Biophysics Reports

2019 Vol. 5, No. 2

Cover Story

The imaging rate of structured illumination microscopy (SIM) reached 188 Hz recently. As the exposure time decreases, the camera detects fewer virtual photons, while the noise level remains the same. As a result, the signal-to-noise ratio (SNR) decreases sharply. Furthermore, the SNR decreases further because of photobleaching and phototoxicity. This decreased quality of SIM raw data may lead to surprising artifacts with various causes, which may confuse a new user of SIM microscopy. We summarize three significant possible sources of severe artifacts in reconstructed super-resolution (SR) images. Ultrafast motion of a biological sample or an uneven illumination pattern is the most difficult to be identified. The estimated parameter could also be incorrect, leading to artifact of regular patterns. Furthermore, reconstruction with the Wiener method generates stochastic artifacts due to the amplification of noise during the deconvolution process. To deal with these problems, we have established a protocol to reconstruct ultrafast SIM raw data obtained in low SNR conditions. First, we checked the quality of the raw data with the ImageJ plugin SIMcheck before reconstruction. Then, a modified parameter estimation method was used to improve the precision of the parameters. Finally, an iterative algorithm was used for SIM reconstruction under low signal-to-noise ratio conditions. This procedure effectively suppressed the artifacts in the super-resolution images reconstructed from raw data of low signal-to-noise ratio.

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Energy coupling mechanism of FO in a rotary ATP synthase: a model update

Xuejun C. Zhang, Min Liu

2019, 5(2): 61-64. doi: 10.1007/s41048-018-0077-8

Abstract(73)

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Abstract:

CHDOCK: a hierarchical docking approach for modeling C_n symmetric homo-oligomeric complexes

Yumeng Yan, Sheng-You Huang

2019, 5(2): 65-72. doi: 10.1007/s41048-019-0088-0

Abstract(292)

PDF(99)

Abstract:
Protein-protein interactions are crucial in many biological processes. Therefore, determining the complex structure between proteins is valuable for understanding the molecular mechanism and developing drugs. Many proteins like ion channels are formed by symmetric homo-oligomers. In this study, we have proposed a hierarchical docking algorithm to predict the structure of C_n symmetric protein complexes, which is referred to as CHDOCK. The symmetric binding modes were first constructed by an FFT-based docking algorithm and then optimized by our iterative scoring function for protein-protein interactions. When tested on a symmetric protein docking benchmark of 212 homooligomeric complexes with C_n symmetry, CHDOCK obtained a significantly better performance in binding mode predictions than three state-of-the-art symmetric docking methods, M-ZDOCK, SAM, and SymmDock. When the top 10 predictions were considered, CHDOCK achieved a success rate of 44.81% and 72.17% for unbound docking and bound docking, respectively in comparison to those of 36.79% and 65.09% for M-ZDOCK, 31.60% and 54.25% for SAM, and 30.66% and 31.60% for SymmDock. CHDOCK is computationally efficient and can normally complete a symmetric docking calculation within 30 min. The CHDOCK can be freely accessed by a web server at http://huanglab.phys.hust.edu.cn/hsymdock/.

A light-addressable microfluidic device for label-free functional assays of bioengineered taste receptor cells via extracellular recording

Liping Du, Wei Chen, Yulan Tian, Ping Zhu, Jian Wang, Wen Cai, Chunsheng Wu

2019, 5(2): 73-79. doi: 10.1007/s41048-019-0085-3

Abstract(124)

PDF(62)

Abstract:
The functional assay of chemical sensitive cells is of great importance for the preparation of sensitive elements towards the development of cell-based biosensors for chemical sensing. In this study, a novel light-addressable microfluidic device was developed by the integration of a light-addressable potentiometric sensor (LAPS) with a microfluidic chip for label-free functional assays of bioengineered taste receptor cells via extracellular recording. Extracellular potential changes of single bioengineered cells were recorded by LAPS. Microfluidic chip was capable of providing stable microenvironments for cell measurements with a well-defined concentration stimulus. Bioengineered taste receptor cells were utilized as a model of chemical sensitive cells and functional assayed by this light-addressable microfluidic device using bitter stimulation. The results indicate that this microfluidic device can efficiently monitor the membrane potential changes originated from bioengineered taste receptor cells in response to specific bitter stimulation. The bioengineered cells with responsive functions can be easily identified via the analysis on the firing rates of extracellular recording data. The expression of bitter receptors in the bioengineered taste receptor cells was further validated by the immunofluorescent staining results, which proved that the expression of specific bitter receptor was the main contribution to the responsive function of bioengineered cells. This microfluidic device can not only be used for the functional assays of chemical sensitive cells towards chemical sensing, but also suitable to be applied for the research on the chemical signal transduction mechanisms.

A protocol for structured illumination microscopy with minimal reconstruction artifacts

Junchao Fan, Xiaoshuai Huang, Liuju Li, Shan Tan, Liangyi Chen

2019, 5(2): 80-90. doi: 10.1007/s41048-019-0081-7

Abstract(473)

PDF(79)

Abstract:
The imaging rate of structured illumination microscopy (SIM) reached 188 Hz recently. As the exposure time decreases, the camera detects fewer virtual photons, while the noise level remains the same. As a result, the signal-to-noise ratio (SNR) decreases sharply. Furthermore, the SNR decreases further because of photobleaching and phototoxicity. This decreased quality of SIM raw data may lead to surprising artifacts with various causes, which may confuse a new user of SIM microscopy. We summarize three significant possible sources of severe artifacts in reconstructed super-resolution (SR) images. Ultrafast motion of a biological sample or an uneven illumination pattern is the most difficult to be identified. The estimated parameter could also be incorrect, leading to artifact of regular patterns. Furthermore, reconstruction with the Wiener method generates stochastic artifacts due to the amplification of noise during the deconvolution process. To deal with these problems, we have established a protocol to reconstruct ultrafast SIM raw data obtained in low SNR conditions. First, we checked the quality of the raw data with the ImageJ plugin SIMcheck before reconstruction. Then, a modified parameter estimation method was used to improve the precision of the parameters. Finally, an iterative algorithm was used for SIM reconstruction under low signal-to-noise ratio conditions. This procedure effectively suppressed the artifacts in the super-resolution images reconstructed from raw data of low signal-to-noise ratio.

A new co-culture method for identifying synaptic adhesion molecules involved in synapse formation

Wei Jiang, Jihong Gong, Yi Rong, Xiaofei Yang

2019, 5(2): 91-97. doi: 10.1007/s41048-019-0084-4

Abstract(115)

PDF(144)

Abstract:
The proper formation of synapses is essential for brain function. Synaptic cell adhesion molecules (CAMs) are thought to play essential roles in the initiation of the synapse formation process. The artificial synapse formation assay, in which non-neuronal cells and neurons are co-cultured, has been shown to be a powerful system for screening CAMs. However, controlling a large number of cell pools in co-culture is complicated, creating a potential barrier for high-throughput screening. This protocol describes a new co-culture method in which cDNA plasmid is transfected into human embryonic kidney 293T cells using polyetherimide 24 h after cells were mixed with neurons, and immunostaining and confocal imaging are employed for analyzing synaptogenesis. This optimized method is simpler and easier to perform than the traditional method for the examination of the synaptogenic activities of individual cell-surface proteins in isolation, and provides an unbiased screening platform for synaptogenic proteins.

Identification of key genes and pathways for Alzheimer's disease via combined analysis of genome-wide expression profiling in the hippocampus

Mengsi Wu, Kechi Fang, Weixiao Wang, Wei Lin, Liyuan Guo, Jing Wang

2019, 5(2): 98-109. doi: 10.1007/s41048-019-0086-2

Abstract(369)

PDF(142)

Abstract:
In this study, combined analysis of expression profiling in the hippocampus of 76 patients with Alzheimer's disease (AD) and 40 healthy controls was performed. The effects of covariates (including age, gender, postmortem interval, and batch effect) were controlled, and differentially expressed genes (DEGs) were identified using a linear mixed-effects model. To explore the biological processes, functional pathway enrichment and protein-protein interaction (PPI) network analyses were performed on the DEGs. The extended genes with PPI to the DEGs were obtained. Finally, the DEGs and the extended genes were ranked using the convergent functional genomics method. Eighty DEGs with q < 0.1, including 67 downregulated and 13 upregulated genes, were identified. In the pathway enrichment analysis, the 80 DEGs were significantly enriched in one Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, GABAergic synapses, and 22 Gene Ontology terms. These genes were mainly involved in neuron, synaptic signaling and transmission, and vesicle metabolism. These processes are all linked to the pathological features of AD, demonstrating that the GABAergic system, neurons, and synaptic function might be affected in AD. In the PPI network, 180 extended genes were obtained, and the hub gene occupied in the most central position was CDC42. After prioritizing the candidate genes, 12 genes, including five DEGs (ITGB5, RPH3A, GNAS, THY1, and SEPT6) and seven extended genes (JUN, GDI1,GNAI2, NEK6, UBE2D3, CDC42EP4, and ERCC3), were found highly relevant to the progression of AD and recognized as promising biomarkers for its early diagnosis.