To be launched on Aug 2015
Sponsored by Biophysical Society of China and Institute of Biophysics, CAS, P.R. China.
Online published by SpringerLink.

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Simulated microgravity potentiates generation of reactive oxygen species in cells
Fanlei Ran, Lili An, Yingjun Fan, Haiying Hang, Shihua Wang
Biophysics Reports    2016, 2 (5-6): 100-105.   DOI: 10.1007/s41048-016-0029-0
Abstract   PDF (500KB)
Microgravity (MG) and space radiation are two major environmental factors of space environment. Ionizing radiation generates reactive oxygen species (ROS) which plays a key role in radiation-induced DNA damage. Interestingly, simulated microgravity (SMG) also increases ROS production in various cell types. Thus, it is important to detect whether SMG could potentiate ROS production induced by genotoxins including radiation, especially at a minimal level not sufficient to induce detectable ROS. In this study, we treated mouse embryonic stem (MES) cells with H2O2 and SMG for 24 h. The concentration of H2O2 used was within 30 μmol/L at which intracellular ROS was the same as that in untreated cells. Exposure of cells to SMG for 24 h did not induce significantly higher levels of intracellular ROS than that of control cells either. Simultaneous exposure of cells to both SMG- and H2O2- induced ROS and apoptosis in MES cells. Although incubation in medium containing 5 or 30 lmol/L H2O2 induced a small enhancement of DNA double-strand breaks (DSBs), the addition of SMG treatment dramatically increased DSB levels. Taken together, SMG can significantly potentiate the effects of H2O2 at a low concentration that induce a small or negligible change in cells on ROS, apoptosis, and DNA damage. The results were discussed in relation to the combined effects of space radiation and MG on human body in this study.
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Regulation of metabolism by the mediator complex
Dou Yeon Youn, Alus M. Xiaoli, Jeffrey E. Pessin, Fajun Yang
Biophysics Reports    2016, 2 (2-4): 69-77.   DOI: 10.1007/s41048-016-0031-6
Abstract   PDF (437KB)
The Mediator complex was originally discovered in yeast, but it is conserved in all eukaryotes. Its bestknown function is to regulate RNA polymerase II-dependent gene transcription. Although the mechanisms by which the Mediator complex regulates transcription are often complicated by the contextdependent regulation, this transcription cofactor complex plays a pivotal role in numerous biological pathways. Biochemical, molecular, and physiological studies using cancer cell lines or model organisms have established the current paradigm of the Mediator functions. However, the physiological roles of the mammalian Mediator complex remain poorly defined, but have attracted a great interest in recent years. In this short review, we will summarize some of the reported functions of selective Mediator subunits in the regulation of metabolism. These intriguing findings suggest that the Mediator complex may be an important player in nutrient sensing and energy balance in mammals.
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Uniporter substrate binding and transport: reformulating mechanistic questions
Xuejun C. Zhang, Lei Han
Biophysics Reports    2016, 2 (2-4): 45-54.   DOI: 10.1007/s41048-016-0030-7
Abstract   PDF (870KB)
Transporters are involved in material transport, signaling, and energy input in all living cells. One of the fundamentalquestionsabout transportersisconcernedwiththepreciseroleoftheirsubstrateindrivingthe transport process. This is particularly important for uniporters, which must utilize the chemical potential of substrateastheonlyenergysourcedrivingthetransport.Thus,uniporterspresentanexcellentmodelforthe understanding of how the difference in substrate concentration across the membrane is used as a driving force. Local conformational changes induced by substrate binding are widely considered as the main mechanism to drive the functional cycle of a transporter; in addition, reducing the energy barrier of the transition state has also been proposed to drive the transporter. However, both points of view require modification to allow consolidation with fundamental thermodynamic principles. Here, we discuss the relationship between thermodynamics and kinetics of uniporters. Substrate binding-induced reduction of the transition-state energy barrier accelerates the transport process in kinetic terms, while the chemical potential of the substrate drives the process thermodynamically.
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Biophysics Reports    2016, 2 (1): 0-0.   DOI:
Abstract   PDF (1714KB)
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Combining biophysical methods to analyze the disulfide bond in SH2 domain of C-terminal Src kinase
Dongsheng Liu, David Cowburn
Biophysics Reports    2016, 2 (1): 33-43.   DOI: 10.1007/s41048-016-0025-4
Abstract   PDF (3116KB)
The Src Homology 2 (SH2) domain is a structurally conserved protein domain that typically binds to a phosphorylated tyrosine in a peptide motif from the target protein. The SH2 domain of C-terminal Src kinase (Csk) contains a single disulfide bond, which is unusual for most SH2 domains. Although the global motion of SH2 domain regulates Csk function, little is known about the relationship between the disulfide bond and binding of the ligand. In this study, we combined X-ray crystallography, solution NMR, and other biophysical methods to reveal the interaction network in Csk. Denaturation studies have shown that disulfide bond contributes significantly to the stability of SH2 domain, and crystal structures of the oxidized and C122S mutant showed minor conformational changes. We further investigated the binding of SH2 domain to a phosphorylated peptide from Csk-binding protein upon reduction and oxidation using both NMR and fluorescence approaches. This work employed NMR, X-ray cryptography, and other biophysical methods to study a disulfide bond in Csk SH2 domain. In addition, this work provides in-depth understanding of the structural dynamics of Csk SH2 domain.
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Symmetry-mismatch reconstruction of genomes and associated proteins within icosahedral viruses using cryo-EM
Xiaowu Li, Hongrong Liu, Lingpeng Cheng
Biophysics Reports    2016, 2 (1): 25-32.   DOI: 10.1007/s41048-016-0024-5
Abstract   PDF (1449KB)
Although near-atomic resolutions have been routinely achieved for structural determination of many icosahedral viral capsids, structures of genomes and associated proteins within the capsids are still less characterized because the genome information is overlapped by the highly symmetric capsid information in the virus particle images. We recently developed a software package for symmetry-mismatch structural reconstruction and determined the structures of the genome and RNA polymerases within an icosahedral virus for the first time. Here, we describe the protocol used for this structural determination, which may facilitate structural biologists in investigating the structures of viral genome and associated proteins.
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Exploring the inside details of virions by electron microscopy
Zheng Liu, Jingqiang Zhang
Biophysics Reports    2016, 2 (1): 21-24.   DOI: 10.1007/s41048-016-0022-7
Abstract   PDF (622KB)
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Radiolabeled cyclic RGD peptides as radiotracers for tumor imaging
Jiyun Shi, Fan Wang, Shuang Liu
Biophysics Reports    2016, 2 (1): 1-20.   DOI: 10.1007/s41048-016-0021-8
Abstract   PDF (3115KB)
The integrin family comprises 24 transmembrane receptors, each a heterodimeric combination of one of 18α and one of 8β subunits. Their main function is to integrate the cell adhesion and interaction with the extracellular microenvironment with the intracellular signaling and cytoskeletal rearrangement through transmitting signals across the cell membrane upon ligand binding. Integrin αvβ3 is a receptor for the extracellular matrix proteins containing arginine-glycine-aspartic (RGD) tripeptide sequence. The αvβ3 is generally expressed in low levels on the epithelial cells and mature endothelial cells, but it is highly expressed in many solid tumors. The αvβ3 levels correlate well with the potential for tumor metastasis and aggressiveness, which make it an important biological target for development of antiangiogenic drugs, and molecular imaging probes for early tumor diagnosis. Over the last decade, many radiolabeled cyclic RGD peptides have been evaluated as radiotracers for imaging tumors by SPECT or PET. Even though they are called "αvβ3-targeted" radiotracers, the radiolabeled cyclic RGD peptides are also able to bind αvβ5, α5β1, α6β4, α4β1, and αvβ6 integrins, which may help enhance their tumor uptake due to the "increased receptor population." This article will use the multimeric cyclic RGD peptides as examples to illustrate basic principles for development of integrin-targeted radiotracers and focus on different approaches to maximize their tumor uptake and T/B ratios. It will also discuss important assays for pre-clinical evaluations of the integrin-targeted radiotracers, and their potential applications as molecular imaging tools for noninvasive monitoring of tumor metastasis and early detection of the tumor response to antiangiogenic therapy.
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Crystal structures of MdfA complexed with acetylcholine and inhibitor reserpine
Ming Liu, Jie Heng, Yuan Gao, Xianping Wang
Biophysics Reports    2016, 2 (2-4): 78-85.   DOI: 10.1007/s41048-016-0028-1
Abstract   PDF (1269KB)
The DHA12 family of transporters contains a number of prokaryotic and eukaryote membrane proteins. Some of these proteins share conserved sites intrinsic to substrate recognition, structural stabilization and conformational changes. For this study, we chose the MdfA transporter as a model DHA12 protein to study some general characteristics of the vesicular neurotransmitter transporters (VNTs), which all belong to the DHA12 family. Two crystal structures were produced for E. coli MdfA, one in complex with acetylcholine and the other with potential reserpine, which are substrate and inhibitor of VNTs, respectively. These structures show that the binding sites of these two molecules are different. The Achbinding MfdA is mainly dependent on D34, while reserpine-binding site is more hydrophobic. Based on sequence alignment and homology modelling, we were able to provide mechanistic insights into the association between the inhibition and the conformational changes of these transporters.
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Structure determination of a human virus by the combination of cryo-EM and X-ray crystallography
Zheng Liu, Tom S. Y. Guu, Jianhao Cao, Yinyin Li, Lingpeng Cheng, Yizhi Jane Tao, Jingqiang Zhang
Biophysics Reports    2016, 2 (2-4): 55-68.   DOI: 10.1007/s41048-016-0027-2
Abstract   PDF (1791KB)
Virus 3D atomic structures provide insight into our understanding of viral life cycles and the development of antiviral drugs. X-ray crystallography and cryo-EM have been used to determine the atomic structure of viruses. However, limited availability of biological samples, biosafety issues due to virus infection, and sometimes inherent characteristics of viruses, pose difficulties on combining both methods in determining viral structures. These have made solving the high resolution structure of some medically important viruses very challenging. Here, we describe our recently employed protocols for determining the high-resolution structure of the virus-like particle of hepatitis E virus (HEV), a pathogen of viral hepatitis in human. These protocols include utilizing recombinant baculovirus system to generate sufficient amount of virus particles, single-particle cryo-EM to get an intermediate resolution structure as a phasing model, and X-ray crystallography for final atomic structure determination. Our protocols have solved the hepatitis E virus structure to the resolution of 3.5 Å. The combined methodology is generally applicable to other human infectious viruses.
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Opinion:hazards faced by macromolecules when confined to thin aqueous films
Robert M. Glaeser, Bong-Gyoon Han
Biophysics Reports    2017, 3 (1-3): 1-7.   DOI: 10.1007/s41048-016-0026-3
Abstract   PDF (584KB)
Samples prepared for single-particle electron cryo-microscopy (cryo-EM) necessarily have a very high surface-to-volume ratio during the short period of time between thinning and vitrification. During this time, there is an obvious risk that macromolecules of interest may adsorb to the air-water interface with a preferred orientation, or that they may even become partially or fully unfolded at the interface. In addition, adsorption of macromolecules to an air-water interface may occur even before thinning. This paper addresses the question whether currently used methods of sample preparation might be improved if one could avoid such interfacial interactions. One possible way to do so might be to preemptively form a surfactant monolayer over the air-water interfaces, to serve as a structure-friendly slide and coverslip. An alternative is to immobilize particles of interest by binding them to some type of support film, which-to continue using the analogy-thus serves as a slide. In this case, the goal is not only to prevent the particles of interest from diffusing into contact with the air-water interface but also to increase the number of particles seen in each image. In this direction, it is natural to think of developing various types of affinity grids as structure-friendly alternatives to thin carbon films. Perhaps ironically, if precautions are not taken against adsorption of particles to air-water interfaces, sacrificial monolayers of denatured protein may take the roles of slide, coverslip, or even both.
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E2-2, a novel immunohistochemical marker for both human and monkey plasmacytoid dendritic cells
Jianping Ma, Haisheng Yu, Xiangyun Yin, Menglan Cheng, Quanxing Shi, Zhao Yin, Xiaohua Nie, Wang Shouli, Liguo Zhang
Biophysics Reports    2015, 1 (3): 139-147.   DOI: 10.1007/s41048-016-0023-6
Abstract   PDF (3481KB)
Plasmacytoid dendritic cells (pDCs) play important roles in initiating and regulating immune responses. pDC infiltration has been documented in multiple pathological lesions including infections, tumors, and autoimmune diseases, and the severity of pDC infiltration correlates with disease progression. However, a specific antibody for identifying pDCs by immunohistochemical staining on paraffin-embedded tissue sections is still lacking. Here, we developed a novel antibody targeted E2-2, a transcription factor preferentially expressed in pDCs. The antibody stains the nuclei of pDCs specifically in immunohistochemical analysis of various tissues from both human and rhesus monkey. This novel antibody will serve as a beneficial tool for pDC-related basic research and clinical investigation.
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Pseudomonas sp. LZ-Q continuously degrades phenanthrene under hypersaline and hyperalkaline condition in a membrane bioreactor system
Yiming Jiang, Haiying Huang, Mengru Wu, Xuan Yu, Yong Chen, Pu Liu, Xiangkai Li
Biophysics Reports    2015, 1 (3): 156-167.   DOI: 10.1007/s41048-016-0018-3
Abstract   PDF (1399KB)
Phenanthrene is one of the most recalcitrant components of crude oil-contaminated wastewater. An efficient phenanthrene-degrading bacterium Pseudomonas sp. strain named LZ-Q was isolated from oil-contaminated soil near the sewage outlet of a petrochemical company. Pseudomonas sp. LZ-Q is able to degrade 1000 mg/L phenanthrene in Bushnell-Hass mineral salt medium. It also degrades other polycyclic aromatic hydrocarbons such as naphthalene, anthracene, pyrene, petrol, and diesel at broad ranges of salinities of 5 g/L to 75 g/L, pHs of 5.0-10.0, and temperatures of 10-42 ℃. Therefore, Pseudomonas sp. LZ-Q could be a good candidate for remediation of polycyclic aromatic hydrocarbon (PAH)-contaminated wastewater. A membrane bioreactor (MBR) was applied to investigate the remediation ability of the strain LZ-Q. Wastewater containing phenanthrene with pH of 8, salinity of 35 g/L, and COD of 500 mg/L was continuously added to the system (HRT = 3 h). Results showed that Pseudomonas sp. LZ-Q is capable of degrading 96% of 20 mg/L phenanthrene and 94% of 500 mg/L COD for 60 days in a continuous mode. These results showed that the MBR system with strain LZ-Q might be a good approach for PAHs' remediation in industrial wastewaters.
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Implications for directionality of nanoscale forces in bacterial attachment
Jan J. T. M. Swartjes, Deepak H. Veeregowda
Biophysics Reports    2015, 1 (3): 120-126.   DOI: 10.1007/s41048-016-0019-2
Abstract   PDF (1432KB)
Adhesion and friction are closely related and play a predominant role in many natural processes. From the wall-clinging feet of the gecko to bacteria forming a biofilm, in many cases adhesion is a necessity to survive. The direction in which forces are applied has shown to influence the bond strength of certain systems tremendously and can mean the difference between adhesion and detachment. The spatula present on the extension of the feet of the gecko can either attach or detach, based on the angle at which they are loaded. Certain proteins are known to unfold at different loads, depending on the direction at which the load is applied and some bacteria have specific receptors which increase their bond strength in the presence of shear. Bacteria adhere to any man-made surface despite the presence of shear forces due to running fluids, air flow, and other causes. In bacterial adhesion research, however, adhesion forces are predominantly measured perpendicularly to surfaces, whereas other directions are often neglected. The angle of shear forces acting on bacteria or biofilms will not be at a 90 angle, as shear induced by flow is often along the surface. Measuring at different angles or even lateral to the surface will give a more complete overview of the adhesion forces and mechanism, perhaps even resulting in alternative means to discourage bacterial adhesion or promote removal.
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Thermodynamics of GPCR activation
Xuejun C. Zhang, Ye Zhou, Can Cao
Biophysics Reports    2015, 1 (3): 115-119.   DOI: 10.1007/s41048-016-0017-4
Abstract   PDF (594KB)
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Radioligand saturation binding for quantitative analysis of ligand-receptor interactions
Chengyan Dong, Zhaofei Liu, Fan Wang
Biophysics Reports    2015, 1 (3): 148-155.   DOI: 10.1007/s41048-016-0016-5
Abstract   PDF (474KB)
The reversible combination of a ligand with specific sites on the surface of a receptor is one of the most important processes in biochemistry. A classic equation with a useful simple graphical method was introduced to obtain the equilibrium constant, Kd, and the maximum density of receptors, Bmax. The entire 125I-labeled ligand binding experiment includes three parts: the radiolabeling, cell saturation binding assays and the data analysis. The assay format described here is quick, simple, inexpensive, and effective, and provides a gold standard for the quantification of ligand-receptor interactions. Although the binding assays and quantitative analysis have not changed dramatically compared to the original methods, we integrate all the parts to calculate the parameters in one concise protocol and adjust many details according to our experience. In every step, several optional methods are provided to accommodate different experimental conditions. All these refinements make the whole protocol more understandable and user-friendly. In general, the experiment takes one person less than 8 h to complete, and the data analysis could be accomplished within 2 h.
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Visualizing the Ensemble Structures of Protein Complexes Using Chemical Cross-Linking Coupled with Mass Spectrometry
Zhou Gong, Yue-He Ding, Xu Dong, Na Liu, E. Erquan Zhang, Meng-Qiu Dong, Chun Tang
Biophysics Reports    2015, 1 (3): 127-138.   DOI: 10.1007/s41048-015-0015-y
Abstract   PDF (1992KB)
Chemical cross-linking coupled with mass spectrometry (CXMS) identifies protein residues that are close in space, and has been increasingly used for modeling the structures of protein complexes. Here we show that a single structure is usually sufficient to account for the intermolecular cross-links identified for a stable complex with sub-μmol/L binding affinity. In contrast,we show that the distance between two cross-linked residues in the different subunits of a transient or fleeting complex may exceed the maximum length of the cross-linker used, and the cross-links cannot be fully accounted for with a unique complex structure. We further show that the seemingly incompatible cross-links identified with high confidence arise from alternative modes of protein-protein interactions. By converting the intermolecular crosslinks to ambiguous distance restraints, we established a rigid-body simulated annealing refinement protocol to seek the minimum set of conformers collectively satisfying the CXMS data. Hence we demonstrate that CXMS allows the depiction of the ensemble structures of protein complexes and elucidates the interaction dynamics for transient and fleeting complexes.
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A new dimethyl labeling-based SID-MRM-MS method and its application to three proteases involved in insulin maturation
Dongwan Cheng, Li Zheng, Junjie Hou, Jifeng Wang, Peng Xue, Fuquan Yang, Tao Xu
Biophysics Reports    2015, 1 (2): 71-80.   DOI: 10.1007/s41048-015-0012-1
Abstract   PDF (2339KB)
The absolute quantification of target proteins in proteomics involves stable isotope dilution coupled with multiple reactions monitoring mass spectrometry (SID-MRM-MS). The successful preparation of stable isotope-labeled internal standard peptides is an important prerequisite for the SID-MRM absolute quantification methods. Dimethyl labeling has been widely used in relative quantitative proteomics and it is fast, simple, reliable, cost-effective, and applicable to any protein sample, making it an ideal candidate method for the preparation of stable isotope-labeled internal standards. MRM mass spectrometry is of high sensitivity, specificity, and throughput characteristics and can quantify multiple proteins simultaneously, including low-abundance proteins in precious samples such as pancreatic islets. In this study, a new method for the absolute quantification of three proteases involved in insulin maturation, namely PC1/3, PC2 and CPE, was developed by coupling a stable isotope dimethyl labeling strategy for internal standard peptide preparation with SID-MRM-MS quantitative technology. This method offers a new and effective approach for deep understanding of the functional status of pancreatic β cells and pathogenesis in diabetes.
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Skeletal intramyocellular lipid metabolism and insulin resistance
Yiran Li, Shimeng Xu, Xuelin Zhang, Zongchun Yi, Simon Cichello
Biophysics Reports    2015, 1 (2): 90-98.   DOI: 10.1007/s41048-015-0013-0
Abstract   PDF (428KB)
Lipids stored in skeletal muscle cells are known as intramyocellular lipid (IMCL). Disorders involving IMCL and its causative factor, circulatory free fatty acids (FFAs), induce a toxic state and ultimately result in insulin resistance (IR) in muscle tissue. On the other hand, intramuscular triglyceride (IMTG), the most abundant component of IMCL and an essential energy source for active skeletal muscle, is different from other IMCLs, as it is stored in lipid droplets and plays a pivotal role in skeletal muscle energy homeostasis. This review discusses the association of FFA-induced ectopic lipid accumulation and IR, with specific emphasis on the relationship between IMCL/IMTG metabolism and IR.
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The application of traditional transmission electron microscopy for autophagy research in Caenorhabditis elegans
Attila L. Kovács
Biophysics Reports    2015, 1 (2): 99-105.   DOI: 10.1007/s41048-015-0014-z
Abstract   PDF (362KB)
Traditional ultrastructural characterization of autophagic processes remains an important approach to be used in parallel with molecular genetics, light microscopy, and other methods. The special nature of Caenorhabditis elegans as an object for transmission electron microscopy makes its introduction into autophagy research a challenging task. The basis of the protocol to prepare C. elegans samples for autophagy studies was worked out around the turn of the millennium and has been used since then in my laboratory with some modifications. The method described here enables the user to prepare samples for systematic morphologic as well as morphometric investigations to characterize autophagy with a high but still realistic investment of effort.
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Detecting peroxiredoxin hyperoxidation by one-dimensional isoelectric focusing
Zhenbo Cao, Neil J. Bulleid
Biophysics Reports    2015, 1 (1): 14-17.   DOI: 10.1007/s41048-015-0007-y
Abstract   PDF (408KB)
The activity of typical 2-cys peroxiredoxin (Prxs) can be regulated by hyperoxidation with a consequent loss of redox activity. Here we developed a simple assay to monitor the level of hyperoxidation of different typical 2-cys prxs simultaneously. This assay only requires standard equipment and can compare different samples on the same gel. It requires much less time than conventional 2D gels and gives more information than Western blotting with an antibody specific for hyperoxidized peroxiredoxin. This method could also be used to monitor protein modification with a charge difference such as phosphorylation.
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A comprehensive procedure for antiviral inhibitor discovery using EV71 as an example
Lin Cao, Shouhai Zhu, Yaxin Wang, Zhiyong Lou, Yuna Sun
Biophysics Reports    2015, 1 (2): 81-89.   DOI: 10.1007/s41048-015-0006-z
Abstract   PDF (2725KB)
The prevalence of chronic viral infectious diseases, the emergence and re-emergence of new viral infections, and in particular, resistance to currently used antiviral drugs have led to increased demand for new antiviral strategies and reagents. Increased understanding of the molecular mechanisms of viral infection has provided great potential for the discovery of new antiviral agents that target viral proteins or host factors. In this work, we introduce a comprehensive system using enteroviruses 71 (EV71) as an example for leading compound discovery to develop new antiviral.
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Transcriptomic analysis of human breast cancer cells reveals differentially expressed genes and related cellular functions and pathways in response to gold nanorods
Teng Zhou, Yipeng Du, Taotao Wei
Biophysics Reports    2015, 1 (2): 106-114.   DOI: 10.1007/s41048-015-0005-0
Abstract   PDF (1703KB)
Breast cancer is the leading cause of cancer deaths inwomen.Recent advances in nanomedicine have shown that gold nanorods (AuNRs), as multifunctional drug delivery and photothermal therapeutic agents, have potential for use in cancer therapy. However, the effect of AuNRs on the transcriptome of breast cancer cells is unknown. In the present study, cells of the triple-negative human breast cancer cell line MDA-MB-231, which has high metastatic activity, were treated with AuNRs for transcriptomic analysis using RNA-seq technology. In total, 3126 genes were found to be up-regulated and 3558 genes were found to be downregulated inAuNR-treatedMDA-MB-231 cells. These differentially expressed genes presumably take part in multiple biological pathways, including glycolysis and regulation of the actin cytoskeleton, and impact a variety of cellular functions, including chemoattractant activity. The distinct gene expression profile of MDA-MB-231 cells treated with AuNRs provides a foundation for further screening and validation of important genes involved in the interaction between AuNRs and MDA-MB-231 cells.
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Phosphorylation and function of DGAT1 in skeletal muscle cells
Jinhai Yu, Yiran Li, Fei Zou, Shimeng Xu, Pingsheng Liu
Biophysics Reports    2015, 1 (1): 41-50.   DOI: 10.1007/s41048-015-0004-1
Abstract   PDF (5854KB)
Aberrant intramuscular triacylglycerol (TAG) storage in human skeletal muscle is closely related to insulin insensitivity. Excessive lipid storage can induce insulin resistance of skeletal muscle, and under severe conditions, lead to type 2 diabetes. The balance of interconversion between diacylglycerol and TAG greatly influences lipid storage and utilization. Diacylglycerol O-acyltransferase 1 (DGAT1) plays a key role in this process, but its activation and phosphorylation requires further dissection. In this study, 12 putative conserved phosphorylation sites of DGAT1 were identified by examining amino acid conservation of DGAT1 in different species. Another 12 putative phosphorylation sites were also found based on bioinformatics predictions and previous reports. Meanwhile, several phosphorylation sites of DGAT1 were verified by phosphorylation mass spectrometry analysis of purified DGAT1 from mouse myoblast C2C12 cells. Using single point mutations, a regulatory role of 3 putative phosphorylation sites was dissected. Finally, using truncation mutations, a potential domain of DGAT1 that was involved in the regulation of enzymatic activity was revealed. This study provides useful information for further understanding DGAT1 activity regulation.
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Biophysics Reports    DOI:
Biophysics Reports: focus on theoretical and technical advances
Tao Xu
Biophysics Reports    2015, 1 (1): 1-1.   DOI: 10.1007/s41048-015-0010-3
Abstract   PDF (301KB)
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Analysis of chromatin bersin Hela cells with electron tomography
Xiaomin Li, Hongli Feng, Jianguo Zhang, Lei Sun, Ping Zhu
Biophysics Reports    2015, 1 (1): 51-60.   DOI: 10.1007/x41048-015-0009-9
Abstract   PDF (2590KB)
The presence and folding pattern of chromatin in eukaryotic cells remain elusive and controversial. In this study, we prepared ultra-thin sections of Hela cells with three different fixation and sectioning methods, i.e., chemical fixation, high pressure freezing with freeze substitution, and cryo-ultramicro- tomy with SEM-FIB (focused ion beam), and analyzed in vivo architecture of chromatin fibers in Hela nuclei with electron tomography technology. The results suggest that the chromatin fibers in eukaryotic Hela cells are likely organized in an architecture with a diameter of about 30 nm.
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An efficient two-step subcellular fractionation method for the enrichment of insulin granules from INS-1 cells
Yan Chen, Zhiping Xia, Lifen Wang, Yong Yu, Pingsheng Liu, Eli Song, Tao Xu
Biophysics Reports    2015, 1 (1): 34-40.   DOI: 10.1007/s41048-015-0008-x
Abstract   PDF (1234KB)
Insulin is one of the key regulators for blood glucose homeostasis. More than 99% of insulin is secreted from the pancreatic β-cells. Within each β-cell, insulin is packaged and processed in insulin secretary granules (ISGs) before its exocytosis. Insulin secretion is a complicated but well-organized dynamic process that includes the budding of immature ISGs (iISGs) from the trans-Golgi network, iISG maturation, and mature ISG (mISG) fusion with plasma membrane. However, the molecular mechanisms involved in this process are largely unknown. It is therefore crucial to separate and enrich iISGs and mISGs before determining their distinct characteristics and protein contents. Here, we developed an efficient two-step subcellular fractionation method for the enrichment of iISGs and mISGs from INS-1 cells: OptiPrep gradient purification followed by Percoll solution purification. We demonstrated that by using this method, iISGs and mISGs can be successfully distinguished and enriched. This method can be easily adapted to investigate SGs in other cells or tissues, thereby providing a useful tool for elucidating the mechanisms of granule maturation and secretion.
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Solubilization and purification of recombinant modified C-reactive protein from inclusion bodies using reversible anhydride modification
Lawrence A. Potempa, Zhen-Yu Yao, Shang-Rong Ji, János G. Filep, Yi Wu
Biophysics Reports    2015, 1 (1): 18-33.   DOI: 10.1007/s41048-015-0003-2
Abstract   PDF (2149KB)
The precise function of C-reactive protein (CRP) as a regulator of inflammation in health and disease continues to evolve. The true understanding of its role in host defense responses has been hampered by numerous reports of comparable systems with contradictory interpretations of CRP as a stimulator, suppressor, or benign contributor to such processes. These discrepancies may be explained in part by the existence of a naturally occurring CRP isoform, termed modified CRP (i.e., mCRP), that is expressed when CRP subunits are dissociated into monomeric structures. The free mCRP subunit undergoes a non-proteolytic conformational change that has unique solubility, antigenicity, and bioactivity compared to the subunits that remain associated in the native, pentameric CRP molecule (i.e., pCRP). As specific reagents have been developed to identify and quantify mCRP, it has become apparent that this isoform can be formed spontaneously in calcium-free solutions. Furthermore, mCRP can be expressed on perturbed cell membranes with as little as 24-48 h incubation in tissue culture. Because mCRP has the same size as pCRP subunits as evaluated by SDS-PAGE, its presence in a pCRP reagent would not be apparent using this technique to evaluate purity. Finally, because many antibody reagents purported to be specific for "CRP" contains some, or substantial specificity tomCRP, antigen-detection techniques using such reagents may fail to distinguish the specific CRP isoform detected. All these caveats concerning CRP structures and measurements suggest that the aforementioned contradictory studies may reflect to some extent on distinctive bioactivities of mCRP rather than on pCRP. To provide a reliable, abundant supply of mCRP for separate and comparable studies, a recombinant proteinwas engineered and expressed in E. coli (i.e., recombinant mCRP or rmCRP). Synthesized protein was produced as inclusion bodies which proved difficult to solubilize for purification and characterization. Herein, we describe a method using anhydride reagents to effectively solubilize rmCRP and allow for chromatographic purification in high yield and free of contaminating endotoxin. Furthermore, the purified rmCRP reagent represents an excellent comparable protein to the biologically produced mCRP and as a distinctive reagent from pCRP. Deciphering the true function of CRP in both health and disease requires a knowledge, understanding, and reliable supply of each of its structures so to define the distinctive effects of each on the body's response to tissue damaging events.
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Phenylboronic acid-functionalized magnetic nanoparticles for one-step saccharides enrichment and mass spectrometry analysis
Xiangdong Xue, Yuanyuan Zhao, Xu Zhang, Chunqiu Zhang, Anil Kumar, Xiaoning Zhang, Guozhang Zou, Paul C. Wang, Jinchao Zhang, Xing-Jie Liang
Biophysics Reports    DOI: 10.1007/s41048-015-0002-3
Abstract   PDF (1530KB)
In this work, 2-(2-aminoethoxy) ethanolblocked phenylboronic acid-functionalized magnetic nanoparticles (blocked PMNPs) were fabricated for selective enrichment of different types of saccharides. The phenylboronic acid was designed for capturing the cis-diols moieties on saccharides molecules, and the 2-(2-aminoethoxy) ethanol can deplete the nonspecific absorption of peptides and proteins which always coexisted with saccharides. For mass spectrometry analysis, the PMNPs bound saccharides can be directly applied onto the MALDI plate with matrix without removing the PMNPs. By PMNPs, the simple saccharide (glucose) could be detected in pmol level. The complex saccharides can also be reliably purified and analyzed; 16 different Nglycans were successfully identified fromovalbumin, and the high-abundance N-glycans can be detected even when the ovalbumin was in very low concentration (2 μg). In human milk, ten different oligosaccharides were identified, and the lactose can still be detected when the human milk concentration was low to 0.01 μL.
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CN 10-1302/Q
ISSN 2364-3439
eISSN 2364-3420
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