Biophysics Reports

2021 Vol. 7, No. 3

Cover Story

Although single-particle cryogenic electron microscopy (cryo-EM) has been applied extensively for elucidating many crucial biological mechanisms at the molecular level, this technique still faces critical challenges, the major one of which is to prepare the high-quality cryo-EM specimen. Aiming to achieve a more reproducible and efficient cryo-EM specimen preparation, novel supporting films including graphene-based two-dimensional materials have been explored in recent years. Here the authors report a robust and simple method to fabricate EM grids coated with single- or few-layer reduced graphene oxide (RGO) membrane in large batch for high-resolution cryo-EM structural determination. The RGO membrane has decreased interlayer space and enhanced electrical conductivity in comparison to regular graphene oxide (GO) membrane. Moreover, they found that the RGO supporting film exhibited nice particle-absorption ability, thus avoiding the air–water interface problem. More importantly, they found that the RGO supporting film is particularly useful in cryo-EM reconstruction of sub-100-kDa biomolecules at near-atomic resolution, as exemplified by the study of RBD-ACE2 complex and other small protein molecules. The authors envision that the RGO membranes can be used as a robust graphene-based supporting film in cryo-EM specimen preparation.

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REVIEW
PROTOCOL
METHOD
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INVITED REVIEW

Emerging two-dimensional material nanozymes for theranostic nanomedicine

Yanling You, Zhongmin Tang, Han Lin, Jianlin Shi

2021, 7(3): 159-172. doi: 10.52601/bpr.2021.210011

Abstract(2629)

PDF(137)

Abstract:
Nanomaterials-based artificial enzymes (nanozymes) with valuable enzyme-like catalytic properties have been booming during the past few years. Promoted by the advances in biological medicine and nanotechnology, nanozymes possess the potential to serve as an emerging agent for biosensing, immunoassays, detection and diagnosis, catalytic therapeutics, and other applications in the biomedicine field. Two-dimensional (2D) nanomaterials are of considerable interest in biomedical applications due to their ultrathin layered structure and unique physiochemical properties. Inspired by the diversified catalytic performance of 2D nanomaterials, scientists extensively have developed 2D materials as bioactive nanozymes for theranostic nanomedicine. Here, recent advances in enzyme-like 2D nanomaterials design and construction are comprehensively presented. Additionally, we exhibit that, with the synergistic effect of catalytic activities and desirable physicochemical performances, 2D nanozymes can serve as versatile platforms with extensive applications from target detection to in vivo theranostic. It is believed that such promising alternatives towards natural enzymes will be of vital significance in the field of nanotechnology and biomedicine.

REVIEW

Latest developments on the mechanism of action of membrane disrupting peptides

Sara Pandidan, Adam Mechler

2021, 7(3): 173-184. doi: 10.52601/bpr.2021.200037

Abstract(1085)

PDF(87)

Abstract:
Antimicrobial peptides (AMPs) are integral components of the innate immune defence system of all complex organisms including plants, insects, and mammals. They have wide range of antibacterial, antifungal, antiviral, and even anticancer activities, therefore AMPs are attractive candidates for developing novel therapeutic approaches. Cationic α-helical membrane disrupting peptides are perhaps the most widely studied subclass of AMPs due to their common fundamental characteristics that allow for detailed structure-function analysis and therefore offer a promising solution to the threat of multidrug resistant strains of bacteria. The majority of the studies of AMP activity focused on the biological and biophysical aspects of membrane disruption; the understanding of the molecular mechanism of action from the physicochemical point of view forms a relatively small subfield. This review will provide an overview of these works, focusing on the empirical and thermodynamic models of AMP action.

Research progress of STC2 in breast cancer

Xuezhi Niu, Yong Zhan, Suhua Zhang, Zixin Liu, Chang Qu

2021, 7(3): 185-192. doi: 10.52601/bpr.2021.210002

Abstract(1367)

PDF(33)

Abstract:
Breast cancer ranks second in the list of most common cancers among women and brings the double burden of economy and health to women. Therefore, it is an urgent and necessary task to study the pathogenic mechanism and the treatment of breast cancer. Glycoprotein hormone is a kind of hormones to promote the growth and the development of cell and stanniocalcin 2 (STC2) is one of them. Research has shown us a various expression of SCT2 in organs and tissues and it can regulate many different pathological and physiological processes. In addition, there are a lot of previous studies that indicated a close correlation between STC2 and the development and metastasis of many cancers, which infers STC2 can serve as biomarker of certain cancers. Until now, the effects of STC2 on breast cancer have been studied widely, but research findings demonstrated two different views, one view is that STC2 plays an oncogenic role and the other is the opposite. In this paper, it will summarize and evaluate the research data and results about mammalian STC2 on breast cancer.

What do we know about IL-6 in COVID-19 so far?

Jingrui Jiang, Jun Wang, Lulu Yao, Shenghan Lai, Xueji Zhang

2021, 7(3): 193-206. doi: 10.52601/bpr.2021.200024

Abstract(1727)

PDF(37)

Abstract:
Interleukin 6 (IL-6) is a cytokine with dual functions of pro-inflammation and anti-inflammation. It is mainly produced by mononuclear macrophages, Th2 cells, vascular endothelial cells and fibroblasts. IL-6 binds to glycoprotein 130 and one of these two receptors, membrane-bound IL-6R or soluble IL-6R, forming hexamer (IL-6/IL-6R/gp130), which then activates different signaling pathways (classical pathway, trans-signaling pathway) to exert dual immune-modulatory effects of anti-inflammation or pro-inflammation. Abnormal levels of IL-6 can cause multiple pathological reactions, including cytokine storm. Related clinical studies have found that IL-6 levels in severe COVID-19 patients were much higher than in healthy population. A large number of studies have shown that IL-6 can trigger a downstream cytokine storm in patients with COVID-19, resulting in lung damages, aggravating clinical symptoms and developing excessive inflammation and acute respiratory distress syndrome (ARDS). Monoclonal antibodies against IL-6 or IL-6R, such as tocilizumab, sarilumab, siltuximab and olokizumab may serve as therapeutic options for COVID-19 infection.

PROTOCOL

How to use open-pFind in deep proteomics data analysis?— A protocol for rigorous identification and quantitation of peptides and proteins from mass spectrometry data

Guangcan Shao, Yong Cao, Zhenlin Chen, Chao Liu, Shangtong Li, Hao Chi, Meng-Qiu Dong

2021, 7(3): 207-226. doi: 10.52601/bpr.2021.210004

Abstract(7355)

PDF(618)

Abstract:
High-throughput proteomics based on mass spectrometry (MS) analysis has permeated biomedical science and propelled numerous research projects. pFind 3 is a database search engine for high-speed and in-depth proteomics data analysis. pFind 3 features a swift open search workflow that is adept at uncovering less obvious information such as unexpected modifications or mutations that would have gone unnoticed using a conventional data analysis pipeline. In this protocol, we provide step-by-step instructions to help users mastering various types of data analysis using pFind 3 in conjunction with pParse for data pre-processing and if needed, pQuant for quantitation. This streamlined pParse-pFind-pQuant workflow offers exceptional sensitivity, precision, and speed. It can be easily implemented in any laboratory in need of identifying peptides, proteins, or post-translational modifications, or of quantitation based on ¹⁵N-labeling, SILAC-labeling, or TMT/iTRAQ labeling.

METHOD

Reduced graphene oxide membrane as supporting film for high-resolution cryo-EM

Nan Liu, Liming Zheng, Jie Xu, Jia Wang, Cuixia Hu, Jun Lan, Xing Zhang, Jincan Zhang, Kui Xu, Hang Cheng, Zi Yang, Xin Gao, Xinquan Wang, Hailin Peng, Yanan Chen, Hong-Wei Wang

2021, 7(3): 227-238. doi: 10.52601/bpr.2021.210007

Abstract(5283)

PDF(324)

Abstract:
Although single-particle cryogenic electron microscopy (cryo-EM) has been applied extensively for elucidating many crucial biological mechanisms at the molecular level, this technique still faces critical challenges, the major one of which is to prepare the high-quality cryo-EM specimen. Aiming to achieve a more reproducible and efficient cryo-EM specimen preparation, novel supporting films including graphene-based two-dimensional materials have been explored in recent years. Here we report a robust and simple method to fabricate EM grids coated with single- or few-layer reduced graphene oxide (RGO) membrane in large batch for high-resolution cryo-EM structural determination. The RGO membrane has decreased interlayer space and enhanced electrical conductivity in comparison to regular graphene oxide (GO) membrane. Moreover, we found that the RGO supporting film exhibited nice particle-absorption ability, thus avoiding the air–water interface problem. More importantly, we found that the RGO supporting film is particularly useful in cryo-EM reconstruction of sub-100-kDa biomolecules at near-atomic resolution, as exemplified by the study of RBD-ACE2 complex and other small protein molecules. We envision that the RGO membranes can be used as a robust graphene-based supporting film in cryo-EM specimen preparation.

RESEARCH ARTICLE

Endogenous reactive oxygen species and nitric oxide have opposite roles in regulating HIF-1alpha expression in hypoxic astrocytes

Qingquan Chen, Wenlan Liu, Xi Sun, Ke Jian Liu, Rong Pan

2021, 7(3): 239-249. doi: 10.52601/bpr.2021.200016

Abstract(1302)

PDF(63)

Abstract:
Ischemic stroke results in cerebral tissue hypoxia and increased expression of hypoxia-inducible factor (HIF), which is critically implicated in ischemic brain injury. Understanding the mechanisms of HIF-1alpha regulation in the ischemic brain has been an important research focus. The generation of both nitric oxide (NO) and reactive oxygen species (ROS) is increased under hypoxic/ischemic conditions and each of them has been independently shown to regulate HIF-1alpha expression. In this study, we investigated the cross-effects of NO and ROS on the expression of HIF-1alpha in hypoxic astrocytes. Exposure of astrocytes to 2 h-hypoxia remarkably increased HIF-1alpha protein levels, which was accompanied by increased NO and ROS production. Decreasing ROS with NAC, NADPH oxidase inhibitor DPI, or SOD mimetic MnTMPyP decreased hypoxia-induced HIF-1alpha protein accumulation and increased NO level in hypoxic astrocytes. The NO synthase (NOS) inhibitor L-NAME inhibited ROS generation, which led to a reduction in hypoxia-induced HIF-1alpha protein expression. Although NOS inhibitor or ROS scavengers alone reduced HIF-1alpha protein levels, the reduction was reversed when NOS inhibitor and ROS scavenger present together. The NO scavenger PTIO increased hypoxia-induced HIF-1alpha protein expression and ROS production, while HIF-1alpha protein level was decreased in the presence of NO scavenger and ROS scavenger together. These results suggest that ROS, NO, and their interaction critically contribute to the regulation of hypoxia-induced HIF-1alpha protein accumulation under hypoxic condition. Furthermore, our results indicate that hypoxia-induced NO generation may represent an endogenous mechanism for balancing ROS-mediated hypoxic stress, as reflected by inhibiting hypoxia-induced HIF-1alpha protein accumulation.