mechanisms. These findings may hold significant implications for developing effective strategies to prevent and mitigate respiratory health risks arising from exposure to such airborne pollutants. However, due to certain limitations of the study, our results should be interpreted with caution.

Small molecule generation via disentangled representation learning.

MOTIVATION: Expanding our knowledge of small molecules beyond what is known in nature or designed in wet laboratories promises to significantly advance cheminformatics, drug discovery, biotechnology and material science. In silico molecular design remains challenging, primarily due to the complexity of the chemical space and the non-trivial relationship between chemical structures and biological properties. Deep generative models that learn directly from data are intriguing, but they have yet to demonstrate interpretability in the learned representation, so we can learn more about the relationship between the chemical and biological space. In this article, we advance research on disentangled representation learning for small molecule generation. We build on recent work by us and others on deep graph generative frameworks, which capture atomic interactions via a graph-based representation of a small molecule. The methodological novelty is how we leverage the concept of disentanglement in the graph variational autoencoder framework both to generate biologically relevant small molecules and to enhance model interpretability. RESULTS: Extensive qualitative and quantitative experimental evaluation in comparison with state-of-the-art models demonstrate the superiority of our disentanglement framework. We believe this work is an important step to address key challenges in small molecule generation with deep generative frameworks. AVAILABILITY AND IMPLEMENTATION: Training and generated data are made available at https://ieee-dataport.org/documents/dataset-disentangled-representation-learning-interpretable-molecule-generation. All code is made available at https://anonymous.4open.science/r/D-MolVAE-2799/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Purely nonlinear amplified ultralow-noise transmission using hybrid second-order DRA and PPLN-based PSA.

We report ultralow-noise transmission over a 102-km single-mode fiber using a purely nonlinear amplification scheme consisting of a second-order distributed Raman amplifier (DRA) and a phase-sensitive amplifier (PSA) based on periodically poled LiNbO3 waveguides. The hybrid DRA/PSA features a broadband gain over the C and L bands and an ultralow-noise advantage, with a less than -6.3 dB effective noise figure in the DRA stage and a 1.6 dB optical signal-to-noise ratio (OSNR) improvement in the PSA stage. Compared with the unamplified link, the OSNR is improved by 10.2 dB for a 20-Gbaud 16QAM signal in the C band, resulting in error-free detection (a bit-error rate of less than 3.8 × 10-3) for the signal with a low link input power of -25 dBm. Mitigation of nonlinear distortion is also achieved by the proposed nonlinear amplified system due to the subsequent PSA.

Single-cell microliter-droplet screening system (MISS Cell): An integrated platform for automated high-throughput microbial monoclonal cultivation and picking.

Solid plates have been used for microbial monoclonal isolation, cultivation, and colony picking since 1881. However, the process is labor- and resource-intensive for high-throughput requirements. Currently, several instruments have been integrated for automated and high-throughput picking, but complicated and expensive. To address these issues, we report a novel integrated platform, the single-cell microliter-droplet screening system (MISS Cell), for automated, high-throughput microbial monoclonal colony cultivation and picking. We verified the monoclonality of droplet cultures in the MISS Cell and characterized culture performance. Compared with solid plates, the MISS Cell generated a larger number of monoclonal colonies with higher initial growth rates using fewer resources. Finally, we established a workflow for automated high-throughput screening of Corynebacterium glutamicum using the MISS Cell and identified high glutamate-producing strains. The MISS Cell can serve as a universal platform to efficiently produce monoclonal colonies in high-throughput applications, overcoming the limitations of solid plates to promote rapid development in biotechnology.

First donor haemovigilance system at a national level in China: Establishment and improvement.

BACKGROUND AND OBJECTIVES: No systematic study has measured the incidence of adverse reactions (ARs) to blood donation at the national level in China before 2019. The objective of this study was to establish an effective reporting system to collect information on ARs to blood donation in China. MATERIALS AND METHODS: The status of donor haemovigilance (DHV) in blood collection facilities in China was evaluated, and an online DHV system was established to collect data on ARs to blood donation in July 2019. The definitions of ARs were based on the International Society of Blood Transfusion (ISBT) standards. The prevalence and data quality of ARs from 2019 to 2021 were analysed. RESULTS: A standard online reporting system has been established for ARs to blood donation. In total, 61, 62 and 81 participating sites were included in this pilot study in 2019, 2020 and 2021, respectively. From July 2019 to December 2021, 21,502 cases of whole-blood-related ARs and 1114 cases of apheresis platelet-related ARs were reported, with an incidence of 3.8‰ and 2.2‰, respectively. Data completeness for key reporting elements improved from 41.7% (15/36) in 2019 to 74.4% (29/39) in 2020. Data quality analysis for the year 2021 yielded similar results as for 2020. CONCLUSION: The construction and continuous improvement of the blood donor safety monitoring system prompted the establishment of the DHV system. Improvements have been made to the DHV system in China, with a significant increase in sentinels and higher data quality.

ISSLS Prize in Bioengineering Science 2023: Age- and sex-related differences in lumbar intervertebral disc degeneration between patients with chronic low back pain and asymptomatic controls.

PURPOSE: Clinical management of disc degeneration in patients with chronic low back pain (cLBP) is hampered by the challenge of distinguishing pathologic changes relating to pain from physiologic changes related to aging. The goal of this study was to use imaging biomarkers of disc biochemical composition to distinguish degenerative changes associated with cLBP from normal aging. METHODS: T1ρ MRI data were acquired from 133 prospectively enrolled subjects for this observational study (80 cLBP, 53 controls; mean ± SD age = 43.9 ± 13.4 years; 61 females, 72 males). The mean T1ρ relaxation time in the nucleus pulposus (NP-T1ρ; n = 650 discs) was used as a quantitative biomarker of disc biochemical composition. Linear regression was used to assess associations between NP-T1ρ and age, sex, spinal level, and study group, and their interactions. RESULTS: NP-T1ρ values were lower in cLBP patients than controls (70.8 ± 22.8 vs. 76.4 ± 22.2 ms, p = 0.009). Group differences were largest at L5-S1 (ΔT1ρcLBP-control = -11.3 ms, p < 0.0001), representing biochemical deterioration typically observed over a 9-12 year period (NP-T1ρ declined by 0.8-1.1 ms per year [95% CI]). Group differences were large in younger patients and diminished with age. Finally, the age-dependence of disc degeneration was stronger in controls than cLBP patients. CONCLUSION: Aging effects on the biochemical composition of the L5-S1 disc may involve a relatively uniform set of factors from which many cLBP patients deviate. NP-T1ρ values at L5-S1 may be highly relevant to clinical phenotyping, particularly in younger individuals.

A randomized controlled study of remote computerized cognitive, neurofeedback, and combined training in the treatment of children with attention-deficit/hyperactivity disorder.

There is an increasing interest in non-pharmacological treatments for children with attention-deficit/hyperactivity disorder (AD/HD), especially digital techniques that can be remotely delivered, such as neurofeedback (NFT) and computerized cognitive training (CCT). In this study, a randomized controlled design was used to compare training outcomes between remotely delivered NFT, CCT, and combined NFT/CCT training approaches. A total of 121 children with AD/HD were randomly assigned to the NFT, CCT, or NFT/CCT training groups, with 80 children completing the training program. Pre- and post-training symptoms (primary outcome), executive and daily functions were measured using questionnaires as well as resting EEG during eyes-closed (EC) and eyes-open (EO) conditions. After 3 months of training, the inattentive and hyperactive/impulsive symptoms, inhibition, working memory, learning and life skills of the three groups of children were significantly improved. The objective EEG activity showed a consistent increase in the relative alpha power in the EO condition among the three training groups. Training differences were not observed between groups. There was a positive correlation between pre-training EO relative alpha power and symptom improvement scores of inattention and hyperactivity/impulsivity, as well as a negative correlation between pre-training inattention scores and change in EO relative alpha. This study verified the training effects of NFT, CCT, and combined NFT/CCT training in children with AD/HD and revealed an objective therapeutic role for individual relative alpha activity. The verified feasibility and effectiveness of home-based digital training support promotion and application of digital remote training.

Symptom-guided multimodal neuroimage fusion patterns in children with attention-deficit/hyperactivity disorder and its potential "brain structure-function-cognition-behavior" pathological pathways.

The "brain-cognition-behavior" process is an important pathological pathway in children with attention-deficit/hyperactivity disorder (ADHD). Symptom guided multimodal neuroimaging fusion can capture behaviorally relevant and intrinsically linked structural and functional features, which can help to construct a systematic model of the pathology. Analyzing the multimodal neuroimage fusion pattern and exploring how these brain features affect executive function (EF) and leads to behavioral impairment is the focus of this study. Based on gray matter volume (GMV) and fractional amplitude of low frequency fluctuation (fALFF) for 152 ADHD and 102 healthy controls (HC), the total symptom score (TO) was set as a reference to identify co-varying components. Based on the correlation between the identified co-varying components and EF, further mediation analysis was used to explore the relationship between brain image features, EF and clinical symptoms. This study found that the abnormalities of GMV and fALFF in ADHD are mainly located in the default mode network (DMN) and prefrontal-striatal-cerebellar circuits, respectively. GMV in ADHD influences the TO through Metacognition Index, while fALFF in HC mediates the TO through behavior regulation index (BRI). Further analysis revealed that GMV in HC influences fALFF, which further modulates BRI and subsequently affects hyperactivity-impulsivity score. To conclude, structural brain abnormalities in the DMN in ADHD may affect local brain function in the prefrontal-striatal-cerebellar circuit, making it difficult to regulate EF in terms of inhibit, shift, and emotional control, and ultimately leading to hyperactive-impulsive behavior.

Optical injection locking based local oscillator regeneration for continuous variable quantum key distribution.

We develop an optical injection locking (OIL) based local oscillator (LO) regeneration for continuous variable quantum key distribution (CVQKD) by sending a weak polarization multiplexed pilot carrier from the transmitter. The OIL at the receiver has superior performance in terms of minimum input power and noise level at offset frequencies to the erbium-doped fiber amplifier (EDFA)-based scheme. The weak pilot carrier is recovered both in power and phase via the OIL while incurring little excess noise to the CVQKD system. The phase-locked LO enables heterodyne detection of a Gaussian modulated quantum signal with a simple data-aided phase recovery without pilot tone. The obtained parameters are compatible with a raw key rate of 0.83 Mbit/s in the asymptotic regime over a 22-km fiber transmission. The technique is expected to be used in more phase-sensitive quantum optical applications.

Paraspinal Muscle in Chronic Low Back Pain: Comparison Between Standard Parameters and Chemical Shift Encoding-Based Water-Fat MRI.

BACKGROUND: Paraspinal musculature (PSM) is increasingly recognized as a contributor to low back pain (LBP), but with conventional MRI sequences, assessment is limited. Chemical shift encoding-based water-fat MRI (CSE-MRI) enables the measurement of PSM fat fraction (FF), which may assist investigations of chronic LBP. PURPOSE: To investigate associations between PSM parameters from conventional MRI and CSE-MRI and between PSM parameters and pain. STUDY TYPE: Prospective, cross-sectional. POPULATION: Eighty-four adults with chronic LBP (44.6 ± 13.4 years; 48 males). FIELD STRENGTH/SEQUENCE: 3-T, T1-weighted fast spin-echo and iterative decomposition of water and fat with echo asymmetry and least squares estimation sequences. ASSESSMENT: T1-weighted images for Goutallier classification (GC), muscle volume, lumbar indentation value, and muscle-fat index, CSE-MRI for FF extraction (L1/2-L5/S1). Pain was self-reported using a visual analogue scale (VAS). Intra- and/or interreader agreement was assessed for MRI-derived parameters. STATISTICAL TESTS: Mixed-effects and linear regression models to 1) assess relationships between PSM parameters (entire cohort and subgroup with GC grades 0 and 1; statistical significance α = 0.0025) and 2) evaluate associations of PSM parameters with pain (α = 0.05). Intraclass correlation coefficients (ICCs) for intra- and/or interreader agreement. RESULTS: The FF showed excellent intra- and interreader agreement (ICC range: 0.97-0.99) and was significantly associated with GC at all spinal levels. Subgroup analysis suggested that early/subtle changes in PSM are detectable with FF but not with GC, given the absence of significant associations between FF and GC (P-value range: 0.036 at L5/S1 to 0.784 at L2/L3). Averaged over all spinal levels, FF and GC were significantly associated with VAS scores. DATA CONCLUSION: In the absence of FF, GC may be the best surrogate for PSM quality. Given the ability of CSE-MRI to detect muscle alterations at early stages of PSM degeneration, this technique may have potential for further investigations of the role of PSM in chronic LBP. LEVEL OF EVIDENCE: 2 TECHNICAL EFFICACY STAGE: 2.

Flexible SERS Substrate with a Ag-SiO2 Cosputtered Film for the Rapid and Convenient Detection of Thiram.

It is very important to build uniform large-area dense hotspots to improve the surface-enhanced Raman scattering (SERS) detection limit. In our research, we designed and prepared a new flexibile SERS substrate with ultradense hot spots that has the advantages of high sensitivity, good repeatability, easy fabrication, and low cost. Due to the special dense hot spot structure, the substrate reaches a SERS enhancement factor of 2.1 × 1011. Because of the excellent physical stability of polydimethylsiloxane, the substrate can be bent at will, and the SERS performance will not change with bending. This is very important to extract effective detection objects on complex surfaces. The substrate has good light transmittance and softness and can be directly attached to the detected agricultural products to realize real-time and rapid SERS monitoring. This structure exhibits extraordinary performance for thiram detection in the ultralow concentration range of 10-13 M.

The contributions of cartilage endplate composition and vertebral bone marrow fat to intervertebral disc degeneration in patients with chronic low back pain.

PURPOSE: The composition of the subchondral bone marrow and cartilage endplate (CEP) could affect intervertebral disc health by influencing vertebral perfusion and nutrient diffusion. However, the relative contributions of these factors to disc degeneration in patients with chronic low back pain (cLBP) have not been quantified. The goal of this study was to use compositional biomarkers derived from quantitative MRI to establish how CEP composition (surrogate for permeability) and vertebral bone marrow fat fraction (BMFF, surrogate for perfusion) relate to disc degeneration. METHODS: MRI data from 60 patients with cLBP were included in this prospective observational study (28 female, 32 male; age = 40.0 ± 11.9 years, 19-65 [mean ± SD, min-max]). Ultra-short echo-time MRI was used to calculate CEP T2* relaxation times (reflecting biochemical composition), water-fat MRI was used to calculate vertebral BMFF, and T1ρ MRI was used to calculate T1ρ relaxation times in the nucleus pulposus (NP T1ρ, reflecting proteoglycan content and degenerative grade). Univariate linear regression was used to assess the independent effects of CEP T2* and vertebral BMFF on NP T1ρ. Mixed effects multivariable linear regression accounting for age, sex, and BMI was used to assess the combined relationship between variables. RESULTS: CEP T2* and vertebral BMFF were independently associated with NP T1ρ (p = 0.003 and 0.0001, respectively). After adjusting for age, sex, and BMI, NP T1ρ remained significantly associated with CEP T2* (p = 0.0001) but not vertebral BMFF (p = 0.43). CONCLUSION: Poor CEP composition plays a significant role in disc degeneration severity and can affect disc health both with and without deficits in vertebral perfusion.

A Foreground-Aware Framework for Local Face Attribute Transfer.

In the context of social media, large amounts of headshot photos are taken everyday. Unfortunately, in addition to laborious editing and modification, creating a visually compelling photographic masterpiece for sharing requires advanced professional skills, which are difficult for ordinary Internet users. Though there are many algorithms automatically and globally transferring the style from one image to another, they fail to respect the semantics of the scene and are unable to allow users to merely transfer the attributes of one or two face organs in the foreground region leaving the background region unchanged. To overcome this problem, we developed a novel framework for semantically meaningful local face attribute transfer, which can flexibly transfer the local attribute of a face organ from the reference image to a semantically equivalent organ in the input image, while preserving the background. Our method involves warping the reference photo to match the shape, pose, location, and expression of the input image. The fusion of the warped reference image and input image is then taken as the initialized image for a neural style transfer algorithm. Our method achieves better performance in terms of inception score (3.81) and Fréchet inception distance (80.31), which is about 10% higher than those of competitors, indicating that our framework is capable of producing high-quality and photorealistic attribute transfer results. Both theoretical findings and experimental results are provided to demonstrate the efficacy of the proposed framework, reveal its superiority over other state-of-the-art alternatives.

Unsupervised Exemplar-Domain Aware Image-to-Image Translation.

Image-to-image translation is used to convert an image of a certain style to another of the target style with the original content preserved. A desired translator should be capable of generating diverse results in a controllable many-to-many fashion. To this end, we design a novel deep translator, namely exemplar-domain aware image-to-image translator (EDIT for short). From a logical perspective, the translator needs to perform two main functions, i.e., feature extraction and style transfer. With consideration of logical network partition, the generator of our EDIT comprises of a part of blocks configured by shared parameters, and the rest by varied parameters exported by an exemplar-domain aware parameter network, for explicitly imitating the functionalities of extraction and mapping. The principle behind this is that, for images from multiple domains, the content features can be obtained by an extractor, while (re-)stylization is achieved by mapping the extracted features specifically to different purposes (domains and exemplars). In addition, a discriminator is equipped during the training phase to guarantee the output satisfying the distribution of the target domain. Our EDIT can flexibly and effectively work on multiple domains and arbitrary exemplars in a unified neat model. We conduct experiments to show the efficacy of our design, and reveal its advances over other state-of-the-art methods both quantitatively and qualitatively.

Li2 MTeO6 (M=Ti, Sn): Mid-Infrared Nonlinear Optical Crystal with Strong Second Harmonic Generation Response and Wide Transparency Range.

Oxide crystals have been widely used in nonlinear optics (NLO) in the ultraviolet-visible and near-infrared regions. Most traditional oxide crystals are restricted to the mid-infrared region due to their narrow transmission window. Hence, attempting to extend infrared cutoff wavelength of oxides has attracted much attention. Herein, we report two new tellurates Li2 TiTeO6 (LTT) and Li2 SnTeO6 (LST) with broad transparent regions of 0.38-6.72 and 0.38-6.86 µm, respectively, as excellent candidates for mid-infrared NLO applications. Both LTT and LST crystallize in the orthorhombic space group Pnn2. The LTT crystal exhibits intense powder second-order generation efficiency (26×KDP) under the fundamental wavelength of 1064 nm. First-principles calculations and dipole moments were used to illustrate the results of the powder second-harmonic generations based on the crystal structures. Our results provide a novel oxide NLO crystal with a strong SHG and wide transparency range. They also pave a way for the design of new oxide mid-IR NLO crystals.

Noise evolution with the phase-sensitive gain in a hybrid fiber phase-sensitive amplifier.

We investigate the noise evolution of a hybrid fiber phase-sensitive amplifier (PSA) with an erbium-doped fiber amplifier (EDFA) as a function of the phase-sensitive gain. The noise performance of the hybrid amplifier with a large overall gain of 45.6 dB is experimentally evaluated in optical and electrical domains. The experimental results are in good agreement with theory, showing that significant improvement of the overall noise figure (NF) of the hybrid amplifier occurs in a low PSA gain region. A combined NF below 3 dB has been obtained in the hybrid amplifier with a PSA gain of 9 dB, which is 1.1 dB better than a conventional EDFA with an equivalent overall gain.

On-chip high-sensitivity photonic temperature sensor based on a GaAs microresonator.

We demonstrate an on-chip high-sensitivity photonic temperature sensor based on a GaAs microdisk resonator. Based on the large thermo-optic coefficient of GaAs, a temperature sensitivity of 0.142 nm/K with a measurement resolution of 10 mK and low input optical power of only 0.5 µW was achieved. It exhibits great potential for chip-scale biological research and integrated photonic signal processing.

Microbial microdroplet culture system (MMC): An integrated platform for automated, high-throughput microbial cultivation and adaptive evolution.

Conventional microbial cell cultivation techniques are typically labor intensive, low throughput, and poorlyparallelized, rendering them inefficient. The development of automated, modular microbial cell micro-cultivation systems, particularly those employing droplet microfluidics, have gained attention for their high-throughput, highly paralellized and efficient cultivation capabilities. Here, we report the development of a microbial microdroplet culture system (MMC), which is an integrated platform for automated, high-throughput cultivation and adaptive evolution of microorganisms. We demonstrated that the MMC yielded both accurate and reproducible results for the manipulation and detection of droplets. The superior performance of MMC for microbial cell cultivation was validated by comparing the growth curves of six microbial strains grown in MMC, conventional shake flasks or well plates. The highest incipient growth rate for all six microbial strains was achieved by using MMC. We also conducted an 18-day process of adaptive evolution of methanol-essential Escherichia coli strain in MMC and obtained two strains exhibiting higher growth rates compared with the parent strain. Our study demonstrates the power of MMC to provide an efficient and reliable approach for automated, high-throughput microbial cultivation and adaptive evolution.

An integrated microfluidic device for single cell trapping and osmotic behavior investigation of mouse oocytes.

Transport properties of oocytes play an important role in the optimization of their cryopreservation. However, there are still no systematical investigations on oocyte transport properties from the viewpoint of single-cell trapping and high precision perfusion, especially with the powerful microfluidic approach. To this end, we developed an easy-to-fabricate and easy-to-use microfluidic chip along with automatic single cell trapping capability to investigate the oocyte membrane transport properties. The experimental results indicate that the device is available and reliable. We further performed a comparative study of the oocyte membrane transport properties between single and multi-step CPA addition protocols and confirmed that the transport property parameters measured by single-step osmotic shift could not be used for prediction of the osmotic responses of oocytes in multi-step CPA addition. This study provides a powerful tool for investigation of oocyte osmotic responses.